Austin Dulak

Potent and selective bivalent inhibitors of BET bromodomains

Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC50 of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.

AZD5153: a novel bivalent BET bromodomain inhibitor highly active against hematologic malignancies

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563–74. ©2016 AACR.

Multi-omic measurement of mutually exclusive loss-of-function enriches for candidate synthetic lethal gene pairs

BackgroundIdentification of synthetic lethal interactions in cancer cells could offer promising new therapeutic targets. Large-scale functional genomic screening presents an opportunity to test large numbers of cancer synthetic lethal hypotheses. Methods enriching for candidate synthetic lethal targets in molecularly defined cancer cell lines can steer effective design of screening efforts. Loss of one partner of a synthetic lethal gene pair creates a dependency on the other, thus synthetic lethal gene pairs should never show simultaneous loss-of-function. We have developed a computational approach to mine large multi-omic cancer data sets and identify gene pairs with mutually exclusive loss-of-function. Since loss-of-function may not always be genetic, we look for deleterious mutations, gene deletion and/or loss of mRNA expression by bimodality defined with a novel algorithm BiSEp.ResultsApplying this toolkit to both tumour cell line and patient data, we achieve statistically significant enrichment for experimentally validated tumour suppressor genes and synthetic lethal gene pairings. Notably non-reliance on genetic loss reveals a number of known synthetic lethal relationships otherwise missed, resulting in marked improvement over genetic-only predictions. We go on to establish biological rationale surrounding a number of novel candidate synthetic lethal gene pairs with demonstrated dependencies in published cancer cell line shRNA screens.ConclusionsThis work introduces a multi-omic approach to define gene loss-of-function, and enrich for candidate synthetic lethal gene pairs in cell lines testable through functional screens. In doing so, we offer an additional resource to generate new cancer drug target and combination hypotheses. Algorithms discussed are freely available in the BiSEp CRAN package at http://cran.r-project.org/web/packages/BiSEp/index.html.

Comparative analysis of primary versus relapse/refractory DLBCL identifies shifts in mutation spectrum

Current understanding of the mutation spectrum of relapsed/refractory (RR) tumors is limited. We performed whole exome sequencing (WES) on 47 diffuse large B cell lymphoma (DLBCL) tumors that persisted after R-CHOP treatment, 8 matched to primary biopsies. We compared genomic alterations from the RR cohort against two treatment-naïve DLBCL cohorts (n=112). While the overall number and types of mutations did not differ significantly, we identified frequency changes in DLBCL driver genes. The overall frequency of MYD88 mutant samples increased (12% to 19%), but we noted a decrease in p.L265P (8% to 4%) and increase in p.S219C mutations (2% to 6%). CARD11 p.D230N, PIM1 p.K115N and CD79B p.Y196C mutations were not observed in the RR cohort, although these mutations were prominent in the primary DLBCL samples. We observed an increase in BCL2 mutations (21% to 38% of samples), BCL2 amplifications (3% to 6% of samples) and CREBBP mutations (31% to 42% of samples) in the RR cohort, supported by acquisition of mutations in these genes in relapsed compared to diagnostic biopsies from the same patient. These increases may reflect the genetic characteristics of R-CHOP RR tumors expected to be enriched for during clinical trial enrollment. These findings hold significance for a number of emerging targeted therapies aligned to genetic targets and biomarkers in DLBCL, reinforcing the importance of time-of-treatment biomarker screening during DLBCL therapy selection.Current understanding of the mutation spectrum of relapsed/refractory (RR) tumors is limited. We performed whole exome sequencing (WES) on 47 diffuse large B cell lymphoma (DLBCL) tumors that persisted after R-CHOP treatment, 8 matched to primary biopsies. We compared genomic alterations from the RR cohort against two treatment-naïve DLBCL cohorts (n=112). While the overall number and types of mutations did not differ significantly, we identified frequency changes in DLBCL driver genes. The overall frequency of MYD88 mutant samples increased (12% to 19%), but we noted a decrease in p.L265P (8% to 4%) and increase in p.S219C mutations (2% to 6%). CARD11 p.D230N, PIM1 p.K115N and CD79B p.Y196C mutations were not observed in the RR cohort, although these mutations were prominent in the primary DLBCL samples. We observed an increase in BCL2 mutations (21% to 38% of samples), BCL2 amplifications (3% to 6% of samples) and CREBBP mutations (31% to 42% of samples) in the RR cohort, supported by acquisition of mutations in these genes in relapsed compared to diagnostic biopsies from the same patient. These increases may reflect the genetic characteristics of R-CHOP RR tumors expected to be enriched for during clinical trial enrollment. These findings hold significance for a number of emerging targeted therapies aligned to genetic targets and biomarkers in DLBCL, reinforcing the importance of time-of-treatment biomarker screening during DLBCL therapy selection.

