Inhibition Of BET Proteins Research Articles

P0515BRD4 PROTEIN REGULATES THE RESPONSES TO HYPOXIA TRIGGERED IN THE EXPERIMENAL RENAL DAMAGE

Abstract Background and Aims Renal hypoxia is not only one of the most common causes of acute kidney injury, but also a critical mediator in the transition to chronic kidney disease. When the kidney is exposed to an insufficient supply of oxygen to meet demand, some adaptive mechanisms are triggered by the cells to maintain homeostasis. Induction of HIF-1α transcription factor and activation of unfolded protein response (UPR) pathway, as consequence of ER dysfunction, are both essential to mediate the cell survival. The UPR pathway is regulated by three major protein sensors (IRE1α, PERK and ATF6) which under ER stress initiate the activation of the XBP1, ATF4 and ATF6 transcription factors, respectively. However, inappropriate activation of these mechanisms could lead to the enhanced transcription of genes involved in key processes in renal damage (inflammation, cell death or autophagy). On the other hand, BRD4 is an epigenetic reader that recognizes acetylated lysine residues on histone and other proteins, and mediates the binding of transcription factors to the transcriptional machinery. Our aim was to investigate whether BRD4-mediated epigenetic mechanisms could modulate the response to hypoxia triggered in acute renal damage. Method Tubular epithelial cell line, HK2, was cultured with thapsigargin (Tg) or in hypoxia chamber (1% O2, 5% CO2). In addition, these cells were treated with specific BET proteins inhibitors (JQ1, I-BET762) and with small interfering RNAs (siRNA BRD4, p300), or were subjected to knockdown of ATF4 and XBP1 by CRISPR/cas9 technology. Transcriptional changes were analyzed in each condition by RNA-sequencing. The binding of BRD4 to target genes and recruitment of the transcriptional machinery was analyzed by chromatin immunoprecipitation (ChIP) with specific antibodies against BRD4, RNA PolII, AcH3 and AcH4. Effect of JQ1 inhibitor was assayed in an ischemia/reperfusion injury (IRI) model, and analysis of gene expression, inflammatory cell infiltration, and epigenetic remodeling was carried out by quantitative PCR, IHQ and ChIP assay, respectively. Results Treatment of HK2 cells with BETs inhibitors, previously cultured with Tg or under hypoxia conditions, inhibits the gene expression of the GPR78 ER chaperon, and the XBP1 and ATF4 transcription factors modulating the downstream signaling pathways. Meanwhile, ATF6 expression remains unchanged. Gene silencing with siRNA and ChIP assays reveal that under activation of the UPR pathway or hypoxia, BRD4 recognizes acetylated histones in the GPR78, ATF4 and XBP1 promoters, recruits the pTEF-b complex and activates RNA-pol II allowing the gene transcription. Additionally, inhibition of BRD4 impairs the HIF-1α stabilization, downregulating the expression of hypoxia-induced genes. Results from whole-genome gene expression assays after stable knockdown of XBP1 and ATF4 reveal that most (86%) of the UPR genes regulated by BET proteins are dependent of XBP1 and only 32% by ATF4. Moreover, almost all genes regulated by ATF4 are also XBP1-dependent. This result may be due to the fact that ATF4 regulates IRE1α expression and thus modulates the XBP1 mRNA splicing. Administration of JQ1 in an IRI model supports that blockage of BRD4 ameliorates the renal damage (reducing BUN and creatinine levels) due to a decreased UPR activation and expression of HIF-1α target genes. As consequence, the expression of inflammatory genes and the inflammatory cell infiltration is diminished. Conclusion Our results show that BRD4 protein regulates two key processes, induction of HIF-1α transcription factor and UPR pathway activation triggered by renal hypoxia. Pharmacological inhibition of BET proteins reduces the activation these pathways, ameliorating renal damage and avoiding its progression.

Apabetalone -BET protein inhibition in cardiovascular disease and Type 2 diabetes.

Apabetalone is the first selectiveBET protein inhibitor in the field of cardiovascular diseases (CVD). BET proteins are epigenetic regulators that link upstream epigenetic modifications to downstream gene expression. Inhibition of BET proteins by apabetalone has been shown to modulate reverse cholesterol transport, coagulation, inflammation and vascular calcification. Furthermore, apabetalone reduces circulating markers of CVD risk and plaque vulnerability. Post-hoc pooled analyses suggest a potential reduction in risk of major adverse cardiac events (MACE) in patients with Type 2 diabetes (T2D) and stable CVD. However, the current cardiovascular outcomes trial BET-on-MACE failed to detect the assumed 30% reduction of MACE by apabetalone in patients with T2D after an acute coronary syndrome.

Mechanistic basis and efficacy of targeting the β-catenin–TCF7L2–JMJD6–c-Myc axis to overcome resistance to BET inhibitors

AbstractThe promising activity of BET protein inhibitors (BETi's) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear β-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the β-catenin–TCF7L2–JMJD6–c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear β-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the β-catenin–TCF7L2–JMJD6–c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells.

