Extra-terminal Bromodomain Research Articles

Exploring Transcriptional Regulation of Beta Cell SASP by Brd4-Associated Proteins and Cell Cycle Control Protein p21.

Type 1 diabetes (T1D) is a metabolic disease resulting from progressive autoimmune destruction of insulin-producing pancreatic beta cells. Although the majority of beta cells are lost in T1D, a small subset undergoes senescence, a stress response involving growth arrest, DNA damage response, and activation of a senescence-associated secretory phenotype (SASP). SASP in beta cells of the nonobese diabetic (NOD) mouse model of T1D and primary human islets is regulated at the level of transcription by bromodomain extra-terminal (BET) proteins, but the mechanisms remain unclear. To explore how SASP is transcriptionally regulated in beta cells, we used the NOD beta cell line NIT-1 to model beta cell SASP and identified binding partners of BET protein Brd4 and explored the role of the cyclin-dependent kinase inhibitor p21. Brd4 interacted with a variety of proteins in senescent NIT-1 cells including subunits of the Ino80 chromatin remodeling complex, which was expressed in beta cells during T1D progression in NOD mice and in human beta cells of control, autoantibody-positive, and T1D donors as determined from single-cell RNA-seq data. RNAi knockdown of p21 during senescence in NIT-1 cells did not significantly impact viability or SASP. Taken together, these results suggest that Brd4 interacts with several protein partners during senescence in NIT-1 cells, some of which may play roles in SASP gene activation and that p21 is dispensable for the SASP in this beta cell model.

Targeting BRD4 attenuates the stemness and aggressiveness of ameloblastoma.

BRD4, belonging to the bromodomain extra-terminal (BET) protein family, plays a unique role in tumor progression. However, the potential impact of BRD4 in ameloblastoma (AM) remains largely unknown. Herein, we aimed to assess the expression and functional role of BRD4 in AM. The expression level of BRD4 was assessed by immunohistochemistry. The proliferation, migration, invasion, and tumorigenic abilities of AM cells were assessed by a series of assays. To explore the molecular expression profile of BRD4-depleted AM cells, RNA sequencing (RNA-seq) was performed. Bioinformatic analysis was performed on AM expression matrices obtained from the Gene Expression Omnibus (GEO). The therapeutic efficacy of BET-inhibitors (BETi) was assessed with AM patient-derived organoids. Upregulation of BRD4 was observed in conventional AMs, recurrent AMs, and ameloblastic carcinomas. Depletion of BRD4 inhibited proliferation, invasion, migration, and tumorigenesis in AM. Administration of BETi attenuated the aggressiveness of AM and the growth of AM patient-derived organoids. Bioinformatic analysis indicated that BRD4 may promote AM progression by regulating the Wnt pathway and stemness-associated pathways. BRD4 increases the aggressiveness and promotes the recurrence of ameloblastoma by regulating the Wnt pathway and stemness-associated pathways. These findings highlight BRD4 as a promising therapeutic target in AM management.

BET proteins inhibitor JQ1 impairs GM-CSF-promoted peritoneal macrophage self-renewal and IL-4-induced alternative polarization

Peritoneal macrophages (PMs), which resided in peritoneal cavity, are crucial to maintain tissue homeostasis and immunity. Macrophage self-renewal and polarization states are critical for PM population homeostasis and function. However, the underlying molecular mechanism that regulates self-renewal and polarization of PMs is still unclear and needs to be explored. Here, we demonstrated that PMs self-renewal was stimulated by granulocyte macrophage colony-stimulating factor (GM-CSF), but not by macrophage colony-stimulating factor (M−CSF). Pharmacological inhibition of Bromodomain & Extraterminal (BET) Proteins by either JQ1 or ARV-825 significantly reduced GM-CSF-dependent peritoneal macrophage self-renewal by abrogating cell proliferation and decreasing self-renewal-related gene expression, such as MYC and Klf4, at transcriptional and protein levels. In addition, transcriptomic analysis showed that JQ1 blocked alternative PMs polarization by downregulating key transcriptional factor IRF4 expression, but not the activation of AKT or STAT6 in PMs. These findings illustrated that the significance of BET family proteins in GM-CSF-induced PMs self-renewal and IL-4-induced alternative polarization.

Abstract LB153: Inhibiting BRD4 as a proposed alternative therapeutic strategy for HPV16-associated head and neck squamous cell carcinoma

