Bromo And Extraterminal Research Articles

Bromodomain Protein Inhibition Protects β-Cells from Cytokine-Induced Death and Dysfunction via Antagonism of NF-κB Pathway.

Cytokine-induced β-cell apoptosis is a major pathogenic mechanism in type 1 diabetes (T1D). Despite significant advances in understanding its underlying mechanisms, few drugs have been translated to protect β-cells in T1D. Epigenetic modulators such as bromodomain-containing BET (bromo- and extra-terminal) proteins are important regulators of immune responses. Pre-clinical studies have demonstrated a protective effect of BET inhibitors in an NOD (non-obese diabetes) mouse model of T1D. However, the effect of BET protein inhibition on β-cell function in response to cytokines is unknown. Here, we demonstrate that I-BET, a BET protein inhibitor, protected β-cells from cytokine-induced dysfunction and death. In vivo administration of I-BET to mice exposed to low-dose STZ (streptozotocin), a model of T1D, significantly reduced β-cell apoptosis, suggesting a cytoprotective function. Mechanistically, I-BET treatment inhibited cytokine-induced NF-kB signaling and enhanced FOXO1-mediated anti-oxidant response in β-cells. RNA-Seq analysis revealed that I-BET treatment also suppressed pathways involved in apoptosis while maintaining the expression of genes critical for β-cell function, such as Pdx1 and Ins1. Taken together, this study demonstrates that I-BET is effective in protecting β-cells from cytokine-induced dysfunction and apoptosis, and targeting BET proteins could have potential therapeutic value in preserving β-cell functional mass in T1D.

Abstract 6049: Cell-based PROTAC screening for cancer drug discovery

Abstract Proteolysis-Targeting Chimeras (PROTACs) have emerged as a promising strategy for selectively degrading specific target proteins, thereby offering a novel approach to precision cancer therapy. Among the most notable oncogenes, KRAS with the G12C mutation is responsible for 45%-50% of KRAS mutations in non-small cell lung carcinomas. Additionally, BRD4, a well-studied member of the Bromo- and Extra-Terminal (BET) protein family, is implicated in various hematological and solid tumors. The implementation of cell-based screening for PROTACs targeting either KRAS(G12C) or BRD4 represents a highly valuable platform for advancing cancer drug discovery. We utilized the NanoBRETTM Ternary Complex Assay, NanoBiTTM Assay, and Western Blot assay to evaluate the efficacy of PROTACs. The NanoBRETTM Ternary Complex Assay revealed that KRAS G12C degrader-1 and LC-2 effectively degrade KRAS(G12C), while dBET6 and MZ-1 demonstrated the ability to degrade BRD4. Notably, the NanoBiTTM assay showed that LC-2 dose-dependently degrades KRAS(G12C) in HEK293 cells co-transfected with KRAS(G12C)-LgBiT and KRAS(G12C)-SmBiT. Moreover, BET6 exhibited a dose-dependent degradation of endogenous BRD4 in HepG2 cells, and LC-2 demonstrated the degradation of endogenous KRAS(G12C) in MiaPaCa2 cells. In summary, the development of a robust cell-based assay platform for high-throughput screening of PROTACs marks a substantial advancement in cancer drug discovery. This approach not only enables the identification of promising PROTACs but also holds the potential for more effective and targeted interventions in the ongoing battle against cancer. Citation Format: Yong Wan, Haiqing Ma, Jianghong Wu. Cell-based PROTAC screening for cancer drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6049.

Establishment of environment-sensitive probes targeting BRD3/BRD4 for imaging and therapy of tumor

The BET (bromo and extra-terminal) family proteins are epigenetic readers and master transcription coactivators, which have attracted great interests as cancer therapeutic targets. However, there are few developed labeling toolkits that can be applied for the dynamic studies of BET family proteins in living cells and tissue slices. In order to label and study the distribution of the BET family proteins in tumor cells and tumor tissues, a novel series of environment-sensitive fluorescent probes (6a-6c) were designed and evaluated for their labeling properties. Interestingly, 6a is capable of identifying tumor tissue slices and making a distinction between the tumor and normal tissues. Moreover, it can localize to the nuclear bodies in tumor slices just like BRD3 antibody. In addition, it also played an anti-tumor role through the induction of apoptosis. All these features render 6a may compatible for immunofluorescent studies and future cancer diagnosis, and guide for the discovery of new anticancer drugs.