Identification of CCR2 and CD180 as Robust Pharmacodynamic Tumor and Blood Biomarkers for Clinical Use with BRD4/BET Inhibitors

Purpose: AZD5153 is a novel BRD4/BET inhibitor with a distinctive bivalent bromodomain binding mode. To support its clinical development, we identified pharmacodynamic (PD) biomarkers for use in clinical trials to establish target engagement. Experimental Design: CCR2 and CD180 mRNAs, initially identified from whole transcriptome profiling, were further evaluated by quantitative PCR in hematologic cell lines, xenografts, and whole blood from rat, healthy volunteers, and patients with cancer. MYC and HEXIM1 mRNAs were also evaluated. Results: RNA-sequencing data showed consistent decreases in CCR2/CD180 expression across multiple hematologic cell lines upon AZD5153 treatment. Evaluation of dose dependence in MV4,11 cells confirmed activity at clinically relevant concentrations. In vivo downregulation of CCR2/CD180 mRNAs (>80%) was demonstrated in MV4,11 and KMS-11 xenograft tumors at efficacious AZD5153 doses. Consistent with in vitro rat blood data, an in vivo rat study confirmed greater inhibition of CCR2/CD180 mRNA in whole blood versus MYC at an efficacious dose. Finally, in vitro treatment of whole blood from healthy volunteers and patients with cancer demonstrated, in contrast to MYC, almost complete downregulation of CCR2/CD180 at predicted clinically achievable concentrations. Conclusions: Our data strongly support the use of CCR2 and CD180 mRNAs as whole blood PD biomarkers for BRD4 inhibitors, especially in situations where paired tumor biopsies are unavailable. In addition, they can be used as tumor-based PD biomarkers for hematologic tumors. MYC mRNA is useful as a hematologic tumor-based biomarker but suboptimal as a whole blood biomarker. Utility of HEXIM1 mRNA may be limited to higher concentrations. Clin Cancer Res; 23(4); 1025–35. ©2017 AACR.

BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer and confers sensitivity to BET inhibitors

BRD4 is a transcriptional co-activator functioning to recruit regulatory complexes to acetylated chromatin. A subset of High-grade Serous Ovarian Cancer (HGSOC) patients are typified by focal, recurrent BRD4 gene amplifications. Despite previously described cancer dependencies, it is unclear whether BRD4 amplification events are oncogenic in HGSOC. We find that physiologically relevant levels of expression of BRD4 isoforms in non-transformed ovarian cells result in cellular transformation. Transcriptional profiling of BRD4-transformed ovarian cells, and BRD4-amplified HGSOC patient samples revealed shared expression patterns, including enriched MYC, and E2F1 gene signatures. Furthermore, we demonstrate that a novel BET inhibitor, AZD5153, is highly active in BRD4-amplified patient derived xenografts and uncover Neuregulin-1 as a novel BRD4 effector. Experiments involving Neuregulin-1 inhibition and exogenous addition, demonstrate Neuregulin-1 as necessary and sufficient for BRD4-mediated transformation. This study demonstrates the oncogenic potential of BRD4 amplification in cancer and establishes BRD4-amplified HGSOC as a potential patient population that could benefit from BET inhibitors.