Effects of triptolide and BET protein inhibitor JQ1 on the proliferation and apoptosis of MLL-rearranged acute myeloid leukemia cells and their mechanisms

Objective To investigate the effects of triptolide (TPL) and BET protein inhibitor JQ1 on proliferation inhibition and apoptosis induction of MLL-rearranged acute myeloid leukemia (AML) cell line MV4-11, and to explore their synergistic mechanisms. Methods MV4-11 cells in logarithmic growth phase were treated with different concentrations (100, 200, 300, and 400 nmol/L) of JQ1, 4 nmol/L TPL or different concentrations of JQ1 combined with 4 nmol/L TPL for 48 h. Cell proliferation was detected by CCK-8 method, apoptosis was detected by flow cytometry (FCM), mitochondrial membrane potential was detected by JC-1 method, and expressions of mitochondrial apoptosis pathway-related proteins were detected by Western blot. Results The 50% inhibitory concentration (IC50) value of MV4-11 cells treated with JQ1 for 48 h was (283.9±10.7) nmol/L. However, 4 nmol/L TPL significantly enhanced the inhibitory effect of JQ1 on proliferation of MV4-11 cells, the IC50 value of MV4-11 cells treated with JQ1 combined with TPL was (148.1±2.6) nmol/L, and the difference was statistically significant (t = 25.31, P = 0.029). The result of FCM assay showed that compared with the JQ1 alone group [(9.6±2.3)%, (12.6±1.4)%, (19.5±3.3)%, and (22.7±2.1)%], 4 nmol/L TPL combined with different concentrations (100, 200, 300, and 400 nmol/L) of JQ1 acted on MV4-11 cells for 48 h, the proportions of apoptotic cells were (16.4±1.9)%, (27.5±2.1)%, (32.9±3.6)%, and (35.5±3.0)%, respectively, the difference was statistically significant (F = 9.25, P < 0.01). After treated with 4 nmol/L TPL and JQ1 for 12 h, the level of cell membrane potential in MV4-11 cells was significantly lower than that of JQ1 single agent group, and the difference was statistically significant (P < 0.05). After treated by 4 nmol/L TPL combined with JQ1 for 24 h, the levels of anti-apoptotic proteins bcl-2 and Mcl-1 decreased, and the level of pro-apoptotic protein bax increased. Conclusion TPL can significantly enhance the proliferation inhibition and apoptosis induction effects of BET protein inhibitor JQ1 on MLL-rearranged AML cells, and the mechanism may be related to enhancing the mitochondrial apoptosis pathway. Key words: Gene rearrangement; Leukemia, myeloid, acute; Triptolide; BET protein inhibitor JQ1

Targeting Oncogenic Super Enhancers in MYC-Dependent AML Using a Small Molecule Activator of NR4A Nuclear Receptors

Epigenetic reprogramming in Acute Myeloid Leukemia (AML) leads to the aberrant activation of super enhancer (SE) landscapes that drive the expression of key oncogenes, including the oncogenic MYC pathway. These SEs have been identified as promising therapeutic targets, and have given rise to a new class of drugs, including BET protein inhibitors, which center on targeting SE activity. NR4A nuclear receptors are tumor suppressors of AML that function in part through transcriptional repression of the MYC-driven oncogenic program via mechanisms that remain unclear. Here we show that NR4A1, and the NR4A inducing drug dihydroergotamine (DHE), regulate overlapping gene expression programs in AML and repress transcription of a subset of SE-associated leukemic oncogenes, including MYC. NR4As interact with an AML-selective SE cluster that governs MYC transcription and decommissions its activation status by dismissing essential SE-bound coactivators including BRD4, Mediator and p300, leading to loss of p300-dependent H3K27 acetylation and Pol 2-dependent eRNA transcription. DHE shows similar efficacy to the BET inhibitor JQ1 at repressing SE-dependent MYC expression and AML growth in mouse xenografts. Thus, DHE induction of NR4As provides an alternative strategy to BET inhibitors to target MYC dependencies via suppression of the AML-selective SE governing MYC expression.

Abstract P3-10-06: Combination of BET and FGFR inhibition as a rational therapeutic strategy for invasive lobular breast cancer

