BRD4 Research Articles

Ultra-Rare BRD9 Loss-of-Function Variants Limit the Antiviral Action of Interferon

The human type I interferon (IFN) system is central to innate immune defense, and is essential to protect individuals against severe viral disease. Consequently, genetic disruption of IFN signaling or effector mechanisms is extremely rare, as affected individuals typically suffer life-threatening infections at an early age. While loss-of-function (LOF) mutations in canonical JAK-STAT signaling genes (such as IFNAR2, TYK2, STAT1, STAT2 and IRF9) have previously been characterized, little is known about the consequences of mutations in other human factors required for IFN signaling. Here, we studied the impact of rare human genetic variants in the recently identified contributor to IFN-stimulated gene expression and antiviral activity, bromodomain-containing protein 9 (BRD9). Using a cell-based BRD9 knock-out and reconstitution model system, we functionally assessed 12 rare human BRD9 missense variants predicted to impair protein function, as well as 3 ultra-rare human BRD9 LOF variants that lead to truncated versions of BRD9. As compared to wild-type BRD9, none of the 12 BRD9 missense variants affected the ability of exogenous IFN to limit virus replication. In contrast, all 3 truncated BRD9 LOF variants failed to allow exogenous IFN to function efficiently, as evidenced by exacerbated replication of an IFN-sensitive virus and diminished IFN-stimulated gene expression. Thus, while no homozygous BRD9 LOF carriers have yet been identified, our results predict that such extremely rare individuals would exhibit a compromised ability to mount a fully protective IFN-mediated antiviral response. Genetic variation in BRD9 could be considered in future studies to understand the infection susceptibility of some individuals.

Transforming Stimulated Clone 22 (TSC-22) Interacts Directly with Bromodomain-Containing Protein 7 (BRD7) to Enhance the Inhibition of Extracellular Signal-Regulate Kinase (ERK) Pathway in Ovarian Cancer.

Bromodomain-containing protein 7 (BRD7) participates in many cellular processes and embryo development. BRD7 is down-regulated in various cancers and evidence of its tumor suppressor function has been accumulating. Here, we identified transforming stimulated clone 22 (TSC-22) as a novel BRD7 interacting protein and show its novel function as a positive regulator of BRD7. We found that TSC-22 expression potentiated the inactivation of the extracellular signal-regulate kinase (ERK) pathway by BRD7. Our data establishes TSC-22 as a modulator of BRD7 and unravels the molecular mechanisms that drive the synergistic tumor-suppressing effects of TSC-22 and BRD7. Our findings may open new avenues for developing novel molecular therapies for tumors exhibiting down-regulated BRD7 and/or TSC-22.

BRPF1-KAT6A/KAT6B Complex: Molecular Structure, Biological Function and Human Disease.

Simple SummaryBRPF1 (also named as BR140) was identified 28 years ago, and it was not until the past 5 years that its mutations in humans caught increasing attention. Those patients with BRPF1 mutations often display intellectual disability or suffer from leukemia or medulloblastoma. BRPF1 is an activator and a scaffold protein of a multiunit complex, with other members being KAT6A/KAT6B, ING5 or ING4 and MEAF6. This review summarizes the molecular structure, biological function and human diseases associated with the BRPF1-KAT6A/KAT6B complex and summarizes the development of inhibitors for targeting specific domains of BRPF1.The bromodomain and PHD finger–containing protein1 (BRPF1) is a member of family IV of the bromodomain-containing proteins that participate in the post-translational modification of histones. It functions in the form of a tetrameric complex with a monocytic leukemia zinc finger protein (MOZ or KAT6A), MOZ-related factor (MORF or KAT6B) or HAT bound to ORC1 (HBO1 or KAT7) and two small non-catalytic proteins, the inhibitor of growth 5 (ING5) or the paralog ING4 and MYST/Esa1-associated factor 6 (MEAF6). Mounting studies have demonstrated that all the four core subunits play crucial roles in different biological processes across diverse species, such as embryonic development, forebrain development, skeletal patterning and hematopoiesis. BRPF1, KAT6A and KAT6B mutations were identified as the cause of neurodevelopmental disorders, leukemia, medulloblastoma and other types of cancer, with germline mutations associated with neurodevelopmental disorders displaying intellectual disability, and somatic variants associated with leukemia, medulloblastoma and other cancers. In this paper, we depict the molecular structures and biological functions of the BRPF1-KAT6A/KAT6B complex, summarize the variants of the complex related to neurodevelopmental disorders and cancers and discuss future research directions and therapeutic potentials.

