BET Inhibitors JQ1 Research Articles

Bromodomain and Extraterminal Inhibition by JQ1 Produces Divergent Transcriptional Regulation of Suppressors of Cytokine Signaling Genes in Adipocytes.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway has cell-specific functions. Suppressors of cytokine signaling (SOCS) proteins are negative-feedback regulators of JAK-STAT signaling. STAT5 plays a significant role in adipocyte development and function, and bromodomain and extraterminal (BET) proteins may be involved in STAT5 transcriptional activity. We treated 3T3-L1 adipocytes with the BET inhibitor JQ1 and observed that growth hormone (GH)-induced expression of 2 STAT5 target genes from the SOCS family, Socs3 and Cish, were inversely regulated (increased and decreased, respectively) by BET inhibition. Chromatin immunoprecipitation analyses revealed that changes in STAT5 binding did not correlate with gene expression changes. GH promoted the recruitment of the BET protein BRD2 to the Cish, but not Socs3, promoter. JQ1 treatment ablated this effect as well as the GH-induced binding of ribonucleic acid polymerase II (RNA Pol II) to the Cish transcription start site. BRD2 knockdown also suppressed GH induction of Cish, further supporting the role of BRD2 in Cish transcriptional activation. In contrast, JQ1 increased the binding of activated Pol II to the Socs3 coding region, suggesting enhanced messenger RNA (mRNA) elongation. Our finding that JQ1 transiently reduced the interaction between the positive transcription elongation factor (P-TEFb) and its inhibitor hexamethylene bis-acetamide inducible 1 (HEXIM1) is consistent with a previously described off-target effect of JQ1, whereby P-TEFb becomes more available to be recruited by genes that do not depend on BET proteins for activating transcription. These results demonstrate substantially different transcriptional regulation of Socs3 and Cish and suggest distinct roles in adipocytes for these 2 closely related proteins.

Drug Repurposing of Bromodomain Inhibitors as Potential Novel Therapeutic Leads for Lymphatic Filariasis Guided by Multispecies Transcriptomics.

To better understand the transcriptomic interplay of organisms associated with lymphatic filariasis, we conducted multispecies transcriptome sequencing (RNA-Seq) on the filarial nematode Brugia malayi, its Wolbachia endosymbiont wBm, and its laboratory vector Aedes aegypti across the entire B. malayi life cycle. In wBm, transcription of the noncoding 6S RNA suggests that it may be a regulator of bacterial cell growth, as its transcript levels correlate with bacterial replication rates. For A. aegypti, the transcriptional response reflects the stress that B. malayi infection exerts on the mosquito with indicators of increased energy demand. In B. malayi, expression modules associated with adult female samples consistently contained an overrepresentation of genes involved in chromatin remodeling, such as the bromodomain-containing proteins. All bromodomain-containing proteins encoded by B. malayi were observed to be upregulated in the adult female, embryo, and microfilaria life stages, including 2 members of the bromodomain and extraterminal (BET) protein family. The BET inhibitor JQ1(+), originally developed as a cancer therapeutic, caused lethality of adult worms in vitro, suggesting that it may be a potential therapeutic that can be repurposed for treating lymphatic filariasis.IMPORTANCE The current treatment regimen for lymphatic filariasis is mostly microfilaricidal. In an effort to identify new drug candidates for lymphatic filariasis, we conducted a three-way transcriptomics/systems biology study of one of the causative agents of lymphatic filariasis, Brugia malayi, its Wolbachia endosymbiont wBm, and its vector host Aedes aegypti at 16 distinct B. malayi life stages. B. malayi upregulates the expression of bromodomain-containing proteins in the adult female, embryo, and microfilaria stages. In vitro, we find that the existing cancer therapeutic JQ1(+), which is a bromodomain and extraterminal protein inhibitor, has adulticidal activity in B. malayi.

Abstract B008: NEO2734, a novel dual BET and P300/CBP bromodomain inhibitor, is more active in NUT midline carcinoma than single agent BET or P300/CBP inhibitors

Abstract Background: NUT midline carcinoma (NMC) is an aggressive subtype of poorly differentiated squamous cell carcinoma, occurring anywhere along the trunk or the head, most commonly along the midline. NMC is characterized by chromosomal translocation resulting in the fusion of the gene nuclear protein in testis (NUT) and bromodomain and extraterminal domain (BET) proteins (BRD2,3,4 and BRDT). The acquired bromodomain region tethers the BRD-NUT fusion protein to acetylated chromatin. The molecular mechanism by which BRD-NUT is involved in the generation of NMC is unclear, although it seems to be related to massive disruption of normal gene regulation at key cell type–specific genes. After binding to acetylated chromatin through bromodomains, the NUT moiety of the fusion protein strongly interacts with and recruits E1A interacting protein of 300 kDa (EP300/P300), initiating cycles of BRD-NUT/P300 recruitment and creating chromatin “megadomains” enriched in histone H3 lysine 27 acetylation (H3K27ac), a specific enhancer and super-enhancer histone mark. The creation of these domains activates transcription of adjacent genes that seem to be critical for the progression of NMC. As BET inhibitors have shown activity in NMC and P300 is important in its pathophysiology, we examined the pre-clinical activity of NEO2734 and NEO1132, novel dual BET and P300/CBP inhibitors (Giles et al Abs #2510 ESMO 2018), in NMC. Results:Considering the important roles of BET proteins and P300 in NMC, two NMC-cell lines (1015 and 14169, C. French: Brigham and Women’s Hospital) were treated with dual BET and P300/CBP inhibitors NEO2734 and NEO1131 (Epigene Therapeutics Inc), BET inhibitors JQ1 and iBET155 (GSK525762) (Sigma-Aldrich) and P300/CBP inhibitor CPI-637 (MedChem Express) for comparison. The average IC50 values of the two molecules were 44.04 nM and 130.73 nM respectively in 1015 cell line and 77.37 nM and 123.95 nM in 14169 cell line. For the two cell lines, the dual inhibitor NEO2734 was more potent than NEO1131, IBET155 and p300 inhibitors. The 14169 cell line, with a higher number of genetic lesions than the 1015 cell line, was more resistant to NEO2734 than the 1015 cell line. Conclusions: The dual inhibitor NEO2734 showed superior in vitroanti-proliferative activity in two NMC cell lines compared with single BET and p300 inhibitors. We also observed the presence of two different cell populations (adherent and suspension cells) in both NMC cell lines with adherent cells being more resistant to NEO2734, Differences in the responses to NEO2734 in NMC are being studied to molecularly identify biomarkers of resistance. These data support the inclusion of patients with NMC in clinical studies of NEO2734. Citation Format: Tian Tian, Marc Cosín, Francis Giles, Irene Braña, Elena Garralda, Sandra Peiró. NEO2734, a novel dual BET and P300/CBP bromodomain inhibitor, is more active in NUT midline carcinoma than single agent BET or P300/CBP inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B008. doi:10.1158/1535-7163.TARG-19-B008