Abstract Integration of Human Papilloma Virus (HPV) 16 into human genomes is an integral event for cancer progression in Head and Neck Squamous Cell Carcinoma (HNSCC). There is a need to differentiate treatment for HPV-16 integrated HNSCC from other subtypes. We hypothesize that by inhibiting Bromodomain Extra Terminal (BET) protein BRD4, a transcriptional co-regulator of host and viral genes can advance treatment in patients with the integrated subtype of HPV16 associated HNSCC. Through small molecule inhibition and knockdown of BRD4 in seven model HPV-16 integrated cell lines, we demonstrated that BRD4 down-regulates the expression of viral genes E6, E7, independent of viral E2 type of integration. Given that E6 expression is mediated by BRD4, E6-p53 interactions was abrogated as revealed through increased expression of p53. Furthermore, BRD4 inhibition caused downregulation of c-Myc. This result also suggests the function of BRD4 in the regulation of proteins in cell cycle regulation, including E2F1 and p21. BRD4 inhibition was also shown to delay DNA damage response (DDR) by reducing the expression of RAD51 in 6 out of 7 cell lines. RNA-sequencing results give further insight regarding the downregulation of genes involved in DDR – RAD51, BRCA1 and BRCA2 in all cell lines. RNA-sequencing data also gives us an understanding of the implication of BRD4-inhibition in other genes involved in cell cycle regulation. Knockdown of BRD4 done through small hairpin RNA (shRNA) constructs phenocopied the effects shown through inhibition of BRD4. Additionally, treating the above-mentioned cell lines with BRD4 inhibitor (JQ1) followed by radiation showed further downregulation of viral oncogenes E6, and E7. This suggests that combining radiation and JQ1 treatment can be a viable combination treatment. De-escalating radiation treatment with BRD4 inhibition has potential to be an effective combination treatment for HPV16 integrated HNSCC. Citation Format: Dhruthi Suresh, Chitrasen Mohanty, Albert R. Wang, Christina Kendziorski, Gopal Iyer. Inhibiting BRD4 as a proposed alternative therapeutic strategy for HPV16-associated head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB153.

A novel dual epigenetic approach targeting BET proteins and HDACs in Group 3 (MYC-driven) Medulloblastoma

BackgroundMedulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor clinical outcomes and respond poorly to current therapies. Epigenetic deregulation is very common in MYC-driven MB. The bromodomain extra-terminal (BET) proteins and histone deacetylases (HDACs) are epigenetic regulators of MYC transcription and its associated tumorigenic programs. This study aimed to investigate the therapeutic potential of inhibiting the BET proteins and HDACs together in MB.MethodsUsing clinically relevant BET inhibitors (JQ1 or OTX015) and a pan-HDAC inhibitor (panobinostat), we evaluated the effects of combined inhibition on cell growth/survival in MYC-amplified MB cell lines and xenografts and examined underlying molecular mechanism(s).ResultsCo-treatment of JQ1 or OTX015 with panobinostat synergistically suppressed growth/survival of MYC-amplified MB cells by inducing G2 cell cycle arrest and apoptosis. Mechanistic investigation using RNA-seq revealed that co-treatment of JQ1 with panobinostat synergistically modulated global gene expression including MYC/HDAC targets. SYK and MSI1 oncogenes were among the top 50 genes synergistically downregulated by JQ1 and panobinostat. RT-PCR and western blot analyses confirmed that JQ1 and panobinostat synergistically inhibited the mRNA and protein expression of MSI1/SYK along with MYC expression. Reduced SYK/MSI expression after BET (specifically, BRD4) gene-knockdown further confirmed the epigenetic regulation of SYK and MSI1 genes. In addition, the combination of OTX015 and panobinostat significantly inhibited tumor growth in MYC-amplified MB xenografted mice by downregulating expression of MYC, compared to single-agent therapy.ConclusionsTogether, our findings demonstrated that dual-inhibition of BET and HDAC proteins of the epigenetic pathway can be a novel therapeutic approach against MYC-driven MB.

225 (PB105) - Discovery of novel small molecules that recruit DCAF11 for selective degradation of BRD4

Background: Targeted protein degradation (TPD) using the endogenous ubiquitin (Ub) proteasome system (UPS) is a rapidly growing drug discovery approach to eliminate pathogenic proteins. Strategies for TPD have focused on designing heterobifunctional PROTACs that utilize ligand binding to select E3 ligases (e.g., cereblon and VHL), often resulting in compounds with poor drug-like properties. Monovalent degraders represent an alternative approach, in which small molecules are designed to bind the target protein and induce its degradation through the recruitment of an E3 ligase complex. Monovalent degraders generally have better physico-chemical properties than PROTACs; however, designing compounds to induce degradation is not well precedented and relies on serendipitous discovery. Methods: An E3 ligase agnostic chemical library was screened using our ultra-high throughput cell-based screening (uHTS) platform. Hits were identified by measuring protein degradation of BRD4 using immunofluorescence. A Ub pathway focused CRISPR screen was used to identify the E3 ligase responsible for compound-induced degradation of BRD4. The degradation mechanism was validated using western blot, luciferase-based protein-protein interaction (PPI) and mutational analyses. In vitro antitumor activity was assessed by proliferation and apoptosis assays. NOD/ SCID mice were implanted sub-Q with acute myeloid leukemia (AML) xenograft model MV-4-11 and treated with compounds administered QD. Results: uHTS resulted in identification of selective monovalent degraders that target the bromodomain extra-terminal (BET) protein, BRD4. Degrader series optimization produced PLX-3618, which demonstrated potent degradation of BRD4, without depleting BET family members BRD2 and BRD3. Proteasome and neddylation inhibitors confirmed degradation was mediated via the UPS, and a Ub-focused CRISPR screen identified CUL4DCAF11 as the E3 complex responsible for PLX-3618 induced degradation of BRD4. PPI studies verified a BRD4/PLX-3618/DCAF11 ternary complex; BRD4 point mutational analyses provided further insights into the DCAF11-mediated degradation mechanism. Degradation of BRD4 by PLX-3618 resulted in downregulation of the MYC oncogene and potent anti-proliferative activity against a panel of tumor cell lines, with high sensitivity observed in AML. in vivo, PLX-3618 resulted in complete tumor regression, whereas a pan-BET inhibitor only resulted in tumor growth inhibition without regression. PK/PD analyses provided insight into the unique exposure/response profile of targeted protein degraders. Conclusion: These results demonstrate the efficient discovery of novel monovalent degraders using our proprietary platform. Characterization of the degradation mechanism highlights the discovery of DCAF11 as a novel E3 ligase substrate receptor amenable to recruitment and degradation of BRD4. No conflict of interest.