BRD4 facilitates replication stress-induced DNA damage response

Previous reports have demonstrated that select cancers depend on BRD4 to regulate oncogenic gene transcriptional programs. Here we describe a novel role for BRD4 in DNA damage response (DDR). BRD4 associates with and regulates the function of pre-replication factor CDC6 and plays an indispensable part in DNA replication checkpoint signaling. Inhibition of BRD4 by JQ1 or AZD5153 resulted in a rapid, time-dependent reduction in CHK1 phosphorylation and aberrant DNA replication re-initiation. Furthermore, BRD4 inhibition sensitized cancer cells to various replication stress-inducing agents, and synergized with ATR inhibitor AZD6738 to induce cell killing across a number of cancer cell lines. The synergistic interaction between AZD5153 and AZD6738 is translatable to in vivo ovarian cell-line and patient-derived xenograft models. Taken together, our study uncovers a new biological function of BRD4 and provides mechanistic rationale for combining BET inhibitors with DDR-targeted agents for cancer therapy.

Abstract B21: Brd4 modulates S-phase checkpoint response and sensitizes ovarian cancer cells to ATR inhibition

The BET (bromodomain and extra terminal domain) family proteins function as chromatin readers through interactions with acetylated lysine residues on both histones and transcription factors. BRD4 is the most well-studied member of this group that promotes tumorigenesis by regulating expression of core transcriptional programs; however, the role of Brd4 in other cellular processes is not entirely clear. Here, we show using small molecule inhibition of the BET proteins that Brd4 is required to maintain S-phase checkpoint signaling. Treatment of high-grade serous ovarian and osteosarcoma cancer cell lines with BET inhibitors, AZD5153 and JQ1, results in the accumulation of gH2AX and 53BP1 foci formation, which is suggestive of elevated DNA damage levels. The induction of DNA damage caused by both inhibitors corresponds with cell cycle arrest at the G1/S boundary. Displacement of Brd4 in cell lines under intrinsic or exogenous replication stress led to a time-dependent reduction in phospho-Chk1 first detected within 30 minutes of AZD5153 treatment in U2OS cells. The decrease in Chk1 phosphorylation was observed without a concomitant decrease in total Chk1, and this was not replicated by treatment with pan-transcriptional inhibitors, suggesting a non-transcriptional mechanism linked to Brd4. Furthermore, Brd4 interacts with core DNA replication machinery during S-phase and inhibition of Brd4 leads to aberrant DNA replication without decreasing ATR phosphorylation. Consistent with a role in S-phase signaling, Brd4 inhibition sensitizes replicating cells to replication stress-inducing agents. Finally, we observed that ovarian cancer cell lines are highly-sensitive to the combination of Brd4 and ATR (AZD6738) inhibitors. Concurrent treatment with AZD5153 and AZD6738 induces DNA damage and apoptosis in a synergistic manner. Together, these findings highlight a novel function for Brd4 and provide evidence to support further evaluation of Brd4i combinations with DNA damage repair agents. Citation Format: Jingwen Zhang, Huawei Chen, Austin Dulak. Brd4 modulates S-phase checkpoint response and sensitizes ovarian cancer cells to ATR inhibition. [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 B21.

Abstract 1026: Identify BRD4 as a facilitator of replication stress response signaling

BRD4 is a member of the BET (bromodomain and extraterminal domain) family of chromatin readers that recognize acetylated-lysines on histones and nuclear proteins. Previous reports have demonstrated that select cancers depend on BRD4 to regulate oncogenic gene transcriptional programs. However, whether BRD4 contributes to cancer malignancy through other mechanisms has not been extensively evaluated. Here, we show that BRD4 is important for maintaining an intact DNA replication checkpoint in cancers. Displacement of BRD4 by BET bromodomain inhibitor AZD5153 in cell lines under intrinsic or exogenous replication stress led to a time-dependent reduction in phospho-Chk1 first detected within 30 minutes, and reaching maximum phospho-Chk1 inhibition (~85%) at an hour after AZD5153 treatment in U2OS cells. The decrease in Chk1 phosphorylation was observed without a concomitant decrease in total Chk1, and this was not replicated by treatment with pan-transcriptional inhibitors, suggesting a non-transcriptional mechanism linked to BRD4. Furthermore, BRD4 interacts with the DNA pre-replication complex and inhibition of BRD4 leads to hyperactivation of the pre-replication complex and aberrant DNA replication re-initiation under replication stress conditions. Consistent with a role in S-phase signaling, BETi treatment sensitizes replicating cells to replication stress-inducing agents. Finally, we observed that ovarian cancer cell lines are highly-sensitive to the combined inhibition of BRD4 and ATR. Coadministration of AZD5153 and AZD6738 (ATR inhibitor) significantly inhibited tumor growth in OVCAR3 ovarian xenograft model (TGI after 21 day dosing: AZD5153, 52%; AZD6738, 46%; Combo, 84%). Sustained tumor growth delay was observed after combination treatment cessation. Together, our study uncovered a novel function for BRD4 in regulating DNA replication stress response, and provide mechanistic rational for combining BETi with DNA damage-targeted agents for cancer therapies. Citation Format: Jingwen Zhang, Brandon Willis, Maureen Hattersley, Alan Lau, Corinne Reimer, Michael Zinda, Stephen Fawell, Gordon Mills, Austin Dulak, Huawei Chen. Identify BRD4 as a facilitator of replication stress response signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1026. doi:10.1158/1538-7445.AM2017-1026

