Features

By Kyla Trkulja

Liver cancer is one of the most difficult types of cancer to treat, making it the third most common cause of cancer death around the world.1 The prognosis for patients diagnosed with the disease is poor, with only 35% of patients with early-stage disease surviving 5 years after diagnosis—and a mere 3-12% of patients surviving this long if the cancer spreads.1 It is difficult to treat because many of these patients often also have liver disease, making them unable to tolerate chemotherapy or surgery.

Dr. Mamatha Bhat, a hepatologist and clinician-scientist at Toronto General Hospital, is using creative and innovative approaches to try to improve these grim statistics and the lives of patients with liver disease. After completing her medical education and residency in internal medicine and gastroenterology at McGill University, she pursued a research fellowship and obtained her PhD in medical biophysics from the University of Toronto. She currently runs a translational research program using an interdisciplinary approach that aims to improve the outcomes of patients after liver transplantation—of which 35-40% are cancer patients.

Dr. Bhat describes the ability to perform a transplant for those with liver cancer as a “unique paradigm,” as transplantation is not a method of treatment for other types of cancer, with the exception of cancers originating in

“ “ ...a way of overcoming one of the biggest challenges in oncology

the blood. Clinically, she looks after 350 patients who are referred for a liver transplant before, during, and after their surgery. On the research side, she aims to uncover ways to improve patient outcomes post-transplant—specifically by using nanoparticles to prevent and treat recurrence of liver cancer.

Nanoparticles to treat liver disease and cancer were developed in collaboration with Dr. Gang Zheng at the Princess Margaret Cancer Centre. Put simply, these types of nanoparticles contain proteins and drugs that can treat the liver disease and cancer a patient may have. They bind to receptors that are specifically expressed on the liver and its cancer cells, such as the receptors used to take up cholesterol. This new and innovative approach allows the treatment to be targeted and delivered to the specific cells in need.

“With this approach, we can very selectively target the liver cancer cells,” Dr. Bhat explains. “Beyond that, we might be able to target specific molecular profiles, and also [optimize] the dose, depending on the degree of liver dysfunction that the patient has, so this is a very unique consideration in cancer where people have underlying liver disease.”

Dr. Bhat has demonstrated the efficacy of this treatment in animal models through her collaboration with Dr. Zheng, which has been supported by the Terry Fox Research Institute and the Canadian Liver Foundation. Dr. Zheng’s nanoparticle technology will soon be studied in a clinical trial with oncologists at the Princess Margaret Cancer Center to test this therapy in a variety of cancer types. If the trial is safe and effective, it could be brought to the clinic as a way to treat those with liver cancer and hopefully improve their disease outcomes.

The use of nanoparticles to target and treat liver cancer is a way of overcoming one of the biggest challenges in oncology, which Dr. Bhat describes as treating cancer with a “one-sizefits-all” approach. “Everyone has their own background, their own clinical characteristics, their own disease manifestation,” she explains. “Each person is unique … but we still go based on clinical guidelines, and

DR. MAMATHA BHAT

MD, PhD; Hepatologist and Clinician Scientist, UHN Multi Organ Transplant Program, Toronto General Hospital Research Institute; Assistant Professor, Division of Gastroenterology, Department of Medicine at the University of Toronto

Photo Credit: University Health Network

clinical guidelines may be good for the population as a whole, but they may not be good for individuals within that population.” By tailoring both the dose and type of treatment inside the liver-specific nanoparticle, this challenge is being addressed to make cancer treatments both safer and more effective while considering the patients’ underlying conditions such as chronic liver disease and cirrhosis.

Dr. Bhat stressed the importance of collaboration in making this research possible, both between researchers and between research sites. “The world has become much more complicated, and it isn’t possible for one single person to have the whole skill set to address a specific problem,” she said. “It used to be that you could be one lab doing your own thing. But I think things evolved and changed over time.” She is now utilizing tools she has learned from other disciplines, such as bioinformatics and machine learning, to aid her research in personalizing treatment.

