2015 Van Andel Research Institute Scientific Report

IT STARTS HERE Van Andel Research Institute Scientific Report 2015

Compiled and edited by David E. Nadziejka and Leah A. Sienkowski.

Published January 2015.

Copyright 2015 by the Van Andel Institute; all rights reserved.

Van Andel Institute, 333 Bostwick Avenue, N.E. Grand Rapids, Michigan 49503, U.S.A.

In 2014, Van Andel Research Institute started a new chapter in its history. Our commitment is to become a global center for epigenetics, a burgeoning frontier in biomedical research. We see great potential to expand and enrich the exceptional work already underway at VARI in cancer and neurodegenerative disease research.

Advancements will take shape in many ways, including new collaborations with colleagues around the globe (including collaborations on clinical trials) and an emphasis on strategic recruitment.

In short, VARI is poised for significant growth.

Our dedication to translating basic research into clinical applications is stronger than ever, and in the coming years we expect to continue making a significant difference for those who matter most— the people affected by the diseases we study.

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Director's Introduction 1 Laboratory Reports Center for Cancer and Cell Biology

Table of Contents

Arthur S. Alberts, Ph.D. Laboratory of Cell Structure and Signal Integration

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Nicholas S. Duesbery, Ph.D. Laboratory of Cancer and Developmental Cell Biology

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Carrie R. Graveel, Ph.D. Breast Cancer Signaling and Therapeutics

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Brian B. Haab, Ph.D. Laboratory of Cancer Immunodiagnostics

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Yuanzheng (Ajian) He, Ph.D. Structural Science and Molecular Signaling

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Xiaohong Li, Ph.D. Laboratory of Tumor Microenvironment and Metastasis

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Jeffrey P. MacKeigan, Ph.D. Laboratory of Systems Biology

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Karsten Melcher, Ph.D. Laboratory of Structural Biology and Biochemistry

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Cindy K. Miranti, Ph.D. Laboratory of Integrin Signaling and Tumorigenesis

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Lorenzo F. Sempere, Ph.D. Laboratory of microRNA Diagnostics and Therapeutics

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Matthew Steensma, M.D. Laboratory of Musculoskeletal Oncology

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George F. Vande Woude, Ph.D. Laboratory of Molecular Oncology

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Bart O. Williams, Ph.D. Laboratory of Cell Signaling and Carcinogenesis

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Ning Wu, Ph.D. Laboratory of Cancer Signaling and Metabolism

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Qian Xie, M.D., Ph.D. Molecular Oncogenesis and Targeted Therapy

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H. Eric Xu, Ph.D. Laboratory of Structural Sciences

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Tao Yang, Ph.D. Laboratory of Skeletal Biology

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Yu-Wen Zhang, M.D., Ph.D. Signal Transduction and Molecular Medicine

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Laboratory Reports

continued

Center for Epigenetics Peter A. Jones, Ph.D., D.Sc. Laboratory of Epigenetic Therapies

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Stefan Jovinge, M.D., Ph.D. Laboratory of Cardiovascular Research

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Peter W. Laird, Ph.D. Laboratory of Cancer Epigenetics 51 Gerd Pfeifer, Ph.D Laboratory of Epigenetic Pathways in Disease 52 Hui Shen, Ph.D. Laboratory of Epigenomic Analysis in Human Disease

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Piroska E. Szabó, Ph.D. Laboratory of Developmental Reprogramming 54 Steven J. Triezenberg, Ph.D. Laboratory of Transcriptional Regulation

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Michael Weinreich, Ph.D. Laboratory of Genome Integrity and Tumorigenesis 58

Center for Neurodegenerative Science Lena Brundin, M.D., Ph.D. Laboratory of Behavioral Medicine

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Patrik Brundin, M.D., Ph.D. Laboratory of Translational Parkinson’s Disease Research

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Jiyan Ma, Ph.D. Laboratory of Prion Mechanisms in Neurodegeneration

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Darren Moore, Ph.D. Laboratory of Molecular Neurodegeneration

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Jeremy M. Van Raamsdonk, Ph.D. Laboratory of Aging and Neurodegenerative Disease

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Core Technologies and Services

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Ting-Tung (Anthony) Chang, Ph.D. Small-Animal Imaging Facility

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Bryn Eagleson, B.S., RLATG Vivarium and Transgenics Core

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Timothy Feinstein, Ph.D. Confocal Microscopy and Quantitative Imaging Core

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Scott D. Jewell, Ph.D. Pathology and Biorepository Core

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Heather Schumacher, B.S., MT(ASCP) Flow Cytometry Core

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Mary E. Winn, Ph.D. Bioinformatics and Biostatistics Core

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Award for Scientific Achievement

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Jay Van Andel Award for Outstanding Achievement in Parkinson’s Disease Research

Educational and Training Programs

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Van Andel Institute Graduate School Postdoctoral Fellowship Program Grand Rapids Area Pre-College Engineering Program Student Internship Program

Seminar Series

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Jay Van Andel Seminar Series VARI Seminar Series

Organization 97 Boards Office of the Director Administrative Organization VAI Organizational Structure

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Peter A. Jones, Ph.D., D.Sc. Research Director and Chief Scientific Officer, Van Andel Research Institute

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T

was a period of tremendous progress and growth for Van Andel Research Institute. Although my appointment as VARI’s research director began in early 2014, I had been following the research conducted here for some time. It is heartening to know I have joined an organization so dedicated to producing excellent basic and translational research and so generously supported by the Van Andel family and our network of donors.

HE past year

The VARI laboratories have been restructured into three overlapping centers: the Center for Cancer and Cell Biology, the Center for Epigenetics, and the Center for Neurodegenerative Science, all of which are supported by Core Technologies and Services. While the Centers have discrete areas of focus, each will also pursue research at the intersections of epigenetics, cancer, and neurodegeneration.

Our plan is to apply the power of epigenetics to the basic discovery and translational research being carried out on cancer and neurodegenerative disorders. We expect the synergy that develops among the laboratories, the Centers, and the research fields will be significant and will result in major contributions to our goal of improving human health.

VARI scientists have made great strides toward this goal, studying new biomarkers that could improve the detection of cancer and Parkinson’s disease; identifying new compounds that combat tumor growth while protecting healthy tissue; and improving our understanding of how Parkinson’s disease spreads through certain areas of the brain. Other research helped elucidate the link between inflammation and the severity of fatigue, anxiety, depression, and cognitive impairment in people with Parkinson’s disease. Another advance was the creation of a blueprint for designing selective activators of the neurotransmitter serotonin, which holds promise for the treatment of anxiety, migraines, and depression.

causes nonmalignant tumors throughout the body that can produce severe health complications. We hope this collaborative effort will result in clinical trials and new, effective treatments for patients with this disease.

Events and awards In September 2014, VARI had the honor of hosting Parkinson’s disease experts for its third Grand Challenges in Parkinson’s Disease symposium. During the symposium, the Institute presented the Jay Van Andel Award for Outstanding Achievement in Parkinson’s Disease Research to Andrew John Lees, M.D., F.R.C.P., FMedSci, in recognition of his groundbreaking work, including the use of apomorphine for late-stage treatment of Parkinson’s.

At the annual Origins of Cancer symposium, which is sponsored by the Foundation for Advanced Cancer Studies, leaders in cancer science and medicine gathered to hear eight excellent speakers address various aspects of cancer’s origins. The 2014 symposium was planned by Drew Howard, Eric Nollet, and Dr. Nikki Thellman as part of their Ph.D. program in the Van Andel Institute Graduate School. Kudos to all for a job well done.

Jeff MacKeigan has established the Pathway of Hope, a research initiative dedicated to studying the molecular mechanisms underlying tuberous sclerosis complex, which

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Faculty

Funding

VARI recently added several top-notch faculty to its team, bringing the Institute closer to critical mass. Lorenzo Sempere leads the Laboratory of microRNA Diagnostics and Therapeutics, where he studies the role of microRNA regulatory networks in carcinogenesis. Darren Moore joined VARI as head of the Laboratory of Molecular Neurodegeneration. Work in his lab focuses on the normal biology and pathobiology of gene products related to inherited Parkinson’s disease, such as LRRK2.

A sample of grant awards to VARI scientists in 2014 includes the following.

• A four-year competitive R01 renewal from the NIH to Eric Xu for his study of Structural Genomics of Orphan Nuclear Receptors • A competitive renewal award to Jeff MacKeigan from the Michigan Economic Development Corporation/Michigan Strategic Fund supporting research on tuberous sclerosis complex • A three-part MEDC award to Peter Jones for a Center of Excellence in Epigenetics; Patrik Brundin for comprehensive research into brain disease modification and repair; and Jeff MacKeigan for translational research on tuberous sclerosis complex • A three-year Department of Defense grant to Cindy Miranti for the study of ING4 Loss in Prostate Cancer Progression • Two grants to Patrik Brundin, from the Michael J. Fox Foundation and from the Cure Parkinson’s Trust • A three-year grant to Qian Xie from the Stephen M. Coffman Charitable Trust for her project on Identifying Genomic Signatures and Determinants for MET-Targeted Therapy in Glioblastoma

The Center for Epigenetics has welcomed four researchers.

Peter Laird’s Laboratory of Cancer Epigenetics studies DNA methylation, with a focus on developing mouse models for the study of epigenetic mechanisms. Hui Shen heads the Laboratory of Epigenomic Analysis in Human Disease, which uses bioinformatics to study the epigenome and its roles in cancer. In the Laboratory of Epigenetic Pathways in Disease, Gerd Pfeifer leads studies of CpG island hypermethylation in cancer and development. And Piroska Szabó heads the Laboratory of Developmental Reprogramming, which studies the mechanisms for resetting the epigenome between generations and specifically in the context of genomic imprinting.

I offer my sincerest thanks to former VARI Professor Dr. Brian Nickoloff, who led the Center for Translational Medicine, and to George Vande Woude, who led the Center for Cancer and Cell Biology prior to our restructuring. I also would like to recognize the Research Leadership Council—Patrik Brundin, George Vande Woude, and Jana Hall—for its excellent stewardship of VARI during the search for a new research director. Each of these individuals has contributed immensely to VARI’s success, and I truly appreciate their hard work.

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Parting thoughts In 2014, a new chapter started for VARI with our commitment to becoming a global center for epigenetics, a burgeoning frontier in biomedical research. We see great potential to expand the exceptional work already underway at VARI in cancer and neurodegenerative diseases. Advancements will take shape in many ways, including new collaborations with colleagues around the globe (including collaborations on clinical trials) and an emphasis on strategic recruitment. In short, VARI is poised for significant growth. Our dedication to translating basic research into clinical applications is stronger than ever, and in the coming years we expect to continue making a significant difference for those who matter most—the people affected by the diseases we study.

VARI SCIENTIFIC STRUCTURE Center for Epigenetics

Center for Neurodegenerative Science

Center for Cancer and Cell Biology

Core Services

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LABORATORY REPORTS

Center for Cancer and Cell Biology Bart O. Williams, Ph.D. Director

The Center’s scientists study the basic mechanisms and molecular biology of cancer and other diseases, with the goal of developing better diagnostics and therapies.

The confocal microscopy image at left shows staining that characterizes cellular and molecular heterogeneity. miRNA and protein biomarkers are shown in a breast cancer tissue core from an ER-negative tissue microarray using a fully automated 6-plex ISH/IHC assay. This innovative technology was recently implemented as a collaborative project between VARI research labs and the pathology core. Cytokeratin 19 (orange) highlights cancer cells, and miR-21 (green) marks tumor-associated fibroblasts. U6 snRNA (red) is expressed in many nuclei, and collagen I (light blue) highlights the tissue vasculature. Image by Tim Feinstein and Lorenzo Sempere; sample provided by Galen Hostetter.

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Arthur S. Alberts, Ph.D. Laboratory of Cell Structure and Signal Integration Dr. Alberts earned his degrees in biochemistry and cell biology (B.A., 1987) and in physiology and pharmacology (Ph.D., 1993) from the University of California, San Diego. He was a postdoctoral fellow in Richard Treisman’s laboratory from 1994 to 1997 at the Imperial Cancer Research Fund (now Cancer Research UK London Research Institute) as a Howard Hughes Medical Institute International Scholar. From 1998 to 1999 he was the Carol Franc Buck Fellow in Frank McCormick’s lab at the Helen Diller Comprehensive Cancer Center at UC San Francisco. Dr. Alberts joined VARI in January 2000; he was promoted in 2006 to Associate Professor and to Professor in 2009.

Dr. Alberts also directs the Flow Cytometry core facility.

From left: Alberts, Howard, Goosen, Turner

Staff

Student

Visiting Scientist

Susan Goosen, M.B.A. Andrew Howard, B.A. Heather Schumacher, B.S., MT(ASCP)

Kristin Rybski

Julie Davis Turner, Ph.D.

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Alberts

Research Interests • To gain a full understanding of how cells spatially and temporally organize the signaling networks required for cell growth control and differentiation.

• To uncover how defects in cell structure and disruptions in signaling create vulnerabilities in the acquisition of cancer traits.

In 2013 we focused our basic research on the intersection of Rho and Wnt signaling to the nucleus and the cytoskeletal remodeling apparatus. We focused our translational research on targeted therapies that reinforce and/or repair cell infrastructure. We are developing compounds called intramimics that activate the molecular machinery that builds the cell infrastructure in support of normal cell function and/or repairs cellular defects that trigger or support malignant progression. Our disease research relies upon genetic models of blood cancers that arise from cells of the bone marrow. We use these models to test ideas generated by our molecular studies and to inform the development of novel diagnostic and therapeutic tools.

Recent Publications Ercan-Sencicek, A. Gulhan, Samira Jambi, Daniel Franjic, Sayoko Nishimura, Mingfeng Li, Paul El-Fishawy, Thomas M. Morgan, Stephan J. Sanders, et al. In press. Homozygous loss of DIAPH1 is a novel cause of microcephaly in humans. European Journal of Human Genetics.

Pan, Jiajia, Larissa Lordier, Deborah Meyran, Philippe Rameau, Yann Lecluse, Susan Kitchen-Goosen, Idinath Badirou, Hayat Mokrani, Shuh Narumiya, et al. In press. The formin DIAPH1 (mDia1) regulates megakaryocyte proplatelet formation by remodeling the actin and microtubule cytoskeletons. Blood.

Kher, Swapnil S., Amanda P. Struckhoff, Arthur S. Alberts, and Rebecca A. Worthylake. 2014. A novel role for p115RhoGEF in regulation of epithelial plasticity. PLoS One 9(1): e85409.

Lash, Leanne L., Bradley J. Wallar, Julie D. Turner, Steven M. Vroegop, Robert E. Kilkuskie, Susan M. Kitchen-Goosen, H. Eric Xu, and Arthur S. Alberts. 2013. Small-molecule intramimics of formin auto-inhibition: a new strategy to target the cytoskeletal remodeling machinery in cancer cells. Cancer Research 73(22): 6793–6803.

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Nicholas S. Duesbery, Ph.D. Laboratory of Cancer and Developmental Cell Biology Dr. Duesbery received a B.Sc. (Hon.) in biology (1987) from Queen’s University, Canada, and both his M.Sc. (1990) and Ph.D. (1996) degrees in zoology from the University of Toronto under the supervision of Yoshio Masui. Before his appointment at VARI in 1999, he was a postdoctoral fellow in George Vande Woude’s laboratory at the National Cancer Institute, Frederick Cancer Research and Development Center, Maryland. Dr. Duesbery was promoted to Associate Professor in 2006, and he chairs VARI’s Undergraduate Internship Program Committee.

From left, seated: Kuk, Naidu; standing: Bromberg-White, Boguslawski, Minard, Andersen, Rocky, Duesbery, Marritt, Kramer

Staff

Students

Adjunct Faculty

Nicholas Andersen, Ph.D. Elissa Boguslawski Jenn Bromberg-White, Ph.D. Michelle Minard

Holly Kramer Cynthia Kuk, B.S. Eimi Marritt Agni Naidu, B.S., B.A.

Christopher Chambers, M.D., Ph.D. Lou Glazer, M.D. Barbara Kitchell, D.V.M., Ph.D., DACVIM

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Duesbery

Research Interests Several years ago we discovered that turning off mitogen-activated protein kinase kinases (MKKs) reduces blood vessel density in tumors and causes them to stop growing or to shrink. Since then we have focused our efforts on defining the role of MKK signaling in blood vessel function in cancer and vascular disease, whose pathogeneses rely upon abnormal blood vessel formation. Our goal is to develop new therapies to control blood vessel growth in disease. Our scientific projects are organized around four diseases of blood vessels, affording a unique opportunity to compare and contrast the relationship between MKK signaling and blood vessel growth during development and disease.

Project 1) W e discovered that MKK inhibition by anthrax lethal toxin causes tumor hemorrhage, reduces blood flow, and lowers blood vessel density. To explain this, we are testing the scientific hypothesis that anthrax lethal toxin acts like a hemorrhagic toxin, disrupting tumor blood vessel function by breaking elements that hold blood vessels together. Our goal is to develop new ways to prevent tumor blood vessel growth and shrink tumors.

Project 2) W e have found that MKK inhibition blocks growing blood vessels in the retina but does not affect fully grown blood vessels. We also discovered that the vitreous of patients with proliferative diabetic retinopathy has higher amounts of several small inflammatory proteins. To explain clinical resistance to current therapy, we are testing the scientific hypothesis that one or more of these small inflammatory proteins activates MKK signaling and turns on retinal blood vessel growth. Our goal is to develop new anti-MKK therapies to prevent abnormal blood vessel growth in diseases like proliferative diabetic retinopathy.

Project 3) W e discovered that turning on MKK drives the growth of a rare tumor type called angiosarcoma. Using mouse models, we showed that treatment of angiosarcoma with drugs causes these tumors to shrink. We are currently testing MKK inhibitors in combination with other anti-cancer drugs, working to develop more effective therapies for treating angiosarcoma and other sarcomas.

Project 4) W e have found that MKK is essential for the regrowth of blood vessels following injury or artery blockage. We are testing the hypothesis that activation of MKK in the cells that form blood vessels makes those cells divide and grow to expand existing blood vessels as well as to make new ones. Our goal is to better understand this process in order to develop therapies that will speed the recovery of blood vessel function following injury or blocking of the arteries.

Recent Publications Andersen, Nicholas J., Brian J. Nickoloff, Karl J. Dykema, Elissa A. Boguslawski, Roman I. Krivochenitser, Roe E. Froman, Michelle J. Dawes, Laurence H. Baker, Dafydd G. Thomas, et al. 2013. Pharmacologic inhibition of MEK signaling prevents growth of canine hemangiosarcoma. Molecular Cancer Therapeutics 12(9): 1701–1714.

Bromberg-White, Jennifer L., Louis Glazer, Robert Downer, Kyle Furge, Elissa Boguslawski, and Nicholas Duesbery. 2013.

Identification of VEGF-independent cytokines in proliferative diabetic retinopathy vitreous. Investigative Ophthalmology and Visual Science 54(10): 6472–6480.

Ding, Ximin Chen, Jin Zhu, Nicholas S. Duesbery, Xunjia Cheng, and Brian Cao. 2013. A human/murine chimeric Fab antibody neutralizes anthrax lethal toxin in vitro. Clinical and Developmental Immunology 2013: 475809.

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Carrie R. Graveel, Ph.D. Breast Cancer Signaling and Therapeutics Dr. Graveel earned her Ph.D. in cellular and molecular biology from the University of Wisconsin–Madison in 2002. She then served as a postdoctoral fellow in the laboratory of George Vande Woude at VARI from 2002 to 2007.

In 2007, she became a Research Scientist and in 2010 was promoted to Senior Research Scientist. Dr. Graveel is currently a Research Assistant Professor in VARI and an Instructor in the VAI Graduate School.

From left: Linklater, Graveel, Essenburg

Staff

Student

Curt Essenburg, B.S. Erik Linklater, B.S.

Josh Castle

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Graveel

Research Interests Receptor tyrosine kinase signaling can promote the growth, invasion, and survival of both normal and cancerous cells. In cancer, altered receptor tyrosine kinases frequently drive tumor initiation, progression, and metastasis. Understanding how these receptors signal in breast cancer cells is crucial to developing successful therapeutic strategies. The goal of the team is to identify novel therapeutic targets and prognostic signatures of therapy resistance in breast cancer. Dr. Graveel is focused on the role of the MET receptor and the ERBB receptor family in breast cancer progression and therapy resistance.

Triple-negative breast cancer (TNBC) accounts for 15–20% of breast cancers and generally has an advanced stage at diagnosis and a poorer outcome than other breast cancer subtypes. Tyrosine kinases are promising druggable targets due to their high expression in TNBC, but in the clinic, tyrosine kinase inhibitors have had limited success. Recent clinical studies of non-small-cell lung cancer (NSCLC) suggest that combined inhibition of MET and EGFR improves and prolongs therapeutic efficacy in a subset of patients. The goals of our research are to determine MET and EGFR expression, activation, and signaling in TNBC; to determine the efficacy of MET and EGFR inhibition on TNBC progression in vivo; and to identify unique molecular drivers of African-American and European-American TNBC. Numerous studies have demonstrated that paracrine interactions between breast cancer cells and the tumor microenvironment (including stromal fibroblasts and inflammatory cells) promote tumor progression and metastasis. To investigate how oncogenic signaling from the microenvironment influences the progression of TNBC, we are using the MET and IL-6 pathways as a paradigm of oncogenic/inflammatory interactions. In collaboration with Bonnie Sloane (Wayne State University) and Patricia LoRusso (Karmanos Cancer Institute), we are investigating MET and IL-6 inhibitors to determine whether combined inhibition of these pathways will be beneficial to TNBC patients. Our goals are to determine MET and IL-6 activation and signaling in TNBC; to investigate the effect of MET and/or IL-6 inhibition on TNBC cell invasion using 3D co-culture models; and to determine the efficacy of MET and IL-6 inhibition on TNBC progression and metastasis in vivo.

Recent Publications Mackenzie, Todd A., Gary N. Schwartz, Heather M. Calderone, Carrie R. Graveel, Mary E. Winn, Galen Hostetter, Wendy A. Wells, and Lorenzo F. Sempere. In press. Stromal expression of miR-21 identifies high-risk group in triple-negative breast cancer. American Journal of Pathology.

Graveel, Carrie R., David Tolbert, and George F. Vande Woude. 2013. MET: a critical player in tumorigenesis and therapeutic target. Cold Spring Harbor Perspectives in Biology 5(7): a009209.

Paulson, Amanda K., Erik S. Linklater, Bree D. Berghuis, Colleen A. App, Leon D. Oostendorp, Jayne E. Paulson, Jane E. Pettinga, Marianne K. Melnik, George F. Vande Woude, and Carrie R. Graveel. 2013. MET and ERBB2 are coexpressed in ERBB2+ breast cancer and contribute to innate resistance. Molecular Cancer Research 11(9): 1112–1121.

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Brian B. Haab, Ph.D. Laboratory of Cancer Immunodiagnostics Dr. Haab obtained his Ph.D. in chemistry from the University of California at Berkeley in 1998. He then served as a postdoctoral fellow in the laboratory of Patrick Brown in the Department of Biochemistry at Stanford University.

Dr. Haab joined VARI as a Special Program Investigator in May 2000, became a Scientific Investigator in 2004, was promoted to Senior Scientific Investigator in 2007, and is now a VARI Professor. He is also an Adjunct Professor in the Department of Biochemistry and Molecular Biology and in the Genetics Program at MSU.