Abstract TMIM-085: BROMODOMAIN INHIBITION IN OVARIAN CANCER AND THE TUMOR MICROENVIRONMENT

Abstract BACKGROUND: Ovarian cancer is the most lethal gynecologic malignancy. While women with BRCA deficient tumors show sensitivity to PARP inhibitors (PARPi), new treatment options are urgently needed for patients with PARPi-resistant tumors. An emerging strategy to improve PARPi response is combination therapy with epigenetic drugs. A recently recognized epigenetic drug target in ovarian cancer is the bromodomain and extraterminal (BET) family of proteins. BET proteins such as BRD4 promote oncogenic transcription of genes promoting cell growth, survival and DNA repair. One example is the well-established link between inflammation and cancer, nuclear factor-kappaB (NF-κB). In syngeneic mouse ovarian cancer models, M2-like pro-tumor macrophages are a prominent component of the ovarian cancer tumor microenvironment (TME). NF-κB inhibition can reduce the M2 macrophage population; however, more sustained treatment with a systemic NF-κB inhibitor leads to more ascites and reduced survival time. Thus, BET inhibitors (BETi) have the potential to induce transcriptional reprogramming in tumors and macrophages that may be beneficial or harmful depending on context. OBJECTIVE: To determine the cellular and molecular effects of combining BETi and PARPi in mouse ovarian cancer cells and peritoneal macrophages. METHODS: Cultured ID8 mouse ovarian cancer cells, PMJ2-PC mouse peritoneal macrophages and immortalized bone marrow-derived macrophages were treated with vehicle, the PARPi olaparib, the BETi JQ1 or the JQ1/olaparib combination for 24-72h. Sulforhodamine B (SRB) assays assessed cell growth in vitro. C57BL/6 mice injected intra-peritoneally (IP) with ID8 cells were treated with JQ1 (30 days, 50mg/kg by IP injection) with volume of ascites fluid, weight of harvested tumor, and number of tumor implants assessed. Markers of apoptosis (cleaved PARP or cleaved caspase-3), and DNA damage (pH2AX) were measured by immunohistochemistry or western blot. NF-κB activity was measured by luciferase assays of a NF-κB reporter plasmid. Expression of M1 (CCL3) and M2 (CD206, arginase-1) macrophage markers was measured by quantitative real-time RT-PCR (QPCR). RESULTS: In culture, JQ1 treatment sensitized ID8 ovarian cancer cells to olaparib-induced growth inhibition, DNA damage and apoptosis. However, despite modest stimulatory effects on DNA damage and apoptosis of long-term JQ1 treatment in ID8 tumors in vivo, JQ1 unexpectedly increased ascites formation without reducing overall tumor burden. In macrophages, there were also contrasting effects between JQ1 treatment in vitro and in vivo. JQ1 alone or combined with olaparib reduced NF-κB activity in cultured macrophage cell lines, and increased expression of CCL3, and reduced CD206 and arginase-1 expression. In contrast, increased ascites due to JQ1 treatment in vivo was accompanied by a pronounced M2 macrophage shift. CONCLUSIONS: Sustained inhibition of NF-κB activity in macrophages and potentially other cells in the ovarian TME could have overall deleterious effects on tumor progression. Our results strongly suggest that the therapeutic effects of BETi in ovarian cancer, and any therapeutic agent with the potential to alter cell function in the tumor microenvironment, need to be more thoroughly tested in the context of functional immune system before being translated to patients. This is particularly relevant since BETi are currently being tested in human patients in early clinical trials. Citation Format: Andrew J. Wilson, Alyssa Hoover, Whitney Harris, Esther Liu, Dineo Khabele, Fiona E. Yull. BROMODOMAIN INHIBITION IN OVARIAN CANCER AND THE TUMOR MICROENVIRONMENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-085.

PROTAC induced-BET protein degradation exhibits potent anti-osteosarcoma activity by triggering apoptosis

Targeting oncogenic proteins for degradation using proteolysis-targeting chimera (PROTAC) recently has drawn increasing attention in the field of cancer research. Bromodomain and extra-terminal (BET) family proteins are newly identified cancer-related epigenetic regulators, which have a role in the pathogenesis and progression of osteosarcoma. In this study, we investigated the in vitro and in vivo anti-osteosarcoma activity by targeting BET with a PROTAC molecule BETd-260. The results showed that BETd-260 completely depletes BET proteins and potently suppresses cell viability in MNNG/HOS, Saos-2, MG-63, and SJSA-1 osteosarcoma cell lines. Compared with BET inhibitors HJB-97 and JQ1, the activity of BETd-260 increased over 1000 times. Moreover, BETd-260 substantially inhibited the expression of anti-apoptotic Mcl-1, Bcl-xl while increased the expression of pro-apoptotic Noxa, which resulted in massive apoptosis in osteosarcoma cells within hours. In addition, pro-oncogenic protein c-Myc also was substantially inhibited by BETd-260 in the OS cells. Of note, BETd-260 induced degradation of BET proteins, triggered apoptosis in xenograft osteosarcoma tumor tissue, and profoundly inhibited the growth of cell-derived and patient-derived osteosarcoma xenografts in mice. Our findings indicate that BET PROTACs represent a promising therapeutic agent for human osteosarcoma.