Modeling binding plasticity in BET proteins through simulations and NMR.

The bromo and extraterminal (BET) family of proteins play an important role in regulating the expression of immune response genes—and has also been associated to multiple types of cancers and virus attack. The extra terminal domain (ET) of BET proteins acts as an interaction hub to accommodate binding to multiple host and viral proteins. Surprisingly, it interacts with these proteins through multiple binding modes, where peptide epitopes can bind as single strands, hairpins in different orientations and helices.

CBX5 loss drives EGFR inhibitor resistance and results in therapeutically actionable vulnerabilities in lung cancer

Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFRi) are approved for treating EGFR-mutant lung adenocarcinoma (LUAD), emergence of acquired resistance limits their clinical benefits. Several mechanisms for acquired resistance to EGFRi in LUAD have been identified; however, the molecular basis for this resistance remains unknown in ~30% of LUAD. Chromatin and DNA modifiers and their regulators play important roles in determining response to anticancer therapies. Therefore, to identify nongenetic mechanisms of EGFRi resistance in LUAD, we performed an epigenome-wide shRNA screen targeting 363 human epigenetic regulator genes. This screen identified loss of the transcriptional repressor chromobox homolog 5 (CBX5) as a driver of EGFRi resistance in EGFR-mutant LUAD. Loss of CBX5 confers resistance to multiple EGFRi in both cell culture and mice. We found that CBX5 loss in EGFR-mutant LUAD cells leads to increased expression of the transcription factor E2F1, which in turn stimulates expression of the antiapoptotic gene BIRC5 (survivin). This E2F1-mediated upregulation of BIRC5 in CBX5-knockdown LUAD cells attenuates apoptosis induction following EGFRi treatment. Consistent with these results, knockdown of E2F1 or BIRC5 partly rescues CBX5-knockdown-induced EGFRi resistance in cell culture and mice. EGFRi-resistant LUAD cell lines show reduced CBX5 expression compared to parental lines; however, bromo- and extra-terminal (BET)-domain inhibitors (BETi) restore CBX5 expression in these cells and sensitize them to EGFRi/BETi combination therapy. Similarly, treatment with a BIRC5 inhibitor suppresses growth of EGFRi-resistant LUAD cells. Collectively, these studies identify CBX5 loss as a driver of EGFRi resistance and reveal therapeutic opportunities for treating EGFRi-resistant LUAD.

Combination bromo- and extraterminal domain and poly (ADP-ribose) polymerase inhibition synergistically enhances DNA damage and inhibits neuroblastoma tumorigenesis

PurposeJQ1 is a bromo- and extraterminal (BET) domain inhibitor that downregulates MYC expression and impairs the DNA damage response. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent DNA damage sensing and repair. We hypothesized that JQ1 would promote a DNA repair-deficient phenotype that sensitizes neuroblastoma cells to PARP inhibition.MethodsFour human neuroblastoma cell lines were examined: two MYCN-amplified (BE(2)-C and IMR-32), and two non-MYCN-amplified (SK-N-SH and SH-SY5Y). Cells were treated with JQ1 (BET inhibitor), Olaparib (PARP inhibitor), or in combination to assess for therapeutic synergy of JQ1 and Olaparib. Treated cells were harvested and analyzed. Quantitative assessment of combination treatment synergy was performed using the median effect principle of Chou and Talalay.ResultsCombination treatment with Olaparib decreased the IC50 of JQ1 by 19.9-fold, 2.0-fold, 12.1-fold, and 2.0-fold in the BE(2)-C, IMR-32, SK-N-SH, and SH-SY5Y cell lines, respectively. In the MYCN-amplified cell lines, BE(2)-C and IMR-32, combination treatment decreased gene expression of MYCN relative to single-drug treatment alone or control. Combination treatment decreased protein expression of DNA repair proteins Ku80 and RAD51, led to accumulation of DNA damage marker phospho-histone H2A.X, and increased caspase activity. In the non-MYCN-amplified cell lines, SK-N-SH and SH-SY5Y, combination treatment induced G0/G1 cell cycle arrest.ConclusionsCombination BET and PARP inhibition synergistically inhibited neuroblastoma tumorigenesis in vitro. In MYCN-amplified neuroblastoma cells, this effect may be induced by downregulation of MYCN transcription, defects in DNA repair, accumulation of DNA damage, and apoptosis. In non-MYCN-amplified cell lines, combination treatment induced cell cycle arrest.