Abstract Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor-positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required. As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 (uniquely among BRD family members) was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression (and not other BRD members) was associated with poor recurrence-free survival (p&amp;lt;0.01, HR=3.16, CI 1.24-8.03). Using a two ILC cell lines (SUM44PE and MDA-MB134VI) we found that ILC cells were relatively resistant to the anti-estrogen therapies tamoxifen and fulvestrant compared to those derived from IDC. Next, we tested whether the ILC cell lines were sensitive to BET protein inhibition using the pan-BET family inhibitor JQ1. Interestingly, while JQ1 inhibited cell growth in both ILC cell lines tested, apoptosis was only induced in SUM44PE cells, while MDA-MB134VI cells exhibited G1 arrest. Dynamic BH3 profiling was used to dissect the underlying anti-apoptotic dependencies in each ILC cell type and showed that in the JQ1-resistant MDA-MB134VI cells, survival was dependent on sustained Bcl-Xl expression and transcriptional rewiring inducing Fibroblast Growth Factor Receptor 1 (FGFR1). This increase in FGFR1 was also evident in invasive ductal carcinoma (IDC) cell lines that also did not respond to JQ1. The combination of JQ1 and FGFR1 inhibitor, or JQ1 and the Bcl2/Bcl-Xl/Bcl-W inhibitor navitoclax (ABT-263), were highly effective at inhibiting growth in both 2D and 3D models of ILC and IDC. Interestingly, we found in the RATHER cohort of 61 ILC patients that 20% had FGFR1 amplification suggesting that this therapeutic approach may have utility in a large number of ILC patients. Conclusions: We provide evidence that BETi either alone or in combination with FGFR1 inhibitors or BH3 mimetics may be a useful therapeutic strategy for recurrent ILC patients. Citation Format: Darran P O'Connor, Louise Walsh, Kathryn Haley, Bruce Moran, Brian Mooney, Stephen Madden, Sudipto Das, Oscar Rueda, Catriona Dowling, Damir Vareslija, Suet-Feung Chin, Sabine Linn, Leonie Young, Karin Jirstrom, John P Crown, Rene Bernards, Carlos Caldas, William M Gallagher, Triona Ni Chonghaile. Combination of BET and FGFR inhibition as a rational therapeutic strategy for invasive lobular breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-06.

Targeting BRD/BET proteins inhibits adaptive kinome upregulation and enhances the effects of BRAF/MEK inhibitors in melanoma

BackgroundBRAF-mutant melanoma patients respond to BRAF inhibitors and MEK inhibitors (BRAFi/MEKi), but drug-tolerant cells persist, which may seed disease progression. Adaptive activation of receptor tyrosine kinases (RTKs) has been associated with melanoma cell drug tolerance following targeted therapy. While co-targeting individual RTKs can enhance the efficacy of BRAFi/MEKi effects, it remains unclear how to broadly target multiple RTKs to achieve more durable tumour growth inhibition.MethodsThe blockage of adaptive RTK responses by the new BET inhibitor (BETi), PLX51107, was measured by RPPA and Western blot. Melanoma growth was evaluated in vitro by colony assay and EdU staining, as well as in skin reconstructs, xenografts and PDX models following BRAFi, MEKi and/or PLX51107 treatment.ResultsTreatment with PLX51107 limited BRAFi/MEKi upregulation of ErbB3 and PDGFR-β expression levels. Similar effects were observed following BRD2/4 depletion. In stage III melanoma patients, expression of BRD2/4 was strongly correlated with ErbB3. PLX51107 enhanced the effects of BRAFi/MEKi on inhibiting melanoma growth in vitro, in human skin reconstructs and in xenografts in vivo. Continuous triple drug combination treatment resulted in significant weight loss in mice, but intermittent BETi combined with continuous BRAFi/MEKi treatment was tolerable and improved durable tumour inhibition outcomes.ConclusionsTogether, our data suggest that intermittent inhibition of BET proteins may improve the duration of responses following BRAFi/MEKi treatment in BRAF-mutant melanoma.

An epigenetic screening determines BET proteins as targets to suppress self-renewal and tumorigenicity in canine mammary cancer cells

Targeting self-renewal and tumorigenicity has been proposed as a potential strategy against cancer stem cells (CSCs). Epigenetic proteins are key modulators of gene expression and cancer development contributing to regulation and maintenance of self-renewal and tumorigenicity. Here, we have screened a small-molecule epigenetic inhibitor library using 3D in vitro models in order to determine potential epigenetic targets associated with self-renewal and tumorigenicity in Canine Mammary Cancer (CMC) cells. We identified inhibition of BET proteins as a promising strategy to inhibit CMC colonies and tumorspheres formation. Low doses of (+)-JQ1 were able to downregulate important genes associated to self-renewal pathways such as WNT, NOTCH, Hedgehog, PI3K/AKT/mTOR, EGF receptor and FGF receptor in CMC tumorspheres. In addition, we observed downregulation of ZEB2, a transcription factor important for the maintenance of self-renewal in canine mammary cancer cells. Furthermore, low doses of (+)-JQ1 were not cytotoxic in CMC cells cultured in 2D in vitro models but induced G2/M cell cycle arrest accompanied by upregulation of G2/M checkpoint-associated genes including BTG2 and CCNG2. Our work indicates the BET inhibition as a new strategy for canine mammary cancers by modulating the self-renewal phenotype in tumorigenic cells such as CSCs.