Abstract 3067: OHM-581, a dual JAK2-BET inhibitor for the treatment of myelofibrosis and other hematologic malignancies

Abstract Ruxolitinib is the only JAK2 inhibitor approved for myelofibrosis (MF). Although JAK2 inhibitor therapy improves splenomegaly and systemic symptoms, it does not significantly reduce the MF clone or alter the natural history and survival in most patients. The novel small molecule OHM-581 not only inhibits JAK2, but also BET (bromodomain and extra terminal) proteins such as BRD4. Dual JAK2 and BET inhibition using other molecules has shown superior efficacy in murine MF models compared with JAK2 inhibition alone including amelioration of fibrosis, synergistic induction of apoptosis of primary patient, post-MF secondary acute myeloid leukemia (sAML) blasts, and significant improvement of the survival of mice engrafted with human sAML cells. OHM-581 is being developed for the treatment of MF and other hematologic malignancies to leverage these advantages of dual JAK2-BET inhibition. Results: In vitro biochemical assays show OHM-581 to inhibit BRD4 BD1 and BD2 with IC50s of 189 and 116 nM, respectively, and JAK2 with an IC50 of 7.1 nM (JAK1=244 nM; JAK3=76 nM). No significant hERG liability was seen. OHM-581 displays significant anti-proliferative activity against multiple liquid cancer cell lines. Notably, the GI50 values for MV4-11 (AML, MLL fusion+), HEL 92.1.7 (AML, JAK2V617F+) and UKE-1 (AML, JAK2V617F+) are 32, 87 and 140 nM, respectively. Consistent with its mechanism of action, OHM-581 robustly down-regulates cMYC expression (cMYC mRNA down-regulation IC50=18.6 nM compared with 41.4 nM for BET inhibitor JQ1) and JAK2 signaling. Moreover, the compound triggers apoptosis in MV4-11 cells (~7-fold increase in Caspase 3/7 activity at 24-h). OHM-581 does not reflect an efflux liability, the efflux ratio being nearly 1.8. OHM-581 is soluble in PBS pH7.4 at 26 µM and metabolically stable with nearly 51%, 67% and 88% remaining in mouse, rat and human liver microsomes, respectively, after 30 min. OHM-581 exhibits an oral bioavailability of ~40%. As compared to the small molecule fedratinib, which is also being developed for MF, OHM-581 does not inhibit thiamine uptake. Dose proportionality with a greater than proportional increase in both Cmax and AUC at doses up to 60 mg/kg, and a saturation of Cmax at doses of 60 mg/kg and higher, are observed for OHM-581. In vivo efficacy studies using an MV4-11 xenograft model demonstrate robust tumor inhibition (~75-90%) following doses of 30 and 60 mg/kg po BID for 20 days. Conclusion: OHM-581 is a novel dual inhibitor of JAK2 and BET that has potential as a therapeutic agent for MF and other hematologic malignancies. In addition to IND-enabling studies, current OHM-581 development activities are focused on further proof-of-concept analyses using preclinical models of MF and other myeloproliferative neoplasms, with results to be reported. Citation Format: Ram S. Upadhayaya, Raghava Reddy Kethiri, Stephan W. Morris, H. Michael Shepard, Samuel R. Saks, Michael J. Weickert. OHM-581, a dual JAK2-BET inhibitor for the treatment of myelofibrosis and other hematologic malignancies [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 3067.

Abstract 3827: Antitumor activity of ODM-207, a novel BET bromodomain inhibitor, in nonclinical models of ER+ breast cancer as single agent and as a combination treatment

Abstract Introduction: The bromodomain and extraterminal (BET) family of proteins are chromatin readers that promote the transcription of several important cell identity genes. BET proteins also control expression of many genes that play an essential role in the pathogenesis of human cancer, including cell-cycle and proliferation-regulating genes. The small-molecule BET inhibitors block BET binding to chromatin and have shown antitumor activity in a variety of pre-clinical cancer models. In this study, we evaluated the anticancer activity of the novel BET inhibitor, ODM-207, in ER+ breast cancer models as a single agent and in combination with other cancer drugs. Methods: ER+ breast cancer cell lines were studied for sensitivity to ODM-207 and the in vivo efficacy was assessed using the ER+ Ma3366 patient-derived xenograft model. For gene expression analyses, breast cancer cells were treated with ODM-207 or reference BET inhibitor JQ1 and differentially expressed genes were analyzed by RNA-sequencing. The ability of ODM-207 to regulate anticancer signaling pathways was validated by western blotting. Synergistic drug interactions were profiled using five-concentration dose response matrices. Results: ODM-207 is a novel BET inhibitor structurally distinct from JQ1 that shows antiproliferative activity in a broad panel of cancer cell lines. The strongest antitumor activity could be observed in hormone-dependent prostate and breast cancer models. In this study, we show that ODM-207 effectively inhibits the proliferation of ER+ breast cancer cell lines by inducing cell cycle arrest in G0/G1-phase. Additionally, ODM-207 suppresses the growth of ER+ patient-derived breast cancer xenograft tumors. ODM-207 as well as JQ1 targeted several pathways important for cancer progression such as MYC, estrogen response and cell cycle gene signatures. The inhibition of key cell cycle regulators, such as CDK4 and Cyclin D1, were further verified. The cyclin D1:CDK4/6 axis plays a significant role in the development, and currently, treatment of ER+ breast cancer together with endocrine therapy. Interestingly, ODM-207 was shown to synergize with palbociclib in vitro in ER+ breast cancer cell lines: the combination of ODM-207 and CDK4/6 inhibitor palbociclib achieved greater cell proliferation inhibition than either drug alone at sub IC50 concentrations. Notably, the ODM-207 and palbociclib combination did not cause the induction of an obvious senescent-like phenotype as compared to palbociclib alone, but rather affected cell survival cellular assays. Conclusions: In summary, ODM-207, which is currently in Phase I clinical trials for treating solid tumors, causes significant growth inhibition in pre-clinical models of ER+ breast cancer and enhances antiproliferative activity of palbociclib, providing a rationale for development of a combination therapy. Citation Format: Julia Lindqvist, Mari Björkman, Reetta Riikonen, Daniel Nicorici, Elina Mattila, Mahaboobi Jaleel, Chandrasekhar Abbineni, Anu-Maarit Moilanen. Antitumor activity of ODM-207, a novel BET bromodomain inhibitor, in nonclinical models of ER+ breast cancer as single agent and as a combination treatment [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 3827.