Versatile Nano-PROTAC-Induced Epigenetic Reader Degradation for Efficient Lung Cancer Therapy.

Recent evidence has indicated that overexpression of the epigenetic reader bromodomain‐containing protein 4 (BRD4) contributes to a poor prognosis of lung cancers, and the suppression of its expression promotes cell apoptosis and leads to tumor shrinkage. Proteolysis targeting chimera (PROTAC) has recently emerged as a promising therapeutic strategy with the capability to precisely degrade targeted proteins. Herein, a novel style of versatile nano‐PROTAC (CREATE ( CR V‐LLC m e mbrane/DS‐PLG A /dB ET 6)) is developed, which is constructed by using a pH/GSH (glutathione)‐responsive polymer (disulfide bond‐linked poly(lactic‐co‐glycolic acid), DS‐PLGA) to load BRD4‐targeted PROTAC (dBET6), followed by the camouflage with engineered lung cancer cell membranes with dual targeting capability. Notably, CREATE remarkably confers simultaneous targeting ability to lung cancer cells and tumor‐associated macrophages (TAMs). The pH/GSH‐responsive design improves the release of dBET6 payload from nanoparticles to induce pronounced apoptosis of both cells, which synergistically inhibits tumor growth in both subcutaneous and orthotopic tumor‐bearing mouse model. Furthermore, the efficient tumor inhibition is due to the direct elimination of lung cancer cells and TAMs, which remodels the tumor microenvironment. Taken together, the results elucidate the construction of a versatile nano‐PROTAC enables to eliminate both lung cancer cells and TAMs, which opens a new avenue for efficient lung cancer therapy via PROTAC.

Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140

BackgroundSP140 is a bromodomain-containing protein expressed predominantly in immune cells. Genetic polymorphisms and epigenetic modifications in the SP140 locus have been linked to Crohn’s disease (CD), suggesting a role in inflammation.ResultsWe report the development of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in macrophages. We show that SP140 is highly expressed in CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte-to-inflammatory macrophage differentiation and lipopolysaccharide (LPS)-induced inflammatory activation, while inducing the generation of CD206+ regulatory macrophages that were shown to associate with a therapeutic response to anti-TNF in CD patients. SP140 preferentially occupies transcriptional start sites in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduces SP140 chromatin binding and thereby expression of SP140-regulated genes. GSK761 inhibits the expression of cytokines, including TNF, by CD14+ macrophages isolated from CD intestinal mucosa.ConclusionsThis study identifies SP140 as a druggable epigenetic therapeutic target for CD.

Functional networks of the human bromodomain-containing proteins.

Background: Bromodomains are a structurally conserved epigenetic reader domain that bind to acetylated lysine residues in both histone and non-histone proteins. Bromodomain-containing proteins (BRD proteins) often function as scaffolding proteins in the assembly of multi-protein complexes to regulate diverse biological processes. BRD proteins have been classified based on biological and functional similarity, however the functions of many BRD proteins remains unknown. PPI network analysis is useful for revealing organizational roles, identifying functional clusters, and predicting function for BRD proteins. Results: We used available data to construct protein-protein interaction networks (PPINs) to study the properties of the human bromodomain protein family. The network properties of the BRD PPIN establishes that the BRD proteins serve as hub proteins that are enriched near the global center to form an inter-connected PPIN. We identified dense subgraphs formed by BRD proteins and find that different BRD proteins share topological similarity and functional associations. We explored the functional relationships through clustering and Hallmark pathway gene set enrichment analysis and identify potential biological roles for different BRD proteins. Conclusion: In our network analysis we confirmed that BRD proteins are conserved central nodes in the human PPI network and function as scaffolds to form distinctive functional clusters. Overall, this study provides detailed insight into the predictive functions of BRD proteins in the context of functional complexes and biological pathways.