Abstract NT-108: TARGETING BROMODOMAIN PROTEINS AS A THERAPEUTIC STRATEGY FOR CLEAR CELL CARCINOMA OF THE OVARY

Abstract Background and Objectives: Clear cell carcinoma of the ovary (OCCC) is more resistant to platinum-based chemotherapy than high-grade serous ovarian cancer (HGSOC). Also, the indication for PARP inhibitors seems to be limited in OCCC because of the lower frequency of homologous recombination deficiency. As genomic background significantly differs among subtypes, therapeutic strategies should be optimized in response to each subtype or unique gene alterations. This study was designed to find novel therapeutic targets for OCCC by utilizing siRNA screens and unique patient-derived ovarian cancer cells that phenocopy original tumors. Materials and Methods: Novel patient-derived ovarian cancer cells were established and kindly provided by Dr. Tan Ince at University of Miami. Genomic alterations of these cells were determined by DNA sequencing. siRNA screens targeting 2,172 unique genes were performed with ARID1A-mutated OCCC cell line OCI C5x, and high-grade serous ovarian cancer cell line OCI P5x. Genes inhibition of which significantly suppressed cell proliferation were selected as hits for each subtype independently. The difference of cell viability between OCI C5x and P5x was also compared, and subtype-specific hits were additionally determined. Hits were applied to Gene Ontology (GO) enrichment analysis to comprehend the gene set enrichment. Several prioritized targets for OCCC were validated with additional RNAi experiments and small molecule inhibitors. Results: 114 and 108 genes were selected as hits specific to OCI C5x and OCI P5x, respectively. GO enrichment analysis revealed OCCC is potentially more vulnerable to epigenetic interventions than HOGSC. The following inspection revealed bromodomain-containing proteins were enriched in the hits for OCI C5x. Among these, we highlighted bromodomain and extra-terminal domain (BET) family proteins BRD2 and BRD3. Subsequent RNAi experiments validated BRD2 and BRD3 are highly potent therapeutic targets for OCCC. Conclusions: siRNA screening results suggest different approaches are required according to subtypes for the targeted therapy of ovarian cancer. We propose that epigenetic intervention such as BET protein inhibition as a potential therapeutic option for OCCC, 50-60% of which harbor ARID1A mutation. Citation Format: Shogo Shigeta, Reid Shaw, Russell Moser, Kay Gurley, Goldie Lui, Carla Grandori, Christopher Kemp. TARGETING BROMODOMAIN PROTEINS AS A THERAPEUTIC STRATEGY FOR CLEAR CELL CARCINOMA OF THE OVARY [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 NT-108.

Mechanistic Basis and Efficacy of Targeting β-Catenin-TCF7L2-JMJD6-MYC Axis to Overcome Resistance to BET Inhibitors

Hematopoietic progenitor cells of myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit constitutive activation of JAK-STAT5/3 and NFkB signaling. Transformation of MPN-MF to AML (post-MPN sAML) occurs in up to 15% of patients with MPN-MF. Standard induction anti-AML chemotherapy and the JAK1 &amp; 2 inhibitor (JAKi) ruxolitinib are ineffective in post-MPN sAML. BET protein BRD4 is a non-oncogene addiction target in AML, and treatment with acetyl-lysine mimetic BET protein inhibitor (BETi) disrupts binding of BRD4 to acetylated chromatin and transcription factors (TFs). This attenuates transcription of super-enhancer regulated oncogenes, including MYC, Bcl-xL, PIM1 and CDK4/6, inhibiting growth and survival of post-MPN sAML blasts. BETi treatment also inhibits binding of BRD4 to acetylated RELA (NFkB-p65), inhibiting its transcriptional activity and attenuating levels of its target cytokines. However, BETi treatment induces BRD4, potentially reducing BETi activity in repressing oncogenes. Preclinical and clinical studies have demonstrated that innate or adaptive BETi-resistance is common in sAML cells. To model BETi-resistance, we repeatedly exposed (10 times) secondary (s) AML SET2 and HEL92.1.7 (HEL) cells to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, thus generating BETi persister-resistant (BETi-P/R) SET2-P/R and HEL-P/R cells. These cells showed &amp;gt; 10-fold resistance to OTX015 and cross-resistance to other BETis. Compared to the parental controls, BETi-P/R cells lacked additional genetic alterations or altered levels of TRIM33, SPOP, DUB3 or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). However, ATAC-Seq and ChIP-Seq (H3K27Ac mark) analyses demonstrated that, as compared to their parental controls, BETi-P/R cells showed gain of peaks and active enhancers with enrichment of STAT5, MYC, PU.1 and GATA2 binding sites. Newly gained peaks were in the enhancers of JAK1/2, RUNX1, PU.1, MYC and BCL2L1. RNA-Seq determined mRNA level alterations, included induction of gene-sets involving MYC/MAX, STAT5, NFkB and TCF7L2 targets. QPCR and Western analyses confirmed increase in the mRNA and protein levels of TCF7L2, JMJD6, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Expression of the arginine demethylase JMJD6, recruited by BRD4 to regulate enhancer-mediated transcriptional pause-release, was also increased. This was associated with increased expression of the nuclear β-catenin-TCF7L2 targets, including Cyclin D1, TERT, survivin, c-Myc and PU.1. Patient-derived human AML blasts that exhibited innate resistance ex vivo to BETi, also demonstrated increased expression of TCF7L2, JMJD6 and c-Myc. We next probed the mechanistic role of the β-catenin-JMJD6-TCF7L2-MYC axis in conferring BETi-resistance. CRISPR-Cas9-mediated knockout of TCF7L2 or JMJD6 significantly reversed BETi-resistance in BETi-P/R sAML cells (p &amp;lt; 0.001). Conversely, ectopic overexpression of TCF7L2 or JMJD6 significantly conferred BETi-persister-resistance in HEL and SET2 cells (p &amp;lt; 0.001). Notably, confocal microscopy demonstrated increased binding of β-catenin with TBL1 and TCF7L2 in the nucleus of BETi-P/R sAML cells. BC2059, which disrupts binding of nuclear β-catenin with TBL1 and TCF7L2, depleted β-catenin levels and exerted similar lethality in BETi-P/R sAML and control sAML cells. shRNA-mediated knockdown of BRD4 and treatment with BRD4-PROTAC (proteolysis-targeting chimera) ARV-771 (Arvinas, Inc.) that degrades BRD4/3/2, also induced similar levels of apoptosis in BETi-P/R and control sAML cells. Co-treatment with ARV-771 and BC2059 synergistically induced lethality in BETi-P/R sAML cells as well as in patient-derived, CD34+ sAML BPCs (combination indices &amp;lt; 1.0). This was associated with marked attenuation of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL levels. Also, compared to each agent alone, in vivo treatment with ARV-771 (30 mg/kg SQ daily x 5, per week) and BC2059 (30 mg/kg IP BIW per week) for 3 weeks, significantly reduced sAML burden and improved survival of NSG mice engrafted with HEL-P/R cells (p &amp;lt; 0.01). Collectively, these findings underscore that increased levels and activity of β-catenin-TCF7L2-JMJD6-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BRD4 degrader and β-catenin-TCF7L2 inhibitor is a promising therapeutic strategy against BETi-P/R sAML BPCs. Disclosures Bhalla: Beta Cat Pharmaceuticals: Consultancy. Verstovsek:Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding.

