Single cell analysis and perturbation of the tumor-immune ecosystem
MRA Established Investigator Award
Ido Amit, Ph.D., Weizmann Institute of Science
Melanoma is one of the chief health concerns worldwide. Yet the complexity, dynamics, and intricacy of melanoma pose a major challenge to the medical community—from prevention and early detection to diagnosis and therapy. Novel immunotherapies –namely, 'turning on' the immune system against tumors- are currently at the forefront of revolutionary melanoma treatments. By developing some of the newest tools in single cell genomics, big data analysis algorithms, and functional genomics research, we are creating detailed road maps of the cross-talk between the immune system’s and melanoma. These discoveries are vital to lead to new, personalized approaches of immunotherapy. By conducting in-depth basic research into the processes in which tumors evolve to actively escape the defensive mechanisms of the immune system; We are seeking to identify ways to reactivate tumor-specific T-cells in melanoma. Our findings are likely to provide tools for predicting tumor response, and optimization of immunotherapeutic strategies, which will greatly increase the success rate of therapy. Our aims within this program are to develop unbiased and high-resolution characterization of the malignant and immune cell populations within the tumor microenvironment using advanced single cell techniques. Furthermore, we will describe the cell states and markers that stratify patients for effective therapeutic intervention. The aims of this proposal are therefore threefold: 1). To understand the states of T cell in melanoma; 2). To reveal how these states are determined by either surrounding tumor or immune cells, and 3). To determine which cell states do or do not react to checkpoint blockade. As a clear and immediate spin-off, aims 2 and 3 will identify both target cell types and target molecules for therapeutic manipulation. Together, our research will provide tools for predicting tumor response, markers for tumor characterization, and identification of novel immune modulatory pathways.
DNA-based biomarkers for melanoma diagnosis and prognostication
MRA Established Investigator Award
Boris Bastian, M.D., University of California, San Francisco
Despite improvements in melanoma therapy over the last decade, metastatic melanoma remains potentially deadly. After the primary melanoma has been removed, subsequent treatments are initiated when new tumors are detected. At that time the number of tumor cells has increased substantially and some cells may have evolved to be resilient to therapy. The odds of cure could be improved if patients at high risk of developing metastatic disease could be treated immediately after removal of their primary tumor. Current methods fail to identify all patients in need of treatment and falsely identify patients who don’t need treatment, putting them at risk for harm. My group studies the genetic evolution of primary melanomas found that their genetic alterations accumulate over time and arise in a specific sequence. We will evaluate the potential of this novel information as to how far it can estimate the likelihood that a given primary melanoma leads to metastases later on. We will adapt an already existent clinical next-generation sequencing assay to interrogate genes recurrently mutated in melanoma. We will perform a retrospective study and compare mutations in primary melanomas of 400 patients from our institution of which 200 will have advanced to stage IV disease or died of melanoma to those of primary melanomas from 200 patients with similar risk profiles that have not progressed after at least 10 years of follow-up. We will identify genetic alterations and combinations thereof that are significantly associated with outcome independent from known variable and formulate a candidate algorithm that distinguishes high-risk and low-risk primaries. We will validate this algorithm on an independent cohort of 300 primary melanomas with the goal to demonstrate that it outperforms current clinical parameters of prognostication.
Melanocyte stem cells and their progression to malignancy and metastasis
MRA Established Investigator Award
Elaine Fuchs, Ph.D., The Rockefeller University
Often arising in sun-damaged skin, melanomas are among the most aggressive and life-threatening cancers world-wide. BRAFV600E mutations are found in ~70% of human melanomas. Although BRAF inhibitors initially appeared promising, chemo-resistance soon surfaced. While immuno-therapy offers new promise, only some patients respond. To expand therapy prospects, researchers have analyzed individual cells within bulk melanoma tumors, yielding important information about the genes expressed by melanoma cells. State-of-the-art genomics and transcriptomics on cultured melanoma cells revealed further hints about how melanoma cells respond to immunotherapy. While elegant, we still don't know how melanoma-propagating cells are spatially organized within these tumors, and whether their neighbors and/or their intrinsic properties determine whether a melanoma cell will respond to chemo or immune therapies. We also know precious little about the molecular drivers that trigger a normal melanocyte along the path to malignancy, metastasis and immune evasion. This proposal addresses these important, as yet unanswered issues. To do so, we have devised a strategy to isolate and characterize the melanocyte stem cells, which exist within the adult skin and which are responsible for generating the pigmented differentiated melanocytes which transfer melanin pigment to differentiating hair cells to give hair its color. As long-lived stem cells, these are the cells which over time can accumulate the mutations that lead to melanomas. By comparing the properties of these cells to their differentiated progeny and to melanocyte stem cells as they acquire BRAFV600E and other mutations that lead to malignancy, we will unveil new and important insights that could lead to early detection of melanoma and accelerate the rate of developing new and improved therapies based upon the various backup survival strategies that melanoma cells employ to evade existing chemo and immune therapies.
Molecular mechanism of UV-induced mutagenesis in melanoma
MRA Established Investigator Award
Chengyu Liang, M.D., Ph.D., University of Southern California
In 2016, approximately 76,380 new melanomas will be diagnosed in the U.S., and about 10,130 people will die from it. Moreover, the incidence of melanoma is on the rise in the U.S. and around the globe. The main risk factor for melanoma is exposure to natural/artificial ultraviolet (UV) light, which elicits DNA photodamage. If left unrepaired, this damage results in the accumulation of “UV-signature” mutations and induction of melanoma. UV-induced damage is repaired through the nucleotide excision repair (NER) pathway. However, the regulatory mechanisms that safeguard NER and its high importance for cumulative UV-like mutagenesis in melanoma remain elusive. This project will fill this gap, capitalizing on our recent discovery of the UV-protecting aspect of an autophagic modulator UVRAG (UV irradiation-resistance associated gene) and its inverse association with genetic instability in skin melanoma. We have identified UVRAG as a bona fide guardian of NER, antagonizing photodamage to prevent melanoma. We postulate that inadequate NER represents an important mechanistic link between UV-exposure and melanoma development. Hence, we will first delineate the molecular basis responsible for the UV-induced mutagenesis in melanoma, and then investigate the contribution of NER in antagonizing UV-induced oncogene-driven melanoma. Finally, given that cumulative mutagenesis provides non-self neo-antigens activating T cells, we will test the hypothesis that NER status may influence melanoma response to immune checkpoint therapy. This study will illuminate an unrecognized mechanism of NER in melanoma genetics and pathogenesis, having important implications for effective prevention and personalized treatment of this fatal disease.
Identification of active drugs for NF1-mutant, BRAF/NRAS-wildtype melanoma
Leveraged Finance Fights Melanoma-MRA Established Investigator Award
A. Thomas Look, M.D., Dana-Farber Cancer Institute
Melanoma accounts for the vast majority of skin cancer deaths. To date, most efforts for developing or improving targeted therapies have been focused on melanomas with mutations of the cancer-causing BRAF or RAS genes. However, these genes remain normal in at least 9% of aggressive cutaneous melanomas, which are caused by mutations of the NF1 tumor suppressor gene. Patients with this particular subtype of melanomas are unlikely to benefit from the newly approved BRAF inhibitors such as dabrafenib and vemurafenib, because the BRAF genes are normal in this subtype. There is a clear need to develop new precise therapies for these patients. The goal of this project is to fill the gap of our understanding of this understudied type of skin cancer and identify effective drugs that can be rapidly moved into clinical trials in patients with this disease. Capitalizing on our zebrafish model of the NF1-mutant subtype of melanoma, we have recently developed methods for rapid analysis of the effects of small molecule drugs on the tumor cells in the living fish. In our system, we test the drugs by adding them to the fish water for 6 days, with the drugs replenished every other day. In this project, we will use these zebrafish to test a collection of 978 FDA-approved drugs as well as drugs in phase I/II trials for other cancers to test their ability to kill NF1-mutant melanoma cells. These drugs include compounds that inhibit many different physiologic pathways. Our hypothesis is that inhibition of one or more of these pathways is likely to cooperate with inhibition of known targeted pathway inhibitors to kill NF1-mutant melanoma cells. We anticipate identifying one or more drugs that will show activity against the very aggressive NF1-mutant melanoma. The identified drugs can be rapidly “repurposed” for testing in melanoma patients because they are already in human use to treat other types of diseases or conditions.
Granzyme B imaging to predict efficacy of immunotherapy in melanoma
MRA Established Investigator Award
Umar Mahmood, M.D., Ph.D., Massachusetts General Hospital
Immunotherapy represents a promising breakthrough for melanoma patients who may not have other viable alternatives. Despite the promising success stories, the harsh reality is that in the best cases only 60% of patients will respond. One of the core challenges facing oncologists is who will receive what treatment. Currently, there is no way to predict who will respond to immunotherapy, which puts patients at risk for potentially severe side effects, including death. Because immunotherapies utilize a patient’s own immune system, there can be dramatic changes in the tumor in relatively short time periods, which make trying to predict who will respond by biopsy or genetic screening very difficult. Because of this, we set out to utilize a technique already utilized to detect millions of cancers, termed positron emission tomography (PET) to help predict response to immunotherapy. Traditional PET uses radioactive sugar to detect cancer cells that need more sugar to grow, but immune cells that invade the tumor also cause increases in sugar uptake. We therefore had to find a different way to measure response to immunotherapy, so we chose a molecule termed granzyme B, which is released by immune cells and directly kills tumor cells. When we looked at granzyme B levels by PET imaging in mouse cancer immunotherapies, we were able to predict with great accuracy those mice that would survive, even before changes in the tumor size. We also checked human melanoma samples and found that granzyme B was higher in patients who responded, and that our PET agent could detect it. We plan to investigate different combinations of immunotherapy in mouse models of melanoma, in addition to determining the best time point to perform PET imaging in a planned clinical trial. We believe that granzyme B PET imaging may allow more effective therapeutic prescription and prevent unnecessary suffering in melanoma patients in the near future.
