Julien Sage - US grants

[unreadable] DESCRIPTION (provided by applicant): The retinoblastoma (RB) gene is a potent suppressor of human cancers. While RB is thought to have multiple functions during the cell cycle, the specific functions of RB that are critical for its tumor suppression activity are still unclear. We propose to test the idea that RB and its two related family members p107 and p130 play a pivotal role in preventing cancer initiation in vivo by controlling the transition between the GO and G1 phase of the cell cycle. Specifically, we hypothesize that the acute somatic loss of RB family function leads to defects in both cell cycle exit (G1->GO) and cell cycle re-entry (GO->G1) in critical cell populations, and that these defects may result in the initiation of tumorigenesis. We will test our hypothesis using mouse models with combinations of germline and conditional mutations of RB family genes. This mouse genetic approach will enable us to determine the role of RB family genes in spatially and temporally-defined cell populations. These mouse models will also allow us to recapitulate in vivo the early stages of cancer in humans. Studies in the first Specific Aim will characterize the function of RB family genes in cell cycle exit during embryonic development. RB patients develop retinoblastomas, which are retinal neural tumors, as early as the fetal stage. We will focus our studies on neural progenitors to explore the consequences of loss of RB family function on cell cycle exit and differentiation defects leading to cancer initiation. Studies in the second Specific Aim will determine if the RB family proteins are required for maintenance of differentiation in vivo. Specifically, we will investigate the consequences of acute loss of function of RB family genes on cell cycle re-entry of differentiated hepatocytes in vivo. These studies will be important to determine if some terminally differentiated cells may be at the origin of cancer. Studies in the third Specific Aim will combine molecular approaches and RNA interference in vivo to understand the molecular mechanisms underlying cell cycle re-entry upon loss of RB family function in vivo. We will focus our studies on candidate mediators of cell cycle re-entry in RB family mutant hepatocytes, including the E2F transcriptional activator and the Id2 transcriptional repressor. Understanding the molecular bases and the cellular consequences of the loss of RB family tumor suppressors in vivo will give important and novel insights into the mechanisms of cancer initiation. These studies will further provide the foundation for the detection and treatment of early stages of human cancer. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Abstract Since the mid-1990s, approximately 150,000 Americans have died of lung cancer every year, and the upward trend in total cancer deaths is largely due to the increasing rate of lung cancer mortality. Even if we could prevent cigarette smoking and exposure to other carcinogens today, hundreds of thousands of lung cancer cases would still need to be treated in the next decades. A major current effort in the lung cancer field is to detect and treat lung cancer at earlier stages to improve the survival of patients. In addition, identifying novel drug targets would allow for the development of more efficacious therapeutic strategies against lung tumors. Finally, lung cancer patients are treated following well-established protocols that most often do not take into account the genetic diversity of their tumors, and very little is known about prognostic factors for individual lung cancer patients. A better knowledge of the molecular events in lung cancer development would help to identify diagnostic and prognostic markers in lung cancer patients. Rb, p53 and Kras are among the most frequently mutated genes in human cancer. In particular, combinations of mutations in these three genes are often found in human lung cancer and define important clinical subtypes. Using advanced gene-targeting approaches, we and others have generated genetically engineered mice with mutations in these genes. These mutant mice develop tumors that closely resemble human lung tumors and provide a genetically tractable system to study lung tumorigenesis in vivo. Here, we propose to use comparative gene expression analysis to define genotype-specific oncogenic signatures using these mouse models of lung cancer. Our specific goals are: - To develop gene expression signatures from mouse tumors and compare them to human data to identify new human lung cancer subtypes. The validation of subtype-specific genes from these signatures will be performed using human tissue arrays. - To identify key regulators and "drivers" of these gene expression signatures using conventional bioinformatics approaches as well as a novel "event" centered gene network that we will develop. In particular, we will introduce the notion of ordered, causal events in lung cancer gene networks to identify key nodes in these gene networks. - To functionally analyze