Thomas Franklin Westbrook - US grants

The Genome-wide RNAi Analysis Core is a newly established resource at BCM facilitated by the generous support of the Kleberg Foundation, BCM, and the Dept. of Biochemistry &Molecular Biology. While this Core became operational in May 2008, its support of the research programs of BCM and BCM-IDDRC investigators can already be seen by successful genetic screens ongoing in the Core. For instance. Dr. Westbrook is currently conducting a genetic screen for modifiers of REST, a master regulator of neural differentiation programs. This screen is designed to identify new pathways in nervous system function and development using the pooled shRNA-barcoding approach described above. In addition, Dr. Zoghbi's laboratory is in the process of performing a genetic screen for potential therapeutic targets in Spinocerebellar Ataxia type 1 (SCA1). This screen, which combines the shRNA library resource, high-throughput robotics, and flow cytometry of the Core, will identify new therapeutic targets for SCA1 and serve as a roadmap for finding treatments in other neurologic disorders. The potential for the Genome-wide RNAi Analysis Core to facilitate the objectives and research plans for numerous BCM-IDDRC investigators is evident from the proposed utilization by projects listed below. To gauge the level of demand for this newly proposed core among currently supported BCM-IDDRC investigators, a poll was taken. Nearly three quarters of investigators expressed enthusiasm for the core. Its availability will allow future BCM-IDDRC investigators to propose future projects that will take advantage of this type of screening facility. For those projects listed below in proposed utilization, there is immediate demand. It is likely based on the survey of BCM-IDDRC investigators who anticipate using the Core that demand will increase in future years. It is likely that additional

Genetic screens have been a powerful approach for defining signaling and developmental pathways in model organisms, but historically have not been exploited in mammalian cancer biology because of a lack of systematic tools. Recent advances in RNA interference (RNAi) have begun to facilitate such approaches, and these genetic tools have become essential components of basic and translational cancer biology. The Genome-wide RNAi Screening and Analysis (GRSA) Shared Resource was established through philanthropic and institutional support in 2008 to facilitate investigators in their use of new RNAi technologies and genetic screening methods. The Shared Resource is directed by Drs. Thomas Westbrook and Dan Liu who have extensive expertise in developing genetic technologies (e.g. RNAi libraries) and in mammalian genetic screening methods. Combined with this expertise, the GRSA provides all essential elements for single-gene analyses to whole-genome genetic screens including genome-wide short-hairpin RNA (shRNA) libraries, multiple automated robotic platforms for library manipulation, high-throughput analyzers for phenotypic analysis, and data processing infrastructure for mammalian genetic screens, making this DLDCC Shared Resource unique among NCI Cancer Centers. Specific services provided by the Shared Resource include (1) performing whole-genome or sub-genome scale RNAi screens, (2) utilizing individual lentivirus-based shRNA vectors, (3) large-scale automated manipulation and preparation of shRNA libraries, (4) automated mammalian cell transfection and lentivirus production, (5) high-throughput cell-based assays using automated microscopy or flow cytometry, and (6) data analysis, storage, and management. By housing these resources in a single, cohesive Shared Resource, the GRSA enables investigators to employ genetic approaches that are often cost- and labor-prohibitive or simply not feasible for individual laboratories. In the current application, the GRSA is proposed as a new Shared Resource for the DLDCC grant application.

DESCRIPTION (provided by applicant): Triple-negative breast cancer (TNBC) is a common and aggressive subtype of breast cancer that is refractory to current targeted therapies. A major barrier to developing TNBC therapies is the paucity in our understanding of the molecular drivers of TNBC. Identifying the signaling networks whose dysregulation drives TNBC would have enormous impact on our understanding of the disease and how we treat afflicted patients. Recently, we discovered a tumor suppressor role for the PTPN12 tyrosine phosphatase in TNBC (Sun et al, Cell 2011). Our data indicate PTPN12 is compromised in many epithelial cancers including more than 70% of TNBCs. Loss of endogenous PTPN12 leads to hyper-activation of specific proto-oncogenic tyrosine kinases and consequent transformation of human mammary epithelial cells (HMECs). PTPN12 is frequently inactivated in TNBC by post-transcriptional mechanisms, and restoring PTPN12 function dramatically impairs tumor progression and metastasis in TNBCs. These studies suggest PTPN12 functions as a suppressor of human TNBC. However, the pathways controlling PTPN12 function and the mechanisms by which PTPN12 suppresses epithelial cancers like TNBC are poorly understood. We propose to define the molecular framework of the PTPN12 tumor suppressor network and exploit these mechanisms as therapeutic entrypoints in TNBC. Specifically, we will address the following critical questions: Aim 1: How is the PTPN12 tumor suppressor protein hyper-degraded in TNBC? Our preliminary data suggest that PTPN12 is prominently inactivated in TNBC at the protein level by hyper-degradation. Our evidence indicates PTPN12 is ubiquitylated and highly unstable. We will exploit newly developed genetic screening tools that we developed to identify components of the regulatory network controlling PTPN12 ubiquitination and stability, and test their role in cellular transformation and TNBC survival. Aim 2: Can reactivation of PTPN12 in murine and human triple-negative breast cancers suppress tumor progression in vivo? PTPN12 protein is frequently lost in human TNBCs, and the tumorigenic and metastatic properties of PTPN12-deficient TNBC are impaired in response to restoring PTPN12. We will develop and test complementary models of PTPN12-deficient human and murine TNBC, and use these models to delineate the mechanisms of PTPN12 anti-TNBC properties. Aim 3: How does PTPN12 dysfunction regulate survival of TNBCs? Restoring PTPN12 expression is cytotoxic and impairs TNBC tumor progression, suggesting PTPN12 inhibits key survival pathways in TNBCs. Mechanistically, loss of PTPN12 leads to combined hyper-activation of the tyrosine kinases (TKs) cMET and PDGFR-¿. We will test the hypothesis that PTPN12 impairs TNBC progression by combined inhibition of cMET and PDGFR-¿, and these PTPN12-regulated TKs cooperate to confer tumor survival in PTPN12- deficient TNBCs.