Selective bromodomain and extra-terminal bromodomain inhibitor inactivates macrophages and hepatic stellate cells to inhibit liver inflammation and fibrosis

ABSTRACT Liver fibrosis occurs following inflammation triggered by the integrated actions of activated liver-resident macrophages (Kupffer cells) and hepatic stellate cells (HSCs), and the multiplicity of these mechanisms complicates drug therapy. Here, we demonstrate that the selective bromodomain and extra-terminal (BET) bromodomain inhibitor compound38 can block both the Janus kinase-signal transducer and activator of transcription and mitogen-activated protein kinase signaling pathways in macrophages, which decreased their secretion of proinflammatory cytokines in a dose-dependent manner. The inactivation of macrophages attenuated lipopolysaccharide-induced injurious inflammation concurrent with a reduction in F4/80+ cells, proinflammatory cytokine levels, and neutrophil infiltration. Moreover, compound 38 inhibited the Wnt/β-catenin and transforming growth factor-beta/SMAD signaling pathways to abolish the activation of HSCs. In vivo, compound 38 significantly decreased the collagen deposition and fibrotic area of a CCl4-induced liver fibrosis model, and restored the deficiency of activated HSCs and the upregulation of liver inflammation. These results highlight the potential role of compound 38 in treating liver fibrosis considering its simultaneous inhibitory effects on liver inflammation and related fibrosis.

Design and Synthesis of Hybrid Compounds as Epigenetic Modifiers.

Epigenetic modifiers acting through polypharmacology mechanisms are promising compounds with which to treat several infectious diseases. Histone deacetylase (HDAC) enzymes, mainly class I, and extra-terminal bromodomains (BET) are involved in viral replication and the host response. In the present study, 10 compounds were designed, assisted by molecular docking, to act against HDAC class I and bromodomain-4 (BRD4). All the compounds were synthesized and characterized by analytical methods. Enzymatic assays were performed using HDAC-1, -4, and -11 and BRD4. Compounds (2–10) inhibited both HDAC class I, mainly HDAC-1 and -2, and reduced BRD4 activity. For HDAC-1, the inhibitory effect ranged from 8 to 95%, and for HDAC-2, these values ranged from 10 to 91%. Compounds (2–10) decreased the BRD4 activity by up to 25%. The multi-target effects of these compounds show desirable properties that could help to combat viral infections by acting through epigenetic mechanisms.

Epigenetically Enhanced MED12L in ETO2-GLIS2 Positive Pediatric Acute Megakaryoblastic Leukemia Is Associated with Resistance to the CDK8 Inhibitors