Efficacy of Targeted Therapy in Novel Pre-Clinical in Vitro and In Vivo Models of Richter Transformation-Diffuse Large B-Cell Lymphoma (RT-DLBCL)

Richter Transformation (RT) is defined as the development of aggressive DLBCL (mostly ABC-type) in up to ~15% of patients with antecedent or concurrent diagnosis of CLL. Based on the comparison of immunoglobin gene rearrangements, approximately 80% of RT-DLBCL arise due to a direct clonal evolution of the underlying CLL clone, i.e., clonally related (CLR) RT-DLBCL, which exhibit poor median survival (MS) of one year. Approximately 20% of RT-DLBCLs are clonally unrelated (CUR) to the underlying CLL, arising most likely due to branched clonal evolution from a common pre-CLL progenitor. CUR RT-DLBCLs exhibit a better MS of 5 years. Although chemo-immunotherapy and treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib or anti-apoptotic BCL2 inhibitor venetoclax can induce remissions, they fail to induce prolonged disease-free survival in RT-DLBCL. Majority of patients relapse with therapy-refractory disease. Lack of availability of in vitro cultured RT-DLBCL cells or PD xenograft models has prevented pre-clinical testing and development of novel targeted agents against RT-DLBCL. Here, we report the establishment of 3 patient-derived xenograft (PDX) models of RT-DLBCL. Based on immunoglobulin heavy chain (IGH) clonality testing by NGS, the RT-DLBCL RT17 was CLR, RT15 was CUR and RT5 was of indeterminate clonality. The PDXs were generated by tail-vein infusion and engraftment of luciferase-transduced CD19+ RT-DLBCL cells in NSG mice. The RT-DLBCL PDXs grew in the bone marrow and spleen, causing marked splenomegaly, requiring euthanasia 4 to 6 weeks after engraftment. All three RT-DLBCL PDX cells were EBV-negative by genomic and EBNA2 protein expression analyses. NextGen DNA sequencing of RT17, RT15, and RT5 cells showed large numbers of genetic mutations, including mutations in TP53, ATM, NOTCH2, TET2 and MLL3 genes with a high variant allelic frequencies. Array-CGH showed DNA copy gains or losses in multiple chromosomes, including 3, 8, 9, 11, 12, 17 and 18. A 5'-MYC amplification was detected by FISH analysis in RT5 DLBCL cells. ATAC-Seq showed increased signal intensity representing increased chromatin accessibility in the RT-DLBCL cells compared to CD34+ normal progenitors. High peak numbers were detected in specific loci, including TCF4, PAX5, IRF4, MYB, MYC, BCL2L1 and BCL-2. Anti-H3K27Ac ChIP-Seq analysis showed increased average, normalized read-densities at super-enhancers/enhancers (SEs/Es), including those of TCF4, PAX5, IRF4, BCL2 (RT17 and RT15) and MYC (RT5). Western analyses showed that all three RT-DLBCL PDX cells expressed TCF4, c-Myc, and BRD4, with highest expression in RT5 cells. Accordingly, RT5 cells were more sensitive than cells RT17 and RT15 cells to the BET protein inhibitor (BETi) OTX015-induced apoptosis. This was associated with greater, OTX015-mediated, depletion of c-Myc in RT5 cells. RT17 and RT15 expressed high levels of BCL2, Bcl-xL and MCL1, whereas RT5 lacked BCL2 expression. Consistent with this, RT17 and RT15 cells were significantly more sensitive than RT5 cells to venetoclax-induced apoptosis (p &lt; 0.01). RT17 and RT15, but not RT5 cells, expressed NFkB2 (p52), consistent with activation of non-canonical NFkB signaling. This was associated with resistance of RT17 and RT15 cells to ibrutinib-induced apoptosis (p &lt; 0.001). However, co-treatment with OTX015 and ibrutinib or venetoclax induced synergistic lethality in all RT-DLBCL cells (combination indices &lt; 1.0). BET-PROTAC ARV-825 and ARV-771 treatment depleted BRD4, leading to marked reduction in c-Myc levels and apoptosis of all RT-DLBCL cells. Treatment with the ATP-competitive, CDK9 inhibitor NVP2 also dose-dependently induced apoptosis in RT-DLBCL cells associated with depletion of c-Myc, Bcl-xL, and MCL1 protein levels. These findings highlight the activity and support further in vitro and in vivo evaluation of BETi, BET-PROTAC or CDK9i-based combinations with ibrutinib or venetoclax against genetically-profiled RT-DLBCL cells that are clonally-related or clonally-unrelated to the antecedent CLL. Disclosures Maiti: Celgene: Other: research funding. Bhalla:Beta Cat Pharmaceuticals: Consultancy. Khoury:Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding.