This openness to new approaches and collaborations is described by Dr. Bhat as one of the most important characteristics to have as a researcher, as “the world has become much more interdisciplinary.” This has been exceptionally important in her role as a clinician-scientist, where broad and creative thinking are necessary to tackle an issue seen in the clinical setting. “It’s not just hard work,” she says. “It’s also working smarter.”

Using her clinical experience to inform on her research projects is an exceptionally rewarding part of her career, despite the challenges that go along with managing the dual roles. She explains that a positive attitude, interest and passion for the work being done, and motivation to dedicate extra years to acquiring the skills necessary for both roles are essential to succeeding as a clinician-scientist. If you have the passion and openness, the role is an excellent way to tackle the gaps in healthcare since you can inform projects from your own clinical experiences.

Using these skills, Dr. Bhat is aiming to play a role in the future of liver disease and cancer research and care, which she envisions as moving away from the one-size-fits-all approach to treatment and embracing the potential for new and innovative targeted therapies. She believes that these are key to improving outcomes for patients with liver cancer and reducing fatalities, similar to what has been done for other types of common cancer such as breast and colorectal. Nanoparticle-based therapies are one example of how this may be done, so she looks forward to bringing these from bench to bedside in the coming years.

References

1. American Society of Clinical Oncology. (2022, February 24). Liver cancer - statistics. Cancer.Net. Retrieved May 20, 2022, from https:// www.cancer.net/cancer-types/liver-cancer/statistics

Balancing Acts

Function & Dysfunction in the Immune System

By Beatrix Wang

We rely on our immune system to fight off pathogenic microbes and guard against the growth of malignant cells.1 In recent decades, much progress has been made in mapping out the body’s strategy to defend against infection and immune disease, but fundamental questions remain within the field. How exactly are immune responses regulated? And, just as importantly, what are the mechanisms that lead to immune dysfunction and consequent disease?

“Everything,” Dr. Katherine Siminovitch says, “is about balance.” A healthy and functional immune system exists in a state of homeostasis wherein tightly regulated pathways are able to respond to invading pathogens appropriately.2 This response involves a fine-tuned combination of immune activation and deactivation to ensure that the system neither over- nor under-reacts to stimuli. When this balance is disrupted, immune cells may begin attacking the body—a phenomenon known as autoimmunity—or lose their ability to respond to external (antigenic) stimuli, both of which can lead to debilitating health issues.

Dr. Siminovitch, a Professor of Medicine and Immunology at the University of Toronto and Senior Investigator at the Lunenfeld-Tanenbaum Research Institute, has made it the goal of her research career to identify the cellular and molecular pathways modulating immune function and involved in immune dysfunction, with a particular focus on autoimmune disorders. “Major gaps still exist in our understanding of how autoimmune diseases develop and persist,” Dr. Siminovitch says. For some conditions, such as systemic sclerosis or vasculitis, the precise mechanisms underlying pathogenesis remain almost entirely unknown. Furthermore, even for extensively studied disorders such as lupus, there is a lack of therapies that go beyond symptom management and immunosuppression.

Identifying drivers of disease and discovering potential therapeutics have been complicated by the extensive heterogeneity that exists within individual immunologic conditions. “Different molecular pathways can lead to the same clinical phenotype,” Dr. Siminovitch says. “Clinical heterogeneity is very obvious, for example, in lupus. It becomes very challenging to find effective treatments when lupus patients are grouped into a study without initial sub-classification or sub-categorization, ignoring the likelihood the disease-causal pathways may differ among these patients and, as such, may need to be managed differently.”

Enormous efforts are continuing to elucidate the mechanisms by which different genes and molecules lead to the immune dysfunction underpinning disease. Dr. Siminovitch has played a vital role in this research. During her postdoctoral training, she became interested in an oftenfatal immune deficiency disorder known as Wiskott-Aldrich syndrome (WAS). During this time, she contributed significantly to identifying the genetic cause of the illness, mapping the gene responsible, and characterizing various gene mutations that lead to WAS.3 The gene in question turned out to encode Wiskott-Aldrich syndrome protein (WASp), a large adaptor protein that mediates signal transduction, directs immune cell cytoskeletal activity, and is an essential immune regulator.4 Furthermore, these findings allowed for the development of genetic tests to diagnose WAS and identify carriers of the mutant gene.

