Sarki Abdulkadir - US grants

DESCRIPTION (Applicant's Description): Prostate cancer is thought to initiate as non-invasive prostatic intraepithelial neoplasia (PIN), which progresses to locally invasive carcinoma, then to metastatic disease. The molecular genetic changes associated with prostate cancer progression are not completely understood. The broad goal of this proposal is to examine the role of the transcription factor Early Growth Response Gene 1 (Egr1) in the progression of prostate cancer from PIN to invasive carcinoma. Egr1 is highly expressed in prostate cancer, and its levels correlate with the tumor grade. Preliminary results show that Egr1 regulates the expression of multiple genes that enhance tumor progression in prostate cancer cells. Additionally, preliminary results from the study of Egr1 knockout mice show impaired prostate tumor progression. The hypotheses to be examined are: 1) that prostate cancer progression is delayed in transgenic mice lacking Egr1, and 2) that Egr1 induces the expression of genes that promote prostate cancer progression. Three specific aims are proposed. The first aim is establish and characterize a transgenic mouse model for prostate cancer in the Egr1 null background. The Cr2-Tag mice, which develop prostate cancer as a result of the expression of the SV40 T antigen in the prostate, will be bred to Egr1/- mice to obtain Cr2-Tag/Egr1-/- and Cr2-Tag/Egr1 +/+ animals. We will monitor cancer progression in these mice by detailed histopathological analysis. The second aim is to determine the effect of Egr1 overexpression on the ability of prostate carcinoma cells to invade a reconstituted basement membrane. We will infect human prostate carcinoma cells with adenovirus overexpressing Egr1 and measure the ability of the cells to invade a reconstituted basement membrane matrix. The induced genes that are important for this process will also be determined. The third aim is to identify the genes that are differentially expressed in the prostate tumors of Egr1 knockout and wild type mice, by using high density oligonucleotide microarray hybridization analysis (" gene chip"). The immediate goal is to train in a high-quality research environment and to develop tools and techniques that will prepare the Principal Investigator for a successful academic pathology career.

Most current transgenic mouse models of prostate cancer suffer from the drawback that the initiating oncogene (the SV40 T antigen) has no known role in the pathogenesis of the human disease. Thus it is critical to generate an improved murine model of prostate cancer that is based on the genetic lesions commonly observed in human prostate carcinoma. Two of the most common chromosomal aberrations observed in prostate carcinoma cells include loss of sequences from the short arm of chromosome 8 and gain of sequences on its long arm. A strong candidate for a tumor suppressor located at 8p21 is Nkx3.1, a homeobox- containing protein whose expression is lost in many prostate tumors and prostatic intraepithelial neoplasia (PIN) lesions. We have used Cre/loxP-mediated recombination to generate mice with deletion of Nkx3.1 in the adult prostate. These mice develop preinvasive PIN lesions. The Myc oncogene maps to 8q24, and is the target for amplification in many prostate tumors. Overrepresentation of Myc gradually increases in PIN lesions, primary carcinomas and metastases, indicating that Myc overexpression is associated with tumor progression. Intriguingly, gain of 8q24 is often accompanied by loss of 8p21 in prostate carcinomas, and concurrent loss of 8p and gain of 8q is associated with poor patient prognosis. The overall goal of this proposal is to generate and characterize mice with conditional loss of Nkx3.1 and gain of Myc in the adult prostate. We hypothesize that cooperation between loss of Nkx3.1 and gain of Myc will result in the progression of PIN lesions to invasive carcinoma and ultimately metastatic disease. The following Specific Aims are proposed: 1) To generate and characterize transgenic mice that overexpress Myc upon Cre- mediated recombination in the prostate. 2) To generate and characterize mice with concurrent loss of Nkx3.1 and overexpression of Myc in the prostate. 3) To examine the genetic pathways altered in prostate tumorigenesis in these models, with particular emphasis on pathways known to be commonly altered in human prostate carcinoma. This proposal seeks to develop an in vivo genetic model of prostate cancer that recapitulates the human disease. This will provide an important tool for studying the molecular events that lead to prostate cancer.