BET Bromodomain Inhibitors Suppress Inflammatory Activation of Gingival Fibroblasts and Epithelial Cells From Periodontitis Patients.

BET bromodomain proteins are important epigenetic regulators of gene expression that bind acetylated histone tails and regulate the formation of acetylation-dependent chromatin complexes. BET inhibitors suppress inflammatory responses in multiple cell types and animal models, and protect against bone loss in experimental periodontitis in mice. Here, we analyzed the role of BET proteins in inflammatory activation of gingival fibroblasts (GFs) and gingival epithelial cells (GECs). We show that the BET inhibitors I-BET151 and JQ1 significantly reduced expression and/or production of distinct, but overlapping, profiles of cytokine-inducible mediators of inflammation and bone resorption in GFs from healthy donors (IL6, IL8, IL1B, CCL2, CCL5, COX2, and MMP3) and the GEC line TIGK (IL6, IL8, IL1B, CXCL10, MMP9) without affecting cell viability. Activation of mitogen-activated protein kinase and nuclear factor-κB pathways was unaffected by I-BET151, as was the histone acetylation status, and new protein synthesis was not required for the anti-inflammatory effects of BET inhibition. I-BET151 and JQ1 also suppressed expression of inflammatory cytokines, chemokines, and osteoclastogenic mediators in GFs and TIGKs infected with the key periodontal pathogen Porphyromonas gingivalis. Notably, P. gingivalis internalization and intracellular survival in GFs and TIGKs remained unaffected by BET inhibitors. Finally, inhibition of BET proteins significantly reduced P. gingivalis-induced inflammatory mediator expression in GECs and GFs from patients with periodontitis. Our results demonstrate that BET inhibitors may block the excessive inflammatory mediator production by resident cells of the gingival tissue and identify the BET family of epigenetic reader proteins as a potential therapeutic target in the treatment of periodontal disease.

Bromodomain and Extraterminal (BET) Protein Inhibition Restores Redox Balance and Inhibits Myofibroblast Activation.

Background and Objective Progressive pulmonary fibrosis is the main cause of death in patients with systemic sclerosis (SSc) with interstitial lung disease (ILD) and in those with idiopathic pulmonary fibrosis (IPF). Transforming growth factor-β (TGF-β) and NADPH oxidase- (NOX-) derived reactive oxygen species (ROS) are drivers of lung fibrosis. We aimed to determine the role of the epigenetic readers, bromodomain and extraterminal (BET) proteins in the regulation of redox balance in activated myofibroblasts. Methods In TGF-β-stimulated fibroblasts, we investigated the effect of the BET inhibitor JQ1 on the mRNA expression of the prooxidant gene NOX4 and the antioxidant gene superoxide dismutase (SOD2) by quantitative RT-PCR, the antioxidant transcription factor NF-E2-related factor 2 (Nrf2) activity by a reporter assay, and intracellular ROS levels by dichlorofluorescein staining. Myofibroblast activation was determined by α-smooth muscle actin immunocytochemistry. The role of specific BET protein isoforms in NOX4 gene regulation was studied by siRNA silencing and chromatin-immunoprecipitation. Results and Conclusions Affymetrix gene array analysis revealed increased NOX4 and reduced SOD2 expression in SSc and IPF fibroblasts. SOD2 silencing in non-ILD control fibroblasts induced a profibrotic phenotype. TGF-β increased NOX4 and inhibited SOD2 expression, while increasing ROS production and myofibroblast differentiation. JQ1 reversed the TGF-β-mediated NOX4/SOD2 imbalance and Nrf2 inactivation and attenuated ROS production and myofibroblast differentiation. The BET proteins Brd3 and Brd4 were shown to bind to the NOX4 promoter and drive TGF-β-induced NOX4 expression. Our data indicate a critical role of BET proteins in promoting redox imbalance and pulmonary myofibroblast activation and support BET bromodomain inhibitors as a potential therapy for fibrotic lung disease.

The Bromodomain and Extra-Terminal Protein Inhibitor OTX015 Suppresses T Helper Cell Proliferation and Differentiation.

Dynamic epigenetic alterations accompanying CD4+ T helper cell differentiation have been implicated in multiple autoimmune diseases. The bromodomain and extra-terminal (BET) proteins are epigenetic regulators that recognize and bind to acetylated histones in chromatin and are targets for pharmacological inhibition. In this study we tested a new BET inhibitor under clinical development, OTX015, to interrogate its effects on key CD4+ T cell subsets associated with autoimmunity. Naïve and memory murine and human CD4+ T cells were isolated and differentiated into populations characterized by the expression of interferon (IFN)-γ and interleukin (IL)-17. Cultured cells were then exposed to varying concentrations of OTX015 in vitro, and its impact on cytokine expression was quantified by flow cytometry. In parallel, the expression of the transcription factors TBX21 and RORC was quantified by PCR. A previously studied BET inhibitor JQ1 was used as a pharmacological control. OTX015 suppressed both murine and human CD4+ T cell proliferation. Its impact on cytokine expression varied in murine and human naïve and memory subsets. OTX015 was similarly effective as JQ1 in the suppression of cytokines and T helper cell proliferation. Higher concentrations of OTX015 also had a greater impact on the viability of murine versus human cells. IL-17 and IFN-γ expression was not altered in murine memory CD4+ T cells, whereas in human memory CD4+ T cells, OTX015 inhibited IL-17, but not IFN-γ. Across all human T cell subsets OTX015 suppressed IL-17 more effectively than IFN-γ. Our studies demonstrate that OTX015 has anti-inflammatory effects by suppressing murine and human CD4+ T cell proliferation and subset-dependent proinflammatory cytokine expression, including the selective suppression of IL-17 in human memory CD4+ T cells.