BRD7 restrains TNF-α-induced proliferation and migration of airway smooth muscle cells by inhibiting notch signaling

Objective: Bromodomain-containing protein 7 (BRD7) is a key component of the switch/sucrose non-fermentable complex that participates in chromatin remodeling and transcriptional regulation. Although the emerging role of BRD7 in the pathophysiology of various diseases has been observed, its role in asthma remains unknown. Here, we assessed the function of BRD7 as a mediator of airway remodeling in asthma using an in vitro model. Methods: Airway smooth muscle cells (ASMCs) were challenged with tumor necrosis factor-α (TNF-α) to establish an in vitro airway remodeling model. Protein levels were examined using western blotting. Cell proliferation was measured using the cell counting kit-8 and 5-ethynyl-2′-deoxyuridine assays. Cell migration was assessed using a transwell migration assay. Results: Exposure to TNF-α dramatically decreased BRD7 levels in ASMCs. BRD7 remarkably decreased TNF-α-induced proliferation and migration of ASMCs. In contrast, ASMCs with BRD7 deficiency were more sensitive to TNF-α-induced pro-proliferative and pro-migratory effects. Mechanistically, BRD7 could repress the expression of Notch1 and block the Notch pathway in TNF-α-challenged cells. Notably, reactivation of Notch signaling substantially reversed the BRD7 overexpression-mediated effects, whereas restraining Notch signaling abolished BRD7-depletion-mediated effects on TNF-α-challenged cells. Conclusions: BRD7 inhibits the proliferation and migration of ASMCs elicited by TNF-α by downregulating the Notch pathway. This study indicates that BRD7 may exert a suppressive effect on airway remodeling during asthma.

NUT Carcinoma-An Underdiagnosed Malignancy.

NUT carcinoma (NC) is a rare and highly aggressive malignancy with a dismal prognosis and a median survival of 6–9 months only. Although very few cases of NC are reported each year, the true prevalence is estimated to be much higher, with NC potentially widely underdiagnosed due to the lack of awareness. NC primarily occurs in midline structures including thorax, head, and neck; however, other sites such as pancreas and kidney are also affected, albeit at lower frequencies. NC is characterized by a single translocation involving the NUTM1 (NUT midline carcinoma family member 1) gene and different partner genes. The resulting fusion proteins initiate tumorigenesis through a mechanism involving BET (bromo-domain and extra-terminal motif) proteins such as Bromodomain-containing protein 4 (BRD4) and inordinate acetylation of chromatin, leading to the dysregulation of growth and differentiation genes. While no clinical characteristics are specific for NC, some histologic features can be indicative; therefore, patients with these tumor characteristics should be routinely tested for NUTM1. The diagnosis of NC using immunohistochemistry with a highly specific antibody is straightforward. There are currently no standard-of-care treatment options for patients with NC. However, novel therapies specifically addressing the unique tumorigenic mechanism are under investigation, including BET inhibitors. This review aims to raise awareness of this underdiagnosed cancer entity and provide all patients the opportunity to be properly diagnosed and referred to a clinical study.