JQ1 attenuates neuroinflammation by inhibiting the inflammasome-dependent canonical pyroptosis pathway in SAE

Sepsis-associated encephalopathy (SAE) manifests clinically in hyperneuroinflammation. Pyroptosis, which can induce an inflammatory cascade response, has been considered to be a causative factor of SAE. Evidence has shown that the bromo- and extraterminal (BET) proteins (including BRD2, BRD3, BRD4 and BRDT) inhibitor JQ1 can inhibit inflammation and suppress pyroptosis in various diseases. Therefore, we examined the effect of JQ1 on inflammasome-induced pyroptosis in the hippocampus in a mouse model of sepsis induced by lipopolysaccharide (LPS) injection. The results showed that JQ1 treatment alleviated sepsis-related symptoms, protected the blood–brain barrier (BBB), as indicated by upregulated expression of the tight junction proteins occludin and ZO-1, and remarkably rescued neuronal damage in SAE mice. Mechanistically, we demonstrated that JQ1 intervention inhibited the expression of BRD proteins and decreased the expression of inflammasomes by blocking phospho-nuclear factor kappa B (p-NF-κB) signalling, attenuating the canonical pyroptosis (mediated by cleaved-Caspase1/11) pathway and the release of proinflammatory factors in the hippocampus of septic mice. Interestingly, we also found that JQ1 selectively suppressed the activation of hippocampal microglia in SAE mice. Thus, JQ1 protected the hippocampal BBB and neuronal damage through the attenuation of neuroinflammation by inhibiting the inflammasome-dependent canonical pyroptosis pathway induced by LPS injection in mice, and JQ1 may be a promising target for the prevention of SAE.

BET-ting on histone proteomics in schizophrenia

In a recent study, Farrelly, Zheng, and colleagues used a histone proteomics approach and patient-derived neurons to show increase in histone variant H2A.Z acetylation associated with schizophrenia (SCZ). They identified the bromo- and extraterminal (BET) protein BRD4 as an H2A.Z acetylation 'reader', and showed that a BRD4 inhibitor ameliorated the SCZ-associated transcriptional signature, revealing a new candidate target for treatment.

Abstract P1-04-12: Pathway analysis of immune checkpoint gene regulation as altered in Type 2 diabetes: Implications for breast cancer patients treated with checkpoint inhibitors

Abstract Background: Mediators of immune exhaustion are an active area of breast cancer research. Type II diabetes mellitus (T2D), the most common metabolic disorder, both increases breast cancer incidence and decreases survival. Despite this clear link, impacts of the T2D immune phenotype on cancer remain poorly understood. We have previously demonstrated that the chronic inflammatory state of T2D leads to immune exhaustion, but how the T2D breast microenvironment interacts with T cells is unclear. Exosomes are crucial components of intercellular communication and are associated with increased breast cancer aggressiveness. Given this function, we hypothesized that T2D adipocyte-derived exosomes drive T cell exhaustion. Here, we characterize expression patterns and regulatory networks driving T2D immune phenotypes. Methods: Exosomes were first isolated from culture media of mature human primary breast adipocytes from, either insulin-sensitive (IS), or rendered insulin-resistant (IR) by ex vivo TNFα-treatment. Human primary peripheral blood mononuclear cells (PBMCs) from nondiabetic (ND) and T2D donors were stimulated ex vivo with plate bound anti-CD3 (5 ug/ml) and soluble CD28 (2 ug/ml) for 48 hours and treated with exosomes. Different small molecule inhibitors of the bromo and extraterminal (BET) protein family, including the pan-BET inhibitor JQ1 and the BRD4-selective PROTAC degrader MZ-1, were used to identify BET protein-regulated targets. We also used the AMP-activating protein kinase (AMPK) inhibitor Compound-C to identify the role of this pathway in activating this major epigenetic player. Multicolor flow cytometry was subsequently performed with an LSRII cytometer to assess expression of inhibitory receptors PD-1, CTLA-4, TIM-3 and TIGIT on immune subsets. Events for live cells were analyzed in FlowJo. Cytokines were collected from conditioned media and analyzed via Th17 cytokine staining panel. Results: We observed that exosomes derived from IR breast adipocytes increase expression of immune exhaustion markers in CD4+ and CD8+ T cells, compared to IS or ND matched controls. Additionally, we define signal transduction among BET proteins, AMPK signaling, and immune checkpoint expression. Lastly, we identify changes in cytokine profile between IS and IR treated groups. Taken together, our findings suggest a network of immune regulation imparted by exosomes. Impacts: Metabolic health does not inform the current standard of care in breast medical oncology, which contributes to a large, underserved population in which treatment plans are not well established or optimized for their comorbidities. Our findings offer a deeper understanding of immune checkpoint regulation in T2D and suggest new insights into treatment of diabetic breast cancer patients. Citation Format: Christina S. Ennis, Pablo LLevenes, Manohar Kolla, Naser Jafari, Anna C Belkina, Gerald V. Denis. Pathway analysis of immune checkpoint gene regulation as altered in Type 2 diabetes: Implications for breast cancer patients treated with checkpoint inhibitors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-12.