potential key regulators of these lung cancer gene expression signatures. To this end, we will first use gene expression-based high throughput screening to identify such regulators and we will then test their functional role in lung cancer development in vivo. The overall goal of our work is to begin to define critical pathways that are required for genotype-specific oncogenesis. Characterization of these pathways may provide a useful approach for identification of new approaches for diagnosis, prognosis, and targeted therapy in lung cancer patients. PUBLIC HEALTH RELEVANCE: We propose to use a novel gene network to identify molecular events downstream of key oncogenic "driver" mutations for lung cancer by comparing gene expression profiles in lung tumors from genetically defined mouse models to gene expression profiles from human lung tumors. We will test the functional role of candidate regulators of lung cancer in mouse models and human tumor cell lines and tissues. Our experiments will lay the foundation needed for the development of novel strategies to detect and treat lung cancer, the number one cancer killer in both men and women in the United States.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The retinoblastoma tumor suppressor (RB) is a central cell cycle regulator and tumor suppressor. RB cellular functions are known to be regulated by a diversity of post-translational modifications such as phosphorylation and acetylation, raising the possibility that RB may also be methylated in cells. Here we demonstrate that RB can be methylated by SMYD2 at lysine 860, a highly conserved and novel site of modification. This methylation event occurs in vitro and in cells, and it is regulated during cell cycle progression, cellular differentiation, and in response to DNA damage. Furthermore, we show that RB monomethylation at lysine 860 provides a direct binding site for the methyl-binding domain of the transcriptional repressor L3MBTL1. These results support the idea that a code of post-translational modifications exists for RB and helps guide its functions in mammalian cells.

DESCRIPTION (provided by applicant): Merkel Cell Carcinoma (MCC) is a skin cancer that is thought to arise from the uncontrolled growth of so-called Merkel cells, which serve as mechanoreceptors and osmoreceptors essential for light-touch response. MCC incidence is about 1,500 US cases per year, and this number is continually increasing. MCC is an aggressive cancer type, highly metastatic, and it is only cured when detected early. Critical issues in the field are to better understand the mechanisms of MCC development to be able to detect this deadly cancer earlier and to treat patients more effectively. Such issues are extremely challenging to address in human patients due to limitations in acquiring and studying tumor samples from these patients and also because many cases of MCC are detected late in the course of the disease. The exact cause of MCC is still unclear, but its development may be linked to sun exposure and immunosuppression. MCC development also correlates very strongly with infection by MCPyV (Merkel Cell PolyomaVirus), a virus that was first described in 2008. The current model is that, in many cases of MCC, cellular or organism stress creates a favorable environment for activation of the MCPyV genome, resulting in the induction of proliferation and cancer initiation. Based on virology studies with other polyomaviruses in various species and recent data in MCC primary tumors and cell lines, we hypothesize that inhibition of the RB and p53 tumor suppressor pathways is critical in the early stages of MCC development. Our first aim is to test this hypothesis by deleting Rb family genes and p53 in Merkel cells in the skin of mice using advanced tools of mouse genetics. We also hypothesize that MCC initiation correlates with changes in transcriptional profiles in Merkel cells, in part because of inactivation of the RB and p53 transcriptional regulators. Our second specific aim is to examine changes in gene expression in Merkel cells upon loss of function of RB and p53 function; to detect these changes; we will use novel high-throughput RNA sequencing methods. If successful, these experiments will provide the first pre-clinical model of Merkel Cell Carcinoma and will identify novel candidate biomarkers and potential therapeutic targets to detect and treat patients. PUBLIC HEALTH RELEVANCE: Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine type of skin cancer whose incidence is increasing and mortality risk very high. Emerging evidence indicates that the RB and p53 tumor suppressors are inactivated in MCC cells. We propose to delete the Rb and p53 genes in Merkel cells in the skin of adult mice to generate and analyze a mouse model for this deadly cancer. The development of mouse lesions resembling MCC in patients would provide a pre-clinical system to investigate the molecular and cellular mechanisms underlying MCC development and may ultimately provide novel means to prevent and treat MCC in patients.