Development of YAP inhibitors to modulate regulatory T cell in melanoma
MRA Established Investigator Award
Fan Pan, M.D., Ph.D., Johns Hopkins University-School of Medicine
In the United States, metastatic melanoma cases and death rates are increasing at a disturbing rate, prompting efforts to develop more effective treatments. A strategy called “immunotherapy” involves compelling a patient’s own immune system to recognize and kill tumors. Unfortunately, obstacles to effective immunotherapy arise from both tumor cells themselves and certain cells of immune system. Through a number of poorly understood processes, tumors promote their own growth while suppressing the immune system’s tumor-killing capacity. Regulatory T cells (“Tregs”) are a cell type that is an important component of these “anti-inflammatory” responses. Research has shown that depletion of these cells can cause severe autoimmune diseases. These cells, however, are also employed by the tumor to evade the immune system. Depletion of Tregs in the tumor microenvironment has been shown to improve anti-tumor immunity in melanoma patients and have profound effects on clinical outcomes. Therefore, precisely targeting Tregs to enhance anti-tumor immunity without destroying the natural function of these cells represents an attractive alternative strategy to improve patient outcomes. Recently, we have identified a molecule named “YAP”, which is highly expressed in the Treg subset, and is critical for Treg function. We have also found that mice without Yap develop very small or no tumors, indicating that inhibition of Yap may be a promising therapeutic strategy. In line with these results, we propose to test novel therapeutic regimens in a mouse model of a highly aggressive melanoma. In addition, we believe that Yap inhibitors may be used effectively in combination with existing immunotherapeutic agents, such as checkpoint blockade, which is a highly effective immunotherapy currently in clinical use. In the process of testing this approach, we will shed light on the enigmatic tumor-immune system interaction.
Non-invasive imaging of the anti-melanoma immune response
Leveraged Finance Fights Melanoma-MRA Established Investigator Award
Hidde Ploegh, Ph.D., Children's Hospital Boston
Immunotherapy has changed how melanoma is treated today: moving from an academic specialty to what is now first line treatment, the use of antibody-based therapy has produced remissions and even cures. Disappointingly, not all melanoma patients respond to this form of treatment, but the reasons for such failure have yet to be identified. A positive factor is the presence of the target recognized by the antibody used for therapy. For example, when anti-PD-L1 antibodies are used, the presence of PD-L1 itself can be a positive prognostic indicator, with the possibility of a favorable therapeutic outcome. We now know that the presence of killer T cells bearing the CD8 marker in the tumor is probably the best positive prognostic indicator. This type of analysis requires taking tissue samples and analyzing each sample microscopically. In the case of metastatic melanoma, it is not usually possible to sample each individual metastasis, a further complication being the heterogeneity even within a single lesion. We have developed small antibody fragments that can be equipped with a radioactive tracer, thus making them suitable for use in positron emission tomography/computer assisted tomography (PET/CT). Our preliminary data show that the distribution of CD8 T cells within the tumor can predict the response to therapy with checkpoint-blocking antibodies. In this proposal we seek to extend these observations to other markers on immune cells, in the context of tumor development and in response to immunotherapy with antibodies. The results should help develop a strategy that would allow better prognosis and improved therapeutic outcomes without the need for invasive surgical procedures.
Advancing SBI-756, a translation initiation inhibitor, for melanoma therapy
Sokoloff Family-MRA Established Investigator Award
Ze'ev Ronai, Ph.D., Technion Israel Institute of Technology
Here, we propose an alternative approach to killing tumor cells that exploits one of the critical cellular machines; namely, the control of RNA translation to proteins. While essential for the survival of every cell, tumors “abuse” the translation machinery by using its “turbo mode.” Thus, a number of initiatives focused on identifying drugs that can turn off the turbo mode. Because normal cell function does not require the turbo-mode, such drugs could cut off translation events that drive the tumor without affecting normal cells. Work in our lab over the past 7 years, initially supported by an MRA award, has led to the development of a small molecule named SBI-756, that inhibit melanoma regardless the types of genetic mutations. SBI-756 also inhibits the development of vemurafenib resistance in melanoma in animal models. SBI-756 acts by interfering with one of the key components of the translation initiation complex. To date, we have developed more than 100 analogues of SBI-756 to improve its specificity and efficacy. However, there is an urgent need to understand precisely how SBI-756 binds to its cellular target, eIF4G1. Mapping the interaction at the molecular level will help to advance the development of SBI-756 and establish its utility for clinical evaluation. This application proposes to use genetic models to map the specific site(s) on eIF4G1 required for binding of SBI-756 and to use this information to create melanoma cells that produce a mutant form of eIF4G1 that cannot be inhibited by SBI-756. Such cells will teach us about the precise mechanism used by SBI-756 to affect the translation machinery. The cells will also provide us with more precise tools to understand and advance the development of SBI-756 analogues. The drugs will be evaluated using mouse models of melanoma with different genetic mutations. This work will advance an innovative and attractive therapeutic modality for overcoming therapy resistance and tumor heterogeneity in melanoma.
Cell of origin as a driver of heterogeneity in melanoma
Anna-Maria and Stephen Kellen Foundation-MRA Established Investigator Award
Lorenz Studer, Memorial Sloan-Kettering Cancer Center
Melanoma is characterized by its striking heterogeneity at clinical, histological and genetic levels that underlies drug resistance and metastasis, the major causes of death from the disease. The mechanisms underlying heterogeneity are poorly understood. One factor that has received little attention is the cell of origin in melanoma. Melanocytes, long thought to be cell of origin, are now recognized to exist along a phenotypic spectrum within the adult epidermis, ranging from early neural crest-like progenitors, to melanoblasts and fully differentiated melanocytes. In this proposal, we will address how the cell of origin impacts the molecular identity of melanoma. We will utilize two complementary systems: Human pluripotent stem cell (hPSC) and zebrafish models of melanoma. The hPSC system, developed in the Studer lab, allows for precise derivation of neural crest/melanoblast/melanocyte cells, which can be transformed using oncogenic drivers such as BRAFV600E. In parallel, the White lab has established zebrafish models of melanoma, in which BRAFV600E is driven by stage-specific promoters representing neural crest/melanoblast/melanocyte cells. Based on the complementary strength of the two approaches, we will perform profiling studies to determine whether cell of origin impacts the molecular identity of the resulting tumors. We will further stratify the stage-specific tumors for molecular and clinical parameters observed in primary melanoma including transcriptional subclasses, patient survival data, BRAF responsiveness and immune cell response. Our study sets the stage for a new experimental approach to study melanoma heterogeneity and a platform to test novel targeted therapeutic approaches.
Multiplex biomarkers for response to PD-1/PD-L1 checkpoint blockade
Leveraged Finance Fights Melanoma-MRA Established Investigator Award
Janis M. Taube, M.D., Johns Hopkins University School of Medicine
Melanomas may express the PD-1 and PD-L1 molecules that are normally expressed by the immune system to turn off an immune response. New therapies have been designed to block these molecules, protecting the immune system and often resulting in an anti-tumor immune response. Notably, studies have shown that features such as PD-L1 expression by tumor, the number of mutations in the underlying tumor, and the density of anti-tumor immune cells are associated with response to anti-PD-1. Most of these predictive features have been promoted as single factors and none are absolute. We plan to expand on previous analyses by mapping the location of a number of different immune cell types, their associated immunoactive proteins, such as PD-L1 and other checkpoint proteins (PD-1, LAG-3, TIM-3, PD-L2, etc), and their secreted factors to specific coordinates in the tumor microenvironment. We will also characterize the mutations in the underlying tumor. We will then associate these features with response or resistance to anti-PD-1 using multifactorial models. As the coordinate mapping of multiple parameters as well as the associated genomic analyses generate very large datasets, we are collaborating with JHU Astrophysicists who handle open-access, spatially-resolved petabyte data from Deep Sky Survey and Hubble Space Telescope images. Big Data statistical approaches will be used to query the characterized features on a discovery set of melanomas. The resultant multiplex model will then be tested on a separate, validation cohort. Finally, the validated model will be applied to melanomas from patients who are recognized as “hard to treat”, such as transplant patients who are immunosuppressed to assess whether they could also be candidates for anti-PD-1. A better understanding of the multiple factors associated with response to anti-PD-1 treatment will allow for improved patient selection as well as the rational combination of anti-PD-1 with other therapeutic agents.