BRD4 in physiology and pathology: ‘‘BET’’ on its partners

Bromodomain-containing 4 (BRD4), a member of Bromo and Extra-Terminal (BET) family, recognizes acetylated histones and is of importance in transcription, replication, and DNA repair. It also binds non-histone proteins, DNA and RNA, contributing to development, tissue growth, and various physiological processes. Additionally, BRD4 hasbeenimplicated in driving diverse diseases, ranging from cancer, viral infection, inflammation to neurologicaldisorders. Inhibiting its functions with BET inhibitors (BETis) suppresses the progression of several types of cancer, creating an impetus for translating these chemicals to the clinic. The diverse roles of BRD4 are largely dependent on its interaction partners in different contexts. In this review we discuss the molecular mechanisms of BRD4 with its interacting partners in physiology and pathology. Current development of BETis is also summarized. Further understanding the functions of BRD4 and its partners will facilitate resolving the liabilities of present BETis and accelerate their clinical translation.

JQ1 as a BRD4 Inhibitor Blocks Inflammatory Pyroptosis-Related Acute Colon Injury Induced by LPS.

Endotoxemia is a severe inflammation response induced by infection especially bacterial endotoxin translocation, which severely increases mortality in combination with acute colon injury. Bromodomain-containing protein 4 (BRD4) is an important Bromo and Extra-Terminal (BET) protein to participate in inflammatory responses. However, it is still unknown about the specific connection between BRD4 and inflammation-related pyroptosis in endotoxemia colon. Here, through evaluating the mucous morphology and the expression of tight junction proteins such as occludin and ZO1, we found the upregulation of BRD4 in damaged colon with poor tight junction in an endotoxemia mouse model induced by lipopolysaccharides (LPS). Firstly, the BRD4 inhibitor JQ1 was used to effectively protect colon tight junction in endotoxemia. As detected, high levels of pro-inflammation cytokines IL6, IL1β and IL18 in endotoxemia colon were reversed by JQ1 pretreatment. In addition, JQ1 injection reduced endotoxemia-induced elevation of the phosphorylated NF κB and NLRP3/ASC/caspase 1 inflammasome complex in colon injury. Furthermore, activated pyroptosis markers gasdermins in endotoxemia colon were also blocked by JQ1 pretreatment. Together, our data indicate that BRD4 plays a critical role in regulating pyroptosis-related colon injury induced by LPS, and JQ1 as a BRD4 inhibitors can effectively protect colon from endotoxemia-induced inflammation injury.