Hypothetical bromodomain-containing protein 5 is required for the growth of Toxoplasma gondii

Bromodomain (BRD) is a highly conserved structural module domain, found in various proteins, including chromatin-related proteins, nucleus acetyltransferases, and transcription-associated proteins. Toxoplasma gondii, a zoonotic protozoan, encodes at least 12 predicted BRD-containing proteins (BDPs). Here, we investigated the subcellular location and regulatory role of a hypothetical protein BDP that we named TgBDP5. The BRD of TgBDP5 did not contain the conserved Asn and Tyr residues required for acetyl-lysine recognition. TgBDP5 localized in the nucleus of the parasite and remained unchanged during parasite replication. Conditional ablation of TgBDP5 through an auxin-inducible degron-based knockdown strategy caused a growth defect in parasite replication. Depletion of TgBDP5 led to changes in the expression level of 179 genes, suggesting it as an important target for drugs acting against T. gondii.

The Functional Role and Regulatory Mechanism of Bromodomain-Containing Protein 9 in Human Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is the most common type of uterine sarcoma associated with poor prognosis, high rates of recurrence, and metastasis. There is currently limited information about uLMS molecular mechanisms of origin and development. Bromodomain (BRD)-containing proteins are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD protein dysfunction has been linked to many diseases including tumorigenesis. However, the role of BRD proteins in the pathogenesis of uLMS is unknown. Here, we show for the first time that BRD9 is aberrantly overexpressed in uLMS tissues compared to adjacent myometrium. BRD9 expression is also upregulated in uLMS cell lines compared to benign uterine fibroid and myometrium cell lines. Inhibition of BRD9 using the specific inhibitor (TP-472) suppressed uLMS cell proliferation via inducing apoptosis and cell cycle arrest. To further characterize the mechanistic basis for TP-472 inhibition of uLMS cell growth, we performed a comparative RNA-seq analysis of vehicle-treated and TP-472-treated uLMS cells (n = 4 each). Bioinformatics analysis revealed that TP-472 treatment distinctly altered the uLMS cell transcriptome. Gene set enrichment analysis identified critical pathways altered by BRD9 inhibition, including interferon-alpha response, KRAS signaling, MYC targets, TNF-a signaling via NFkB, and MTORC1 signaling. Parsimonious gene correlation network analysis identified nine enriched modules, including cell cycle and apoptosis modules. Moreover, the ENCODE Histone Modifications gene set and TargetScan microRNA analysis in Enrichr suggested that TP-472-induced BRD9 inhibition may alter the uLMS cell transcriptome by reprograming the oncogenic epigenome and inducing miRNA-mediated gene regulation. Therefore, BRD9 constitutes a specific vulnerability in malignant uLMS, and targeting non-BET BRD proteins in uLMS may provide a promising and novel strategy for treating patients with this aggressive uterine cancer.

BRD9 degraders as chemosensitizers in acute leukemia and multiple myeloma

Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.

Targeting sphingosine kinase 1/2 by a novel dual inhibitor SKI-349 suppresses non-small cell lung cancer cell growth

Sphingosine kinase 1 (SphK1) and sphingosine kinase (SphK2) are both important therapeutic targets of non-small cell lung cancer (NSCLC). SKI-349 is a novel, highly efficient and small molecular SphK1/2 dual inhibitor. Here in primary human NSCLC cells and immortalized cell lines, SKI-349 potently inhibited cell proliferation, cell cycle progression, migration and viability. The dual inhibitor induced mitochondrial depolarization and apoptosis activation in NSCLC cells, but it was non-cytotoxic to human lung epithelial cells. SKI-349 inhibited SphK activity and induced ceramide accumulation in primary NSCLC cells, without affecting SphK1/2 expression. SKI-349-induced NSCLC cell death was attenuated by sphingosine-1-phosphate and by the SphK activator K6PC-5, but was potentiated by the short-chain ceramide C6. Moreover, SKI-349 induced Akt-mTOR inactivation, JNK activation, and oxidative injury in primary NSCLC cells. In addition, SKI-349 decreased bromodomain-containing protein 4 (BRD4) expression and downregulated BRD4-dependent genes (Myc, cyclin D1 and Klf4) in primary NSCLC cells. At last, SKI-349 (10 mg/kg) administration inhibited NSCLC xenograft growth in nude mice. Akt-mTOR inhibition, JNK activation, oxidative injury and BRD4 downregulation were detected in SKI-349-treated NSCLC xenograft tissues. Taken together, targeting SphK1/2 by SKI-349 potently inhibits NSCLC cell growth in vitro and in vivo.