Single-cell biomarkers for engineering T-cell function and metabolism
MRA Established Investigator Award
Navin Varadarajan, Ph.D., University of Houston
Melanoma is one of the leading causes of cancer related deaths within the United States. Immunotherapy that aims to harness the impressive capabilities of the human immune system has emerged as one of the frontline therapies for metastatic melanoma. With the FDA approval of antibodies against the CTLA-4 and PD-1, T-cell immunotherapy is now an established cancer therapy. The success of these antibodies targeting immune checkpoints highlights the clinical potential of reversing T-cell inhibition; however, mechanistically, these immunomodulatory antibodies critically rely on the existence of infiltrating tumor resident T cells. To address this dependency, adoptive cell therapy (ACT), based on the infusion of expanded and/or modified T cells, has the potential to target tumors with poor immune cell infiltrates (non-inflamed, or “cold” tumors). ACT that works directly to harness the cancer-eliminating properties of patient T-cells has shown considerable promise with clinical response rates of ~20-30% even in stage IV melanoma refractory to all other treatment methods. Regrettably however, this methodology results in a wide range of patient outcomes from complete remission to no identifiable benefit. The unpredictable and variable nature of the clinical effectiveness of ACT is a major disadvantage to an otherwise promising therapeutic treatment. One of the major barriers associated with successful ACT is the tumor microenvironment that known to harbor gradients of nutrients essential for the survival of all cells including T cells. This proposal aims to overcome this barrier by engineering T cells that have the capacity to execute anti-tumor functions under conditions of nutrient starvation. This will enable the clinical translation of modified T cells in ACT that can lead to predictable patient outcomes.
Adipocytes in the melanoma microenvironment
MRA Established Investigator Award
Richard White, M.D., Ph.D., Memorial Sloan Kettering Cancer Center
It is increasingly clear that cancer cells do not exist in a vacuum – they are surrounded by numerous other cell types in the body that exert dominant effects on their behavior. Collectively, all of the cells surrounding the cancer cells are called the tumor microenvironment. The most compelling evidence for the importance of the tumor microenvironment is the dramatic success of immunotherapy in melanoma. Outside of the immune system, little attention has been paid to the large number of other cell types in the tumor microenvironment, and we feel that many of these other cell types would make excellent therapeutic targets. To address this question, my laboratory has developed an unusual model for studying melanoma – the zebrafish. Although not a typical model for cancer, the zebrafish is transparent, offering the unique capacity to visualize cancer from the moment the tumor begins all the way through to metastatic disease. Using this model, we have uncovered an unexpected interaction between melanoma cells and the fat cells that are normally present beneath the skin. We have shown that these fat cells, also called adipocytes, strongly promote melanoma cell growth and invasion, making the tumor cells more metastatic by directly transferring fat droplets into the melanoma cells. We have identified a specific protein that mediates this fat transfer and have found a chemical that can block it, thereby stopping growth of the tumors. In this grant, we wish to fully explore how these microenvironmental adipocytes interact with the melanoma cells, using both our zebrafish models but also human tissue specimens. We want to determine whether the chemical we discovered has the potential as a new therapeutic target in melanoma, and in which patients it is likely to be most useful. We believe this proposal takes advantage of a novel system for studying melanoma, and will lead to clinically meaningful insights that can be used to target a new member of the tumor microenvironment.
Targeting TEAD autopalmitoylation in YAP-dependent uveal melanoma
The Samuel Ming-Sum Fisher Memorial Award-MRA Established Investigator Award
Xu Wu, Ph.D., Massachusetts General Hospital
Uveal melanoma is a highly malignant form of cancer developed in the eye, and is the most common primary cancer of the eye. Although the primary tumor can be treated effectively, majority of the tumors relapse and metastasize to distal organs, such as liver. Metastatic uveal melanoma lacks effective treatment and carries very poor prognosis. Therefore, it is very important to develop new and effective therapies to treat metastatic uveal melanoma. Recent genetic studies have revealed that more than 80% of uveal melanoma carries mutations on GNAQ/GNA11 genes, which resulted constitutive active G proteins (Gq/11). It has been shown that activated G proteins promote tumor growth by activate oncogene YAP. In our lab, we have been dedicated to develop new strategies to inhibit cancer promoting YAP. We recently found that the partners of YAP, called TEADs, possess “enzyme-like” activities, and could be “druggable”. We have identified novel TEAD inhibitors, which effectively block YAP-TEAD activities in uveal melanoma cells. We propose to further understand how TEAD-YAP is involved in metastatic uveal melanoma, and to test our inhibitors in uveal melanoma cell in animal models. Our work could potentially provide new therapeutics for YAP-dependent uveal melanoma.
BRAF fusions as a therapeutic target in melanoma
Boris Bastian, M.D., University of California, San Francisco
Melanoma therapy has been improved by the introduction of drugs that inhibit mutated genes such as BRAF. However, a significant subset of melanomas does not seem to harbor similar alterations. We and others recently described BRAF fusion genes in a portion of these ‘pan-negative’ melanomas. BRAF fusions are composed of a portion of BRAF encoding the active part at the end which is fused to a range of different genes in the front. The resulting chimeric genes are constitutively active kinases that promise to be good targets for therapy. However, sensitive methods for their detection in patient samples are lacking and our understanding of their method of action is limited. We address these shortcomings by (1) Establishing clinical-grade detection methods for BRAF fusions by increasing the sensitivity of sequencing methods of DNA and RNA, developing improved fluorescence in situ hybridization (FISH) tests and single molecule expression analysis approaches (Nanostring nCounter) applicable to clinical samples, (2) Determining the clinical, histologic and genetic correlates of melanomas driven by BRAF fusions using a cohort of 400 patients with ‘pan-negative’ melanoma, which we are profiling by targeted sequencing of DNA and RNA as part of a separately funded clinical trial (3) Establishing principles to guide treatment by using our expanding collection of currently 6 melanoma cell lines with BRAF fusions as well as suitable cell models in which we introduce the different BRAF fusions genes to elucidate how they activate key signaling pathways that make cells grow in an uncontrolled fashion. We will treat the melanoma cell lines with different types of RAF and MEK inhibitors. We will validate the most active compounds and/or combinations in a mouse model using the melanoma cell lines with BRAF fusions we have identified. Our proposal addresses an important unmet need, which is how to identify patients whose melanomas are driven by BRAF fusions and how to treat them.
Harnessing the epigenome for melanoma oncogene discovery
Mount Sinai-MRA Established Investigator Award
Emily Bernstein, Ph.D., Icahn School of Medicine at Mount Sinai
Cutaneous melanoma is a deadly form of skin cancer that originates from specialized pigment-producing cells called melanocytes. Despite preventive campaigns and medical advances in treatments, melanoma remains an incurable disease. Historically, cancer has been studied at the DNA level and key mutations of the DNA sequence have been linked to melanoma biology. However, these alterations in gene products cannot entirely explain the processes that cause melanocytes to grow out of control and gain the potential to spread through the body - both hallmarks of cancer. It is now clear that chromatin factors (the proteins that package DNA) are important in cancer development as well. Unfortunately, these chromatin-associated mechanisms remain poorly understood. Here we propose to use sophisticated chromatin-based methods to identify ‘enhancer regulatory elements’ in melanoma – these elements are important for regulating genes during human development as well as in cancer. In fact, recent studies in other cancers (such as leukemia), have utilized similar enhancer studies to identify known as well as novel genes that are important for promoting cancer. The proposed study aims to characterize enhancer elements genome-wide using innovative chromatin and computational approaches, and we will do so using cells derived directly from human tissues that closely resemble the tumor biology. Using normal melanocytes as a control for these studies, we will elucidate the genes that enhancers control in the development of malignant melanoma. These studies will lead to the identification of new factors that promote melanoma – an instrumental step in the development of novel targeted treatment strategies for this disease.
Enhancing immune therapy for brain metastases with focused ultrasound
UVA-MRA Established Investigator Award
Timothy N Bullock, Ph.D., The Rector and Visitors of the University of Virginia
The effectiveness of immunotherapies for melanoma is exciting and revolutionary, but has yet to be fulfilled in the clinically challenging realm of brain metastases. This hurdle is related to aspects of the relationship between the brain, tumor metastasis and the immune system that limits immune cell and antibody access. Until effective delivery strategies are developed and translated to the clinic, a considerable fraction of cancer patients will receive little to no benefit from emerging immunotherapies. Access to the brain metastases by drugs and cells is restrained by the blood-brain barrier, which regulates the trafficking of molecules to the brain, and the blood-tumor barrier, which (due to the malformed blood vessels of the tumor) leads to high interstitial pressures that reduce the natural flow of molecules into the tumor. We propose to deploy a non-invasive novel technology in melanoma brain metastases: image guided microbubble-mediated focused ultrasound. Focused ultrasound, in conjunction with gas-filled microbubbles, uses sound waves to induce damage specifically in the tumor microenvironment. We intend to develop this technology to disrupt an environment that normally suppresses immune response and does sufficient damage to the tumor to increase the natural immune response to tumors. We will subsequently determine whether brain metastases can be further controlled by co-deploying leading edge immunotherapies that increase the activation, survival and function of tumor-killing immune cells.