From left: Murphy, Partyka, Weaver, Hsueh, Singh, Tang, Ensink, Yadav, Haab

Staff

Students

Visiting Scientists

Katie Partyka, B.S. Sudhir Singh, Ph.D. Huiyuan Tang, Ph.D. Laura Weaver, A.A.S. Jessica Yadav, M.S.

Alexander Clegg Elliot Ensink Peter Hsueh, B.S. Anne Murphy, B.S. Alexandria Rogers, B.A. Arkadeep Sinha, B.S.

Birendra KC, M.D. David VonEhr, B.S., M.Ed.

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Haab

Research Interests The Haab laboratory is working to develop molecular tests that will contribute to improved outcomes for cancer patients. Many cancer patients receive late or inappropriate treatment due to lack of accurate information about their disease. Molecular tests promise to improve that situation. We are developing experimental and bioinformatics methods to identify the carbohydrates and proteins produced only in cancer tissues and are working to define the particular molecular species that are most valuable in clinical tests. We work closely with our clinical partners on pancreatic cancer, addressing problems involving early detection, diagnosis, and treatment decisions.

Early diagnosis of pancreatic cancer A blood test that distinguishes early-stage pancreatic cancer from other, benign diseases of the pancreas could have a significant impact on patient care. It could allow patients with early-stage disease to get the appropriate treatment as rapidly as possible; it could be portable to locations distant from major hospitals, thus benefitting a broader population; it could be applied to high-risk individuals as part of a surveillance program to guide the use of more expensive and invasive methods; and it could spare some patients from unnecessary procedures or surgery. We have made good strides toward developing such a test.

Carbohydrates, or glycans, cover the surfaces of epithelial cells and decorate nearly all secreted proteins. It has long been recognized that cancer cells rework the glycosylation of their proteins and that the detection of the altered glycans could form the basis of accurate biomarkers. In fact, the current best biomarker for pancreatic cancer is a glycan, a tetrasaccharide known as the sialyl Lewis A (sLeA) antigen. Although the blood level of sLeA is strongly associated with pancreatic cancer—it is elevated in 70–80% of pancreatic cancer patients—it is not satisfactory for diagnosing cancer because the risk of both false negative and false positive diagnoses is too high.

The main question we have been asking is, do the cancers that are low in sLeA make other glycans that could be useful biomarkers? If we could find such glycans, they could be used in combination with sLeA to create an effective clinical test.

Our recent work provides evidence that this goal is achievable. Some evidence comes from mass spectrometry analyses of pancreatic tissue and gene expression analyses of cell lines, but the best evidence comes from experiments using the antibodylectin sandwich array. With this array, we can efficiently examine all combinations of capture and detection antibodies—each antibody targeting a different glycan—to obtain precise measurements of many glycans in a sample. A study of samples from pancreatic cancer patients and control subjects revealed that the sLeA-low cancers increased their production of several glycans related to sLeA, including a structural isomer of sLeA. We acquired antibodies against those glycans and used them to optimize the detection of the sLeA-low cancers. Furthermore, in collaboration with the Melcher and Xu labs, we produced glycan-binding proteins that provided improved glycan detection in sLeA-low pancreatic cancers.

We are consolidating these results into a single, validated test for the detection of early-stage pancreatic cancer. In collaboration with Spectrum Health and others, we are analyzing samples from patients being evaluated for pancreatic issues, and we are performing detailed comparisons between our marker and conventional methods at each stage of patient care. Successful validation of this blood test could be the first step toward achieving a breakthrough in detecting early-stage pancreatic cancer.

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Detection of preinvasive pancreatic cysts Another class of pancreatic disease we are working on is pancreatic cysts. These cysts develop from a variety of causes; some that are not cancerous arise through inflammatory events, and others arise through the growth of neoplastic, pre-malignant cells. The latter type could be life threatening if the cysts develop into cancer, so the removal of cysts with high malignant potential is crucial. Surgery can remove the threat, but physicians do not have accurate methods for distinguishing dangerous cysts from benign ones. Furthermore, surgeons do not want to remove cysts unnecessarily because pancreatic surgery carries significant risks and physical stress.

We are developing molecular biomarkers that help distinguish between life-threatening cysts and nonmalignant ones. As we found in our studies on pancreatic adenocarcinoma, certain glycans produced by the cysts are valuable biomarkers. One protein, MUC5AC, is particularly informative. Like most other proteins secreted by the cells of epithelial layers, MUC5AC has several different glycans attached to it. The informative feature is that the glycans on MUC5AC from pre-malignant cells differ from those produced by nonmalignant cells. We can use this difference to our advantage: the detection of the specific glycoform of MUC5AC produced by pre-malignant cells forms the basis of an accurate molecular test. We have also identified glycoforms of two proteins called MUC3A and endorepellin that contribute to promising biomarker panels. We have performed two blinded validation studies that have confirmed initial results, and we are performing further validation, optimization, and characterization of the markers. We hope to develop a clinical assay useful to doctors and patients, ultimately leading to more accurate diagnosis of the pre-malignant cysts and improved outcomes for patients.

Novel tools and methods that advance biomarker and glycobiology research Our work required the development of several novel approaches and tools. One such tool is a database and analysis program, GlycoSearch, which was built by our collaborators at the Palo Alto Research Center and houses analyzed data and metadata from glycan array experiments. Researchers can use the software to find lectins or glycan-binding antibodies that bind specific targets, to delve into the details of specific proteins, or to explore biological relationships. We foresee significant value of this system for many research applications, especially in combination with microarray methods for the molecular profiling of clinical samples.

Recent Publications Kletter, Doron, Bryan Curnutte, Kevin Maupin, Marshall Bern, and Brian B. Haab. In press. Exploring the specificities of glycan-binding proteins using glycan array data and the GlycoSearch software. Methods in Molecular Biology.

Sinha, Arkadeep, David Cherba, Heather Bartlam, Elizabeth Lenkiewicz, Lisa Evers, Michael T. Barrett, and Brian B. Haab. In press. Mesenchymal-like pancreatic cancer cells harbor specific genomic alterations more frequently than their epithelial-like counterparts. Molecular Oncology.

Gbormittah, Francisca Owusu, Brian B. Haab, Katie Partyka, Carolina Garcia-Ott, Marina Hincapie, and William S. Hancock.

2014. Characterization of glycoproteins in pancreatic cyst fluid using a high performance multiple lectin affinity chromatography platform. Journal of Proteome Research 13(1): 289–299.

Partyka, Katie, Shuangshuang Wang, Ping Zhao, Brian Cao, and Brian B. Haab. 2014. Array-based immunoassays with rolling-circle amplification detection. In Molecular Toxicology Protocols, Phouthone Keohavong and Stephen G. Grant, eds.

Methods in Molecular Biology series, Vol. 1105. New York: Humana Press, pp. 3–15.

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Yuanzheng (Ajian) He, Ph.D. Structural Science and Molecular Signaling After graduating from the Chinese Academy of Sciences’ Shanghai Institute of Biochemistry in 2000, Dr. He worked as a postdoctoral fellow in Stoney Simons’ lab at the National Institute of Diabetes and Digestive and Kidney Diseases in Maryland. While there, he studied the mechanism of steroid hormone receptor–regulated gene expression, focusing on the glucocorticoid receptor. In 2008, Dr. He was recruited to Van Andel Research Institute to work on drug discovery for the glucocorticoid receptor and the structural relationship between ligand recognition and receptor function. He is currently a Research Assistant Professor.

Research Interests Glucocorticoids play important roles in regulating many essential processes of the human body, including energy metabolism, immune modulation, stress responses, cell proliferation and differentiation, the circadian clock, and memory functions.

Glucocorticoids are also the most effective and widely used anti-inflammatory agents for the treatment of diseases such as asthma and arthritis. However, long-term use of glucocorticoids can cause side effects such as obesity, diabetes, and bone loss. My research goals are to understand the structural mechanism of glucocorticoids in regulating cellular pathways and to use the knowledge to develop novel “dissociated glucocorticoids” that retain only the beneficial anti-inflammatory effects.

Over the past year, we have made the following progress toward our goals.

• We developed dissociated glucocorticoids based on the findings that the dissociation of transrepression from transactivation can be achieved through interfering with the dimerization interface of the glucocorticoid receptor (GR).

• We solved the structure of the cortisol-bound and the mometasone furoate-bound GR ligand-binding domain (LBD), discovering that the high potency of glucocorticoids can be achieved through a lipophilic group such as a furoate ester in the C-17a position to fully occupy the hydrophobic cavity in the GR ligand-binding pocket.

• We have designed and developed several highly potent glucocorticoids based on our structure of the mometasone furoate-bound LBD. These are ideal for the treatment of asthma, as they can be used at low dose to minimize unwanted systemic effects.

Recent Publications He, Yuanzheng, Wei Yi, Kelly Suino-Powell, X. Edward Zhou, W. David Tolbert, Xiaobo Tang, Jing Yang, Huaiyu Yang, Jingjing Shi, et al. 2014. Structures and mechanism for the design of highly potent glucocorticoids. Cell Research 24(6): 713–726.

Zhi, Xiaoyong, X. Edward Zhou, Yuanzheng He, Christoph Zechner, Kelly M. Suino-Powell, Steven A. Kliewer, Karsten Melcher, David J. Mangelsdorf, and H. Eric Xu. 2014. Structural insights into gene repression by the orphan nuclear receptor SHP. Proceedings of the National Academy of Sciences U.S.A. 111(2): 839–844.

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Xiaohong Li, Ph.D. Laboratory of Tumor Microenvironment and Metastasis Dr. Li received her Ph.D. from the Institute of Zoology, Chinese Academy of Sciences in Beijing in 2001, and since 2000 has pursued postdoctoral training in the laboratories of David Ong and Neil Bhowmick at Vanderbilt University in Nashville, Tennessee. She was a research assistant professor in the Department of Cancer Biology from 2009 to 2012, mentored by Lynn Matrisian. Dr. Li joined VARI as an Assistant Professor in September 2012.

From left: Ganguly, Li, Lewis, Vander Ark, Meng

Staff

Students

Sourik Ganguly, Ph.D. Priscilla Lee, B.S. Diana Lewis, A.S. Xiangqi (Neil) Meng, Ph.D. Alexandra Vander Ark, M.S.

Samuel Ameh Yiqing Dong Rebecca Emery Peter Hsueh, B.S. Julienne Louters, B.S. Demarcus Williams

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Li

Research Interests Our laboratory is committed to understanding cancer metastases, particularly bone metastases. Most people who die of cancer have metastases somewhere in their body, but metastases of certain cancers (such as breast, prostate, and lung) are more likely to be found in bone. Once in the bone, cancer cells induce either osteolytic (bone resorption) or osteoblastic (abnormal bone formation) lesions, which cause fractures, spinal cord compression, hypercalcemia, and extreme bone pain.

Current treatments for bone-metastasis patients reduce some symptoms such as pain but do not increase survival time. In order to develop early diagnostic and targeted therapeutic strategies, our long-term goal is to determine the mechanisms through which different cancers cause distinct types of bone lesions. To achieve this goal, we study the cancer cells and the microenvironments of both the primary site and the bone-metastasis site. The microenvironment of the bone is a rich reservoir of growth factors and cytokines, such as transforming growth factor (TGF)- and hepatocyte growth factor (HGF). These factors play crucial roles in both cancerous and healthy bone. More importantly, their effects are highly context-dependent spatially and temporally. Our short-term goal is to delineate the context-dependent effects of the important factors in bone metastases. Our current projects are as follows.

1) Determine the influence of the primary microenvironment on the development of prostate cancer osteoblastic bone lesions. The objectives are to determine how TGF- from the prostate contributes to bone lesion development, to identify the cytokines induced by the loss of TGF- signaling that mediate prostate cancer bone metastasis, and to determine the prognostic and therapeutic value of the identified cytokines for bone metastases. The Prostate Cancer Research Program of the Department of Defense has funded this project from 2012 until 2016.

2) Identify the cell-specific effects of TGF- signaling from the bone microenvironment on bone metastases. Using genetically engineered mouse models, we have been able to knock out TGF- signaling in certain bone cells.

Context-dependent TGF- effects on osteolytic or osteoblastic bone metastases from different types of cancers will be investigated.

3) Establish animal models of osteoblastic bone metastasis and identify the signaling pathway that drives this process, in collaboration with VARI’s Cindy Miranti.

4) Determine the stromal HGF signaling effect on heterogenous cancer cell bone metastasis, in collaboration with VARI’s George Vande Woude.

5) Test new applications of bone-tropic and tumor-specific agents in early detection of and drug delivery for cancer bone metastasis, in collaboration with VARI’s Anthony Chang.

6) Screen new lymphangiogeneis factors as potential therapeutic targets of cancer metastases, in collaboration with Dr. Miles Qian, Sun Yat-sen University, China.

Recent Publication Banerjee, J., R. Mishra, X. Li, R.S. Jackson II, A. Sharma, and N.A. Bhowmick. 2014. A reciprocal role of prostate cancer on stromal DNA damage. Oncogene 33(41): 4924–4931.

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Jeffrey P. MacKeigan, Ph.D. Laboratory of Systems Biology Dr. MacKeigan received his Ph.D. in microbiology and immunology at the University of North Carolina Lineberger Comprehensive Cancer Center in 2002 and then held a postdoctoral fellowship in the Department of Cell Biology at Harvard Medical School. In 2004, he joined Novartis Institutes for Biomedical Research in Cambridge, Massachusetts, as an investigator and project leader in the Molecular and Developmental Pathways expertise platform. Dr. MacKeigan joined VARI in 2006 as an Assistant Professor and was promoted to Associate Professor in 2010. He leads the Laboratory of Systems Biology and directs the Pathway of Hope research initiative.

From left, back row: MacKeigan, Burgenske, Merrill, Doppel, Sisson, Webb, Solitro, Nelson, Bagchi; kneeling: Kortus, Lanning, Kerk

Staff Nicole Doppel, B.S. Vanessa Fogg, Ph.D. Sam Kerk, B.S. Jennifer Kordich, M.S. Matt Kortus, M.S. Nate Lanning, Ph.D. Brendan Looyenga, Ph.D. 18

Katie Martin, Ph.D. Amy Nelson Juliana Sacoman, Ph.D. Aaron Sayfie, B.A. Kellie Sisson, B.S. Jennifer Webb, B.A.

Students

Adjunct Faculty

Aditi Bagchi, M.D. Dani Burgenske, B.S. Megan Goodall, Ph.D. Nate Merrill, B.S. Anna Plantinga, B.S. Abbey Solitro, B.S. Laura Westrate, Ph.D.

Brian Lane, M.D., Ph.D.

MacKeigan

Research Interests Systems biology integrates disciplines such as biochemistry, mathematics, and genetics to investigate biological questions.

The Laboratory of Systems Biology focuses on identifying and understanding the genes and signaling pathways that, when mutated, contribute to the pathophysiology of cancer and neurodegeneration. Our lab has two major research programs: cancer metabolism and the evasion of apoptosis, and PI3K-mTOR and the autophagy signaling network. We use tools such as RNA interference (RNAi), quantitative proteomics, and in silico screening to investigate the kinases and phosphatases that mediate the pro-apoptotic and cell survival functions of mitochondria, as well as those that regulate lipid signaling and autophagy.

The laboratory’s primary scientific objectives are to investigate the molecular details of cancer and tumor diseases; develop therapeutics for high-priority targets; reposition drugs into cancer and neurodegenerative diseases; and map genes to disease.

Cancer metabolism and the evasion of apoptosis A wealth of experimental evidence connects the regulation of cellular metabolism with the development of cancer. Metabolic changes are considered key events in the transition from a normal cell to a cancer cell. Such changes reprogram cells to provide the fuel and energy required for rapid malignant proliferation. To identify genes crucial in cancer cell metabolism, we developed a novel, high-throughput method to comprehensively screen all known nuclear-encoded genes whose protein products localize to mitochondria. Our screen also included other metabolic genes and used cellular ATP levels as a readout. The screen was performed under both glycolytic and oxidative phosphorylation–restricted conditions to define genes contributing to ATP production in each bioenergetic state.

From our screen, we identified several genes that drive cancer cell bioenergetics and that cancer cells rely on for survival and proliferation. A substantial proportion of the genes we identified as novel targets were dysregulated in tumors from glioma patients, and their expression and copy number significantly correlated with patient survival. One such gene was for a mitochondrial adenylate kinase (AK4), which regulates cellular ATP levels and AMPK signaling. We also identified a mechanism by which electron transport chain changes under glycolytic conditions increased ATP production through enhanced glycolytic flux, highlighting the cellular potential for metabolic plasticity. This study has comprehensively mapped the bioenergetic landscape of all mitochondrial proteins in the context of varied metabolic substrates and thus has begun to link key metabolic genes to clinical outcomes.

PI3K-mTOR and the autophagy signaling network Autophagy is a cellular catabolic process that generates internal nutrients by targeting portions of cytosol for lysosomal degradation. During times of stress, autophagy is activated as a way to generate energy, clear damaged organelles, and delay or prevent cell death. Accordingly, autophagy is often activated by oncogenic transformation (e.g., KRAS) and is crucial for tumor survival and progression as an adaptation to stressors such as chemotherapy. Mechanistically, autophagy is directly inhibited by mTOR; therefore, molecular-targeted therapies that block PI3K-AKT-mTOR signaling induce autophagy, providing a counterproductive mechanism of cell survival and drug resistance. In addition, compounds not directly impinging on that pathway can generate intracellular stress signals that activate autophagy by less direct mechanisms. Understanding the effects of a compound on autophagy is crucial to improving its therapeutic efficacy.

We have identified a potent activator of autophagy in cancer cells. To further this discovery, we used RNAi to target the human kinome and also a set of genes encoding proteins having likely roles in the regulation of autophagy. We developed a robust, microscopy-based assay to quantitatively measure autophagy and we demonstrated that drug-induced autophagy required two genes. One was an expected finding due to its central role in autophagy; the other was a novel finding with no previous connections to autophagy. We used these two genes as leads in a second RNAi screen. We confirmed that knockdown of the novel gene selectively decreased the viability of the oncogenic KRAS line, making this gene a promising target for further research.

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Tuberous sclerosis complex Tuberous sclerosis complex (TSC) is an autosomal-dominant multisystem disorder characterized by benign tumors in the brain, skin, heart, kidneys, and lung. These tumors cause a diverse set of clinical problems including epilepsy, learning difficulties, behavioral problems, and renal failure. TSC is caused by mutations within the TSC1 or TSC2 genes. These mutations inactivate the genes’ tumor suppressive function and drive tumor cell growth. TSC1 and TSC2 interact in a protein signaling complex in the mTOR-S6K pathway, and our lab has launched a multifaceted TSC project as part of our PI3K-mTOR and autophagy program. This research works toward new treatment options for TSC patients and will likely have broad implications for cancer patients as well.

To identify novel genes and investigate their cellular dynamics, we optimized both compound and RNAi screening platforms.

From our screens, we identified genes which when knocked down decreased cell viability, including genes involved in cell cycle control. We are confirming our results in additional cell lines and are exploring the outcomes in the presence of rapamycin, an effective tumor suppressor for TSC patients. Further, we are using whole-exome sequencing on TSC tissue samples and analyzing patient genomes. We have obtained 150 tissue samples from national collaborators, and samples meeting quality and yield criteria were sent for next-generation sequencing. We are collaborating with the VARI Bioinformatics core to analyze the sequencing data for meaningful genetic variants.

We have opened a feasibility study at two clinical sites to pilot a personalized-medicine approach for TSC. We have used patient biopsies, genome technologies, and a team of experts to design a therapeutic protocol specific to each enrolled participant. Five patients have enrolled in the feasibility study. Our Molecular Tumor Board has reviewed patient genomic data, evaluated the molecular alterations, discussed relevant targeted therapies, and developed recommended treatment plans specific to each individual genome. Although the feasibility study does not involve patient treatment, we are working toward a clinical trial in which physicians may implement our personalized medicine approach as part of the treatment strategy.

Recent Publications Goodall, Megan L., Tong Wang, Katie R. Martin, Matthew G. Kortus, Audra L. Kauffman, Jeffrey M. Trent, Steven Gately, and Jeffrey P. MacKeigan. 2014. Development of potent autophagy inhibitors that sensitize oncogenic BRAF V600E mutant melanoma tumor cells to vemurafenib. Autophagy 10(6): 1120–1136.

Lanning, Nathan J., Brendan D. Looyenga, Audra L. Kauffman, Natalie M. Niemi, Jessica Sudderth, Ralph J. DeBerardinis, and Jeffrey P. MacKeigan. 2014. A mitochondrial RNAi screen defines cellular bioenergetic determinants and identifies an adenylate kinase as a key regulator of ATP levels. Cell Reports 7(3): 907–917.

Niemi, Natalie M., Juliana L. Sacoman, Laura M. Westrate, L. Alex Gaither, Nathan J. Lanning, Katie R. Martin, and Jeffrey P. MacKeigan. 2014. The pseudophosphatase MK-STYX physically and genetically interacts with the mitochondrial phosphatase PTPMT1. PLoS One 9(4): e93896.

Westrate, Laura M., Jeffrey A. Drocco, Katie R. Martin, William S. Hlavacek, and Jeffrey P. MacKeigan. 2014. Mitochondrial morphological features are associated with fission and fusion events. PLoS One 9(4): e95265.

Westrate, Laura M., Aaron D. Sayfie, Danielle M. Burgenske, and Jeffrey P. MacKeigan. 2014. Persistent mitochondrial hyperfusion promotes G2/M accumulation and caspase-dependent cell death. PLoS One 9(3): e91911.

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This fluorescence intensity profile shows the effects of the antimalarial agent chloroquine on sarcoma cells; the effects can be followed over time. In this image, the nucleus is stained blue, endosomes green, and lysosomes red. Chloroquine inhibits autophagy by deacidifying lysosomes so they can no longer digest cellular materials, leading eventually to cell death. Yellow peaks suggest that some lysosomes undergo autophagy even after chloroquine treatment. Image by Megan Goodall of the MacKeigan laboratory.

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Scientific Report 2015

Karsten Melcher, Ph.D. Laboratory of Structural Biology and Biochemistry Dr. Melcher earned his master’s degree in biology and his Ph.D. degree in biochemistry from the Eberhard Karls Universität in Tübingen, Germany, after which he was a postdoctoral fellow at the University of Texas Southwestern Medical Center in Dallas. He was then an independent investigator at the University of Ulster in Coleraine, U.K., and at Goethe University in Frankfurt. Dr. Melcher was recruited to VARI in 2007, serving as a Research Scientist within the Laboratory of Structural Sciences. In 2011, he became Assistant Professor and Head of the Laboratory of Structural Biology and Biochemistry, and in 2013 he was promoted to Associate Professor.

From left: Gu, Wang, Sridharamurthy, Li, Kovach, de Waal, Melcher, Grant

Staff

Students

Parker de Waal, B.S. Stephanie Grant, B.S. Xin Gu, M.Sc. Amanda Kovach, B.S. Xiaoyin (Edward) Zhou, Ph.D.

Mark Farha Xiaodan Li, B.S. Kelvin Searose-Xu Madhuri Sridharamurthy, B.S. Adam Thelen Lili Wang, B.S.

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Melcher

Research Interests The Laboratory of Structural Biology and Biochemistry studies the structure and function of proteins that have central roles in cellular signaling. To do so, we employ X-ray crystallography in combination with biochemical and cellular methods to identify structural mechanisms of signaling at high resolution.