Introduction. ETO2-GLIS2 (aka CBFA2T3-GLIS2) is the most common (30%) alteration in pediatric de novo acute megakaryoblastic leukemia (AMKL). These patients have poor response to induction therapy, a high incidence of relapse (~90%), and dismal 5-year survival rates (<20%). Previous studies suggest that ETO2-GLIS2 induces leukemia through abnormal enhancer formation as a single oncogenic “hit”. Because ETO2-GLIS2 expression induces formation of leukemia-specific neo-superenhancer (SE) elements, we hypothesize that mediator (MED) proteins are involved in linking neo-SE elements to distal gene expression, and thus could be a therapeutic target. Here, we analyzed expression of MED-family genes in ETO2-GLIS2 positive AMKL patients in combination with enhancer-histone marks, and evaluated impact of MED-kinase inhibition with selective CDK8 inhibitors (CDKi) against cell viability.Methods. To address our hypothesis, we analyzed published RNA sequencing dataset (Smith et al. 2020) for human MED-genes expression from a pan-pediatric AML cohort (N=1476). The cohort consisted of subgroups expressing fusions of ETO2-GLIS2 (N=40), CBFB-MYH11 (N=174), DEK-NUP214 (N=49), KMT2A-ELL (N=50), KMT2A-MLLT10 (N=86), KMT2A-MLLT3 (N=114), KMT2A-MLLT4 (N=49), NUP98-NSD1 (N=107), RUNX1-RUNX1T1 (N=210) and no detectable fusions (N=526), compared to normal bone marrow (NBM) samples (N=71). Enrichment of histone marks overlapping MED-genes was analyzed from published chromatin immunoprecipitation (ChIP) sequencing in ETO2-GLIS2 positive M-07e AMKL line (Thirant et al. 2017). We tested efficacy of CDK8 inhibition with BI-1347 and CCT251545 against M-07e cells to determine their activity in the context of marked MED12L overexpression.Results. We examined expression of 29 MED-genes comprising the 4 major (head, middle, tail, and kinase) MED-modules. MED gene expression was variable across AML subtypes and NBM. However, MED genes were more commonly over-expressed in the ETO2-GLIS2 group, in particular MED 17, MED1, MED10, MED27, and MED12L (paralog of MED12) were upregulated in the subgroup. Most notably, we noted exceptional upregulation of MED12L (FC 4.9, log2), compared to NBM (Figure 1). Because MED12/12L plays an intrinsic biological role in establishing oncogenic enhancer-expression loops in hematopoietic stem or leukemic cells, we investigated the overlap of enhancer bound histones such as H3K27ac and H3K4me1 to MED12L in M-07e cells, compared to umbilical cord blood-derived normal megakaryoblasts (MK) (S004BT; Blueprint epigenome database). We found enrichment of H3K4me1 at MED12L transcription start site (TSS) and upstream promoter both in MK and M-07e cells. In addition, we observed a large region of H3K27ac enrichment spanning 89 Kb (16 kb upstream and 73 kb downstream) across MED12L TSS in M-07e cells, suggesting neo-enhancer activity at this locus. Considering the dependency of MED12 on CDK8 for MED-kinase activities (Klatt et al. 2020), we treated M-07e cells with CDK8i(s), to test our hypothesis if perturbation of epigenetically enhanced MED12L expression can impact leukemic growth. However, we observed a poor correlation between M-07e cell viability and IC 50 of BI-1347 (IC 50: 0.87 µM, R 2: 0.36) or CCT251545 (IC 50: 0.4 µM, R 2: 0.48). In contrast, ETO2-GLIS2 negative MV4-11 AML cells were susceptible to both BI-1347 (IC 50: 0.44 µM, R 2: 0.88) and CCT251545 (IC 50: 0.12 µM, R 2: 0.87). Given the inefficacy of CDK8i against M-07e, and cooperativity of bromodomain extra-terminal (BET)-BRD4 and MED12/12L in forming enhancer complexes, we tested possible inhibitory impact of BRD4 inhibitor JQ1 on MED12L expression and leukemic growth in target cells. We observed effective reduction in M-07e cell viability (IC 50:0.3 µM, R 2: 0.93) with concomitant reduction not only in BRD4 protein-expression, but diminished MED12L protein expression at IC 50 and higher doses of JQ1.Conclusion. Our findings revealed that MED12L is highly overexpressed and overlapped with strong neo-enhancer chromatin-marks in ETO2-GLIS2 positive cells, while maintaining resistance to CDK8i. Future studies will contribute to deeper insights into the preferential recruitment and role of MED12L in ETO2-GLIS2 bound enhancers and potential mechanisms of resistance to CDK8 inhibition in the disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Synergistic efficacy of inhibiting MYCN and mTOR signaling against neuroblastoma

BackgroundNeuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB.MethodsUsing small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses.ResultsCo-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy.ConclusionsTogether, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.

Abstract 1283: Discovery of selective and potent BRD4 protein degraders using Plexium's DELPhe platform

Abstract Targeted protein degradation using the endogenous Ubiquitin Proteasome System (UPS) represents a fundamentally new approach to drug discovery that potentially allows proteins that cannot be modulated by conventional small molecule inhibitors to be brought under therapeutic control. Plexium has developed the DELPhe platform, which combines solid phase synthesis of DNA encoded libraries with high-throughput ultra-miniaturized cell-based assays, to readily and cost-effectively identify both bifunctional (PROTAC) and monovalent degraders for traditionally undruggable targets including scaffolding proteins, protein-protein interactions and transcription factors. In addition to modulating previously “undruggable” proteins, degradation can lead to greater efficacy and a prolonged downstream signaling response, thereby addressing common obstacles seen with small molecule inhibitors. The bromodomain extra-terminal (BET) protein family are epigenetic readers that have been targeted using small molecule inhibitors. Compounds currently in development typically inhibit multiple BET family members. The bromodomain protein BRD4 is a transcriptional and epigenetic regulator that associates at super-enhancers, driving the expression of oncogenic proteins such as MYC that are critical for the pathogenesis of cancer. We describe here the use of Plexium's DELPhe platform to sample extensive chemical space and discover small molecule monovalent degraders that demonstrate selective and sustained degradation of BRD4. Near complete degradation was observed within 4 hours and DC50 potency of &amp;lt;10 nM was achieved for BRD4 without any appreciable degradation of the highly homologous BRD2 and BRD3 proteins. Degradation of BRD4 resulted in down regulation of MYC protein levels and potent anti-proliferative activity (20-200 nM) against a panel of tumor cell lines with activity superior to inhibition with the pan-BET inhibitor JQ1. Co-treatment with either proteosome or neddylation inhibitors blocked BRD4 degradation, suggesting that protein turnover is regulated by a Cullin-RING Ligase (CRL). Biochemical studies verified a direct interaction between the small molecule and BRD4, suggesting that binding promotes a conformational change that exposes key protein motifs to ubiquitination and degradation. Mutational analysis and a ubiquitin ligase-focused CRISPR screen were used to provide insights into defining the principles of degradation. Collectively, these data demonstrate that the Plexium DELPhe platform enables the discovery of selective and potent monovalent degraders of BRD4. Citation Format: Gregory S. Parker, Julia A. Toth, Geoffray Leriche, Simon Bailey, Kenneth Chng, Sara Fish, Aleks Jamborcic, Elizabeth Daniele, Erika Green, Michael Hocker, Adam Kallel, Peggy A. Thompson, Steven D. Brown, Kandaswamy Vijayan. Discovery of selective and potent BRD4 protein degraders using Plexium's DELPhe platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1283.

