2013 Van Andel Research Institute Scientific Report

Van Andel Research Institute Scientific Report 2013

Scientific Report

333 Bostwick Avenue, N.E., Grand Rapids, Michigan 49503 Phone 616.234.5000 Fax 616.234.5001 www.vai.org

13

Van Andel Research Institute Scientific Report 2013

Cryosection of a mouse calvaria.

In using tissue-specific knock-out mouse models, the promoter must have precise specificity. Here we used the mTmG reporter model to demonstrate that Ocn-Cre expresses specifically in the bone cells. Top panel: Cells were stained with DAPI (blue) for nucleic acids. Bone cells are expressing GFP (green), while all other cells are expressing Tomato (red). Lower panel: A differential interference contrast image with DAPI stain of the same area.

Photo by Alex Zhong of the Williams laboratory.

Van Andel Research Institute |

Scientific Report

Published March 2013.

Copyright 2013 by the Van Andel Institute; all rights reserved.

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

ii

VARI | 2013

Introduction

1

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

6

William H. Baer II, M.D., Pharm.D. VARI-ClinXus, LLC 8

John F. Bender, Pharm.D. Clinical Operations 10 Patrik Brundin, M.D., Ph.D. Translational Parkinson’s Disease Research 11 Ting-Tung (Anthony) Chang, Ph.D. Small-Animal Imaging Facility/Translational Imaging 14 Nicholas S. Duesbery, Ph.D. Cancer and Developmental Cell Biology 16

Bryn Eagleson, B.S., RLATG Vivarium and Transgenics 19 Kyle A. Furge, Ph.D. Interdisciplinary Renal Oncology 22

Table of Contents

Brian B. Haab, Ph.D. Cancer Immunodiagnostics 25

Galen H. Hostetter, M.D. Analytical Pathology 28 Scott D. Jewell, Ph.D. Program for Biospecimen Science 30 Xiaohong Li, Ph.D. Tumor Microenvironment and Metastasis 34

Jeffrey P. MacKeigan, Ph.D. Systems Biology 35 Karsten Melcher, Ph.D. Structural Biology and Biochemistry 38 Cindy K. Miranti, Ph.D. Integrin Signaling and Tumorigenesis 41 Mark W. Neff, Ph.D. Canine Genetics and Genomics 44

Brian J. Nickoloff, M.D., Ph.D. Cutaneous Oncology 46 Giselle S. Sholler, M.D. Neuroblastoma Translational Research 47

Matthew Steensma, M.D. Musculoskeletal Oncology 49

Steven J. Triezenberg, Ph.D. Transcriptional Regulation 51 Jeremy M. Van Raamsdonk, Ph.D. Aging and Neurodegenerative Disease 54

iii

Van Andel Research Institute |

Scientific Report

Laboratory Reports, continued George F. Vande Woude, Ph.D. Molecular Oncology

57

Craig P. Webb, Ph.D. Translational Medicine 60 Michael Weinreich, Ph.D. Genome Integrity and Tumorigenesis 63 Bart O. Williams, Ph.D. Cell Signaling and Carcinogenesis 66

H. Eric Xu, Ph.D. Structural Sciences 70

Awards for Scientific Achievement

73

J ay Van Andel Award for Outstanding Achievement in Parkinson’s Disease Research Han-Mo Koo Memorial Award

Postdoctoral Fellowship Program

76

List of Fellows

Student Programs

78

Grand Rapids Area Pre-College Engineering Program Summer Student Internship Program

VARI Seminar Series

82

2011 – 2012 Seminars

Van Andel Research Institute Organization Boards Office of the Director VAI Administrative Organization

iv

85

Introduction

1

Van Andel Research Institute |

Scientific Report

Introduction Phase II of the Van Andel Institute building, which opened in late 2009, added 240,000 square feet to the Institute, nearly tripling the available laboratory space, and it garnered LEED Platinum status from the United States Green Building Council.

This expansion enabled the start of a major new initiative into the study of neurodegenerative diseases and provided the infrastructure to establish the Van Andel Research Institute (VARI) Center for Neurodegenerative Science. The Center is led by Dr. Patrik Brundin, one of the world’s leading researchers in the field of Parkinson’s disease, who arrived from Lund University in Sweden in January 2012. Dr. Brundin holds the Jay Van Andel Endowed Chair in Parkinson’s Research and also serves as VARI Associate Director.

The VARI investigator staff welcomed two other distinguished members into its ranks in 2012. Jeremy Van Raamsdonk’s research focuses on aging, Parkinson’s disease, and Huntington’s disease. He heads the Laboratory of Aging and Neurodegenerative Disease, and in his translational research, positive results from studies in worm and mouse models will be used to identify therapeutic targets for clinical trials. Xiaohong Li leads the Laboratory for Tumor Microenvironment and Metastasis. Her research focuses on the role of stromal transforming growth factor (TGF-b) in the microenvironment of primary and metastatic tumor sites and its effect on bone metastases, with the aim of developing early diagnostic and treatment strategies for breast and prostate cancer metastasis to bone.

The Institute hosted world-renowned researchers in 2012 and honored two of them for their contributions to science. In May 2012, Dr. Phillip A. Sharp was the first recipient of the Institute’s Han-Mo Koo Memorial Award. Dr. Sharp received the 1993 Nobel Prize in Physiology or Medicine for his discovery of RNA splicing, which fundamentally changed the understanding of gene structure. Much of his research has focused on the molecular biology of gene expression relevant to cancer. The Han-Mo Koo Award recipients are selected on the basis of their scientific achievements and contributions to human health and research.

The award is named for one of VAI’s founding scientists who, in 2004 at the age of 40, succumbed to aggressive NK T-cell lymphoma, a rare form of cancer.

The Van Andel Institute held the “Grand Challenges in Parkinson’s Disease” symposium in September 2012, gathering experts from nearly a dozen nations to present the latest research on this devastating disease. Dr. Ted Dawson of Johns Hopkins University and Dr. Roger Barker of the University of Cambridge provided keynote addresses. During the symposium, the Institute presented the inaugural Jay Van Andel Award for Outstanding Achievement in Parkinson’s Disease Research to Dr. Andrew B. Singleton of the National Institutes of Health. Dr. Singleton’s research focuses on the genetic causes of Parkinson’s disease, and he is actively studying the consequences of gene alterations in the context of the aging brain.

VARI researchers in 2012 had much success in terms of funded grant proposals and sponsored research. Major grants included the following: • a four-year R01 renewal from the National Institutes of Health (NIH) to Bart Williams for the project entitled “Analyzing the Role of Wnt Signaling in Bone Development”; • a five-year R01 award to Cindy Miranti for a project on “The Role of a6b1 Integrin in Prostate Cancer”; • a three-year R01 award to Karsten Melcher for “Structural and Functional Analysis of a Dynamic ABA Signaling Complex”; and • a five-year NIH U01 award to Brian Haab for a project on “Targeted Glycomics and Affinity Reagents for Cancer Biomarker Development”.

2

VARI | 2013

In addition, Scott Jewell received several major contracts for the Program for Biospecimen Science, including one for “Research Studies in Cancer and Normal Tissue Acquisition and Processing Variables”. The Program for Biospecimen Science also became one of only seven biorepositories in the nation accredited by the College of American Pathologists (CAP), based on the results of an on-site inspection as part of the CAP Accreditation Program.

VARI has announced an agreement with Dako, the Danish-based, worldwide supplier of cancer diagnostic tools, to license, manufacture, and distribute cancer diagnostics utilizing the MET4 antibody. This antibody, which detects the MET gene in human tumors, works exceptionally well in classical diagnostic procedures. MET4 was developed by the laboratories of George F. Vande Woude and Brian Cao of VARI and Beatrice Knudsen, formerly of the Fred Hutchinson Cancer Research Center.

Among VARI research publications in 2012 was “Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases”, co-authored by Fen-Fen Soon, Karsten Melcher, and Eric Xu and published in a January 2012 edition of Science. Abscisic acid (ABA) is a crucial plant hormone involved in stress adaptation. Activation of the signaling pathway for ABA includes the phosphorylation of pathway proteins by a SnRK kinase. In this paper, the authors determined that the SnRK kinase is turned off by the direct binding of the kinase activation loop into the catalytic cleft of a PP2C phosphatase as part of a two-step inactivation mechanism. The kinase is turned on when it is displaced from the phosphatase by the ABA hormone receptor complex. That displacement is the result of the similarity in PP2C recognition between the kinase molecule and the complex, which allows facile regulation of the kinase’s activity. This study provides a new paradigm of kinase–phosphatase regulation.

Thanks to the achievements of new and existing programs, Van Andel Institute anticipates the continued growth and success of its research programs into cancer and neurodegenerative disease in 2013 and beyond. This growing intellectual capital complements the expansion of the Institute’s state-of-the-art facilities. At full capacity, Phase II will support a $125 million annual research operation that will expand the number of laboratories to more than 50 and provide some 550 additional jobs.

Such growth is made possible, in part, by the Institute’s wide network of dedicated supporters. Thanks to the generous endowment of the Van Andel family, 100% of donor contributions go directly to the laboratories where VARI scientists seek discoveries leading to improved treatments for patients. That’s 100% to Research, Discovery, and Hope!

3

Van Andel Research Institute |

4

Scientific Report

Laboratory Reports

5

Arthur S. Alberts, Ph.D. Laboratory of Cell Structure and Signal Integration Dr. Alberts received his Ph.D. in physiology and pharmacology from the School of Medicine at the University of California, San Diego in 1993. From 1994-1997, he was an HHMI postdoctoral scholar in Richard Treisman’s lab at the Imperial Cancer Research Fund in London. Prior to joining VARI, he was in the laboratory of Frank McCormick at the University of California, 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: Lash-Van Wyhe, Schepers, Goosen, Schumacher, Becker, Alberts, Howard, Rybski, LaGrone, Turner

Staff

Students

Visiting Scientists

Susan Goosen, M.B.A. Leanne Lash-Van Wyhe, Ph.D. Heather Schumacher, MT(ASCP)

Lisa Becker Andrew Howard, B.A. Chantice LaGrone Kristin Rybski Alison Schepers Sarah Sternberger, M.S.

Julie Davis Turner, Ph.D. Brad Wallar, Ph.D.

6

VARI | 2013

Research Interests • To investigate the genetic and molecular basis of disease arising from defects in the cell infrastructure, which comprises the microtubule and microfilament cytoskeletons.

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

We place a basic research focus on the intersection of Rho and Wnt signaling to the nucleus and on the cytoskeletal remodeling apparatus. We place a translational focus on targeted therapies that reinforce and/or repair the cell infrastructure.

Our disease focus is the blood cancers that arise from cells of the bone marrow. We use genetic models of these diseases to test ideas generated by our molecular studies. These models will inform the development of novel diagnostic and therapeutic tools for treating these cancers.

Recent Publications Touré, Fatouma, Günter Fritz, Qing Li, Vivek Rai, Gurdip Daffu, Yu Shan Zou, Rosa Rosario, Ravichandran Ramasamy, Arthur S. Alberts, Shi Fang Yan, et al. 2012. Formin mDia1 mediates vascular remodeling via integration of oxidative and signal transduction pathways. Circulation Research 110(10): 1279–1293.

Alberts, Art, and Michael Way. 2011. Actin motility: formin a SCAry tail. Current Biology 21(1): R27–R30.

He, Yuanzheng, Yong Xu, Chenghai Zhang, Xiang Gao, Karl J. Dykema, Katie R. Martin, Jiyuan Ke, Eric A. Hudson, Sok Kean Khoo, James H. Resau, et al. 2011. Identification of a lysosomal pathway that modulates glucocorticoid signaling and the inflammatory response. Science Signaling 4(180): ra44.

Thomas, S.G., S.D.J. Calaminus, L.M. Machesky, A.S. Alberts, and S.P. Watson. 2011. G-protein coupled and ITAM receptor regulation of the formin FHOD1 through Rho kinase in platelets. Journal of Thrombosis and Haemostasis 9(8): 1648–1651.

7

William H. Baer II, M.D., Pharm.D. VARI-ClinXus, LLC Dr. Baer joined ClinXus in 2009 as Executive Director and Chief Medical Officer. When ClinXus became VARI-ClinXus LLC in January 2011, Dr. Baer was appointed as an Associate Professor within VARI. Dr. Baer received his pharmacy degree from Duquesne University, the Pharm.D. from the West Virginia University, and his M.D. from West Virginia University School of Medicine. He practices internal medicine at Grand Valley Medical Specialists. His areas of interest and research development include pharmacogenetics, disease prevention and wellness, obesity, and nutrition.

From left: Baer, Eckhardt, Rogers

Staff Elizabeth Eckhardt, B.S. Lisa Moore, M.S. Daniel Rogers, B.S., CCRC Heidi Smith-Green, RN, B.S.N., B.S.W. Emily Vander Molen, B.A., CHRC, CIP 8

VARI | 2013

Research Interests VARI-ClinXus, LLC, is a West Michigan translational research organization dedicated to benefiting human health and improving patient’s lives through early-phase and molecular-based trials that are fundamental to personalized medicine. VARI-ClinXus works with community partner institutions that are highly credentialed in areas of health care, early clinical development, clinical research, and academics. Through our network, we are able to provide client organizations with the many advantages of collective expertise to facilitate innovative clinical trials of diagnostics, devices, and biological agents and bring them to market in a more efficient time frame. We offer an integrated suite of services that includes protocol and project design, clinical trial development and implementation, state-of-the-art patient facilities and support, extensive molecular profiling capabilities, and a full breadth of integrated IT infrastructure.

The comprehensive expertise of our partner institutions extends across a wide range of specialties, with an emphasis on oncologic and neurodegenerative medicine. Current partners include Advanced Radiology Services, Borgess Research Institute, Bronson Healthcare, Cancer and Hematology Centers of West Michigan, Ferris State University, Grand Valley Medical Specialists, Grand Valley State University, Innovative Analytics, Jasper Clinical Research & Development, Metro Health Hospital, Michigan Institute for Clinical & Health Research, Michigan State University, MPI Research, Saint Mary’s Health Care, and Spectrum Health hospitals.

We have partnered with the Critical Path Institute’s Predictive Safety Testing Consortium (PSTC) in several capacities, and we provide clinical advice and support for PSTC’s clinical efforts in the evaluation and qualification of new biomarkers to assist in the safety of drug development. The PSTC’s mission is to bring pharmaceutical companies together to validate each other’s safety testing methods.

9

John F. Bender, Pharm.D. Clinical Operations Dr. Bender holds a B.S. in biology from Mount Saint Mary’s College, a B.S. in pharmacy from the University of Maryland, and a Pharm.D. from the University of Utah. He worked at Parke-Davis as director of clinical research – oncology for over 20 years. Dr. Bender also served as senior vice-president of clinical research and of research and development at two biopharmaceutical companies in San Diego that focused on cancer treatments. He is currently the Clinical Operations Director at the Van Andel Research Institute. He is also an Adjunct Assistant Professor of Clinical Pharmacy with the Ferris State College of Pharmacy in Grand Rapids.

Research Interests As VARI Clinical Operations Director, Dr. Bender coordinates the development of oncology clinical trials to accelerate translational research studies in Grand Rapids. He provides translational research support to VARI research, with active projects currently in eight labs. An effort underway is to establish a clinical trial center for VARI. Dr. Bender has an effective network of colleagues within Michigan and beyond, and he fosters productive interactions between VARI researchers, outside investigators, and the pharmaceutical and clinical communities.

Staff Ashley Rodriguez

10

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

He has over 30 years of experience with neurodegenerative diseases, has some 300 publications, and is in the top 0.5% of cited researchers in the field. Much of his research has addressed disease mechanisms in cell culture and animal models of Parkinson’s disease. In addition to managing laboratories at VARI and in Lund, Sweden, he is Associate Director of VARI and the co-editor-in-chief of the Journal of Parkinson’s Disease.

From left: Kaufman, Beauvais, Brundin, Steiner, Cousineau, Ghosh

Staff Genevieve Beauvais, Ph.D. Kim Cousineau, B.S. Martha Escobar, Ph.D. Anamitra Ghosh, Ph.D. Darcy Kaufman, M.S. Jennifer Steiner, Ph.D. 11

Van Andel Research Institute |

Scientific Report

Research Interests The Laboratory for Translational Parkinson’s Disease Research studies cellular and rodent models of neurodegenerative disease. We currently focus on several projects that might lead us to our ultimate goals of 1) understanding why Parkinson’s disease (PD) develops and 2) discovering new methods of treatment that could stop or slow disease progression.

We expect that these experiments will reveal how genetic and other factors are associated with PD pathology.

Many rodent models of PD are based on treating the animals with neurotoxins such as 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) or 6-hydroxydopamine. These toxins lead to select neuronal degeneration within days in brain areas relevant to PD. However, we know that the development of PD in humans is a decades-long process of neuron death, unlike the short time line of days in these models. We have initiated work in a mouse that lacks one copy of a gene known to be expressed in midbrain dopaminergic neurons and that exhibits a progressive degeneration of these cells.

As a consequence, the neurons’ slow degeneration over many weeks into adulthood more closely mirrors PD. In our studies, we are carefully analyzing the morphological and neurochemical changes in the degenerating dopamine neurons and trying to understand the changes in gene expression in the cells during the process. We believe these mice will be a highly relevant model of PD, and we are now planning to treat them with potentially neuroprotective agents over several weeks in attempts to slow down the degenerative process.

We are also using a transformed cell line derived from the immature human ventral midbrain. We can differentiate these cells into mature dopaminergic neurons that exhibit the expected electrical activity and synthesize dopamine. We have previously identified the sensitivity of these human midbrain neurons to cellular toxins or stresses. This unique dopaminergic cell line serves as a starting point for many of our studies with both neurotoxins and neuroprotective agents. We aim to determine whether known neuroprotective drugs, some of which are currently in clinical trials, rescue these dopaminergic cells from PD-relevant challenges. If these human cells respond positively to these drugs, then we will test the agents in the mouse models described earlier. For example, disturbances in mitochondrial function are hypothesized to play an important role in the development of PD. Therefore we will explore whether drugs that modulate mitochondrial function can protect against neurodegeneration. Our current experiments using the genetic mouse models and toxin-based mouse models of PD described above will help us decide whether these mitochondrial modulators may be efficacious in the clinic.