CORE: Cell-Based Assay Screening Services Shared Resource (Cell & in vivo Biology Group) PROJECT SUMMARY The complexity and heterogeneity of cancer requires cell-based technological platforms that enable researchers to uncover key genes, pathways, interactions, and modifications that drive cancer development in order to devise effective and personalized therapeutics. The Cell-Based Assay Screening Service (C-BASS) Shared Resource provides DLDCC members with a unique combination of cutting-edge technologies, advanced instrumentation, and genomic resources. C-BASS houses essential elements for single-gene analyses to whole-genome screens including genome-wide short-hairpin RNA (shRNA) libraries, BiFC-tagged (Bi-molecular Fluorescence Complementation) cDNA libraries and multiple state-of-the-art automated robotic instruments for library manipulation, high-throughput screening for phenotypic analyses, and data processing infrastructure for screens. The Shared Resource is directed Drs. Thomas Westbrook and Dan Liu who have extensive expertise in developing and implementing technologies for genome-wide screens. This combination of expertise and resources greatly facilitates DLDCC investigators in their cancer research efforts. Specific services provided by the C-BASS Shared Resource include (1) performing whole-genome or sub-genome scale BiFC and RNAi screens, (2) utilizing individual cDNA and shRNA vectors, (3) large-scale automated manipulation and preparation of cDNA and shRNA libraries, (4) automated mammalian cell transfection and lentivirus production, (5) high-throughput cell-based assays using automated microscopy or flow cytometry, and (6) data analysis, storage, and management. In the past few months, C-BASS has also added genome- editing services using the CRISPR/Cas9 system. The ever-expanding functionalities of CRISPR/Cas9, which include gene targeting, genome modification, and transcription modulation, promises to be of tremendous added value to DLDCC members. By housing these resources in a single, cohesive facility, C-BASS enables Cancer Center Investigators to employ a variety of genomic and genetic platforms that are often cost- and labor-prohibitive, or simply not feasible for individual researchers.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with more than 200,000 new diagnoses each year. The oncogene Myc is a common driver of TNBC. However, despite important research into the biologic and molecular functions of Myc, no tractable therapies have emerged to inhibit this oncogenic transcription factor. TNBC and other Myc-driven cancers remain recalcitrant to current targeted therapies, underlining the urgent need to identify new vulnerabilities in these malignancies. In addition to promoting tumorigenesis, oncogenes such as Myc also produce unique stresses in cancer cells, collectively termed oncogenic stress. Using parallel whole-genome RNAi screens, we discovered that Myc imposes a new type of oncogenic stress on TNBCs that we term RNA splicing stress, or RSS (Kessler, Science 2012; Hsu, Nature 2015). In many cancer types, Myc (or other oncogenic insults) globally amplifies transcription leading to increased pre-mRNA burden on the spliceosome. Consequently, Myc-driven TNBCs are exquisitely sensitive to modest perturbations in the spliceosome (by enhancing RSS). Importantly, pharmacologic or genetic inhibitors of the spliceosome impair primary and metastatic progression of TNBC and are well tolerated in animal pre-clinical models. While spliceosome inhibitors have entered clinical trials, the mechanisms by which RSS and spliceosome inhibitors selectively kill cancer cells are unknown. Herein, we propose to discover the pathway(s) sensing and responding to RNA splicing stress and to delineate the mechanisms by which RSS triggers cancer cell death. Aim 1: Delineate the dsRNA-sensing pathways (DSP?s) stimulated by RSS in Myc+ TNBCs in vivo. Our preliminary data suggest that RSS induces TNBC apoptosis by triggering innate immune pathways that detect and respond to dsRNAs. By leveraging a TNBC PDX cohort with new genetic and proteomic platforms, we will determine how DSP?s and other stress response pathways are activated in response to spliceosome inhibition. Aim 2: Discover how RSS triggers dsRNA-sensing pathways in TNBCs. Many viral pathogens encode dsRNA genomes that are detected by dsRNA-sensors of the innate immune system. Our studies indicate RSS leads to cytoplasmic accumulation of intron-retained mRNAs, a potential source of dsRNAs. Using new sequencing and single-molecule microscopy methods, we will study the mechanisms by which RSS shapes the secondary structure and subcellular localization of TNBC transcriptomes and stimulates dsRNA-sensors. Aim 3: Delineate how DSPs and other stress response pathways govern TNBC response to spliceosome inhibitors. We hypothesize that there is a coordinated cellular response to RSS that parallels well-studied homeostatic stress responses like the unfolded protein response. By leveraging forward genetics and identifying new regulators of RSS, we propose to unveil a genetic framework for this novel type of cellular stress and provide new therapeutic inroads that exploit this cancer vulnerability.