Targeted BRD4 protein degradation by dBET1 ameliorates acute ischemic brain injury and improves functional outcomes associated with reduced neuroinflammation and oxidative stress and preservation of blood–brain barrier integrity

Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) protein family, plays a crucial role in regulating inflammation and oxidative stress that are tightly related to stroke development and progression. Consequently, BRD4 blockade has attracted increasing interest for associated neurological diseases, including stroke. dBET1 is a novel and effective BRD4 degrader through the proteolysis-targeting chimera (PROTAC) strategy. We hypothesized that dBET1 protects against brain damage and neurological deficits in a transient focal ischemic stroke mouse model by reducing inflammation and oxidative stress and preserving the blood–brain barrier (BBB) integrity. Post-ischemic dBET1 treatment starting 4 h after stroke onset significantly ameliorated severe neurological deficits and reduced infarct volume 48 h after stroke. dBET1 markedly reduced inflammation and oxidative stress after stroke, indicated by multiple pro-inflammatory cytokines and chemokines including IL-1β, IL-6, TNF-α, CCL2, CXCL1 and CXCL10, and oxidative damage markers 4-hydroxynonenal (4-HNE) and gp91phox and antioxidative proteins SOD2 and GPx1. Meanwhile, stroke-induced BBB disruption, increased MMP-9 levels, neutrophil infiltration, and increased ICAM-1 were significantly attenuated by dBET1 treatment. Post-ischemic dBET1 administration also attenuated ischemia-induced reactive gliosis in microglia and astrocytes. Overall, these findings demonstrate that BRD4 degradation by dBET1 improves acute stroke outcomes, which is associated with reduced neuroinflammation and oxidative stress and preservation of BBB integrity. This study identifies a novel role of BET proteins in the mechanisms resulting in ischemic brain damage, which can be leveraged to develop novel therapies.

Alternative Mechanisms for DNA Engagement by BET Bromodomain-Containing Proteins.

Epigenetic reader domains regulate chromatin structure and modulate gene expression through the recognition of post-translational modifications on histones. Recently, reader domains have also been found to harbor double-stranded (ds) DNA-binding activity, which is as functionally critical as histone association. Here, we explore the dsDNA recognition of the N-terminal bromodomain of the bromodomain and extra-terminal (BET) protein, BRD4. Using protein-observed 19F NMR, 1H-15N HSQC NMR, electrophoretic mobility shift assays (EMSA), and competitive-inhibition assays, we establish the binding surface of dsDNA and find it to be largely overlapping with the acetylated histone (KAc)-binding site. Rather than engaging in electrostatic contacts, we find dsDNA to interact competitively within the KAc-binding pocket. These interactions are distinct from the highly homologous BET bromodomain, BRDT. Nine additional bromodomains have also been characterized for interacting with dsDNA, including tandem BET bromodomains. Together, these studies help establish a binding model for dsDNA interactions with BRD4 bromodomains and elucidate the chromatin-recognition mechanisms of the BRD4 protein for regulating gene expression.

Expansion microscopy allows high resolution single cell analysis of epigenetic readers.