In order to study how PD develops, we also study the spreading of abnormal a-synuclein (a-syn) protein. The transmission of a-syn-associated pathology from cell to cell throughout the nervous system is believed to drive the clinical disease state and underlie several PD symptoms, including nonmotor symptoms. We are interested in identifying the mechanisms underlying intercellular a-syn transfer and transport in order to clarify their role(s) in the development of PD. We will partly focus on inter/intracellular transfer involving exosomes. We plan to perform studies using exosomes isolated under specific conditions (e.g., overexpression of a-syn) to determine whether exosomes play a role in a-syn transfer and aggregation. We will also explore the fate of a-syn that has been taken up by neurons. Thus, we will attempt to clarify how the imported a-syn is processed inside the cells and under what conditions it is transported between brain regions in rodents.

In addition, we plan to use Caenorhabditis elegans to identify genes that control a-syn transfer between cells. We will generate transgenic C. elegans strains that will allow us to study a-syn transfer between neurons with the help of fluorescent markers.

12

VARI | 2013

Recent Publications Brundin, Patrik, and Jeffrey H. Kordower. In press. Neuropathology in transplants in Parkinson’s disease: implications for disease pathogenesis and the future of cell therapy. In Functional Neural Transplantation III, Amsterdam: Elsevier.

Rey, Nolwen L., Elodie Angot, Christopher Dunning, Jennifer A. Steiner, and Patrik Brundin. In press. Accumulating evidence suggests that Parkinson’s disease is a prion-like disorder. In Research and Perspectives in Alzheimer’s Disease, Berlin: Springer.

Tomé, Carla M. Lema, Trevor Tyson, Nolwen L. Rey, Stefan Grathwohl, Markus Britschgi, and Patrik Brundin. In press.

Inflammation and a-synuclein’s prion-like behavior in Parkinson’s disease — is there a link? Molecular Neurobiology.

Angot, Elodie, Jennifer A. Steiner, Carla M. Lema Tomé, Peter Ekström, Bengt Mattsson, Anders Björklund, and Patrik Brundin.

2012. Alpha-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo. PLoS One 7(6): e39465.

Jeon, Iksoo, Nayeon Lee, Jia-Yi Li, In-Hyun Park, Kyoung Sun Park, Jisook Moon, Soung Han Shim, Chunggab Choi, Da-Jeong Chang, Jihye Kwon, et al. 2012. Neuronal properties, in vivo effects, and pathology of a Huntington’s disease patient-derived induced pluripotent stem cells. Stem Cells 30(9): 2054–2062.

Paul, Gesine, Ilknur Özen, Nicolaj S. Christophersen, Thomas Reinbothe, Johan Bengzon, Edward Visse, Kararina Jansson, Karin Dannaeus, Catarina Henriques-Oliveira, Laurent Roybon, et al. 2012. The adult human brain harbors multipotent perivascular mesenchymal stem cells. PLoS One 7(4): e35577.

Tyson, Trevor, and Patrik Brundin. 2012. VPS41-mediated neuroprotection in a Caenorhabditis elegans model of Parkinson’s disease. Future Neurology 7(3): 255–258.

13

Ting-Tung (Anthony) Chang, Ph.D. Small-Animal Imaging Facility/Laboratory of Translational Imaging Dr. Chang received a B.S. degree in medical imaging and radiological sciences from Chang Gung University (Taoyuan, Taiwan) and his Ph.D. degree in medical physics (CAMPEP), 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: Bozio, Dieffenbach, Dykstra, Peck, Li, Holly, Chang, Nelson

Staff

Students

Visiting Scientist

Adjunct Faculty

Shihong Li, Ph.D. Amy Nelson Anderson Peck, M.S.E.

Ryan Bozio, B.S. Zachary Dieffenbach Michael Dykstra Brittany Holly Yasmeen Robinson

Samhita Rhodes, Ph.D.

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

14

VARI | 2013

Research Interests The Small-Animal Imaging Facility provides novel imaging and image analysis tools for use with biology specimens and small animals. Our instruments include digital X-ray, high-resolution microCT, microSPECT/CT, microPET/CT, micro-ultrasound, and optical imaging. Our research focuses on the development of new preclinical imaging technologies that can offer significant anatomic and functional information to biomedical investigators.

The Laboratory of Translational Imaging aims at developing imaging technologies capable of monitoring organ/tissue activity at the molecular level. We intend these developments to be highly translatable into clinical use, especially for tumor early detection and staging. Combining tracer development, imaging analysis, and genomic information, we are dedicated to collecting medically useful information through novel, non-invasive imaging technologies that will advance the goal of personalized precision medicine.

Recent Publications Flaten, Gøril Eide, Ting-Tung Chang, William T. Phillips, Martin Brandl, Ande Bao, and Beth Goins. In press. Liposomal formulations of poorly soluble camptothecin: drug retention and biodistribution. Journal of Liposome Research.

Figure 1 Figure 1. Three-dimensional imaging of a kidney cyst in vivo using contrastenhanced computed tomography (CT).

15

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 Council for Research Affairs.

From left: Duesbery, Boguslawski, Bromberg-White, Lewis, Kuk, Andersen, Bhattacharya, Naidu

Staff

Students

Adjunct Faculty

Nicholas Andersen, Ph.D. Poulomi Bhattacharya, Ph.D. Elissa Boguslawski Jenn Bromberg-White, Ph.D. Kara Kits, Ph.D. Diana Lewis, A.S.

Cynthia Kuk, B.S. Agni Naidu, B.S., B.A.

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

16

VARI | 2013

Research Interests Our lab is interested in a family of related proteins called the mitogen-activated protein kinase kinases (MKKs). MKKs are evolutionarily conserved, regulatory protein kinases that play pivotal roles in a wide variety of developmental cellular processes, including growth, division, and differentiation. Our lab is specifically interested in the roles of these kinases in the developmental and pathologic growth of blood vessels.

More than a decade ago we showed that blocking the activity of MKKs in tumors caused decreased blood flow and tumor regression. Since then we have used a variety of experimental approaches to understand how the loss of MKK activity affects the growth of blood vessels. Most recently we discovered that MKK activity was essential for the regrowth of blood vessels in a mouse model of diabetic retinopathy. Our results suggest that the inhibition of MKK activity may be a good strategy for treating eye diseases such as proliferative diabetic retinopathy or wet macular degeneration. We are currently exploring this possibility in collaboration with Grand Rapids ophthalmologist Dr. Louis Glazer.

In some cases the abnormal growth of cells that form blood vessels results in cancer. These tumors, called angiosarcomas, are an extremely rare but deadly form of cancer for which there is no effective treatment. In collaboration with Dr. Barbara Kitchell at the Michigan State University College of Veterinary Medicine, Dr. Laurence Baker at the University of Michigan, and Dr. Gary Schwartz at the Memorial Sloan – Kettering Cancer Center, we have discovered that MKK activity plays an essential role in the growth of these tumors. On-going studies in our lab are using unique mouse models we have developed to identify combinatorial approaches for treating these tumors.

While excessive blood vessel growth is characteristic of cancer and retinal diseases, decreased blood flow is a crucial factor in peripheral arterial disease. This disease, often associated with obesity, diabetes, and smoking, is caused by blood vessel obstruction and a diminished ability to grow or expand existing blood vessels. Together with Dr. Christopher Chambers, a cardiovascular surgeon at the Meijer Heart and Vascular Institute, we have begun an exciting new research project involving human clinical samples to investigate the molecular biology of peripheral arterial disease.

The goals of the lab in the coming years are to • Define the key roles of MKKs in developmental and pathologic growth of blood vessels, using models of retinal disease and peripheral arterial disease • Identify novel anti-angiogenic targets • Discover and validate genetic and biochemical drivers of site-specific disease in angiosarcoma • Translate these findings to improve the clinical care of patients.

17

Van Andel Research Institute |

Scientific Report

Figure 1

Figure 1: MKK activity is essential for blood vessel growth. In a model that mimics diabetic retinopathy, blood vessels in these mouse retina whole mounts show regrowth following oxygen deprivation (left panel). Such regrowth is prevented (right panel) in retinas treated with anthrax lethal toxin, an MKK inhibitor. Such inhibitors may have utility in treating human eye diseases such as proliferative diabetic retinopathy. Photographs by Jennifer Bromberg-White (Bromberg-White et al., 2011, Investigative Ophthalmology and Visual Science 52: 8979); ©Association for Research in Vision and Ophthalmology.

Recent Publications Bromberg-White, Jennifer L., Nicholas J. Andersen, and Nicholas S. Duesbery. 2012. MEK genomics in development and disease. Briefings in Functional Genomics 11(4): 300–310.

Andersen, Nicholas, Roe Froman, B. Ketchell, and Nicholas S. Duesbery. 2011. Angiosarcoma: clinical and molecular aspects. In Soft Tissue Sarcoma, Austria: I-Tech Education and Publishing, pp. 149–174.

Bromberg-White, Jennifer L., Elissa Boguslawski, Daniel Hekman, Eric J. Kort, and Nicholas S. Duesbery. 2011. Persistent inhibition of oxygen-induced retinal neovascularization by anthrax lethal toxin. Investigative Ophthalmology and Visual Science 52(12): 8979–8992.

Lee, Chih-Shia, Karl J. Dykema, Danielle M. Hawkins, David M. Cherba, Craig P. Webb, Kyle A. Furge, and Nicholas S. Duesbery. 2011. MEK2 is sufficient but not necessary for proliferation and anchorage-independent growth of SK-MEL-28 melanoma cells. PLoS One 6(2): e17165.

18

Bryn Eagleson, B.S., RLATG Vivarium and Laboratory of Transgenics Ms. Eagleson 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 Vivarium Director and Transgenics Special Program Manager. She has a B.S. degree in psychology from the University of Maryland University College. Ms. Eagleson is a member of the IACUC and has served two terms as its chair.

Standing, from left: Kefene, Guikema, Boguslawski, Post, Ramsey, Meringa, Baumann, B. Eagleson, Timmer, Stroben, Vrbis, K. Eagleson, Brady, Ehrke Kneeling, from left: Kempston, Rackham, Brandow, Holzgen

Research Technicians

Laboratory Animal Technicians

Animal Caretaker Staff

Audra Guikema, B.S., LVT Tristan Kempston, B.S.

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

Sylvia Timmer, Vivarium Supervisor Crystal Brady Neil Brandow Kendra Eagleson Crystal Ehrke Katie Holzgen Mat Rackham Brandon Stroben Ashlee Vrbis

19

Van Andel Research Institute |

Scientific Report

Research Interests The goal of the VARI vivarium and transgenics core is to develop, provide, and support high-quality mouse modeling services for the VARI investigators, collaborators, and the greater research community. The vivarium is a state-of-the-art facility that includes a high-level containment barrier. All procedures are conducted according to the NIH Guide for the Care and Use of Laboratory Animals. Because we understand the importance of excellence in animal care to producing quality research, we are committed to the highest quality in animal standards, and the Van Andel Research Institute is an AAALAC-accredited institution. The staff provides rederivation, surgery, dissection, necropsy, breeding, weaning, tail biopsies, sperm and embryo cryopreservation, animal data management, and health-status monitoring. Transgenic mouse models are produced on request for project-specific needs.

Transgenics Fertilized eggs contain two pronuclei, one that is derived from the egg and contains the maternal genetic material and one derived from the sperm that contains the paternal genetic material. As development proceeds, these two pronuclei fuse, the genetic material mixes, and the cell proceeds to divide and develop into an embryo. Transgenic mice are produced by injecting small quantities of foreign DNA (the transgene) into a pronucleus of a one-cell fertilized egg. DNA microinjected into a pronucleus randomly integrates into the mouse genome and will theoretically be present in every cell of the resulting organism.

Expression of the transgene is controlled by elements called promoters that are genetically engineered into the transgenic DNA. Depending on the selection of the promoter, the transgene can be expressed in every cell of the mouse or in specific cell populations such as neurons, skin cells, or blood cells. Temporal expression of the transgene during development can also be controlled by genetic engineering. These transgenic mice are excellent models for studying the expression and function of the transgene in vivo.

Gene targeting Mouse models are produced using gene-targeting technology, a well-established, powerful method for inserting specific genetic changes into the mouse genome. The resulting mice can be used to study the effects of these changes in the complex biological environment of a living organism. The genetic changes can include the introduction of a gene into a specific site in the genome (gene “knock-in”) or the inactivation of a gene already in the genome (gene “knock-out”). Since these mutations are introduced into the reproductive cells known as the germline, they can be used to study the developmental aspects of gene function associated with inherited genetic diseases.

The vivarium and transgenics lab can also produce mouse models in which the gene of interest is inactivated in a target organ or cell line instead of in the entire animal. These models, known as conditional knock-outs, are particularly useful in studying genes that, if missing, cause the mouse to die as an embryo.

Our gene-targeting service encompasses three major procedures: DNA electroporation, clone expansion and cryopreservation, and microinjection. Gene targeting is initiated by mutating the genomic DNA of interest and inserting it into mouse embryonic stem (ES) cells via electroporation. The mutated gene integrates into the genome and, by a process called homologous recombination, replaces one of the two wild-type copies of the gene in the ES cells. Clones are identified, isolated, and cryopreserved, and genomic DNA is extracted from each clone and delivered to the client for analysis. Correctly targeted ES cell clones are thawed, established into tissue culture, and cryopreserved in liquid nitrogen. Gene-targeting mutations are introduced by microinjection of the pluripotent ES cell clones into 3.5-day-old mouse embryos (blastocysts). These embryos, containing a mixture of wild-type and mutant ES cells, develop into mice called chimeras. The offspring of chimeras that inherit the mutated gene are heterozygotes possessing one copy of the mutated gene. The heterozygous mice are bred together to produce “knock-out mice” that completely lack the normal gene and have two copies of the mutant gene.

20

VARI | 2013

Embryo/sperm cryopreservation We provide cryopreservation services for archiving and reconstituting valuable mouse strains. These cost-effective procedures decrease the need to continuously breed valuable mouse models, and they provide added insurance against the loss of custom mouse lines due to disease outbreak or a catastrophic event. Mouse embryos at various stages of development, as well as mouse sperm, can be cryopreserved and stored in liquid nitrogen; they can be thawed and used, respectively, by implantation into the oviducts of recipient mice or by in vitro fertilization of oocytes.

Rederivation Mice harboring pathogens can negatively affect research results, and they may pass on those pathogens to other mice within the colony. Strain rederivation, by embryo transfer, is a management tool to clean a mouse line from pathogen infection or to import mice into a barrier facility from outside the vivarium. At VARI, any mice imported from an outside research institution are rederived to ensure the specific pathogen-free status of the animals coming in, and also to ensure that our existing research models remain pathogen-free.

21

Kyle A. Furge, Ph.D. Laboratory of Interdisciplinary Renal Oncology Dr. Furge received his Ph.D. in biochemistry from the Vanderbilt University School of Medicine in 2000. Prior to obtaining his degree, he worked as a software engineer at YSI, Inc., where he wrote operating systems for remote environmental sensors. Dr. Furge did his postdoctoral work in the laboratory of George Vande Woude. He joined VARI in June 2001 and was promoted to Assistant Professor in May 2005. Dr. Furge also heads the Kidney Cancer Research Program.

From left: Ooi, Petillo, Furge, Dykema

Staff

Adjunct Faculty

Karl Dykema, B.A. Aikseng Ooi, Ph.D. David Petillo, Ph.D.

Richard Kahnoski, M.D. Brian Lane, M.D., Ph.D. Bin Teh, M.D., Ph.D.

22

VARI | 2013

Research Interests Renal cell carcinoma (RCC) is the most common type of cancer that arises within the adult kidney, and the tumors can be separated into categories based on the morphology of their cells. Clear cell RCC is the most common subtype, constituting 70–80% of renal tumors. Papillary RCC, which can be divided into type 1 and type 2, is the next most common subtype, representing 10–15%. Chromophobe RCC represents about 5% of renal tumors; other renal cell carcinomas are either unclassifiable by conventional means or represent rare subtypes. The latter include transitional cell carcinoma of the renal pelvis, renal medullary tumor, tubulocystic carcinoma, Xp11.2 translocation-associated tumor, collecting duct tumor, adult Wilms tumor, mixed epithelial and stromal tumor/cystic nephroma, and the usually benign renal oncocytoma and angiomyolipoma.

Several decades of kidney cancer research indicate that the genetic mutations that accumulate within the tumor cells differ depending on the particular subtype. Overall, the laboratory is interested in identifying the genetic mutations present in renal cancer cells and in understanding how the different mutations transform normal cells into cancerous cells. We also want to understand the features associated with the most aggressive renal tumors.

The analysis of papillary type 2 tumors (PRCC2) is one current focus. This is an aggressive subtype that has no effective treatment. Individuals who inherit a rare germline mutation in the fumarate hydratase gene (FH) are predisposed to develop this cancer. However, most PRCC2 tumors arise in the general population and do not contain that mutation. The genetic defects that lead to formation of sporadic PRCC2 tumors in the general population are not known.

We have recently discovered that the transcription factors NRF1 and NRF2 (nuclear factor–erythroid-related factors 1 and 2) are activated in type 2 papillary RCC but not other subtypes of RCC. NRFs are key mediators of the adaptive detoxification response, and they regulate the many aspects of cellular detoxification and cell metabolism. NRF1 and NRF2 become activated as cells are exposed to electrophilic and reactive oxygen insults. NRFs then activate the transcription of a crucial set of enzymes that promotes cell survival by clearing toxic metabolites and xenobiotics.

The FH mutations present in hereditary PRCC2 tumors result in high levels of intercellular fumarate. We have found that the NRF transcription factors become activated as fumarate, a reactive molecule, chemically modifies proteins at their exposed cysteine residues, a process termed succination (Figure 1). The modification of proteins by fumarate leads to NRF activation in these tumors. Sporadic PRCC2 tumors frequently lack FH mutations, so the mechanisms by which NRF is activated in these tumors is unclear. Both the mechanism by which NRF activation occurs in PRCC2 tumors and the functional connection between NRF activation and tumor cell survival are current focuses of the laboratory.

We are also interested in the genetic mechanisms that give rise to the chromophobe subtype of renal tumors. Individuals who inherit a rare germline mutation in the folliculin gene (FLCN) are predisposed to chromophobe renal cancer. The mRNA profiles of tumors from such individuals gave clues that FLCN has a role in the energy sensing network, particularly in mitochondrial function. The connection between FLCN loss of function and tumor cell development is another focus.