Targeted Therapy for AML Expressing RUNX1 Mutation

RUNX1 is the DNA-binding subunit of the core binding factor (CBF) complex and a transcription factor (TF) involved in normal and malignant hematopoiesis. Transcriptionally active full-length RUNX1b isoform is transcribed from 6 exons, encoding a DNA binding ‘Runt’ homology domain and a more C-terminal transcription activation domain. Within its CTCF-anchored loop or TAD (topologically-associating domain), RUNX1 gene has a highly conserved +24-kb enhancer (eR1 in mouse), which is part of a large super-enhancer (>170 kb) covering the entire intron 1 between proximal P2 and distal P1 promoter of RUNX1 and controlling the transcription of RUNX1. TFs bind to super-enhancers/enhancers that loop over to interact with promoters to transcriptionally regulate oncogenes, such as RUNX1 and its targets, as well as multiple other AML-relevant oncogenes involved in growth and survival of AML blast progenitor cells (BPCs). Heatmap of Hi-C interaction scores within the RUNX1 TAD showed triangle shaped regions of high interaction scores highlighting interactions between +24-kb enhancer and the P2 promoter of RUNX1. To functionally validate these findings, we cloned and transfected into HEK293 cells expressing RUNX1, a luciferase reporter construct containing the DNA of the P2 promoter with or without the +24 kb-enhancer of RUNX1. Our findings confirm that, as compared to the promoter or the enhancer alone, the +24 kb-enhancer-driven P2 promoter exhibited superior luciferase induction representing increased transcriptional activity of RUNX1. Somatic mutations in RUNX1 commonly occur in MDS, as well as in the secondary (s) or de novo AML (~10%). Majority of mutant (mt) RUNX1 are missense, large deletions or truncation-mutations, behaving mostly as loss of function mutations. Presence of mtRUNX1 confers relative therapy-resistance and poorer survival in patients with AML. Lack of specific targeted therapy coupled with resistance to standard chemotherapy may account for the poorer outcome in AML expressing somatic or germline mtRUNX1. Therefore, there is a strong rationale and an unmet need to develop novel targeted therapies for AML expressing somatic or germline mtRUNX1. In the present studies, we demonstrate for the first time that shRNA mediated knockdown of RUNX1 expression inhibits growth and induces differentiation and apoptosis of cultured cell line (OCI-AML5) and patient-derived (PD) AML BPCs expressing mtRUNX1. After engraftment, doxycycline-inducible shRNA-mediated in vivo knockdown of RUNX1 in OCI-AML5 cells, compared to controls, markedly reduced AML burden and improved survival of the immune-depleted (NSG) mice engrafted with luciferase transduced OCI-AML5 cells. Interaction of TFs with DNA and transcriptional co-factors such as the histone acetyltransferase p300 at enhancers and promoters leads to acetylation of histone proteins and TFs. BET (Bromodomain Extra-terminal) protein (BETP) BRD4 binds to acetylated histones and TFs at super-enhancers and recruits transcriptional co-factors to promote RNA pol II (RNAP2)-mediated transcription of oncogenes and their targets. ChIP-Seq analysis with anti-BRD4 and anti-H3K27Ac antibody showed that Runx1 super-enhancer and P2 promoter are occupied by BRD4. shRNA mediated knockdown of BRD4 attenuated expression of RUNX1 and its targets, MYC, PU.1 and myeloperoxidase (MPO), inhibited growth and induced apoptosis of OCI-AML5 cells. We also found that BETP-PROTACs (proteolysis targeted chimera) ARV-825 and its pharmacologically superior version ARV-771 (20 to 250 nM) caused degradation and depletion of BRD4, reduced BRD4 occupancy at the+24-kb-enhancer and P2 promoter of RUNX1, repressing RUNX1 and its targets, and potently induced apoptosis of OCI-AML5, Mono-Mac-1 and PD AML BPCs expressing mtRUNX1. Treatment with ARV-771 (30 mg/kg SQ daily for 3 weeks also inhibited AML growth and improved survival of NSG mice engrafted with luciferase-transduced OCI-AML5 cells. By utilizing the RNA-Seq mRNA signature from RUNX1-depleted AML cells we queried for expression mimickers (EMs) through LINCS1000-CMap (Connectivity Mapping) analyses. This uncovered several novel agents that inhibit RUNX1 expression and induce in vitro lethality and reduce in vivo leukemia burden in AML BPCs expressing mtRUNX1. These findings highlight RUNX1-targeted novel agents to be developed and tested as potential therapy for AML expressing mtRUNX1. DisclosuresDiNardo:AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding. Qian:4Arvinas, LLC. New Haven, CT: Employment. Raina:4Arvinas, LLC. New Haven, CT: Employment. Takahashi:Symbio Pharmaceuticals: Consultancy.

Bromodomain Epigenetic Protein Promotes Metastatic Potential in Melanoma Cells through Increased Invasiveness and Decreased Macrophage-Mediated Phagocytosis

Bromodomain Epigenetic Protein Promotes Metastatic Potential in Melanoma Cells through Increased Invasiveness and Decreased Macrophage-Mediated Phagocytosis

Metabolism of JQ1, an inhibitor of bromodomain and extra terminal bromodomain proteins, in human and mouse liver microsomes†.