Enhancer Deregulation in Myeloma

The complexity of gene expression regulation is the result of a composite interplay between promoters, enhancers and other cis-acting regulatory elements bound by transcription factors (TFs) that controls the transcriptional activity of genes. Primary tumor cells, in comparison to their healthy counterparts, are known to display altered enhancer repertoires that are associated with tumor-specific transcription. Large groups of transcriptional enhancers cluster together to form super-enhancers (SEs). These elements have been shown to control genes that are important for maintaining cell identity but are also frequently associated with oncogenes as well as translocations that result in aberrant gene expression in cancer. Immunoglobulin (IGH, IGL, IGK) and non-immunoglobulin (PVT1, FAM46C, DUSP22, etc.) enhancers hijacking by variable genes (MYC, MAF, CCND1/2/3, MMSET, IRF4) is a recognized oncogenic driver event in multiple myeloma (MM). However, the identity of the TFs or transcriptional regulatory complexes binding and regulating the activity of these enhancers remains to be fully elucidated and may yield valuable therapeutic targets. In this regard, the bromodomain and extra-terminal (BET) inhibitors have emerged as promising molecules for the treatment of hematologic malignancies. BET family proteins are chromatin adaptors, functionally linked to important pathways for cellular viability and cancer signaling. In particular, BRD4 has a direct role in the transcription regulation of different genes involved in the cell cycle progression and cellular viability. The BET inhibitor JQ1 selectively inhibits BRD4 by competitively binding to the acetyl-lysine recognition pocket of BET bromodomains from chromatin leading to the inhibition of MYC transcription in a dose- and time-dependent manner. Thus, BRD4 has been recently described as a therapeutic target for MM, among other hematologic diseases. Constitutive activation of MYC signaling is detected in more than 60% of patient-derived MM cells and can be involved in the pathogenesis of MM through different mechanisms. One of the most common somatic genomic aberrations in early and late-stage MM is rearrangement or translocation of MYC. Regardless of whether MYC rearrangements occur at early or late stages of MM pathogenesis, MYC rearrangements may provide one of several critical events contributing to increased autonomy and a more aggressive phenotype. Moreover promiscuous rearrangements of the MYC locus are known to hijack enhancers and super-enhancers to dysregulate MYC expression in MM and are involved in its pathogenesis. The development of the immunomodulatory drugs (IMiDs) has contributed significantly to improve the outcomes of MM patients. They possess pleiotropic anti-MM properties and through CRBN binding they induce Ikaros and Aiolos ubiquitylation and proteasomal degradation with an ensuing transcriptional repression of MYC and IRF4, two essential factors for myeloma cells survival. However, is not clear how IKZF1/IKZF3 regulate MYC transcription and how myeloma cells acquire resistance to IMIDs, “beyond CRBN”. In addition, acquired resistance to IMIDs and the loss of the transcriptional repression of MYC are nearly universal and occur in spite of sustained IKZF1/3 degradation suggesting that transcriptional rewiring may be sustaining hijacked enhancers activity and transcription of driver oncogenes. In this contest we have recently demonstrated that IMiDs are repressors of IKZF1/3-depedent oncogenic enhancers. Transcriptional plasticity with expression of extra-lineage TFs such as the ETS family member ETV4 sustains oncogenic enhancers in MM overcoming IKAROS and AIOLOS dependency and promoting IMiDs resistance. Therefore defining TFs occupancy and their circuitry at enhancers identifies “non-canonical” (aberrant) myeloma TFs dependency that may be linked to potential therapeutic targets. DisclosuresNeri:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.

T cell toxicity of HIV latency reversing agents.

Combination antiretroviral therapy reduces morbidity and mortality in HIV infected patients. However, the cure of HIV infection is hindered by the persistence of the latent HIV reservoir. Latency reversing agents (LRAs) are developed to target the HIV latently infected cells for HIV reactivation. In addition to reversal of HIV latency, the eradication of HIV latently infected cells will require effector HIV-specific CD8+ T cells. Therefore it is imperative we understand how LRAs affect immune cells. We have performed a comparative in depth analysis of the cytotoxicity of several compounds belonging to four LRA classes on T cells, B cells, and NK cells. In addition, the effect of these LRAs on activation and inhibitory receptor expression of CD8+ T cells was examined. We show that the HDAC inhibitors romidepsin and panobinostat are highly cytotoxic for CD4+ and CD8+ T cells, whereas the PKC agonists bryostatin and prostratin and BET inhibitors JQ1 and OXT-015 were less cytotoxic. The BAF inhibitors CAPE and pyrimethamine exhibit no cytotoxicity. Drug-specific cytotoxicity on CD8+ T cells was comparable between healthy controls and cART-treated HIV-infected patients. Bryostatin and both BET inhibitors downregulated the expression of CD279 on CD8+ T cells without affecting their activation. Our comparison of LRAs identified differences in cytotoxicity between LRA classes and members within a class and suggests that some LRAs such as bryostatin and BET inhibitors may also downregulate inhibitory receptors on activated HIV-specific CD8+ T cells. These findings may guide the use of LRAs that have the capacity to preserve or restore CD8+ T cell immunity.

Epigenetic modulation of FOXM1-gene interacting network by BET inhibitors in breast cancer.