BET protein targeting suppresses the PD-1/PD-L1 pathway in triple-negative breast cancer and elicits anti-tumor immune response.

Therapeutic strategies aiming to leverage anti-tumor immunity are being intensively investigated as they show promising results in cancer therapy. The PD-1/PD-L1 pathway constitutes an important target to restore functional anti-tumor immune response. Here, we report that BET protein inhibition suppresses PD-1/PD-L1 in triple-negative breast cancer. BET proteins control PD-1 expression in T cells, and PD-L1 in breast cancer cell models. BET protein targeting reduces T cell-derived interferon-γ production and signaling, thereby suppressing PD-L1 induction in breast cancer cells. Moreover, BET protein inhibition improves tumor cell-specific T cell cytotoxic function. Overall, we demonstrate that BET protein targeting represents a promising strategy to overcome tumor-reactive T cell exhaustion and improve anti-tumor immune responses, by reducing the PD-1/PD-L1 axis in triple-negative breast cancer.

The Hippo pathway modulates resistance to BET proteins inhibitors in lung cancer cells.

Inhibitors of BET proteins (BETi) are anti-cancer drugs that have shown efficacy in pre-clinical settings and are currently in clinical trials for different types of cancer, including non-small cell lung cancer (NSCLC). Currently, no predictive biomarker is available to identify patients that may benefit from this treatment. To uncover the mechanisms of resistance to BETi, we performed a genome-scale CRISPR/Cas9 screening in lung cancer cells. We identified three Hippo pathway genes, LATS2, TAOK1, and NF2, as key determinants for sensitivity to BETi. The knockout of these genes induces resistance to BETi, by promoting TAZ nuclear localization and transcriptional activity. Conversely, TAZ expression promotes resistance to these drugs. We also showed that TAZ, YAP, and their partner TEAD are direct targets of BRD4 and that treatment with BETi downregulates their expression. Noticeably, molecular alterations in one or more of these genes are present in a large fraction of NSCLC patients and TAZ amplification or overexpression correlates with a worse outcome in lung adenocarcinoma. Our data define the central role of Hippo pathway in mediating resistance to BETi and provide a rationale for using BETi to counter-act YAP/TAZ-mediated pro-oncogenic activity.

RUNX1-targeted therapy for AML expressing somatic or germline mutation in RUNX1

AbstractRUNX1 transcription factor regulates normal and malignant hematopoiesis. Somatic or germline mutant RUNX1 (mtRUNX1) is associated with poorer outcome in acute myeloid leukemia (AML). Knockdown or inhibition of RUNX1 induced more apoptosis of AML expressing mtRUNX1 versus wild-type RUNX1 and improved survival of mice engrafted with mtRUNX1-expressing AML. CRISPR/Cas9-mediated editing-out of RUNX1 enhancer (eR1) within its intragenic super-enhancer, or BET protein BRD4 depletion by short hairpin RNA, repressed RUNX1, inhibited cell growth, and induced cell lethality in AML cells expressing mtRUNX1. Moreover, treatment with BET protein inhibitor or degrader (BET–proteolysis targeting chimera) repressed RUNX1 and its targets, inducing apoptosis and improving survival of mice engrafted with AML expressing mtRUNX1. Library of Integrated Network–based Cellular Signatures 1000–connectivity mapping data sets queried with messenger RNA signature of RUNX1 knockdown identified novel expression-mimickers (EMs), which repressed RUNX1 and exerted in vitro and in vivo efficacy against AML cells expressing mtRUNX1. In addition, the EMs cinobufagin, anisomycin, and narciclasine induced more lethality in hematopoietic progenitor cells (HPCs) expressing germline mtRUNX1 from patients with AML compared with HPCs from patients with familial platelet disorder (FPD), or normal untransformed HPCs. These findings highlight novel therapeutic agents for AML expressing somatic or germline mtRUNX1.