Abstract 1751: BPI-23314: A novel orally bioavailable small molecular BET inhibitor with target protein degradation activity

Abstract The Bromo- and Extra-Terminal (BET) proteins, BRD2, BRD3, and especially BRD4, play important roles in transcriptional regulation of many oncogenes like MYC or BCL2, thus makes it an attractive drug target in multiple pathological settings, particularly in cancer. Earlier BET inhibitors, while effectively bind and inhibit the activity of BRD4, adversely induce compensatory accumulation of the target protein, that may account for their limited preclinical and clinical efficacy. Herein, we report BPI-23314, a potent, selective, and orally available small molecular BET bromodomain inhibitor, which possesses potent target inhibition activity as well as target protein degradation property. In vitro, BPI-23314 induced dose-dependent growth inhibition in a wide range of cancer cell lines, including those from AML, MM, DLBCL, NSCLC, TNBC, and CRPC, etc. Quantitative RT-PCR revealed that BPI-23314 markedly affected the transcription of genes encoding MYC, BCL2, and HEXIM1, thus induced G1 cell cycle arrest and apoptosis in MV-4-11 AML cell line. In vivo, oral administration of BPI-23314 led to tumor growth suppression or regression in multiple xenograft models of AML and MM. Transcriptional modulation of MYC, BCL2 and HEXIM1 were confirmed in BPI-23314-treated xenografts and in normal monkey, supporting their potential as pharmacodynamic biomarkers in clinical trials. Unlike previously described BET inhibitors that induced BRD4 accumulation, BPI-23314 serendipitously decreased BRD4 protein in MV-4-11 cells and in tumors from MV-4-11 xenografts. The feature of target-protein degradation differentiates BPI-23314 from the other BET inhibitors, renders it good in vivo efficacy, and suggests a lower risk of drug resistance in future clinical development. In conclusion, our study establishes BPI-23314 as a novel small molecular BET inhibitor with target BRD4 protein degradation property. BPI-23314 has obtained IND approval from Chinese regulatory authority and is planned to enter Phase 1 clinical development in hematologic malignancies in early 2020. Citation Format: Jing Guo, Yan Xu, Dan Yan, Bo Yu, Weiwei Yu, Haibo Chen, Xiaofeng Xu, Xiangyong Liu, Hong Lan, Lieming Ding, Jiabing Wang. BPI-23314: A novel orally bioavailable small molecular BET inhibitor with target protein degradation activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1751.

Effects of epigenetic pathway inhibitors on corticotroph tumour AtT20 cells.

Medical treatments for corticotrophinomas are limited, and we therefore investigated the effects of epigenetic modulators, a new class of anti-tumour drugs, on the murine adrenocorticotropic hormone (ACTH)-secreting corticotrophinoma cell line AtT20. We found that AtT20 cells express members of the bromo and extra-terminal (BET) protein family, which bind acetylated histones, and therefore, studied the anti-proliferative and pro-apoptotic effects of two BET inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, using CellTiter Blue and Caspase Glo assays, respectively. JQ1 and PFI-1 significantly decreased proliferation by 95% (P < 0.0005) and 43% (P < 0.0005), respectively, but only JQ1 significantly increased apoptosis by >50-fold (P < 0.0005), when compared to untreated control cells. The anti-proliferative effects of JQ1 and PFI-1 remained for 96 h after removal of the respective compound. JQ1, but not PFI-1, affected the cell cycle, as assessed by propidium iodide staining and flow cytometry, and resulted in a higher number of AtT20 cells in the sub G1 phase. RNA-sequence analysis, which was confirmed by qRT-PCR and Western blot analyses, revealed that JQ1 treatment significantly altered expression of genes involved in apoptosis, such as NFκB, and the somatostatin receptor 2 (SSTR2) anti-proliferative signalling pathway, including SSTR2. JQ1 treatment also significantly reduced transcription and protein expression of the ACTH precursor pro-opiomelanocortin (POMC) and ACTH secretion by AtT20 cells. Thus, JQ1 treatment has anti-proliferative and pro-apoptotic effects on AtT20 cells and reduces ACTH secretion, thereby indicating that BET inhibition may provide a novel approach for treatment of corticotrophinomas.

Stereoselective synthesis of allele-specific BET inhibitors.