JQ1 is a small-molecule inhibitor of the bromodomain and extra terminal (BET) protein family that potently inhibits the bromodomain testis-specific protein (BRDT), which is essential for spermatogenesis. JQ1 treatment produces a reversible contraceptive effect by targeting the activity of BRDT in mouse male germ cells, validating BRDT as a male contraceptive target. Although JQ1 possesses favourable physical properties, it exhibits a short half-life. Because the details of xenobiotic metabolism play important roles in the optimization of drug candidates and in determining the role of metabolism in drug efficacy, we investigated the metabolism of JQ1 in human and mouse liver microsomes. We present the first comprehensive view of JQ1 metabolism in liver microsomes, distinguishing nine JQ1 metabolites, including three monohydroxylated, one de-tert-butylated, two dihydroxylated, one monohydroxylated/dehydrogenated, one monohydroxylated-de-tert-butylated and one dihydroxylated/dehydrogenated variant of JQ1. The dominant metabolite (M1) in both human and mouse liver microsomes is monohydroxylated on the fused three-ring core. Using recombinant cytochrome P450 (CYP) enzymes, chemical inhibitors and the liver S9 fraction of Cyp3a-null mice, we identify enzymes that contribute to the formation of these metabolites. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is the main contributor to the production of JQ1 metabolites in vitro, and the CYP3A4/5 inhibitor ketoconazole strongly inhibits JQ1 metabolism in both human and mouse liver microsomes. Our findings suggest that JQ1 half-life and efficacy might be improved in vivo by co-administration of a selective CYP inhibitor, thereby impacting the use of JQ1 as a probe for BRDT activity in spermatogenesis and as a probe or therapeutic in other systems.

BET Inhibitors Synergize with Carfilzomib to Induce Cell Death in Cancer Cells via Impairing Nrf1 Transcriptional Activity and Exacerbating the Unfolded Protein Response.

Currently, proteasome inhibitors bortezomib, carfilzomib, and ixazomib are successfully used in clinics to treat multiple myeloma. However, these agents show limited efficacy against solid tumors. Identification of drugs that can potentiate the action of proteasome inhibitors could help expand the use of this therapeutic modality to solid tumors. Here, we found that bromodomain extra-terminal (BET) family protein inhibitors such as JQ1, I-BET762, and I-BET151 synergize with carfilzomib in multiple solid tumor cell lines. Mechanistically, BET inhibitors attenuated the ability of the transcription factor Nrf1 to induce proteasome genes in response to proteasome inhibition, thus, impeding the bounce-back response of proteasome activity, a critical pathway by which cells cope with proteotoxic stress. Moreover, we found that treatment with BET inhibitors or depletion of Nrf1 exacerbated the unfolded protein response (UPR), signaling that was initiated by proteasome inhibition. Taken together, our work provides a mechanistic explanation behind the synergy between proteasome and BET inhibitors in cancer cell lines and could prompt future preclinical and clinical studies aimed at further investigating this combination.

BET Proteins Are Required for Transcriptional Activation of the Senescent Islet Cell Secretome in Type 1 Diabetes.

Type 1 diabetes (T1D) results from the progressive loss of pancreatic beta cells as a result of autoimmune destruction. We recently reported that during the natural history of T1D in humans and the female nonobese diabetic (NOD) mouse model, beta cells acquire a senescence-associated secretory phenotype (SASP) that is a major driver of disease onset and progression, but the mechanisms that activate SASP in beta cells were not explored. Here, we show that the SASP in islet cells is transcriptionally controlled by Bromodomain ExtraTerminal (BET) proteins, including Bromodomain containing protein 4 (BRD4). A chromatin analysis of key beta cell SASP genes in NOD islets revealed binding of BRD4 at active regulatory regions. BET protein inhibition in NOD islets diminished not only the transcriptional activation and secretion of SASP factors, but also the non-cell autonomous activity. BET protein inhibition also decreased the extent of SASP induction in human islets exposed to DNA damage. The BET protein inhibitor iBET-762 prevented diabetes in NOD mice and also attenuated SASP in islet cells in vivo. Taken together, our findings support a crucial role for BET proteins in the activation of the SASP transcriptional program in islet cells. These studies suggest avenues for preventing T1D by transcriptional inhibition of SASP.