Abstract 4705: Therapeutic activity of bivalent BRD4 inhibitor AZD5153 in hematological cancers

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Across a number of tumor models pharmacological targeting of BRD4 bromodomains by small-molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor. AZD5153 is a candidate drug that possesses an unprecedented bivalent binding mode among reported BET inhibitors, which allows AZD5153 to ligate the tandem bromodomains in BRD4. The avidity resulted from the bivalent binding interaction translates into markedly enhanced cellular potency. Although AZD5153 demonstrates broad activity across a cancer cell line panel comprising solid and hematologic subtypes, there is enriched antitumor activity against hematologic cell lines, including acute myeloid leukemia (AML), multiple myeloma (MM), and diffuse large B-cell lymphoma (DLBCL). The activity of AZD5153 in hematologic tumors was further confirmed in five selected xenograft models of AML, MM, and DLBCL where AZD5153 treatment led to tumor stasis or regression, accompanied by concomitant modulation of BRD4 pharmacodynamic markers, such as MYC and HEXIM1. In order to characterize the transcriptional consequences elicited by AZD5153, we carried out transcriptional profiling of 11 hematologic tumor lines and identified the robust modulation of MYC, and E2F transcriptional programs. Moreover, our transcriptional data was used to identify candidate clinical PD biomarkers. The suitability and dynamic range of the top two candidate biomarkers for AZD5153 was confirmed using human whole blood from normal healthy volunteers. To identify protein biomarkers associated with sensitivity to AZD5153 treatment, we deployed reverse-phase protein array (RPPA) technology to quantitatively examine the level of 182 proteins following AZD5153 treatment. Our findings indicate that cell lines sensitive to AZD5153 uniquely exhibit a marked decrease in the level of mTOR-pathway associated proteins following AZD5153 treatment. Conversely, MYC modulation was observed in both sensitive and resistant groups. Thus, these data suggest that in hematologic malignancies, mTOR pathway downregulation may serve as an appropriate biomarker of sensitivity to BRD4 inhibitors such as AZD5153. Our study establishes AZD5153 as a novel and potent BRD4/BET inhibitor possessing a unique bivalent binding property. We have characterized the pharmacological consequences of BRD4/BET inhibition by AZD5153 via unbiased transcriptional and proteome profiling. These efforts are the first to identify mTOR modulation as a putative biomarker of sensitivity to BET bromodomain inhibition in hematologic tumors and may help to inform future clinical evaluation of AZD5153 and other BET bromodomain inhibitors. Citation Format: Huawei (Ray) Chen, Maureen Hattersley, Garrett Rhyasen, Austin Dulak, Wendy Wang, Phil Petteruti, Ian Dale, Tony Cheung, Shenghua Wen, Lilian Castriotta, Deborah Lawson, Mike Collins, Miika Ahdesmaki, Graeme Walker, Al Rabow, Jonathan Dry, Corinne Reimer, Paul Lyne, Steve Fawell, MIke Waring, Mike Zinda, Ed Clark, Ed Clark. Therapeutic activity of bivalent BRD4 inhibitor AZD5153 in hematological cancers. [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 4705.