The tools that we use to study renal tumor development include a blend of computational modeling, molecular biology, and genetics. The genetic analysis of tumor cells typically includes the analysis of large amounts of DNA sequencing, mRNA expression profiling, and DNA copy number data. Therefore, we develop and apply new computational tools that can assist in extracting the significant biological information from these data sets, with a goal of understanding how cancer cells differ from normal cells at the molecular level.

23

Van Andel Research Institute |

Scientific Report

Figure 1

Figure 1: Mechanism of NRF2 activation in hereditary papillary renal cell carcinoma. NRF2 is a transcription factor that can migrate to the nucleus and activate the transcription of detoxification genes such as AKR1B10. Low levels of NRF2 are maintained

by KEAP1 and CUL3. KEAP1 and CUL3 are required for NRF2 ubiquitination and degradation. This process is disrupted in cells with fumarate hydratase (FH) mutations. The normal biochemical activity of fumarate hydratase and succinate dehydrogenase are shown as part of the mitochondrial TCA cycle. In cells with FH mutation, excess fumarate is exported from the mitochondria and reacts with cysteine residues on KEAP1 (rounded rectangle). Modified KEAP1 is ubiquitinated and degraded. This prevents NRF2 from being degraded, and so nuclear levels of NRF2 increase.

Recent Publications Farber, Leslie J., Kyle Furge, and Bin Tean Teh. 2012. Renal cell carcinoma deep sequencing: recent developments.

Current Oncology Reports 14(3): 240–248.

Klomp, Jeff A., and Kyle A. Furge. 2012. Genome-wide matching of genes to cellular roles using guilt-by-association models derived from single sample analysis. BMC Research Notes 5: 370.

Ong, Choon Kiat, Chutima Subimerb, Chawalit Pairojkul, Sopit Wongkham, Ioana Cutcutache, Willie Yu, John R. McPherson, George E. Allen, Cedric Chuan Young Ng, Bernice Huimin Wong, et al. 2012. Exome sequencing of liver fluke-associated cholangiocarcinoma. Nature Genetics 44(6): 690–693.

Zhang, Yu-Wen, Ben Staal, Karl J. Dykema, Kyle A. Furge, and George F. Vande Woude. 2012. Cancer-type regulation of MIG-6 expression by inhibitors of methylation and histone deacetylation. PLoS One 7(6): e38955.

24

Brian B. Haab, Ph.D. Laboratory of Cancer Immunodiagnostics Dr. Haab earned his Ph.D. in chemistry from the University of California, Berkeley in 1998, after which he was a postdoctoral fellow in the laboratory of Patrick Brown in the Department of Biochemistry at Stanford University.

Dr. Haab joined VARI in May 2000 and was promoted to Associate Professor in 2007.

From left, front row: Nelson, Partyka, Bartlam, Tang, Brouhard, Ma; back row: McDonald, Curnutte, Sinha, Haab, Cao, Westra

Staff

Students

Visiting Scientist

Betsy Brouhard, B.S. Zheng Cao, Ph.D. Bryan Curnutte, B.S. Amy Nelson Katie Partyka, B.S. Huiyuan Tang, Ph.D.

Heather Bartlam, B.S. Yinjiao Ma, M.S. Mitch McDonald Arkadeep Sinha, B.S. Hannah Westra

David Nowack, Ph.D.

25

Van Andel Research Institute |

Scientific Report

Research Interests The Haab laboratory studies pancreatic cancer, with the aims of identifying molecular factors that characterize and promote cancer progression and of using this information to more accurately diagnose and guide the treatment of pancreatic cancer.

Diagnostics for pancreatic cancers Modern medicine increasingly relies on detailed molecular information to make accurate diagnoses and treatment decisions.

A molecular-level understanding of healthy versus diseased human tissue promises to provide much more information about the patient than conventional clinical approaches. The development of improved tools for assessing pancreatic cancer is one of our main goals.

For certain patients, there are serious difficulties in distinguishing pancreatic cancer from benign conditions of the pancreas.

Some patients have abnormalities that are difficult to diagnose using imaging and biopsy procedures, and the diagnostic work-up process can be highly invasive, costly, and even after using all available methods, inconclusive. A blood test that could clearly resolve the differences between malignant and benign conditions of the pancreas would alleviate this situation.

We are working to develop such a blood test based on changes to the carbohydrates (glycans) that are abnormally produced in pancreatic cancers. These structures are attached to a variety of proteins, some of which are secreted and detectable in the blood. An FDA-approved test is available for the CA 19-9 antigen, the most common carbohydrate antigen made by pancreatic cancers, but that test has limited value because some 20% of cancers produce low amounts of CA 19-9. Our studies have shown that the cancers that do not produce much CA 19-9 instead overproduce other structures, and we propose that assays to detect the alternate structures plus the CA 19-9 antigen will accurately identify a higher percentage of cancer patients. We are working with our clinical collaborators at the University of Pittsburgh, the University of Michigan, and in Grand Rapids to test this strategy.

Another diagnostic problem is found with patients who have fluid-filled openings, known as pancreatic cysts, within their pancreas. Some cysts are unlikely to ever develop into cancer, while others may progress rapidly to cancer. Current diagnostic methods can not clearly differentiate these types. We are working with our collaborators to analyze the proteins and carbohydrates in fluid collected from the cysts, which could result in tests to determine which patients should have those cysts removed.

We also are applying these approaches to related problems in pancreatic cancer, such as determining which patients should have surgery as opposed to chemotherapy only, and monitoring how well a patient is responding to treatment. A future goal is to use our new markers to detect incipient disease among people at a high risk for developing pancreatic cancer, such as those with predisposing genetic characteristics.

Glycans in pancreatic ductal adenocarcinoma The goals described above will be advanced by further characterizing the changes in glycans as cancer cells develop and by understanding the cellular processes that result in those changes. We are using novel tools (described below) as well as powerful mass-spectrometry methods to compare the carbohydrates between tumors that produce CA 19-9 and those that do not. In addition, we are controlling the production of CA 19-9 in cultured cells or in mouse hosts to identify what carbohydrate structures are produced when CA 19-9 production is reduced. That control is based on manipulating specific genes involved in the production of CA 19-9. Our aim is to determine which genes are most important in producing the glycan structures.

26

VARI | 2013

Genetics and phenotypes of cancer cell subsets Not all cancer cells within a tumor are equivalent. The more advanced and aggressive cells are proposed to be primarily responsible for the migration and spread of cancer (metastasis) and for resistance to chemotherapeutics. An improved understanding of the molecular characteristics and origins of these subtypes could help to specifically eliminate them.

We have approached this problem by comparing the molecular characteristics of pancreatic cancer cells that appear mesenchymal (migratory) to those that appear epithelial (stationary), and we have identified several consistent differences. One difference is the overexpression of the cell surface protein MRC2 in mesenchymal-like cancer cells. MRC2 has a primary function of helping cells to recognize and degrade the extracellular matrix that surrounds them. We now are investigating whether MRC2 is specifically up-regulated in pancreatic cancer cells that are transitioning to a mesenchymal state.

Another difference is in the particular genetic alterations characteristic of mesenchymal-like cancer cells. We are determining which of those alterations are most prevalent in primary tumors and which contribute to the behavioral changes of the cancer cells. We plan to build on these studies to improve methods for assessing and treating pancreatic cancer.

New tools for studying specific carbohydrate structures We are developing novel methods for studying carbohydrates in human tissue samples. In particular, we are developing new molecular reagents that bind specific carbohydrate structures and so can be used to detect and measure them. Such reagents are unavailable for many carbohydrates that may be overexpressed in cancer tissue. We are using new bioinformatics methods developed by us and collaborators that allow us to search publicly available information on naturally occurring proteins that have carbohydrate-binding properties. Once we identify potentially useful reagents, we test them with our antibody and protein array technologies, optimize them, and then evaluate them in the analysis of carbohydrates in clinical specimens. These tools have value for our pancreatic cancer studies and the potential for broader scientific use in various glycobiology studies.

Recent Publications Haab, B. 2012. Using lectins in biomarker research: addressing the limitations of sensitivity and availability. Proteomics Clinical Applications 6(7-8): 346–350.

Partyka, Katie, Kevin A. Maupin, Randall E. Brand, and Brian B. Haab. 2012. Diverse monoclonal antibodies against the CA 19-9 antigen show variation in binding specificity with consequences for clinical interpretation. Proteomics 12(13): 2212–2220.

Partyka, Katie, Mitchell McDonald, Kevin A. Maupin, Randall Brand, Richard Kwon, Diane M. Simeone, Peter Allen, and Brian B. Haab. 2012. Comparison of surgical and endoscopic sample collection for pancreatic cyst fluid biomarker identification.

Journal of Proteome Research 11(5): 2904–2911.

Maupin, Kevin A., Daniel Liden, and Brian B. Haab. 2011. The fine specificity of mannose-binding and galactose-binding lectins revealed using outlier motif analysis of glycan array data. Glycobiology 22(1): 160–169.

Yue, Tingting, Kevin A. Maupin, Brian Fallon, Lin Li, Katie Partyka, Michelle A. Anderson, Dean E. Brenner, Karen Kaul, Herbert Zeh, A. James Moser, et al. 2011. Enhanced discrimination of malignant from benign pancreatic disease by measuring the CA 19-9 antigen on specific protein carriers. PLoS One 6(12): e29180.

27

Galen H. Hostetter, M.D. Laboratory of Analytical Pathology Dr. Hostetter received his M.D. degree from the University of Pennsylvania in 1993, and he is board-certified in pathology. He has completed medical and cancer genetics fellowships at the National Institutes of Health. His primary research interest has been applications of genomic assays and validation in clinical samples using tissue microarrays. He was staff pathologist at the Translational Genomics Research Institute (TGen) from 2003 to 2011. Dr. Hostetter joined VARI in 2011 as an Assistant Professor and head of the Laboratory of Analytical Pathology within the Program for Biospecimen Science (PBS).

Staff

Students

Bree Berghuis, B.S., HTL(ASCP), QIHC Eric Hudson, B.S. Lisa Turner, B.S., ST(ASCP), QIHC

Eric Edewaard Peter Varlan

28

VARI | 2013

Research Interests As head of the Laboratory of Analytical Pathology, Dr. Hostetter provides histology and pathology review for a wide range of tissue-based studies performed in VARI laboratories. Services provided include high-quality histology, diagnostic tissue review, morphometric analysis, immunohistochemistry, in situ hybridization, tissue microarrays, digital imaging and analysis by light, and spectral and confocal microscopy. Arcturus integrated laser-capture microdissection, isolation of nucleic acids and proteins from cells and tissue, and whole-cell antibody-specific isolation/purification are also provided. Zeiss and Nikon confocal microscopes are used. The Zeiss 510 multi-photon scope is equipped with both a Ti-sapphire pulse laser and a Meta-detector, which enables investigators to view simultaneously as many as eight fluorophores at the cellular and molecular level. The Nikon A1 confocal system provides static and live-cell imaging. The lab also has a CRi Nuance spectral imaging system to enable researchers to quantify chromic-dyed histological preparations.

Dr. Hostetter’s research addresses the effects of preanalytic variables in the collection and transport of biosamples. Ongoing research includes the development and validation of novel liquid-based collection media with a focus on macroanalyte yield and integrity. This research contributes to the emerging field of biospecimen science and will determine the extent of experimental biases related to macroanalyte integrity, an ever-constant challenge in both the research laboratory and the clinical laboratory.

Interactions with various core facilities and services include macroanalyte (DNA, RNA, protein) extractions suitable for downstream assays, with a focus on optimized and standardized protocols. Dr. Hostetter works closely with the excellent histotechnical staff within the PBS to provide top-quality, accurate, and interpretable results for use in clinical applications.

For example, an immunohistochemical assay on a tissue section detects expression of a candidate protein identified in the research laboratory; the result is validated with an automated immunostainer that mimics the workflow in the hospital pathology department, thereby translating research findings into potential clinical care practices. Additionally, tissue microarrays can be used to determine the prevalence of a given expressed protein in specific tumor types, and standardized measures of staining intensity can be determined using high-resolution digital image scanners and semi-quantitative algorithms. Interactive collaborative efforts with clinical partners of VARI provide continuing opportunities and challenges that focus on improving patient care.

Recent Publications Demeure, Michael J., Elizabeth Stephen, Shripad Sinari, David Mount, Steven Gately, Paul Gonzales, Galen Hostetter, Richard Komorowski, Jeff Kiefer, Clive S. Grant, et al. 2012. Preclinical investigation of nanoparticle albumin-bound paclitaxel as a potential treatment for adrenocortical cancer. Annals of Surgery 255(1): 140–146.

Stephens, Bret, Stephen P. Anthony, Haiyong Han, Jeffrey Kiefer, Galen Hostetter, Michael Barrett, and Daniel D. Von Hoff.

2012. Molecular characterization of a patient’s small cell carcinoma of the ovary of the hypercalcemic type. Journal of Cancer 3: 58–66.

Weiss, Glen J., Winnie S. Liang, Tyler Izatt, Shilpi Arora, Irene Cherni, Robert N. Raju, Galen Hostetter, Ahmet Kurdoglu, Alexis Christoforides, Shripad Sinari, et al. 2012. Paired tumor and normal whole genome sequencing of metastatic olfactory neuroblastoma. PLoS One 7(5): e37029.

Whitsett, Timothy G., Emily Cheng, Landon Inge, Kaushal Asrani, Nathan M. Jameson, Galen Hostetter, Glen J. Weiss, Christopher B. Kingsley, Joseph C. Loftus, Ross Bremner, et al. 2012. Elevated expression of Fn14 in non-small cell lung cancer correlates with activated EGFR and promotes tumor cell migration and invasion. American Journal of Pathology 181(1): 111–120.

29

Scott D. Jewell, Ph.D. Program for Biospecimen Science Dr. Jewell received his M.S. and Ph.D. degrees in experimental pathology and immunology from The Ohio State University. He has more than 25 years of experience in biorepository and biospecimen services and pathology laboratory services. Dr. Jewell previously served as director for the Human Tissue Resource Network and associate director of the OSU Comprehensive Cancer Center’s Biorepository and Biospecimen Resource. He joined VARI in 2010 as a Professor and Director of Program for Biospecimen Science.

Front row, from left: Khoo, Wiesner, Hilsabeck, Berghuis, Turner, Noyes Back rows, from left: Christensen, Blanski, Koeman, Webster, Feenstra, Hudson, Hostetter, Beck, Filkins, Rohrer, Harbach, Watkins, Jewell

Staff John Beck, B.S. Bree Berghuis, B.S., HTL(ASCP), QIHC Alexander Blanski, B.S. Carrie Christensen, B.S. Kristin Feenstra, B.S. Dana Filkins, B.A., CAPM Phil Harbach, M.S. Renee Hilsabeck, B.S. Eric Hudson, B.S. 30

Sok Kean Khoo, Ph.D. Julie Koeman, B.S., CG(ASCP) Dan Maxim, B.S. Sabrina Noyes, B.S. Dan Rohrer, B.S., M.B.A. Lisa Turner, B.S., HT(ASCP), QIHC Anthony Watkins Timothy Webster, B.A. Cathy Wiesner, M.S.

Students

Adjunct Faculty

Eric Edewaard Mary Goyings Adriane Shorkey Katie Uhl Peter Varlan

Sandra Cottingham, M.D., Ph.D. James Resau, Ph.D.

VARI | 2013

Research Interests Biospecimen science uses evidence-based approaches to study the effects of collection, processing, and storage on the biological parameters of biospecimens in an effort to establish best practices for the collection and control of high-quality human biospecimens for research. In our Program for Biospecimen Science (PBS), two broad categories of interest are the pre- and post-analytical variables that can alter in vivo biological assessments. Model systems are used for the study of the variables that can arise in biospecimen management, and we are working to establish in vitro and in vivo tissue models that can be used to answer specific questions.

Laboratory of Analytical Pathology The Laboratory of Analytical Pathology, directed by Galen Hostetter, M.D., provides histology and morphometric analysis using immunohistochemistry, in situ hybridization, tissue microarray technology, digital imaging and analysis by light and spectral microscopy, confocal microscopy, and diagnostic tissue evaluation. The lab can visualize cells and their components with striking clarity, and the images enable researchers to determine where in a cell particular molecules are located and to quantify the molecules through imaging analysis software. See p. 28 for a complete description.

Laboratory of Microarray Technology The Laboratory of Microarray Technology is directed by Sok Kean Khoo, Ph.D. It provides gene expression arrays, miRNA arrays, and array CGH using the Agilent microarray platform and cDNA platform capabilities. Microarray technology plays an important part in the discovery of genetic signatures, copy number variations, and biomarkers. Genomic DNA or total RNA from a wide range of tissues, including blood and fresh or frozen tissues, can be analyzed. Agilent microarrays in array formats from 4 x 44,000 to 1 x 1 million are used, and whole-genome gene expression (GE) arrays, exon arrays, miRNA arrays, and array CGH are available. Human, mouse, rat, and canine arrays are most frequently processed, but the lab offers GE and custom arrays for more than 20 plant and animal model organisms. Recently the lab has successfully developed a microarray gene expression technique for RNA from newborn blood spots. This technique can detect thousands of gene signatures using low-resolution arrays, enabling clinical research into the origins, epidemiology, and diagnosis of pediatric diseases.

Cytogenetics Core facility Julie Koeman, CG (ASCP), directs the Cytogenetics Core facility, which uses both cytogenetic and molecular genetic techniques to identify structural and numerical chromosomal aberrations associated with mammalian disease. Information about the loss or gain of a gene or about gene amplification can be generated from many sample types, and that information can be used to validate microarray data. Cytogenetic techniques can also be used for species identification, which is especially valuable when working with tumor xenograft models. The cytogenetic services include fluorescence in situ hybridization (FISH), custom FISH probe production, spectral karyotyping (SKY), transgene localization, routine karyotyping (G-banding), chromosomal breakage studies, and mouse embryonic stem cell trisomy 8 screening.