Beta defensin 3: UV induction and effects on melanocyte genome stability
University of Kentucky Markey Cancer Foundation-MRraA Established Investigator Award
John D'Orazio, M.D., Ph.D., University of Kentucky Research Foundation
Though great strides are being made in treating melanoma, the majority of patients with advanced disease still have a very poor long-term prognosis. Our laboratory is focused on determining genetic risk factors that place individuals at risk for melanoma so that preventive therapies can be developed to prevent the disease from developing in the first place. We target our efforts on the melanocortin 1 receptor (MC1R) present on the surface of melanocytes because it is a key molecule that determines melanoma risk. People with inherited defects in MC1R, who number in the millions in our country alone, are sun-sensitive and have a four-fold increased lifetime melanoma risk. The MC1R pathway is an attractive research target because it potentially a good pharmacologic target. When MC1R is activated, melanocytes protect themselves and the skin much better against UV radiation. This is important because UV is a major cause of melanomas, as evidenced by the abundance of UV-signature mutations found in melanomas and the strong correlation between UV exposure and disease. One way in which MC1R protects melanocytes from UV mutations is by enhancing their ability to repair UV DNA damage so that mutations (and melanoma) don’t develop. In this proposal, we will determine how beta defensin 3 (BD3), a relatively newly identified regulator of MC1R activity, is made in response to UV and what impact BD3 has on melanocyte DNA repair responses. The results from these studies will clarify an individual's melanoma risk and will serve as a platform for the development of rational UV-preventive strategies based on targeting the BD3 - MC1R signaling axis naturally present in the skin.
Role of Fucosyltransferases in Melanoma metastasis
NYULMC-MRA Established Investigator Award
Eva M Hernando, Ph.D., New York University School of Medicine
Proteins in the cell membrane often acquire sugar modifications, which shape the interactions between a tumor cell and its environment. For instance, these sugar branches modify the recognition of tumor cells by the immune system and their ability to invade and colonize other organs. We have found that certain enzymes that modify those sugar branches (called fucosyltransferases) are altered in melanoma tissues during melanoma dissemination throughout the body. We plan to elucidate how those enzymes impact the aggressive behavior of melanoma and assess if their inhibition may hold therapeutic benefit for these patients. Moreover, because sugar branches can be highly specific of tumor cells and trigger an immune response, they are ideal candidates for development of biological inhibitors (i.e. vaccines or antibodies). Therefore, our studies have the potential to open an unexplored avenue for melanoma therapy, alternative or complementary to current targeted or immunomodulatory strategies.
Identification and characterization of drivers of melanoma brain metastasis
Leveraged Finance Fights Melanoma-MRA Established Investigator Award
Sheri Holmen, Ph.D., University of Utah
The increasing incidence of melanoma and mortality associated with advanced stages of the disease are cause for concern. Excitingly, the efficacy of new therapies in melanoma have significantly shifted the treatment paradigm for this disease. However, metastatic spread of melanoma to the brain is one of the most common and devastating complications of these therapies; up to 75% of patients with advanced stages of melanoma will develop brain metastases and the prognosis for these patients is very poor. The significance of this problem is emphasized by the finding that more than half of all melanoma deaths are due to brain metastases. It is therefore critical that we define the molecular mechanisms underlying melanoma brain metastasis such that specific factors that augment or suppress the development of these lesions can be identified. Previously, studies of melanoma brain metastases were hampered by the lack of models that spontaneously develop metastases similar to human melanoma. We recently developed a novel mouse model of melanoma that closely mimics the human disease including the development of brain metastases. The goal of this project is to study key proteins implicated in promoting brain metastases and determine whether inhibiting them will suppress melanoma growth and metastasis. An increased understanding of the biology of brain metastases will aid the development of more effective therapies for these patients. We expect our findings to not only have a positive impact for melanoma patients but also patients with other cancers that spread to the brain.
Advancing the understanding and treatment of uveal melanoma using zebrafish
Jacqueline Lees, Ph.D., Massachusetts Institute of Technology
Uveal melanoma is the most common adult primary eye tumor, with about 2500 new cases diagnosed in the US each year. Although successful treatments exist for the primary tumor, approximately 50% of uveal melanoma patients develop metastases within 15 years at which point their average survival is 6 months. Thus, there is desperate need for improved understanding and treatment of this cancer. To this end, we have generated a model of uveal melanoma in zebrafish. Our fish develop skin and uveal melanomas with high frequency and die as a result of these tumors. Importantly, the cancers in our model are driven by the same genetic changes that are responsible for inducing human uveal melanomas, which are activating mutations in the GNAQ and GNA11 genes. The goal of this project is to use our zebrafish model to understand the biological events that are responsible for promoting the formation and progression of uveal melanoma. Specifically, we will identify the changes that occur within the zebrafish tumors and compare them ones that occur in human uveal melanomas. This will allow us to identify events that are fundamentally important in promoting tumor development, including the drivers of high-risk versus of low-risk disease. Finally, we use our zebrafish models to test candidate genes and therapies, and to conduct screens for novel inhibitory drugs. Our key goal is to use our zebrafish model to improve the treatment of uveal melanoma.
Mechanism of action of SLAMF6 and its potential role in immunotherapy
Michal Lotem, M.D., Hadassah Medical Organization
Active immunotherapy represents an approach to the treatment of cancer based on eliciting effective immunity against tumor associated molecules (antigens). An imperative requirement for optimizing immunotherapy involves effective triggering of additional molecules, called co-stimulatory signals. In order to enhance the activity of lymphocytes against cancer, the search for such compounds is currently the focus of much ongoing research. We have recently identified such a co-stimulatory molecule, called SLAMF6 (signaling lymphocytic activation molecules 6). A unique characteristic of this molecule is its ability to stimulate lymphocytes by interaction with other (identical) SLAMF6 molecules. In preliminary studies performed by us, we have identified SLAMF6 self-receptors as a potential target for improving melanoma immunotherapy. The results showed that SLAMF6 markedly reduces lymphocyte cell death due to cell activation and gives preference to the growth of specific anti-melanoma lymphocytes from a population of cells extracted from a tumor biopsy. Importantly, SLAMF6 serves as a substitution for IL-2, a compound necessary during melanoma immunotherapy, which is mandatory for immune cell survival but also very toxic to the patient. These exciting observations, obtained in human and mouse studies, provide the rationale for our overall hypothesis, that SLAMF6 is a highly relevant, albeit unexplored, target for the immunotherapy of melanoma. The aim of this project is to evaluate the efficacy of SLAMF6 for melanoma immunotherapy in animal models, to study its mechanism of action and to develop a molecule which targets SLAMF6 in order to activate it as a novel melanoma treatment. The results from this study may have important implications for cancer immunotherapy, and could lead to clinical application within a relatively short period of time.
CARMA1 in Control of Regulatory T Cell Function in Melanoma
BMS-MRA Established Investigator Award in Immunotherapy
Thorsten Mempel, M.D., Ph.D., Massachusetts General Hospital
Malignant melanoma is a form of cancer that in principle is prone to rejection by the immune system. However, a variety of mechanisms of so-called immunological self-tolerance, which are in place to prevent our immune system from attacking our own healthy tissues, also prevent it from effectively attacking melanoma. Central players in establishing immunological tolerance are so-called regulatory T cells (Treg), which also infiltrate the tumor tissue and prevent other immune cells from killing the cancer cells. Efforts to strengthen anti-tumor immune responses by depleting Treg in cancer patients can be effective, but are limited by the side effects resulting from the concomitant loss of self-tolerance. In this project we are evaluating in a mouse model of malignant melanoma the role of a protein expressed by T cells, called CARMA1, in supporting the activities specifically of those Treg that prevent the immune system from rejecting tumors. We hypothesize that removing this protein from Treg will cause them to lose their functions, which in turn will unleash the activities of other immune cells that can then control or even eliminate the tumors. If our studies confirm our hypothesis, this would suggest that targeting CARMA1 in Treg could be a new therapeutic approach for malignant melanoma.
Development of an equipotent inhibitor of mutant RAF monomers and dimers
Leveraged Finance Fights Melanoma-MRA Established Investigator Award
Neal Rosen, M.D., Ph.D., Memorial Sloan Kettering Cancer Center
Melanoma is a disease that results from mutations in genes that control cell growth. The mutant proteins encoded by these genes are hyperactivated and this leads to the unregulated growth that is characteristic of cancer. The BRAF gene is mutated in more than 50% of melanomas and is probably necessary for their abnormal growth. RAF inhibitors have been developed and are extremely effective in treating melanomas with this mutation. However, these responses are temporary; resistance of the tumor to these drugs almost always develops. We have found that RAF mutants exist in two forms. Current RAF inhibitors work only against the first form, a so-called `monomer.’ They do not work against mutants that take the second `dimer’ form. Furthermore, we find that resistance to the current drugs often occurs because of genetic events that cause the sensitive monomer to become a resistant dimer. We have now identified a drug that inhibits both forms of BRAF. In experimental models, it works against all tumors with activated BRAF mutants and in tumors that have become resistant to the current inhibitors. We believe that this type of drug could be significantly more effective than those that are currently in use and could lead to a major improvement in therapy of this disease. In this proposal we plan to aggressively characterize these compounds in preclinical systems and mouse models so that the best compound can be identified and tested in patients.