DESCRIPTION (Applicant's Description): Prostate cancer is thought to initiate as non-invasive prostatic intraepithelial neoplasia (PIN), which progresses to locally invasive carcinoma, then to metastatic disease. The molecular genetic changes associated with prostate cancer progression are not completely understood. The broad goal of this proposal is to examine the role of the transcription factor Early Growth Response Gene 1 (Egr1) in the progression of prostate cancer from PIN to invasive carcinoma. Egr1 is highly expressed in prostate cancer, and its levels correlate with the tumor grade. Preliminary results show that Egr1 regulates the expression of multiple genes that enhance tumor progression in prostate cancer cells. Additionally, preliminary results from the study of Egr1 knockout mice show impaired prostate tumor progression. The hypotheses to be examined are: 1) that prostate cancer progression is delayed in transgenic mice lacking Egr1, and 2) that Egr1 induces the expression of genes that promote prostate cancer progression. Three specific aims are proposed. The first aim is establish and characterize a transgenic mouse model for prostate cancer in the Egr1 null background. The Cr2-Tag mice, which develop prostate cancer as a result of the expression of the SV40 T antigen in the prostate, will be bred to Egr1/- mice to obtain Cr2-Tag/Egr1-/- and Cr2-Tag/Egr1 +/+ animals. We will monitor cancer progression in these mice by detailed histopathological analysis. The second aim is to determine the effect of Egr1 overexpression on the ability of prostate carcinoma cells to invade a reconstituted basement membrane. We will infect human prostate carcinoma cells with adenovirus overexpressing Egr1 and measure the ability of the cells to invade a reconstituted basement membrane matrix. The induced genes that are important for this process will also be determined. The third aim is to identify the genes that are differentially expressed in the prostate tumors of Egr1 knockout and wild type mice, by using high density oligonucleotide microarray hybridization analysis (" gene chip"). The immediate goal is to train in a high-quality research environment and to develop tools and techniques that will prepare the Principal Investigator for a successful academic pathology career.

DESCRIPTION (provided by applicant): The transcription factor Egr1 (Early Growth Response Gene 1) has been implicated in diverse roles in the regulation of growth, apoptosis and differentiation. In some tumor types, such as lung cancers and gliomas, Egr1 is expressed at low levels, and its overexpression in these tumor cell lines is reported to result in growth suppression. In contrast, several independent studies have shown that Egr1 is overexpressed in prostate tumors and can promote prostate tumor progression. In prostate cancer-prone transgenic mice, lack of Egr1 significantly impairs tumor progression. We have recently observed a functional interaction between Egr1 and the androgen receptor (AR) in prostate carcinoma cells in culture. We therefore hypothesize that the effects of Egr1 on tumorigenesis are critically dependent on the cellular context, and that functional interactions between Egr1 and AR in prostate carcinoma cells contributes to the ability of Egr1 to specifically promote tumor progression in the prostate. To test these hypotheses, we propose the following Specific Aims: I. To determine the consequences of the interaction between Egr1 and AR in prostate carcinoma cells in culture by functional studies, and by target gene analysis. II. To generate and characterize transgenic mice that overexpress, Egr1 in the prostate. Ill. To analyze prostate tumorigenesis and response to castration in prostate cancer-prone animals that constitutively overexpress Egrl. These studies are likely to shed light on our understanding of tissue-specific mechanisms of tumor initiation, progression and maintenance in the prostate.