DESCRIPTION (provided by applicant): More than 600,000 people die from Hepatocellular carcinoma (HCC) worldwide annually. The environmental factors that cause HCC are well known and include infection by hepatitis B and C viruses (HBV and HCV), exposure to Aflatoxin B1, and excessive alcohol intake. However, no effective treatments exist for HCC and the prognosis of HCC patients is usually poor, with an overall median survival of less than one year. The RB tumor suppressor and its family member's p107 and p130 are functionally inactivated in nearly all cases of human HCC. This inactivation is due to increased CDK4 kinase activity resulting from the silencing of the CDK4 inhibitor p16 or from increased expression of the CDK4 partner Cyclin D1. In addition, some proteins produced by HBV and HCV can inactivate RB family members, including by triggering their degradation. We generated a mouse model for human HCC by deleting RB family genes in the liver of adult mice to model this functional inactivation of RB family proteins. RB/p107/p130 triple knockout (TKO) mice develop liver tumors whose histology and gene expression profiles resemble human HCCs. The activity of E2F transcription factors, which are normally inhibited by the RB family, is high in TKO HCC cells. TKO HCCs initiate from progenitor cells and not hepatocytes, which do not divide due to unknown mechanisms that suppress their proliferation. The TKO HCC model provides a unique in vivo system to query the mechanisms of tumorigenesis in the liver and to specifically interrogate how RB/E2F transcriptional regulatory complexes control HCC development. Our general hypothesis is that inactivation of the RB pathway drives cancer initiation at least in part by deregulating E2F activity and compromising the balance between regulatory networks in acutely sensitive cell populations. Specifically, we propose that increased E2F activity engages pathways that promote the expansion of mutant cells, including liver progenitors, but also triggers negative feedback loops preventing cancer initiation from mature hepatocytes and limiting the growth of HCC cells. We will first test the specific hypothesis that activation of p21 by E2F in the TKO model blocks the proliferation of hepatocytes, thereby preventing HCC initiation from these mature cells. Next, we will test the idea that activation of Notch signaling y E2F limits the expansion of HCC cells during tumorigenesis. Finally, we will examine the possibility that activation of the EZH2 methyltransferase by E2F promotes the growth of both liver progenitors and HCC cells. To test these hypotheses, we will manipulate the activity of RB and E2F family members, p21 and CDK2, Notch pathway members, and EZH2 in adult liver progenitor cells, mature hepatocytes, and HCC cells in vivo. These experiments in mutant mice will be complemented by analyses of human liver cells ex vivo. Our studies will identify novel means to diagnose, detect, and treat HCC. In addition, because genetic, epigenetic, and/or viral inactivation of the RB pathway is a nearly universal event in human cancer cells, these studies are generally relevant to a broad cross-section of cancer patients.

DESCRIPTION (provided by applicant): Our overarching goal is to investigate the importance of protein post-translational modification (PTM) networks in cancer. The signaling system defined by the PTM lysine methylation has recently emerged as a potential key regulator of cancer pathways. However, in comparison to enzymes like protein kinases that have well-established roles in tumorigenesis, the mechanisms by which lysine methyltransferase (KMT) enzymes contribute to cancer are poorly understood. We believe that characterizing novel KMTs involved in the post-translational control of cellular proteins in cancer-relevant signaling networks can provide novel insights into the basic mechanisms of cellular transformation and identify new therapeutic targets for cancer treatment. SMYD3 is a KMT whose expression is up-regulated in a wide range of human cancers. We and others have shown that transient overexpression of SMYD3 can promote the tumorigenic potential of several cancer cell lines. However, knowledge of the endogenous substrates of SMYD3 and the overall mode of action of this enzyme in cells and in vivo is still obscure. We hypothesize that SMYD3 promotes the proliferation and the survival of cancer cells by methylating and regulating key cellular effectors including non-histone proteins. We will first test this idea by exploring the mode of action of SMYD3 in cancer in vivo, which has not previously been done. In Aim 1, we will use a novel conditional mutant Smyd3 allele to test the hypothesis that loss of SMYD3 inhibits tumorigenesis. Because SMYD3 levels are more specifically elevated in tumors with oncogenic RAS, we will focus on mouse models of lung adenocarcinoma and pancreas ductal carcinoma, two fatal human cancers in which the RAS pathway is often activated. In Aim 2 we will analyze the molecular mechanisms of SMYD3 action in cancer cells. We recently identified the MEKK2 (MAP3K2) kinase as a SMYD3 substrate. MEKK2 belongs to the same family as RAF proteins and mediates cellular responses to various growth factors, including EGF, to regulate signaling networks such as the JNK and NF-kB pathways. We will test the hypothesis that SMYD3-mediated methylation of MEKK2 constitutes a new mechanism by which cancer cells respond to stress and growth factors. The goal of Aim 3 is to identify new substrates of SMYD3 using a novel chemical biological- proteomic strategy we have developed for proteome-wide discovery of functionally-relevant SMYD3 substrates. The role of the most promising targets in regulation of cancer cell pathways will be investigated using a combination of molecular approaches and mouse genetics. Based on accumulating evidence implicating a role for SMYD3 in cancer, a number of pharmaceutical companies and academic laboratories are developing SMYD3 inhibitors. Our studies have the capacity to identify SMYD3 as an attractive therapeutic target in many cancer types affecting a wide range of patients. Furthermore, the identification of key SMYD3 molecular targets may identify novel biomarkers and promising new candidate therapeutic targets in cancer.

