CREBBP/EP300 acetyltransferase inhibition disrupts FOXA1-bound enhancers to inhibit the proliferation of ER+ breast cancer cells.

Therapeutic targeting of the estrogen receptor (ER) is a clinically validated approach for estrogen receptor positive breast cancer (ER+ BC), but sustained response is limited by acquired resistance. Targeting the transcriptional coactivators required for estrogen receptor activity represents an alternative approach that is not subject to the same limitations as targeting estrogen receptor itself. In this report we demonstrate that the acetyltransferase activity of coactivator paralogs CREBBP/EP300 represents a promising therapeutic target in ER+ BC. Using the potent and selective inhibitor CPI-1612, we show that CREBBP/EP300 acetyltransferase inhibition potently suppresses in vitro and in vivo growth of breast cancer cell line models and acts in a manner orthogonal to directly targeting ER. CREBBP/EP300 acetyltransferase inhibition suppresses ER-dependent transcription by targeting lineage-specific enhancers defined by the pioneer transcription factor FOXA1. These results validate CREBBP/EP300 acetyltransferase activity as a viable target for clinical development in ER+ breast cancer.

Small molecule targeting of chromatin writers in cancer.

More than a decade after the launch of DNA methyltransferase and histone deacetylase inhibitors for the treatment of cancer, 2020 heralded the approval of the first histone methyltransferase inhibitor, revitalizing the concept that targeted manipulation of the chromatin regulatory landscape can have profound therapeutic impact. Three chromatin regulatory pathways-DNA methylation, histone acetylation and methylation-are frequently implicated in human cancer but hundreds of potentially druggable mechanisms complicate identification of key targets for therapeutic intervention. In addition to human genetics and functional screening, chemical biology approaches have proven critical for the discovery of key nodes in these pathways and in an ever-increasing complexity of molecularly defined human cancer contexts. This review introduces small molecule targeting approaches, showcases chemical probes and drug candidates for epigenetic writer enzymes, illustrates molecular features that may represent epigenetic dependencies and suggests translational strategies to maximize their impact in cancer therapy.

Bromodomains: a new target class for drug development.

Less than a decade ago, it was shown that bromodomains, acetyl lysine 'reader' modules found in proteins with varied functions, were highly tractable small-molecule targets. This is an unusual property for protein-protein or protein-peptide interaction domains, and it prompted a wave of chemical probe discovery to understand the biological potential of new agents that targeted bromodomains. The original examples, inhibitors of the bromodomain and extra-terminal (BET) class of bromodomains, showed enticing anti-inflammatory and anticancer activities, and several compounds have since advanced to human clinical trials. Here, we review the current state of BET inhibitor biology in relation to clinical development, and we discuss the next wave of bromodomain inhibitors with clinical potential in oncology and non-oncology indications. The lessons learned from BET inhibitor programmes should affect efforts to develop drugs that target non-BET bromodomains and other epigenetic readers.

Discovery of a Potent and Selective in Vivo Probe (GNE-272) for the Bromodomains of CBP/EP300.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.

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.

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.

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.

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.

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.

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 G(1) 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 Burkitt's 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.

A kinase shRNA screen links LATS2 and the pRB tumor suppressor.

pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB's ability to arrest tumor cells. Here we describe two shRNA screens that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Our results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells.

High-throughput screens in diploid cells identify factors that contribute to the acquisition of chromosomal instability.

Chromosomal instability and the subsequent genetic mutations are considered to be critical factors in the development of the majority of solid tumors, but the mechanisms by which a stable diploid cell loses the ability to maintain genomic integrity are not well characterized. We have approached this critical issue through the use of high-throughput screens in untransformed diploid epithelial cells. In a screen of a cDNA library, we identified 13 kinases whose overexpression leads to increased ploidy. In a series of shRNA screens, we identified 16 kinases whose loss leads to increased ploidy. In both cDNA and shRNA screens, the majority of hits have not been linked previously to genomic stability. We further show that sustained loss of the shRNA screening hits leads to multipolar spindles and heterogeneous chromosome content, two characteristics of chromosomal instability. Loss of several of the kinases leads to loss of contact inhibition and to anchorage-independent growth, vital traits acquired during tumor development. We anticipate that this work will serve as a template for the comprehensive identification of pathways whose dysregulation can drive tumorigenesis through impaired karyotypic maintenance.

Nucleoside diphosphate kinase Nm23-H1 regulates chromosomal stability by activating the GTPase dynamin during cytokinesis.

Chromosomal instability and the subsequent genetic mutations are considered to be critical factors in the development of the majority of solid tumors. Here, we describe how the nucleoside diphosphate kinase Nm23-H1, a protein with a known link to cancer progression, regulates a critical step during cytokinesis. Nm23-H1 acts to provide a local source of GTP for the GTPase dynamin. Loss of Nm23-H1 in diploid cells leads to cytokinetic furrow regression, followed by cytokinesis failure and generation of tetraploid cells. Loss of dynamin phenocopies loss of Nm23-H1, and ectopic overexpression of WT dynamin complements the loss of Nm23-H1. In the absence of p53 signaling, the tetraploid cells resulting from loss of Nm23-H1 continue cycling and develop classic hallmarks of tumor cells. We thus provide evidence that the loss of Nm23-H1, an event suspected to promote metastasis, may additionally function at an earlier stage of tumor development to drive the acquisition of chromosomal instability.

Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis.

Transforming growth factor beta (TGF-beta) induces both apoptosis and cell-cycle arrest in some cell lines, but only growth arrest in others. It is not clear how this differential response to TGF-beta is specified. Smad proteins are critical mediators of TGF-beta signalling. After stimulation by TGF-beta, Smad2 and Smad3 become phosphorylated by the activated TGF-beta receptor kinases, oligomerize with Smad4, translocate to the nucleus and regulate the expression of TGF-beta target genes. Here we report that the sensitivity to TGF-beta induced apoptosis is regulated by crosstalk between the Akt/PKB serine/threonine kinase and Smad3 through a mechanism that is independent of Akt kinase activity. Akt interacts directly with unphosphorylated Smad3 to sequester it outside the nucleus, preventing its phosphorylation and nuclear translocation. This results in inhibition of Smad3-mediated transcription and apoptosis. Furthermore, the ratio of Smad3 to Akt correlates with the sensitivity of cells to TGF-beta induced apoptosis. Alteration of this ratio changes the apoptotic, but not the growth-inhibitory, responses of cells to TGF-beta. These findings identify an important determinant of sensitivity to TGF-beta-induced apoptosis that involves crosstalk between the TGF-beta and phosphatidylinositol-3-OH kinase (PI(3)K) pathways.