Dr. Siminovitch subsequently became interested in other adaptor proteins within the WASp family, including WAVE2, another actin cytoskeletal regulator with the potential to play a role in immune function. “Not many people have been working on WAVE2,” Dr. Siminovitch says, “but my group was very interested in this protein’s functions because it is expressed at high levels in immune cells and because of our ongoing interest in cytoskeletal regulatory protein roles in immunity.”

It wasn’t until relatively recently that Dr. Siminovitch and her team had an opportunity to study this protein’s function in immune cells, her group having generated a conditional knockout mouse in which WAVE2 expression is specifically deleted in T lymphocytes. The results were striking. “The mice have an obvious phenotype of very severe autoimmune disease,” Dr. Siminovitch says. “And paradoxically, along with autoimmune disease, they also exhibit profound immune deficiency. So, the lack of WAVE2 in T cells

DR. KATHERINE SIMINOVITCH

Professor of Medicine; Professor of Immunology; Senior Scientist at Lunenfeld-Tanenbaum

Photo Credit: Dr. Katherine Siminovitch

leads to a disease representing both ends of the immunologic spectrum.” Using this mouse model, Dr. Siminovitch and her team identified WAVE2 as a suppressor of the TOR protein, a key regulator of cell metabolism, proliferation, apoptosis, and multiple other functions.5,6 They found that in the absence of WAVE2, TOR is hyperactivated, leading to spontaneous T cell activation, with loss of immune homeostasis and diminished responsiveness to antigenic stimuli. These abnormalities, in turn, cause the severe immune disease observed in the mice.5

This work has served as an exciting starting point for Dr. Siminovitch and her team. Moving forward, Dr. Siminovitch believes there is much to learn about how WAVE2 functions in other immune cell types and is particularly interested in exploring an unexpected link between WAVE2 and neurodegenerative disease. Furthermore, the link between TOR hyperactivity and immune dysfunction suggests that drugs that inhibit the TOR pathway may have relevance to the treatment of autoimmune and other immune-mediated disease.

Notably, Dr. Siminovitch’s findings also demonstrate how much more there is to learn about proteins and pathways even after they have been assigned specific functions. For both WASp and WAVE2, regulation of the cytoskeleton is only the beginning of the story. “These proteins have been primarily studied as cytoskeletal regulators because they contain domains that allow them to promote actin polymerization,” Dr. Siminovitch says. “But they have additional functions and I believe our current understanding of their functions represents only the tip of the iceberg of their biological contributions.”

Dr. Siminovitch has no doubt that the coming years will bring a wealth of answers to many complex questions about immune disease. “This is an incredible time to be involved in biomedical research,” she says. “I thought the same thing when I first set up my lab, but now the research tools available to scientists— technical and computational—are truly unprecedented in terms of the types of questions you can ask and reasonably hope to answer.” In this context, immunologists like Dr. Siminovitch are poised to address fundamental questions about the immune response and how it becomes dysregulated in disease. The ultimate hope is that this research will illuminate the complex combinations of factors that can trigger autoimmunity and can then be clinically translated to allow improved detection, more effective treatment, and prevention of immune disorders.

References

1. Chaplin DD. Overview of the Immune Response. J Allergy Clin

Immunol. 2010 Feb;125(2 Suppl 2):S3-23. 2. da Gama Duarte J, Woods K, Andrews MC, et al. The good, the (not so) bad and the ugly of immune homeostasis in melanoma.

Immunology & Cell Biology. 2018;96(5):497–506. 3. Kolluri R, Shehabeldin A, Peacocke M, et al. Identification of WASP mutations in patients with Wiskott-Aldrich syndrome and isolated thrombocytopenia reveals allelic heterogeneity at the WAS locus.