Biorepository services Dan Rohrer directs the operations of the biorepository, including database tracking and management of biospecimen inventory; biospecimen kit development and manufacturing; shipping and tracking services; procurement of surgical tissue and biospecimens from patient populations; quality control assessment of operations for the collection and banking of biospecimens; and biospecimen project management. The VARI biorepository contains approximately 2,000 frozen human tissues and a paraffin block archive of human diagnostic tissues currently exceeding 800,000 blocks. Tissue acquisition is in collaboration with West Michigan hospitals, providing fresh-frozen and paraffin-embedded surgical tissues and blood from consenting patients. The biorepository is designed to provide human tumors to investigators with IRB-approved basic and translational research projects.

31

Van Andel Research Institute |

Scientific Report

Comprehensive biospecimen resource for the NCI Cancer Human Biobank The Cancer Human Biobank (caHUB) includes biospecimen source sites, a comprehensive biospecimen resource, a pathology resource center, and a comprehensive data resource to implement the collection and management of high-quality biospecimens for NCI and NIH projects such as the Genotype-Tissue Expression (GTEx) program. VARI’s Program for Biospecimen Science was awarded funding as the comprehensive biospecimen resource for the caHUB. Using a stringent quality management program and project-specific standard operating procedures, we produce biospecimen kits for the collection and management of human tissues and pathology services for caHUB projects. In 2011 and 2012, our Program was awarded major contracts to support the caHUB projects.

Biospecimen resource for the Multiple Myeloma Research Foundation CoMMpass study The Multiple Myeloma Research Foundation launched a genomics study, CoMMpassSM, in collaboration with the Translational Genomics Research Institute (TGen), our Program for Biospecimen Science, and Spectrum Health Medical Center. The primary aim of CoMMpass is to collect biospecimens from 1,000 multiple myeloma patients for genomic analysis to assess changes associated with major clinical events, treatment response, and disease progression. This data will fuel therapeutic target discovery, drug development, and biomarker validation. Biospecimen kits are designed by the VARI PBS for the collection of bone marrow aspirate and peripheral blood. The kits, which are tracked from design through shipment and use, maintain biospecimens at 2–8 °C during shipment to Spectrum, where they are characterized by flow cytometry and BRAF sequencing in a clinical diagnostic laboratory. The PBS isolates CD138+ tumor cells and nucleic acids from patient samples for molecular sequencing and analysis at TGen. CoMMpass biospecimen management includes kit design, distribution, tracking, processing, and biobanking. Since July 2011, 200 patient cases have been processed, of which 28 have completed full genomic analysis.

In 2011 the PBS was awarded an eight-year contract for this project.

Recent Publications Jewell, Scott D. 2012. Perspective on biorepository return of results and incidental findings. Minnesota Journal of Law, Science and Technology 13(2): 655–667.

Resau, James H., Nhan T. Ho, Karl Dykema, Matthew S. Faber, Julia V. Busik, Radoslav Z. Nickolov, Kyle A. Furge, Nigel Paneth, Scott Jewell, and Sok Kean Khoo. 2012. Evaluation of sex-specific gene expression in archived dried blood spots (DBS). International Journal of Molecular Sciences 13(8): 9599–9608.

Glaser, Ronald, Rebecca Andridge, Eric V. Yang, Arwa Y. Shana’ah, Michael Di Gregorio, Min Chen, Sheri L. Johnson, Lawrence A. De Renne, David R. Lambert, Scott D. Jewell, et al. 2011. Tumor site immune markers associated with risk for subsequent basal cell carcinomas. PLoS One 6(9): e25160.

Moore, Helen M., Andrea Kelly, Scott D. Jewell, Lisa M. McShane, Douglas P. Clark, Renata Greenspan, Pierre Hainaut, Daniel F. Hayes, Paula Kim, Elizabeth Mansfield, et al. 2011. Biospecimen reporting for improved study quality. Biopreservation and Biobanking 9(1): 57–70.

32

Figure 1

Figure 2

Figure 3

Differentiated prostate epithelial cells.

Figure 1 shows basal cells (the lowest layer of cells) stained for integrin a6; the red stain is largely on the periphery of the cells. Figure 2 shows secretory cells (the upper layer), which have differentiated from the basal cells. The green stain in the secretory cells, which have lost integrin expression, is for the ING4 molecule in the nucleus. Figure 3 shows a composite image of both stains, plus DAPI stain (blue) for DNA. Images by Penny Berger and Elly Park of the Miranti lab.

33

Xiaohong Li, Ph.D. Laboratory for Tumor Microenvironment and Metastasis Dr. Li received her Ph.D. from the Chinese Academy of Sciences in Beijing in 2000, and she moved to Vanderbilt University in the same year. Dr. Li was a postdoctoral fellow in the laboratory of David Ong until 2005 and in the laboratory of Neil Bhowmick from 2005 to 2009. She was promoted to research assistant professor in the Department of Urologic Surgery in 2009.

Dr. Li joined VARI as an Assistant Professor in September 2012.

Research Interests The laboratory is committed to understanding cancer and metastasis. We study not only the cancer cells, but also the contributions of the tumor microenvironment, aiming to develop early diagnostic and treatment strategies for breast and prostate cancer metastasis to bone. Our research focuses on the role of stromal transforming growth factor (TGF-b) in the microenvironment of primary and metastatic tumor sites, as well as its effects in bone metastases, and on the development of animal models of cancer-induced osteolytic and osteoblastic bone disease.

We have recently been funded by the Department of Defense Prostate Cancer Research Program to study the influence of the primary microenvironment on the development of prostate cancer osteoblastic bone lesions. The objectives are to determine the contribution of prostate mesenchymal TGF-b to lesion development and to determine whether chemokines induced by the loss of TGF-b signaling mediate prostate cancer bone metastasis. Other developing projects include the creation of animal models for studying prostate osteoblastic bone metastases and mechanisms; study of the role of TGF-b on the development of breast cancer-induced osteolytic bone lesions; and the evaluation of anti-TGF-b combination therapies on cancer-induced bone disease.

Staff Priscilla Lee, B.S. Diana Lewis, A.S. Jared Murdoch, B.S.

34

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, followed by a postdoctoral fellowship with John Blenis 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, who joined VARI in 2006, is an Associate Professor.

From left: MacKeigan, Niemi, Burgenske, Martin, Westrate, Doppel, Lanning, Goodall, Looyenga, Fogg, May, Nelson, Karnes, Kauffman

Staff

Students

Visiting Scientist

Nicole Doppel, B.S. Vanessa Fogg, Ph.D. Audra Kauffman, M.S. Nate Lanning, Ph.D. Brendan Looyenga, Ph.D. Katie Martin, Ph.D. Brett May, B.S. Amy Nelson

Dani Burgenske, B.S. Megan Goodall, B.S. Matt Harder Jonathan Karnes, M.S. Natalie Niemi, Ph.D. Anna Plantinga Aaron Sayfie Laura Westrate, B.A., B.S.

Aaron Putzke, Ph.D.

35

Van Andel Research Institute |

Scientific Report

Research Interests “Systems biology” integrates multiple disciplines such as biochemistry, mathematics, and genetics to investigate unanswered 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. The lab has two major research programs: cancer metabolism and the PI3K-mTOR autophagy signaling network. We employ 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 Parkinson’s disease; develop therapeutics for high-priority targets; and reposition drugs for use against cancer and neurodegenerative diseases.

Cancer metabolism and cellular energetics Evasion of apoptosis is a significant problem in a variety of cancers. In order to identify novel regulators of apoptosis, the lab performed an RNAi screen against all kinases and phosphatases in the human genome. A possible regulator we identified was MK-STYX (encoded by the STYXL1 gene), a catalytically inactive phosphatase with homology to the MAPK phosphatases.

Despite this homology, MK-STYX knockdown failed to modulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX prevented cells from undergoing apoptosis induced by cellular stressors, activating mitochondrial-dependent apoptosis. This MK-STYX phenotype mimicked the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential: cells without MK-STYX expression were unable to release cytochrome c.

The overexpression of pro-apoptotic Bcl-2 proteins was unable to trigger cytochrome c release in MK-STYX knockdown cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP).

MK-STYX localizes to the mitochondria, but it is neither released from the mitochondria upon apoptotic stress nor localized proximal to the machinery currently known to control MOMP. Thus, MK-STYX regulates the chemoresistance potential of cancer cells through the control of MOMP, but in distinct fashion from currently characterized mechanisms. Additionally, we have determined that MK-STYX interacts with a mitochondrial phosphatase, PTPMT1. The loss of PTPMT1 in MK-STYX knockdown cells resensitizes the cells to chemotherapy and cytochrome c release, demonstrating a genetic interaction between these two proteins. Ongoing studies are focused on characterizing this MK-STYX–PTPMT1 interaction and on gaining insight into the metabolic and apoptotic capacity of cancer cells.

A wealth of experimental evidence clearly connects the regulation of cellular metabolism with the development of cancer.

Metabolic changes in cancer cells are considered a key event in the transition from a normal cell to a cancer cell. Such changes cause cancer cells to be metabolically reprogrammed to provide the fuel and energy required for rapid proliferation. To identify genes crucial for 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. We identified several genes that drive cancer cell bioenergetics and upon which cancer cells rely 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 status significantly correlated with patient survival. Current experiments seek to answer questions about the cellular interactions involving these target genes and how these interactions affect the metabolic programs of normal and cancer cells.

36

VARI | 2013

PI3K-mTOR and the autophagy signaling network Autophagy is a cellular recycling program essential for homeostasis and survival during cytotoxic stress. When cancer cells encounter environmental stressors such as nutrient starvation or chemotherapy, autophagy is dramatically up-regulated, resulting in cellular adaptation to the stress and subsequent survival. The autophagy process, which has an emerging role in disease etiology and treatment, is executed in four stages through the coordinated action of more than 30 proteins. An effective strategy for studying this complicated process involves the construction and analysis of computational models. When developed and refined from experimental knowledge, these models can be used to interrogate signaling pathways, formulate novel hypotheses about systems, and make predictions about cell signaling changes induced by specific interventions.

In conjunction with collaborators at Los Alamos National Laboratory, we developed a computational model describing autophagic vesicle dynamics in a mammalian system. We used time-resolved live-cell microscopy to measure the synthesis and turnover of autophagic vesicles in single cells. The stochastically simulated model was consistent with data acquired during conditions of both basal and chemically induced autophagy. The model was tested by genetic modulation of the autophagic machinery and it accurately predicted the vesicle dynamics observed experimentally. Furthermore, the model generated an unforeseen prediction about vesicle size that is consistent with both published findings and our experimental observations. Thus, we have developed an accurate and useful model that can serve as the foundation for future efforts to quantitatively characterize autophagy. Ongoing efforts include building and refining a computational model of autophagy that will make reliable predictions about complex cancer cell behavior; verifying the predictions in cellular and preclinical models; and ultimately using the model to develop effective strategies for therapeutically targeting autophagy in cancer.

Recent Publications Martin, Katie R., Dipak Barua, Audra L. Kauffman, Laura M. Westrate, Richard G. Posner, William S. Hlavacek, and Jeffrey P. MacKeigan. 2013. Computational model for autophagic vesicle dynamics in single cells. Autophagy 9(1): 74–92.

Niemi, Natalie M., Nathan J. Lanning, Laura M. Westrate, and Jeffrey P. MacKeigan. 2013. Downregulation of the mitochondrial phosphatase PTPMT1 is sufficient to promote cancer cell death. PLoS One 8(1): e53803.

Klionsky, Daniel J., Fabio C. Abdalla, Hagai Abeliovich, Robert T. Abraham, Abraham Acevedo-Arozena, Khosrow Adeli, Lotta Agholme, Maria Aganello, Patrizia Agostinis, Julio A. Aguirre-Ghiso, et al. 2012. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8(4): 445–544.

Looyenga, Brendan D., Danielle Hutchings, Irene Cherni, Chris Kingsley, Glen J. Weiss, and Jeffrey P. MacKeigan.

2012. STAT3 is activated by JAK2 independent of key oncogenic driver mutations in non-small cell lung carcinoma.

PLoS One 7(2): e30820.

Looyenga, Brendan D., and Jeffrey P. MacKeigan. 2012. Characterization of differential protein tethering at the plasma membrane in response to epidermal growth factor signaling. Journal of Proteome Research 11(6): 3101–3111.

Stark, Mitchell S., Susan L. Woods, Michael G. Gartside, Vanessa F. Bonazzi, Ken Dutton-Regester, Lauren G. Aoude, Donald Chow, Chris Sereduk, Natalie M. Niemi, Nanyun Tang, et al. 2012. Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing. Nature Genetics 44(2): 165–169.

37

Karsten Melcher, Ph.D. Laboratory of Structural Biology and Biochemistry Dr. Melcher earned his master’s in biology and his Ph.D. 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 has been 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.

From left: deWaal, Zhou, Li, Wang, Melcher, Kovach, Merrill, Weber

Staff

Students

Amanda Kovach, B.S. Stephanie Weber, B.S. Xiaoyin (Edward) Zhou, Ph.D.

Parker deWaal Xiaodan Li, B.S. Nate Merrill, B.S. Lili Wang, B.S.

38

VARI | 2013

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窶電rug 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 diabetes, cancer, and neurological disorders.

Two areas of focus in the lab are the adenosine-monophosphate (AMP)-activated protein kinase (AMPK), a cellular energy and nutrient sensor, and the receptors and key signaling proteins for a plant hormone, abscisic acid (ABA).

AMP-activated protein kinase 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 a sensor of the energy status 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 programs.

To adjust energy balance, AMPK regulates 窶｢ 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) 窶｢ Whole-body energy balance (appetite regulation in the hypothalamus via leptin, adiponectin, ghrelin, and cannabinoids) 窶｢ Many nonmetabolic processes (cell growth and proliferation, mitochondrial homeostasis, autophagy, aging, neuronal activity, and cell polarity).

Due to its central roles in the uptake and metabolism of glucose and fatty acids, AMPK is an important pharmacological target for the treatment of 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.

39

Van Andel Research Institute |

Scientific Report

Abscisic acid Abscisic acid is an ancient signaling molecule that is 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. We have determined the structure of ABA receptors in the free state and bound to ABA. Using computational receptor docking 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.

Recent Publications Pal, Kuntal, Karsten Melcher, and H. Eric Xu. 2012. Structure and mechanism for recognition of peptide hormones by Class B G-protein-coupled receptors. Acta Pharmacologica Sinica 33(3): 300–311.

Soon, Fen-Fen, Ley-Moy Ng, X. Edward Zhou, Graham M. West, Amanda Kovach, M. H. Eileen Tan, Kelly M. Suino-Powell, Yuanzheng He, Yong Xu, Michael J. Chalmers, et al. 2012. Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases. Science 335(6064): 85–88.

Soon, Fen-Fen, Kelly M. Suino-Powell, Jun Li, Eu-Leong Yong, H. Eric Xu, and Karsten Melcher. 2012. Abscisic acid signaling: thermal stability shift assays as tool to analyze hormone perception and signal transduction. PLoS One 7(10): e47857.

Zhou, X. Edward, Karsten Melcher, and H. Eric Xu. 2012. Structure and activation of rhodopsin. Acta Pharmacologica Sinica 33(3): 291–299.

Zhou, X. Edward, Fen-Fen Soon, Ley-Moy Ng, Amanda Kovach, Kelly M. Suino-Powell, Jun Li, Eu-Leong Yong, Jian-Kang Zhu, H. Eric Xu, and Karsten Melcher. 2012. Catalytic mechanism and kinase interactions of ABA-signaling PP2C phosphatases.

Plant Signaling & Behavior 7(5): 581–588.

Ng, Ley-Moy, Fen-Fen Soon, X. Edward Zhou, Graham M. West, Amanda Kovach, Kelly M. Suino-Powell, Michael J. Chalmers, Jun Li, Eu-Leong Yong, Jian-Kang Zhu, et al. 2011. Structural basis for basal activity and autoactivation of abscisic acid (ABA) signaling SnRK2 kinases. Proceedings of the National Academy of Sciences U.S.A. 108(52): 21259–21264.

Zhi, Xiaoyong, X. Edward Zhou, Karsten Melcher, Daniel L. Motola, Verena Gelmedin, John Hawdon, Steven A. Kliewer, David J. Mangelsdorf, and H. Eric Xu. 2011. Structural conservation of ligand binding reveals a bile acid–like signaling pathway in nematodes. Journal of Biological Chemistry 287(7): 4894–4903.

40

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 Dr. 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: Frank, Cooper, Zarif, Berger, Nollett, Hildebrandt, Schulz, Miranti, Park

Staff

Students

Teacher Intern

Penny Berger, B.S. Elly Park, Ph.D. Veronique Schulz, B.S.

Alexis Bergsma, B.S. Jason Cooper, B.S. Amanda Erwin Sander Frank, B.A. Erin Hildebrandt, B.S. Eric Nollet, B.S. Jelani Zarif, M.S.

Erin Combs, M.S.

41

Van Andel Research Institute |

Scientific Report

Research Interests Our objective is to understand how cell adhesion and the tumor microenvironment promote prostate cancer progression and metastasis. Our work focuses on three major questions: 1) how do the androgen receptor (AR) and integrin interactions with the tumor microenvironment cooperate to promote prostate cancer bone metastasis? 2) how do oncogenes disrupt integrin signaling and prostate epithelial differentiation to promote tumorigenesis? and 3) how does the metastasis suppressor gene CD82/KAI1 regulate the tumor microenvironment to suppress prostate cancer metastasis? Our strategy is to develop cell- and animal-based models that accurately reflect the in vivo biology of human prostate cancer as observed in the clinic and use them to develop therapeutic strategies for prostate cancer.

The AR/a6b1 integrin axis The human prostate gland contains basal cells which express and use integrins to adhere to laminin matrix. Basal cells do not express AR, but they differentiate into AR-expressing secretory cells that detach from matrix and lose integrin expression. In prostate cancer, the AR-expressing tumor cells retain abnormal expression of integrin a6b1. We hypothesize that abnormal cross-talk between AR and integrin a6b1 is crucial for prostate cancer development and progression to castration-resistant disease.

We found that AR binds directly to the integrin a6 promoter to stimulate its transcription, while simultaneously decreasing the expression of other integrins. Control of integrin a6 expression by AR requires the fusion gene, TMPRSS2-Erg, suggesting cross-talk between AR and Erg. We discovered that AR stimulation of a6b1 expression activates a laminin-dependent survival pathway involving NF-kB/RelA activation and subsequent increased transcription of Bcl-xL. To understand the mechanisms that promote the survival of castration-resistant cancer, we screened tumors cells for NF-kB target genes whose expression depends on AR and integrin a6b1. We identified and validated BNIP3 as such a gene.