Abstract 293: Inhibition of BET proteins augments nab-paclitaxel-gemcitabine-based chemotherapy response in preclinical models of pancreatic cancer

Abstract BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) often presents with metastatic activity, leading to an extremely poor prognosis. Nanoparticle albumin-bound paclitaxel (NPT) in combination with gemcitabine (NPT+GEM) is a standard treatment for PDAC patients, resulting in a survival of ~8.5 months. Bromodomain and extraterminal domain (BET) proteins are epigenetic regulators of gene expression and are involved in cancer pathogenesis. Targeted inhibition of BET protein is currently under investigation for several cancers. We hypothesize that BET protein pathway inhibition by iBet-762 will enhance nab-paclitaxel-gemcitabine-based standard chemotherapy response in PDAC. METHODS: In vitro cell proliferation assays were performed using WST-1 reagent. Protein expressions were determined by Western Blot analysis. In vivo animal survival and tumor growth experiments were performed in NOD-SCID mice. RESULTS: BET inhibitor (iBET-762) and standard chemotherapy (NPT+Gem) had a dose-dependent in vitro growth inhibitory effect on several PDAC cell lines tested. Inhibition in cell proliferation at 1 μM drug concentration in NPT+Gem, iBET-762 and NPT+Gem+iBet-762 treatment groups was 64%, 27%, 76% in AsPC-1; 43%, 13%, 69% in Panc-1; and 42%, 51%, 75% in MIA PaCa-2 cells. Immunoblot analysis demonstrated that iBET-762 decreased the expression of oncogenic proteins c-Myc, β-catenin, vimentin and phospho-AKT, while increasing the expression of apoptosis-related proteins such as cleaved PARP-1 and cleaved caspase-3 and cell cycle inhibitor proteins P21 and P27. In subcutaneous xenografts, compared to controls, NPT+Gem and iBet-762 decreased tumor growth by 72% and 57%, respectively (p&amp;lt;0.02). Importantly, the combination therapy group (NPT+Gem+iBet-762) had an additive effect on tumor growth inhibition (98%, p&amp;lt;0.0001). In a peritoneal dissemination model, median animal survival compared to controls (21 days) was increased by NPT+Gem (33 days, a 57% increase) and iBet-762 (30 days, a 43% increase) therapy. This was further increased in the combination therapy group NPT+Gem+iBET-762 (44 days, a 110% increase). CONCLUSION: These findings demonstrate that nab-paclitaxel-gemcitabine-based standard chemotherapy response can be enhanced through specific inhibition of BET proteins by iBET-762 in preclinical models of PDAC. The data support the potential of this combinatorial therapeutic strategy for clinical PDAC therapy. Citation Format: Niranjan Awasthi, Sandeep Singh, Ross D. McCauley, Johann RE Schwarz, Margaret A. Schwarz, Roderich E. Schwarz. Inhibition of BET proteins augments nab-paclitaxel-gemcitabine-based chemotherapy response in preclinical models of pancreatic cancer [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 293.

Abstract 1071: Targeted therapy evaluation in novel pre-clinical in vitro and in vivo models of Richter transformation

Abstract Richter Transformation (RT) is the development (in up to 15%) of aggressive DLBCL in patients with antecedent or concurrent CLL. By comparing immunoglobin gene rearrangements, 80% of RT-DLBCL (mostly ABC type) are clonally related (CR) to the underlying CLL, exhibiting poor median survival (MS) of 1 year, whereas 20% of RT-DLBCLs are clonally unrelated (CUR), exhibiting MS of 5 years. Lack of availability of in vitro cultured RT-DLBCL cells or PD xenograft models had prevented pre-clinical testing of novel targeted agents against RT-DLBCL. Here, we report the establishment of 3 patient-derived xenograft (PDX) models of RT-DLBCL (CR: RT15 and RT17, CUR: RT5) generated by tail-vein infusion and engraftment of luciferase-transduced CD19+ RT-DLBCL cells in NSG mice. RT-DLBCL xenografts grew in the bone marrow and spleen, causing marked splenomegaly and requiring euthanasia 4 to 6 weeks after engraftment. All three RT-DLBCL cells were EBV-negative by genomic and EBNA2 protein expression analyses. NextGen DNA sequencing of RT5 and RT15 cells showed mutations in TP53, ATM, NOTCH2, TET2 and MLL3 genes with a high VAF. Array-CGH showed DNA copy gains or losses in multiple chromosomes including 3, 8, 9, 11, 12, 17 and 18. Anti-H3K27Ac ChIP-Seq analysis showed increased average, normalized read-densities across all super-enhancers/enhancers (SEs/Es), with several SEs/Es exhibiting high scores. ATAC-Seq showed markedly increased signal intensity in RT-DLBCL cells compared to CD34+ normal progenitors, with high peak numbers in specific loci. Western analyses showed that RT15 and RT17 expressed high levels of BCL2, Bcl-xL and MCL1, whereas RT5 lacked BCL2 expression. RT15 and RT17 cells were more sensitive than RT5 cells to anti-BCL2 venetoclax-induced apoptosis (p &amp;lt; 0.01). RT15 and RT17, but not RT5 cells, expressed NFkB2 (p52), consistent with activation of non-canonical NFkB signaling. This was associated with resistance of RT15 and RT17 cells to ibrutinib-induced apoptosis. RT15 and RT17 cells were also less sensitive than RT5 cells to the BET protein inhibitor (BETi) OTX015-induced apoptosis. However, co-treatment with OTX015 and ibrutinib or venetoclax induced synergistic lethality in all RT-DLBCL cells (combination indices &amp;lt; 1.0). BET-PROTAC ARV-825 and ARV-771 treatment depleted BRD4, leading to marked reduction in c-Myc levels and apoptosis of RT-DLBCL cells. Treatment with the ATP-competitive, CDK9 inhibitor NVP2 dose-dependently induced apoptosis in RT-DLBCL cells which was associated with depletion of c-Myc, Bcl-xL and MCL1 protein levels. These findings highlight the activity and support further evaluation of in vitro and in vivo BETi, BET-PROTAC or CDK9i-based combinations with ibrutinib or venetoclax against genetically profiled RT-DLBCL cells that are clonally-related or clonally-unrelated to the antecedent CLL. Citation Format: Warren C. Fiskus, Abhishek Maiti, Dyana T. Saenz, Christopher P. Mill, Joseph D. Khoury, Kapil N. Bhalla. Targeted therapy evaluation in novel pre-clinical in vitro and in vivo models of Richter transformation [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 1071.

