Andrew R. Conery

Targeting MYC dependence in cancer by inhibiting BET bromodomains.

The MYC transcription factor is a master regulator of diverse cellular functions and has been long considered a compelling therapeutic target because of its role in a range of human malignancies. However, pharmacologic inhibition of MYC function has proven challenging because of both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein–DNA interactions. Here, we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small molecule inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was observed in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain–promoter interactions and subsequent reduction of MYC transcript and protein levels resulted in G1 arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from cell cycle arrest and growth suppression by BET inhibitors. MYC suppression was accompanied by deregulation of the MYC transcriptome, including potent reactivation of the p21 tumor suppressor. Treatment with a BET inhibitor resulted in significant antitumor activity in xenograft models of Burkitts lymphoma and acute myeloid leukemia. These findings demonstrate that pharmacologic inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clinical utility given the widespread pathogenetic role of MYC in cancer.

Fragment-Based Discovery of a Selective and Cell-Active Benzodiazepinone CBP/EP300 Bromodomain Inhibitor (CPI-637).

CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.

Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma

Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. Here, we demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Selective targeting of multiple myeloma cell lines through CBP/EP300 bromodomain inhibition is the result of direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4, which is essential for the viability of myeloma cells, and the concomitant repression of the IRF4 target gene c-MYC. Ectopic expression of either IRF4 or MYC antagonizes the phenotypic and transcriptional effects of CBP/EP300 bromodomain inhibition, highlighting the IRF4/MYC axis as a key component of its mechanism of action. These findings suggest that CBP/EP300 bromodomain inhibition represents a viable therapeutic strategy for targeting multiple myeloma and other lymphoid malignancies dependent on the IRF4 network. DOI: http://dx.doi.org/10.7554/eLife.10483.001

Regulatory T Cell Modulation by CBP/EP300 Bromodomain Inhibition

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.

Preclinical Anticancer Efficacy of BET Bromodomain Inhibitors Is Determined by the Apoptotic Response

Small-molecule inhibitors of the bromodomain and extraterminal (BET) family of proteins are being tested in clinical trials for a variety of cancers, but patient selection strategies remain limited. This challenge is partly attributed to the heterogeneous responses elicited by BET inhibition (BETi), including cellular differentiation, senescence, and death. In this study, we performed phenotypic and gene-expression analyses of treatment-naive and engineered tolerant cell lines representing human melanoma and leukemia to elucidate the dominant features defining response to BETi. We found that de novo and acquired tolerance to BETi is driven by the robustness of the apoptotic response, and that genetic or pharmacologic manipulation of the apoptotic signaling network can modify the phenotypic response to BETi. We further reveal that the expression signatures of the apoptotic genes BCL2, BCL2L1, and BAD significantly predict response to BETi. Taken together, our findings highlight the apoptotic program as a determinant of response to BETi, and provide a molecular basis for patient stratification and combination therapy development.

Regulation of GLI underlies a role for BET bromodomains in pancreatic cancer growth and the tumor microenvironment

Purpose: The initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) results from the interplay of genetic and epigenetic events. While the genetic alterations of PDAC have been well characterized, epigenetic pathways regulating PDAC remain, for the most part, elusive. The goal of this study was to identify novel epigenetic regulators contributing to the biology of PDAC. Experimental Design: In vivo pooled shRNA screens targeting 118 epigenetic proteins were performed in two orthotopic PDAC xenograft models. Candidate genes were characterized in 19 human PDAC cell lines, heterotopic xenograft tumor models, and a genetically engineered mouse (GEM) model of PDAC. Gene expression, IHC, and immunoprecipitation experiments were performed to analyze the pathways by which candidate genes contribute to PDAC. Results: In vivo shRNA screens identified BRD2 and BRD3, members of the BET family of chromatin adaptors, as key regulators of PDAC tumor growth. Pharmacologic inhibition of BET bromodomains enhanced survival in a PDAC GEM model and inhibited growth of human-derived xenograft tumors. BET proteins contribute to PDAC cell growth through direct interaction with members of the GLI family of transcription factors and modulating their activity. Within cancer cells, BET bromodomain inhibition results in downregulation of SHH, a key mediator of the tumor microenvironment and canonical activator of GLI. Consistent with this, inhibition of BET bromodomains decreases cancer-associated fibroblast content of tumors in both GEM and xenograft tumor models. Conclusions: Therapeutic inhibition of BET proteins offers a novel mechanism to target both the neoplastic and stromal components of PDAC. Clin Cancer Res; 22(16); 4259–70. ©2016 AACR.

Abstract B19: Targeting dependencies within apoptotic pathways through inhibition of BET bromodomains

Cancer is driven in large part by dysregulated transcriptional programs that allow for the acquisition of the many ‘Hallmarks of Cancer.9 Multiple regulatory factors are essential for the establishment and maintenance of these cancer-specific transcriptional programs, and this dependence creates vulnerabilities that can be therapeutically targeted. Here we describe a selective dependence on the bromodomain and extraterminal (BET) family of proteins for the reprogramming of apoptotic signaling networks and demonstrate how this dependence can be predicted prior to therapeutic intervention. We demonstrate in phenotypically sensitive cell lines that BET inhibition results in a rapid and robust transcriptional response among regulators of apoptosis, and that this transcriptional response is correlated with changes in the apoptotic threshold of target cells and subsequent apoptosis. We show that the robustness of the apoptotic response, and not that of the cytostatic response, predicts phenotypic sensitivity to BETi. Consistent with this, we observed that acquired BETi tolerance in two disparate cellular models is driven by dysregulated expression of anti-apoptotic BCL2 family proteins, and that genetic or pharmacological manipulation of apoptotic signaling can modify the phenotypic response to BETi. We further demonstrate that the basal expression levels of a set of apoptotic factors significantly predict preclinical response to BETi, and in particular note that BETi preferentially targets those cells that are dependent on BCL2 for survival. Our findings suggest that tumor cells have acquired a dependence on BET bromodomain function to evade apoptosis, and highlight opportunities to exploit this dependence in the clinic through rational patient selection and drug combination strategies. Citation Format: Andrew R. Conery, Richard C. Centore, Kerry L. Spillane, Nicole E. Follmer, Archana Bommi-Reddy, Charlie Hatton, Barbara M. Bryant, Patricia Greninger, Arnaud Amzallag, Cyril H. Benes, Jennifer A. Mertz, Robert J. Sims, III. Targeting dependencies within apoptotic pathways through inhibition of BET bromodomains. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B19.