In addition to their fundamental physiological roles, most signaling proteins are also important targets of therapeutic drugs. Determination of the three-dimensional structures of protein–drug complexes at atomic resolution allows a detailed understanding of how a drug binds its target and modifies its activity. This knowledge allows the rational design of new and better drugs against diseases such as cancer, diabetes, and neurological disorders.

Three areas of focus in the lab are the adenosine monophosphate (AMP)–activated protein kinase (AMPK); the receptors and key signaling proteins for a plant hormone, abscisic acid (ABA); and the folate receptors.

AMP-activated protein kinase (AMPK) Cells use ATP to drive energy-consuming cellular processes such as muscle contraction, cell growth, and neuronal excitation.

AMPK is a three-subunit protein kinase that functions as an energy sensor and regulator of homeostasis in human cells. Its kinase activity is triggered by energy stress (i.e., a drop in the ratio of ATP to AMP/ADP), activating ATP-generating pathways and reducing energy-consuming metabolic pathways and cell proliferation.

To adjust energy balance, AMPK regulates • Almost all cellular metabolic processes (activation of ATP-generating pathways such as glucose and fatty acid uptake and catabolism, and inhibition of energy-consuming pathways such as the synthesis of glycogen, fatty acids, cholesterol, proteins, and ribosomal RNA) • Whole-body energy balance (appetite regulation in the hypothalamus via leptin, adiponectin, ghrelin, and cannabinoids) • Many nonmetabolic processes (cell growth and proliferation, mitochondrial homeostasis, autophagy, aging, neuronal activity, and cell polarity) Because of its central roles in the uptake and metabolism of glucose and fatty acids, AMPK is an important pharmacological target for treating diabetes and obesity. Moreover, AMPK activation restrains the growth and metabolism of tumor cells and has thus become an exciting new target for cancer therapy. In this project we strive to determine the structural mechanisms of AMPK regulation by direct binding of AMP, ADP, ATP, drugs, and glycogen, in order to provide a structural framework for the rational design of new therapeutic AMPK modulators.

Abscisic acid Abscisic acid is an ancient signaling molecule found in plants, fungi, and metazoans ranging from sponges to humans. In plants, ABA is an essential hormone and is also the central regulator protecting plants against abiotic stresses such as drought, cold, and high salinity. These stresses—most prominently, the scarcity of fresh water—are major limiting factors in crop production and therefore major contributors to malnutrition. Malnutrition affects an estimated one billion people and contributes to more than 50% of human disease worldwide, including cancer and infectious diseases.

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We have determined the structure of ABA receptors in their free state and while bound to ABA. Using computational receptordocking experiments, we have identified and verified synthetic small-molecule receptor activators as new chemical scaffolds toward the development of new, environmentally friendly, and affordable compounds that will protect plants against abiotic stresses. We have also identified the structural mechanism of the core ABA signaling pathway, which will allow modulation of this pathway through genetic engineering of crop plants.

Folate receptors Folates (folic acid and derivatives) are one-carbon donors required for the synthesis of DNA. Rapidly dividing cells, such as cancer cells, require rapid DNA synthesis and are therefore selectively dependent on high folate levels. This vulnerability has been therapeutically exploited since the 1940s, when toxic folate analogs (antifolates) were used as the first chemotherapeutic agents. However, current antifolates have severe side effects such as immunosuppression, nausea, and hair loss, because they also kill nonmalignant proliferative cells.

Cells can take up folates in two main ways: by a ubiquitous, high-capacity, low-affinity uptake system known as RFC and by folate receptors, which are cysteine-rich cell surface glycoproteins that allow high-affinity uptake of folates by endocytosis but that do not take up the current antifolate drugs. While folate receptors are expressed at very low levels in most tissues, they are “hijacked” and expressed at high levels in numerous cancers. This selective expression has been therapeutically and diagnostically exploited by the administration of antibodies against folate receptor , of folate-based imaging agents, and of folate-conjugated drugs and toxins. We expect that targeting antifolates for uptake by folate receptors, but not by the RFC, would greatly reduce the side effects of antifolate chemotherapy.

We have determined the structure of folate receptor  in complex with folic acid. The structure, validated by systematic mutations of pocket residues and quantitative folic acid binding assays, has provided a detailed map of the extensive interactions between folic acid and FR. It also provides a structural framework for the design of novel antifolates that are selectively taken up by folate receptors. Our short-term goal is to determine the structures of novel, preclinical chemotherapeutic antifolates, bound to folate receptors and bound to the folate-metabolizing enzymes they inhibit, in order to rationally design novel antifolates that selectively target cancer cells.

Recent Publications Li, Xiaodan, Lili Wang, Edward X. Zhou, Jiyuan Ke, Parker W. de Waal, Xin Gu, M.H. Eileen Tan, Dongye Wang, Donghai Wu, et al. In press. Structural basis of AMPK regulation by adenine nucleotides and glycogen. Cell Research.

He, Yuanzheng, Wei Yi, Kelly Suino-Powell, X. Edward Zhou, W. David Tolbert, Xiaobo Tang, Jing Yang, Huaiyu Yang, Jingjing Shi, et al. 2014. Structures and mechanism for the design of highly potent glucocorticoids. Cell Research 24(6): 713–726.

Ng, Ley Moy, Karsten Melcher, Bin Tean Teh, and H. Eric Xu. 2014. Abscisic acid perception and signaling: structural mechanisms and applications. Acta Pharmacologica Sinica 35(5): 567–584.

Zhi, Xiaoyong, X. Edward Zhou, Yuanzheng He, Christoph Zechner, Kelly M. Suino-Powell, Steven A. Kliewer, Karsten Melcher, David J. Mangelsdorf, and H. Eric Xu. 2014. Structural insights into gene repression by the orphan nuclear receptor SHP. Proceedings of the National Academy of Sciences U.S.A. 111(2): 839–844.

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Cindy K. Miranti, Ph.D. Laboratory of Integrin Signaling and Tumorigenesis Dr. Miranti received her M.S. in microbiology from Colorado State University and her Ph.D. in biochemistry from Harvard Medical School.

She was a postdoctoral fellow in the laboratory of Joan Brugge at ARIAD Pharmaceuticals, Cambridge, Massachusetts, and in the Department of Cell Biology at Harvard Medical School. Dr. Miranti joined VARI in January 2000, where she is currently an Associate Professor. She is also an Adjunct Professor in the Department of Physiology at Michigan State University.

From left: Bergsma, Frank, Nollet, Schulz, Ganguly, Berger, Miranti, Jensen

Staff

Students

Penny Berger, B.S. Sourik Ganguly, Ph.D. Veronique Schulz, B.S.

Alexis Bergsma, B.S. Sander Frank, B.A. Corbin Jensen Eric Nollet, B.S. Khristal Thomas McLane Watson Jelani Zarif, M.S 25

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Scientific Report 2015

Research Interests Our objective is to define the roles of integrins and the tumor microenvironment in prostate cancer development, hormonal resistance, and metastasis. Our approach is to understand the normal biology of the prostate gland and its microenvironment, as well as the bone environment, to inform on the mechanisms by which tumor cells remodel and use that environment to develop, acquire hormonal resistance, and metastasize. Our research is focused in three primary areas: 1) developing in vitro and in vivo models that recapitulate human disease based on clinical pathology, 2) identifying signal transduction pathway components that could serve as both clinical markers and therapeutic targets, and 3) defining the genetic/epigenetic programming involved in prostate cancer development.

Clinical significance • Prostate cancer remains the second-leading cancer killer of men due to the inability to cure hormone-resistant metastatic disease. Our laboratory is focused on defining the mechanisms of hormone resistance and metastasis, and we hypothesize that the tumor microenvironment plays a major role.

• We have shown that drugs which initially show promise in laboratory settings fail in clinical trials because the existing models for prostate cancer fail to adequately address the role of the tumor microenvironment. We have developed a rational approach to defining how the tumor microenvironment affects cell survival and drug resistance.

• Many men develop prostate cancer that will not progress to lethal disease, but we lack the ability to predict which tumors will progress, resulting in overdiagnosis and unnecessary treatment. We need to identify specific steps in oncogenesis that lead to aggressive disease, and we are addressing the lack of adequate models for primary disease progression by developing new ones.

• Over 80% of prostate cancers metastasize to the bone. We are developing better models to understand both normal bone development and bone/cancer cell interactions.

The AR/61 integrin axis The human prostate gland contains basal cells which express and use integrins to adhere to the laminin matrix in the tissue microenvironment. Basal cells do not express the androgen receptor (AR), but they differentiate into AR-expressing secretory cells that detach from the matrix and lose integrin expression. In prostate cancer, the AR-expressing tumor cells retain expression of integrin 61. We hypothesize that abnormal cross talk between AR and integrin 61 is crucial for prostate cancer development and progression to castration-resistant disease.

We identified a novel AR survival pathway in androgen-responsive tumors whereby AR in combination with the oncogenic TMPRSS2-Erg fusion protein directly stimulates integrin 61 transcription and expression. Engagement of integrin 61 by laminin in turn stimulates NF-B/RelA activation and subsequently increases the transcription of Bcl-xL to promote survival. In castration-resistant tumors, this same pathway in coordination with HIF1/2 induces another pathway, one that involves BNIP3 and promotes survival in the presence of high oxidative stress by specifically targeting mitochondrial degradation through autophagy.

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Miranti

The loss of Pten, which leads to enhanced PI3K signaling, occurs in 60% of advanced prostate cancers, yet PI3K inhibitors are not effective in patients. When plated on laminin to engage integrin 61, tumor cells were resistant to PI3K inhibition.

Blocking PI3K in combination with blocking AR, integrin 61, RelA, Bcl-xL, or BNIP3 resensitized the cells to such inhibition.

Thus, interaction with the tumor microenvironment through AR/61 is an important mechanism by which prostate tumor cells escape their reliance on PI3K signaling, and disrupting this pathway will be necessary for effectively blocking prostate cancer in vivo.

Differentiation and oncogenesis The prostate cancer field is hampered by the lack of cell models that reflect in vivo events. We developed an in vitro differentiation model in which basal epithelial cells are differentiated into secretory cells that behave similarly to those in vivo; i.e., the secretory cells are marked by their loss of integrin expression and loss of adhesion to matrix. When we engineered these cells to simultaneously overexpress Myc and TMPRSS2/Erg and inhibit Pten, we generated tumorigenic cells that co-expressed integrin 61 and AR, analogous to what is seen in vivo. Moreover, these tumor cells were unable to differentiate due to loss of the chromatin-modifying protein ING4. The expression of this protein is lost in more than 60% of human prostate cancers. We are currently determining how ING4 expression regulates chromatin and epigenetic programming to suppress tumorigenesis.

CD82/KAI1 in bone development CD82/KAI1 is encoded by a metastasis suppressor gene whose loss in primary prostate tumors correlates with poor patient prognosis. CD82 is one of 33 tetraspanins whose functions remain enigmatic but are linked to cell adhesion. We generated CD82-null mice to better understand the normal function of CD82. Their most striking phenotype was enhanced platelet clotting and reduced bleeding, as well as a twofold increase in total platelets. The increase in platelets was due to changes in megakaryocyte differentiation within the bone marrow. Other changes within the bone included an increased number of adipocytes and a reduced number of hematopoietic stem cells, as well as an increased bone density. CD82 has been implicated in osteoclast differentiation; correspondingly, there are fewer TRAP osteoclasts. We are currently determining what aspect of osteoclast differentiation is affected by CD82 loss.

Recent Publications Berger, Penny L., Sander B. Frank, Veronique V. Schulz, Eric A. Nollet, Mathew J. Edick, Brittany Holly, Ting-Tung A. Chang, Galen Hostetter, Suwon Kim, et al. 2014. Transient induction of ING4 by MYC drives prostate epithelial cell differentiation and its disruption drives prostate tumorigenesis. Cancer Research 74(12): 3357–3368.

Miranti, Cindy K., Alexis Bergsma, and Annemiek B. van Spriel. 2014. Tetraspanins as master organizers of the plasma membrane. In Cell Membrane Nanodomains: From Biochemistry to Nanoscopy, A. Cambi and D.S. Lidke, eds. Boca Raton, FL: CRC Press, pp. 59–86.

Akfirat, Canan, Xiaotun Zhang, Aviva Ventura, Dror Berel, Mary E. Coangelo, Cindy K. Miranti, Maryla Krajewska, John C. Reed, Celestia S. Higano, et al. 2013. Tumour cell survival mechanisms in lethal metastatic prostate cancer differ between bone and soft tissue metastases. Journal of Pathology 230(3): 291–297.

Frank, Sander B., and Cindy K. Miranti. 2013. Disruption of prostate epithelial differentiation pathways and prostate cancer development. Frontiers in Oncology 3: 273.

Nollet, Eric A., and Cindy K. Miranti. 2013. Integrin and matrix regulation of autophagy and mitophagy. In Autophagy, Yannick Bailly, ed. New York: InTech, pp. 465–485.

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Lorenzo F. Sempere, Ph.D. Laboratory of microRNA Diagnostics and Therapeutics Dr. Sempere obtained his B.S. in biochemistry at Universidad Miguel Hernรกndez, Elche, Spain. He trained in the laboratory of Victor Ambros at Dartmouth College and is currently an Assistant Professor in the Center for Cancer and Cell Biology. He has expertise in diverse areas of microRNA research, including evolutionary and developmental biology, molecular and cellular biology, and immunology and cancer biology. He is the editor of the journal microRNA Diagnostics and Therapeutics.

From left: Westerhuis, Calderone, Grit, Weaver, Sempere

Staff Heather Calderone, Ph.D. Jamie Grit, B.S. Laura Weaver Jenni Westerhuis, M.S.Ed., M.S. 28

Sempere

Research Interests Our laboratory pursues complementary lines of translational research to explain the etiological role of microRNAs and to unravel microRNA regulatory networks during carcinogenesis. We mainly investigate these questions in clinical samples and preclinical models of breast cancer and pancreatic cancer. MicroRNAs can regulate and modulate the mRNA expression of hundreds of target genes, some of which are components of the same signaling pathways or biological processes. Accordingly, functional modulation of a single microRNA can affect multiple target mRNAs (i.e., one drug, multiple hits), unlike therapies based on small interfering RNAs, antibodies, or small-molecule inhibitors.

The laboratory has active projects in the areas of cancer biology and tumor microenvironment, with a translational focus on molecular and cellular heterogeneity and its clinical implications for improving diagnostic applications and therapeutic strategies. Knowledge of microRNAs is integrated into collaborative efforts with VARI researchers and cores, as well as into new technologies being developed for microRNA studies. Work in the laboratory includes the following.

• Molecular pathology with innovative human tissue–based multiplex ISH/IHC assays to implement diagnostic applications of microRNA biomarkers in a clinical setting. Tissue samples are the direct connection between cancer research and cancer medicine. Detailed cellular and molecular characterization of tumors presents a unique opportunity to translate scientific knowledge into useful clinical information.

– Clinically validate tumor compartment–specific expression of miR-21 as a novel prognostic marker in breast cancer.

There is a focused interest in stromal expression of miR-21 for risk assessment and stratification of patients with triple-negative breast cancer, for which prognostic markers and effective targeted therapies are lacking.

– Develop integrative diagnostic applications for pancreatic cancer and precursor lesions using information derived from cancer-associated microRNAs and glycosylation biology. Integration of informative microRNA and protein markers should enhance diagnostic power and interpretation.

– Implement innovative technological platforms for high-content tissue-based marker analysis. Our goal is a fully automated pipeline from tissue stain to image analysis that we can use to characterize tumor features and interrogate the biology of tumor compartment–specific events such as molecular alterations in cancer cells, paracrine signaling by tumor-associated fibroblasts, and anti-tumor immune cell responses.

• Genetic engineering of models to assess the role of microRNAs within tumor microenvironment compartments.

– Evaluate the miR-21 activity required in cancer cell and tumor stroma compartment(s) to support aggressive and metastatic features in animal models of breast and pancreatic cancers.

– Replenish miR-155 immunostimulatory activity in combination with immune checkpoint regulators to boost antitumor immunity in preclinical models of pancreatic cancer.

• Molecular biology and cellular biology studies to identify microRNA targets and regulatory networks.

– Develop methods for isolating microRNA/target mRNA interactions in in vitro and in vivo systems.

– Identify tumor compartment–specific microRNA-regulated target networks in preclinical models and clinical specimens.

– Evaluate tumor compartment–specific delivery of synthetic microRNA activity modulators in preclinical cancer models and patient-derived cells (e.g., cancer cells, tumor-associated fibroblasts, dendritic cells).

Recent Publications Mackenzie, T.A., G.N. Schwartz, H.M. Calderone, C.R. Graveel, M.E. Winn, G.Hostetter, W.A. Wells, and L.F. Sempere. In press. Stromal expression of miR-21 identifies high-risk group in triple-negative breast cancer. American Journal of Pathology.

Sempere, Lorenzo F. 2014. Tissue slide-based microRNA characterization of tumors: how detailed could diagnosis become for cancer medicine? Expert Review of Molecular Diagnostics 14(7): 853–869.

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Matthew Steensma, M.D. Laboratory of Musculoskeletal Oncology Dr. Steensma received his B.A. from Hope College and his M.D. from Wayne State University School of Medicine in Detroit. He then completed research training in the laboratory of George Vande Woude (VARI) prior to a musculoskeletal oncology fellowship at Memorial Sloan-Kettering Cancer Center in New York. Upon completion of his surgical training, Dr. Steensma worked in the laboratory of Steve Goldring, studying mechanisms of pathologic bone resorption. There Dr. Steensma further developed his interest in the molecular and cellular mechanisms underlying bone and soft-tissue sarcomas. Dr. Steensma is a practicing surgeon in the Spectrum Health Medical Group, and he joined VARI as an Assistant Professor in 2010.

From left: Schmidt, Lewis, Scholten, Smith, Steensma, Peacock, Kampfschulte, Pelle, Mooney, Foley.

Staff

Students

Visiting Scientists

Kevin Kampfschulte, B.A. Diana Lewis, A.S. Jacqueline Peacock, Ph.D.

Marie Mooney, M.S. Akash PremKumar Courtney Schmidt D.J. Scholten, B.A. Mallory Smith

Jessica Foley, M.D. Dominic Pelle, M.D.

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Steensma

Research Interests The Laboratory of Musculoskeletal Oncology conducts research which aims to develop new treatment strategies for sarcomas.

Specifically, we are interested in determining the mechanisms underlying tumor formation in sporadic bone and soft tissue sarcomas and in neurofibromatosis type 1, a hereditary disorder caused by mutations in the neurofibromin 1 (NF1) gene.

Neurofibromin is considered a tumor suppressor that suppresses Ras activity by promoting Ras GTP hydrolysis to GDP. People with mutations in the neurofibromin 1 gene develop benign tumors called neurofibromas and have an elevated risk of malignancies ranging from solid tumors to leukemia, including highly aggressive sarcomas. The disease affects 1 in 3000 people in the United States, of whom 8-13% will ultimately develop a neurofibromatosis-related sarcoma in their lifetime. These aggressive tumors typically arise from benign neurofibromas, but the process of benign to malignant transformation is not well understood and treatment options are limited, leading to poor five-year survival rates.

Our current sarcoma-related research efforts include the development of genetically engineered mouse of models of neurofibromatosis type 1 tumor progression; the identification of targetable patterns of intratumoral and intertumoral heterogeneity through next-generation sequencing; genotype-phenotype correlations in neurofibromatosis type 1 and related diseases; and mechanisms of chemotherapy resistance in bone and soft-tissue sarcomas.

Recent Publications Pelle, Dominic W., Jonathan W. Ringler, Jacqueline D. Peacock, Kevin Kampfschulte, Donald J. Scholten II, Mary M. Davis, Deanna S. Mitchell, and Matthew R. Steensma. In press. Targeting receptor-activator nuclear kappa beta ligand in aneurysmal bone cysts: verification of target and therapeutic response. Translational Research.

Puhaindran, M., S. Schlumbohm, K. Hamilton, M. Rich, D. Mitchell, and M.R. Steensma. In press. Radiation-induced osteosarcoma of the hand. Journal of Hand Surgery.

Scholten, D.J., II, C.M. Timmer, J. Peacock, D.W. Pelle, B. Williams, and M.R. Steensma. In press. Hypoxia-mediated downregulation of Wnt/î ˘-catenin signaling is a viable target for chemoresistance in human osteosarcoma cells. PLoS One. Monks, Noel R., David M. Cherba, Steven G. Kamerling, Heather Simpson, Anthony W. Rusk, Derrick Carter, Emily Eugster, Marie Mooney, Robert Sigler, et al. 2013. A multi-site feasibility study for personalized medicine in canines with osteosarcoma.

Journal of Translational Medicine 11: 158.

Peacock, Jacqueline D., David Cherba, Kevin Kampfschulte, Mallory K. Smith, Noel R. Monks, Craig P. Webb, and Matthew R. Steensma. 2013. Molecular-guided therapy predictions reveal drug resistance phenotypes and treatment alternatives in malignant peripheral nerve sheath tumors. Journal of Translational Medicine 11: 213.

Steensma, Matthew, and John H. Healey. 2013. Trends in the surgical treatment of pathologic proximal femur fractures among Musculoskeletal Tumor Society members. Clinical Orthopaedics and Related Research 471(6): 2000–2006.

Steensma, Matthew R., Wakenda K. Tyler, Allison G. Shaber, Steven R. Goldring, F. Patrick Ross, Bart O. Williams, John H. Healey, and P. Edward Purdue. 2013. Targeting the giant cell tumor stromal cell: functional characterization and a novel therapeutic strategy. PLoS One 8(7): e69101.

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George F. Vande Woude, Ph.D. Laboratory of Molecular Oncology Dr. Vande Woude received his M.S. and Ph.D. degrees from Rutgers University. In 1972, he joined the National Cancer Institute as head of the Human Tumor Studies and Virus Tumor Biochemistry sections. In 1983, he became director of the Advanced Bioscience Laboratories–Basic Research Program at the Frederick Cancer Research and Development Center, a position he held until 1998. From 1995, Dr. Vande Woude first served as special advisor to the director, and then as director, of the Division of Basic Sciences at NCI. In 1999, he was recruited as the founding Director of VARI. In 2009, Dr. Vande Woude stepped down as Director while retaining his leadership of the Laboratory of Molecular Oncology as a Distinguished Scientific Fellow and Professor. Dr. Vande Woude is a member of the National Academy of Sciences (1993) and a Fellow of the American Association for the Advancement of Science (2013).

From left, standing: Gao, Kang, Koo, Yerrum; seated: Essenburg, Vande Woude, Kaufman

Staff

Adjunct Faculty

Curt Essenburg, B.S. Chongfeng Gao, Ph.D. Liang Kang, B.S. Dafna Kaufman, M.S. Kay Koo Yanli Su, A.M.A.T. Smitha Yerrum, M.S.

Brian Cao, M.D. Henry B. Skinner, Ph.D.

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Vande Woude

I am pleased to report that in the past year, three senior scientists from my lab—Drs. Yu-Wen Zhang, Carrie Graveel, and Qian Xie—were promoted to the position of Research Assistant Professor, an independent leadership position at VARI. Drs. Zhang, Graveel, and Xie joined my laboratory as postdoctoral fellows in the early 2000s, and it has been a privilege to mentor them over the years as they have grown and developed their unique scientific paths. Each one has demonstrated outstanding research abilities that have contributed tremendously to the Institute and toward fulfilling the VARI mission as a whole.