BET protein inhibition regulates cytokine production and promotes neuroprotection after spinal cord injury

BackgroundSpinal cord injury (SCI) usually causes a devastating lifelong disability for patients. After a traumatic lesion, disruption of the blood-spinal cord barrier induces the infiltration of macrophages into the lesion site and the activation of resident glial cells, which release cytokines and chemokines. These events result in a persistent inflammation, which has both detrimental and beneficial effects, but eventually limits functional recovery and contributes to the appearance of neuropathic pain. Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that regulate the expression of inflammatory genes by interacting with acetylated lysine residues. While BET inhibitors are a promising therapeutic strategy for cancer, little is known about their implication after SCI. Thus, the current study was aimed to investigate the anti-inflammatory role of BET inhibitors in this pathologic condition.MethodsWe evaluated the effectiveness of the BET inhibitor JQ1 to modify macrophage reactivity in vitro and to modulate inflammation in a SCI mice model. We analyzed the effects of BET inhibition in pro-inflammatory and anti-inflammatory cytokine production in vitro and in vivo. We determined the effectiveness of BET inhibition in tissue sparing, inflammation, neuronal protection, and behavioral outcome after SCI.ResultsWe have found that the BET inhibitor JQ1 reduced the levels of pro-inflammatory mediators and increased the expression of anti-inflammatory cytokines. A prolonged treatment with JQ1 also decreased reactivity of microglia/macrophages, enhanced neuroprotection and functional recovery, and acutely reduced neuropathic pain after SCI.ConclusionsBET protein inhibition is an effective treatment to regulate cytokine production and promote neuroprotection after SCI. These novel results demonstrate for the first time that targeting BET proteins is an encouraging approach for SCI repair and a potential strategy to treat other inflammatory pathologies.

BET bromodomain targeting suppresses the PD-1/PD-L1 pathway in triple-negative breast cancer and elicits anti-tumor immune response

Abstract Therapeutic strategies aiming to leverage anti-tumor immunity are being intensively investigated because they show promising results in several cancer therapies. In tumors able to evade immune clearance, the PD-1/PD-L1 pathway constitutes an important target to restore a functional anti-tumor immune response. Here, we report that BET protein inhibition suppresses the PD-1/PD-L1 axis in triple-negative breast cancer. BET proteins control PD-1 expression in effector T cells, and PD-L1 in breast cancer cells. BET protein targeting reduces T cell-derived interferon-gamma production and signaling, thereby suppressing PD-L1 sustained induction in breast cancer cells. Moreover, BET protein inhibition improves tumor cell-specific T cell cytotoxic function. Overall, we demonstrate that BET protein targeting represents a promising strategy to overcome tumor-reactive T cell exhaustion and improve anti-tumor immune responses, by reducing the PD-1/PD-L1 axis in triple-negative breast cancer.

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.

Inhibition of BET Proteins and Histone Deacetylase (HDACs): Crossing Roads in Cancer Therapy.

Histone DeACetylases (HDACs) are enzymes that remove acetyl groups from histones and other proteins, regulating the expression of target genes. Pharmacological inhibition of these enzymes re-shapes chromatin acetylation status, confusing boundaries between transcriptionally active and quiescent chromatin. This results in reinducing expression of silent genes while repressing highly transcribed genes. Bromodomain and Extraterminal domain (BET) proteins are readers of acetylated chromatin status and accumulate on transcriptionally active regulatory elements where they serve as scaffold for the building of transcription-promoting complexes. The expression of many well-known oncogenes relies on BET proteins function, indicating BET inhibition as a strategy to counteract their activity. BETi and HDACi share many common targets and affect similar cellular processes to the point that combined inhibition of both these classes of proteins is regarded as a strategy to improve the effectiveness of these drugs in cancer. In this work, we aim to discuss the molecular basis of the interplay between HDAC and BET proteins, pointing at chromatin acetylation as a crucial node of their functional interaction. We will also describe the state of the art of their dual inhibition in cancer therapy. Finally, starting from their mechanism of action we will provide a speculative perspective on how these drugs may be employed in combination with standard therapies to improve effectiveness and/or overcome resistance.