Abstract CT095: A Phase Ib/IIa study of the BET bromodomain inhibitor ZEN-3694 in combination with enzalutamide in patients with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Abiraterone (ABI) and enzalutamide (ENZ) have significant activity in mCRPC yet demonstrate frequent cross-resistance limiting efficacy of sequential androgen receptor (AR) targeting. Bromodomain extra terminal (BET) inhibitors (BETi) down-regulate the expression of putative drivers of ABI/ENZ resistance. ZEN-3694 is an orally bioavailable, potent, and selective BETi with significant anti-tumor activity in ENZ-resistant pre-clinical models. The safety and efficacy of ZEN-3694 in combination with ENZ was evaluated in a phase 1b/2a study in mCRPC (NCT02711956). Methods: Patients (pts) were required to have progressive mCRPC, prior resistance to ABI and/or ENZ, and no prior chemotherapy for mCRPC. A 3 plus 3 dose escalation schema was utilized, with a starting daily oral dose of ZEN-3694 36 mg plus ENZ 160 mg. Dose expansion was conducted in parallel cohorts at low and high-dose ZEN-3694 (48 and 96 mg daily, respectively). The primary objective was determination of maximally tolerated dose (MTD); key secondary endpoints included time to radiographic progression (TTP) and pharmacokinetic (PK) parameters. Pharmacodynamic (PD) markers included whole blood RNA expression of BETi targets including MYC, IL-8, CCR1, and IL1RN. Results: 64 pts were enrolled. The median age and PSA at study entry was 70 (range 47 - 89) and 25.9 (range 0.1 - 1701.8), respectively. At study entry, 24 (37.5%) of pts were resistant to ABI, 29 (45.3%) were resistant to ENZ, and 11 (17.2%) to both. ZEN-3694 dose levels ranged from 36 mg to 144 mg daily without reaching a MTD. The most common treatment-related adverse events (AEs) (any grade) included transient photophobia (66%), nausea (40%), fatigue (31%), decreased appetite (22%), and dysgeusia (16%). Grade ≥ 3 related AEs (N = 8) and dose-limiting toxicities (N = 1 at 96 mg dose level) were uncommon. No Grade ≥ 3 thrombocytopenia was observed. Exposure to ZEN-3694 increased with dose without significant drug-drug interaction with ENZ. PD analyses demonstrated exposure-dependent, up to 4-fold decrease in expression of BETi targets. RNA-Seq of paired tumor biopsies demonstrated suppression of BET-dependent genes. The overall median TTP was 44.4 weeks, and was similar in subgroups with prior ABI vs. ENZ resistance. Durable responses were observed, including 3 pts with disease primarily refractory to ABI on study treatment for 21.3 +, 20.8 +, and 17.3 months, respectively, with &amp;gt; 90% decline in serum PSA. Early transitory serum PSA increases were associated with longer TTP. Conclusions: ZEN-3694 demonstrates an acceptable safety and PK profile, robust target modulation, and encouraging disease stabilization in combination with ENZ in ABI/ENZ-refractory mCRPC. Analysis of paired metastatic tumor biopsies, circulating tumor cells and ctDNA is ongoing. Further investigation of the combination is warranted. Citation Format: Rahul Aggarwal, Wassim Abida, Michael Schweizer, Allan Pantuck, David Nanus, Elisabeth Heath, Sanjay Lakhotia, Henrik Hansen, Michael Silverman, Lisa Bauman, Margo Snyder, Eric Campeau, Karen Norek, Sarah Attwell, Marie O'Farrell, Steve Smith, Philip Wegge, Ravi Jahagirdar, Joshi Alumkal. A Phase Ib/IIa study of the BET bromodomain inhibitor ZEN-3694 in combination with enzalutamide in patients with metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT095.

Abstract 3836: Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML

Abstract Standard AML chemotherapy and JAK inhibitor (ruxolitinib) treatments are ineffective against post-myeloproliferative neoplasm (MPN) sAML. Mutations in JAK2, MPL and calreticulin, and co-epimutations, result in a dysregulated transcriptome, which is responsible for transformation of MPN to sAML and for its therapy-refractoriness. CDK9 is the catalytic subunit of pTEFb that phosphorylates serine 2 in the heptad-repeats of the C-terminal domain of RNA pol II (RNAP2). CDK9 also phosphorylates and inactivates negative transcript elongation factors (NELF and SPT5), which induces promoter-proximal pause-release of RNAP2 to enable productive mRNA transcript elongation. The bromodomain extra-terminal (BET) protein BRD4 recruits pTEFb to the chromatin to promote RNAP2-mediated elongation of transcripts, including those of c-Myc, Bcl-xL, PIM1 and MCL1, important for cell growth and survival of post-MPN sAML cells. Here, we determined the effects of inhibiting BRD4-CDK9-RNAP2 axis in post-MPN sAML cells. Treatment with the CDK9 inhibitor (CDK9i) NVP2 or BAY1143572 (B) dose-dependently induced apoptosis of ruxolitinib-sensitive (Rux-S) sAML cultured cells lines HEL92.1.7 (HEL) and SET2, as well as of patient-derived sAML blast progenitor cells (BPCs), while sparing normal CD34+ progenitor cells (HPCs). CDK9i-induced lethality in sAML cells was associated with attenuation of protein levels of S2-phosphorylated RNAP2, as well as depletion of mRNA and protein levels of c-Myc, PIM1, MCL1, Bcl-xL and XIAP. ATAC-Seq analyses demonstrated that treatment with NVP2 or B caused marked alterations in chromatin accessibility, including large peak losses on the chromatin with binding sites of TFs such as ERG, PU.1, STAT5, c-Myc, and RELA. This was associated with attenuation of mRNA expression of cMyb, c-Myc, RUNX1, LMO2, RELB, NFKB2, c-FLIP, MCL1, CDK6 and Bcl-xL. Following engraftment of luciferase transduced HEL cells into NSG mice, daily treatment with B at 10 mg/kg for 3 wks significantly reduced sAML burden and improved survival of the NSG mice (p &amp;lt; 0.002). Co-treatment of NVP2 or B with ruxolitinib or BCL2/Bcl-xL inhibitor ABT263 synergistically induced apoptosis of ruxolitinib-sensitive sAML cells (CI values of &amp;lt; 1.0). Treatment with NVP2 or B, or with BETi OTX015, also induced apoptosis of ruxolitinib persister/resistant (Rux-P) sAML cells (which demonstrate &amp;gt; 10-fold higher ruxolitinib IC50 values than Rux-S cells). This was associated with attenuated protein levels of p-RNAP2, c-Myc, PIM1, Bcl-xL and XIAP. Co-treatment with OTX015 and NVP2 or B synergistically induced apoptosis of not only HEL and SET2 but also HEL-RuxP and SET-RuxP cells, as well as of patient-derived sAML BPCs (CI values &amp;lt; 1.0). These findings confirm that targeted inhibition of BRD4-CDK9-RNAP2 leads to high level of pre-clinical efficacy against ruxolitinib-sensitive and ruxolitinib-refractory sAML cells. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Christopher P. Mill, Joseph D. Khoury, Srdan Verstovsek, Kapil N. Bhalla. Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3836.