The creativity, broad knowledge, and unique experiences of these scientists have led to many important publications and professional collaborations with scientists around the globe. I couldn’t be more proud of the accomplished colleagues that they have become, and I wish them continued success on their respective journeys to scientific discovery. To learn more about their research projects and current interests, please see their individual pages.

Research Interests MET is overexpressed in many types of human cancer, and its expression correlates with aggressive disease and poor prognosis (visit http://www.vai.org/met/). Since discovering the MET receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF/SF), in the mid 1980s, the Vande Woude lab has focused on investigating the paramount role these molecules play in malignant progression and metastasis. As part of the ongoing effort to further our understanding of this signaling system, the lab focuses on the mechanisms responsible for tumor progression under the hypothesis that phenotypic switching and chromosome instability can drive tumor progression. In addition, we continue to develop and characterize novel animal research models that are used in preclinical evaluation of new inhibitors that target MET in a variety of human cancers.

Tumor phenotypic switching: mechanism and therapeutic implications In human carcinomas, the acquisition of an invasive phenotype requires a breakdown of intercellular junctions with neighboring cells, a process termed the epithelial-to-mesenchymal transition (E-MT). Upon arriving at secondary sites, the mesenchymal cells revert to an epithelial phenotype via a mesenchymal-to-epithelial transition (M-ET). Human carcinoma tissues and cells typically show extensive heterogeneity in both phenotype and genotype, suggesting a role for genetic instability in cell type determination. To test this possibility, we have developed methods to continually isolate phenotypic variants from epithelial or mesenchymal subclones of carcinoma cell lines. We have explored the signal pathway underlying E-MT/M-ET phenotypic switching by gene expression analysis, spectral karyotyping (SKY), and fluorescent in situ hybridization (FISH). We found that changes in chromosome content are associated with phenotypic switching. We further showed that these changes dictated the expression of specific genes, which in E-MT events are mesenchymal and in M-ET events are epithelial. Our results suggest that chromosome instability can provide the diversity of gene expression needed for tumor cells to switch phenotype.

In vivo research models: model development and preclinical treatment evaluation Anti-cancer therapy based on blocking the HGF-Met signaling pathway has emerged as an important goal of pharmaceutical research. One of the limitations of studying the altered Met–HGF/SF signaling of human cancers grafted in mouse models has been that the murine HGF/SF protein has a low affinity for human MET. To overcome this, our lab developed the transgenic human HGF-SCID mouse model (hHGFtg-SCID), which generates a human-compatible HGF/SF protein and thus allows for the propagation of human tumors. This model has proven to be a valuable preclinical tool for in vivo study of MET-dependent cancers and is used to evaluate treatment strategies that aim to target this pathway.

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Recent Publications Staal, Ben, Bart O. Williams, Frank Beier, George F. Vande Woude, and Yu-Wen Zhang. 2014. Cartilage-specific deletion of Mig-6 results in osteoarthritis-like disorder with excessive articular chrondrocyte proliferation. Proceedings of the National Academy of Sciences U.S.A. 111(7): 2590–2595.

Graveel, Carrie R., David Tolbert, and George F. Vande Woude. 2013. MET: a critical player in tumorigenesis and therapeutic target. Cold Spring Harbor Perspectives in Biology 5(7): a009209.

Merchant, Mark, Xiaolei Ma, Henry R. Maun, Zhong Zheng, Jing Peng, Mally Romero, Arthur Huang, Nai-ying Yang, Merry Nishimura, et al. 2013. Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent. Proceedings of the National Academy of Sciences U.S.A. 110(32): E2987–E2996.

Paulson, Amanda K., Erik S. Linklater, Bree D. Berghuis, Colleen A. App, Leon D. Oostendorp, Jayne E. Paulson, Jane E. Pettinga, Marianne K. Melnik, George F. Vande Woude, et al. 2013. MET and ERBB2 are coexpressed in ERBB2+ breast cancer and contribute to innate resistance. Molecular Cancer Research 11(9): 1112–1121.

Xie, Qian, Yanli Su, Karl Dykema, Jennifer Johnson, Julie Koeman, Valeria De Giorgi, Alan Huang, Robert Schlegel, Curt Essenburg, et al. 2013. Overexpression of HGF promotes HBV-induced hepatocellular carcinoma progression and is an effective indicator for Met-targeting therapy. Genes & Cancer 4(7-8): 247–260.

Zhang, Yu-Wen, Ben Staal, Curt Essenburg, Steven Lewis, Dafna Kaufman, and George F. Vande Woude. 2013. Strengthening context-dependent anticancer effects on non–small cell lung carcinoma by inhibition of both MET and EGFR. Molecular Cancer Therapeutics 12(8): 1429–1441.

Zhang, Yu-Wen, and George F. Vande Woude. 2013. MIG-6 and SPRY2 in the regulation of receptor tyrosine kinase signaling: balancing act via negative feedback loops. In Future Aspects of Tumor Suppressor Genes, Yue Chang, ed. Rijeka, Croatia: InTech, pp. 199–221.

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Bart O. Williams, Ph.D. Laboratory of Cell Signaling and Carcinogenesis Dr. Williams received his Ph.D. in biology from Massachusetts Institute of Technology in 1996 where he trained with Tyler Jacks. For three years, he was a postdoctoral fellow at the National Institutes of Health in the laboratory of Harold Varmus, former Director of NIH. Dr. Williams joined VARI as a Scientific Investigator in July 1999. He is now a Professor and the Director of the Center for Cancer and Cell Biology.

From left: Droscha, Lewis, Maupin, Ethen, Diegel, Nguyen, Valkenburg, VanHouten, McDonald, Williams

Staff

Students

Adjunct Faculty

Visiting Scientist

Cassandra Diegel, B.S. Diana Lewis, A.S. Mitch McDonald, B.S.

Casey Droscha, B.S. Nicole Ethen Kevin Maupin, B.A., B.S. Rebecca Nguyen Ken Valkenburg, B.S.

Clifford Jones, M.D. Debra Sietsema, Ph.D., RN

Chad VanHouten, M.S.

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Research Interests Our laboratory is interested in understanding how alterations in the Wnt signaling pathway cause human disease. Wnt signaling is an evolutionarily conserved process that functions in the differentiation of most tissues within the body. Given its central role in growth and differentiation, it is not surprising that alterations in the pathway are among the most common events associated with human cancer. In addition, several other human diseases, including osteoporosis, cardiovascular disease, and diabetes, have been linked to altered regulation of this pathway. A specific focus of our work is characterizing the role of Wnt signaling in bone formation. Our interest is not only from the perspective of normal bone development, but also in trying to understand whether aberrant Wnt signaling plays a role in the predisposition of some common tumor types (for example, prostate, breast, lung, and renal tumors) to metastasize to and grow in bone. The long-term goal of this work is to provide insights useful in developing strategies to lessen the morbidity and mortality associated with skeletal metastasis.

Wnt signaling in normal bone development Mutations in the Wnt receptor Lrp5 have been causally linked to alterations in human bone development. We have characterized a mouse strain deficient in Lrp5 and have shown that it recapitulates the low-bone-density phenotype seen in human patients who have a Lrp5 deficiency. We have further shown that mice carrying mutations in both Lrp5 and the related Lrp6 protein have even more-severe defects in bone density. To test whether Lrp5 deficiency causes changes in bone density due to aberrant signaling through -catenin, we created mice carrying an osteoblast-specific deletion of -catenin (OC-Cre;-cateninflox/flox mice). We are addressing how other genetic alterations linked to Wnt/-catenin signaling affect bone development and osteoblast function. We have generated mice with conditional alleles of Lrp6 and Lrp5 that can be inactivated via Cre-mediated recombination and demonstrated that both Lrp5 and Lrp6 function within osteoblasts to regulate normal bone development and homeostasis. We have also created mice which lack the ability to secrete Wnts from osteoblasts and shown that these mice also have extremely low bone mass, establishing that the mature osteoblast is an important source of Wnts for establishing and maintaining normal bone mass. We are also examining the effects of chemical inhibitors of the enzyme porcupine on normal bone development and homeostasis, which is required for the secretion and activity of all Wnts. Given that such inhibitors are currently in human clinical trials for treatment of several tumor types, their side effects related to the lowering of bone mass must be evaluated.

Wnt signaling in mammary development and cancer We are also addressing the relative roles of Lrp5 and Lrp6 in Wnt1-induced mammary carcinogenesis. A deficiency in Lrp5 dramatically inhibits the development of mammary tumors, and a germline deficiency for Lrp5 or Lrp6 results in delayed mammary development. Because Lrp5-deficient mice are viable and fertile, we have focused our initial efforts on these mice.

We have also found that Lrp6 plays a key role in mammary development, and we are focusing on the mechanisms underlying this role. We are particularly interested in how these pathways may regulate the proliferation of normal mammary progenitor cells, as well as of tumor-initiating cells.

Wnt signaling in prostate development and cancer Two hallmarks of advanced prostate cancer are the development of skeletal osteoblastic metastasis and the ability of the tumor cells to become independent of androgen for survival. We have created mice with a prostate-specific deletion of the Apc gene. These mice develop fully penetrant prostate hyperplasia by four months of age, and these tumors progress to frank carcinomas by seven months. We have found that these tumors initially regress under androgen ablation but show signs of androgen-independent growth some months later.

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Williams

Genetically engineered mouse models of bone disease We have also focused on developing mouse models of osteoarthritis and of fracture repair. In addition, we are interested in identifying novel genes that play key roles in skeletal development and maintenance of bone mass. For example, current work is focused on the role of galectin-3, a member of the lectin family, in this context.

Recent Publications Collins, C.J., J.F. Vivanco, S. Sokn, B. Williams, T. Burgers, and H.L. Ploeg. In press. Fracture healing in mice lacking Pten in osteoblasts: a micro-computed tomography image-based analysis of the mechanical properties of the femur. Journal of Orthopaedic Research.

Lim, W.H., B. Liu, D. Cheng, B.O. Williams, S.J. Mah, and J.A. Helms. In press. Wnt signaling regulates homeostasis of the periodontal ligament. Journal of Periodontal Research.

Scholten, D.J., II, C.M. Timmer, J. Peacock, D.W. Pelle, B. Williams, and M.R. Steensma. In press. Hypoxia-mediated down-regulation of Wnt/-catenin signaling is a viable target for chemoresistance in human osteosarcoma cells. PLoS One. Zhong, Zhendong, Nicole J. Ethen, and Bart O. Williams. In press. WNT signaling in bone development and homeostasis.

Wiley Interdisciplinary Reviews: Developmental Biology.

Hoffmann, F. Michael, Travis Burgers, James J. Mason, Bart O. Williams, Debra L. Sietsema, and Clifford B. Jones. 2014.

Biomechanical evaluation of fracture fixation constructs using a variable-angle locked periprosthetic femur plate system.

Injury 45(7): 1035–1041.

Joiner, Danese M., Kennen D. Less, Emily M. Van Wieren, Yu-Wen Zhang, Daniel Hess, and Bart O. Williams. 2014.

Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 (Mig-6) after ligament and meniscus injury. Arthritis Research and Therapy 16(2): R81.

Kabiri, Zahra, Gediminas Greicius, Babita Madan, Steffan Biechele, Zhendong Zhong, Hamed Zaribafzadeh, Edison, Jamal Aliyed, Yonghui Wu, et al. 2014. Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts. Development 141(11): 2206–2215.

Lim, Won Hee, Bo Liu, Du Cheng, Daniel J. Hunter, Zhendong Zhong, Daniel M. Ramos, Bart O. Williams, Paul T. Sharpe, Claire Bardet, et al. 2014. Wnt signaling regulates pulp volume and dentin thickness. Journal of Bone and Mineral Research 29(4): 892–901.

Staal, Ben, Bart O. Williams, Frank Beier, George F. Vande Woude, and Yu-Wen Zhang. 2014. Cartilage-specific deletion of Mig-6 results in osteoarthritis-like disorder with excessive articular chrondrocyte proliferation. Proceedings of the National Academy of Sciences U.S.A. 111(7): 2590–2595.

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Ning Wu, Ph.D. Laboratory of Cancer Signaling and Metabolism Dr. Wu received her Ph.D. in the Department of Biochemistry of the University of Toronto in 2002. She then served as a research associate at the Scripps Research Institute in the Department of Chemistry. In 2004, she joined the Beth Israel Deaconess Medical Center, a teaching hospital of Harvard Medical School, as a research fellow in a lab studying the signaling pathways that regulate normal mammalian cell growth and the defects that cause cell transformation. Dr. Wu joined VARI in 2013 as an Assistant Professor.

Research Interests Our laboratory investigates the interface between cellular metabolism and signal transduction. The generation of two daughter cells depends on the proper uptake and use of nutrients that are often limited in the tumor environment. The distribution of these nutrients is controlled not only by the intrinsic catalytic rate and allosteric regulation of the enzymes, but also by posttranslational modifications of these enzymes by signaling molecules. At the same time, signaling molecules must respond to cellular nutrient status and other cues such as environmental stresses and growth factors. Our laboratory focuses on key metabolic steps in glucose and lipid catabolism and aims to understand the mutual regulation between metabolites and signaling during cell replication.

Fundamentally, cancer is a disease of uncontrolled cell growth. Relative to normal cells, tumor cells have aberrant metabolic addictions that differ depending on the cell’s tissue of origin and genetic mutations. By understanding the energy requirements and regulatory pathways of tumor cells, more-effective treatments can be developed. Our projects include unraveling the molecular mechanisms that regulate glucose uptake in cancers, investigating the effect of glucose on mitochondrial activity, and exploring the role of glucose as the link between metabolic syndrome and cancer incidence.

From left: Nelson, Wu, Weston

Staff

Students

Amy Nelson

Kelsey Veldkamp Erin Weston, B.A.

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Qian Xie, M.D., Ph.D. Molecular Oncogenesis and Targeted Therapy Dr. Xie received her M.D. from Fudan University Shanghai Medical College, China, in 1995. She then joined Fudan’s Zhongshan Hospital to study the molecular mechanisms of cancer invasion and metastasis. She received her Ph.D. degree in oncology in 2002 and then joined George Vande Woude as a postdoctoral fellow. She is now a Research Assistant Professor.

Research Interests Our work focuses on the molecular mechanisms of MET drugs, and we were the first to report that HGF-autocrine activation may be a molecular feature that can predict the sensitivity of glioblastoma patients to MET inhibitors. We validated that finding in liver cancer models, in which HGF-autocrine activation is also related to MET inhibitor sensitivity. We plan to explore the opportunity to translate this finding to the clinical side.

The goal of targeted therapy requires animal models that mimic the human cancer phenotype and genotype. We have invested great effort in characterizing patient-derived xenograft models of glioblastoma that show invasive tumor growth and in developing human-HGF transgenic mice that produce spontaneous hepatocellular carcinomas having genomic profiles similar to those of the human disease. Real-time imaging procedures have been established to monitor tumor growth. From these studies, we have learned key principles for establishing and optimizing preclinical models for targeted therapy.

Recent Publication Xie, Qian, Yanli Su, Karl Dykema, Jennifer Johnson, Julie Koeman, Valeria De Giorgi, Alan Huang, Robert Schlegel, Curt Essenburg, et al. 2013. Overexpression of HGF promotes HBV-induced hepatocellular carcinoma progression and is an effective indicator for Met-targeting therapy. Genes & Cancer 4(7-8): 247–260.

From left: Kang, Johnson, Xie

Staff Jennifer Johnson, M.S. Liang Kang, A.S. 39

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H. Eric Xu, Ph.D. Laboratory of Structural Sciences Dr. Xu went to Duke University and the University of Texas Southwestern Medical Center, where he earned his Ph.D. in molecular biology and biochemistry. Following a postdoctoral fellowship with Carl Pabo at MIT, he moved to Glaxo Wellcome in 1996 as a research investigator of nuclear receptor drug discovery. Dr. Xu joined VARI in July 2002 and was promoted to Professor in March 2007. He is also the Primary Investigator and Distinguished Director of the VARI/SIMM Research Center in Shanghai, China.

From left: Grant, Kang, Pal, Reynolds, Kovach, Powell, Xu, Ke, Sridharamurthy, Duan, Li, J. Chen, de Waal, Q. Chen, Zhi, Gu, Gao, Wang, Zhou

Staff Xiang Gao, Ph.D. Stephanie Grant, B.S. Xin Gu, M.S. Yanyong Kang, Ph.D. Jiyuan Ke, Ph.D.

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Amanda Kovach, B.S. Kuntal Pal, Ph.D. Kelly Powell, B.S. Xiaoyong Zhi, Ph.D. Xiaoyin (Edward) Zhou, Ph.D.

Students

Visiting Scientists

Christian Cavacece Parker de Waal, B.S. Xiaodan Li, B.S. Madhuri Sridharamurthy, B.S. Eileen Tan, B.S. Lili Wang, B.S. Feng Zhang, B.S.

Jian Chen, Ph.D. Qin Chen, M.S. Xiaoqun Duan, Ph.D. Ross Reynolds, Ph.D.

Xu

Research Interests Hormone signaling is essential to eukaryotic life. Our research is focused on the signaling mechanisms of physiologically important hormones, striving to answer fundamental questions that have a broad impact on human health and disease. The overall goals of the research program are to seek new biological paradigms through structural and functional analysis of key hormone signaling complexes and to develop therapeutic applications using the structural information we obtain. We currently focus on two families of proteins, the nuclear hormone receptors and the G protein–coupled receptors, because these proteins, beyond their fundamental roles in biology, are important drug targets for treating major human diseases.

Nuclear hormone receptors Nuclear hormone receptors are a large family comprising ligand-regulated and DNA-binding transcription factors, which include receptors for classic steroid hormones such as estrogen, progesterone, androgens, and glucocorticoids, as well as receptors for peroxisome proliferator activators, vitamin D, vitamin A, and thyroid hormones. One distinguishing fact about these classic receptors is that they are among the most successful targets in the history of drug discovery. Every receptor has one or more cognate synthetic ligands being used as medicines. Nuclear receptors also include a class of “orphan” receptors for which no ligand has been identified. In the last five years, we have developed the following projects centering on the structural biology of nuclear receptors.

Peroxisome proliferator–activated receptors

The peroxisome proliferator–activated receptors (PPAR, , and γ) are the key regulators of glucose and fatty acid homeostasis and, as such, are important therapeutic targets for treating cardiovascular disease, diabetes, and cancer. Millions of patients have benefited from treatment with novel PPARγ ligands (rosiglitazone and pioglitazone) for type II diabetes. To understand the molecular basis of ligand-mediated signaling by PPARs, we have determined crystal structures of each PPAR’s ligand-binding domain (LBD) bound to many diverse ligands, including fatty acids, the lipid-lowering drugs called fibrates, and the new generation of anti-diabetic drugs, the glitazones. We have also determined the crystal structures of the receptors bound to coactivators or co-repressors and the crystal structure of PPARγ bound to a nitrated fatty acid. These structures have provided a framework for understanding the mechanisms of agonists and antagonists, as well as the recruitment of co-activators and corepressors in gene activation and repression. Furthermore, these structures serve as a molecular basis for understanding the potency, selectivity, and binding mode of diverse ligands and have provided crucial insights for designing the next generation of PPAR medicines. We have discovered a number of natural ligands of PPARγ. Our plan is to test their physiological roles in glucose and insulin regulation in order to unravel their molecular and structural mechanisms of action and to develop them into therapeutics for diabetes and dislipidemia.

The human glucocorticoid receptor

The human glucocorticoid receptor (GR), the prototype steroid hormone receptor, is crucial for a wide spectrum of human physiology including immune/inflammatory responses, metabolic homeostasis, and control of blood pressure. GR is a wellestablished target for drugs, and those drugs have an annual market of over $10 billion. GR ligands such as dexamethasone and fluticasone propionate are used to treat asthma, leukemia, and autoimmune diseases. However, the clinical use of these ligands is limited by undesirable side effects partly associated with their receptor cross-reactivity or low potency. The discovery of potent and more-selective GR ligands—so-called “dissociated glucocorticoids” that have the potential to separate the good effects from the bad—remains a major goal of pharmaceutical research.

We have determined a number of GR crystal structures bound to unique ligands and have found an unexpected regulatory mechanism: degradation by lysosomes. We also are studying the molecular and structural mechanisms of the dissociated glucocorticoids identified by our research.

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Structural genomics of nuclear receptor LBDs

The ligand-binding domain of a nuclear receptor contains key structural elements that mediate ligand-dependent regulation of the receptors and, as such, it has been the focus of intense structural studies. Crystal structures for more than half of the 48 human nuclear receptors have been determined. These structures have illustrated the details of ligand binding, the conformational changes induced by agonists and antagonists, the basis of dimerization, and the mechanism of co-activator and co-repressor binding. The structures also have provided many surprises regarding the identity of ligands, the size and shape of the ligand-binding pockets, and the structural implications of the receptor signaling pathways. There are only a few orphan nuclear receptors for which the LBD structure remains unsolved; in the past few years, we have determined the crystal structures of the LBDs of CAR, SHP, SF-1, COUP-TFII, and LRH-1. Our structures have helped to identify new ligands and signaling mechanisms for orphan nuclear receptors.

G protein–coupled receptors (GPCRs) The GPCRs form the largest family of receptors in the human genome. They receive a diverse set of signals carried by photons, ions, small chemicals, peptides, and large protein hormones. These receptors account for over 40% of drug targets, but their structures remain a challenge because they are seven-transmembrane receptors. There are only a few crystal structures for class A GPCRs, and many important questions regarding GPCR ligand binding and activation remain unanswered. From our standpoint, GPCRs are similar to nuclear hormone receptors with respect to regulation by protein-ligand and proteinprotein interactions. Currently we are focused on class B GPCRs, which includes receptors for parathyroid hormone (PTH), corticotropin-releasing factor (CRF), glucagon, and glucagon-like peptide-1. We have determined crystal structures of the ligand-binding domain of the PTH receptor and the CRF receptor, and we are developing hormone analogs for treating osteoporosis, depression, and diabetes. In addition, we are developing a mammalian overexpression system and plan to use it to express full-length GPCRs for crystallization and structural studies.

Recent Publications Fu, T., S. Seok, S. Choi, Z. Huang, K. Suino-Powell, H.E. Xu, B. Kemper, and J.K. Kemper. In press. miR-34a inhibits beige and brown fat formation in obesity in part by suppressing adipocyte FGF21 signaling and SIRT1 function. Molecular and Cellular Biology.

Li, X., L. Wang, E.X. Zhou, J. Ke, P.W. de Waal, X. Gu, M.H.E. Tan, D. Wang, D. Wu, H.E. Xu, et al. In press. Structural basis of AMPK regulation by adenine nucleotides and glycogen. Cell Research.

He, Yuanzheng, Wei Yi, Kelly Suino-Powell, X. Edward Zhou, W. David Tolbert, Xiaobo Tang, Jing Yang, Huaiyu Yang, Jingjing Shi, et al. 2014. Structures and mechanism for the design of highly potent glucocorticoids. Cell Research 24(6): 713–726.

Zhi, Xiaoyong, X. Edward Zhou, Yuanzheng He, Christoph Zechner, Kelly M. Suino-Powell, Steven A. Kliewer, Karsten Melcher, David J. Mangelsdorf, and H. Eric Xu. 2014. Structural insights into gene repression by the orphan nuclear receptor SHP. Proceedings of the National Academy of Sciences U.S.A. 111(2): 839–844.

Chen, Chen, Jiyuan Ke, X. Edward Zhou, Wei Yi, Joseph S. Brunzelle, Jun Li, Eu-Leong Yong, H. Eric Xu, and Karsten Melcher.