Interactions between epigenetic readers and histone modifications play a pivotal role in gene expression regulation and aberrations can enact etiopathogenic roles in both developmental and acquired disorders like cancer. Typically, epigenetic interactions are studied by mass spectrometry or chromatin immunoprecipitation sequencing. However, in these methods, spatial information is completely lost. Here, we devise an expansion microscopy based method, termed Expansion Microscopy for Epigenetics or ExEpi, to preserve spatial information and improve resolution. We calculated relative co-localization ratios for two epigenetic readers, lens epithelium derived growth factor (LEDGF) and bromodomain containing protein 4 (BRD4), with marks for heterochromatin (H3K9me3 and H3K27me3) and euchromatin (H3K36me2, H3K36me3 and H3K9/14ac). ExEpi confirmed their preferred epigenetic interactions, showing co-localization for LEDGF with H3K36me3/me2 and for BRD4 with H3K9/14ac. Moreover addition of JQ1, a known BET-inhibitor, abolished BRD4 interaction with H3K9/14ac with an IC50 of 137 nM, indicating ExEpi could serve as a platform for epigenetic drug discovery. Since ExEpi retains spatial information, the nuclear localization of marks and readers was determined, which is one of the main advantages of ExEpi. The heterochromatin mark, H3K9me3, is located in the nuclear rim whereas LEDGF co-localization with H3K36me3 and BRD4 co-localization with H3K9/14ac occur further inside the nucleus.

Abstract 1093: BRD9 inhibition overcomes epithelial to mesenchymal transition (EMT)-associated tyrosine kinase inhibitor (TKI) tolerance in epidermal growth factor receptor (EGFR) mutant lung cancer

Abstract Advanced lung cancer patients that present with activating mutations in the epidermal growth factor receptor (EGFR) are treated with EGFR tyrosine kinase inhibitors (EGFR TKIs). While initially effective, all patients eventually develop therapeutic resistance and experience disease progression. Strategies to prevent EGFR TKI resistance are needed to improve patient outcomes. We chronically exposed H1975 cells (EGFR-L858R/T790M) to EGFR TKI osimertinib to study emergence of resistance. H1975 cells that were expanded under drug treatment (designated H1975OR) acquired an EMT phenotype and were re-sensitized to EGFR TKI upon prolonged drug withdrawal. These features led us to classify H1975OR as a model of drug tolerance rather than a model of stable drug resistance. Bulk RNA-sequencing revealed significant dysregulation of chromatin modifying genes in H1975OR. Bromodomain containing protein 9 (BRD9) was among the set of significantly upregulated chromatin regulators and was selected for further investigation as a mediator of drug tolerance. Although BRD9 is known to control stemness and EMT, its role in promoting EMT-associated TKI resistance is unknown. To test the contribution of BRD9 to EGFR TKI tolerance, pharmacological inhibition of BRD9 by the selective inhibitor, I-BRD9 significantly increased sensitivity to TKI in models with EMT phenotypes (IC50 reduced 3-4 fold). In contrast, I-BRD9 did not affect TKI sensitivity in two models where EGFR TKI resistance was genetically fixed. To gain mechanistic insights, H1975OR cells were treated with osimertinib +/-I-BRD9 and subjected to RNA-sequencing. Combination of osimertinib with I-BRD9 resulted in a significant decrease in EMT-related genes, including MMP9, Zeb2, PDGFRb and IL6. Further, use of I-BRD9 in these models diminished the mesenchymal phenotype, as measured in cell invasion assays. Genetic knockdown of BRD9 via shRNA phenocopied effects of I-BRD9 treatment, supporting BRD9 as the therapeutic target of I-BRD9 in our cell line models. To further establish a role for BRD9 in the emergence of drug tolerant cells, we exposed treatment naïve H1975 cells to EGFR TKI ± I-BRD9. I-BRD9 significantly decreased the size of the EGFR TKI tolerant population. In time course studies, I-BRD9 also delayed onset of TKI resistance. In conclusion, our data identifies BRD9 as a novel mediator of EMT-associated EGFR-TKI tolerance in EGFR-mutant lung cancer. Our data further implicates BRD9 inhibition as a novel strategy to delay the emergence of drug tolerant cells that eventually give rise to stable drug resistance. This work was supported by the Roswell Park Alliance Foundation and National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park Comprehensive Cancer Center’s Genomics and Bioinformatics Shared Resources. Citation Format: Hannah Calkins, Tatiana Shaurova, David W. Goodrich, Mukund Seshadri, Candace S. Johnson, Pamela A. Hershberger. BRD9 inhibition overcomes epithelial to mesenchymal transition (EMT)-associated tyrosine kinase inhibitor (TKI) tolerance in epidermal growth factor receptor (EGFR) mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1093.