? DESCRIPTION (provided by applicant): Small Cell Lung Cancer (SCLC) is a neuroendocrine subtype of lung cancer. SCLC tumors have unique biology, with a high proliferative index and aggressive metastatic potential. Treatment options for SCLC have remained virtually unchanged for the past 30 years, with corresponding little improvement in survival rates. The world's population continues to increase and the number of smokers still follows. Thus, SCLC, which kills more than 200,000 patients every year - most of them heavy smokers - will continue to be a major health issue in the decades to come. We aim to develop an understanding of the molecular and cellular mechanisms underlying SCLC progression to aid in the identification of novel therapeutic approaches. Here we specifically propose to investigate the mechanisms driving the metastatic spread of SCLC. Given the lack of human specimens for metastatic SCLC, we have developed a genetically engineered mouse model of SCLC in which we can isolate pure populations of cancer cells from primary tumors and metastases. Using this murine model and novel genomic methodologies, we identified genome-wide transcriptional and chromatin changes in metastatic SCLC cells. These unbiased approaches uncovered several candidate regulators of the metastatic process in SCLC. In particular, we found increased expression of the NFIB transcription factor in metastatic SCLC cells, which correlates with a stabilization of open chromatin at a very large number of enhancer regions containing NFIB binding sites across the genome. Based on these observations, we hypothesize that increased levels of NFIB drive chromatin and transcriptional changes that promote metastasis in SCLC cells. Our first goal is to determine the specific stage(s) of the metastatic cascade at which NFIB may act. To this end, we will perform a series of experiments in culture and in mice. Our second goal is to conduct additional unbiased genomic analyses to define: (i) the gene programs regulated by NFIB, (ii) the molecular mechanisms by which NFIB drives the increased accessibility of a genomic regions and (iii) how NFIB induces gene expression programs that promote metastatic ability. In particular, we will investigate transcription factors involved in th biology of neural cells that may act as co-factors with NFIB to promote SCLC metastasis. Finally, we will examine the role of the NFIB target and neuronal cell surface molecule Lingo1 in SCLC metastasis. These experiments will include pre-clinical assays blocking Lingo1 to determine if Lingo1 may become a clinical target to prevent the metastatic spread of SCLC cells in patients. These experiments will provide novel insights into the basic mechanisms of metastatic progression and identify innovative strategies for targeted therapy for SCLC - the most lethal form of lung cancer.