2013. Structural basis for molecular recognition of folic acid by folate receptors. Nature 500(7463): 486–489.

Wang, Chong, Yi Jiang, Jinming Ma, Huixian Wu, Daniel Wacker, Vsevolod Katritch, Gye Won Han, Wei Liu, Xi-Ping Huang, et al. 2013. Structural basis for molecular recognition at serotonin receptors. Science 340(6132): 610–614.

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Tao Yang, Ph.D. Laboratory of Skeletal Biology Dr. Yang received his Ph.D. in biochemistry at the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, in 2001. He then joined Baylor College of Medicine as a postdoctoral fellow, working with Paul Overbeek (2002–2004) and Brendan Lee (2004–2009). In 2009, he was appointed an instructor in the Department of Molecular and Human Genetics at Baylor. Dr. Yang joined VARI as an Assistant Professor in February 2013.

Research Interests The skeletal system is the largest reservoir of mesenchymal stem cells (MSCs) in postnatal life. Large numbers of MSCs, in niches such as the marrow and periosteum, are necessary for the growth and maintenance of bone, which undergoes ceaseless remodeling and micro-damage repair. Dysregulations in MSC proliferation, lineage specification, and differentiation are common causes of skeletal fragility and of developmental or chronic diseases. Our long-term interest is to study how signals and cellular processes regulate MSC activity during skeletal development and homeostasis and how they affect skeletal disorders and aging. We have established in vivo genetics models and an ex vivo cell culture system that allow us to explore the mechanisms of AKT/ERK signaling integration in MSC renewal and differentiation, and the role of the sumoylation pathway in MSC renewal and senescence. Our studies could provide insights into the prevention or treatment of osteoporosis and osteoarthritis and into the healing and regeneration of skeletal tissues.

Recent Publication Grafe, Ingo, Tao Yang, Stefanie Alexander, Erica P. Homan, Caressa Lietman, Ming Ming Jiang, Terry Bertin, Elda Munivez, Yuqing Chen, Brian Dawson, et al. 2014. Excessive transforming growth factor- signaling is a common mechanism in osteogenesis imperfecta. Nature Medicine 20(6): 670–675.

From left: McMillan, Yang, Li, Lu, Lewis, Weaver

Staff Diana Lewis, A.S. Jianshuang Li, B.S.

Student Di Lu, M.S. Kevin Weaver, B.S.

Adam McMillan

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Yu-Wen Zhang, M.D., Ph.D. Signal Transduction and Molecular Medicine Dr. Zhang received his M.D. degree from Fudan University Shanghai Medical College, China, in 1989 and his Ph.D. degree in medical science from Kyoto University, Japan, in 1997. He served as a postdoctoral fellow in the laboratory of Yoshiaki Ito at the Institute for Virus Research of Kyoto University between 1997 and 1999, and then in the laboratory of George Vande Woude at VARI from 1999 to 2002. In 2002 he became a Research Scientist, and in 2010, a Senior Research Scientist at VARI. He was promoted to Research Assistant Professor in April 2013.

From left: Zhang, Staal, Rietberg

Staff

Student

Ben Staal, M.S.

Skyler Rietberg

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Zhang

Research Interests Receptor tyrosine kinases (RTKs) are crucial for many developmental and physiological processes, and their inappropriate activation can lead to cancer and other diseases. The intensity and duration of an RTK signal determines how cells will respond and what the biological outcome will be. Under normal conditions, RTK signaling needs timely attenuation via mechanisms such as receptor turnover, phosphatase-mediated dephosphorylation, and negative feedback inhibition.

We are interested in understanding how signaling driven by the RTKs, especially EGFR and MET, is regulated and why deregulation of such signaling affects disease development, progression, and therapy response.

Both EGFR and MET are often aberrantly activated in human cancers. In non-small-cell lung cancer (NSCLC) cells, these two RTKs are often coexpressed and may cross-talk in driving tumorigenesis and metastasis. Using NSCLC xenograft models, we have found that combined inhibition of MET and EGFR results in a better therapeutic efficacy by enhancing antiproliferation, inducing apoptosis, or overcoming drug resistance in a cellular context–dependent manner. We will continue to study how receptor cross talk and tumor heterogeneity influence malignant processes and drug resistance.

Mitogen-inducible gene-6 (Mig-6) inhibits the signaling driven by RTKs via a negative feedback loop: EGF induces Mig-6 expression through activation of the EGFR-RAS-MAPK signaling cascade, and Mig-6 attenuates the signaling by modulating the activity of EGFR and its downstream molecules. Mig-6 expression is altered in various human cancers, and we have identified Mig-6 mutations in several lung cancer samples (although this is rare). A tumor suppressor role for Mig-6 is also supported by the fact that its deletion in mice results in neoplasia in multiple organs/tissues. Mig-6 is essential for maintaining joint homeostasis, and mice with a Mig-6 deficiency develop degenerative joint disease in multiple synovial joints.

We are investigating how Mig-6 exerts its tumor suppressor activity and why its loss in mice leads to joint degeneration.

Recent Publications Pest, Michael A., Bailey A. Russell, Yu-Wen Zhang, Jae-Wook Jeong, and Frank Beier. In press. Loss of mitogen-inducible gene 6 results in disturbed cartilage and joint homeostatis. Arthritis and Rheumatology.

Joiner, Danese M., Kennen D. Less, Emily M. Van Wieren, Yu-Wen Zhang, Daniel Hess, and Bart O. Williams. 2014. Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 (Mig-6) after ligament and meniscus injury. Arthritis Research and Therapy 16(2): R81.

Staal, Ben, Bart O. Williams, Frank Beier, George F. Vande Woude, and Yu-Wen Zhang. 2014. Cartilage-specific deletion of Mig-6 results in osteoarthritis-like disorder with excessive articular chrondrocyte proliferation. Proceedings of the National Academy of Sciences U.S.A. 111(7): 2590–2595.

Merchant, Mark, Xiaolei Ma, Henry R. Maun, Zhong Zheng, Jing Peng, Mally Romero, Arthur Huang, Nai-ying Yang, Merry Nishimura, et al. 2013. Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent. Proceedings of the National Academy of Sciences U.S.A. 110(32): E2987–E2996.

Zhang, Yu-Wen, Ben Staal, Curt Essenburg, Steven Lewis, Dafna Kaufman, and George F. Vande Woude. 2013. Strengthening context-dependent anticancer effects on non–small cell lung carcinoma by inhibition of both MET and EGFR. Molecular Cancer Therapeutics 12(8): 1429–1441.

Zhang, Yu-Wen, and George F. Vande Woude. 2013. MIG-6 and SPRY2 in the regulation of receptor tyrosine kinase signaling: balancing act via negative feedback loops. In Future Aspects of Tumor Suppressor Genes, Yue Chang, ed. Rijeka, Croatia: InTech, pp. 199–221.

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LABORATORY REPORTS

Center for EPIGENETICS Peter A. Jones, Ph.D., D.Sc. Director

The Center’s researchers study epigenetics and epigenomics in health and disease, with the ultimate goal of developing novel therapies to treat cancer and neurodegenerative diseases.

The Center collaborates extensively with other VARI research groups and with external partners to maximize its efforts to develop therapies that target epigenetic mechanisms.

A strand of DNA (pink) is enclosed by DNA methyltransferase 1 (blue). The addition of methyl groups to DNA by this enzyme can silence and regulate genes without changing the genetic sequence. DNA methylation is being studied in relation to cancer.

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Peter A. Jones, Ph.D., D.Sc. Laboratory of Epigenetic Therapies Dr. Jones was born in South Africa, was raised and attended college in Rhodesia (now Zimbabwe), and received his Ph.D. from the University of London. He joined the University of Southern California in 1977, attaining the rank of professor in 1985 and distinguished professor in 1999, and serving as director of the USC Norris Comprehensive Cancer Center between 1993 and 2011. His laboratory discovered the effects of 5-azacytidine on DNA methylation and linked this process to the activation of silenced genes. He shared with Stephen Baylin the Kirk A. Landon Award for Basic Cancer Research from AACR in 2009 and the Medal of Honor from the American Cancer Society in 2011. Dr. Jones joined VARI in 2014 as its Research Director, Chief Scientific Officer, and Director of the Center for Epigenetics.

Research Interests Epigenetics may be defined as mitotically heritable changes in gene expression that are not caused by changes in the DNA sequence itself. Epigenetic processes establish the differentiated state of cells and govern how genes are used to allow organs and cells to function correctly and inherit their properties through cell division. In the case of diseases such as cancer, these processes can go wrong, changing the behavior of cells to adverse effect. Importantly, many of these changes are potentially reversible by treatment with appropriate drugs. Because epigenetic processes are at the root of biology, they have implications in all of human development and disease.

Our laboratory studies the mechanisms by which epigenetic processes become misregulated in cancer and contribute to the disease phenotype. We focus on the role of DNA methylation in controlling the expression of genes during normal development and in cancer. Our work has shifted to a holistic approach in which we are interested in the interactions between processes such as DNA methylation, histone modification, and nucleosomal positioning in the epigenome, and we want to determine how mutations in the genes which modify the epigenome contribute to the cancer phenotype. We have had a long-term interest in the mechanism of action of DNA methylation inhibitors, both in the lab and in the clinic. We are working with several major institutions to bring epigenetic therapies to the forefront of cancer medicine.

Research Publications Taberlay, Phillippa C., Aaron L. Statham, Theresa K. Kelly, Susan J. Clark, and Peter A. Jones. 2014. Reconfiguration of nucleosome-depleted regions at distal regulatory elements accompanies DNA methylation of enhancers and insulators in cancer. Genome Research 24(9): 1421–1432.

Yang, Xiaojing, Han Han, Daniel D. De Carvalho, Fides D. Lay, Peter A. Jones, and Gangning Liang. 2014. Gene body methylation can alter gene expression and is a therapeutic target in cancer. Cancer Cell 26(4): 577–590.

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Hitoshi Otani, Ph.D.

Anthony Popkie, Ph.D.

Stefan Jovinge, M.D., Ph.D. Laboratory of Cardiovascular Research Dr. Jovinge received his M.D. (1991) and his Ph.D. (1997) at Karolinska Institute in Stockholm. He served as a research fellow at Cedars-Sinai Hospital UCLA (1997–1999). After completing his internship, residencies, and fellowships, he become a critical care cardiologist. Dr. Jovinge was the medical director of the Cardiac Intensive Care Unit at Scania University Hospital, Lund, Sweden (2008–2012). Since December 31, 2013, he has been a critical care cardiologist at Spectrum Health and the Medical Director of Research at the Fred Meijer Heart and Vascular Institute. He also is Director of the DeVos Cardiovascular Research Program. Dr. Jovinge serves as a Professor at Michigan State University and at VARI, and he holds the Chair in Regenerative Cardiology at Lund University.

From left: Jovinge, Ellis, Neering, Tarnawski, Davidson, Dylewski, Eugster

Staff Paula Davidson, M.S. Dawna Dylewski, B.S. Ellen Ellis Emily Eugster, M.S. Sarah Neering, M.S. Laura Tarnawski, M.S. 49

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Research Interests The DeVos Cardiovascular Research Program is a joint effort between VARI and Spectrum Health. The basic science lab is the Laboratory of Cardiovascular Research at VARI, and a corresponding clinical research unit resides within the Fred Meijer Heart and Vascular Institute.

Cardiovascular diseases are among the major causes of death and disability worldwide. While the incidence of ischemic heart diseases has started to decline, congestive heart failure is still rising. Medical treatment is supportive, and the only available replacement therapy is heart transplantation.

The regeneration of myocardium after disease or damage is one of the major challenges in medicine. The Jovinge group is working on true heart muscle regeneration along three axes. The most robust source for generating heart muscle cells has been pluripotent stem cells, either from an embryonic stem cell (ESC) system or from reprogrammed pluripotent stem cells (iPSCs). The main drawback to this approach is that all germ layers are generated from the heterogeneous cell population and therefore hold the potential to create tumors. ESCs represent an external source, with the need for lifelong immunosuppressive treatment. iPSCs, however, could be generated from the patient’s own cells. To be able to use these sources, we have developed strategies, like those for bone-marrow cells, to help select homogenous and safe populations to transplant.

Although adult human heart muscle cells are to a small extent generated after birth, the internal source for this generation is still unknown. Some data indicate that cardiac progenitors could be involved, and other data suggest that differentiated heart muscle cells might be the source. We have shown that the generation of heart muscle cells is greatly impaired at the time when those cells start to become bi-nucleate. Recent data indicate that in the neonatal phase, when murine heart muscle cells are mono-nucleate, they have a complete regenerative capacity that relies on generation of new heart muscle cells from mature cells. We and our collaborators have rejected the view that adult heart muscle cells are not capable of undergoing a complete cell division. With the use of 14C dating, the adult heart has been shown to have a regenerative capacity. This has opened a completely new field of induced local generation of heart muscle cells, which is now being explored.

Our program’s eventual aims are clinical concept studies of heart muscle cell regeneration in patients, either by cell transplantation or stimulation of endogenous sources. The program’s clinical side involves a multistep process to prepare for these studies.

We have been studying the inflammatory response to myocardial infarction and the possibility of new biological anti-inflammatory regimens in order to develop options for enhancing cell endogenous and exogenous survival in myocardial infarction. We have used unique magnetic resonance sequences to identify the threatened ischemic volume of the heart. Indirectly we then can estimate the “hostility” of the local environment: the higher the proportion of threatened volume that actually infarcts, the more hostile the environment. New biological compounds that modify the inflammatory response will be studied to optimize the environment for cell transplantation.

Patients with the most severe heart disease, i.e., those needing mechanical support, are being studied to optimize treatments that will be used in later safety studies. Because the studies will enroll fewer than 200 patients, end points other than mortality and myocardial infarction will be identified. These will be based mainly on biomarkers and various imaging technologies.

The final phase of patient studies will involve the administration of cells or compounds to stimulate endogenous regeneration.

To prepare cells for transplantation into humans, an accredited Good Manufacturing Practice facility will be established, and the first safety studies (Phase II) will be followed by studies evaluating the best route for delivering the treatment and the best timing. In the final stage, randomized prospective clinical trials will be launched.

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Peter W. Laird, Ph.D. Laboratory of Cancer Epigenetics Dr. Laird earned his B.S. and M.S. from the University of Leiden and his Ph.D. in 1988 from the University of Amsterdam with Piet Borst. He received postdoctoral training from Anton Berns at the Netherlands Cancer Institute and from Rudolf Jaenisch at the Whitehead Institute for Biomedical Research. Dr. Laird was a faculty member at the University of Southern California from 1996 to 2014, where he served as professor of surgery, biochemistry and molecular biology; as Skirball-Kenis Professor of Cancer Research; as a program leader in epigenetics and regulation for the Norris Comprehensive Cancer Center; and as director of the USC Epigenome Center. He joined VARI as a Professor in September 2014.

Research Interests Our goal is to develop a detailed understanding of the molecular basis of human disease, with a particular emphasis on the role of epigenetics in cancer. Cancer is often considered to have a primarily genetic basis, with contributions from germline variations in risk and somatically acquired mutations, rearrangements, and copy number alterations. However, it is clear that nongenetic mechanisms can exert a powerful influence on cellular phenotype, as evidenced by the marked diversity of cell types within our bodies, which virtually all contain an identical genetic code. This differential gene expression is controlled by tissue-specific transcription factors and variations in chromatin packaging and modification, which can provide stable phenotypic states governed by epigenetic, not genetic, mechanisms. It seems intrinsically likely that an opportunistic disease such as cancer would take advantage of such a potent mediator of cellular phenotype. Our laboratory is dedicated to understanding how epigenetic mechanisms contribute to the origins of cancer and how to translate this knowledge into more-effective cancer prevention, detection, treatment, and monitoring.

One of the best understood epigenetic marks is the covalent modification of DNA as 5-methylcytosine in CpG dinucleotides. We use a multidisciplinary approach to understand the role of DNA methylation in cancer, relying on newly developing technology, mechanistic studies in model organisms and cell cultures, clinical and translational collaborations, genome-scale and bioinformatic analyses, and epidemiological studies. In recent years, we participated in the generation and analysis of high-dimensional epigenetic data sets, including the production of all epigenomic data for The Cancer Genome Atlas (TCGA), and the application of next-generation sequencing technology to single-base-pair-resolution, whole-genome DNA methylation analysis. We are leveraging this epigenomic data for translational applications and hypothesis testing in animal models. A major focus of our laboratory at VARI is to develop mouse models for investigating epigenetic mechanisms and drivers of cancer and to develop novel strategies for single-cell epigenomic analysis.

Staff Kelly Foy, B.S. Toshinori Hinoue, Ph.D. KwangHo Lee, Ph.D. JinA Park, M.S. Zhouwei Zhang, M.S. 51

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Gerd Pfeifer, Ph.D. Laboratory of Epigenetic Pathways in Disease Dr. Pfeifer earned his M.S. in pharmacology in 1981 and his Ph.D. in biochemistry in 1984 from Goethe University in Frankfurt, Germany. In 1988, following a postdoctoral fellowship at the University of Frankfurt Medical School, he joined Beckman Research Institute at City of Hope in Duarte, California. In 1993, he became full member of City of Hope Comprehensive Cancer Center, and in 1999 he was promoted to professor of biology at Beckman Research Institute. He also served as chair of the Department of Biology (2001–2008) and as chair of the Department of Cancer Biology (2008–2012). Most recently, he held the Lester M. and Irene C. Finkelstein Chair in Biology at City of Hope. He joined VARI in 2014 as a Professor.

Research Interests The laboratory studies epigenetic mechanisms of cancer, with a focus on DNA methylation and the role of 5-hydroxymethylcytosine in cancer. Specifically, the lab studies hypermethylation in cancer genes with the intent of elucidating the mechanisms and significance of CpG island methylation. The work centers on the hypothesis that CpG island hypermethylation in tumors is driven by one or a combination of the following: carcinogenic agents, inflammation, imbalances in methylation and demethylation pathways, oncogene activation leading to epigenetic changes, and the polycomb repression complex.

To better study genome-wide DNA methylation, Dr. Pfeifer and his team developed the methylated CpG island recovery assay (MIRA), which is used to identify commonly methylated genes that are present in human cancers. The work also focuses on epigenomic changes following exposure to environmental factors such as ultraviolet radiation and on aging and epigenomic instabilities.

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Hui Shen, Ph.D. Laboratory of Epigenomic Analysis in Human Disease Dr. Shen received her Ph.D. from University of Southern California in genetic, molecular, and cellular biology. She was appointed as a research associate at the USC Epigenome Center in 2013 and joined VARI in September 2014 as an Assistant Professor.

Research Interests The laboratory focuses on the epigenome and its interaction with the genome in various diseases, with a specific emphasis on female cancers and cross-cancer comparisons. We use bioinformatics as a tool to understand the etiology, cell of origin, and epigenetic mechanisms of various diseases and to devise better approaches for cancer prevention, detection, therapy, and monitoring. We have extensive experience with genome-scale DNA methylation profiles in primary human samples, and we have made major contributions to epigenetic analysis within The Cancer Genome Atlas (TCGA).

DNA methylation is ideally suited to deconstruct heterogeneity among cell types within a tissue sample. In cancer research, this approach can be used for cancer cell clonal evolution studies or for quantifying normal cell infiltration and stromal composition.

The latter can provide insights into the tumor microenvironment, and in noncancer studies it can be a useful tool for accurately estimating cell populations and providing insights into lineage structures and population shifts in disease. In addition, we are interested in translational applications of epigenomic technology. To this end, we bring markers emerging from our bioinformatics analysis into clinical assay development, marker panel assembly, and optimization, with the ultimate goal of clinical testing and validation.

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Piroska E. Szabó, Ph.D. Laboratory of Developmental Reprogramming Dr. Szabó earned an M.Sc. in biology and a Ph.D. in molecular biology from József Attila University, Szeged, Hungary. She joined Beckman Research Institute at City of Hope, Duarte, California, in 1992 as a postdoctoral fellow.

She then was an assistant research scientist and associate research scientist before becoming an assistant professor in the Department of Molecular and Cellular Biology at Beckman Research Institute in 2006; she was promoted to associate professor in 2011. She joined VARI in 2014 as an Associate Professor in the Center for Epigenetics.

Research Interests The laboratory studies the molecular mechanisms responsible for resetting the epigenome between generations in general and specifically in the context of genomic imprinting. Both the soma–germline and the germline–soma transitions involve global erasure and reestablishment of DNA methylation patterns. At the soma–germline transition, the paternally and maternally inherited sets of chromosomes are prepared separately in the male and female germlines. The chromosomes in the sperm and egg contribute to the next generation when they join at fertilization. Soon after fertilization, at the germline–soma transition, the two half genomes undergo another wave of global remodeling initiating somatic development. The chromosomes inherited from the sperm or the egg carry with them into the soma an epigenetic memory of the male or female germlines, which is detectable in the parental-allele-specific transcription of imprinted genes.

We are interested in the mechanisms of how DNA methylation is erased in primordial germ cells and in the zygote. We also study the patterning of de novo DNA methylation in fetal male germ cells. We use the tools of molecular biology and mouse genetics to map the changes in the epigenome and to identify the specific molecules that take part in these global and imprinted locus-specific processes. In addition, we test whether the natural epigenetic reprogramming processes of the germline are sensitive to environmental insults, potentially leading to transgenerational epigenetic inheritance.

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Steven J. Triezenberg, Ph.D. Laboratory of Transcriptional Regulation Dr. Triezenberg received his bachelor’s degree in biology and education at Calvin College in Grand Rapids, Michigan. His Ph.D. training in cell and molecular biology at the University of Michigan was followed by postdoctoral research in the laboratory of Steven L. McKnight at the Carnegie Institution of Washington. Dr. Triezenberg was a faculty member of the Department of Biochemistry and Molecular Biology at Michigan State University for more than 18 years, where he also served as associate director of the Graduate Program in Cell and Molecular Biology. In 2006, Dr. Triezenberg was recruited to VAI to serve as the founding President and Dean of the Van Andel Institute Graduate School. He succeeded Dr. Gordon Van Harn as the Director of the Van Andel Education Institute in January 2009. Dr. Triezenberg is also a VARI Professor.

From left: Alberts, Sutherland, Thellman, Triezenberg, Botting, Vander Schaaf

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Glen Alberts, B.S. Carolyn Botting, M.S. Kristie Kioshi, B.S. Nicole Vander Schaaf, B.S.

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Research Interests Our research explores the mechanisms that control how genes are expressed inside cells. Some genes must be expressed more or less constantly throughout the life of any eukaryotic cell; others must be turned on (or off) in particular cells at specific times or in response to specific signals or events. Regulation of gene expression helps determine how a given cell will function.

Our laboratory explores the mechanisms that regulate the first step in that flow, the process of transcription. We use infection by herpes simplex virus as an experimental context for exploring the mechanisms of transcriptional activation in human cells.

Transcriptional activation during herpes simplex virus infection Herpes simplex virus type 1 (HSV-1) causes the common cold sore or fever blister. The initial lytic or productive infection by HSV-1 results in the obvious symptoms in the skin and mucosa, typically in or around the mouth. Like all viruses, HSV-1 relies on the molecular machinery of the infected cell to express viral genes so that the infection can proceed and new copies of the virus can be made. This process is triggered by a viral protein known as VP16, which stimulates the initial expression of viral genes in the infected cell. Much of our work over the years has explored how VP16 activates these genes during lytic infection.