FOXM1 is a transcription factor that has been implicated in the genesis of several tumors by regulating the expression of genes involved in the mitotic process. Transcriptomic enrichment analysis was performed to evaluate deregulated pathways in breast cancer, and relapse-free survival associated with the upregulated genes of FOXM1 signature was explored using the KM Plotter online tool. Treatment with bromodomain and extraterminal (BET) inhibitor JQ1 was explored in breast cancer cell lines to evaluate FOXM1 by qPCR and proliferation by MTT colorimetric assays. The FOXM1 gene signature was clearly deregulated in breast cancer patients, particularly in the basal-like subgroup where it was linked with detrimental prognosis. Treatment with the BET inhibitor JQ1 reduced the expression of FOXM1, decreasing cell proliferation in a panel of cell lines, being more active in the basal-like subtype, MDA-MB-231 and HS-578T. Knockdown of FOXM1 or treatment with JQ1 reduced genes included in the FOXM1 signature. Similarly, genes downregulated by the FOXM1 small interfering RNA approach were associated with detrimental outcome in breast cancer patients. Finally, we observed that FOXM1 was amplified in the triple-negative breast cancer subtype in around 15% of patients. Our study demonstrates that activation of the FOXM1 pathway has a prognostic role in breast cancer. JQ1 can modulate the expression of the FOXM1-gene interacting network, opening the opportunity for the evaluation of this compound in breast cancer patients.

Epigenomic Control of Cardiac Fibrosis by Bet Bromodomain Proteins in Dilated Cardiomyopathy

Pathologic gene expression is a hallmark of DCM. Mice carrying a human DCM mutation in phospholamban (PLNR9C) develop fibrosis, DCM, HF and premature death. RNAseq showed fibrotic gene expression to be a key early driver of DCM in PLNR9C mice. Recently, bromodomain and extraterminal (BET) epigenetic reader proteins have been identified as key regulators of pathologic gene expression in the heart. Using a chemical genetic strategy, we studied the role of BET proteins on the temporal regulation of gene expression in DCM. PLNR9C and age-matched wild type (WT) mice were treated longitudinally with the BET inhibitor JQ1 or vehicle. Vehicle-treated PLNR9C mice developed progressive DCM with reduced LV function, chamber dilation and LV hypertrophy; this was blunted with JQ1 (fractional shortening 15±3% vs. 27±3%, p

Abstract 626: Bromodomain inhibition in ovarian cancer and the tumor microenvironment to improve PARP inhibitor response

Abstract Background: Ovarian cancer is the most lethal gynecologic malignancy. While women with BRCA-deficient tumors show sensitivity to PARP inhibitors (PARPi), new treatment options are needed for PARPi-resistant tumors. An emerging strategy to improve PARPi response is combination therapy with epigenetic drugs. A newly recognized epigenetic drug target in ovarian cancer is the bromodomain and extraterminal (BET) protein family. BET proteins such as BRD4 promote oncogenic transcription of progrowth and survival genes, including the established link between inflammation and cancer, nuclear factor-kappaB (NF-κB). A complementary strategy to targeting cancer cells with cytotoxic drugs is to activate normal immune processes in the tumor microenvironment (TME). In syngeneic mouse ovarian cancer models, we have shown that M2-like protumor macrophages are a prominent component of the TME, and that NF-κB inhibition reduces the M2 population. Thus, BET inhibitors (BETi) have the potential to induce transcriptional reprogramming in both tumors and macrophages for therapeutic benefit. Objective: To determine the cellular and molecular effects of combining BETi and PARPi in mouse ovarian cancer and peritoneal macrophage cell lines. Methods: Cultured wild-type and CRISPR-modified (TP53 and TP53/BRCA2 knockout) ID8 mouse ovarian cancer cells, and PMJ2-PC mouse peritoneal macrophages, were treated with vehicle, the PARPi olaparib, the first-in-class BETi JQ1 or the JQ1/olaparib combination for 24-72h. Sulforhodamine B (SRB) assays assessed cell growth. Immunofluorescence assays assessed adherent cell number, DNA damage (pH2AX) and cell cycle indices. Protein levels of pH2AX and the apoptosis marker cleaved PARP were assessed by Western blot. NF-κB activity was measured by luciferase assays of a transiently transfected reporter plasmid. Results: Combined JQ1 and olaparib treatment synergistically reduced cell growth in SRB assays in wild-type and TP53 knockout ID8 cells. TP53/BRCA2 knockout cells showed greater responses to PARPi alone and no synergism was observed. Consistent with these results, the JQ1/olaparib combination cooperatively reduced the number of adherent cells and cells in S phase, and increased the G0/G1 population, DNA damage and apoptosis. In contrast, the drug combination had minimal effects on DNA damage or apoptosis in PMJ2-PC macrophages, while NF-κB activity was reduced in both cancer cells and macrophages. Conclusions: BETi sensitize mouse ovarian cancer cells to the cytotoxic effects of PARPi. Combined drug treatment also has potential to inhibit NF-κB in both cancer cells and macrophages. Our novel immunomodulatory strategy will be tested in ID8 syngeneic ovarian cancer models. We believe BETi combination treatment could expand the use of PARPi in ovarian cancer patients, with the potential to benefit a substantial number of women with this devastating disease. Citation Format: Andrew J. Wilson, Alyssa Hoover, Whitney Harris, Esther Liu, Dineo Khabele, Fiona Yull. Bromodomain inhibition in ovarian cancer and the tumor microenvironment to improve PARP inhibitor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 626.