Myc deregulation is a hallmark of cancer and promotes tumor aggression across multiple tumor types. When deregulated, levels of the full length Myc (MYC, MYCN, or MYCL) transcription factor are drastically increased, leading to a global remodeling of gene expression that is poorly understood. Here we show evidence for and propose ?Myc enhancer invasion? as a novel mechanism in which excess Myc protein invades distal cis-regulatory enhancers that are not normally bound at physiological Myc levels. As enhancers control tissue specific gene expression, Myc invasion creates aberrant regulatory interactions that drive tumor progression and can be targeted as a therapeutic strategy. Specifically, we have developed and provide evidence for a model of Myc enhancer invasion in which 1) a subset of enhancers contain weak Myc binding motifs that are accessed when Myc is deregulated. 2) Invaded enhancers act as reservoirs for excess Myc binding and drive the Myc responsive transcription of target genes. 3) Tumor specific enhancer landscapes specify the context of Myc enhancer invasion leading to different enhancer invasion responsive genes in different tumors. 4) Enhancer invaded pathways and other transcription factors that form enhancers can be targeted to interdict tumor specific Myc transcriptional control. To investigate this model for Myc enhancer invasion, we propose the following specific aims. 1) To map and model Myc enhancer invasion in neuroblastoma and osteosarcoma in order to identify genomic parameters that predicate enhancer invasion. 2) To investigate transcriptional consequences of Myc enhancer invasion in vitro and in vivo in order to functionally validate that tumor specific Myc enhancer invaded target genes are dynamically and selectively responsive to Myc perturbation. 3) To identify and target oncogenic Myc enhancer regulation to establish proof of concept for the therapeutic targeting of Myc enhancer regulation. We will perform this work in pediatric neuroblastoma and osteosarcoma models and primary tumors. In these diseases, Myc deregulation is associated with high risk disease, metastasis, increased tumor aggression, and poor responsiveness to existing treatments. As no targeted therapies exist for Myc deregulated neuroblastoma or osteosarcoma, there is a critical unmet need for novel strategies to identify dependencies and effectors of Myc deregulation. In our preliminary data, we find that the enhancer transcription factor TWIST1 acts as a co-factor of MYCN enhancer invasion and is a specific dependency of MYCN driven neuroblastoma. These data highlight the utility of the proposed approach to connect mechanistic investigation of Myc transcriptional regulation to novel frameworks for the identification and validation of tumor specific therapeutic targets in Myc driven neuroblastoma and osteosarcoma.

PROJECT SUMMARY Despite extensive efforts to characterize the genomes and proteomes of triple-negative breast cancer (TNBC), no dominantly-acting mutated RTK has emerged as therapeutic target in TNBC. Instead, TNBCs exhibit broad, rather than selective, hyper-activation of RTK signaling, with several proto-oncogenic RTKs hyper-active in the same tumor. Recent discoveries have revealed that the coordinate activation of RTKs observed in TNBC is a result of inactivation of the protein tyrosine phosphatase PTPN12. In the current proposal, we aim to translate our new knowledge of how RTKs are aberrantly activated and drive TNBC progression into a new therapeutic approach for TNBC patients. Data from our team and confirmed by others indicates that PTPN12 functions as a tumor suppressor by restraining the activity of a select set of proto-oncogenic RTKs, including MET and PDGFR?, binding these receptors and suppressing downstream signaling. PTPN12 is compromised in 45-55% of TNBCs, and we have shown in in vitro and pre-clinical models of TNBC that inactivation of PTPN12 leads to hyper-activation of MET, PDGFR?, and a small subset of other RTKs. Importantly, this provokes the therapeutic hypothesis that PTPN12-deficient TNBCs can be treated by combined targeting of RTKs like MET, PDGFR, and potentially other RTKs that are locked in the chronically active state. To translate these pre-clinical findings into a new therapeutic approach, we will evaluate the efficacy of the well-tolerated MET/PDGFR inhibitor sitravatinib as monotherapy in metastatic TNBC patients, develop rational strategies to combine sitravitinib with standard of care therapies, and explore new ways to capitalize on the anti-tumoral and pro-immune effects of sitravatinib in the context of immune checkpoint therapies. Herein, our mechanistic, pre-clinical, and clinical studies will evaluate the proof-of-concept for the use of sitravatinib to treat PTPN12-deficient TNBC and lay the groundwork for the next generation of combination approaches for TNBC and other RTK-dependent cancers.