After the initial infection resolves, HSV-1 finds its way into nerve cells, where the virus can remain in a latent mode for long periods of time—essentially for the entirety of the host organism’s life. Occasionally, some triggering event (such as emotional stress or damage to the nerve from a sunburn or a root canal operation) will cause the latent virus to reactivate, producing new viruses in the nerve cell and sending them back to the skin to cause a recurrence of the cold sore. We are investigating the role that VP16 might play during such reactivation.

Chromatin-modifying coactivators in reactivating latent herpes simplex virus The strands of DNA in which the human genome is encoded are much longer than the diameter of a typical human cell. To help fit the DNA into the cell, cellular DNA is typically packaged as chromatin, in which the DNA is wrapped around “spools” of histone proteins and then further arranged into higher-order structures. When genes need to be expressed, they are partially unpackaged by the action of chromatin-modifying coactivator proteins, which either chemically change the histone proteins or physically slide the histones along the DNA. Transcriptional activator proteins such as VP16 can recruit these chromatin-modifying coactivator proteins to specific genes.

We have shown that this process is not very important during lytic infection, because the viral DNA in either the viral particle or the infected cell is not effectively packaged into chromatin. However, in the latent state, few viral genes are expressed because the viral DNA is packaged much like the silent genes of the host cell. Our present hypothesis is that the coactivators recruited

by VP16 are required to open up chromatin as an early step in reactivating the viral genes from latency. We are currently testing this hypothesis in quiescent infections of cultured human nerve cells.

Regulating the regulatory proteins: posttranslational modifications of VP16 The activity of a given protein is not only dependent on being expressed at the right time, but also on chemical modifications of that protein. Proteins can be posttranslationally modified by adding chemical groups, including phosphates, sugars, methyl or acetyl groups, lipids, or small proteins such as ubiquitin. Each of these modifications can affect how the protein folds, how it interacts with other proteins, and how stable it remains in the cell.

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We know that VP16 can be phosphorylated, and we have already defined several sites within the VP16 protein where this happens. We are now testing whether these or other modifications affect how VP16 functions, either as a transcriptional activator protein or as a structural protein of the HSV-1 virion. In some experiments, we make mutations that either prevent phosphorylation or that introduce an amino acid that mimics phosphorylation, and then we test the effects of these mutations on VP16 functions. In other experiments, we inhibit the enzymes, such as kinases, that apply the modifications. We expect that this work will lead to new ideas about ways to selectively inhibit the modification of VP16 using small-molecule drugs and thereby prevent or shorten infection.

Other cellular regulators of HSV infections HSV, like all viruses, makes use of many cellular proteins to promote its infection. In response, the infected cells take defensive measures to inhibit the virus. We would like to find ways to block the cellular proteins that support the virus or boost the cellular proteins that inhibit it. We have found that some of the cellular proteins that normally repair damaged DNA in the host cell also contribute to the replication of viral DNA, but we are still working out just how that happens. We have also found that a number of protein kinases from the host cell help with early steps in the infection process. Some of those seem to be involved in the entry of the virus into the cell; others clearly affect viral infection, but we don’t yet know why. We are exploring each of these potential participants to find out what roles they play in virus infection and whether drugs that block these kinases might be useful in treating viral infection in humans.

Recent Publication Danaher, Robert J., Ross K. Cook, Chunmei Wang, Steven J. Triezenberg, Robert J. Jacob, and Craig S. Miller. 2013. Cterminal trans-activation sub-region of VP-16 is uniquely required for forskolin-induced herpes simplex virus type 1 reactivation from quiescently infected-PC12 cells but not for replication in neuronally differentiated-PC12 cells. Journal of Neurovirology 19(1): 32–41.

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Michael Weinreich, Ph.D. Laboratory of Genome Integrity and Tumorigenesis Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsin–Madison. He did a postdoctoral fellowship at Cold Spring Harbor Laboratory, New York, before joining VARI as a Scientific Investigator in March 2000. He is currently an Associate Professor.

From left: Chang, Sasi, Minard, Weinreich

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FuJung Chang, M.S. Michelle Minard Nanda Kumar Sasi, B.S.

Nick Beam Clare Laut

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Research Interests We are studying several proteins that initiate DNA replication to learn how cells accurately maintain their genetic information.

Cancer is caused by genetic and epigenetic mutations in DNA, and early mutations that impair cellular surveillance mechanisms can promote an increased mutation rate. The cumulative mutational burden can lead to the development of cancer as cells escape the normal growth, immunological, and proliferation controls. In addition, many of the proteins required for the initiation of DNA replication also have important roles in DNA repair pathways and other cell-cycle transitions.

The Dbf4-dependent kinase (DDK; also known as Cdc7-Dbf4 kinase) is needed to initiate DNA replication at independent origins throughout the genome. It accomplishes this by phosphorylating and activating the MCM helicase, previously loaded in an inactive form at all origins during the G1 phase. DDK is also required for the accurate segregation of chromosomes during stress via a poorly understood mechanism. We recently reported that Dbf4 interacts with the Polo-like kinase Cdc5 to maintain the spindle position checkpoint.

In yeast, Cdc5 facilitates chromosome separation during metaphase, entry into anaphase, spindle elongation, the exit from mitosis, and cytokinesis. We found that DDK binds to and inhibits Cdc5 when the mitotic spindle apparatus is not properly aligned between mother and daughter cells. Loss of this regulation can cause a significant increase in chromosome segregation errors and cell death; if the mitotic process were to continue with this misalignment, it would produce one multinucleate cell and one anucleate cell. However, the mechanism and regulation of Cdc5 by DDK is not well understood.

Polo kinases such as Cdc5 contain two domains, a C-terminal polobox domain (PBD) that targets the kinase to its substrates and an N-terminal kinase domain. The PBD recognizes phosphorylated serine or threonine residues within a conserved S-pS/ pT-P/X motif. Although we previously defined a short peptide on Dbf4 that binds to the PBD, the PBD surface that interacts with Dbf4 was not known. We therefore used saturation mutagenesis to define residues within the PBD that are key to binding Dbf4, and in a second round of mutagenesis, we mapped a well-defined surface on the PBD—distinct from its pS/pT binding pocket—that binds to Dbf4. We can now investigate conserved interactions in human cells that might affect chromosome replication or segregation.

Human cells contain four Polo-like kinases (Plks), several of which have increased expression in tumor cells. Dbf4 has evolved a checkpoint effector role to prevent chromosome segregation defects through binding and inhibiting Polo kinase. Our studies detailing the mechanism of Cdc5 binding to Dbf4 have defined a fundamentally new type of interaction, one perhaps used by other Cdc5 binding proteins. Many types of tumors show increased levels of both DDK and the Polo-like kinase Plk1 which, when inhibited, cause the death of many tumor cell types but not of normal cells. Because the ability of DDK to control multiple aspects of chromosome metabolism is likely conserved, it is crucial to understand these pathways for further development of highly effective chemotherapeutic agents and interventions.

Recent Publications Corbi, Daniel, Sham Sunder, Michael Weinreich, Aikaterini Skokotas, Erica S. Johnson, and Edward Winter. 2014. Multisite phosphorylation of the Sum1 transcriptional repressor by S-phase kinases controls exit from meiotic prophase in yeast.

Molecular and Cellular Biology 34(12): 2249–2263.

Hiraga, Shin-ichiro, Gina M. Alvino, FuJung Chang, Hui-yong Lian, Akila Sridhar, Takashi Kubota, Bonita J. Brewer, Michael Weinreich, M.K. Raghuraman, et al. 2014. Rif1 controls DNA replication by directing protein phosphatase 1 to reverse Cdc7mediated phosphorylation of the MCM complex. Genes and Development 28(4): 372–383.

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LABORATORY REPORTS

Center for NEURODEGENERATIVE SCIENCE Patrik Brundin, M.D., Ph.D. Director

The Center’s laboratories focus on the development of novel treatments that slow or stop the progression of neurodegenerative disease, in particular Parkinson’s disease.

The work revolves around three main goals: disease modification, biomarker discovery, and brain repair.

The image shows embryonic dopaminergic neurons (stained green for the enzyme tyrosine hydroxylase) that have been grafted into the rat brain striatum. Red staining indicates virally induced overexpression of the human -synuclein protein; neuron nuclei are stained blue with DAPI. This rodent system is used to model Parkinson’s disease in the laboratory.

Image by Sonia George of the Brundin laboratory.

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Lena Brundin, M.D., Ph.D. Laboratory of Behavioral Medicine Dr. Brundin earned her Ph.D. in neurobiology and her M.D. from Lund University, Sweden. Prior to moving to Grand Rapids in 2012, she was an associate professor in experimental psychiatry at Lund University as well as a clinical psychiatrist. She holds a joint appointment as Associate Professor in the Michigan State University College of Human Medicine, Department of Psychiatry and Behavioral Medicine, and as Associate Professor at VARI.

From left: Sauro, Christensen, Weaver, Brundin, Bryleva, Keaton, Rajamani

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Elena Bryleva, Ph.D. Keerthi T. Rajamani, Ph.D. Analise Sauro, B.Sc. Laura Weaver, A.A.S.

Kyle Christensen, B.S. Sarah Keaton, M.S. Amissa Sei

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Research Interests The Laboratory of Behavioral Medicine at VARI works with the hypothesis that inflammation in the brain is a cause of important psychiatric symptoms such as depression and thoughts of suicide. This hypothesis stems from the fact that people with infections such as the flu often develop behavioral symptoms known as sickness behavior. Our laboratory has shown that individuals who attempt suicide have high levels of inflammation and toxic products of inflammation both in the blood and in the cerebrospinal fluid. The higher the degree of inflammation, the more depressed and suicidal is the affected patient. Therefore, we think that the biological mechanisms of sickness behavior and the disease traditionally known as psychiatric depression are similar, involving activation of the inflammatory response in the brain and subsequent effects on nerve cells. In a recent clinical study, we showed that when depression is successfully treated, it is associated with a significant decrease of inflammation products in the blood.

The laboratory is conducting both clinical studies on patients in the Grand Rapids area and translational experiments in the laboratory at VARI, trying to detail what inflammatory mechanisms are responsible for the effects on emotion and behavior.

Such mechanisms could be the foundation of novel treatments directed at depression and suicidal behavior. The medications used today are based on principles identified about 50 years ago in the monoamine hypothesis of depression. Unfortunately, these medications help only about 50% of affected patients. If anti-inflammatory agents could be used to treat depressive and suicidal symptoms, it would be a huge step toward helping patients suffering from so-called treatment-resistant depression.

In recent years, we have identified some infections and genetic variants associated with a higher risk for suicidal behavior and depression. Intriguingly, we found that infection with the parasite Toxoplasma gondii is associated with a sevenfold risk of attempted suicide. Some 10-20% of all Americans are infected with this parasite, which was previously considered harmless to everyone except pregnant women and immunocompromised individuals. After initial infection by ingesting undercooked meat or contaminated soil, the parasite enters the brain and resides in nerve cells. This parasite may be the cause of subtle behavioral changes in the infected hosts, perhaps due to low-grade chronic brain inflammation. Toxoplasma infection may be treatable using current medications, but it still needs to be proved in clinical trials that such treatment has a beneficial effect on depressive and suicidal behavior.

This year, our laboratory is launching two clinical studies in Grand Rapids. The first is a collaborative study of depression with Pine Rest Christian Mental Health, Spectrum Health, and Michigan State University. This multi-institutional NIH-sponsored effort, led by Dr. Brundin, investigates the possible role of the placenta in the development of depression in pregnant women.

The goals of the study are to understand the cause of depression during pregnancy, something that is currently unknown, and to find biomarkers in the blood to identify women who are at risk for depression during and after pregnancy. If we know which women are at risk, they can be closely monitored during pregnancy for symptoms and receive prompt support and help.

Finally, if we uncover the trigger of depression in pregnancy, we will be optimally positioned for developing novel therapies to target the cause of the disease.

The second clinical study is called the Heart Failure and Inflammation in Depression (HFIND) study. With Spectrum Health, we will look at the co-morbidity of cardiovascular disease and depression. We predict that patients suffering from heart failure who have a high level of inflammatory products in their blood will also suffer from depression. Our hypothesis is that if we treat the inflammation, the patient’s mood and cardiovascular status will both improve, giving a doubly beneficial effect.

Finally, we have found that patients who have a certain gene for the inflammatory factor C-reactive protein may be at higher risk for suicidal behavior. We plan to investigate whether this holds true in larger patient samples and to analyze the actions of individual inflammatory mediators in the central nervous system. In particular, a study we published last year showed that suicidal patients have very high levels of quinolinic acid in their cerebrospinal fluid. Quinolinic acid is produced in the brain by inflammation, and it binds directly to receptors on nerve cells. In particular, it increases the degree of activity of glutamate nerve cells. We think this may be an important mechanism in producing depressive and suicidal symptoms. Therefore, we are trying to identify medications that counteract the effects of quinolinic acid on nerve cells.

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Recent Publications Bay-Richter, Cecillie, Klas R. Linderholm, Chai K. Lim, Martin Samuelsson, Lil Träskman-Bendz, Gilles J. Guillemin, Sophie Erhardt, and Lena Brundin. In press. A role for inflammatory metabolites as modulators of the glutamate N-methyl-D-aspartate receptor in depression and suicidality. Brain, Behavior, and Immunity.

Cook, Thomas B., Lisa A. Brenner, C. Robert Cloninger, Patricia Langenberg, Ajirioghene Igbide, Ina Giegling, Annette M. Hartmann, Bettina Konte, Marion Friedl, Lena Brundin, et al. In press. "Latent" infection with Toxoplasma gondii: association with trait aggression and impulsivity in healthy adults. Journal of Psychiatric Research.

Grudet, Cécile, Johan Malm, Åsa Westrin, and Lena Brundin. In press. Suicidal patients are deficient in vitamin D, associated with a pro-inflammatory status in the blood. Psychoneuroendocrinology.

Dahl, Johan, Heidi Ormstad, Hans Christian D. Aass, Ulrik Fredrik Malt, Lil Träskman Bendz, Leiv Sandvik, Lena Brundin, and Ole A. Andreassen. 2014. The plasma levels of various cytokines are increased during ongoing depression and are reduced to normal levels after recovery. Psychoneuroendocrinology 45: 77–86.

Wennström, Malin, Shorena Janelidze, Cecillie Bay-Richter, Lennart Minthon, and Lena Brundin. 2014. Pro-inflammatory cytokines reduce the proliferation of NG2 cells and increase shedding of NG2 in vivo and in vitro. PLoS One 9(10): e109387.

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Patrik Brundin, M.D., Ph.D. Laboratory of Translational Parkinson’s Disease Research Dr. Brundin earned both his M.D. and Ph.D. at Lund University in Sweden.

He has over 30 years of experience in neurodegenerative disease research, has authored some 300 publications, and is in the top 0.5% of cited researchers in the field. Much of Dr. Brundin’s current research focuses on identifying mechanisms of Parkinson’s disease pathogenesis. In addition to managing laboratories at VARI and at Lund, Dr. Brundin serves as a Professor and Associate Director of VARI and is co-editor-in-chief of the Journal of Parkinson’s Disease.

From left: Brundin, Lamberts, Sasidharan, Kaufman, Cousineau, Beauvais, Rey, Hildebrandt, George, Ghosh, Tyson

Staff Genevieve Beauvais, Ph.D. Kim Cousineau, B.S., M.P.A. Martha Escobar, Ph.D. Sonia George, Ph.D. Anamitra Ghosh, Ph.D. Darcy Kaufman, M.S.

Jennifer Lamberts, Ph.D. Brendan Looyenga, Ph.D. Nolwen Rey, Ph.D. Chakrapani Sasidharan, Ph.D. Jennifer Steiner, Ph.D. Trevor Tyson, Ph.D.

Student

Adjunct Faculty

Erin Hildebrandt, B.S.

William Baer, M.D., Pharm.D.

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Research Interests The mission of the Laboratory for Translational Parkinson’s Disease Research is to 1) understand why Parkinson's disease (PD) develops and 2) use relevant animal PD models to discover new treatments that slow or stop disease progression. To achieve this goal, the laboratory has several ongoing, externally funded projects that use both cellular and rodent models to study various pathogenic processes associated with PD. One main focus of the laboratory is to understand how PD develops. Current evidence suggests that mutations in the gene SNCA, which encodes the neuronal protein -synuclein (-syn), play an important role in disease progression. These mutations cause -syn to adopt an abnormal conformation and aggregate in nerve cells, including in the dopamine-producing nerve cells that are known to be crucial for the motor functions affected in PD. Further, the transmission of abnormal -syn protein between nerve cells in the brain is believed to drive the progression of symptoms. Several projects in the laboratory are designed to identify the mechanisms underlying -syn transmission and to clarify the role of this process in the development of PD. The first set of projects uses isolated cell models to better understand the process of -syn transmission. Through a generous gift from the Peter C. and Emajean Cook Foundation, we are currently designing and characterizing a system with which to visualize the transport of -syn within neurons. This novel imaging system will allow us to study -syn transport in living cells, a process that has thus far been underrepresented in the scientific literature. This project is complemented by another imagingbased study focused on evaluating -syn transfer between cells. Together, these projects will identify important mechanisms involved in -syn transmission and help explain how -syn acts in a prion-like fashion during PD progression.

Another project uses a mouse model to evaluate -syn transmission in an organism. In this model, mice are first engineered to express large amounts of human -syn protein in the brain (host), and then young neurons that do not produce human -syn (graft) are transplanted into the mice’s brain. Some time later, the transplanted neurons can be shown to contain -syn, which could have only arisen by transmission from the host to the graft. This model recapitulates the situation observed in human PD patients grafted with young, healthy neurons. By genetically and/or pharmacologically manipulating the mouse model, the mechanisms that contribute to the transmission of -syn can be characterized.

Given that -syn transmission between cells is thought to drive the disease process in PD, interfering with this transmission may produce a therapeutic effect. To test this hypothesis, the laboratory has partnered with GISMO Therapeutics Inc. and obtained funding from the Michael J. Fox Foundation to evaluate the ability of heparan sulfate proteoglycan (HSPG) inhibitors to prevent the transfer of -syn between cells, using the experimental models described above. HSPGs are cell surface proteins responsible for the intercellular transfer of material. The HSPG inhibitors developed by GISMO are a novel class of compounds that prevent the specific interaction between -syn and HSPGs and are therefore predicted to inhibit -syn transfer from cell to cell. If -syn transfer indeed underlies the disease process, these inhibitors may be able to slow disease progression.

The second primary focus of the laboratory is to use relevant animal models to identify new therapeutics for the treatment and management of PD. To this end, a team of postdoctoral fellows has carefully characterized a genetic mouse model in which dopamine-producing nerve cells degenerate slowly (over weeks to months). In this model, the first signs of degeneration are observed in the terminals of the dopamine nerve cells, and the process of autophagy seems to be affected. These features, along with the slow progression of the degenerative process, make this mouse model particularly relevant.

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As part of the search for new treatments that might slow disease progression, we are exploring the compound MSDC0160, developed originally by Metabolic Solutions Development Company in Kalamazoo as an anti-diabetic agent.

Thanks to funding from the Cure Parkinson’s Trust UK and the Campbell Foundation, we are evaluating the efficacy of MSDC-0160 against neurodegeneration in the MPTP (methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of PD. MPTP is a neurotoxin that specifically destroys dopamine-producing nerve cells in humans, monkeys, and mice, leading to motor impairments. Treatment with MSDC-0160 of mice exposed to MPTP reduced both neurodegeneration and motor dysfunction, suggesting that this compound might be neuroprotective in the context of PD. Now we are examining effects of the drug in other mouse models, including our slowly progressing genetic model. Given the favorable safety profile of MSDC-0160, the drug is already under consideration for PD clinical trials, demonstrating its high potential for clinical translation.

A related project focuses on the use of MSDC-0160 to treat L-dopa-induced dyskinesia, a debilitating side effect of long-term treatment with the most commonly used symptomatic therapy for PD. In collaboration with Metabolic Solutions Development Company and through a grant from the Michael J. Fox Foundation, we are testing the ability of MSDC-0160 to prevent and/or reverse L-dopa-induced dyskinesia in a rat model of PD. The results thus far are promising.

Recent Publications Janelidze, Shorena, Ulrika Nordström, Sebastian Kügler, and Patrik Brundin. In press. Pre-existing immunity to AAV2 limits transgene expression following intracerebral AAV2-based gene delivery in a 6-OHDA model of Parkinson's disease.

Journal of Gene Medicine.

Lamberts, Jennifer T., Erin N. Hildebrandt, and Patrik Brundin. In press. Spreading of -synuclein in the face of axonal transport deficits in Parkinson's disease: a speculative synthesis. Neurobiology of Disease.

Nordström, Ulrika, Geneviève Beauvais, Anamitra Ghosh, Baby Chakrapani Pulikkaparambil Sasidharan, Martin Lundblad, Julia Fuchs, Rajiv L. Joshi, Jack W. Lipton, Andrew Roholt, et al. In press. Progressive nigrostriatal terminal dysfunction and degeneration in the engrailed1 heterozygous mouse model of Parkinson's disease. Neurobiology of Disease.

Reyes, Juan F., Tomas T. Olsson, Jennifer T. Lamberts, Michael J. Devine, Tilo Kunath, and Patrik Brundin. In press. A cell culture model for monitoring -synuclein cell-to-cell transfer. Neurobiology of Disease.

Derkinderen, Pascal, Kathleen M. Shannon, and Patrik Brundin. 2014. Gut feelings about smoking and coffee in Parkinson's disease. Movement Disorders 29(8): 976–979.

George, Sonia, Annica Rönnbäck, Gunnar K. Gouras, Géraldine H. Petit, Fiona Grueninger, Bengt Winblad, Caroline Graff, and Patrik Brundin. 2014. Lesion of the subiculum reduces the spread of amyloid beta pathology to interconnected brain regions in a mouse model of Alzheimer's disease. Acta Neuropathologica Communications 2: 17.

Kurowska, Z., P. Brundin, M.E. Schwab, and J.-Y. Li. 2014. Intracellular Nogo-A facilitates initiation of neurite formation in mouse midbrain neurons in vitro. Neuroscience 256(1): 456–466.

Reyes, Juan F., Nolwen L. Rey, Luc Bousset, Ronald Melki, Patrik Brundin, and Elodie Angot. 2014. Alpha-synuclein transfers from neurons to oligodendrocytes. Glia 62(3): 387–398.

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Jiyan Ma, Ph.D. Laboratory of Prion Mechanisms in Neurodegeneration Dr. Ma received his medical training at Shanghai Medical University, China, and earned his Ph.D. in biochemistry and molecular biology from the University of Illinois at Chicago. He was a postdoctoral fellow in Susan Lindquist’s laboratory at Howard Hughes Medical Institute in the University of Chicago, and in 2002, he joined the faculty of the Department of Molecular and Cellular Biochemistry at Ohio State University. Dr. Ma joined VARI in 2013 as a Professor.

From left: Becker, X. Wang, Rodriguez, Ma, F. Wang, Vaughan

Staff

Student

Romany Abskharon, Ph.D. Katelyn Becker, M.S. Ashley Rodriguez Amandine Roux, Ph.D. Robert Vaughan, B.S. Fei Wang, Ph.D. Xinhe Wang, Ph.D.

Toni Divic

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Ma

Research Interests Protein aggregation is a key pathological feature of a large group of late-onset neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Protein misfolding and aggregation are intimately related to pathogenic progress. Our overall goal is to elucidate the molecular events leading to protein misfolding in the aging central nervous system, to understand the relationship between misfolded protein aggregates and neurodegeneration, and, most importantly, to develop approaches to prevent, stop, or reverse protein aggregation and neurodegeneration in these devastating diseases.

