Christopher R. Vakoc, Ph.D. - US grants

DESCRIPTION (provided by applicant): The central goal of this project is to understand the role of BRD4 as a therapeutic target in acute myeloid leukemia. As a regulator of chromatin, BRD4 is a member of an emerging class of anti-cancer drug targets for which little is understood of its therapeutically-relevant molecular function. This proposal seeks to address this issue by identifying the critical biochemical mechanism employed by BRD4 in leukemia cells that accounts for its desirable properties as a therapeutic target. This will include evaluating which regions of BRD4 are most crucial for its disease-related functions, as well as identifying the key protein constituents of the BRD4 complex that are necessary for leukemia maintenance in experimental mouse models. Efforts will also be made to identify effective therapeutic combinations that can synergize with BRD4 inhibitors to suppress leukemia progression in preclinical leukemia models. This project will rely on integrative approaches, including biochemical, genetic, proteomic, and epigenomic strategies. This research will make extensive use of genetically-engineered mouse models of chemotherapy-resistant leukemia, which will be used to evaluate the in vivo efficacy of various therapeutic manipulations of BRD4 or components of its protein complex. In summary, the long-term goal of this research will be to maximize the clinical benefit of targeting BRD4 in leukemia through an understanding of the detailed mechanism of how this protein works as a regulator of chromatin state.

PROJECT SUMMARY-CORE B Core B supports every project within the Program by providing access to state-of-the-art CRISPR-Cas9 and RNAi tools. Short hairpin RNAs (shRNAs) were developed with the support of this program, and ongoing innovations by Core B and Program investigators have helped to make these extremely powerful biological tools. During the past funding period, the Core devised a novel algorithm for predicting shRNA potency, which led to the generation of a 5th version of RNAi libraries corresponding to annotated protein coding genes in humans and mice. Technology has also been developed for using CRISPR-Cas9 screening to expose functionally important protein domains. During the upcoming period of requested support, the Core proposes to aid Program investigators through five general aims. First, the program will continue providing access to state- of-the-art RNAi vectors and CRISPR-Cas9 vectors and procedures for analyzing either single gene knockdowns or for performing pooled RNAi/CRISPR screens. Second, the Core will provide Program investigators with access to the mouse and human version 5 shRNA library and will compile sub-libraries upon request. Third, the Core will construct custom CRISPR-scanning libraries for performing structure-function analysis on genes of interest within each Program. Fourth, the Core will produce custom domain-focused CRISPR libraries to allow for interrogation and discovery cancer drug targets in various contexts. Fifth, the Core will carry on its efforts to improve CRISPR-Cas9 technologies, and make those innovations available to the Program and to the scientific community at large.

PROJECT SUMMARY - PROJECT 4 Emerging evidence has implicated transcriptional coactivators and enhancer elements as having a central role in the pathogenesis of human cancer. The research outlined in Project 4 will develop transcriptional coactivators as proteins that support the aberrant capabilities of cancer cells, thus revealing a novel class of epigenetic drug targets in oncology. This research will explore the role of SWI/SNF and TFIID coactivator complexes in the pathogenesis of Acute Myeloid Leukemia (AML) and will identify novel roles for coactivators in supporting the progression of late-stage Pancreatic Ductal Adenocarcinoma (PDA). The first Aim of this proposal will analyze the anti-leukemia effects of chemical inhibitors of the bromodomain of BRD9, which are novel compounds we generated in collaboration with Boehringer-Ingelheim. The major objective of this research will be to develop drug combinations that augment the therapeutic effects of BRD9 inhibition in AML. This will include use of CRISPR-scanning and domain-focused CRISPR screening, which are recently developed genetic tools to allow deep mechanistic investigation of endogenous protein complexes and to reveal opportunities for drug repurposing. This research has a direct potential to motivate clinical studies of novel drug combinations in AML patient populations. The second Aim will develop the TAF12/TFIID complex as a novel dependency in AML and reveal its underlying mechanism in supporting oncogene-mediated leukemic transformation. This will also include identifying suitable routes for direct chemical inhibition of this complex, an objective that has yet to be achieved in the cancer epigenetics field, largely owing to our incomplete understanding about how TFIID supports cancer pathogenesis. The third Aim of Project 4 will be to understand how modulation of nucleosome structure by writers, readers, and erasers of post-translational histone marks might endow PDA cells with the capacity to undergo metastasis. This will include domain- focused CRISPR screening to interrogate chromatin regulators that allow distal metastasis in mouse models of PDA. Since early metastasis is a major contributor to the high mortality of PDA patients, this research may significantly impact our basic understanding of the lethal form of this disease, and carries the potential for revealing epigenetic drug targets for this malignancy. Importantly, the research outlined in Project 4 will provide a deep mechanistic investigation of transcriptional mechanisms that support AML and PDA, which will rely on extensive use of epigenomic tools (e.g. ChIP-seq-based measurement of enhancer activity), and biochemical approaches. Project 4 will also feature extensive collaborations with the other Projects and Cores to evaluate molecular mechanisms and in vivo biology of malignant cells. This basic research will reveal novel vulnerabilities in lethal malignancies, which could provide new routes for therapy.