[unreadable] DESCRIPTION (provided by applicant): The serine-threonine kinase Pim-1 is an established oncogene in lymphomagenesis, where it shows dramatic cooperativity in tumor induction with the Myc oncogene. The mechanisms of Pim-1-induced tumorigenicity and the basis for its cooperativity with Myc are largely unknown. Recent studies have shown that Pim-1 is overexpressed in a significant proportion of human prostate cancers and prostatic intraepithelial neoplasia (PIN) lesions. We have recently found that chronic overexpression of Pim-1 in human prostate and breast epithelial cell lines leads to chromosomal instability. Our hypothesis is that Pim-1 overexpression dysregulates expression of mitotic molecules including Cyclin B1, disrupting accurate chromosomal segregation and cytokinesis, culminating in chromosomal instability (CIN). CIN is a common feature of human malignancies, and plays a major role in the generation of genetic abnormalities in tumors. In this proposal, our goals are to elucidate the mechanisms of regulation of Cyclin B1 and mitotic checkpoint molecules by Pim-1 and to establish the role of these molecular alterations in Pim-1-induced chromosomal instability and tumorigenesis. In addition, we will establish and characterize a novel prostate-specific Pim-1 transgenic mouse model to validate these ideas in vivo. Work will be conducted according to the following Specific Aims: Aim 1: To elucidate the mechanism(s) underlying altered mitotic regulation and CIN in Pim-1 overexpressing cells. Aim 2: To elucidate the roles of Pim-1 and Pim-1-induced chromosomal instability in prostate tumorigenesis by using human prostate cell lines and transgenic mice. Aim 3: To test the hypothesis that Pim-1 and Myc cooperate to promote prostate tumorigenesis through the use of human prostate cell lines and transgenic mice. These studies should enhance our understanding of the molecular basis for genomic instability in prostate cancer, establish if Pim-1 is a valid molecular target for prostate cancer therapy and generate new models with relevance to the human disease. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Studies of human and mouse prostate cancer indicate that the homeodomain-containing transcription factor Nkx3.1 is an important haplo-insufficient tumor suppressor gene involved in prostate tumor initiation. Nkx3.1 protein expression is lost in human and mouse prostate tumors, and a recent study has found Nkx3.1 mutations in hereditary prostate cancer. We have generated and characterized conventional and conditional Nkx3.1 mutant mice. These mice develop prostatic epithelial hyperplasia and dysplasia and have been extensively studied as a model of prostatic intraepithelial neoplasia (PIN). In these animals, the exit of differentiating prostate luminal epithelial cells from the cell cycle is delayed, resulting in prostatic epithelial hyperplasia and subsequently PIN. Further analysis of Nkx3.1 mutant mice has revealed several features that begin to shed light on the mechanisms of tumor suppression by this protein. Nkx3.1 regulates a class of genes in a dosage-sensitive and stochastic manner, a phenomenon that may underlie haplo-insufficiency and which appears to be dictated by the differential chromatin states of target genes. Nkx3.1 regulates the expression of cell cycle regulators as well as a unique class of androgen target genes. Further, loss of Nkx3.1 dysregulates the expression of pro- and anti-oxidant enzymes (including peroxiredoxin 6, glutathione peroxidase 3 and sulfhydryl oxidase 6) resulting in loss of protection against oxidative damage. It is our hypothesis that loss of Nkx3.1 promotes prostate tumor initiation by deregulating multiple gene programs that alter cell cycle exit, androgen signaling, and the anti-oxidant response. We have outlined specific experiments to test this hypothesis according to the following aims: 1) To examine the regulation and function of dosage-sensitive Nkx3.1 target genes. 2) To define the regulation and function of genes induced by androgen specifically in Nkx3.1-deficient cells. 3) To test the notion that disruption of the anti-oxidant defense system in Nkx3.1 mutant mice leads to the accumulation of genetic mutations. PUBLIC HEALTH RELEVANCE: The significance of these studies lies in their potential to increase our understanding of the mechanisms of prostate tumor initiation and to generate molecular targets for diagnosis and prevention in a preclinical model. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A major clinical problem in prostate cancer is that of tumor recurrence following initial apparently successful therapy. It is widely believed that recurrent tumors may arise from a small number of cancer stem-like cells that survive the initial therapeutic intervention and which have the capacity to regenerate the tumor. However, this idea has been difficult to test in vivo without manipulation of the cancer cells outside their native environment in the animal. Here we propose a lineage-tracing strategy to examine the competence of specific prostate epithelial cell types (castration-resistant Nkx3.1-expressing cells or CARNs and Bmi1+ cells) to regenerate tumors following androgen ablation in mice (Aim 1). We will further examine whether recurrent tumors driven by specific oncogenic mutations preferentially arise from particular cell populations of stem-like cells within a regressed tumor and the role of the androgen receptor (Aim 2). Finally, we will employ lineage ablation of specific cell types within a regressed tumor to assess the relative contributions of specific stem-like cells (CARNs and Bmi1+ cells) to prostate cancer relapse (Aim 3). In these studies will use novel approaches to define the significance of specific prostate cell types in tumor relapse following androgen ablation. This proposal if successful will have a major impact on our understanding of the cellular origins of recurrent prostate cancer and will facilitate effors aimed at successfully eradicating the tumor.