Prognostic and therapeutic impact of lymphovascular niches in melanoma
Maria Soengas, Ph.D., Spanish National Cancer Research Centre
The long-term goal of this proposal is to identify long-range signals induced by melanomas at early stages of development as a strategy to discover new progression biomarkers and therapeutic targets. The rationale stems from the need to address one of the key defining features of this disease, namely, the fact that seemingly thin lesions bear high risk for dissemination to neighboring (sentinel) lymph nodes, and ultimately, to distal sites. We will center on the ability of aggressive melanoma cells to remodel the lymphatic nodal vasculature and promote the so-called lymphovascular niche. This is a protective microenvironment that avoids recognition and attack by the immune system. Intriguingly, while lymph node removal is considered a standard of care for thick melanomas, this procedure cannot consistently improve patient survival. Therefore, alternative mechanisms of melanoma metastasis have been suggested, but their identity remains elusive. A translationally-oriented experimental plan has then been designed to address the following fundamental questions: (i) how are distal metastases established in melanoma, (ii) are lymphovascular niches engaged at these sites, and if so, (iii) how to deactivate them without secondary toxicities? Our experimental plan combines unique mouse models for whole-body imaging of melanoma progression, with a large set of serum specimens and tissue biopsies obtained in the context of active clinical trials for FDA-approved and experimental compounds. First, we will validate the prognostic value of specific proteins we found with unexpected roles in distal lymphangiogenesis. Secondly, we will define the mode of action of novel pharmacological agents that deactivate lymphovascular niches and synergize with therapeutically-relevant immune-checkpoint blockers. We expect to contribute to the melanoma field with new indicators of metastatic risk, and by setting the proof of concept for the clinical testing of new investigational compounds.
Elucidating and Targeting Super-enhancers In Melanoma
Leveraged Finance Fights Melanoma-MRA Established Investigator Award
Hensin Tsao, M.D., Ph.D., Massachusetts General Hospital
Extraordinary progress has been made in advancing treatments for metastatic melanoma. There is now unequivocal evidence that drugs which inhibit melanoma signaling proteins (BRAF, MEK) or antibodies which stimulate the immune system (anti-PD1/PDL1) can both prolong survival. Nevertheless, the early control achieved with molecular therapies is often transient while the extraordinary response to the immune agents is far from universal. Many studies have implicated other melanoma promoting proteins (i.e. oncogenes), which represent opportunities for therapy. Yet, these oncogenes are often “undruggable” - i.e. the function is unyielding to small molecule inhibitors or that the protein is inaccessible to therapeutic antibodies. Fortunately, recent breakthroughs may be ushering in a sea-change. Some cancer cells appear to mass produce key oncogenes through “super-enhancer” elements. These super-enhancers ramp up the production of growth proteins thereby sustaining the cancer’s “addiction” to these oncogenes. A novel small molecule inhibitor (THZ1) against the CDK7 protein was recently shown to preferentially dismantle these super-enhancers, shut down the oncogene source flow (i.e. “oncogene starvation”) and potently kill leukemia cells. Preliminary data from the Applicant document for the first time the clear presence of super-enhancers within one of the “master” oncogenes in melanoma- MITF. Moreover, treatment of melanoma cells with the novel CDK7 inhibitor led to a rapid depletion of MITF and massive melanoma cell death. As the landscape of oncogene production and super-enhancers is poorly mapped in melanoma, this grant will fill in this landscape in by (i) focusing in on one master oncogene (i.e. MITF) in greater mechanistic detail, (ii) performing a comprehensive analysis of super-enhancers in a broad range of melanomas and (iii) advancing the therapeutic goal of oncogene “starvation” through CDK7 suppression.
The Tgfb2/Rrp15 locus is an innate modifier of melanoma development
Graeme Walker, Ph.D., QIMR Berghofer Medical Research Institute
Treatments for melanoma are aimed at late stage disease. It would be important to develop treatments that can strengthen a person’s ability to prevent growth of the tumor. This would be particularly useful after excision of early stage melanomas, where undetectable tumor cells can later grow to cause life-threatening disease. One could approach this by determining the physiological mechanisms by which the body normally works against melanoma development. This should be feasible using a genetic strategy, but it is difficult to determine why some high risk individuals (e.g. with many nevi) develop melanoma, and some do not, because of large individual differences in lifestyle and particular, different interventions and treatments. We have embarked on a novel strategy to find such differences. In an MRA-funded pilot study, we used our novel collection of hundreds of mouse strains that enable rapid mapping of disease genes. We have crossed many of these strains with our model that carries two melanoma-causing mutations. Remarkably, we found that some strains developed melanoma very quickly, and others very slowly or not at all (the latter were resistant because they had naturally-occurring genes that prevent melanoma). We will find the genetic and biological differences between the susceptible and resistant groups of mice. These natural differences will be used to develop ways to prevent or slow melanoma growth. One gene we have discovered greatly exacerbates melanoma development, and we now plan to characterize it to help us understand how it influences tumor growth. This gene is also associated with melanoma survival in human patient cohorts. We will utilize the flexibility of the mouse system to reveal natural biological processes that modulate melanoma growth. Our aim is to validate the gene and its mode of action, then to test candidate therapeutic options in the animal model, opening the way for initial clinical trials in high-risk patients.
Kinetics and effects of vemurafenib on intratumoral immunity
Michael Atkins, M.D., Georgetown University
Approximately 40-50% of malignant melanomas contain a mutation in B-Raf called (BRAFV600E). This results in activation of a critical intracellular pathway leading to unrestrained tumor growth. Recently, highly selective inhibitors of BRAFV600E have shown significant antitumor activity in patients with BRAFV600E mutant melanoma and one agent, vemurafenib, received FDA approval in August 2011. Despite this major treatment breakthrough, few patients experience complete regression of tumor and all patients will eventually develop disease progression. Thus, achievement of more meaningful clinical results will require identification of ways to extend or enhance the efficacy of drugs like vemurafenib. Immunotherapy (eg interleukin 2, ipilimumab, and PD1 antibody) represents an alternative treatment strategy for patients with melanoma that can induce durable tumor regressions in a small subset of patients. Preliminary evidence suggests that blocking BRAF enhances immune recognition of melanoma. These results have stimulated interest in combining BRAF inhibitors with immunotherapy as a means to sustain their efficacy. However, critical information about the timing and nature of the immune effects of BRAF inhibition is needed to be able to rationally design such combination protocols. The present proposal will study the effects of vemurafenib on tumor immunity (mechanisms and timing of immune cell infiltration, specificity and function of these cells, and the effect of therapy on immune regulatory pathways, particularly those associated with treatment response); thereby laying the groundwork for such combination studies.
Development of effective melanoma combination therapies
MRA-Melanoma Research Foundation Established Investigator Award
Marcus Bosenberg, M.D., Ph.D., Yale University
In 2010 for the first time, two different treatments were shown to make more than half of treated melanoma patients live longer. One of the therapies is anti-CTLA4 (also known as Ipilimumab or Yervoy), which works by helping the immune system fight melanoma. The second therapy is vemurafenib (also known as PLX4032), which blocks the BRAF protein that is altered in about half of melanomas. Despite these two very important advances, neither of these therapies cure the vast majority of advanced melanoma patients. It is possible that combining these two therapies will result in higher response rates in melanoma patients. Clinical trials for melanoma patients of some combinations involving these two agents are currently being planned and will likely begin soon. In order to better understand how the combinations work, we propose to comprehensively test combinations of these promising therapies in our melanoma models. We hope to understand why some patients respond better than others and why some combinations are more effective than others. This will help to design more effective future clinical trials that result in more frequent and longer responses and to better predict which patients are likely to benefit most from combination therapies.
Targeting inducible invasive cells in melanoma
Jonathan Cebon, Ph.D., FRACP, Ludwig Institute for Cancer Research,
Although newly emerging therapies have shown early successes in melanoma, treatment failure is common. Many recent reports have identified mechanisms of resistance and the development of new gene mutations can explain some cases. Commonly, however, resistance often develops without mutation. Here we demonstrate that treatment-resistant cells can be identified among melanoma cells growing in the laboratory. The features of these cells have been studied in detail and it appears that they are more likely to invade and therefore perhaps form secondary tumors. We have identified the role of molecules that are able to switch these cells on, and propose that blocking the emergence of these invasive resistant cells may prevent treatment failure. The aims of this project are therefore to: (1) further investigate the mechanisms which switch these cells on, (2) study the mechanisms that make them resistant to drug treatments and immune therapies, (3) block the principal switching mechanisms thereby identifying potentially new treatment options and (4) to test these approaches in melanoma-bearing mice to establish proof-of-principle before proceeding to clinical trials.
Targeting N-RAS as a therapeutic approach for melanoma
Douglas V. Faller, M.D., Ph.D., Boston University, B U Medical Campus
Current therapies for melanoma are inadequate. Activating mutations of a protein called N-RAS, or RAS-related pathways, are found more than 90% of melanomas. A novel therapeutic modality selectively targeting melanomas with activation of N-RAS signaling would make a significant impact on the way melanoma cancer is treated. Mutated, activated N-RAS is an attractive target for therapy of melanoma, but approaches aimed directly at RAS or its critical signaling pathways (which are required for the viability of normal cells) have had very limited success. Our "synthetic lethality" approach, however, exploits an Achilles' heel of melanoma cells containing a mutated, activated N-RAS - i.e., their absolute requirement for a survival pathway mediated by PKC-delta. In contrast, normal cells and tissues do not require PKC-delta for growth and development. This novel approach therefore "hijacks" the RAS signaling pathway, which normally promotes melanoma growth and progression, and redirects it to promote tumor cell death. Furthermore, it is likely that this targeted approach would also show activity in melanoma cells with oncogenic activation of a RAS downstream signaling pathway (BRAF), and in melanoma cells that have become resistant to the new BRAF inhibitors, thereby further broadening its potential application in melanoma.