Abstract LB-191: BET inhibitors kill colorectal cancer cells by activating death receptor 5 through the ER stress response pathway

Abstract Bromodomain and extra-terminal (BET) family proteins are epigenetic "readers" that are frequently activated in cancer and play an important role in regulating gene expression and oncogenic signaling. Targeting BET proteins using small-molecule inhibitors represents an attractive therapeutic strategy. Several small-molecule BET inhibitors have shown promises in recent preclinical and clinical studies. However, the mechanisms by which BET inhibitors selectively kill cancer cells are not well understood. In this study, we found that the BET inhibitors JQ1 and I-BET151 induce mRNA and protein expression of Death Receptor 5 (DR5) in a variety of colorectal cancer (CRC) cells with different genetic backgrounds. This is an on-target effect as knockdown of the BET family member BRD4 also induces DR5 expression. The induction of DR5 by the BET inhibitors is mediated by C/EBP homologous protein (CHOP) through the endoplasmic reticulum (ER) stress response pathway. Furthermore, knockout of DR5 in colon cancer cells suppresses apoptosis and caspase activation induced by the BET inhibitors, and abrogates their chemosensitization effects in CRC cells. Collectively, our results uncover a key role of DR5 in apoptosis induced by the BET inhibitors in CRC cells, and suggest activation of the death receptor pathway as a novel anticancer mechanism of BET-targeting agents. Citation Format: Xiao Tan, Jingshan Tong, Jian Yu, Liangfang Shen, Lin Zhang. BET inhibitors kill colorectal cancer cells by activating death receptor 5 through the ER stress response pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-191.

Abstract 3970: Therapeutic targeting of estrogen receptor positive breast cancer with the BET bromodomain inhibitor ODM-207

Abstract Introduction: The bromodomain and extraterminal (BET) family of proteins are chromatin readers that recognize and bind to specific acetylated histones and promote the transcription of several important cell identity genes. BET bromodomain inhibitors have shown promising antitumor activity in a variety of pre-clinical cancer models, as BET inhibition abrogates the transcription of several key oncogenes in a cell type-specific manner. It is known that inhibition of BET proteins effectively inhibits the proliferation of estrogen receptor positive (ER+) breast cancer cells, at least in part through repression of ER and MYC signaling. However, many additional cancer-associated genes are likely to underlie the growth inhibitory effects of BET inhibitors in breast cancer. The purpose of this study was to determine the anticancer activity of the novel BET bromodomain inhibitor ODM-207 in pre-clinical ER+ breast cancer models, and further, to look for cancer-associated signaling pathways suppressed by BET inhibitors. Methodology and results: ODM-207 is a novel, highly selective BET bromodomain inhibitor structurally distinct from JQ1 and its benzodiazepine-related derivatives. In this study, we show that ODM-207 effectively inhibits the proliferation of ER+ breast cancer cell lines when measured by cell viability assays as well as suppresses the growth of patient-derived xenograft tumors. Furthermore, we wanted to investigate the anticancer signaling pathways regulated by ODM-207 as well as the prototypical BET inhibitor JQ1 in breast cancer cells. For this purpose, we performed RNA sequencing on two ER+ breast cancer cell lines after 24h treatment with the aforementioned BET inhibitors. We found that both BET inhibitors targeted several genes and pathways important for breast cancer progression. For example, the targets included CDK4 and CDK6, two cell cycle kinases fundamental for the development and treatment of ER+ breast cancer. The RNA sequencing results were further validated in vitro, and were utilized as a basis for combination therapy assessment. Conclusions: Our results indicate that the novel BET bromodomain inhibitor ODM-207, which is currently in Phase I clinical trials for treating solid tumors, causes significant growth inhibition and cell cycle arrest in pre-clinical models of ER+ breast cancer, and regulates multiple crucial signaling pathways involved in breast cancer cell cycle and survival. Citation Format: Julia Lindqvist, Mari Björkman, Reetta Riikonen, Daniel Nicorici, Elina Mattila, Chandrasekhar Abbineni, Mahaboobi Jaleel, John Eriksson, Pekka Kallio, Anu-Maarit Moilanen. Therapeutic targeting of estrogen receptor positive breast cancer with the BET bromodomain inhibitor ODM-207 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3970.

Targeting BRD4 proteins suppresses the growth of NSCLC through downregulation of eIF4E expression

ABSTRACTLung cancer is the leading cause of cancer-related death worldwide. Bromodomain and extraterminal domain (BET) proteins act as epigenome readers for gene transcriptional regulation. Among BET family members, BRD4 was well studied, but for its mechanism in non-small cell lung carcinoma has not been elucidated. eIF4E regulates gene translation and has been proved to play an important role in the progression of lung cancer. In this study, we first confirmed that BET inhibitors JQ1 and I-BET151 suppressed the growth of NSCLCs, in parallel with downregulated eIF4E expression. Then we found that knockdown of BRD4 expression using siRNAs inhibited the growth of NSCLCs as well as decreased eIF4E protein levels. Moreover, overexpression of eIF4E partially abrogated the growth inhibitory effect of JQ1, while knockdown of eIF4E enhanced the inhibitory effect of JQ1. Furthermore, JQ1 treatment or knockdown of BRD4 expression decreased eIF4E mRNA levels and inhibited its promoter activity by luciferase reporter assay. JQ1 treatment significantly decreased the binding of eIF4E promoter with BRD4. Finally, JQ1 inhibited the growth of H460 tumors in parallel with downregulated eIF4E mRNA and protein levels in a xenograft mouse model. These findings suggest that inhibition of BET by JQ1, I-BET151, or BRD4 silencing suppresses the growth of non-small cell lung carcinoma through decreasing eIF4E transcription and subsequent mRNA and protein expression. Considering that BET regulates gene transcription epigenetically, our findings not only reveal a new mechanism of BET-regulated eIF4E in lung cancer, but also indicate a novel strategy by co-targeting eIF4E for enhancing BET-targeted cancer therapy.