? DESCRIPTION (provided by applicant): Small Cell Lung Carcinoma (SCLC) is a neuroendocrine subtype of lung cancer. The 5-year survival of SCLC patients is dismal, in large part due to the very transient effects of chemotherapy and the absence of targeted therapies against this cancer type. The world's population continues to increase and the number of smokers quickly follows. Thus, SCLC, which kills more than 200,000 patients every year - most of them heavy smokers - will continue to be a major health issue in the decades to come. Our overarching goal is to gain a better understanding of the mechanisms underlying SCLC development to aid in the development of novel therapeutic strategies in the future. Notch signaling controls cell fate and differentiation in multiple lineages. Interestingly, the Notch pathway is also implicated in various cancer types, and an increasing number of therapeutic tools are being developed to target this pathway in tumor cells. However, the role of Notch signaling is context-dependent, acting as an oncogene or a tumor suppressor in different cell lineages. Thus, it is critical to thoroughly investigate the mode of action of the Notch pathway in specific contexts before any clinical strategy may be safely implemented. Accumulating evidence suggests that active Notch signaling may play a tumor suppressive role in SCLC. In part, these effects may be due to the inhibition of neuroendocrine differentiation by Notch, similar to what is seen during lung development and in the adult lung epithelium in response to injury. However, the exact role of the Notch pathway in SCLC development and response to therapy has not been determined. We will use a combination of mouse genetics, primary mouse and human cancer cells, and molecular, biochemical, and genomics tools to address these key questions. Specifically, we have made the intriguing observation that a contingent of Notch pathway-active tumor cells is naturally present in SCLC tumors, defining a new level of tumor heterogeneity in SCLC. We will investigate how these Notch pathway-active tumor cells interact with the rest of the tumor, including possible tumor-promoting roles for these cells. We have also found that ectopic activation of Notch in all tumor cells initially suppresses the growth of SCLC but, in a second phase, reprograms SCLC neuroendocrine tumor cells towards a non-neuroendocrine state in which Notch becomes oncogenic. Building upon these data, we will determine the optimal conditions for Notch activation to inhibit SCLC cells without triggering this oncogenic switch. Finally, we will use advanced molecular and genomic tools to elucidate the molecular mechanisms by which Notch activation reprograms SCLC cells to a non-neuroendocrine fate. These experiments will provide novel insights into the basic mechanisms of Notch signaling and may help identify novel strategies for targeted therapy directed at SCLC, the most lethal form of lung cancer.

? DESCRIPTION (provided by applicant): The establishment of the thymic microenvironment during development and early in life is crucial to enable the production of functional T cells. Conversely, thymic degeneration (involution) in aging organisms results in decreased T cell output, increased susceptibility to opportunistic infection, auto-immunity, cancer, and a decreased capacity to respond to vaccination. In addition, thymic involution prevents proper restoration of immune function after chemotherapy, ionizing radiation exposure, and infection. Our long-term objective is to gain a better understanding of the mechanisms underlying the deterioration of the thymic microenvironment during aging, focusing on the biology of thymic epithelial cells (TECs), which provide a microenvironment conducive to T cell expansion and differentiation. Little is known about the mechanisms that regulate thymus involution during aging and in response to stress, but we have made observations that indicate that certain manipulations of the thymic stroma can prevent involution. In particular, our most recent studies and accumulating evidence in many cell types during aging suggest that thymic involution is accompanied and caused by changes in the chromatin structure of TECs. These epigenetic changes may not only control normal involution but also may hamper therapeutic attempts to regenerate thymic function during aging. In the thymus, TECs are largely outnumbered by T cells. A major challenge in studies of the thymic epithelium is the low number of cells that can be harvested per mouse, which has prevented genomic analyses of these populations thus far. We will use two approaches to query changes in chromatin structure (ATAC- seq) and DNA methylation (HELP-GT) that occur during aging in TEC populations in mice. These two approaches can be performed with low number of cells (thousands, instead of millions). Combined with gene expression analyses (RNA-seq), these experiments will provide the first detailed report of gene expression and chromatin structure changes in the aging thymic epithelium. Performing these studies in control mice and mice mutant for pathways involved in thymic involution (e.g. the Rb pathway and the Foxn1 transcription factor) will help elucidate molecular mechanisms of thymic involution. Better knowledge of the mechanisms controlling gene expression in TECs may ultimately lead to novel means to control the thymic microenvironment and boost immune function in a wide range of aging patients.