The development of rational therapeutic regimens for NRAS-mutant melanoma
Levi A. Garraway, M.D., Ph.D., Dana-Farber Cancer Institute
Treatment options are still very limited for the >50% of metastatic melanoma patients whose tumors lack BRAF gene mutations. A large proportion of those tumors contain mutations in a different gene, called NRAS. Cancer-associated NRAS mutations bring about biological effects that are somewhat similar to BRAF mutations; however, the new melanoma drug vemurafenib is completely ineffective against tumors with NRAS mutations. The goal of this project is to identify new drug combinations capable of suppressing the growth of NRAS-mutant tumors. Leveraging the integration of "bleeding edge" experimental approaches, we will identify biological processes that can be effectively targeted in NRAS-mutant melanomas using existing and emerging medicines. Since some of the tumor-promoting effects of BRAF and NRAS mutations are similar, we will focus our discovery efforts around an exciting new class of drugs-called MEK and ERK inhibitors-which are capable of intercepting those effects. However, we will utilize both global and focused experimental approaches to discover new drug targets and candidate drugs that might form the basis of highly effective drug combinations. Finally, we will leverage in-depth genetic and molecular studies of clinical specimens to evaluate the validity of top-tier candidate drug targets and combinations. Upon completion of this research, we expect to identify new drug combinations that could move rapidly into clinical trials of patients with NRAS-mutant melanoma, thereby directly supporting the development of more effective treatment options for many melanoma patients.
Delineating BRAF inhibitor resistance mechanisms using proteomic profiling
Thomas G. Graeber, Ph.D., University of California, Los Angeles
An exciting advance in cancer therapies has been the development of drugs that specifically target the function of mutated genes. Regression of tumors targeted by these drugs has been remarkable. Drugs targeted at inhibiting the BRAF mutation in certain melanomas have led recent news headline reports due to spectacular tumor regression. Because of the specificity of these 'molecularly targeted' drugs, they have fewer clinical side effects. Unfortunately, most patients develop drug resistance in less than a year, and this traumatic aspect has also led news headlines. Cancer is a complex mix of interconnected events gone awry through mutations. We know much about the individual events, but we need a better understanding of how they function together, as a system, to cause malignancy. The future of molecular therapies relies on targeting multiple events, thus making it exponentially more difficult for tumor cells to gain the multiple mutations required to escape this coincident drug assault. This is somewhat analogous to anti-HIV drug cocktail therapies. In our work, we use technologies that concurrently measure thousands of events within cancer cells. In particular, we use mass spectrometry-based 'proteomics' to measure the activity of signaling proteins that drive aberrant tumor cell growth and survival. We then analyze the data using computational algorithms to identify points of susceptibility in the system. We are applying this approach to build a 'systems biology' perspective of how melanoma cells become resistant to BRAF inhibitors. These studies have broader implications since the BRAF V600E mutation occurs in 7% of all cancers.
Mechanisms of Cooperation between BRAF & PI3'-kinase Signaling in Melanoma
The Safeway Foundation is providing partial support for this project
Martin McMahon, Ph.D., University of California, San Francisco
In the past year, two new drugs have been approved for treatment of patients with advanced, metastatic melanoma: Vemurafenib and IpiIimumab. Although this represents a sterling success in treatment of patients with advanced disease, the holy grail of melanoma therapy would be an adjuvant regimen, administered after surgery, that eradicates residual melanoma cells thereby preventing metastatic disease from occurring. The current adjuvant regimen, high dose interferon, has marginal efficacy and severe side effects in many patients. Although we are starting to understand the inner workings of the melanoma cell, it remains unclear how best to eradicate melanoma cells after surgical removal of the primary tumor. Moreover, although there are many potential new melanoma drugs being developed, it is unknown which will work best as single agents and how such drugs might best be combined for maximum patient benefit. We have acquired access to a portfolio of promising new drugs to treat melanoma. Using bona fide human melanoma cells we will evaluate the role of various signal transduction proteins in the aberrant behavior of melanoma cells. In addition, using genetically engineered mice, we have invented a mouse model of melanoma, which will be used to test the ability of drugs administered in the adjuvant setting to prevent recurrence of metastatic melanoma. Studies proposed here offer the long-term prospect of evidence-based, effective and comparatively less toxic adjuvant chemotherapy for melanoma patients based on a molecular understanding of how signal transduction pathways contribute to the aberrant biology and biochemistry of the melanoma cell.
Enhancing immunotherapeutic activity of agonistic anti-CD40 antibodies
Jeffrey Ravetch, M.D., Ph.D., The Rockefeller University
Immunotherapy of melanoma has emerged as an area of great promise in the treatment of this disease. Activation of the patient's own immune response against his tumor is hampered by the ability of the tumor to suppress normal immune activation. To overcome this barrier approaches have been developed to induce immune cell activation by bypassing the normal controls that prevent T cells from becoming activated and eliminating the tumor cell. We have discovered that an important immune modulating molecule called CD40 can be triggered to induce killer T cells to eliminate melanoma through a pathway that involves co-engagement of an inhibitory molecule. This novel pathway results in considerable enhancement of tumor killing. We propose to investigate how this pathway operates and how to exploit this pathway to develop novel immunotherapeutics to treat melanoma.
Combinatorial approaches to treating mutant BRAF melanomas
Neal Rosen, M.D., Ph.D., Memorial Sloan-Kettering Cancer Center
In approximately 60% of melanomas, a gene called BRAF is mutated. The mutant BRAF protein is activated and responsible for the unregulated abnormal growth of these tumors. New drugs that inhibit the mutant BRAF protein have had remarkable therapeutic effects. They arrest the growth of the metastases and cause them to get smaller in almost all patients with mutant BRAF melanomas, an unprecedented result in this disease. However, only rare tumors are eradicated and the therapeutic effects are temporary; most tumors eventually recur. This proposal is aimed at devising new combination therapies for mutant BRAF melanoma that cause the tumor to regress more completely and for a longer time and that are effective after resistance to the RAF inhibitor develops. Our recent results suggest strategies to accomplish these goals. We have shown that treatment of tumors with RAF inhibitors causes other compensatory pathways to become activated, which attenuate the antitumor activity of the drugs. Our data suggest that more complete inhibition of RAF in combination with drugs that inhibit these compensatory pathways will be much more effective than either therapy alone. We have also recently attained a better understanding of how tumors become resistant to RAF inhibition, which suggests logical means of attacking the resistant cells. We now propose to use these strategies to attempt to get more complete, long-lasting control of mutant BRAF tumors and to develop treatments that are active in patients with resistant disease. The overarching goal is to radically change the natural history of melanoma.
Network models of signaling pathways and combinatorial therapy in melanoma
Chris Sander, Ph.D., Memorial Sloan-Kettering Cancer Center
Melanoma treatment has experienced recent breakthroughs in targeted therapy with the introduction of specific RAF inhibitors. The specific inhibition of mutant forms of BRAF in melanomas have generated particular excitement, only to be tempered as resistance evolved to these new therapies. This motivates us to pursue the design of combinatorial therapies to target melanomas that are insensitive to RAF inhibitor treatment. For this purpose, we have developed a novel network pharmacology approach. Our strategy combines systematic drug perturbation experiments, high-throughput protein array technologies and advanced computational algorithms to model cell type specific regulatory mechanisms leading to cancer formation. Using our theoretical cellular models, we are predicting novel combinatorial therapies tailored to achieve desired therapeutic responses in melanoma. In addition, we are carrying out comprehensive analysis of genome-wide molecular data from different stages and subtypes of melanoma in order to select a representative panel of cell lines from hundreds of established human melanoma cell lines at MSKCC to be used in our drug perturbation and modeling studies. Our analysis will be a resource for the research community by elucidating molecular signatures of melanoma stages/subtypes. By applying the computational biology methods we have adapted from statistical physics, we can construct pathway diagrams of the protein networks in selected melanoma cell lines. These pathway models can then be analyzed to nominate new drug combinations to test experimentally. Drug combinations that we discover can be rapidly translated to the clinic through our clinical collaborators.
Targeting XIAP for the treatment of melanoma
Hermann Steller, The Rockefeller University
All human cells have the ability to self-destruct by activating an intrinsic cell suicide program when they are damaged or no longer needed. The execution of this cell death program leads to a distinct form of cell death termed apoptosis. Apoptosis provides a crucial natural defense against cancer, and most cancer therapeutics activate this cell suicide program. Unfortunately, tumor cells find ways to escape apoptosis and continue to grow. A common mechanism by which tumor cells block apoptosis is through expressing high levels of anti-apoptotic proteins. Anti-apoptotic proteins normally act as safeguards to prevent unwanted cell loss, but cancer cells can exploit this mechanism to acquire resistance towards apoptosis. In particular, high levels of X-linked Inhibitor of Apoptosis Protein (XIAP), a potent anti-apoptotic protein, have been found in both primary and metastatic melanoma and are thought to play an important role in therapeutic resistance. The goal of this proposal is to develop small-molecule inhibitors of XIAP and test their suitability for the selective killing of melanoma cells. For this purpose, we will take two distinct approaches. First, we will exploit our extensive knowledge of how natural IAP-antagonists function to rationally design novel compounds that target XIAP with high specificity. Second, we will identify compounds that can inactivate XIAP by systematically screening small-molecule libraries. We will initially test and optimize our compounds in cell-based models to identify leads that are suitable for subsequent clinical development. This project has the potential to radically transform the treatment of malignant melanoma.