Abstract 691: DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation

Abstract Background: Neuroblastoma (NB) is the most common extracranial solid pediatric tumor and is associated with MYCN amplification. MYCN is a regulator of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis. Inhibition of this pathway in MYCN-amplified NB tumors has been shown to be a target for treatment. Alpha-difluoromethylornithine (DFMO) inhibits ODC and is currently being used in a Phase II clinical trial for NB. BET inhibitors JQ1 and OTX-015 have been shown to be effective against MYCN-amplified cancers; it is hypothesized that they downregulate MYCN as well as cancer stem cell (CSC) signaling. We propose that inhibiting MYCN with a BET inhibitor, coupled with inhibition of ODC with DFMO, will result in enhanced inhibition of NB growth and tumor-initiating properties. Methods: Single and combination drug treatments were conducted on BE(2)-C, SMS-KCNR, CHLA90, and one patient-derived cell line. CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability in 96-well plates. IC50 values were calculated with a four-parameter variable-slope dose response curve using GraphPad Prism v.5 software. Drug combination studies were conducted in MYCN amplified tumors BE(2)-C and SMS-KCNR using ray designs to evaluate for synergism. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity and apoptosis in NB cells. Western blots measured protein levels of apoptosis markers (cleaved caspase 3 and cleaved PARP) and CSC markers (Nanog, Sox2, NF-kB, CXCR4, Lin28B, and MYCN). Neurosphere assays were used to evaluate tumor initiation via sphere formation. Results: Cell viability of MYCN amplified NB showed an IC50 range of 1.48-1.69 μM for JQ1 and 839.3 nM-2.36 μM for OTX-015; MYCN non-amplified NB showed an IC50 range of 4.22-11.58 μM for JQ1 and 2.99-11.03 uM for OTX-015. Combination treatment in MYCN NB had a synergistic effect, based on Loewe-additivity as the null hypothesis, for cell viability suppression by ray design experiments for BE(2)-C and SMS-KCNR. Western blots showed greater expression of apoptosis markers and decreased expression of CSC markers in combination relative to single drug treatments. A greater than 50% decrease in neurosphere formation with combination treatment in BE(2)-C, SMS-KCNR, and the patient cell line provides evidence for reduced CSC activity. IncuCyte imaging showed an increase in cell death with time post-treatment. Conclusion: The combination of DFMO with BET inhibitors has a synergistic effect in treating MYCN amplified NBs as shown by a decrease in cell viability. This combination targets CSC pathways and decreases tumor-initiating ability. Given the lack of effective treatment options for children with relapsed or refractory high-risk NB, this combination may be a promising novel therapy. Citation Format: Sarah DeCou, Ping Zhao, Tracey Avequin, Abhinav Nagulapally, Jeffrey Bond, Giselle Saulnier Sholler. DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation [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 691. doi:10.1158/1538-7445.AM2017-691

Discovery of Selective Dual Inhibitors of Bromodomain Protein BRD4 and JAK2 for Treatment of Hematologic Malignancies

The Bromodomain and Extra-Terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are functional readers of acetylated lysine residues of histones, and have emerged as potential therapeutic targets in hematologic cancers and solid tumors characterized by dysregulated epigenetic processes. Targeted inhibition of BET proteins has proven to be an effective strategy for transcriptional downregulation of c-MYC, an oncogene that is frequently activated or overexpressed in leukemias, lymphomas, and multiple myeloma. Of the BET family members BRD4 is the most extensively studied for its role in cancer, furthermore C-MYC downregulation by BET inhibitors is attributed to inhibition of enhancer binding by BRD4 (Delmore et al., Cell. 2011. 146:904-17). BRD4 is a critical factor in AML disease maintenance (Zuber et al., Nature. 2011. 478:524-8), and its suppression is the dominant mechanism of BET inhibitor JQ1 activity in AML (Rathert et al., Nature. 2015. 525:543-7). Recent reports have shown that BRD4/BET inhibitors and kinase inhibitors act synergistically in a range of cancer types (Sun et al., Blood. 2015, 126:1565-74; Stratikopoulos et al., Cancer Cell. 2015, 27:837-51). Therefore, optimizing for this synergy by prospectively designing and developing multi-targeting BRD4-kinase inhibitors may prolong therapeutic efficacy and overcome tumor resistance of single-activity BET and oncogenic kinase inhibitors.Dual inhibitors of BET proteins and Janus kinase 2 (JAK2), initially developed by Moffitt Cancer Center (Reuther et al., ASH 2015 Poster, Abstract #2826), demonstrated an opportunity for novel, potent dual inhibitors of BRD4 and JAK for treatment of myeloproliferative neoplasms (MPNs) and other disorders driven by the constitutively active somatic mutation, JAK2-V617F. Although JAK inhibitors such as ruxolitinib show clinical benefits in MPNs, these molecules demonstrate limited ability to induce remissions and are associated with significant toxicities such as myelosuppression. Therefore, selective JAK2 inhibitors that also target BRD4 hold promise as treatments of hematologic malignancies with improved activity and less off-target toxicity. While the dual inhibitor candidates from Moffitt exhibit strong potency, they possess a sub-optimal profile for inhibition of the thiamine transporter and other properties. Herein, we report that next generation, novel dual inhibitors of BRD4 and JAK2 have been discovered by Aptose, in a collaboration with Laxai Avanti Life Sciences (LALS), without observed thiamine transporter inhibition but with favourable drug-like properties.In biochemical assays, the Aptose dual inhibitor compounds exhibit IC50 values ranging from 82-678 nM for BRD4 (BD1) and 0.7-35 nM for JAK2. Compounds exhibited selectivity of 2-7 fold for BRD4 compared to other BRD isoforms, and 3-152 fold selectivity for JAK2 over JAK1 or JAK3. Cellular IC50 values of the compounds in antiproliferative assays ranged from 6.6 - 118 nM in AML (MV4-11) and from 171-721 nM in myeloma (MM1.S) cell lines. Candidate structures show good solubility and metabolic stability in human, rat and mouse liver microsomes. Structure-activity relationship studies are ongoing to improve biochemical and antiproliferative activity and drug like properties. Collectively, we demonstrate that a rational design approach can be used to generate potent molecules with combined BRD4/JAK2 inhibitory activities for treatment of hematologic cancers. DisclosuresNo relevant conflicts of interest to declare.