BNIP3 expression is higher in castration-resistant cells and correlates with disease progression and poor patient outcome.

Furthermore, loss of BNIP3 induces cell death. BNIP3 promotes mitochondrial-specific degradation through autophagy, and we hypothesize that BNIP3 promotes the emergence and survival of castration-resistant tumors by enhancing such mitophagy.

The loss of Pten, which leads to enhanced PI3K signaling, occurs in 60% of advanced prostate cancers; however, PI3K inhibitors are not effective in patients. When plated on laminin to engage integrin a6b1, tumor cells were resistant to PI3K inhibition. Blocking PI3K in combination with blocking AR, integrin a6b1, RelA, or Bcl-xL resensitized the cells to such inhibition.

Thus, interactions with the tumor microenvironment through AR/a6b1 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. As is seen in vivo, the secretory cells are marked by their loss of integrin expression and loss of adhesion to matrix. In fact, the competency to activate AR requires the loss of matrix adhesion. Differentiation is accompanied by a dramatic increase in E-cadherin expression and increased cell-cell adhesion. At the same time, there is a switch in the basal cells from dependence on integrins and MAPK for survival, to E-cadherin and PI3K in the secretory cells.

42

VARI | 2013

Based on our observations that differentiation begins prior to complete loss of integrin a6b1 and that Myc controls integrin a6b1 transcription in epithelial cells, we hypothesize that prostate oncogenesis occurs within a subpopulation of transiently differentiating cells in which AR is partially stabilized but the cells still retain a6b1. Using normal cells engineered to overexpress two known prostate oncogenes, Myc and TMPRSS2/Erg, and an shRNA to Pten, we generated tumorigenic cells that coexpress integrin a6b1 and AR. Surprisingly, these oncogene-modified cells were unable to differentiate. Thus, we developed an in vitro model for studying prostate tumorigenesis that recapitulates many of its in vivo aspects and links prostate cancer to defects in differentiation.

CD82/KAI1 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. Our hypothesis is that CD82 suppresses metastasis by limiting signal transduction pathways that promote integrin-based migration and invasion while simultaneously increasing cell-cell adhesion.

CD82 suppresses both integrin- and ligand-based activation of the tyrosine kinases Met and Src; it also suppresses their ability to stimulate prostate tumor cell migration and invasion in vitro, as well as metastasis in vivo. Other tetraspanins, CD9 and CD151, are required for CD82-dependent suppression of Met. CD82 expression also increases E-cadherin-based cell-cell adhesion. Several CD82 mutants were generated to decipher how CD82 suppresses Met-dependent metastasis and promotes cell-cell adhesion.

The reexpression of CD82 in metastatic tumor cells is sufficient to suppress metastasis. However, using a conditional null CD82 mutant mouse, we found that loss of CD82 alone in a mouse primary prostate tumor model was not sufficient to induce metastasis. To address the possibility that loss of other genes is also needed, we are crossing floxed CD82 mice with mice that are null for another metastasis suppressor gene, RKIP. RKIP regulates miRNAs that are involved in controlling Myc and MAPK signaling, pathways that are not influenced by CD82.

We generated CD82-null mice to better understand the normal function of CD82. The most striking phenotype is enhanced platelet clotting and reduced bleeding, as well as a twofold increase in total platelets. The increase in platelets is due to changes in megakaryocyte differentiation, which is controlled by tyrosine kinase and cytokine signaling and is tightly linked to the cytoskeleton. CD82-null mice also have increased bone density, defects in toll receptor signaling, and reduced capacity to stimulate T-cell signaling. Thus, the in vivo data support our in vitro work, suggesting the major function of CD82 is to regulate cell signaling, and further suggesting CD82 regulates cell differentiation.

Recent Publications Nollet, Eric A., and Cindy K. Miranti. In press. Integrin and matrix regulation of autophagy and mitophagy. In Autophagy, Yannick Bailly, ed. New York: InTech.

Klionsky, Daniel J., Fabio C. Abdalla, Hagai Abeliovich, Robert T. Abraham, Abraham Acevedo-Arozena, Khosrow Adeli, Lotta Agholme, Maria Aganello, et al. 2012. Guidelines for the use and interpretation of assays for monitoring autophagy.

Autophagy 8(4): 445–544.

Lamb, Laura E., Jelani C. Zarif, and Cindy K. Miranti. 2011. The androgen receptor induces integrin a6b1 to promote prostate tumor cell survival via NF-kB and Bcl-xL independently of PI3K signaling. Cancer Research 71(7): 2739–2749.

43

Mark W. Neff, Ph.D. Laboratory of Canine Genetics and Genomics Dr. Neff received his Ph.D. in biological sciences from the University of Virginia and completed a postdoctoral fellowship in canine genetics and genomics at the University of California, Berkeley. Most recently, he served as associate director of the Veterinary Genetics Laboratory at the University of California, Davis. Dr. Neff joined VARI in 2009 as an Associate Professor and Director of the Program for Canine Health and Performance.

From left: Minard, Neff, Hodges, Kefene, Borgman, Roemer

Staff

Students

Lisa Kefene, B.S. Michelle Minard

Andrew Borgman, B.S. Jenea Chesnic Daniel Hodges, M.A. Alex Roemer

44

VARI | 2013

Research Interests I apply a classical genetic perspective and genomic discovery platforms to a unique animal model. Dogs suffer the same inherited disorders as humans, including cancers and neurodegenerative diseases. In veterinary medicine, canine disorders are detected with human diagnostics and treated with human medicines, so it stands to reason that naturally occurring diseases in the dog are models of human disease counterparts. Genetic analysis can identify, in an unbiased way and owing to the strengths of breed isolates, the causal mechanisms underlying complex disease susceptibilities. DNA risk signatures enable predictive genetic epidemiology with corresponding clinical benefits—early intervention and prevention—and they inform on aberrant biological processes. Clinical trials can be performed more rapidly, more powerfully, and more economically in veterinary medicine owing to lesser regulatory constraints and accelerated patient time frames. Pet dogs also serve as a model for lifestyle management (e.g., exercise, appetite, and behavioral modification), creating the opportunity to offset inherited risks. The dog is arguably the best patient model for evidence-based, personalized, and preventive medicine. Over the years, our group has developed the subject recruitment, genomic, and statistical analysis pipelines needed for advancing robust, informative, and efficient experiments in canine genetics research. Just as importantly, we have developed strong relationships with the dog owner and breeder community, without which research in this field would not be possible.

Our lab studies naturally occurring diseases in the dog. We apply the perspective of genetics and the tools of genomics to tie complex phenotypes to causal genotypes. We exploit the strengths of breeds as genetic isolates to identify identicalby-descent mutations from within large ancestral haplotype blocks. These mutations can then be functionally characterized in model organisms or cell culture. Our collaborations over the past two years have included projects on osteosarcoma, hemangiosarcoma, essential head tremor, obsessive-compulsive disorder, agoraphobic-like behavior, cervical spondylopathy, adult onset hearing loss, and idiopathic pulmonary fibrosis.

Recent Publications Wong, A.K., A.L. Ruhe, K.R. Robertson, E.R. Loew, D.C. Williams, and M.W. Neff. In press. A de novo mutation in KIT causes white spotting in a subpopulation of German Shepherd dogs. Animal Genetics.

Neff, Mark W., John S. Beck, Julie M. Koeman, Elissa Boguslawski, Lisa Kefene, Andrew Borgman, and Alison L. Ruhe. 2012.

Partial deletion of the sulfate transporter SLC13A1 is associated with an osteochondrodysplasia in the miniature poodle breed.

PLoS One 7(12): e51917.

Wong, Aaron K., Alison L. Ruhe, Shameek Biswas, Kathryn R. Robertson, Ammar Ali, Joshua M. Akey, and Mark W. Neff. 2012.

Marker panels for genealogy-based mapping, breed demographics, and inference-of-ancestry in the dog. Animal Biotechnology 23(4): 241–252.

Yokoyama, Jennifer S., Ernest T. Lam, Alison L. Ruhe, Carolyn A. Erdmann, Kathryn R. Robertson, Aubrey A. Webb, D. Colette Williams, Melanie L. Chang, Marjo K. Hytönen, Hannes Lohi, et al. 2012. Variations in genes related to cochlear biology is strongly associated with adult-onset deafness in Border collies. PLoS Genetics 8(9): e1002898.

45

Brian J. Nickoloff, M.D., Ph.D. Laboratory of Cutaneous Oncology Dr. Nickoloff received his M.D. and Ph.D. (biochemistry) from Wayne State University, and he completed an internship in Internal Medicine at Harbor General – UCLA Hospital. He is the former director of the Skin Disease Research Laboratory at Loyola University Chicago Medical Center. In 2003, he became the director of Loyola’s Oncology Institute and deputy director of the Cardinal Bernardin Cancer Center. In 2011, he relocated to Grand Rapids to become Professor and Division Director of Dermatology at the College of Human Medicine, Michigan State University. He also holds an appointment as Professor and head of the Laboratory of Cutaneous Oncology at the Van Andel Research Institute. Most recently he became the Medical Director of Dermatopathology at St. Mary’s Hospital in the Skin Cancer Clinic.

Research Interests Our primary interest is in finding new and better methods for diagnosing melanoma using genomics and treatment options in the setting of personalized medicine. Current efforts focus on overcoming treatment resistance and relapse in melanoma patients treated with targeted therapy. We are using human metastatic melanoma xenografts in immunodeficient mice.

We have established a vemurafenib-resistant model system and also combination therapies to overcome this resistance.

Another project is exploring the altered metabolomics in melanoma, using PET/CT imaging to develop novel approaches for targeting BRAF mutant and wild-type tumors.

Recent Publications Monsma, David J., Noel R. Monks, David M. Cherba, Dawna Dylewski, Emily Eugster, Jahn Hailey, Sujata Srikanth, Stephanie B. Scott, Patrick J. Richardson, Robin E. Everts, et al. 2012. Genomic characterization of explant tumorgraft models derived from fresh patient tumor tissue. Journal of Translational Medicine 10: 125.

Nickoloff, Brian J., and George Vande Woude. 2012. Hepatocyte growth factor in the neighborhood reverses resistance to BRAF inhibitor in melanoma. Pigment Cell & Melanoma Research 25(6): 758–761.

Qin, Jianzhong, Hong Xin, and Brian J. Nickoloff. 2012. Specifically targeting ERK1 or ERK2 kills melanoma cells. Journal of Translational Medicine 10: 15.

46

Giselle S. Sholler, M.D. Laboratory of Neuroblastoma Translational Research Dr. Sholler received her M.S. in microbiology and immunology from McGill University, Montreal, Quebec, and her M.D. from New York Medical College. She worked in the Division of Pediatric Hematology/Oncology at the University of Vermont before joining VAI in 2011 as Associate Professor and Co-Director of the Pediatric Oncology Program. Dr. Sholler has a joint appointment with the Helen DeVos Children’s Hospital as the Haworth Family Director of the Innovative Therapeutics Clinic in the Division of Pediatric Oncology.

From left: Sholler, Ellis, Dutta, Vander Werff, McClung, Bender, Eckardt, Kendzicky, Zhao

Staff Mary Bender, RN Genevieve Bergendahl, RN, B.S.N. Akshita Dutta, M.S. Alexandra Eckardt, B.S. Ellen Ellis

Ann Kendzicky, B.S. Heather McClung, Ph.D. Alyssa Vander Werff, M.S. Ping Zhao, Ph.D.

47

Van Andel Research Institute |

Scientific Report

Research Interests Our laboratory is committed to pushing forward cures for childhood cancers by identifying and exploiting new therapies for neuroblastoma and medulloblastoma, which continue to be therapeutic challenges in pediatrics. Our research aims at understanding the specific biological and genomic profiles of patients and using the information from patient-derived xenograft models and laboratory studies to identify and deliver new therapies to, and improve outcomes for, children with relapsed disease. Through the Neuroblastoma and Medulloblastoma Translational Research Consortium, which Dr. Sholler chairs, clinical trials are being conducted, for example, on molecular guided therapy for refractory/recurrent neuroblastoma and on a-diflouromethylornithine (DFMO) for patients with high-risk neuroblastoma in remission.

Recent Publications Eslin, Don, Umesh T. Sankpal, Chris Lee, Robert M. Sutphin, Pius Maliakal, Erika Currier, Giselle Sholler, Moeez Khan, and Riyaz Basha. In press. Tolfenamic acid inhibits neuroblastoma cell proliferation and induces apoptosis: a novel therapeutic agent for neuroblastoma. Molecular Carcinogenesis.

Sholler, Giselle L. Saulnier, William Ferguson, Genevieve Bergendahl, Erika Currier, Shannon R. Lenox, Jeffrey Bond, Marni Slavik, William Roberts, Deanna Mitchell, Don Eslin, et al. In press. A pilot trial testing the feasibility of using molecularguided therapy in patients with recurrent neuroblastoma. Journal of Cancer Therapy.

Sun, Yujing, Girja Shukla, Stephanie C. Pero, Erika Currier, Giselle Sholler, and David Krag. 2012. Single tumor imaging with multiple antibodies targeting different antigens. BioTechniques Rapid Dispatches, doi 10.2144/000113855.

48

Matthew Steensma, M.D. Laboratory of Musculoskeletal Oncology Dr. Steensma received his BA from Hope College and his M.D. from Wayne State University School of Medicine in Detroit. He was admitted into the fellowship program in musculoskeletal surgical oncology at Memorial Sloan-Kettering Cancer Center in New York, obtaining subspecialty training in surgical management of musculoskeletal tumors. Upon completion of this training, Dr. Steensma worked in the laboratory of Dr. Steve Goldring, one of the world’s leading orthopaedic researchers. There Dr. Steensma further developed his interest in understanding the molecular and cellular mechanisms underlying bone and soft-tissue sarcomas. Dr. Steensma is a practicing physician, treating patients in his musculoskeletal oncology clinic, and he joined VARI in 2010 as an Assistant Professor.

From left: Steensma, Scholten, Kampfshulte, Ringler, Peacock, Pelle

Staff

Students

Visiting Scientist

Diana Lewis, A.S. Jacqueline Peacock, Ph.D. Jonathan Ringler, M.S.

Kevin Kampfshulte, B.A. D.J. Scholten, B.A.

Dominic Pelle, M.D.

49

Van Andel Research Institute |

Scientific Report

Research Interests Our laboratory is particularly interested in defining the mechanisms of tumor initiation and disease progression for a rare type of cancer called sarcoma. In doing so, we seek to identify novel diagnostic and therapeutic targets for the disease. The lab centers its efforts around two disease entities: the primary bone cancer, called osteosarcoma, and Type 1 neurofibromatosis (NF1), also called Von Recklinghausen’s disease.

Osteosarcoma affects predominantly children and young adults; it arises directly from bone and is highly aggressive. Advances in treatment have been slow over the last four decades, particularly with respect to metastatic osteosarcoma, which is largely incurable. Our lab is studying mechanisms whereby certain cells within the primary tumor resist chemotherapy, spread to a distant site, and reinitiate tumor formation (i.e., the process of metastasis). This subpopulation resembles mesenchymal stem cells in that they are capable of continuous self-renewal and multipotent differentiation. As a group, these cells are often referred to as tumor-initiating cells. The role of the microenvironment in the formation of these cells within the primary tumor and metastatic lesions is poorly understood. We are examining the effect of up-regulated hypoxia-inducible factor (HIF) signaling on tumor-initiating cell formation to determine whether HIF antagonists are useful adjuncts in preventing latent recurrence of osteosarcoma. We are also conducting genomic profiling studies of osteosarcomas to identify novel biomarkers and drug targets. This work is in collaboration with Drs. Craig Webb and Giselle Scholler. By comparing gene expression and mutational profiles of tumor-initiating cells with those of the bulk tumor, we aim to identify novel therapeutic targets specific to the most treatment-resistant cell populations.

NF1 is an inherited disease that predisposes the affected individuals to both benign and malignant tumors. The lifetime incidence of sarcoma development in NF1 is about 10%, which is nearly 10,000-fold higher than for non-affected individuals. NF1-related sarcomas are highly aggressive and do not respond well to chemotherapy. Individuals with NF1 carry a mutation in one of two copies of the gene encoding neurofibromin (NF1), which results in deregulated RAS signaling. Loss of the second copy of NF1 is necessary for cancer to develop, but other factors have also been shown to be important for malignant transformation.

Specifically, the lab is examining how HGF/MET signal activation drives both neurofibroma and neurofibrosarcoma development in the context of NF1. This work is being accomplished using novel, genetically engineered mouse models. Through a collaboration with Craig Webb, we are also applying a systems biology approach for analyzing clinical samples in anticipation of an NF1 personalized medicine trial.

Recent Publications Steensma, Matthew, and John H. Healey. In press. Trends in the surgical treatment of pathologic proximal femur fractures among Musculoskeletal Tumor Society members. Clinical Orthopaedics and Related Research.

Steensma, M.R., and C. Morris. In press. Ewing’s sarcoma. In Orthopaedic Knowledge Update, S. Biermann, ed. Rosemont, IL: American Academy of Orthopaedic Surgeons.

Valkenburg, Kenneth C., Matthew R. Steensma, Bart O. Williams, and Zhendong Zhong. In press. Skeletal metastasis: treatments, mouse models, and Wnt signaling. Chinese Journal of Cancer.

Zhong, Zhendong, Bart O. Williams, and Matthew R. Steensma. 2012. The activation of b-catenin by Gas contributes to the etiology of phenotypes seen in fibrous dysplasia and McCune-Albright syndrome. IBMS BoneKEy 9: 113.

50

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 with 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 as the founding President and Dean of the Van Andel Institute Graduate School and as a researcher in VARI. He succeeded Dr. Gordon Van Harn as the Director of the Van Andel Education Institute in January 2009.

From left: Akuli, Testori, Triezenberg, Klomp, Alberts, Thellman, Pikaart

Staff

Students

Visiting Scientist

Amy Akuli Glen Alberts, B.S. Jennifer Klomp, M.S. Marian Testori, B.S.