PROJECT 3: PROJECT SUMMARY/ABSTRACT A major clinical problem in the management of prostate cancer is the difficulty associated with treating aggressive cancers, especially those that are highly castration resistant. The androgen signaling pathway remains a key therapeutic target for advanced prostate cancer but resistance to agents targeting this pathway is common, highlighting the need to develop novel therapeutic approaches. Mouse prostate cancer modeling has elucidated molecular pathways of aggressive, castration-resistant prostate cancer (CRPC) which include loss of the tumor suppressors PTEN and TP53 and overexpression of the MYC oncogene. Using these spontaneous mouse models of prostate cancer we have identified Ephrin receptor EphB4 as a potential therapeutic target. EPHB4 is a receptor tyrosine kinase that with its ligand ephrin B2, are not expressed in normal prostate gland, but are expressed in a majority of human prostate cancers. EPHB4 is induced by multiple pathways important for CRPC development, including loss of PTEN and TP53 as well as activation of the PI3K pathway downstream of EGFR and IGF1R. In turn, EPHB4 activation engages multiple signaling pathways, including the PI3 kinase/AKT and MAPK pathways known to modulate the androgen receptor and drive CRPC development. To test the significance of EphB4, we generated conditional EphB4 knockout mouse. We found that genetic deletion of EphB4 or its inhibition using a soluble antagonist (sEPHB4) profoundly inhibited prostate tumorigenesis driven by loss of Pten and led to the regression of established tumors in transgenic mice. This was associated with inhibition of PI3K/AKT signaling and apoptosis. Notably, sEpBh4 antagonist and EphB4 knockdown led to markedly lower levels of androgen receptor (AR) protein. These functional genetic data lead us to hypothesize that EPHB4 is a novel pharmacologic target with high therapeutic potential in prostate cancer, including CRPC. We will explore this hypothesis by targeting EphB4 in genetically complex mouse models (loss of Pten, Tp53 and Myc over-expression) and human xenograft models of prostate cancer and CRPC, singly or in combination with AR-targeted therapy (including enzalutamide, abiraterone). We will examine human prostate tumor samples including metastases and CRPCs for the expression of EphB4, EphrinB2, and downstream markers. A soluble decoy EPHB4 receptor ? human serum albumin fusion protein (sEPHB4HSA) antagonist is in early human trials in other tumors, and has been found to be remarkably safe in Phase I study. We will therefore implement a feasibility clinical trial of sEPHB4HSA aimed at determining the therapeutic efficacy of targeting EPHB4 in men with CRPC. Successful completion of the preclinical and early clinical studies we propose in this application could lead to a rapid translation of soluble EPHB4 antagonist as a treatment for advanced prostate cancer.  

? DESCRIPTION (provided by applicant): Androgen and its receptor AR, which is a member of the human nuclear receptor (NRs) superfamily, play critical roles in prostate cancer (PC) and castrate resistant prostate cancer (CRPC) that kills thousands of people world-wide. We posit that a better understanding of NR signaling pathway and its integration in epigenomic signaling are keys for development of future therapeutics of this deadly disease. The long term goal of our research is to discover novel components of NR function in transcriptional and epigenomic regulation with strong translational implications in human diseases. The protein kinase termed PKN1 plays a critical role in AR dependent gene regulation by establishing an epigenomic marking (histone H3 threonine 11 phosphorylation (H3T11P)) on nucleosomes at AR target genes. We hypothesize that the PKN1-H3T11P- WDR5 signaling module plays a previously unrealized and critical role in castrate resistant prostate cancer (CRPC). The goal of this present work is to perform in depth and integrative mechanistic, genome-wide, cellular and animal based studies to dissect the role of this newly discovered signaling module in AR function in CRPC. To achieve our research goals, we will utilize interdisciplinary knowledge and complementary expertise of the co-investigators spanning biochemistry, patho-physiology, genome biology, animal and cellular models of PC and molecular biology. In Specific Aims, we will examine in extensive molecular details the role of WDR5, and PKN1 in gene regulation, and CRPC-cell function in vitro and in vivo using cell and animal based models. We will also determine the gene-specific and genome-wide localization profiles of WDR5 and H3T11P in CRPC cells. Epigenomic markings and identification of their effector proteins in hormone action are new and rapidly evolving concepts and we believe our work lies at the limit of current knowledge of hormone signaling. Our findings will thus significantly advance the field by providing a better understanding of the role of epigenomic modifications and their effector proteins in nuclear receptor action and in human diseases including CRPC.