One project in the lab is the study of protein aggregates in prion diseases, which are also known as transmissible spongiform encephalopathies. This is the only true infectious disease among the late-onset neurodegenerative disorders; it can spread from individual to individual and cause endemic outbreaks. We have established an in vitro system that can reconstitute the prion infectivity with bacterially expressed prion protein plus defined cofactors. We use this system to dissect the essential components and the structural features of an infectious prion and to uncover the molecular mechanisms responsible for the prion strain and species barrier. We have shown that misfolded prion protein in the endoplasmic reticulum is retro-translocated to the cytosol for proteasome degradation, and that in mice, when the cytosolic prion protein escapes degradation, it aggregates and causes neurodegeneration. We are studying how the neurotoxicity caused by cytosolic prion protein contributes to the neurodegeneration found in prion disease.

Recently, the concept of prions has expanded to encompass the pathogenesis of Parkinson’s and Alzheimer’s diseases. Proteins such as -synuclein have been suggested to spread the disease pathology in a prion-like manner from a sick cell to healthy ones. We are interested in understanding the similarities and differences between prions and these amyloidogenic proteins that have prion-like properties. Further, we want to determine the role of these protein aggregates in disease pathogenesis and to develop strategies against these fatal disorders.

Recent Publication Ma, Jiyan, and Fei Wang. 2014. Prion disease and the "protein-only hypothesis". Essays in Biochemistry 56: 181–191.

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Darren Moore, Ph.D. Laboratory of Molecular Neurodegeneration Dr. Moore earned a Ph.D. in molecular neuroscience from the University of Cambridge, U.K., in 2001 in the laboratory of Piers Emson. He completed postdoctoral training with Ted Dawson in the Department of Neurology at the Johns Hopkins University School of Medicine. Dr. Moore joined Johns Hopkins in 2005 as an instructor and became assistant professor in 2006.

In 2008, he moved to the Swiss Federal Institute of Technology (EPFL) in Lausanne as an assistant professor in the Brain Mind Institute. In 2014, Dr. Moore joined the faculty at VARI as an Associate Professor.

From left: Lee, Nguyen, Moore, Kordich

Staff Xi Chen, Ph.D. Kim Cousineau, B.S., M.P.A. Jennifer Kordich, M.S. Caroline Lee, Ph.D. An Phu Tran Nguyen, Ph.D. 70

Moore

Research Interests We investigate the molecular pathophysiology of Parkinson's disease, a chronic, progressive neurodegenerative movement disorder. Although most of Parkinson’s disease cases are sporadic, 5–10% of cases are inherited, with causative mutations identified in at least eight genes. Our laboratory studies the normal biology and pathobiology of gene products that cause inherited Parkinson’s disease, including the common leucine-rich repeat kinase 2 (LRRK2, PARK8), the retromer component vacuolar protein sorting 35 homolog (VPS35, PARK17), the E3 ubiquitin ligase parkin (PARK2), and the lysosomal P5-type ATPase ATP13A2 (PARK9).

We seek to explain the normal biological function of these proteins in the mammalian brain and the molecular mechanism(s) through which disease-associated variants induce neuronal dysfunction and eventual neurodegeneration in inherited forms of Parkinson’s. We also study the molecular pathogenesis of Parkinson’s disease with the goal of developing novel targeted disease-modifying therapies and neuroprotective strategies. Through a clear understanding of the physiological function and pathological dysfunction of these proteins, we hope to gain insights into the molecular mechanisms and cellular pathways underlying neurodegeneration in inherited and idiopathic forms of the disease.

We use a translational approach to identify and validate novel therapeutic strategies that may slow or halt progressive neurodegeneration in Parkinson’s disease. This approach includes experimental models of molecular, cellular, and biochemical techniques in systems such as human cell lines, primary neuronal cultures, Saccharomyces cerevisiae, human brain tissue, and transgenic, knockout, and viral-mediated gene-transfer rodent models.

Recent Publication Tsika, Elpida, Meghna Kannan, Caroline Shi-Yan Foo, Dustin Dikeman, Liliane Glauser, Sandra Gellhaar, Dagmar Galter, Graham W. Knott, Ted M. Dawson, Valina L. Dawson, et al. In press. Conditional expression of Parkinson's disease-related R1441C LRRK2 in midbrain dopaminergic neurons of mice causes nuclear abnormalities without neurodegeneration. Neurobiology of Disease.

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Jeremy M. Van Raamsdonk, Ph.D. Laboratory of Aging and Neurodegenerative Disease Jeremy Van Raamsdonk received a B.Sc. (Honours) in biochemistry from the University of British Columbia in 1993. After completing an M.Sc. in medical science at McMaster University in 1999, he returned to the University of British Columbia, completing a Ph.D. in medical genetics in 2005. Subsequently, he was a postdoctoral fellow in the Department of Biology at McGill University until joining VARI as an Assistant Professor in 2012.

From left: Machiela, Van Raamsdonk, Senchuk, Dues, Spielbauer, Cooper

Staff

Students

Dylan Dues, B.S. Ashley Rodriguez Megan Senchuk, Ph.D.

Emily Andrews Jason Cooper, B.S. Ugomma Eze Emily Machiela, B.S. Claire Schaar Katie Spielbauer, B.S. McLane Watson

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Research Interests As the average human life span continues to increase, the likelihood of an individual developing a neurodegenerative disease also increases. Thus, there is a need to understand the aging process and its role in the development of age-onset disorders such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. Our research is focused on gaining insight into the aging process and the pathogenesis of such diseases. Beyond benefit to the individual, this work has potential benefits for society by decreasing health care costs and helping to maintain productivity and independence to a later age.

The free radical theory of aging (FRTA) proposes that aging results from the accumulation of oxidative damage caused by reactive oxygen species (ROS) generated during normal metabolism. However, recent work in the worm Caenorhabditis elegans has indicated that the relationship between ROS and life span is more complex. Superoxide dismutase (SOD) is an enzyme that decreases the levels of ROS, but the deletion of SOD genes (individually or in combination) does not decrease life span. In fact, quintuple-mutant worms lacking all five sod genes live as long as wild-type worms despite a markedly increased sensitivity to oxidative stress. Thus, it appears that while oxidative damage increases with age, it does not cause aging.

Recent evidence suggests that increased levels of superoxide can act as a pro-survival signal that leads to increased longevity.

This is demonstrated by life-span increases following the deletion of the mitochondrial gene sod-2 and the treatment of wild-type worms with the superoxide generator paraquat. The fact that sod quintuple-mutant worms have a normal life span despite their increased sensitivity to oxidative stress suggests a balance between the pro-survival signaling and the toxic effects of superoxide. Thus, one of the main goals of this work is to elucidate the mechanism by which superoxide-mediated pro-survival signaling leads to increased longevity. We explore how increased levels of superoxide trigger the signal, how the signal is transmitted, and which changes the signal introduces lead to increased life span. These experiments use a combination of genetic mutants and RNA interference to gain insight into the signaling mechanism.

The role of aging in Parkinson’s disease The greatest risk factor for developing Parkinson’s disease (PD) is advanced age. Even individuals with the inherited forms of PD live decades without exhibiting symptoms or neuronal loss, despite the fact that the disease-causing mutation is already present at birth. This suggests that changes taking place during normal aging make cells more susceptible to the mutations implicated in PD. This conclusion is supported by the fact that the onset of the disease in animal models is proportional to the life span of the organism and is not related to chronological time. Moreover, several changes known to take place during the aging process have been shown to affect functions implicated in the pathogenesis of PD. This work will be conducted using C. elegans PD models, because these worms offer many advantages as a genetic model organism. Importantly, C. elegans has orthologs to almost all of the genes implicated in PD, including PARK2 (pdr-1), PINK-1 (pink-1), LRRK-2 (lrk-1), DJ-1 (djr-1.1,-1.2), UCHL-1 (ubh-1), ATP13A2 (catp-6), VPS-35 (vps-35), and GBA (gba-1-4). In fact, a number of worm models of PD have been developed, including chemical models such as 6-OHDA and MPTP transgenic worms expressing -synuclein, transgenic worms expressing mutant LRRK2, and deletion mutants of pdr-1, pink-1, djr-1.1, and catp-6. These models exhibit a number of PD-related phenotypes, including aggregation of -synuclein, decreased mobility, decreased adaption to food (a response mediated by dopamine neurons), and, importantly, degeneration of dopamine neurons. The main goals of this work will be 1) to determine whether genes that extend life span are beneficial in the treatment of worm models of PD and 2) to determine whether processes that show decreased function with age specifically exacerbate PD-like features in worm models. We will use genetic crosses to generate double mutants and will use RNA interference via feeding to specifically knock down the gene of interest. The health of the resulting worms will be compared with that of PD control worms to determine whether the aging gene affects the disease-like abnormalities. By examining the role of aging in PD, this project will provide new insight into the mechanism underlying the disease. This knowledge will provide novel therapeutic targets that may lead to an effective treatment.

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Migrating cancer cells of the pancreas are stained for the proteins EpCAM (green) and MRC2 (red), both of which may have roles in tumor development and metastasis. Cells expressing both proteins appear as yellow. Comparing the counts of these dual-labeled cells in tissue sections from cancer patients may provide a way to improve the accuracy of clinical prognoses. Image by Arkadeep Sinha of the Haab laboratory and the staff of the Confocal Microscopy and Quantitative Imaging core.

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CORE TECHNOLOGIES AND SERVICES

Scott D. Jewell, Ph.D. Director

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Ting-Tung (Anthony) Chang, Ph.D. Small-Animal Imaging Facility Dr. Chang received his B.S. degree in medical imaging and radiological sciences from Chang Gung University (Taoyuan, Taiwan) and his Ph.D. degree in medical physics specializing in diagnostic imaging physics from the University of Texas Health Science Center at San Antonio. He received advanced imaging training at Yale University and at the Vanderbilt University Institute of Imaging Science. Dr. Chang joined VARI in 2010 as a Research Assistant Professor and Director of the Small-Animal Imaging Facility.

From left: Chang, Radhakrishnan, Peck, Li, Holly

Staff

Students

Visiting Scientist

Adjunct Faculty

Shihong Li, Ph.D. Lori Moon Anderson Peck, M.S.E.

Priya Balasubramanian, B.S. Ryan Bozio, B.S. Kaiya Bryant Kyle Dean Zachary Dieffenbach, B.S. Michael Dykstra Brittany Holly, B.S. Nicole Niewenhuis Vineeth Radhakrishnan, B.S. Grace Walter, B.S. Eboni White

Samhita Rhodes, Ph.D.

Ewa Komorowska-Timek, M.D. Zheng (Jim) Wang, Ph.D.

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Services The Small-Animal Imaging Facility (SAIF) focuses on the development of preclinical imaging technologies that offer anatomic and functional information to biomedical investigators. The SAIF also aims to develop imaging technologies capable of monitoring organ/tissue activity at the molecular level in order to advance clinical applications such as early detection and staging of cancer. By combining new tracers, imaging analysis, and genomic information, we are assisting investigators in non-invasive imaging technologies for translational research. Our technologies include digital X-ray, high-resolution microCT, microSPECT/CT, microPET/CT, micro-ultrasound, optical imaging, radiochemistry, and custom tracers. Our comprehensive facility management system was designed to provide real-time analysis capabilities for imaging studies. This system allows researchers to group mice based on results of previous time points, enhancing the study’s overall quality and making effective use of resources.

We have recently developed 1) an automated system to quantify tail residual activity for correction of standard uptake value– related calculations in PET and SPECT imaging; 2) a QA/QC protocol to evaluate whether an optical imager with certain characteristics is adequate for Cherenkov luminescence imaging acquisition; 3) an in vivo, non-invasive, high-resolution imaging method for Kupffer cell migration in response to early liver metastasis; 4) a method to reduce respiratory artifacts in microCT imaging by using a high-frequency oscillatory ventilation system; 5) a multifunctional liposome-based drug delivery and imaging system; and 6) a method for self-assembly of periodic concentric layered magnesium carbonate microparticles without the use of a template.

Recent Publications Berger, Penny L., Sander B. Frank, Veronique V. Schulz, Eric A. Nollet, Mathew J. Edick, Brittany Holly, Ting-Tung A. Chang, Galen Hostetter, Suwon Kim, et al. 2014. Transient induction of ING4 by MYC drives prostate epithelial cell differentiation and its disruption drives prostate tumorigenesis. Cancer Research 74(12): 3357–3368.

Li, Shihong, Zheng Jim Wang, and Ting-Tung Chang. 2014. Temperature oscillation modulated self-assembly of periodic concentric layered magnesium carbonate microparticles. PLoS One 9(2): e88648.

Flaten, Gøril Eide, Ting-Tung Chang, William T. Phillips, Martin Brandl, Ande Bao, and Beth Goins. 2013. Liposomal formulations of poorly soluble camptothecin: drug retention and biodistribution. Journal of Liposome Research 23(1): 70–81.

Wang, Zheng J., Ting-Tung A. Chang, and Richard Slauter. 2013. Use of imaging for preclinical evaluation. In A Comprehensive Guide to Toxicology in Preclinical Drug Development, Ali S. Faqi, ed. Waltham, Massachusetts: Academic Press, pp. 759–776.

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Bryn Eagleson, B.S., RLATG Vivarium and Transgenics Core Ms. Eagleson earned a B.S. degree in psychology from the University of Maryland University College. She began her career in laboratory animal services with Litton Bionetics at the National Cancer Institute’s Frederick Cancer Research and Development Center (NCI-FCRDC) in Maryland. She later worked at the Johnson & Johnson Biotechnology Center in San Diego, California. In 1988 she returned to the NCI-FCRDC as manager of the transgenic mouse colony. In 1999, Ms. Eagleson was recruited to VARI as the Director of Vivarium and Transgenics.

Services The goal of the VARI Vivarium and Transgenics core is to develop, provide, and maintain high-quality mouse modeling services. The vivarium is a state-of-the-art facility that includes a high-level containment barrier. The Van Andel Research Institute is an AAALAC-accredited institution, and it was most recently reaccredited in September 2013. All procedures are conducted according to the NIH Guide for the Care and Use of Laboratory Animals. The staff provides rederivation, surgery, dissection, necropsy, breeding, weaning, tail biopsies, sperm and embryo cryopreservation, animal data management, project management, and health-status monitoring. Transgenic mouse models are produced on request for project-specific needs.

We also provide therapeutic testing and preclinical model development services. Project types include pharmacological testing, target validation testing, patient-derived xenograft (PDX) development, orthotopic engraftment model development, and subcutaneous xenograft/allograft model development.

From left, front row: B. Eagleson, Guikema, Meringa, Ramsey, Brandow, Timmer, Post, Holzgen, Baumann, Verbis; back rows: Boguslawski, Kempston, K. Eagleson, Rackham, Levine, Budnick, Monsma, Weaver

Staff

Laboratory Animal Technicians

Animal Caretaker Staff

Audra Guikema, B.S., LVT, Laboratory Manager Tristan Kempston, B.S. David Monsma, Ph.D.

Kristen Baumann, B.S. Elissa Boguslawski Susan Budnick, B.S. Lisa Kefene, B.S. Tina Meringa, A.S. Janelle Post, B.S. Lisa Ramsey, A.S., LVT

Neil Brandow Kendra Eagleson Katie Holzgen Nate Levine Mat Rackham Sylvia Timmer, Vivarium Supervisor Ashlee Verbis William Weaver

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Timothy Feinstein, Ph.D. Confocal Microscopy and Quantitative Imaging Core Dr. Timothy Feinstein joined VARI as confocal core manager in 2013, bringing 12 years of experience in confocal microscopy and quantitative imaging. He obtained his Ph.D. at Carnegie Mellon University and was a postdoctoral fellow at the University of Pittsburgh. He has published a number of high-impact papers challenging current models of trafficking and signaling by G protein–coupled receptors.

Services Established in October 2013, the Confocal Microscopy and Quantitative Imaging Core provides optical imaging services for the Van Andel Research Institute and collaborating institutions, as well as the expertise and analytical tools to use them effectively.

Our services include live-cell imaging and biosensor studies of cell signaling in cancer and brain tissue; the measurement of gene expression, protein transport, and protein-protein interactions; and 3D reconstruction of large fluorescent structures in blocks of intact tissue. Training in these techniques and in the proper management of the obtained data is provided through workshops or ad hoc sessions with individual researchers. We have two scanning confocal microscopes, a Nikon A1-RSi with coded stage and a Zeiss 510 META-MP instrument. The core has collaborations with academic partners at nearby universities, including Michigan State University and Western Michigan University.

The core allows users of all experience levels to perform quantitative research in line with or exceeding the current professional standards of their field. We are in the process of implementing a comprehensive solution for the collection, management, and processing of all imaging data for researchers at VARI. Users can store and manage their work on the server-based datamanagement platform called OMERO. This system stores all types of imaging work on a central server with software that gives clients simplified access, modification, and download capabilities, reducing the burden on users’ computers and providing the ability to work with proprietary image data outputs.

A suite of commercial and open-source image analysis programs on a powerful Z620 workstation is available with direct access to OMERO. Options include deconvolution and complex 3D visualization (Huygens Professional), neuron tracing (IMARIS Suite), high-throughput phenotype quantitation, machine learning (CellProfiler and CPAnalyst), sophisticated mathematical analysis options (MATLAB), and image manipulation or figure preparation software such as Fiji/ImageJ, Photoshop, and GIMP.

Recent Publication Nordström, U., G. Beauvais, A. Ghosh, B.C.P. Sasidharan, M. Lundblad, J. Fuchs, R.L. Joshi, J.W. Lipton, A. Roholt, et al.

In press. Progressive nitrostriatal terminal dysfunction and degeneration in the engrailed1 heterozygous mouse model of Parkinson's disease. Neurobiology of Disease.

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Scott D. Jewell, Ph.D. Pathology and Biorepository Core Dr. Jewell earned his master’s and doctoral degrees in experimental pathology and immunology from The Ohio State University. He served at Ohio State University as director for the Human Tissue Resource Network and as associate director of the OSU Comprehensive Cancer Center’s Biorepository and Biospecimen Resource, where his dedicated, creative efforts led to the development of a state-of-the-art tissue procurement and biorepository system. He was elected President of the International Society for Biological and Environmental Repositories (ISBER) for 2009– 2010 and joined VARI in 2010 as a Professor and as Director of the Program for Technologies and Cores and of the Program for Biospecimen Science. He is a member of the College of American Pathologists' (CAP) Biorepository Accreditation Program and is on the editorial board of the journal Biopreservation and Biobanking.

From left, back row: Koeman, Watkins, Valley, Smith, Hudson, Rohrer, Montroy, Moon, Jeltema; middle row: Hodges, Joynt, Feenstra, Berghuis, Turner, Harbach, Wang; kneeling: Jewell, McPeak, Feinstein, Hostetter

Staff John Beck, B.S. Bree Berghuis, B.S., HTL(ASCP), QIHC Alexander Blanski, B.S. Eric Collins, B.S. Melissa DeHollander, B.S., M.B.A. Kristin Feenstra, B.S. Timothy Feinstein, Ph.D. Philip Harbach, M.S. 80

Students Renee Hilsabeck, B.S. Meghan Hodges, B.S. Galen Hostetter, M.D. Eric Hudson, B.S. Carrie Joynt, B.S. Julie Koeman, B.S., CG(ASCP) Edward McPeak Craig Meyer, B.S., B.A.

Rob Montroy, B.S. Lori Moon, M.B.A. Dan Rohrer, B.S., M.B.A. Brian Smith, B.S. Lisa Turner, B.S., HT(ASCP), QHIC Dana Valley, B.A. Xihne Wang, M.D., Ph.D. Anthony Watkins, A.S.

Maria Espinoza Devon Jeltema

Jewell

Research Interests The Pathology and Biorepository Core integrates anatomic pathology expertise with biorepository and biospecimen science for assisting in VARI’s research. We build upon historical strengths in standard histology, microscopy, and biobanking, and we use novel technologies to test and apply best practices in biospecimen science. The pathology discipline provides complementary emphasis on high-quality biospecimens and interpretable results with which to validate experimental models and extend them to clinical samples, thereby advancing our common translational mission.

Dr. Jewell, with his expertise in experimental pathology, immunology, and biobanking, and Dr. Hostetter, who is board-certified in anatomic pathology, together provide a wide range of expertise to the VARI laboratories. Currently, they are studying the effects of preanalytical variables in tissue collection and transport on the integrity of downstream analytes. The assessment of tumor suppressors and immunomodulators in tumor tissues and the application of genomic and epigenomic assays for biospecimens are among the services provided by the core. The VARI biorepository is nationally and internationally recognized, serving as the NCI Comprehensive Biospecimen Resource for the Cancer Human Biobank (caHUB) and providing biorepository services for the Multiple Myeloma Research Foundation. The biorepository is accredited by the College of American Pathologists.

Pathology Core services • Histology and diagnostic tissue services, including morphology, immunohistochemistry, in situ hybridization, and multiplex fluorescent IHC assays

• Pathology review and annotation of clinical samples from VARI’s prospective and retrospective tissue collections

• Design and construction of tissue microarrays

• Digital imaging and spectral microscopy coupled with image analysis tools

• Cell fractionation and biospecimen processing

• Laser capture microdissection

• Cytogenetics

• Ion Torrent genomic technology

Biorepository Core services • Biobanking services for VARI investigators, the National Cancer Institute, and the Multiple Myeloma Research Foundation

• Biospecimen kit construction, shipping, and tracking

• Clinical trials biobanking coordination

• Quality management program

Recent Publications Hostetter, G., E. Collins, P. Varlan, E. Edewaard, P.R. Harbach, E.A. Hudson, K.J. Feenstra, L.M. Turner, B.D. Berghuis, et al.

2014. Veterinary and human biobanking practices: enhancing molecular sample integrity. Veterinary Pathology 51(1): 270–280.

Robb, James A., Margaret L. Gulley, Patrick L. Fitzgibbons, Mary F. Kennedy, L. Mark Cosentino, Kay Washington, Rajesh C. Dash, Philip A. Branton, Scott D. Jewell, et al. 2014. A call to standardize preanalytic data elements for biospecimens.

Archives of Pathology and Laboratory Medicine 138: 526–537.

Lonsdale, John, Jeffrey Thomas, Mike Salvatore, Rebecca Phillips, Edmund Lo, Saboor Shad, Richard Hasz, Gary Walters, Fernando Garcia, et al. 2013. The Genotype-Tissue Expression (GTEx) project. Nature Genetics 45(6): 580–585

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Heather Schumacher, B.S., MT(ASCP), Manager Arthur S. Alberts, Ph.D., Director Flow Cytometry Core

Heather Schumacher has a Bachelor of Science in medical technology from Ferris State University and is certified by the American Society of Clinical Pathologists as a generalist (MT). She worked in the pharmaceutical industry for over seven years, performing immunohematology procedures in advanced-level reference laboratories. She has over 12 years of experience in hematology/flow cytometry and is proficient on three major vendor platforms, including seven different flow cytometers.

Every position she has held has involved training of staff members, competency assessment, technical writing, preparing for inspections from regulatory agencies, and overseeing operations related to compliance/quality assurance.