Hum Mol Genet. 1995 Jul;4(7):1119–26. 4. Ngoenkam J, Paensuwan P, Wipa P, et al. Wiskott-Aldrich Syndrome

Protein: Roles in Signal Transduction in T Cells. Frontiers in

Cell and Developmental Biology [Internet]. 2021 [cited 2022 Jun 5];9. Available from: https://www.frontiersin.org/article/10.3389/ fcell.2021.674572 5. Liu M, Zhang J, Pinder BD, et al. WAVE2 suppresses mTOR activation to maintain T cell homeostasis and prevent autoimmunity.

Science. 2021 Mar 26;371(6536):eaaz4544. 6. Zou Z, Tao T, Li H, et al. mTOR signaling pathway and mTOR inhibitors in cancer: progress and challenges. Cell & Bioscience. 2020 Mar 10;10(1):31.

Alpha synuclein nanobodies in the brain and gut

Novel therapeutic laboratory techniques in the treatment of Parkinson’s Disease

By Nayaab Punjani

Parkinson’s Disease (PD) is a neurodegenerative disorder that affects more than 100,000 Canadians.1 This condition is often characterized through motor symptoms such as tremors and non-motor symptoms including constipation and sleep disturbances.2 PD pathology involves the build-up of a protein (alpha synuclein or α-Syn) in the brain, along with the loss of dopaminergic neurons.3 Trying to find a way to reduce the expression of α-Syn, and stopping early PD progression is key to improving the patients’ quality of life. Dr. Anurag Tandon, Scientist at the Tanz Centre for Research in Neurodegenerative Diseases and Associate Professor in the Department of Medicine, Division of Neurology, uses unique approaches to address this pervasive disease.

As a cell biologist, Dr. Tandon began to understand PD through studying the role of α-Syn in vesicular trafficking and neurotransmission. Understanding of PD pathology has evolved and we now recognize the prion-like spread of α-Syn pathology between neurons. Novel therapies for PD aim to reduce α-Syn expression through either targeting its gene expression by studying viral gene knockdown vectors,4 or to decrease the concentration of the protein to reduce aggregation.

When developing immune therapies for PD, Dr. Tandon discusses the challenges of targeted delivery to the brain. Antibodies are often composed of long polypeptide chains, thus when delivered intravenously, only 0.5% pass the blood-brain-barrier (BBB) and enter the cerebrospinal fluid. However, these large proteins are very specific to the target protein. Therefore, being able to take the gene sequence for Neurons in culture (shown in red) expressing the anti-α-Syn nanobody developed less pathology (green) than neurons expressing a non-specific control nanobody.

Photo Credit: Dr. Anurag Tandon

these antibodies, bridging immunotherapy with gene therapy, and express them in the brain, is a promising therapeutic avenue.

Dr. Tandon employs the use of small molecules called nanobodies. These molecules were developed through research conducted by his collaborators, Dr. David Butler and Dr. Anne Messer.5 The process of creating nanobodies involves isolating the variable domain, which recognizes α-Syn, on the heavy chain of a single chain polypeptide antibody from a human blood cell library. Human cells normally do not express single chain antibodies, however, through isolating this sequence and placing it in an expression vector, the nanobody can now bind to α-Syn, allowing it to be degraded and reduce the accumulation of this protein. In order to further improve delivery of the nanobodies using viral vectors across the BBB in PD relevant brain regions, Dr. Tandon is also exploring the use of focused ultrasound, a technique which helps large molecules to cross the BBB and can be used multiple times, to reduce α-Syn levels.

The in vitro cell work, done by graduate student Sabrina Armstrong, demonstrates the benefit of this therapy to help reduce intracellular pathology induced by the presence of α-Syn fibrils, so his current research has shifted to examine this in animal models. Dr. Tandon explains, “The other incredible aspect of this is once there is proof of principle that it works in one model, any neurodegenerative disease could be targeted, and I do not think this is a single treatment approach, it may work in combination with other treatments as well but having a brain cell produce the therapeutic factor continuously has a certain appeal to it.” This research also opens many pathways to consider targeting α-Syn misfolding and pathological spread between cells.