Project Summary Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, which is associated with a high mitotic rate, early metastatic spread, and a rapid evolution of chemotherapy resistance. We recently discovered a novel form of SCLC that resembles the tuft cell lineage, which can be distinguished from the classical neuroendocrine form of this disease through immunohistochemical staining of POU2F3. Importantly, we have identified several molecular vulnerabilities that are specific to the variant form of this disease. In this research proposal, we seek to advance personalized therapies that exploit the unique lineage program present in the tuft cell variant of SCLC. Our innovative functional genomics strategy has already uncovered actionable dependencies that are unique to tuft cell variant of SCLC, such as the kinase IGF1R. In addition, we discovered a profound addiction of tuft cell variant SCLC tumors to POU2F3. Here we will investigate the molecular basis of POU2F3 addiction in SCLC, with the explicit intent to develop small molecules that interfere with POU2F3 function. The first Aim of this proposal will build upon the extensive epigenomic analyses we have performed in SCLC, which has defined a unique enhancer landscape sustained by POU2F3 in this disease. We will now employ two independent functional approaches to elucidate the critical POU2F3 binding sites/enhancers in the genome of SCLC cells, which will be leveraged to pinpoint the critical components of the tuft cell lineage circuit that might be targeted therapeutically. The second Aim will evaluate POU2F3 cofactors, which we have already nominated via an innovative ChIP-SICAP-mass spectrometry analysis of endogenous POU2F3 binding sites. We will perform CRISPR exon scanning and biochemical analysis of each cofactor to define the critical POU2F3:cofactor interactions that selectively support this malignancy. The final Aim of this proposal will employ functional genomics to devise drug combinations with the IGF1R inhibitor linsitinib that are rational and exploit synthetic- lethal genetic interactions. We will also employ our latest CRISPR innovation, homolog co-targeting CRISPR screens, to expose redundant kinase vulnerabilities that are linked with neuroendocrine versus tuft cell variants of SCLC. In summary, we estimate that the tuft cell-like variant is present in ~18% of SCLC cases, which corresponds to approximately 5,000 newly diagnosed SCLC cases and approximately 3,500 deaths in the United States alone each year. Hence, the proposed research could lead to a sustained impact that affects a large patient population for which novel medicines are desperately needed.

Project Summary/Abstract The central goal of this project is to develop strategies to epigenetically reprogram pancreatic cancer cells to diminish metastatic spread. This objective is based on our recent demonstration that pancreatic cancer metastasis is accompanied by a stereotypical pattern of enhancer activation. We implicated the pioneer transcription factor FOXA1 as a driver of enhancer reprogramming and of metastatic spread in this context. In this proposal, we seek to define the enhancer-metastasis connection. In doing so, we seek to nominate a new class of epigenetic targets, which might be uniquely capable of eliminating metastatic potential. In the first Aim of this proposal, we will employ a functional genomics approach to perturb every FOXA1-regulated gene and enhancer and determine whether FOXA1-dependent metastasis can be suppressed. This approach will take advantage of our recent innovations in domain-focused CRISPR screening and will deepen our understanding of the pro-metastatic components of this epigenetic program. In the second Aim of this proposal, we will investigate the earliest steps of the enhancer reprogramming process that occur prior to FOXA1 upregulation. This effort builds from our unexpected observation that metastasis-specific enhancers are already present in an accessible chromatin state in pre-metastatic pancreatic tumor cells. This suggests that additional molecular events occur prior to FOXA1 upregulation to set the stage for enhancer reprogramming during metastasis. We will investigate the transcription factor FOXA2, which our experiments suggest is the critical bookmark that opens up metastasis-specific enhancers in pre-metastatic cancer cells. In addition, we will determine how repressive Polycomb complexes act to restrain enhancer activation prior to FOXA1 upregulation. By evaluating the consequences of FOXA2/Polycomb perturbation, we will provide a proof-of-concept that metastasis- associated enhancers can be effaced at early stages of pancreatic cancer progression. The final Aim of this proposal will be to extend our enhancer mapping studies into the squamous-subtype of pancreatic cancer, which is a recently defined disease entity associated with a particularly dismal prognosis. We will employ a newly characterized complement of patient-derived pancreatic cancer organoids to compare enhancer profiles of squamous-subtype versus the more classical form of this disease. We will identify master-regulators of this squamous transcriptional program, and perform genetic experiments to determine the role of such factors in promoting tumor progression and metastatic spread. We will also determine whether squamous cell identity in PDA is associated with unique epigenetic dependencies. Collectively, the proposed research will provide a mechanistic framework for developing epigenetic therapies that target the unique enhancer configuration of metastatic pancreatic cancer cells.