Developing stereoselective synthetic routes that are efficient and cost-effective allows easy access to biologically active molecules. Our previous syntheses of allele-selective bumped inhibitors of the Bromo and Extra-Terminal (BET) domain proteins, Brd2, Brd3, Brd4 and BrdT, required a wasteful, late-stage alkylation step and expensive chiral separation. To circumvent these limitations, we developed a route based on stereocontrolled alkylation of an N-Pf protected aspartic acid derivative that was used in a divergent, racemisation-free protocol to yield structurally diverse and enantiopure triazolodiazepines. With this approach, we synthesized bumped thienodiazepine-based BET inhibitor, ET-JQ1-OMe, in five steps and 99% ee without the need for chiral chromatography. Exquisite selectivity of ET-JQ1-OMe for Leu-Ala and Leu-Val mutants over wild-type bromodomain was established by isothermal titration calorimetry and X-ray crystallography. Our new approach provides unambiguous chemical evidence for the absolute stereochemistry of the active, allele-specific BET inhibitors and a viable route that will open wider access to this compound class.

Systematic bromodomain protein screens identify homologous recombination and R-loop suppression pathways involved in genome integrity.

Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.

Oncogenic Cooperation between EGR1 and BRD4 in Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) grows rapidly and represents 30% to 40% of newly diagnosed lymphomas, and comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). ABC DLBCL is more aggressive with a current cure rate of only ~40%. The B cell antigen receptor (BCR) signaling pathway is known for its critical role in the survival and proliferation of ABC DLBCL cells. Our recent studies revealed that IL6- and IL10-mediated JAK1/STAT3 activation enhances the survival and proliferation of ABC DLBCL cells. Furthermore, we discovered that EGR1, a transcription factor known to be a downstream effector of BCR signaling, is also a downstream target of JAK1 signaling in DLBCL. While EGR1 has been characterized as a tumor suppressor in leukemia, the role of EGR1 in human lymphoma remains unclear. Initial immunohistochemical analysis of 102 DLBCL cases revealed that not only is the level of EGR1 elevated in DLBCL as compared to normal human tonsils, lymph nodes and mantle cell lymphomas, but its level is higher in ABC DLBCL (non-GCB) than GCB DLBCL, indicating functional significance of EGR1 in DLBCL. In addition, we found that 7 out of 8 ABC DLBCL cell lines are sensitive to EGR1 knockdown, whereas only half of 8 GCB DLBCL cell lines are affected. Our genomics studies discovered dual functions of EGR1 in regulating three signaling pathways. EGR1 mediates transcriptional activation associated with two hallmark oncogenic pathways, JAK1/STAT3 and BCR signaling. Our EGR1 ChIP-seq results revealed a large overlap of EGR1 genomic occupancy with that of BRD4, a highly expressed bromo and extraterminal (BET) protein in DLBCL known to induce MYC expression and activation of MYC target genes. This agrees with the RNA-seq results showing that multiple oncogenic pathways including MYC are activated by EGR1 in ABC DLBCL. The synergistic inhibition of cell growth was observed between EGR1 shRNA and JQ1, an inhibitor of BRD4. Surprisingly, the RNA-seq data and pathway analyses also revealed EGR1-mediated suppression of the type I interferon pathway, which otherwise causes cancer cell death. The majority of the interferon pathway genes are up-regulated upon EGR1 knockdown. EGR1-modulated suppression of the interferon pathway appears to be functionally significant as EGR1 knockdown by shRNA synergizes with the type I interferon inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro and in a xenograft mouse model. EGR1 is known to bind the promoter of p300 and activate p300 expression. P300 catalyzes H3K27 acetylation (H3K27ac), an active histone mark that recruits BRD4 for transcription of MYC and MYC pathway genes, among others. Indeed, immunoblot analysis revealed that EGR1 knockdown reduces H3K27ac expression in ABC DLBCL cells, leading to reduced expression of MYC and MYC target genes based on H3K27ac ChIP qPCR analysis. The results provide a molecular basis for oncogenic cooperation between EGR1 and BRD4 in ABC DLBCL. In summary, our study demonstrates that EGR1 is an oncogenic driver orchestrating multiple important signaling pathways. Notably, EGR1 creates therapeutic vulnerability in ABC DLBCL because both EGR1-mediated transcriptional activation and repression provide growth advantage to the cancer cells. Disclosures No relevant conflicts of interest to declare.