PROJECT SUMMARY Small cell lung cancer (SCLC) is a highly lethal malignancy for which new therapeutic strategies are desperately needed. One promising avenue is the use of immunotherapy (IMT) agents such as PD-1/PD-L1 pathway inhibitors. Despite its high mutation burden, however, data from our group and others indicate that SCLC paradoxically has an immunosuppressed phenotype with relatively low levels of infiltrating T-cells, reduced antigen presentation, and increased levels of CD47, a suppressor of myeloid function. Furthermore, initial clinical testing suggests that most SCLC tumors often express low or very low levels of PD-L1 and fail to respond to PD-1 inhibitor monotherapy; IMT resistance also inevitably emerges in responding tumors by mechanisms that have not yet been characterized. Thus, immunosuppressive mechanisms other than the PD- 1/PD-L1 pathway are likely to play a major role in SCLC, and novel therapeutic approaches and combination therapies are needed to realize the potential of IMT in SCLC. The goal of this proposal is to address this issue by identifying new IMT targets and novel combination regimens, and to rapidly translate them into the clinic. Our team already has promising leads. First, we identified that SCLC is highly vulnerable to drugs targeting DNA damage repair (DDR) including PARP and Chk1 inhibitors, a finding now supported by early clinical results. Our preliminary data further suggest that DDR inhibition may increase PD-L1 expression and, by increasing the production of tumor-associated neoantigens (TAA), may sensitize tumors to IMT. In Aim 1, we will test whether DDR inhibitors can increase the expression of TAAs, and enhance the efficacy of PD-1/PD-L1 inhibitors. Second, we have developed a novel strategy for protecting immune cells from the cytotoxic effects of chemotherapy by using inhibitors of CDK4/6, which can be used to protect immune cells, but not RB-deficient SCLC cells. In Aim 2, we will test whether CDK4/6 inhibition can enhance the anti-tumor effects of immune cells by protecting them from chemotherapy-induced cytotoxicity and enable improved chemotherapy/IMT combinations in SCLC. Third, we have identified the ?don't-eat-me? signal CD47 as a novel IMT target for SCLC; blockade of CD47 effectively promotes the phagocytosis of SCLC cells by macrophages and inhibits tumor growth. In Aim 3, we will test whether targeting this CD47 myeloid checkpoint can enhance antitumor immunity and the efficacy of PD-1/PD-L1 blockade and chemotherapy in vivo in SCLC models. The overall hypothesis tested here is that antitumor immunity can be enhanced in SCLC by targeting all these processes, leading to more effective IMT combination regimens. These studies will be facilitated by novel immune-competent pre-clinical murine SCLC models that we have developed and by a multidisciplinary team including clinical and laboratory investigators, immunologists, pathologists, and others with a record of innovation in SCLC and IMT and a track record of translating laboratory findings into the clinic.

PROJECT SUMMARY This application requests funds to continue the highly successful Cancer Biology training program at the Stanford University School of Medicine entitled ?Cancer Etiology, Prevention, Detection and Diagnosis?. This Interdisciplinary Program provides our faculty, especially those in non-degree granting departments (e.g., Radiation Oncology, Pediatrics, Medicine, or Pathology), the opportunity to recruit and mentor top-notch graduate students. The goal of this program is to provide the very best training for its predoctoral trainees so that they become successful and independent leaders in the field of cancer research. The program accomplishes this goal by providing each trainee with a broad and comprehensive curriculum, a vast array of educational resources such as seminars, lectures, conferences and workshops specifically geared towards the biology of cancer, a faculty comprised of 47 exceptional preceptors spanning 16 departments with extensive experience in cancer research mentoring, and an unparalleled research environment. A key strength of the program is its true multidisciplinary approach to cancer research, incorporating such fields as molecular biology, genetics, cell biology, computational biology, developmental biology, tumor biology, and biotechnology, to understand cancer and to help develop improved cancer diagnostics and therapeutics. The success of the Cancer Biology training program is demonstrated by its track record of attracting outstanding and talented predoctoral candidates to Stanford University and placing graduates of the program in high profile competitive cancer research positions in academia, industry, and medicine. To aid in the further development of the training program, we have recently created internal and external advisory committees consisting of highly accomplished scientists and mentors at Stanford and peer institutions. During the next five-year period, we will continue to enhance the program to allow students to navigate the increasing complexity of cancer biology. We will develop an improved curriculum designed to provide trainees with a solid core of cancer biology coursework while increasing flexibility in electives to match individual needs in areas of specialization such as computational biology and immunology. We will take various measures to increase diversity in the program, as well as to enhance participation and interaction between faculty and trainees. We will ensure accessibility of career workshops and facilitate trainee internships to provide exposure to different potential career options. These collective experiences will provide trainees with a strong foundation in cancer biology to prepare them for independent careers in this field.