Project Abstract: Rhabdomyosarcoma is a highly metastatic soft tissue malignancy of childhood for which new therapies are desperately needed. The clinical management of RMS patients has been largely unchanged over the past three decades, and is currently limited to surgical resection, radiotherapy, and combination chemotherapy. In this context, a mechanism-based targeted therapy would have potential for a transformative impact on RMS patient outcomes. The most common genetic event in RMS pathogenesis is a chromosomal rearrangement that produces the PAX-fusion oncoprotein, which is a chimeric transcription factor that deregulates chromatin and transcription to promote transformation. Our domain-focused CRISPR screens validate that RMS tumors retain a powerful addiction to the PAX-fusion, yet strategies for direct or indirect targeting of this ?undruggable? protein have yet to be successful. One obstacle in this endeavor is our incomplete understanding of the upstream and downstream factors that support the function of the PAX-fusion, which we seek to address with the research proposed here. Through deep molecular profiling of RMS cell lines depleted of the PAX-fusion, we have recently developed reporters which are compatible with flow cytometry-based measurements and cell sorting. This now allows us to perform saturating genetic screens to delineate all components of the PAX-fusion pathway in this disease. In the first aim of this study, we will perform CRISPR exon-scanning of the endogenous PAX-fusion locus, which is an assay we previously developed for exposing functionally important domains of cancer maintenance genes. These experiments will define the critical subregions of the fusion oncoprotein that deregulate transcription to sustain the block in myo-differentiation. The second aim of this proposal will leverage our recently developed paralog domain co-targeting methodology to expose all of the critical genes, and redundant paralogous gene pairs, that are critical for the PAX-fusion to carry out its function. The final aim of this project will identify the critical E3 ligase that acts to restrain PAX-fusion expression in RMS cells, whose function could be stimulated to degrade this oncoprotein. This two-year research project will employ the latest innovations in CRISPR-based genetic screening to establish an important resource for the RMS field; a genetic foundation for mechanism-based research of the PAX-fusion oncoprotein that will enable its pharmacological modulation with therapeutic intent.

Functional Genomics and Genetics Shared Resource - Project Summary/Abstract The Functional Genomics and Genetics Shared Resource provides members of the CSHL Cancer Center with access to advanced RNAi and CRISPR screening technologies and services deriving genetically engineered cells and animals. This Shared Resource was recently established through reorganization and integration of services from the Microarray and RNA Quantitation Shared Resource, the Gene Targeting Shared Resource, and new technologies for shRNA and CRISPR screening. The recent revolution in sequencing technology has facilitated the collection of massive amounts of human genomic and expression data. To understand the human cancer genome and reveal therapeutic opportunities, a complementary set of functional genomics tools is required to systematically discover pathway relationships among genes. It is the goal of the Functional Genomics and Genetics Shared Resource to leverage its innovative approaches to RNAi and genome editing to promote discovery of new therapeutic targets and to create disease relevant models for improving human health. A major focus of this Shared Resource is in assisting investigators with the design, implementation, and data analysis of multi-plexed RNAi/CRISPR screens in mammalian cells. Functional genomic screening is primarily provided as a Shared Resource-assisted service with some full-service screening options. In addition, the Shared Resource also offers full-service construction of custom RNAi/CRISPR plasmid libraries. In order to extend functional assessment of genetic contributions in the context of genetically engineered cells and mice, this Shared Resource provides production of genetically modified mice using Cas9 and single guide RNA (sgRNA) microinjection into 1-cell mouse zygotes. A range of other gene targeting services, including gene targeting in mouse embryonic stem (ES) cells, derivation of ES cells from mouse models, production of chimeric mice by blastocyst injection, production of transgenic mice by pronuclear injection, cryopreservation/recovery of embryo and sperm, and in vitro fertilization are also available. Re-derivation of animals through this Shared Resource is also a key service for maintaining a pathogen-free animal facility. This Shared Resource will be vital to the Cancer Center because it will enable scientists to use genetic screening tools to discover novel cancer genes, therapeutic targets, and mechanisms of drug resistance and for developing new animal models of cancer for pre-clinical studies. Over the last grant period, 18 Cancer Center members (49% of the membership) used the combined services of the new Shared Resource.