Abstract Prostate specific antigen (PSA) screening is an established and useful tool for prostate cancer detection, however, it has no predictive prognostic value at diagnosis. For early diagnosed localized prostate cancer, the major clinical challenge is the treatment decision, in whether a patient should receive invasive intervention or be managed as ?watchful waiting? active surveillance. Consequently, patients with indolent prostate cancer can be unnecessarily over-treated; or conversely, patients with prostate cancer of an aggressive nature may miss out on needed treatment, which ultimately leads to mortality. Therefore, it is an urgent need to develop prognostic biomarkers for localized prostate cancer to guide clinical decision making that is most beneficial to each patient. The objective of this proposal is to address the imminent clinical need by developing and validating a panel of potential prostate cancer prognostic biomarkers. Based on the literature and our compelling preliminary findings, we hypothesize that serum levels of the soluble NKG2D ligand MIC (sMIC) in combination with tumor-associated glycan profiles can provide the predictive biomarker capacity for prostate cancer prognosis. We have assembled large cohorts of prostate cancer tissues and matching serum collected from men diagnosed with localized prostate cancer at the time of prostatectomy. These samples have annotated clinical information including follow up PSA biochemical recurrence (BCR) status. These samples will be used to develop a unique panel of prognostic biomarkers and validate their specificity and sensitivity. Findings will be further validated with independent cohorts of serum samples from clinically-defined prostate cancer patients. There are four Specific Aims: 1) Determine the sensitivity and specificity of tissue MIC and serum sMIC in predicting BCR; 2) Determine the sensitivity and specificity of tissue and serum multi- fucosylated glycan panels in predicting BCR; 3) Determine the prognostic capacity of serum and tissue biomarker panel 4) Validate prognostic capacity of the identified panel of serum biomarkers with independent cohorts of patient samples. The proposed study will be accomplished through a collaborative effort led by a team of well-established investigators.

CORE A: ABSTRACT The SPORE in Prostate Cancer brings together a highly motivated multidisciplinary team of investigators from Northwestern University and the University of Chicago, with contributions from NorthShore University HealthSystem (UC affiliate). The team includes laboratory scientists, urologists, medical oncologists, pathologists, biostatisticians and bioinformaticians, all of whom are dedicated to conducting state-of-the-art translational research to develop and test interventions that improve the outcome of patients diagnosed with prostate cancer. The Administrative, Leadership Development and Advocacy Core has the following Specific Aims: Specific Aim 1: To provide integration within the SPORE, and with the Robert H. Lurie Comprehensive Cancer Center and the University of Chicago Comprehensive Cancer Center Specific Aim 2: To provide strong oversight of research projects and cores as well as financial oversight through planning and evaluation activities Specific Aim 3: To oversee the Developmental Research (DRP) and Career Enhancement (CEP) Programs of the SPORE to encourage investigators to conduct translational prostate cancer research Specific Aim 4: To lead an advocacy group and diversity education initiatives in the community Specific Aim 5: To train the next generation of leaders in prostate cancer research Specific Aim 6: To facilitate collaborations and integration with NIH and other nationally funded programs such as the U54 CA203000 (The Chicago Collaborative to Promote and Advance Cancer Health Equity), Department of Defense and Prostate Cancer Foundation.

DEVELOPMENTAL RESEARCH PROGRAM (DRP): ABSTRACT The Developmental Research Program (DRP) is an essential component of the Prostate SPORE, providing yearly funding to 4-6 projects that are peer reviewed and deemed to have promising translational potential. Funding is flexible, supporting projects for a period of two years or less. It is expected that DRP investigators will develop and advance their pilots sufficiently to either replace main projects that are not progressing or will secure independent funding in prostate cancer research. Of note, all three projects presented in the renewal have emanated from pilot projects and propose translating discoveries made in the investigators? labs to the clinic. The DRP follows a highly structured process to solicit projects from investigators who are interested in developing projects in the area of prostate cancer, to review projects according to NIH review criteria and to monitor projects for scientific progress. The DRP is maintained throughout the entire period of SPORE funding. The specific aims of the Developmental Research Program are: ? Specific Aim 1: To solicit, review and select innovative and translational pilot projects through the highly structured process. A request for applications (RFA) is sent out to investigators at the Northwestern University (NU), University of Chicago and its affiliated NorthShore University HealthSystems. The Scientific Review Committee reviews pilot projects based on the quality and importance to the overall SPORE mission. ? Specific Aim 2: To monitor progress of pilot projects within the context of the overall SPORE goals. Pilot project investigators are required to present their results twice per year at SPORE investigators meeting. They also meet twice per year with the Directors of the DRP to obtain translational insight and to ensure that scientific milestones are being achieved. DRP investigators are required to submit a written progress report that is rigorously evaluated by the SPORE PI and members of the Executive Committee. ? Specific Aim 3: To encourage and recruit additional investigators to pursue research in prostate cancer through a SPORE web site and by widely distributing the RFA announcing the availability of funds. Women and minority investigators are strongly encouraged to submit pilot projects. ? Specific Aim 4: To maintain the administrative process for the Developmental Research Program throughout the SPORE funding period.