Jamie Grit Nikki Thellman, D.V.M.

Michael Pikaart, Ph.D.

51

Van Andel Research Institute |

Scientific Report

Research Interests Our research is focused on the mechanisms that control whether genes are turned on or turned off inside cells. The genetic information encoded in DNA must first be copied, in the form of RNA, before it can be translated into the proteins that do most of the work in a cell. Some genes must be expressed more or less constantly throughout the life of any eukaryotic cell, while others must be turned on (or turned off) in particular cells either at specific times or in response to a specific signal or event. 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 known as 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. After the initial infection resolves, HSV-1 finds its way into nerve cells, where the virus can hide in a latent mode for long times—essentially for the lifetime of the host organism. Occasionally, some trigger event (such as emotional stress, 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 those viruses back to the skin to cause a recurrence of the cold sore.

The DNA genome of HSV-1 encodes approximately 80 different proteins. However, the virus does not have its own machinery for expressing those genes; instead, the virus must divert the gene expression machinery of the host cell. That process is triggered by a viral regulatory protein designated VP16, whose function is to stimulate transcription of the first viral genes to be expressed in the infected cell (the immediate-early, or IE, genes).

Chromatin-modifying coactivators in herpes virus infections: a paradox leads to a hypothesis and yields an unexpected answer 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 space of a cell, eukaryotic DNA is typically packaged as chromatin, in which the DNA is wrapped around “spools” of histone proteins, and these spools are then further arranged into higher-order structures. This elaborate packaging creates a problem when access is needed to the information carried in the DNA, such as when particular genes need to be expressed. This problem is solved in part by chromatin-modifying coactivator proteins, which either chemically change the histone proteins or else slide or remove them.

Transcriptional activator proteins such as VP16 can recruit these chromatin-modifying coactivator proteins to target genes.

We have shown this to be true for the viral genes that VP16 activates during an active infection. Curiously, however, the DNA of herpes simplex virus is not wrapped in histones inside the viral particle, and it also seems to stay relatively free of histones inside the infected cell. That observation leads to a paradox: why would VP16 recruit chromatin-modifying coactivators to the viral DNA, if the viral DNA doesn’t have much chromatin to modify?

We took several approaches to test whether the coactivators recruited to viral DNA by the VP16 activation domain really play a significant role in transcriptional activation. In some experiments, we knocked down expression of given coactivators using short interfering RNAs (siRNAs). In other experiments, we used cell lines that have mutations disrupting the expression or activity of a given coactivator. We expected to find that viral gene expression was inhibited, but the experiments yielded unexpected results: in each case, expression of the viral genes was essentially unaffected. We were forced to conclude that our initial hypothesis was wrong; the coactivators, although present, are not required for viral gene expression during lytic infection.

52

VARI | 2013

The death of one hypothesis, however, gives life to new ideas. After the initial infection of a cold sore subsides, herpes simplex virus establishes a life-long latent infection in sensory neurons. In the latent state, the viral genome is essentially quiet; very few viral genes are expressed. Moreover, the viral genome becomes packaged in chromatin much like the silent genes of the host cell. So our new hypothesis is that the coactivators recruited by VP16 are required to reactivate the viral genes from the latent or quiescent state. We now have evidence that VP16 is likely the very first viral gene to be expressed during the reactivation process. We want to test whether the ability of VP16 to recruit coactivators is essential for subsequent events of reactivation. We will test this hypothesis both in quiescent infections in cultured cells and in animal models with genuinely latent herpesvirus infections.

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 its amino acids and on its interactions with other proteins. 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 might affect the protein in different ways, including how the protein folds, how it interacts with other proteins, and how stable it remains in the cell.

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 modifications matter for how VP16 functions, either as a transcriptional activator protein or as a structural protein of the HSV-1 virion. In some experiments, we create 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 that apply the modifications (for phosphorylation, these enzymes are known as protein kinases). We expect that this work will lead to new ideas about ways that we can selectively inhibit modification of VP16 using small-molecule drugs, and thereby prevent or shorten the infection process by HSV.

Recent Publications Danaher, Robert J., Ross K. Cook, Chunmei Wang, Steven J. Triezenberg, Robert J. Jacob, and Craig S. Miller. In press.

C-terminal 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.

Silva, Lindsey, Hyung Suk Oh, Lynne Chang, Zhipeng Yan, Steven J. Triezenberg, and David M. Knipe. 2012. Roles of the nuclear lamina in stable nuclear association and assembly of a herpesviral transactivator complex on viral immediate-early genes. mBio 3(1): e00300–11.

Sawtell, Nancy M., Steven J. Triezenberg, and Richard L. Thompson. 2011. VP16 serine 375 is a critical determinant of herpes simplex virus exit from latency in vivo. Journal of Neurovirology 17(6): 546–551.

53

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 to complete a Ph.D. in medical genetics in 2005. Subsequently, he became a postdoctoral fellow in the Department of Biology at McGill University until joining the Van Andel Research Institute as an Assistant Professor in 2012.

Staff Kim Cousineau, B.S. Keith Dufendach, B.S. Megan Senchuk, Ph.D. 54

VARI | 2013

Research Interests As the average human life span continues to rise, the likelihood of an individual developing a neurodegenerative disease also increases. Thus, there is an increasing 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. Research in this laboratory is focused on gaining insight into the aging process and the pathogenesis of such diseases. In addition to the obvious benefit to the individual, this work has the potential to be a great benefit to society by decreasing health care costs and helping to maintain productivity and independence to a later age.

Oxidative stress and longevity The widely accepted 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. Recent work in the worm Caenorhabditis elegans has indicated that the relationship between ROS and life span is more complex than anticipated. Decreasing the antioxidant defense through the deletion of individual, or combinations of, superoxide dismutase (SOD) genes does not decrease life span. This is contrary to expectations, because SOD is an enzyme that decreases the levels of ROS. In fact, quintuplemutant 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 the fact that either deletion of the mitochondrial superoxide dismutase gene sod-2 or treatment of wild-type worms with the superoxide generator paraquat results in increased life span. The fact that sod quintuple-mutant worms exhibit a normal life span despite markedly increased sensitivity to oxidative stress suggests a balance between superoxide-mediated 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: how increased levels of superoxide trigger the signal, how the signal is transmitted, and what changes that 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 neurodegenerative disease Advancing age is the greatest risk factor for the development of neurodegenerative disease. In the familial forms of these diseases, the mutation that causes the disease is present from birth, and yet the symptoms do not appear for several decades.

This suggests that changes during normal aging may make cells more susceptible to the disease-causing mutations. This is supported by the fact that the onset of these disorders in animal models is proportional to the life span of the organism, indicating disease progression according to biological age and not chronological time. In fact, multiple changes are known to occur during normal aging that likely reduce the ability of cells to protect themselves against the effects of toxic diseasecausing proteins. In support of this concept, interventions that are known to extend life span, such as caloric restriction, have shown benefit in both worm and mouse models of Huntington’s disease. Thus, by gaining insight into the aging process and examining its role in the pathogenesis of neurodegenerative disease, it may be possible to develop treatments for these devastating disorders.

55

Van Andel Research Institute |

Scientific Report

Huntington’s disease (HD) is an adult-onset neurodegenerative disorder characterized by motor dysfunction, cognitive deficits, and neuropsychiatric abnormalities. Disease onset typically occurs between the ages of 35 and 55 and progresses inevitably to death approximately 15 years later. The disease is caused by a trinucleotide CAG repeat expansion in the HD gene, which codes for the protein huntingtin (HTT). The CAG repeat sequence is translated into a polyglutamine tract in the HTT protein, and thus HD belongs to a group of at least nine polyglutamine toxicity disorders. Interestingly, while the size of the CAG repeat is polymorphic in unaffected individuals (ranging from 9 to 35 repeats), the disease range begins at precisely 35 CAG repeats, and the severity of the disease is correlated with the length of the repeat.

Both worms and mouse models of HD have been created through transgenic expression of varying lengths of the huntingtin protein with a disease-length polyglutamine tract. The worm models express the mutant polyglutamine sequence either in body wall muscle or in neurons. These worms exhibit numerous abnormal phenotypes—including decreased life span, slow development, and decreased mobility—that are not observed in worms expressing a normal length repeat. Mouse models of HD have been shown to recapitulate almost all features of human HD, including motor deficits, cognitive deficits, and selective neurodegeneration.

Our project examines 1) whether genes that increase life span will be beneficial in worm models of HD (i.e., will the increased longevity imparted by the aging gene reduce the severity of the polyglutamine toxicity phenotypes?), and 2) whether specific changes that take place during normal aging and that have been implicated in neurodegenerative disease contribute to pathogenesis in worm models of HD (i.e., do the higher levels of oxidative stress in older individuals contribute to pathogenesis?).

Both of these objectives are being studied using two complementary approaches: genetic crosses to generate double mutants, and specific knockdown of gene expression using RNAi. The results from the worm screen will be used to prioritize the genes that will be studied in mouse models, which provide more physiologically accurate models of HD. Similar experiments are being conducted in animal models of Parkinson’s disease. By comparing the results, it will be possible to identify both overlapping and disease-specific mechanisms in these two neurodegenerative disorders.

56

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 Academy of Arts and Sciences (2006).

From left: Xie, Graveel, Su, Gao, Kang, Essenburg, Vande Woude, Linklater, Yerrum, Staal, Johnson, Zhang, Kaufman

Staff Curt Essenburg, B.S. Chongfeng Gao, Ph.D. Carrie Graveel, Ph.D. Jennifer Johnson, M.S. Liang Kang, B.S. Dafna Kaufman, M.S.

Eric Linklater, B.S. Ben Staal, M.S. Yanli Su, A.M.A.T. Qian Xie, M.D., Ph.D. Smitha Yerrum, M.S. Yu-Wen Zhang, M.D., Ph.D

Student

Adjunct Faculty

Caroline Muhoro

Brian Cao, M.D.

57

Van Andel Research Institute |

Scientific Report

Research Interests Targeting the MET pathway in glioblastoma Glioblastoma multiforme (GBM) is one of the most devastating cancers. Its hallmark is the invasiveness of the tumor cells infiltrating into normal brain parenchyma, making it virtually impossible to remove the tumor completely by surgery and inevitably leading to recurrent disease. Progress in understanding GBM pathobiology and in developing novel antitumor therapies could be greatly accelerated with animal model systems that display characteristics similar to human GBM and that enable noninvasive tumor imaging in real time. We have established GBM patient-derived xenograft models that preserve tumor genotypes and phenotypes during in vivo passage, and we have isolated stem cell–like cancer populations for preclinical testing of drugs to block tumor growth and invasion. High-throughput, real-time, non-invasive imaging using bioluminescence (BLI) technology can detect orthotopic brain tumor growth before and after treatment. These studies have led to the conclusion that GBM with HGF-autocrine activation acts as if it were MET addicted and displays very high sensitivity to MET inhibitors. A combination of MET inhibitor and the EGFR inhibitor erlotinib showed better anti-tumor efficacy than either drug alone. We are planning further in vivo drug combination studies to try to develop drug strategies that will be more effective in treating MET expression in MET paracrine tumor systems.

The role of MET in aggressive breast cancers Understanding the signaling pathways that drive aggressive breast cancers is crucial to the development of effective therapeutics. High expression of the oncogene MET is associated with decreased survival in breast cancer, yet the role it plays in the various breast cancer subtypes is unclear. We are investigating the role of MET in breast cancer progression and metastasis.

Using a mouse model and analyses of human tissues, we have found that high MET expression correlates with estrogen receptor-negative/ERBB2-negative tumors and with basal breast cancers. We believe that MET is a key in the development of aggressive breast cancer subtypes and may be a significant therapeutic target. Currently, we are investigating how MET signaling interacts with the ERBB family of receptors in the progression and therapeutic resistance of ERBB2-positive and triple-negative breast cancers.

MET as a therapeutic target in human cancers Aberrant activation of the HGF-MET signaling pathway is found in many human cancers, and it promotes cell proliferation, invasion and metastasis. Targeting this pathway is a promising approach to cancer intervention. We are using our unique human-HGF transgenic SCID mice to explore how effective such targeting may be in treating human cancers such as non-small cell lung cancer both in vitro and in vivo. Various MET drugs have been developed, and we are interested in identifying parallel pathways that cross-talk with MET or that are crucial in driving cancer cell resistance to MET drugs. We are also studying the benefits of combination treatments using MET inhibitors together with agents such as EGFR inhibitors.

The role of Mig-6 in cancer and joint disease Mig6 is a tumor suppressor gene that functions as a negative feedback regulator in receptor tyrosine kinase signaling, either by direct binding to EGFR/ERBB family receptors or by interactions with signaling molecules downstream of the RTKs. Mig-6 plays an important role in stress responses and tissue homeostasis, and its disruption in mice results in the development of neoplasia and degenerative joint disease. We have shown that Mig6 can be epigenetically silenced and differentially regulated in lung cancer and melanoma cells. Currently, we are investigating the roles and mechanisms of Mig-6 in cancer development and in the maintenance of joint homeostasis.

58

VARI | 2013

Tumor phenotypic switching: mechanism and therapeutic implications In human carcinomas, acquisition of an invasive phenotype requires a breakdown of intercellular junctions with neighboring cells, a process termed the epithelial-to-mesenchymal transition (E-MT). Paradoxically, metastatic carcinomas often exhibit an epithelial phenotype, leading to the hypothesis that E-MT is a transient process induced by microenvironmental factors.

Upon arriving at secondary sites, the mesenchymal cells revert to an epithelial phenotype (mesenchymal-to-epithelial transition; M-ET). Typically, human carcinoma tissues and cells exhibit 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 isolate phenotypic variants from epithelial or mesenchymal subclones of carcinoma cell lines, as well as to identify subclones that switch phenotypically. 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.

Recent Publications Gherardi, Ermanno, Walter Birchmeier, Carmen Birchmeier, and George Vande Woude. 2012. Targeting MET in cancer: rationale and progress. Nature Reviews Cancer 12(2): 89–103.

Kentsis, Alex, Casie Reed, Kim L. Rice, Takaomi Sanda, Scott J. Rodig, Eleni Tholouli, Amanda Christie, Peter J.M. Valk, Ruud Delwel, Vu Ngo, et al. 2012. Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia.

Nature Medicine 18(7): 1118–1122.

Nickoloff, Brian J., and George Vande Woude. 2012. Hepatocyte growth factor in the neighborhood reverses resistance to BRAF inhibitor in melanoma. Pigment Cell & Melanoma Research 25(6): 758–761.

Xie, Qian, George F. Vande Woude, and Michael E. Berens. 2012. RTK inhibition: looking for the right pathways toward a miracle. Future Oncology 8(11): 1397–1400.

Zhang, Yu-Wen, Ben Staal, Karl J. Dykema, Kyle A. Furge, and George F. Vande Woude. 2012. Cancer-type regulation of MIG-6 expression by inhibitors of methylation and histone deacetylation. PLoS One 7(6): e38955.

Xie, Qian, Robert Bradley, Liang Kang, Julie Koeman, Maria Libera Ascierto, Andrea Worschech, Valeria De Giorgi, Ena Want, Lisa Kefene, Yanli Su, et al. 2011. Hepatocyte growth factor (HGF) autocrine activation predicts sensitivity to MET inhibition in glioblastoma. Proceedings of the National Academy of Sciences U.S.A. 109(2): 570–575.

Xie, Qian, Robert Wondergem, Yuehai Shen, Greg Cavey, Jiyuan Ke, Ryan Thompson, Robert Bradley, Jennifer DaughertyHoltrop, Yong Xu, Edwin Chen, et al. 2011. Benzoquinone ansamycin 17AAG binds to mitochondrial voltage-dependent anion channel and inhibits cell invasion. Proceedings of the National Academy of Sciences U.S.A. 108(10): 4105–4110.

59

Craig P. Webb, Ph.D. Laboratory for Translational Medicine Dr. Webb received his Ph.D. in cell biology from the University of East Anglia, England, in 1995. From 1995 to 1999, he was a postdoctoral fellow with George Vande Woude at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland. Dr. Webb joined VARI in October 1999 and was promoted to Professor in 2008. He is also coDirector of the Pediatric Cancer Translational Research Program.

From left: Webb, Moon, Popkie, Davidson, Eugster, Dylewski, Orey, Monsma, Scott, Montroy, Monks, Cherba, Mooney

Staff David Cherba, Ph.D. Paula Davidson, M.S. Dawna Dylewski, B.S. Emily Eugster, M.S. Noel Monks, Ph.D. 60

Students David Monsma, Ph.D. Rob Montroy, B.E. Lori Moon, M.B.A. Anthony Popkie, Ph.D. Stephanie Scott, B.S.

Marie Mooney, M.S. Stephen Orey, B.S.

Visiting Scientists

Adjunct Faculty

Jessica Foley, M.D. Eric Kort, M.D. Debra Weist, Ph.D.

Eric Lester, M.D. Laurence McCahill, M.D.

VARI | 2013

Research Interests The Laboratory of Translational Medicine (LTM) is a multidisciplinary group with both basic and applied research components.

Our basic research is focused on deciphering the molecular basis of solid tumor metastasis, with particular emphasis on the role of the putative cancer stem cell and tumor-host interactions during the early establishment and subsequent progression of metastases in critical organs such as the liver and lung. We focus on pancreatic cancer, triple-negative breast cancer, melanoma, adult and pediatric brain tumors, and pediatric osteosarcoma. Our applied research efforts have resulted from our development of the translational research infrastructure needed to permit real-time, precision medicine (PMed) clinical trials for patients with metastatic and/or refractory disease. Through our expertise and resources in bioinformatics, genomics, preclinical models, clinical trial design, and regulatory affairs, the lab is currently supporting prospective PMed trials in pediatric and adult human patients, as well as in canines with advanced-stage tumors. Given these collective capabilities, the laboratory is initiating collaborative efforts to repurpose existing drugs for specific patient populations.

Pancreatic cancer Pancreatic cancer (PCa) is the fourth leading cause of cancer-related mortality in the United States, with an estimated 37,000 deaths per year and a dismal 5-year survival of less than 6% that has not improved greatly over the past 30 years. As in other cancers, the development of secondary metastases within critical organs, notably the liver, accounts for the majority of PCa-related morbidity and mortality. Identifying the key determinants that drive the early establishment and progression of liver metastases is paramount to improving long-term outcomes for patients. Current efforts within the LTM include investigating the interaction between PCa cells and the host macrophages (Kupffer cells) and stellate cells within the micro-metastatic niche of the liver.