Abstract 4749: Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma

Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. We demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Selective targeting of multiple myeloma cell lines through CBP/EP300 bromodomain inhibition is the result of direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4, which is essential for the viability of myeloma cells, and the concomitant repression of the IRF4 target gene c-MYC. Ectopic expression of either IRF4 or MYC antagonizes the phenotypic and transcriptional effects of CBP/EP300 bromodomain inhibition, highlighting the IRF4/MYC axis as a key component of its mechanism of action. These findings suggest that CBP/EP300 bromodomain inhibition represents a viable therapeutic strategy for targeting multiple myeloma and other lymphoid malignancies dependent on the IRF4 network. Citation Format: Patricia J. Keller, Andrew R. Conery, Richard C. Centore, Archana Bommi-Reddy, Karen E. Gascoigne, Barbara M. Bryant, Jennifer A. Mertz, Robert J. Sims. Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4749.

Abstract A20: A leukemic model of acquired resistance to BET inhibition reveals a role for CBP/EP300 bromodomains in the regulation of c-MYC expression

Pharmacological modulation of c-MYC expression has recently become possible through small molecule engagement of the BET bromodomain proteins. As multiple BET inhibitors (BETi) progress through clinical trials, understanding possible mechanisms of acquired resistance becomes imperative to maximize therapeutic efficacy, assess possible drug combination strategies, and identify characteristics of next generation BET inhibitors. In the present study, we describe a cellular model of BETi resistance, which demonstrates a significantly blunted phenotypic and molecular response to BETi. The expression of c-MYC mRNA, highly susceptible to BETi in the parental cells, is restored in resistant cells through transcriptional bypass of BET-mediated co-activation. Through the use of a novel CBP/EP300 bromodomain inhibitor (CBP/EP300i), we show that c-MYC expression in BETi-resistant cells is dependent on CBP/EP300 bromodomain function, and that co-treatment with CBP/EP300i restores phenotypic sensitivity. CBP/EP300i was additionally found to transcriptionally silence MYC expression in numerous myeloma and leukemia derived cell lines in a manner comparable to BETi, identifying a novel modality to pharmacologically target the MYC oncogenic axis. While both BET and CBP/EP300 bromodomains regulate the expression of MYC, their transcriptional and phenotypic effects are otherwise distinct, suggesting that CBP/EP300 bromodomain inhibition may represent an alternative or complementary therapeutic option to BET bromodomain inhibition. Citation Format: Andrew R. Conery, Richard C. Centore, Charlie Hatton, Adrianne Neiss, Hon-Ren Huang, Patricia J. Keller, Alexander M. Taylor, Alexandre Cote, Michael C. Hewitt, Christopher G. Nasveschuk, Yves Leblanc, Shihua Yao, Eneida Pardo, Laura Zawadzke, Florence Poy, Hari Jayaram, Shivangi Joshi, Peter Sandy, Anthony Romero, Terry Crawford, Richard Pastor, Tommy Lai, Kevin Chen, Jian Wang, Steven Magnuson, Brian K. Albrecht, Steve Bellon, Barbara M. Bryant, Robert J. Sims, III. A leukemic model of acquired resistance to BET inhibition reveals a role for CBP/EP300 bromodomains in the regulation of c-MYC expression. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A20.

Abstract 2327: Defining the determinants of sensitivity and resistance to BET bromodomain inhibition

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Selective inhibition of BET bromodomains by small molecule inhibitors has emerged as a promising therapeutic strategy for the treatment of cancer. Accordingly, we have recently initiated clinical trials to assess this mechanism in patients. Recent evidence demonstrates that BET bromodomain inhibition leads to anti-proliferative activity in pre-clinical models of hematological malignancies and solid tumors. This anti-proliferative activity involves down-regulation of the transcription factor MYC, among other cancer-relevant pathways. However, heterogeneity exists in the response to BET bromodomain inhibition at both the molecular and phenotypic level. Given this complexity, the molecular determinants of sensitivity and resistance to BET bromodomain inhibition remains poorly understood. Current efforts are aimed at addressing this key issue by integrating the following: (1) phenotypic endpoints observed upon BET bromodomain inhibition with transcriptional changes, including down-regulation of MYC and other transcription factors important for regulating proliferation and cell death, such as MYB and BCL-2,(2) transcriptional response with BET protein chromatin binding and eviction, and (3) phenotypic endpoints and transcriptional response with genomic context. Our data potentially stratify indications within hematologic malignancies and solid tumors, and provide insight into patient selection strategies. Citation Format: Nicole Follmer, Jennifer Mertz, Andrew Conery, Barbara Bryant, Charlie Hatton, Richard Centore, Hon-Ren Huang, Kerry Spillane, Robert Sims. Defining the determinants of sensitivity and resistance to BET bromodomain inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2327. doi:10.1158/1538-7445.AM2014-2327