PROJECT 1: ABSTRACT MYC oncoproteins (including c-MYC, L-MYC and N-MYC) have been implicated in up to 70% of all human cancers. In prostate cancer, elevated levels of MYC protein expression are observed across all grades. In castration-resistant prostate cancer (CRPC), there is evidence of further upregulation of c-MYC levels with gene amplification occurring in 45% of cases. In late-stage, therapy-resistant neuroendocrine prostate cancer (NEPC), N-MYC is overexpressed in 40% of cases. In preclinical studies, inhibition of MYC can effectively kill CRPC and NEPC cells. A viable therapeutic strategy to inhibit MYC is therefore likely to have a significant impact on this disease and to fulfill the ongoing need for novel impactful therapies spanning the spectrum of castration resistant prostate cancer. Despite its recognition as an attractive cancer target, MYC has proved difficult to target, and there are currently no clinically viable small molecule MYC inhibitors (MYCi) available. By employing a pharmacophore-based in silico screen of a large chemical library (32 million compounds) coupled to a rapid in vivo screen in mice, we identified a series of novel small molecule inhibitors. These MYC inhibitors are highly drug-like and have shown excellent pharmacokinetic, toxicological and anti-tumor activity profiles in MYC-driven models of prostate cancer and leukemia. The compounds engage MYC inside cells as shown by the cellular thermal shift assay (CETSA); disrupt MYC/MAX complex formation which is required for MYC activity; and inhibit MYC-driven target gene expression. Furthermore, the MYCi compounds enhance phosphorylation of MYC on threonine-58 (T58P) which promotes MYC degradation via a well-characterized ubiquitin-proteasome pathway. Consequently, treatment with MYCi impaired tumorigenicity in vitro and in vivo. The goals of this project are to develop the lead MYC inhibitor, MYCi975, for clinical application in the treatment of prostate cancer and to characterize the mechanisms of MYCi-induced degradation of c-MYC and N-MYC oncoproteins. We will implement the following Specific Aims: Aim 1 is to investigate the mechanisms of MYCi975 regulation of c- MYC and N-MYC phosphorylation and stability and the potential of MYC pT58 as a pharmacodynamic marker. Aim 2 will assess MYCi anti-tumor efficacy and impact on pharmacodynamic biomarkers in preclinical models of c-MYC and N-MYC driven prostate cancer. Aim 3 will seek to develop MYCi975 for use in patients by conducting formal IND-enabling toxicology studies and initiate a phase 1 trial in mCRPC patients. Impact: Successful completion of these studies could lead to first-in-class therapies for lethal prostate cancers dependent on c-MYC/N-MYC activity. This benefit can extend to other human cancers as well because of the pervasive role MYC proteins play in cancers of all types.