PS967 PRECLINICAL ACTIVITY OF THE NOVEL ORAL DUAL BET‐CBP/P300 INHIBITORS, NEO1132 AND NEO2734, IN ACUTE MYELOID LEUKEMIA

Background:For decades, combination chemotherapy has been the therapeutic strategy to treat acute myeloid leukemia (AML). Although this treatment induces complete remission in most patients, only 30–40% of patients survive five years after diagnosis. These low cure rates are mainly due to relapse caused by the survival of a small subpopulation of leukemia cells with stem cell features, named “leukemic stem cells” (LSCs). The bromodomain (BRD) extra‐terminal (BET) protein Brd4 is a regulator of c‐Myc and an important therapeutic target in AML. In vivo, inhibition of Brd4 eradicates AML progenitors and reduces AML burden in mice. While treatment with BET inhibitors induces clinical responses in AML, these are usually partial and transient. Cyclic AMP response element‐binding protein (CREB)‐binding protein (CBP) and E1A interacting protein of 300 kDa (EP300 or P300) are homologues BRD‐containing transcriptional co‐activators that regulate transcription through their acetyltransferase activity. We studied NEO2734 and NEO1132, orally‐bioavailable, first‐in‐class dual BET and CBP/P300 inhibitors in preclinical models of AML.Aims:To evaluate the efficiency of NEO2734 and NEO1132 in AML (stem/progenitor) cell elimination.Methods:We studied the effect of treatment with NEO2734, NEO1132 and the CBP/P300 inhibitor tool compound, CPI637, on AML blast differentiation, apoptosis, engraftment potential in a patient‐derived xenograft AML mouse model (NSG mice), and on the clonogenic capacity of long‐ and short‐term surviving AML cells ex vivo and in vivo.Results:To assess the biological activity of NEO2734, we treated a panel of AML cell lines with various concentrations of NEO2734, NEO1132 and CPI637. Nanomolar concentrations of NEO2734 inhibited cell proliferation with IC50 values ranging from 32 nM to 141 nM. Next, we showed that NEO2734 inhibited cell proliferation and induced apoptosis in primary AML CD45dim blasts across a variety of genotypes (example Figure 1a and 1b)(IC50 between 30–100 nM for 5 samples tested). The potency of NEO2734 to inhibit proliferation and to induce apoptosis in AML cell lines and primary AML blasts was higher than that of NEO1132 and much higher than CPI637 (Figure 1a and 1b). Moreover, we observed no increase in the myeloid differentiation marker CD11b in primary AML blasts after NEO2734 treatment.To evaluate whether NEO2734 efficiently reduces human AML burden in the bone marrow in vivo, patient cells were transplanted into mice and the mice were treated with NEO2734 or NEO1132 (10 mg/kg, orally daily 10 times). The percentage of leukemic engraftment was reduced in the treated mice as compared to the controls (Figure 2). NEO2734 treatment inhibited leukemic engraftment more efficiently than NEO1132. Finally, we investigated whether NEO2734 treatment could inhibit clonogenic capacity of leukemic progenitors and long‐term surviving cells from primary AML cases. In all cases studied, a significant reduction in number of colonies after 1 week and 4 weeks of NEO2734 treatment was observed. Importantly, NEO2734 also reduced survival of immunophenotypically defined LSCs, CD34+CD38− cells. The potency of NEO2734 to inhibit LSC and leukemic progenitor survival was higher than that of NEO1132 and much higher than that of CPI637.imageSummary/Conclusion:The dual BET‐CBP/P300 inhibitor NEO2734 efficiently eliminates AML blasts and LSCs/progenitors, suggesting that AML patients should be included in clinical studies of NEO2734.

APABETALONE, AN EPIGENETIC BET INHIBITOR IN A PHASE 3 TRIAL, INHIBITS VASCULAR INFLAMMATION AND CELLULAR ADHESION LEADING TO BENEFICIAL OUTCOMES IN CVD PATIENTS

Apabetalone (RVX-208) is a small molecule bromodomain & extraterminal (BET) protein inhibitor, targeting the second bromodomain (BD2) of BET proteins. In cardiovascular disease (CVD) patients enrolled in phase 2 trials apabetalone treatment reduced the relative risk of a CV event by 44% (Nicholls