Metastatic melanoma Patients who develop metastatic melanoma (MM) have a poor prognosis, with a median survival of 6–9 months and a 3-year survival rate of 10–15%. The tumors of approximately 40% of MM patients harbor an activating mutation in the BRAF gene which confers sensitivity to B-Raf inhibitors such as the recently approved agent vemurafenib. Through the award of a StandUp-2-Cancer grant, we are enhancing the lab’s PMed bioinformatics framework to incorporate next-generation sequencing and phosphoproteomic technologies. The goal of this project is to identify, in real time, the key molecular drivers of B-Raf wild-type MM and align these findings to a series of experimental agents from biopharmaceutical companies; patients will be treated on the basis of these real-time findings.

In patients whose MM harbors an oncogenic BRAF mutation, the tumors initially show an impressive response to B-Raf inhibitors such as vemurafenib. However, the synchronous regrowth of tumors after a period of treatment is a common occurrence. To investigate the molecular mechanism of drug resistance, the lab has developed a large number of primary patient tumorgrafts for many solid tumors (including MM) that closely resemble the patient’s tumor at the molecular, histopathological, and treatment-response levels. These models preserve a number of key aspects of the tumor-host microenvironment. We are using these tumorgraft models to investigate the role that the innate immune systems play in the onset of drug resistance in MM and developing combination treatment strategies to treat vemurafenib-resistant MM.

61

Van Andel Research Institute |

Scientific Report

Triple-negative breast cancer The breast cancers referred to as triple negative (ER – , PR – , HER2 – ) represent a highly aggressive subtype for which no effective therapies exist. Thus, patients with triple-negative breast cancer (TNBrCa) have a poor prognosis. Within a heterogeneous tumor there resides a subpopulation of cells with stem cell–like properties known as cancer stem cells (CSCs). According to the CSC hypothesis, a hierarchical tumor organization exists in which deregulated, self-renewing CSCs drive tumorigenesis.

CSCs are believed to be the key malignant cell contributing to metastasis and drug resistance, and targeting these cells therefore represents an excellent therapeutic opportunity against multiple tumor types including TNBrCa. Through a Komen Promise grant, the lab is working to characterize the CSCs from TNBrCa patients of different ethnic backgrounds at the genetic, epigenetic, and genomic levels to identify candidate targets for therapy.

Adult and pediatric glioblastoma Glioblastoma (GBM) represents a group of highly aggressive and often recurrent brain tumors that affect both adults and children. Adult and pediatric GBM are largely indistinguishable by morphology or pathology, and their treatment regimens have been similar, with overall poor success. Some recent molecular characterization of GBMs from the two patient populations suggests that the molecular drivers of disease may be quite distinct, warranting different treatment considerations. Efforts in the lab include the identification of key signaling pathways in both adult and pediatric GBM and the evaluation of combinational treatment strategies for each.

Osteosarcoma Osteosarcoma (OSA) is the most common primary bone malignancy in children, with a high rate of local recurrence and metastasis to the lungs. We have recently initiated efforts to characterize the CSCs within pediatric OSA with the goal of identifying CSC-directed therapies. These efforts will soon be expanded to implement a prospective PMed clinical trial in pediatric patients with OSA. As the most common primary bone tumor in dogs, canine OSA is comparable to the human disease at many levels, including its high propensity to metastasize to the lungs. We are also assessing our PMed approach for canine OSA patients to determine the feasibility of genomically profiling the disease in real time to support therapy selection by veterinarians.

Recent Publications Sholler, Giselle L. Saulnier, William Ferguson, Genevieve Bergendahl, Erika Currier, Shannon R. Lenox, Jeffrey Bond, Marni Slavik, William Roberts, Deanna Mitchell, Don Eslin, et al. In press. A pilot trial testing the feasibility of using molecularguided therapy in patients with recurrent neuroblastoma. Journal of Cancer Therapy.

Mazzarella, Richard, and Craig P. Webb. 2012. Computational and bioinformatic strategies for drug repositioning. In Drug Repositioning: Bringing New Life to Shelved Assets and Existing Drugs, Michael J. Barratt and Donald E. Frail, eds. New York: Wiley and Sons, pp. 91–128.

Monsma, David J., Noel R. Monks, David M. Cherba, Dawna Dylewski, Emily Eugster, Hailey Jahn, Sujata Srikanth, Stephanie B. Scott, Patrick J. Richardson, Robin E. Everts, et al. 2012. Genomic characterization of explant tumorgraft models derived from fresh patient tumor tissue. Journal of Translational Medicine 10: 125.

Lee, Chih-Shia, Karl J. Dykema, Danielle M. Hawkins, David M. Cherba, Craig P. Webb, Kyle A. Furge, and Nicholas S. Duesbery.

2011. MEK2 is sufficient but not necessary for proliferation and anchorage-independent growth of SK-MEL-28 melanoma cells. PLoS One 6(2): e17165.

62

Michael Weinreich, Ph.D. Laboratory of Genome Integrity and Tumorigenesis Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsin–Madison, after which he was a postdoctoral fellow in the laboratory of Bruce Stillman, director of Cold Spring Harbor Laboratory, New York. Dr. Weinreich joined VARI in March 2000 and is currently an Associate Professor.

From left: Weinreich, Chang, Minard, Chen, Kenworthy, Tiwari

Staff

Students

FuJung Chang, M.S. Jessica Kenworthy, B.S. Michelle Minard Kanchan Tiwari, M.S.

Ying-Chou Chen, M.S. Nanda Kumar Sasi, B.S. Sandya Subramanian Raymond Yeow 63

Van Andel Research Institute |

Scientific Report

Research Interests The goal of our research is to understand how cells stably and accurately maintain their genetic information. Many diseases, including cancer, are caused by mutations in DNA, and it is now clear that the development of cancer requires multiple independent mutations. Early mutations often impair cellular surveillance mechanisms (checkpoints) that maintain genetic stability, and, in the absence of such checkpoints, additional mutations and genetic alterations become more frequent. This cumulative burden can ultimately lead to cancer as cells escape the normal growth and proliferation controls. Genetic instability also explains why cancer treatments often fail: tumors have such high mutation rates that they can readily develop resistance to chemotherapeutic drugs.

The two-subunit Dbf4-dependent kinase (DDK) that we study (also known as Cdc7-Dbf4 protein kinase) is critical for the accurate replication and segregation of chromosomes. DDK is required for the initiation of DNA replication at multiple 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 G1 phase. It is clear that DDK also affects replication fork stability and DNA repair processes during S phase, although the mechanisms for these activities are poorly understood. We recently reported that Dbf4 interacts with the yeast Polo-like kinase, Cdc5, to maintain the spindle position checkpoint. Polo kinases are master regulators of mitotic events. For example, Cdc5 promotes the loss of chromosome cohesion during metaphase, entry into anaphase, spindle elongation, exit from mitosis, and cytokinesis. Because of its essential role during mitosis, Cdc5 is the target of multiple checkpoint mechanisms to ensure the accurate segregation of chromosomes. We found that DDK 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 mis-segregation events and cell death.

The DNA damage and replication checkpoints are critical regulators of chromosome stability. The checkpoints facilitate repair of DNA damage, suppress late-origin firing, and also prevent premature entry into mitosis, which would be catastrophic with damaged or incompletely replicated chromosomes. The Rad53 protein kinase of yeast, the ortholog of the human tumor suppressor Chk2, is an essential regulator of these checkpoints and directly interacts with Dbf4. Rad53 phosphorylates Dbf4 to prevent the activation of late origins when replication forks stall, and our genetic data imply that Rad53 and DDK also cooperate in another (unknown) pathway that is essential for cell survival.

We have recently investigated the basis of the molecular interaction between Dbf4 and Rad53. Rad53 likely binds Dbf4 using multiple protein-protein contacts in the Dbf4 N-terminus. Interestingly, loss of the Rad53-Dbf4 regulation leads to activation of late-origin firing during periods of replication stress. It is unknown how Rad53 phosphorylation prevents late-origin activation, since we have shown that Rad53 phosphorylation does not disrupt the Dbf4-Cdc7 interaction and results in only a modest decrease in DDK activity. The Rad53 protein binds to the Dbf4 N-terminus but phosphorylates critical residues in the Dbf4 C-terminus to prevent late-origin activation.

In summary, work over the last several years has shown that Dbf4 acts as a molecular scaffold to bind three separate protein kinases: Cdc7, Cdc5, and Rad53 (Figure 1). Binding of Cdc7 occurs through essential middle and C-terminal Dbf4 residues.

Binding of Cdc5 and Rad53 occurs through Dbf4 N-terminal residues that have evolved a checkpoint effector role to mediate the response to DNA damage, replication fork stalling, and chromosome segregation defects. Many different types of tumors show increased levels of DDK, and inhibiting DDK causes the death of many types of tumor cells, but not normal cells. Because the ability of DDK to control multiple aspects of chromosome metabolism is likely conserved, it is crucial to understand these pathways in order to further the development of highly effective chemotherapeutic agents and interventions.

64

VARI | 2013

Figure 1

Figure 1: Dbf4 is a molecular scaffold for three protein kinases and controls genome integrity at multiple levels. Dbf4 binds Cdc7 kinase through C-terminal sequences to initiate DNA replication. Dbf4 binds Cdc5 (Polo kinase) and Rad53 (Chk2 kinase) through adjacent N-terminal sequences to control cell cycle progression in response to spindle and/or genomic stresses.

Recent Publications Chang, FuJung, Caitlin D. May, Timothy Hoggard, Jeremy Miller, Catherine A. Fox, and Michael Weinreich. 2011. Highresolution analysis of four efficient yeast replication origins reveals new insights into the ORC and putative MCM binding elements. Nucleic Acids Research 39(15): 6523–6535.

65

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 under the supervision of Tyler Jacks. He joined VARI in July 1999 and was promoted to Associate Professor in 2006. Prior to his recruitment, he was a postdoctoral fellow at the National Institutes of Health in the laboratory of Harold Varmus.

From left: Valkenburg, Maupin, Zahatnansky, Van Wieren, Williams, Burgers, Haider, Joiner, Diegel, Lewis, Droscha

Staff Travis Burgers, Ph.D. Cassie Diegel, B.S. Danese Joiner, Ph.D. Diana Lewis, A.S. 66

Emily Van Wieren, B.S. Juraj Zahatnansky, M.D. Alex Zhong, Ph.D.

Students

Adjunct Faculty

Casey Droscha, B.S. Rida Haider, M.S. Kevin Maupin, B.S. Ken Valkenberg, B.S.

Clifford Jones, M.D. Madhuri Kakarala, M.D., Ph.D. Charlotta Lindvall, M.D., Ph.D. Debra Sietsema, Ph.D., RN

VARI | 2013

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. Wnt proteins initiate several signaling pathways, including one that results in the activation of the b-catenin protein and its downstream signaling targets.

Given its central role in growth and differentiation, it is not surprising that alterations in the Wnt pathway are among the most common events associated with human cancer. In addition, other human diseases including osteoporosis, cardiovascular disease, neurodegenerative diseases, and diabetes have been linked to altered regulation of this pathway. Our main approach toward gaining insights into the mechanisms of Wnt signaling in development and disease is to create and characterize genetically engineered mouse models. We have pursued studies in three key areas outlined below. In addition, we are interested in understanding the molecular mechanisms by which specificity is generated by Wnts.

Wnt signaling in skeletal development and disease A specific focus of our work is characterizing the role of Wnt signaling in skeletal development and disease. Mutations in the Wnt receptor Lrp5 have been causally linked to alterations in human bone development. Several years ago, we characterized a mouse strain carrying a germline deletion in Lrp5 and found that it recapitulated the low-bone-density phenotype seen in human patients who have a LRP5 deficiency. We further found that mice carrying germline deletions in both Lrp5 and the related Lrp6 protein have even more-severe defects in bone density. We next created mice carrying an osteoblast-specific deletion of b-catenin. Those mice have severely diminished bone mass and elevated osteoclastogenesis associated with changes in the expression of RANKL and osteoprotegerin. Our next step was to create and evaluate mice carrying osteoblastspecific deletions of Lrp6 and Lrp5. We have found that mice carrying deletions in either gene alone have reduced bone mass, and mice lacking both genes in osteoblasts have more-severe phenotypes.

More recent studies have focused on gaining insight into the cell type(s) that secrete the Wnts necessary for normal bone development. Our strategy has used mice carrying osteoblast-specific deletions of the Wntless/Gpr177 (Wls) gene. Wls encodes a protein specifically required for secretion of all mammalian Wnts, and a mouse strain carrying a Wls allele that can be conditionally inactivated was developed by our collaborator, Richard Lang. We have generated mice carrying an osteoblast-specific deletion of this gene and found that mature osteoblasts are a crucial source of the Wnts required for normal skeletal development.

Current work is also focusing on evaluating the roles of Wnt signaling in osteoarthritis and fracture repair, as well examining how other signaling pathways integrate with Wnt/b-catenin signaling to control osteoblast differentiation and function. Two such examples are the effects of parafibromin on regulating transcriptional outputs through its interaction with b-catenin and the potential role of galectin-3 in this process.

67

Van Andel Research Institute |

Scientific Report

Wnt signaling in mammary development and cancer Activation of the Wnt signaling pathway has been linked to the development of some types of breast tumors. We are using genetically engineered mouse models to assess the roles of Wnt signaling in mammary development and carcinogenesis.

Mice carrying conditional deletions of Lrp5 and / or Lrp6 in mammary epithelial cells have been developed and are being characterized. We are evaluating the role that activation of Wnt signaling plays in establishing and maintaining tumor-initiating cells within the mammary gland. We are also examining the source of Wnts necessary for normal mammary development and for the maintenance of some types of breast tumors.

Wnt signaling in prostate development and cancer A hallmark of advanced prostate cancer is the development of skeletal osteoblastic metastases. The association of Wnt signaling with bone growth makes Wnt signaling an attractive candidate for explaining some phenotypes associated with advanced prostate cancer. As a first step to understanding the role of Wnt signaling in prostate carcinogenesis, we have generated mice carrying prostate-epithelial-specific deletion of Apc. We have found that mice carrying conditional deletions induced by either probasin-Cre or Nkx3.1-Cre develop prostate tumors having similar latency and pathology. Further, we are directly examining the role of Wnt signaling by assessing the effects of inhibiting the secretion of Wnts in models of skeletal metastases. We also have a specific interest in examining the role of Wnt5a in this process.

Recent Publications Joiner, Danese M., Jiyuan Ke, Zhendong Zhong, H. Eric Xu, and Bart O. Williams. 2013. LRP5 and LRP6 in development and disease. Trends in Endocrinology and Metabolism 24(1): 31–39.

Fortin, Shannon P., Matthew J. Ennis, Cassie A. Schumacher, Cassandra R. Zylstra-Diegel, Bart O. Williams, Julianna T.D. Ross, Jeffrey A. Winkles, Joseph C. Loftus, Marc H. Symons, and Nhan L. Tran. 2012. Cdc42 and the guanine nucleotide exchange factors Ect2 and Trio mediate Fn14-Rac1-induced migration and invasion of glioblastoma cells. Molecular Cancer Research 10(7): 958–968.

Ke, Jiyuan, Chenghai Zhang, Kaleeckal G. Harikumar, Cassandra R. Zylstra-Diegel, Liren Wang, Laura E. Mowry, Laurence J. Miller, Bart O. Williams, and H. Eric Xu. 2012. Modulation of b-catenin signaling by glucagon receptor activation. PLoS One 7(3): e33676.

Li, Yi, Andrea Ferris, Brian C. Lewis, Sandra Orsulic, Bart O. Williams, Eric C. Holland, and Stephen H. Hughes. 2012. The RCAS/TVA somatic gene transfer method in modeling human cancer. In Genetically Engineered Mice for Cancer Research, Jeffrey E. Green and Thomas Ried, eds. Berlin: Springer Verlag, pp. 83–112.

Zhong, Zhendong., and Bart O. Williams. 2012. Integration of cellular adhesion and Wnt signaling: interactions between N-cadherin and LRP5 and their role in regulating bone mass. Journal of Bone and Mineral Research 27(9): 1849–1851.

Zhong, Zhendong, Bart O. Williams, and Matthew R. Steensma. 2012. The activation of b-catenin by Gas contributes to the etiology of phenotypes seen in fibrous dysplasia and McCune-Albright syndrome. IBMS BoneKEy 9: 113.

Zhong, Zhendong, Cassandra R. Zylstra-Diegel, Cassie A. Schumacher, Jacob J. Baker, April C. Carpenter, Sujata Rao, Wei Yao, Min Guan, Jill A. Helms, Nancy E. Lane, et al. 2012. Wntless functions in mature osteoblasts to regulate bone mass.

Proceedings of the National Academy of Sciences U.S.A. 109(33): E2197–E2204.

68

Improving pancreatic cancer markers.

These plots show two sets of results from a test of new markers to aid in the accurate diagnosis of pancreatic cancer. The current best marker (data set M1) is the total amount of a glycan called CA 19-9. We are testing a combination panel of the CA 19-9 assay with two additional markers (data sets M2 and M3) of CA 19-9 bound to specific proteins. Each column represents a patient sample, and a yellow square indicates a higher level of a marker than normal. If any of the panel markers are elevated in a given sample, the sample is classified as cancer, indicated by a yellow square in the bottom (classification) row. The three-marker panel has more true positives (TP) and fewer false negatives (FN) than the CA 19-9 assay alone, while maintaining low false-positive (FP) and high true-negative (TN) results. Plots provided by the Haab laboratory.

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 GlaxoWellcome 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. Dr. Xu is also the Primary Investigator and Distinguished Director of the VARI/SIMM Research Center in Shanghai, China.

From left: Zhi, Gao, Ke, Cheng, Sridharamurthy, Lili Wang, Weber, Xu, Kang, He, Pal, Li, Liren Wang, Hou, deWaal

Staff Xiang Gao, Ph.D. Yuanzheng (Ajian) He, Ph.D. Yanyong Kang, Ph.D. Jiyuan Ke, Ph.D. 70

Students Kuntal Pal, Ph.D. Kelly Powell, B.S. Stephanie Weber, B.S. Xiaoyong Zhi, Ph.D.