ABSTRACT MYC oncoproteins (including c-MYC, L-MYC and N-MYC) play critical roles in the initiation, progression and recurrence of many human malignancies. Extensive studies indicate that MYC is required to maintain tumor cell survival and proliferation. We have recently used a novel approach that combined computer-aided modeling with a rapid in vivo screen to develop a new series of direct small molecule inhibitors (MYCi?s) that show excellent selectivity, potency and tolerability in multiple MYC-driven cancer models. These compounds demonstrate a dual mechanism of action. First, direct binding of MYCi to MYC in the basic helix-loop-helix (bHLH) region disrupts complex formation with MYC which is required for MYC transcriptional activity. Secondly, binding of MYCi enhances MYC phosphorylation on threonine-58 (pT58) which promotes MYC degradation via the ubiquitin-proteasome pathway. However the key downstream effectors of these events and how they might impact cellular function are unknown. Reduction of MYC protein and enhanced pT58MYC may be expected to have profound effects on MYC family protein interactions with each other and with chromatin. In this regard, we have observed in preliminary studies that MYCi leads to selective loss of MYC at genomic loci enriched for master chromatin regulators (CTCF and FOX), suggesting disruption of the 3D architecture of the MYC-bound genome in response to MYCi. Additionally, unfolded MYC due to MYCi binding and/or enhanced MYC degradation may provoke a cellular stress response. Using unbiased ATAC-seq and RNA-seq approaches, we found that MYCi treatment activates the ATF4/CHOP stress response pathway. Importantly, activation of ATF4/CHOP by MYCi is an on-target, MYC-dependent effect. ATF4 mediates MYCi antitumor activity as ATF4 depletion partially ameliorates the antitumor effects of MYCi. Furthermore, we propose that MYCi-induced ATF4 cytokines modulate the tumor microenvironment. Activation of the ATF4 pathway by MYCi exposes potential therapeutic vulnerabilities for rational combination approaches, such as combination of MYCi with proteasome inhibitors that activates ATF4. Based on the preliminary findings, our central hypotheses is that MYCi inhibits MYC-dependent tumorigenesis by a dual-pronged mode of action. First, MYCi affects MYC family target gene expression by disrupting MYC/MAX interaction and by promoting MYC degradation. Secondly, binding of MYCi to MYC and/or MYC degradation activates an ATF4/CHOP stress response pathway that suppresses tumor cell viability. We propose the following specific aims to test these hypotheses: Aim 1). To investigate the mechanisms by which MYC inhibitor modulates MYC transcriptional activity and the epigenetic landscape. We will investigate the consequences of MYCi treatment on the recruitment of MYC, pT58MYC, and associated factors to chromatin; changes to 3D chromatin architecture; as well as the effects on MYC-driven transcriptional output in tumor cells vitro and in vivo. Aim 2). To define the mechanisms and functional consequences of ATF4/CHOP pathway activation by MYCi. We will determine mechanism of ARF4 upregulation by MYCi; define the role of MYCi- induced ATF4 in regulating target gene expression, cell viability and tumorigenicity; and assess strategies that exploit the consequences of ATF4 activation as a means of enhancing MYCi anti-tumor efficacy. These studies are significant as MYC is implicated in the majority of human cancers. The studies advance the use of MYCi as chemical probes to unmask distinct biology that complements the knowledge derived from genetic manipulations of MYC proteins. The findings will contribute to the efforts aimed at developing small molecule MYCi as potential therapeutics. Specifically, this work indicates that small-molecule MYC inhibitors have an additional anti-tumor effect due to the activation of the ATF4 pathway beyond the antitumor effects of suppressing MYC function. Finally, understanding this on-target ATF4 response provoked by small-molecule MYCi will provide rational strategies for combination therapy to enhance MYCi efficacy.

OVERALL: ABSTRACT This application is a request for renewal funding of the SPORE in Prostate Cancer P50 CA180995 at the Robert H. Lurie Comprehensive Cancer Center of Northwestern University in collaboration with the University of Chicago Comprehensive Cancer Center and NorthShore University HealthSystem, an affiliate of the University of Chicago. The SPORE first received support from the National Cancer Institute in 2001; a renewal application was funded in 2009. In 2015, we were the recipients of a new SPORE and we are now requesting continued funding for this program. Our SPORE unites basic scientists, clinicians, pathologists, biostatisticians, bioinformaticists and advocates from our academic institutions, all of whom are dedicated to advancing translational prostate cancer research. In this application we propose three highly translational and innovative research projects that have both basic science and clinical science co-leadership: Project 1: Targeting the MYC Pathway in Prostate Cancer (Abdulkadir, Hussain); Project 2: Re-directing the sensitivity of metastatic castration-resistant prostate cancer to immunotherapy (Wu, Sosman, Morgans); Project 3: STING Activation to Overcome Resistance to Immune Checkpoint Blockade in PTEN-deficient Prostate Cancer (Patnaik, Gajewski, Stadler). Three core facilities support the proposed research projects: Administrative, Leadership and Advocacy (Abdulkadir, Hussain, Stadler); Biostatistics/Bioinformatics (Kocherginsky, Zhao) and Biospecimen (Yang). Internal and External Advisory Boards provide a source of scientific input to members of the SPORE team on a biannual and yearly basis, respectively. Our Advocacy Group are well established in the SPORE arena and make a valued contribution to our success. The SPORE includes Developmental Research and Career Enhancement Programs, both of which provide a source of innovation and new discoveries. All together, we anticipate that the results obtained from our research endeavors will have a significant impact on the health of patients diagnosed with prostate cancer.