Dr. Alberts earned his degrees in biochemistry and cell biology (B.A., 1987) and in physiology and pharmacology (Ph.D., 1993) from the University of California, San Diego. He was a postdoctoral fellow in Richard Treisman’s laboratory from 1994 to 1997 at the Imperial Cancer Research Fund (now Cancer Research UK London Research Institute) as a Howard Hughes Medical Institute International Scholar. From 1998 to 1999 he was the Carol Franc Buck Fellow in Frank McCormick’s lab at the Helen Diller Comprehensive Cancer Center at UC San Francisco. Dr. Alberts joined VARI in January 2000; he was promoted in 2006 to Associate Professor and to Professor in 2009.

Services The Flow Cytometry Core provides comprehensive flow cytometry analysis and sorting services in support of VARI research.

Additional services include the development of tools for access and training designed to teach skills pertaining to blood analysis cytology. Our instrumentation includes two flow cytometers, a Beckman Coulter MoFlo Astrios and a Partec CyFlow Space.

Other equipment for blood analysis includes a VetScan instrument, a VetScan HMII, and a Shandon Cytospin 3.

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Mary E. Winn, Ph.D. Bioinformatics and Biostatistics Core Dr. Winn is the manager of the VARI Bioinformatics and Biostatistics Core.

She earned her Ph.D. from the University of California, San Diego and completed a postdoctoral fellowship at VARI before moving into the core manager position in 2013. She also serves as an instructor at Van Andel Institute Graduate School. Her research focus, in part, is on the translation of genomics to the clinic through the development of diagnostic and prognostic biomarkers of disease.

From left: Winn, Cherba, Borgman, Dykema

Staff Andrew Borgman, M.S. David M. Cherba, Ph.D. Karl J. Dykema, B.S. 83

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Services Established in April 2013, the Bioinformatics and Biostatistics Core serves the analytical needs of VARI by providing efficient, high-quality computational and statistical services for VARI research labs wrestling with the analysis and interpretation of data.

The BBC is a consolidation of expertise from a number of different laboratories with the new, unified goal of strengthening and maintaining bioinformatics and biostatistics techniques across all VARI laboratories.

The BBC maintains sequencing pipelines for processing and analyzing genomic data sets; develops custom tools and applications for quantitative image analysis; provides access to a variety of proprietary and open-source resources; oversees the maintenance and development of high-performance computing capabilities; supports the design, planning, conduct, analysis, and reporting of research; and more.

We provide statistical consulting, experimental design (including research proposal development, sample size determination, and randomization procedures), manuscript preparation and data deposition, genomic variant detection and annotation, transcript/ isoform differential expression, DNA copy number determination, systems-level analysis such as gene-set or network-based analysis, custom application and tool development, and the analysis, interpretation, and presentation of small and large data sets. We also support the greater educational mission of the Institute, helping students and staff develop an analytic approach and skills in experimental design through seminars, lectures, and tutorials.

In addition to supporting VARI faculty, the BBC maintains external collaborations with various academic and industrial partners, including the Henry Ford Health Systems, Michigan State University, and Transmed Systems.

Recent Publications Mackenzie, Todd A., Gary N. Schwartz, Heather M. Calderone, Carrie R. Graveel, Mary E. Winn, Galen Hostetter, Wendy A. Wells, and Lorenzo F. Sempere. In press. Stromal expression of miR-21 identifies high-risk group in triple-negative breast cancer. American Journal of Pathology.

Sinha, Arkadeep, David Cherba, Heather Bartlam, Elizabeth Lenkiewicz, Lisa Evers, Michael T. Barrett, and Brian B. Haab. In press. Mesenchymal-like pancreatic cancer cells harbor specific genomic alterations more frequently than their epithelial-like counterparts. Molecular Oncology.

Berezovsky, Artem D., Laila M. Poisson, David Cherba, Craig P. Webb, Andrea D. Transou, Nancy W. Lemke, Xin Hong, Laura A. Hasselbach, Susan M. Irtenkauf, et al. 2014. Sox2 promotes malignancy in glioblastoma by regulating plasticity and astrocytic differentiation. Neoplasia 16(3): 193–206.e25.

Monsma, David J., David M. Cherba, Patrick J. Richardson, Sean Vance, Sanjeet Rangarajan, Dawna Dylewski, Emily Eugster, Stephanie B. Scott, Nicole L. Beuschel, et al. 2014. Using a rhabdomyosarcoma patient-derived xenograft to examine precision medicine approaches and model acquired resistance. Pediatric Blood & Cancer 61(9): 1570–1577.

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Fluorescent reporters allow us to identify cells carrying genetic modifications engineered into the mouse prostate. Mice carrying a reporter gene that directs the expression of either the Tomato protein (red) or Green Fluorescent Protein are crossed to mice expressing Cre recombinase in the prostate epithelium. Cells in which Cre has been active express GFP, while those not exposed to Cre retain Tomato expression. Crossing mice having such reporters with mice lacking the tumor suppressor gene Apc may help in developing more-accurate models for tracking prostate cancer cell growth. Image by Ken Valkenburg of the Williams laboratory.

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AWARD FOR SCIENTIFIC ACHIEVEMENT

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Jay Van Andel Award for Outstanding Achievement in Parkinson’s Disease Research This award was established to honor distinguished researchers in the field of Parkinson’s disease and is named after Van Andel Institute founder Jay Van Andel, who passed away in 2004 after a long struggle with the disease. Awardees are selected on the basis of their scientific achievements and renown as a leader in Parkinson’s research or in research on closely related neurodegenerative disorders.

Award Recipient

Andrew John Lees, M.D., F.R.C.P., FMedSci Dr. Lees is Professor of Neurology, The National Hospital for Neurology and Neurosurgery, Queen Square. He has achieved international recognition for his work on Parkinson’s disease and abnormal movement disorders. Dr. Lees introduced continuous subcutaneous apomorphine infusion as an effective treatment for latestage Parkinson's disease. In 2006, he was awarded the Movement Disorders Research Award by the American Academy of Neurology. He received the Stanley Fahn Lectureship Award at MDS Dublin 2012, and was awarded the German Society of Neurology’s 2012 Dingebauer Prize for outstanding scientific attainment in the field of Parkinson’s disease and neurodegenerative disorders.

Dr. Lees delivering his Jay Van Andel Award address.

Prior Recipients Andrew B. Singleton, Ph.D. Alim Louis Benabid, M.D., Ph.D.

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educational and Training Programs

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Van Andel Institute Graduate School Steven J. Triezenberg, Ph.D., Dean The Van Andel Institute Graduate School welcomed its first class of students in 2007 and graduated its first Ph.D. recipients in 2012. Students join a scientific community of distinguished professionals committed to improving human health and wellbeing through the translation of research knowledge and technology. The Ph.D. program is designed as an environment of problem-based learning through which students engage in biomedical research and develop the technical, interpersonal, and communication skills that equip them to pursue careers in scientific discovery.

The Van Andel Institute Graduate School (VAIGS) is accredited by the Higher Learning Commission, a commission of the North Central Association of Colleges and Schools (www.ncahlc.org; 1-800-621-7440).

From left: Bentley, Kioshi, Love, Turner, Nelson, Triezenberg, Withrow, Rappley

Staff

Intern

Kathy Bentley, B.S. Kristie Kioshi, B.A. Michelle Love, M.A. Michelle Nelson, Ph.D. Carol Rappley Julie Davis Turner, Ph.D., Assistant Dean

Dana Withrow, B.S.

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Postdoctoral Fellowship Program The Van Andel Research Institute provides postdoctoral training opportunities to Ph.D. scientists beginning their research careers. The fellowships help promising scientists advance their knowledge and research experience while at the same time supporting the research endeavors of VARI. The fellowships are funded by the laboratories to which the fellow is assigned; by the VARI Office of the Director; or by outside agencies. Each fellow is assigned to a faculty member who oversees the progress and direction of research. Fellows who worked in VARI laboratories in late 2013 and in 2014 are listed below.

Nicholas Andersen

Danese Joiner

Jackie Peacock

University of Iowa VARI mentor: Nicholas Duesberry

University of Michigan VARI mentor: Bart Williams

University of Miami VARI mentor: Matthew Steensma

Genevieve Beauvais

Yanyong Kang

Nolwen Rey

University of Paris Descartes VARI mentor: Patrik Brundin

Institute of Biophysics, Chinese Academy of Sciences VARI mentor: Eric Xu

University of Lyon, France VARI mentor: Patrik Brundin

Travis Burgers University of Wisconsin–Madison VARI mentor: Bart Williams

Zheng Cao University of Maryland, College Park VARI mentor: Brian Haab

Vanessa Fogg Washington University in St. Louis VARI mentor: Jeffrey MacKeigan

Sourik Ganguly University of Kentucky VARI mentor: Cindy Miranti/Xiaohong Li

Anamitra Ghosh Iowa State University VARI mentor: Patrik Brundin

Juliana Sacoman Jennifer Lamberts University of Michigan VARI mentor: Patrik Brundin

Chakrapani Sasidharan Nate Lanning University of Michigan VARI mentor: Jeffrey MacKeigan

Xiangqi (Neil) Meng Sun Yat-sen University, China VARI mentor: Xiaohong Li

An Phu Tran Nguyen Universität Tübingen, Germany VARI mentor: Darren Moore

University of Freiberg, Germany VARI mentor: Patrik Brundin

Sudhir Singh University of Nebraska Medical Center VARI mentor: Brian Haab

Huiyan Tang Michigan State University VARI mentor: Brian Haab

Xiaoyong Zhi Kuntal Pal National University of Singapore VARI mentor: Eric Xu

From left: Ghosh, Singh, Sasidharan, Lamberts, Lanning, Peacock, Rey, Ganguly, Pal

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Michigan State University VARI mentor: Jeffrey MacKeigan

University of Texas Southwestern Medical Center VARI mentor: Eric Xu

Grand Rapids Area Pre-College Engineering Program The Grand Rapids Area Pre-College Engineering Program (GRAPCEP) is administered by Davenport University and jointly sponsored and funded by Schering Plough and VARI. The program is designed to provide selected high school students, who have plans to major in science or genetic engineering in college, with the opportunity to work in a research laboratory. In addition to research methods, the students also learn workplace success skills such as teamwork and leadership.

The four 2014 GRAPCEP students from Innovation Central High School were Maria Espinoza (Jewell/Hostetter) Eunice Eyamba (Chang) Holly Kramer (Duesbery) Demarcus Williams (Li)

From left: Espinoza, Williams, Kramer, Eyamba

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Student Internship Program The VARI student internships were established to provide college students with an opportunity to work with professionals in their fields of interest, to use state-of-the-art equipment and technologies, and to learn valuable interpersonal and communications skills. Student positions last from 10 to 12 weeks, with the student research internships culminating in oral presentations or poster presentations. In 2014, VARI hosted more than 38 students from 19 colleges and universities in formal research internships, administrative department internships, and other student positions. VARI also hosted three students in research internships through the United Negro College Fund (UNCF), one of whom was the recipient of a UNCF–Merck Undergraduate Science Research Scholarship award.

Generous support from the Frederik and Lena Meijer Student Internship Program funds 15 to 18 research internships each year; Meijer interns are denoted by an asterisk (*).

2014 Interns

Hope College, Holland, Michigan

Calvin College, Grand Rapids, Michigan

*Claire Schaar (Van Raamsdonk) *Courtney Schmidt (Steensma) *McLane Watson (Miranti)

Leah Sienkowski, B.A. (Office of the Director) *Kelsey Veldkamp (Wu)

Minnesota State University, Moorhead *Samuel Ameh (Li)

Davenport University, Grand Rapids, Michigan Leah Postema (Finance)

Drew University, Madison, New Jersey Ugomma Eze (Van Raamsdonk)

Ferris State University, Big Rapids, Michigan

Johns Hopkins University, Baltimore, Maryland Akash PremKumar (Steensma)

Lane College, Jackson, Tennessee Khristal Thomas (Miranti)

Michigan State University, East Lansing *Clare Laut (Weinreich) Chad Porterfield (Finance) *Adam Thelen (Melcher) Wade VanConant (Facilities)

Annie Murphy (Haab)

Florida State University, Tallahassee

Purdue Univerity, West Lafayette, Indiana Weston Brander, B.S., M.S. (Business Development)

*Mason Sutherland (Triezenberg)

Grand Valley State University, Allendale, Michigan Kevin Albrecht (Business Development) Elliot Ensink (Haab) Joan Giffels (D, C, and M) Luke McManus (Finance) *Adam McMillan (Yang) Rebecca Nguyen (Williams) Ally Rogers (Haab) Dana Withrow, B.S. (VAI Graduate School)

Hampton University, Hampton, Virginia Amissa Sei (L. Brundin)

Harvard College, Cambridge, Massachusetts *Toni Divic (Ma) Mounir Jamal (D, C, and M)

Wheaton College, Wheaton, Illinois *Devon Jeltema (Jewell/Hostetter)

University of Michigan, Ann Arbor *Josh Castle (Graveel) *Christian Cavacece (Xu) *Alexandar Clegg (Haab) *Nicole Ethen (Williams) *Kristin Rybski (Alberts) *Skyler Rietberg (Zhang)

University of Nevada, Reno *Eimi Marritt (Duesbery)

University of Puget Sound, Tacoma, Washington Yiqing Dong (Li)

Wayne State University, Detroit, Michigan Mallory Smith, B.A. (Steensma) 92

2014 Interns. From left to right, kneeling: VanConant, Laut, Schaar, Sienkowski, Rogers, Smith, Clegg, Divic; standing: Premkumar, Sutherland, McMillan, Nguyen, Ethen, Rietberg, Veldkamp, Porterfield, Jeltema, McManus, Sei, Cavacece, Rybski, Murphy, Ameh, Koster, Withrow, Watson, Jamal, Marritt, Castle, Thelen, Dong, Schmidt, Thomas, Eze

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VARI Seminar Series

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Jay Van Andel Seminar Series May 2013

Vania Broccoli, University of Alabama at Birmingham “Neuronal cell reprogramming: from basic principles to in vivo applications”

David Standaert, University of Alabama at Birmingham “Neuroinflammation and the progression of Parkinson’s disease”

Xianmin Zeng, Buck Institute for Research on Aging, Novato, California

“PSC-derived dopaminergic neurons for therapy and screening for Parkinson’s disease” August

R. Jeremy Nichols, The Parkinson’s Institute and Clinical Center, Sunnyvale, California “Regulators of LRRK2 phosphorylation before and after LRRK2 inhibition”

October

Anders Bjorklund, Lund University, Sweden

“Nurr1 as a therapeutic target for neuroprotection and disease modification in Parkinson´s disease”

Roger A. Barker, University of Cambridge, U.K.

“The future of experimental therapeutics as it will translate to the clinic for Parkinson’s disease”

January 2014

Omar M.A. El-Agnaf, Ph.D., United Arab Emirates University “Exploiting -synuclein as a biomarker candidate for Parkinson’s disease and related disorders”

November

Ronald Melki, Federative Research Institute 115: Genomes, Transcriptomes, Proteomes, Gif-sur-Yvette, France

“Nature, structural and functional characterization of alpha-synuclein infectious assemblies”

VARI Seminar Series March 2014

Laurie S. Kaguni, Michigan State University, East Lansing

“Clustering of POLG disease mutations into functional modules in the human mitochondrial replicase, Pol , establishes predictive genotype-phenotype correlations for the complete spectrum of POLG syndromes”

Conor C. Lynch, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida

“New approaches to breaking the ‘vicious cycle’ of osteogenic prostate to bone metastases”

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May

Robert Schwarcz, University of Maryland School of Medicine, Baltimore

“The kynurenine pathway of tryptophan degradation: links to schizophrenia and other major brain diseases”

Anne Eichmann, Yale University, New Haven, Connecticut

“Guidance of vascular patterning: lessons from the nervous system”

June

Jeffrey H. Kordower, Rush University Medical Center, Chicago

“Rethinking experimental therapeutics: do not screw up new opportunities with old mistakes”

David C Rubinsztein, Cambridge Institute for Medical Research, Cambridge, U.K.

“Autophagy: from neurodegeneration to the plasma membrane”

July

Todd R. Golub, Broad Institute of Harvard and MIT, Cambridge, Massachusetts

“Genomic approaches to cancer”

Anna Spagnoli, University of North Carolina at Chapel Hill

“Control the fire: from joint development to homeostasis”

August

Jongsook Kim Kemper, University of Illinois at Urbana-Champaign

“Metabolic functions and mechanisms of the nuclear receptors FXR and SHP”

Gerald W. Hart, Johns Hopkins University School of Medicine, Baltimore

“Nutrient regulation of signaling and transcription by O-GlcNAcylation”

October

Larry C. Walker, Yerkes National Primate Research Center, Emory University, Atlanta

“Alzheimer’s disease and the many faces of pathogenic A”

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ORGANIZATION

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David L. Van Andel Chairman and CEO, Van Andel Institute

VARI Board of Trustees

David L. Van Andel, Chairman and CEO James Fahner, M.D. W. Gary Tarpley, Ph.D. George F. Vande Woude, Ph.D. Ralph Weichselbaum, M.D.

Board of Scientific Advisors The Board of Scientific Advisors advises the CEO and the Board of Trustees, providing recommendations and suggestions regarding the overall goals and scientific direction of VARI. The members are

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Michael S. Brown, M.D., Chairman Richard Axel, M.D. Joseph L. Goldstein, M.D. Tony Hunter, Ph.D. Phillip A. Sharp, Ph.D.

Office of the Director Van Andel Research Institute

Peter A. Jones, Ph.D., D.Sc. Research Director

Patrick Brundin, M.D., Ph.D. Associate Director

Staff David Cabrera, M.S., Science Policy and Administrative Manager Kim Cousineau, B.S., M.P.A., Senior Administrative Assistant Jens Forsberg, Ph.D., Scientific Project Leader Kayla Habermehl, B.A., B.S., Science Writer Laura Holman, Executive Assistant Jennifer Holtrop, B.S., Scientific Administrator

David Nadziejka, M.S., Science Editor Aaron Patrick, B.S., Research Operations Supervisor Bonnie Petersen, Senior Administrative Assistant Beth Resau, B.A., M.B.A., Scientific Meeting Planner Leah Sienkowski, B.A., Editorial Assistant

From left: Resau, Cousineau, Holtrop, Forsberg, Patrick, Holman, Habermehl, Petersen, Sienkowski, Cabrera

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Van Andel Institute Administrative Organization The departments listed below provide administrative support to both the Van Andel Research Institute and the Van Andel Education Institute.

Executive

Facilities

David Van Andel, Chairman and CEO Christy Goss, Senior Executive Assistant

Samuel Pinto, Manager Amber Baldwin Consuela Bradshaw Rob Cairns Maria Cavasos Jeff Cooling Deb Dale Jason Dawes Guadalupe Delgado Ken DeYoung Art Dorsey Kristi Gentry Hodilia Jimenez Matthew Jump Todd Katerberg

Operations Jana Hall, Ph.D., M.B.A., Chief Operations Officer Ann Schoen, Senior Executive Assistant

Law David Whitescarver, Vice President and Chief Legal Officer Erin Maag, Senior Administrative Assistant

Business Development and Extramural Administration Jerry Callahan, Ph.D., M.B.A., Vice President Marilyn Becker Carolyn Hudson, Ph.D. Thomas DeKoning Andrea Poma, M.P.A. Robert Garces, Ph.D. Norma Torres

Compliance Gwenn Oki, Director Paula Williamson DeBoe Angie Jason Erin Maag

Jonathan Olszewski Daniel Rogers

Tracy Lewis Lewis Lipsey Maria Lopez Dave Marvin Samantha Meekie Kevin Morton Angela Nobel Karen Pittman Richard Sal Jose Santos Amber TenBrink Richard Ulrich Pete VanConant Jeff Wilbourn

Finance Timothy Myers, Vice President and Chief Financial Officer Heather Zak, Controller Tess Kittridge Stephanie Birgy Angie Lawrence Theresa Brown Leah Lunger Sandi Dulmes Susan Raymond Katie Helder Cindy Turner Rich Herrick Kim Scott

Human Resources Development, Marketing, and Communications Love Collins III, Vice President Anne Benson Frank Brenner Breanna Bueche Cori Curtis Justin Ewald Beth Hinshaw Hall Matt Hirsh Sarah Hop

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Nancy Kooienga Sarah Murphy Nikki Outhier Patrick Placzkowski Audrey Rillema Angie Stumpo Ericka Zelasko

Linda Zarzecki, Vice President Stacey Booth Eric Miller Ryan DeCaire Pamela Murray Deidre Griffin John Shereda Brianna Knuth

Information Technology Bryon Campbell, Ph.D., Chief Information Officer David Drolett, Manager Sean Haak Candy Wilkerson, Manager Kenneth Hoekman Sandra Badini Jason Kotecki Bill Baillod Ben Lewitt Terry Ballard Deb Marshall Tom Barney Randy Mathieu Phil Bott Matt McFarlane James Clinthorne Thad Roelofs Dan DeVries Ken Selleck Michael Stolsky

Investments Office

Security

Kathy Vogelsang, Chief Investment Officer Benjamin Carlson Karla Mysels Ted Heilman

Kevin Denhof, CPP, Director Amy Davis Jonathan Fey

Materials Management

Sponsored Research

Richard M. Disbrow, C.P.M., Director Josie Collins Shannon Moore Stefanie Corey Monono Negash Matt Donahue Amy Poplaski Heather Frazee Bob Sadowski Chris Kutschinski Lindsey Sun Jeannette Mendez John Waldon

David Ross, Director Sara O’Neal, Manager Marilyn Becker Anita Boven Nathan Gras

Katee McCarthy Andriana Vincent

Kathy Koehler Michele Quick Heather Wells

Contract Support Caralee Lane, Librarian (Grand Valley State University)

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Van Andel Institute Van Andel Institute Board of Trustees David Van Andel, Chairman John C. Kennedy Mark Meijer Ralph W. Hauenstein (emeritus)

Board of Scientific Advisors Michael S. Brown, M.D., Chairman Richard Axel, M.D. Joseph L. Goldstein, M.D. Tony Hunter, Ph.D. Phillip A. Sharp, Ph.D.

Van Andel Research Institute Board of Trustees

Chief Executive Officer David Van Andel

Van Andel Education Institute Board of Trustees

David Van Andel, Chairman James Fahner, M.D. W. Gary Tarpley, Ph.D. George F. Vande Woude, Ph.D. Ralph Weichselbaum, M.D.

David Van Andel, Chairman James E. Bultman, Ed.D. Donald W. Maine Juan R. Olivarez, Ph.D. Gordon L. Van Harn, Ph.D.

Van Andel Research Institute Research Director

Van Andel Education Institute Director

Peter A. Jones, Ph.D., D.Sc.

Steven J. Triezenberg, Ph.D.

Vice President and Chief Legal Officer

VP Development, Communications, and Marketing

David Whitescarver

Chief Operations Officer

Love Collins III

Jana Hall, Ph.D., M.B.A.

VP Business Development

VP and Chief Financial Officer

Jerry Callahan, Ph.D.

Timothy Myers

VP Human Resources

Chief Information Officer

Linda Zarzecki

Bryon Campbell, Ph.D.

Facilities

Materials Management

Samuel Pinto

Richard Disbrow

Security Kevin Denhof

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The Van Andel Institute and its affiliated organizations (collectively the “Institute�) support and comply with applicable laws prohibiting discrimination based on race, color, national origin, religion, gender, age, disability, height, weight, marital status, U.S. military veteran status, or other personal characteristics covered by applicable law. The Institute also makes reasonable accommodations required by law. The Institute’s policy in this regard covers all aspects of the employment relationship, including recruiting, hiring, training, and promotion.

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