PROJECT SUMMARY The retinoblastoma protein (Rb) pathway is a critical regulator of cell proliferation and a promising target for cancer therapeutics. Rb normally inhibits the transcription program for cell division driven by E2F transcription factors and thus promotes cell cycle arrest in G0/G1. Rb is commonly inactivated by Cyclin-dependent kinase (Cdk) phosphorylation in cancer cells, including by Cdk4/6-CycD complexes. Key unanswered questions in this central cellular pathway include how Cdk4/6 activity is regulated, how specific Rb phosphorylation events mediate E2F activation, why is Rb a more potent tumor suppressive than its close paralogs p107 and p130, and what are the mechanisms of resistance to chemical Cdk4/6 inhibitors. These inhibitors have shown promise in the clinic, but we need to better exploit how they act and what factors influence their response. We will apply our unique combined expertise in biochemical and genetic approaches to answer previously intractable questions about the Cdk4/6-Rb pathway and tumor suppression. Our first goal is to uncover the mechanisms of Cdk4/6-CycD activation in cancer cells. We will use structural, biochemical, and cellular assays to investigate the critical role of the p27 protein in modulating Cdk4/6 activity and the cellular response to Cdk4/6 small molecule inhibitors. Our second goal is to reveal the key molecular changes that occur upon inactivating phosphorylation and the key molecular features that confer tumor suppressor potency to Rb. We will examine how specific Cdk phosphorylation events in Rb lead to its inactivation in cells. We will also explore a small domain in Rb that we hypothesize confers unique tumor suppressive ability compared to p107 and p130. Finally, our third goal is to identify new regulators of the Cdk4/6-Rb pathway using unbiased screening approaches. These new regulators may dictate how we use Cdk inhibitors as therapeutics and innovate new strategies for targeting cancer cell division. These experiments will address fundamental issues in the field of cell-cycle regulation and will transform our understanding of Rb tumor suppressor function, how it is regulated, and how it may be rescued to arrest cancer growth.

SUMMARY My laboratory has been interested in the mechanisms that control the identity and the fate of cancer cells during tumor evolution, including in response to treatment. We have made important contributions to this field of research in the past decade. Our work on the retinoblastoma tumor suppressor Rb in stem cells and cancer models has identified a new function role for Rb in the control of cell identity and plasticity, which explains in part why Rb-mutant cancer cells often fail to respond to therapy. Our pioneering work on Rb-mutant small cell lung cancer (SCLC) has provided fundamental novel insights into the biology of this neuroendocrine cancer. SCLC is the most lethal form of lung cancer. Treatment options have remained virtually unchanged for the past 30 years. SCLC kills ~250,000 patients worldwide every year. As the number of heavy smokers worldwide continues to grow, SCLC will remain a major health issue this century. With unique tools to study SCLC in vivo and a highly resourceful network of collaborators, we are uniquely placed to continue to greatly impact the SCLC field by confronting key issues that few investigators address. Importantly, our research combines technically innovative approaches that will allow us to address questions about SCLC progression and maintenance that are difficult, if not impossible, to tackle using traditional human tumor-derived cell lines, previous mouse models, or cancer patient samples. We have developed rapid and accurate mouse models of human SCLC. We have used these models and patient-derived xenografts to identify the cell of origin of SCLC and biomarkers for early detection, as well as drivers of the tumorigenic phenotype of SCLC and their mechanisms of action. We have also contributed to the elucidation of the genomic landscape of mouse and human SCLC tumors. Notably, our findings have led to the implementation of clinical trials in SCLC patients. In the next 7 years, we will continue to use SCLC as a paradigm to elucidate the mechanisms that determine the identity of cancer cells, their plasticity, and their fate. We will perform these studies in the context of our recent breakthroughs investigating inter- and intra-tumoral heterogeneity in primary mouse and human SCLC tumors. Our model is that SCLC tumors, which have very few stromal cells, generate their own microenvironment to support their growth, in part through activation of Notch signaling. This intra-tumoral heterogeneity may critically contribute to the lack of response of tumors to therapies. A second major focus of our work is to elucidate the mechanisms that underlie the striking metastatic ability of SCLC to multiple organs, including the brain. We propose that the switch to a more neuronal differentiation state that accompanies the gain of metastatic ability of neuroendocrine SCLC cells is a key aspect of this high metastatic potential. We will test these ideas in vivo and ex vivo using a combination of unique genetic, molecular, and cellular approaches.