DR. ANURAG TANDON

Scientist, Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto; Associate Professor, Department of Medicine, Division of Neurology; Full Member, Institute of Medical Science

Photo Credit: Dorsa Derakhshan

animal models which can be challenging. “We have models that replicate aspects of the disease but we do not have an allencompassing model”. Furthermore, “We do not fully appreciate where the disease begins in humans. There is quite a bit of research suggesting that it begins in the peripheral nervous system particularly in the gastrointestinal (GI) tract. So enteric neurons certainly show pathological changes very early on, but that may not be the case for all patients. Some patients may have a disease that starts in the brain, others may be peripheral”.

Patients generally present with PD when it has progressed to neuron loss in the brain, while the early peripheral symptoms are often missed. Thus, it is important to target this primary origin of the disease to stop progression and preserve function. Dr. Tandon is also investigating this approach by adapting animal models of enteric pathology developed by other labs, which include overexpression of α-Syn fibrils in the GI tract. Dr. Tandon aims to explore the pathology transfer from these enteric neurons to higher cortical regions of the brain, which results in dementia-like pathology of late-stage PD. Optimization of this research is ongoing and various factors will need to be considered to enhance delivery of the nanobody gene therapy vectors to peripheral neurons.

Through targeting the early underlying PD pathology in the peripheral nervous system, Dr. Tandon mentions, “Now you might have the ability to first block the spread of the pathology and then the second approach would be to see if we can rescue the existing neurons that are on the verge. We may or may not be able to rescue cells that have died completely, the distances in the brain are very challenging because to have a neuron project from the substantia nigra to the striatum, these are many centimeters, which is not a minor thing in either an animal model or humans where all the neurons are already beautifully laid out, so re-establishing connections may be a challenge, but we might be able to strengthen the existing ones”.

If your family has been impacted by Parkinson’s Disease, Dr. Tandon highlights many avenues where you can seek more information. One, is by reading magazine articles that provide updates on the current state of science in the field. Second, the Parkinson Society of Canada is a great resource to support you and assist in understanding the condition, while also connecting you with researchers. Finally, he indicates the importance of funding agencies, both government-based (Canadian Institutes of Health Research or CIHR) and private philanthropy operations like the Weston Brain Institute, which support cutting-edge research.

Dr. Tandon encourages graduate students to read about the research areas they are interested in and also examine crossdisciplinary applications. He highlights how essential collaboration is to advance scientific discoveries. Despite not having a strong virology or immunology background, through collaboration he is able to incorporate these fields into his work. He recalls, “I do not need to know how to make these things, I just need to know how I could reach the person who made them and ask them to collaborate with us on a project.” Dr. Tandon stresses the importance of humility and being open to sharing your knowledge and collaborating with scientists in other fields.

There is a lot that is yet to be explored in this field, particularly if we consider personalized medicine and assessing individual progression of PD pathology. Cross-disciplinary collaboration will be key to advancing therapies for PD and other neurodegenerative disorders.

References

1. Parkinson’s Disease [Internet]. Parkinson Canada. [cited 2022 Jun 2]. Available from: https://www.parkinson.ca/about-parkinsons/ 2. Symptoms of Parkinson’s [Internet]. Parkinson Canada. [cited 2022

Jun 2]. Available from: https://www.parkinson.ca/about-parkinsons/ symptoms/ 3. Gómez-Benito, M., Granado, N., García-Sanz, P., et al. Modeling

Parkinson’s Disease With the Alpha-Synuclein Protein. Front

Pharmacol. 2020 Apr; 11: 356. 4. Menon S, Kofoed RH, Nabbouh F, Xhima K, Al-Fahoum Y, Langman T, et al. Viral alpha-synuclein knockdown prevents spreading synucleinopathy. Brain Communications. 2021 Oct 1;3(4):fcab247. 5. Messer A, Butler DC. Optimizing intracellular antibodies (intrabodies/nanobodies) to treat neurodegenerative disorders. Neurobiol

Dis. 2020 Feb;134:104619.