Synergistic Targeting of Inhibitory T cell Pathways in Melanoma
Kai W. Wucherpfennig, M.D., Ph.D., Dana-Farber Cancer Institute
T cells, an important population of immune cells, can kill melanoma cells. Unfortunately, the activity of T cells is frequently blocked by the tumor microenvironment. Recent exciting work has shown that blockade of an inhibitory receptor on T cells - CTLA-4 - with the antibody ipililumab enhances the activity of T cells against melanomas and thereby induces regression of metastatic disease in a subset of patients. These results raise an important question: how can the activity of this antibody be enhanced so that a larger fraction of patients with metastatic melanoma can benefit? We have developed a novel approach to identify targets for immunotherapy of melanoma. One of the most important recent discoveries in biomedical research is the RNAi pathway, which is used by cells to regulate the activity of many genes. The principles of RNAi have opened many new possibilities for the identification of therapeutic targets. We developed a novel approach to identify new therapeutic targets for melanoma using an in vivo RNAi screen. The goal of this project is to determine which of the new molecules identified in the RNAi screen show synergistic benefit with an antibody to CTLA-4, an approved treatment for melanoma. We will examine which combination therapies induce optimal expansion of T cells in tumors and are most effective in eradicating established melanomas.
Targeting the Bap1 tumor suppressor gene in a mouse model of melanoma
MRA-Melanoma Research Foundation Established Investigator Award
J. William Harbour, M.D., University of Miami
Metastasis, the spread of cancer cells to distant parts of the body, is the most common cause of death in patients with melanoma and most other cancers. Yet, our understanding of how cancer cells acquire the ability to metastasize remains very limited, making it difficult to tailor therapies that are specific to metastatic tumors. Uveal melanoma (UM) is the most common primary cancer of the eye and the second most common form of melanoma. UMs are notoriously metastatic, resistant to conventional chemotherapy and often fatal. UMs that metastasize can be distinguished from those that do not by their distinct patterns of gene expression, and are classified into "class 1" tumors, which have a low risk of spreading outside of the eye, and "class 2" tumors, which have a very high risk of spreading. Using state-of-the-art genome sequencing techniques, we have identified the gene BAP1 that is located at chromosome 3p21 and that is mutated in almost all class 2 tumors but not in class 1 tumors. BAP1 meets several of the major criteria expected for a metastasis suppressor gene, but very little is known about the role of BAP1 in the spread of melanoma to distant sites. Thus, one objective of this research proposal is to study the effects of BAP1 mutation in melanoma cells using animal models. We expect to find that disabling BAP1 in UM cells increases their ability to metastasize. More importantly, we will use this animal model to identify potential therapeutic agents that block the spread of melanoma cells that have lost BAP1. Overall, the project is expected to lead to innovative new strategies for treating metastatic melanoma.
Optimal T cell receptor affinity for adoptive T cell therapy of melanoma
MRA Established Investigator Award (Anonymous Donor)
David Kranz, Ph.D., University of Illinois at Urbana-Champaign
T cells are white blood cells that are capable of killing cancer cells without harming nearby normal cells. The T cells recognize and respond to the cancer cells using a molecule on their surface that binds to a specific antigen on the tumor cells. The molecule is called a T cell antigen receptor, or T cell receptor (TCR). There has been recent progress in using our knowledge of TCRs in a therapeutic strategy called adoptive T cell therapy. In this approach, T cells are isolated from the peripheral blood of an individual with cancer, and the T cells are expanded in the lab and infused with a TCR that recognizes the cancer. These T cells are then reintroduced into the patient in an effort to have the tumor specifically destroyed by the T cells, without the side effects associated with many cancer treatments. This approach could be improved if information were available about what properties of the TCRs will work most effectively. The goal of our project is to use a mouse model for melanoma, in which we are able to test various TCRs that we have engineered in the lab against a specific model cancer antigen. The mouse model involves both subcutaneous tumors and brain tumors in order to simulate the situation in humans in which melanoma often metastasizes to the brain. The results of the study will provide a guide for the use of TCRs in human therapies.
Combined inhibition of NF-kappaB and AKT for melanoma treatment
Genentech, Inc. is providing funding for this project
Ze'ev Ronai, Ph.D., Sanford-Burnham Medical Research Institute
The recent advances in understanding mechanisms underlying melanoma biology have promoted the development of several drugs that offer, for the first time, effective and specific therapy with limited side effects. Despite this major advance, we recognize the limitations of these current approaches, given the development of resistance in patients that initially respond to mono-specific B-Raf inhibitors. It is now recognized that mono-therapy in melanoma will not be sufficient and combination therapy is warranted. Here we offer to test a novel compound, which exhibits promising results in initial preclinical assessment. Notably, this inhibitor, namely BI-69-A11, targets two of the most prominent pathways that are activated in melanoma and which contribute to the notorious resistance of this tumor to therapy. While initial data suggest that treatment with this newly discovered inhibitor suffices for inhibition of melanoma, we anticipate that it would perform even better when combined with specific existing inhibitors. Our proposed studies will allow comprehensive assessment of this inhibitor, alone and in combination with B-Raf/MEK inhibitors in three complementary experimental systems. Our studies will also gauge possible emergence of tumors that are resistant to such treatment(s) and determine their underlying mechanism. Successful completion of the proposed studies is expected to result in further evaluation of this compound in clinical trials of melanoma.
Defining tumor-host interactions in regional advanced melanoma
Douglas Tyler, M.D., Duke University
Melanoma is currently increasing in incidence faster than other cancers in the United States. For advanced stages of melanoma, there are few effective treatment strategies. This proposal aims to understand more clearly the interactions of treatment on tumor characteristics and the immune system so that patients can be treated with therapies on a more rational, scientific basis. One model of advanced melanoma is in-transit disease, when a patient develops multiple tumor deposits (often >5) in the extremity. Regional chemotherapy (RC) delivers chemotherapy to an isolated extremity (with use of a tourniquet) such that the systemic circulation is NOT exposed to the chemotherapy. While L-phenylalanine mustard (LPAM) is currently utilized for this treatment, preclinical data suggests that temozolomide (TMZ) may be more effective. In this proposal, we will collect tumor, lymph node, and blood samples from 20 patients (10 treated with TMZ and 10 treated with LPAM). Several novel technologies will then be used to characterize the genomic and immunologic makeup of the specimens. Traditional methods like tumor pathology and drug analysis will also be performed. Insight from treating patients regionally will have application to not only systemic therapeutic strategies for melanoma patients but also help identify patients in whom additional targeted approaches may be applied. Because the studies are performed in patients and analysis does not require any long-term follow-up, the projected time from the application of concepts learned from this work to having the potential to impact patient-related outcomes could be fewer than five years.
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Multipeptide vaccination with or without IL - 12 and Daclizumab
Thomas Gajewski, M.D., Ph.D., The University of Chicago
Melanoma vaccines offer an opportunity for instructing the immune system to selectively kill melanoma cancer cells in patients, thus comprising a treatment with minimal side effects. Vaccines developed to date frequently induce activated T cells against tumor antigens, yet only a minority of melanoma patients have tumor regressions. Metastatic melanoma tumors often contain CD4+CD25+ FoxP3+ regulatory T cells (Tregs), which have been shown to suppress the immune response that is induced. We propose that depleting such Tregs from patients will relieve this suppression and thus improve efficacy of melanoma vaccines in patients. The overall goal of this proposal is to test two strategies for vaccination, then deplete Tregs using a monoclonal antibody called Daclizumab. This study also will probe the tumor microenvironment in search for molecular details of the tumor that predict clinical benefit with this treatment.
Entrapment and deletion of melanoma-specific T cells at vaccination sites
Willem W. Overwijk, Ph.D., University of Texas M.D. Anderson Cancer Center
While cancer vaccines can stimulate the body's defenses to fight cancer, this stimulation often isn't strong enough to cause complete cure. Recently, vaccination with a mineral oil-based vaccine increased the lifespan of patients with metastatic melanoma undergoing standard therapy. Much room for improvement remains, and we here show results that suggest inherent limitations to mineral oil-based melanoma vaccines. Specifically, we find that melanoma-specific killer cells travel to the vaccine injection site and not to the melanoma where they are needed to destroy tumor cells. In addition, the killer cells die at these vaccine injection sites. In this proposal we will investigate what causes killer cells to travel to vaccine injection sites rather than to melanoma tumors, and what causes them to die there. We will also develop new, water-based vaccines that we predict will not cause killer cells to traffic to vaccine sites and die. The findings from this proposal will directly result in new therapeutic anti-melanoma vaccines with superior anti-melanoma activity for the treatment of melanoma patients.