BRD4 suppression alleviates cerebral ischemia-induced brain injury by blocking glial activation via the inhibition of inflammatory response and pyroptosis

Ischemic stroke is a major cause of death and disability worldwide. Hyperneuroinflammation significantly contributes to ischemic stroke. Bromodomain-containing protein 4 (BRD4) is a member of the Bromo and Extra-Terminal (BET) family, and promotes inflammatory response in various types of tissue and cells. Thereby, we examined the contribution of BRD4 after cerebral ischemic/reperfusion (I/R) injury in a mouse middle cerebral artery occlusion (MCAO) model. Here, we showed that BRD4 expression was correlated with glial activation and cerebral I/R injury after MCAO in mice. Intriguingly, we found that BRD4 inhibition using its selective inhibitor, JQ1, showed a protective role in cerebral I/R injury in mice. Suppressing BRD4 by JQ1 reduced the infarction volume, brain water contents and neurological deficit score of MCAO mice. In addition, MCAO-induced glial activation was also blunted by JQ1, as proved by the significantly reduced expression of glial fibrillary acidic protein (GFAP) and Iba-1. Consistently, JQ1 treatment decreased the expression of pro-inflammatory factors by blocking nuclear factor kappa B (NF-κB) signaling. Furthermore, inflammasome activation and pyroptosis found in MCAO mice were markedly attenuated by JQ1, which were through suppressing the expression of NLRP3 (nucleotide-binding domain, leucine-rich repeat containing protein 3), ASC (apoptosis-associated speck-like protein containing a CARD), Caspase-1 and GSDMD (gasdermin D). The protective effects of BRD4 inhibition on cerebral ischemia-induced brain injury were verified in astrocytes and microglial cells via the inhibition of inflammation and pyroptosis. In summary, blocking BRD4 expression might serve as a potential therapeutic strategy for stroke therapy.

Intrinsic Resistance to MEK Inhibition through BET Protein-Mediated Kinome Reprogramming in NF1-Deficient Ovarian Cancer.

Mutation or deletion of Neurofibromin 1 (NF1), an inhibitor of RAS signaling, frequently occurs in epithelial ovarian cancer (EOC), supporting therapies that target downstream RAS effectors, such as the RAF-MEK-ERK pathway. However, no comprehensive studies have been carried out testing the efficacy of MEK inhibition in NF1-deficient EOC. Here, we performed a detailed characterization of MEK inhibition in NF1-deficient EOC cell lines using kinome profiling and RNA sequencing. Our studies showed MEK inhibitors (MEKi) were ineffective at providing durable growth inhibition in NF1-deficient cells due to kinome reprogramming. MEKi-mediated destabilization of FOSL1 resulted in induced expression of receptor tyrosine kinases (RTK) and their downstream RAF and PI3K signaling, thus overcoming MEKi therapy. MEKi synthetic enhancement screens identified BRD2 and BRD4 as integral mediators of the MEKi-induced RTK signatures. Inhibition of bromo and extra terminal (BET) proteins using BET bromodomain inhibitors blocked MEKi-induced RTK reprogramming, indicating that BRD2 and BRD4 represent promising therapeutic targets in combination with MEKi to block resistance due to kinome reprogramming in NF1-deficient EOC. IMPLICATIONS: Our findings suggest MEK inhibitors will likely not be effective as single-agent therapies in NF1-deficient EOC due to kinome reprogramming. Cotargeting BET proteins in combination with MEKis to block reprogramming at the transcriptional level may provide an epigenetic strategy to overcome MEKi resistance in NF1-deficient EOC.

Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains

SummaryTargeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.

Modeling combination therapy for breast cancer with BET and immune checkpoint inhibitors

CTLA-4 is an immune checkpoint expressed on active anticancer T cells. When it combines with its ligand B7 on dendritic cells, it inhibits the activity of the T cells. The Bromo- and Extra-Terminal (BET) protein family includes proteins that regulate the expression of key oncogenes and antiapoptotic proteins. BET inhibitor (BETi) has been shown to reduce the expression of MYC by suppressing its transcription factors and to down-regulate the hypoxic transcriptome response to VEGF-A. This paper develops a mathematical model of the treatment of cancer by combination therapy of BETi and CTLA-4 inhibitor. The model shows that the two drugs are positively correlated in the sense that the tumor volume decreases as the dose of each of the drugs is increased. The model also considers the effect of the combined therapy on levels of myeloid-derived suppressor cells (MDSCs) and the overexpression of TNF-α, which may predict gastrointestinal side effects of the combination.