Hao Cheng, B.S. Parker deWaal Li Hou, M.S. Xiaodan Li, B.S.

Madhuri Sridharamurthy, B.S. Lili Wang, B.S. Liren Wang, B.S. Zhongshan Wu, B.S.

VARI | 2013

Research Interests Hormone signaling is essential to eukaryotic life. Our research is focused on the signaling mechanisms of physiologically important hormones, striving to solve fundamental questions that have a broad impact on human health and disease. The overall goal of my research program is 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. My current research programs are focused 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 transcriptional 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 a, d, and g) 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 the novel PPARg 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 ligandbinding 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 these receptors bound to co-activators or co-repressors, and the crystal structure of PPARg 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 co-repressors 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 PPARg, and our plan is to test their physiological roles in glucose and insulin regulation, 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 (Dex) and fluticasone propionate (FP) 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—called “dissociated glucocorticoids”, which can separate the good effects from the bad—remains an intensive goal of pharmaceutical research.

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

71

Van Andel Research Institute |

Scientific Report

Structural genomics of nuclear receptor ligand-binding domains

The LBD of a nuclear receptor contains key structural elements that mediate ligand-dependent regulation of the receptors and as such 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 those for 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 my group is 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 Ke, Jiyuan, Chenghai Zhang, Kaleeckal G. Harikumar, Cassandra R. Zylstra-Diegel, Liren Wang, Laura E. Mowry, Laurence J. Miller, Bart O. Williams, and H. Eric Xu. 2012. Modulation of b-catenin signaling by glucagon receptor activation.

PLoS One 7(3): e33676.

Pal, Kuntal, Karsten Melcher, and H. Eric Xu. 2012. Structure and mechanism for recognition of peptide hormones by Class B G-protein-coupled receptors. Acta Pharmacologica Sinica 33(3): 300–311.

Soon, Fen-Fen, Ley-Moy Ng, X. Edward Zhou, Graham M. West, Amanda Kovach, M.H. Eileen Tan, Kelly M. Suino-Powell, Yuanzheng He, Yong Xu, Michael J. Chalmers, et al. 2012. Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases. Science 335(6064): 85–88.

Soon, Fen-Fen, Kelly M. Suino-Powell, Jun Li, Eu-Leong Yong, H. Eric Xu, and Karsten Melcher. 2012. Abscisic acid signaling: thermal stability shift assays as tool to analyze hormone perception and signal transduction. PLoS One 7(10): e47857.

Yu, Shanghai, and H. Eric Xu. 2012. Couple dynamics: PPARg and its ligand partners. Structure 20(1): 2–4.

Zhou, X. Edward, Fen-Fen Soon, Ley-Moy Ng, Amanda Kovach, Kelly M. Suino-Powell, Jun Li, Eu-Leong Yong, Jian-Kang Zhu, H. Eric Xu, and Karsten Melcher. 2012. Catalytic mechanism and kinase interactions of ABA-signaling PP2C phosphatases. Plant Signaling & Behavior 7(5): 581–588.

72

Awards for Scientific Achievement

VARI | 2013

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 B. Singleton, Ph.D.

Dr. Andrew Singleton during his lecture as the inaugural Jay Van Andel Award recipient.

74

VARI | 2013

Han-Mo Koo Memorial Award

Dr. Han-Mo Koo joined the Van Andel Research Institute in 1999 as one of its founding investigators. Heading the Laboratory of Cancer Pharmacogenetics, Dr. Koo established important projects focused on identifying genetic targets for anti-cancer drugs against melanoma and pancreatic cancer, and he worked tirelessly to contribute to the Institute’s mission to improve health and enhance lives. In May 2004, Dr. Koo passed away following a six-month battle with cancer. To honor his memory and scientific contributions, the Han-Mo Koo Memorial Award and Lecture was established in 2010.

Awardees are selected based upon their scientific achievements and their contributions to human health and research that align with the scientific legacy of Han-Mo Koo.

Award Recipient

Phillip A. Sharp, Ph.D.

Dr. Phillip Sharp delivering the inaugural Han-Mo Koo Memorial Lecture.

75

Postdoctoral Fellowship Program

76

VARI | 2013

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 scientific investigator who oversees the progress and direction of research. Fellows who worked in VARI laboratories in 2012 are listed below.

Nicholas Andersen

Yanyong Kang

Electa Park

University of Iowa VARI mentor: Nicholas Duesberry

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

Louisiana State University Health Sciences Center, New Orleans VARI mentor: Cindy Miranti

Nate Lanning

Jackie Peacock

University of Michigan VARI mentor: Jeffrey MacKeigan

University of Miami VARI mentor: Matthew Steensma

Leanne Lash-Van Whye

Anthony Popkie

University of Texas Medical Branch, Galveston VARI mentor: Arthur Alberts

The Ohio State University VARI mentor: Craig Webb

Genevieve Beauvais University of Paris Descartes VARI mentor: Patrik Brundin

Poulomi Bhattacharya Illinois State University VARI mentor: Nicholas Duesberry

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

Anamitra Ghosh Iowa State University VARI mentor: Patrik Brundin

Danese Joiner University of Michigan VARI mentor: Bart Williams

Heather McClung Wayne State University VARI mentor: Giselle Sholler

Aikseng Ooi University of Malaya, Kuala Lumpur VARI mentor: Kyle Furge

Kuntal Pal National University of Singapore VARI mentor: Eric Xu

Juliana Sacoman Michigan State University VARI mentor: Jeffrey MacKeigan

Huiyan Tang Michigan State University VARI mentor: Brian Haab

Xiaoyong Zhi University of Texas Southwestern Medical Center VARI mentor: Eric Xu

Alex Zhong Sun Yat-sen University, Guangzhou, China VARI mentor: Bart Williams

From left: Ghosh, Cao, Burgers, Pal, Lanning, McClung, Popkie, Peacock, Kang, Fogg, Park, Joiner, Lash-Van Wyhe, Ooi, Sacoman, Beauvais

77

Student Programs

78

VARI | 2013

Grand Rapids Area Pre-College Engineering Program The Grand Rapids Area Pre-College Engineering Program (GRAPCEP) is administered by Davenport University and is sponsored and funded by VAEI. 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 2012 GRAPCEP students from Creston High School were

Jamilah Fields (Hostetter/Jewell) Jasmine Jones (Weinreich) Chantice LaGrone (Alberts) Yasmeen Robinson (Chang)

From left: LaGrone, Robinson, Jones, Fields

79

Van Andel Research Institute |

Scientific Report

Summer Student Internship Program The VARI student internships were established to provide college students with an opportunity to work with professional researchers in their fields of interst, to use state-of-the-art equipment and technologies, and to learn valuable interpersonal and communications skills. At the completion of the 10-week program, the students summarize their projects in an oral presentation or poster.

From January through August 2012, the Van Andel Institure hosted more than 49 students from over 16 colleges and universities in formal summer internships under the Frederik and Lena Meijer Student Internship Program and in other student positions during the year. An asterisk (*) indicates a Meijer student intern.

Standing, from left: Dieffenbach, Langerak, Sayfie, Grit, Dykstra, Dills, Edewaard, Shorkey, deWaal, Uhl, Varlan, Reimink, Muhoro, Hanchon, M. Smith, Searose-Xu, Subramanian, Orey, Rybski.

Kneeling, from left: McMasters, Parker, Vanderlinde, Bergsma, Goyings, Westra, Waslawski, Hotaling, Quinn.

80

VARI | 2013

Aquinas College, Grand Rapids, Michigan

Lauren Smith* (Sholler)

Calvin College, Grand Rapids, Michigan

Eric Edewaard (Jewell) Caroline Muhoro (Vande Woude) Anna Plantinga* (MacKeigan) Allison Schepers (Alberts) Tyler Spiering (Williams)

Central Michigan University, Mount Pleasant, Michigan

Amanda Erwin* (Miranti) Sabrina Parker* (Office of the Director) Adriane Shorkey (Jewell)

Grand Valley State University, Allendale, Michigan

Andrew Borgman (Neff) Jenea Chesnic (Neff) Michael Dykstra* (Chang) Daniel Hodges (Neff) Kevin Kampfschulte, B.S. (Steensma) Justin Langerak (Duesbery) Mitch McDonald (Haab) Brittany Holly (Chang) Stephen Orey (Webb) Alexander Roemer (Neff) Katie Uhl (Jewell) Hannah Westra* (Haab) Raymond Yeow (Weinreich)

Hope College, Holland, Michigan

Jamie Grit* (Triezenberg) Aaron Sayfie (MacKeigan) Mallory Smith (Steensma) Emily Van Wieren (Williams)

Huston Tillotson University, Austin, Texas

Nahome Bete (Haab)

Johns Hopkins University, Baltimore, Maryland

Sandya Subramanian* (Weinreich)

Kalamazoo College, Kalamazoo, Michigan

Michigan State University, East Lansing

Zach Dieffenbach (Chang) Kelvin Searose-Xu (Melcher) Sheila Waslawski* (Duesbery)

Michigan Technological University, Houghton

Nathan Dills* (Melcher)

University of Mannheim, Germany

Lisa Becker (Alberts)

University of Michigan, Ann Arbor

Alexis Bergsma (Miranti) Kristin Rybski* (Alberts)

Vanderbilt University, Nashville, Tennessee

Peter Varlan* (Jewell)

Other Van Andel Institute Interns Calvin College, Grand Rapids, Michigan

Calvin Wiersma (Finance)

Davenport University, Grand Rapids, Michigan

Sarah Kozal (Development) Andrew Lau (Finance)

Ferris State University, Big Rapids, Michigan

Sheri Orlekoski (Compliance)

Grand Valley State University, Allendale, Michigan

Jordan Hanchon (Finance) Christina Middaugh (Van Andel Education Institute) Jessica Reimink (Finance) Holly Vanderlinde (Development)

University of Michigan, Ann Arbor

Ellen Junewick (Business Development)

Parker de Waal (Xu) Mary Goyings* (Jewell)

Livingstone College, Salisbury, North Carolina

Ashley McMasters (Melcher)

Loyola University, Chicago, Illinois

Hudson Hotaling* (Williams) Monique Quinn* (Steensma)

81

VARI Seminar Series

82

VARI | 2013

VARI Seminar Series September 2011

Brooke McCartney, Carnegie Mellon University

“ At the intersection of Wnt signaling and cytoskeletal dynamics: a model systems approach to the study of the enigmatic tumor suppressor adenomatous polyposis coli”

W. James Nelson, Stanford University

“Evolution of epithelia and cadherin-based cell-cell adhesion”

October

Dan Klinosky, University of Michigan

“If you only have time to attend one talk today on autophagy, this is the one”

Robert Maki, Mount Sinai School of Medicine

“Slugs, snails, and puppy dogs’ tails: what sarcomas are made of and how they are treated”

November

Dinshaw Patel, Memorial Sloan – Kettering Cancer Center

“Structural biology of gene and epigenetic regulation”

Andrew Dillin, Salk Institute for Biological Studies

“Immortality, stem cells, and humoral signals of longevity”

Alan Hall, Memorial Sloan – Kettering Cancer Center

“Rho GTPases controlling epithelial morphogenesis and migration”

Jennifer Cross, University of Virginia

“The inflammatory cytokine MIF is an immune-modulating therapeutic target in tumor growth and metastasis” February 2012

Shylam Biswal, Johns Hopkins University

“Nrf2 as a target for cancer therapy”

March

Regis J. O’Keefe, University of Rochester

“Stem cell populations and their regulation in bone repair”

Di Chen, Rush University Medical Center

“TGF-b signaling and osteoarthritis”

David Marc Virshup, Duke University

“Regulating Wnts at the source — basic biology and potential therapy”

April

Aik Choon Tan, University of Colorado

“Translational bioinformatics: from bytes to bench and back”

May

Vicki Rosen, Harvard University

“BMP-2 links appositional bone growth and fracture repair”

Phillip A. Sharp, Massachusetts Institutte of Technology Han-Mo Koo Memorial Lecture

“Transcription and functions of microRNAs and other non-coding RNAs”

Tom Shenk, Princeton University

“Metabolomic analysis: fat management by human cytomegalovirus” 83

Van Andel Research Institute |

Scientific Report

June

Richard Youle, National Institutes of Health

“Molecular mechanisms of mitochondrial quality control through autophagy in Parkinson’s disease”

July

Collin Duckett, University of California, Los Angeles

“IAP proteins in neoplasia and immunodeficiency”

Anna Wu, David Geffen School of Medicine at UCLA

“Engineered antibodies for immunoPET detection of cancer”

August

Hideho Okada, University of Pittsburgh

“Type-1 polarizing vaccines for adult and pediatric gliomas”

Sean Culter, University of California, Riverside

“Chemical and genetic dissection of ABA signaling”

Jennifer Gillette, University of New Mexico

“Regulation of hematopoietic stem cell communication with the bone marrow niche”

Bill Weis, Stanford University

“The interplay of a-catenin and the actin cytoskeleton in cell adhesion and cell polarity”

September

Ralph J. DeBerardinis, M.D., Ph.D., University of Texas Southwestern Medical Center

“Cancer metabolism — biological insights and translational opportunities”

C. Titus Brown, Michigan State University

“An efficient framework for throwing away most of your next-gen sequencing data”

November

Roger K. Sunahara, Univeristy of Michigan Medical School

“Structural basis for G protein activation by GPCRs”

John Kuriyan, University of California, Berkeley

“Allosteric mechanisms in the activation of the EGF receptor”

December

Prasad Jallepalli, Memorial Sloan–Kettering Cancer Center

“Surfing mitosis and cell division with chemical genetics”

84

Van Andel Research Institute Organization

85

Van Andel Research Institute |

Scientific Report

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.

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

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

Scientific Advisory Board The Scientific Advisory Board advises the VARI Director, providing recommendations and suggestions specific to the ongoing research. It also coordinates and oversees the scientific review process for the Institute’s research programs.

The members are

86

Alan Bernstein, Ph.D. Joan Brugge, Ph.D. Webster Cavenee, Ph.D. Frank McCormick, Ph.D.

VARI | 2013

Office of the Director Van Andel Research Institute Research Leadership Council

Patrick Brundin, M.D., Ph.D.

George Vande Woude, Ph.D.

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

Office Staff

John Bender, Clinical Operations Director Kim Cousineau, Senior Administrative Assistant Jens Forsberg, Scientific Project Leader Laura Holman, Executive Assistant Jennifer Holtrop, Scientific Administrator

David Nadziejka, Science Editor Aaron Patrick, Administrative Manager Bonnie Petersen, Senior Administrative Assistant Beth Resau, Senior Administrative Assistant Ashley Rodriguez, Administrative Assistant

From left: Petersen, Holtrop, Bender, Forsberg, Patrick, Rodriguez, Resau, Cousineau, Holman

87

Van Andel Research Institute |

Scientific Report

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 (continued)

David Van Andel, Chairman and CEO Jana Hall, Ph.D., M.B.A., Chief Operations Officer David Whitescarver, Vice President and Chief Legal Officer Christy Goss, Executive Assistant Ann Schoen, Executive Assistant

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

Business Development Jerry Callahan, Ph.D., M.B.A., Vice President Marilyn Becker Andrea DeJonge Thomas DeKoning Carolyn Hudson, Ph.D. Brent Mulder, Ph.D., M.B.A. Norma Torres

Compliance Gwenn Oki, Director Paula Williamson DeBoe Angie Jason Stacy Kuiken Shelly Novakowski Sheri Orlekoski

Development, Marketing, and Communications Love Collins III, Vice President Tim Hawkins Sarah Hop Nancy Kooienga Sarah Lamb Gerilyn May Patrick Placzkowski Angie Stumpo Anthony Thompson Nicky Wilkerson Nadina Williams

Facilities Samuel Pinto, Manager Amber Baldwin Rob Cairns Maria Cavasos Jeff Cooling Deb Dale Jason Dawes Guadalupe Delgado Ken DeYoung Kristi Gentry Matthew Jump Todd Katerberg

88

Finance Timothy Myers, Vice President and Chief Financial Officer Heather Zak, Controller Stephanie Birgy Theresa Brown Cory Cooper Raji Daniel Sandi Dulmes Katie Helder Rich Herrick Angie Lawrence Susan Raymond Cindy Turner Jamie VanPortfleet

Grants and Contracts David Ross, Director Sara ONeal, Manager Marilyn Becker Anita Boven Nathan Gras Kathy Koehler Tanja Lumpp Michele Quick

Human Resources Linda Zarzecki, Vice President Stacey Booth Margie Hoving Eric Miller Pamela Murray Carol Sheldon John Shereda

VARI | 2013

Information Technology

Materials Management

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

Richard M. Disbrow, CPM, Director Eddie Cortadillo, Supervisor Bob Sadowski, Supervisor Matt Donahue Susanne Dubois Heather Frazee Chris Kutschinski Shannon Moore Monono Negash Amy Poplaski Marlene Sal John Waldon

Investments Office

Jodi Tyron, Librarian (Grand Valley State University)

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

Security Kevin Denhof, CPP, Director Amy Davis Kate Harrison Andriana Vincent Chris Wilson

Contract Support

89

Van Andel Research Institute |

Scientific Report

Van Andel Institute

Van Andel Institute Board of Trustees David Van Andel, Chairman Michael Jandernoa John C. Kennedy 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.

David Van Andel, Chairman Donald W. Maine Juan R. Olivarez, Ph.D. Gordon Van Harn, Ph.D.

Van Andel Research Institute Research Director

Van Andel Education Institute Director

Open

Steven J. Triezenberg, Ph.D.

Chief Administrative Officer and General Counsel

VP Development, Communications, and Marketing

David Whitescarver

Love Collins III

Chief Operations Officer Jana Hall, Ph.D., M.B.A.

90

VP Human Resources

VP and Chief Financial Officer

VP Business Development

Linda Zarzecki

Timothy Myers

Jerry Callahan, Ph.D.

VARI | 2013

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.

91

Van Andel Research Institute |

Scientific Report

Printed by Wolverine Printing Company

92

Van Andel Research Institute Scientific Report 2013

Scientific Report

333 Bostwick Avenue, N.E., Grand Rapids, Michigan 49503 Phone 616.234.5000 Fax 616.234.5001 www.vai.org

13