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Development of intra-tumoral prognostic biomarkers for primary melanoma
Dr. Lynda Chin, Dana Farber Cancer Institute, Harvard University
Chin identified six "proinvasion oncogenes" that are significantly overexpressed in metastatic lesions in preclinical models. The protein expression level of one of them, called APC5, was correlated with shorter survival of melanoma patients and, thus, may be a prognostic biomarker in human melanomas. This work is being clinically developed by a company to build prognostic tests based on the molecular characteristics of early stage melanoma. If successful, this will transform the way patients with early stage disease are treated and managed.
A phase I trial of bevacizumab plus ipilimumab in melanoma patients
Dr. F. Stephen Hodi, Dana-Farber Cancer Institute, Harvard University
Hodi conducted a Phase I trial investigating ipilimumab with bevacizumab in stage IV melanoma patients and worked to define the mechanism of action for this drug combination. Based on this work, he secured a NIH R21 grant to continue the trial. In this small group of patients, some have experienced durable clinical benefits and side effects have been manageable.
Targeted strategy for treatment of melanoma
Dr. Roya Khosravi-Far, Beth Israel Deaconess Medical Center
Disseminated melanoma is one of the most treatment-resistant and deadly cancers. Increasing incidence of melanoma worldwide in the absence of effective treatments makes a search for therapeutic strategies of vital importance. Dr. Khosravi-Far has discovered a novel biologic that potently induces death (apoptosis) of tumor cells in multiple cancer cell lines and in tumor models by targeting the anti-apoptotic factor c-FLIP. She hypothesizes that this peptide can be used either as a sensitizer or a stand-alone therapeutic agent for treatment of melanoma. Given this success, apoptosis-activating peptides could become a new class of agents for treatment of malignant melanoma.
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Targeting the IGF1R pathway in melanoma
Dr. Alexander Levitzki, Hebrew University of Jerusalem
Melanoma is one of the most aggressive cancers in humans and remains one of the leading causes of cancer death in developed countries. At present there are no clinical protocols to treat metastatic melanomas, although progress has been made in the development of signal transduction inhibitors as well as immune therapy. Preliminary results suggest that Dr. Levitzki has discovered a new family of molecules that target at the same time the IGF1R signaling pathway as well as Stat3, which in combination may hold great promise for the treatment of metastatic melanoma.
Targeting signaling pathways for therapy in a new mouse model of melanoma
Dr. Martin McMahon, University of California, San Francisco
In melanoma, the earliest and most frequently altered oncogene is BRAF. However, since mutation of BRAF is insufficient to promote melanoma on its own, it is likely that BRAF cooperates with mutations in tumor suppressor genes such as INK4A/ARF or PTEN to promote malignant melanoma. Using a mouse model of melanoma based on expression of oncogenic BRAF V600E combined with silencing of the tumor suppressor PTEN, Dr. McMahon explored the essential role of PI3-kinase and AKT in melanoma initiation and maintenance. Although experiments are ongoing, the data generated to date highlight an essential role for PI3-kinase but are ambivalent regarding the role of AKT in melanomagenesis. These data have potential implications for melanoma therapy with agents that target the PI3-kinase AKT signaling pathway.
Treatment of melanoma combining cancer gene therapy and immunotherapy
Dr. TC Wu, Johns Hopkins University
Melanomas are a highly deadly disease and malignant melanoma accounts for 75 percent of all deaths associated with skin cancer. There is an urgent need for innovative therapies for the control of melanomas. Suicidal DNA vectors have emerged as an important strategy for cancer gene therapy since they express high levels of the encoded protein for a prolonged period and cause the transfected cells to undergo apoptic cell death, making them potentially suitable for inducing cancer cell death. Suicidal DNA vectors can be used to encode an immune-stimulatory protein capable of triggering potent immune responses against tumors. Heat shock proteins (HSP) represent ideal proteins for enhancing immunity against tumors and can be encoded by suicidal DNA vectors for cancer immunotherapy. HSP70 has been shown to activate dendritic cells and prime tumor antigen-specific T cells, eliciting strong antitumor responses, representing a potentially ideal protein to be encoded by suicidal DNA vector for cancer gene therapy. One major limitation of this strategy is the limited transfection efficiency in vivo, which may limit the potency of suicidal DNA. Low-energy laser beam treatment represents a novel approach to increase the efficiency of the delivery of DNA to the tumor. In the current proposal, Dr. Wu plans to test whether intratumoral injection of naked suicidal DNA encoding a secreted form of heat shock protein 70 (sHSP70) followed by laser treatment will lead to wide-spread tumor cell death and potent tumor-specific immunity, leading to the control of melanomas in animal models.
The novel melanoma oncogene GNAQ provides new opportunities for therapeutic intervention
Boris Bastian, M.D.
University of California, San Francisco
Dr. Bastian discovered that a vast majority (83 percent) of uveal (ocular) melanomas have mutations in two genes - GNAQ and GNA11. Functional studies showed that mutations induce spontaneously metastasizing tumors in a mouse model and activate the MAP kinase pathway. The functional similarities between these two genes form the basis to develop mechanism-based therapies for most uveal melanomas, and Dr. Bastian will continue this work in a 2010 MRA Team Science Award.
Systemic MFG-E8 blockade as melanoma therapy
Glenn Dranoff, M.D.
Dana Farber Cancer Institute
Dr. Dranoff showed that the protein MFG-E8 is expressed at high levels when melanomas advance to the stage at which they acquire the capacity for invasion and metastasis. In addition to its direct action on melanoma cells, MFG-E8 aids in tumor angiogenesis and inhibits anti-melanoma immunity. His lab found that a combination of antibodies to MFG-E8 and chemotherapy killed tumors in mice and that blocking MFG-E8 enhanced the function of human T cells in vitro. Next steps include translating these findings into phase 1 clinical testing.
Targeted strategies for melanoma treatment and prevention
David Fisher, M.D., Ph.D.
Massachusetts General Hospital
Dr. Fisher studied the mechanism of action of imatinib (Gleevec) on a cell line model for Kit mutated cancer (some melanomas harbor activating mutations in the c-Kit receptor tyrosine kinase) and found that the programmed cell death protein BIM plays an important role. These insights will be important for developing strategies to address resistance to c-KIT targeted therapies. Dr. Fisher has also identified several candidate drugs for topical use to prevent melanoma in fair skinned individuals. This work will continue with funding from the NIH.
Synthetic lethality to MAP kinase pathway inhibition in BRAF-mutant melanoma
Levi Garraway, M.D., Ph.D.
Dana Farber Cancer Institute
In recent years, genetic studies have revealed that most cutaneous melanomas contain activating point mutations in the BRAF or NRAS oncogenes, indicating a profound reliance on the MAP kinase pathway for carcinogenesis. However, results from clinical trials of single agents targeting the MAP kinase pathway have had limited success. Dr. Garraway's research aims to identify a candidate set of melanoma genes that may provide rationale for combining therapeutics targeting the MAP kinase pathway. Dr. Garraway is continuing this work under a 2009 MRA Team Science Award with Michael Weber.
Genetics of melanoma metastasis
Daniel Pinkel, Ph.D.
University of California, San Francisco
A better understanding of the metastatic potential (prognosis) of a newly diagnosed (primary) tumor is important for treatment recommendations. Dr. Pinkel's study will analyze primary melanomas and their associated metastases to detect genetic factors within the tumors that are associated with metastasis, assess their function, and test their utility for prognostication.
Platform for MHC-exchange based T cell therapy for melanoma
Ton Schumacher, Ph.D.
The Netherlands Cancer Institute
Dr. Schumacher is developing and applying technology to better understand melanoma T cell reactivity and to selectively isolate these cells for improved adoptive T cell therapy. This research led to the development of a method that allows the generation of large collections of protein complexes that can be used to detect melanoma-specific T cell populations in small amounts of patient material. A clinical grade reagent was developed in order to purify melanoma-reactive T cells that recognize an epitope of interest. In addition, this is in use to monitor T cell reactivity during other immunotherapeutic treatments, in particular anti-CTLA-4.
A pathway to rational combination therapies for melanoma: Synthetic lethal screening with small molecule inhibitors, guided by phosphoproteome analysis
Michael Weber, Ph.D.
University of Virginia Cancer Center
Dr. Weber will clarify how the MAP kinase pathway is networked to other cell regulatory systems. He will use an assembled library of over 100 small molecule inhibitors to search for "super-additive" inhibition of growth when used in combination. Reverse Phase Protein Arrays (RPPAs) before and after treatment will profile the phosphorylation-driven cell signaling networks of the cell lines, xenografts and patient materials. A subset of promising drugs and drug combinations that are identified will be tested in xenograft and ex vivo patient samples to assess promise as therapy. Dr. Weber is continuing this work under a 2009 MRA Team Science Award.
Immunologic signatures of response to ipilimumab
Jedd Wolchok, M.D., Ph.D.
Wolchok's research goal is to extend the preliminary observations regarding changes in immune parameters occurring after CTLA-4 blockade to a larger set of melanoma patients. More detailed knowledge of the T cell phenotype and antigen recognition found in patients who experience clinical response or immune related adverse events will improve treatment results in patients with melanoma. Wolchok is continuing this work under a 2009 MRA Team Science Award.
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