Abstract 421: PLX-3618, a potent, selective monovalent BRD4 degrader demonstrates activity in models of prostate cancer

Abstract Prostate cancer is the second most common cause of cancer related deaths in men in the United States. Pathogenesis is driven by the androgen receptor (AR), which has led to front-line treatment modalities that are based on androgen deprivation therapy (ADT). About 10-20% of all prostate cancers evolve to resist ADT and are classified as castration-resistant prostate cancer (CRPC) indicating the continued need for new treatment options. Bromodomain-containing protein 4 (BRD4) is an acetylated-chromatin associating protein that is involved in transcriptional elongation, mRNA splicing, epigenetic bookmarking, and super-enhancer activity. The BRD4 protein has been shown to both bind and colocalize with AR at androgen response elements (AREs) on chromatin. Furthermore, elevated BRD4 expression is prognostic of increased prostate specific antigen (PSA) levels following radical prostatectomy and is correlated with higher Gleason scores and poor overall survival. The intertwined activity of BRD4 with multiple essential driver mechanisms of prostate cancer suggests it may be a key target for developing novel therapeutics. Using our ultra-high throughput cell-based screening platform, which directly measures degradation of pathogenic proteins upon exposure to diverse chemical libraries, we identified a series of novel monovalent BRD4 degraders that was optimized to produce our lead compound PLX-3618. A cancer cell panel screen for antiproliferative effects of PLX-3618 indicated enhanced sensitivity in subsets of prostate cancer lines. PLX-3618 elicited selective, rapid, and deep degradation of BRD4 protein in prostate cancer cell models, without degrading the closely related BRD2 and BRD3 proteins. Addition of either proteosome or neddylation inhibitors blocked BRD4 degradation indicating a ubiquitin-proteosome system mediated clearance mechanism. Degradation of BRD4 led to sustained multimodal inhibition of the AR pathway and disruption of key oncogene enhancer networks. When compared to pan-BET inhibitors, treatment of select prostate cancer cell lines with PLX-3618 resulted in increased levels of the tumor suppressors p53 and p21, an aberrant DNA damage response, and substantially amplified apoptosis. Finally, in in vivo preclinical models of prostate cancer, PLX-3618 showed far superior efficacy over a pan-BET inhibitor. Taken together, the selective degradation of BRD4 via the potent monovalent degrader PLX-3618 represents a novel strategy in treating prostate cancer. Citation Format: Kenneth Steadman, Gregory S. Parker, Geoffray Leriche, Sarah Fish, Julia Toth, Mary E. Spalding, Elizabeth Daniele, Aleksandar Jamborcic, Xiaoming Li, E Adam Kallel, Farhana Barmare, Kenneth Chng, Erika Green, Michael Hocker, Elliot Imler, Yi Zhang, Peggy A. Thompson, Simon Bailey. PLX-3618, a potent, selective monovalent BRD4 degrader demonstrates activity in models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 421.

Abstract 2136: Prediction of small molecule-protein binding events for BRD4 and EGFR inhibitors using HR-LiP, a novel structural proteomics approach

Abstract Background: Beyond phenotypic efficacy and safety categorization, high resolution profiling of drug-protein interactions and binding mechanisms remains a major hurdle during lead selection and optimization. A key milestone in structure-based drug design is compound binding site identification and characterization. Structure-activity relationship (SAR) studies utilize techniques such as nuclear magnetic resonance (NMR), x-ray crystallography (X-ray), cryo-electron microscopy (cryo-EM) and the mass spectrometry-based hydrogen-deuterium exchange (HDX) to address these hurdles but they are labor, time and cost intensive. Further, SAR studies are often complicated by protein size (i.e. large proteins) and location (i.e. membrane proteins), which can lead to protocol adaptations (e.g. recombinant protein usage and/or protein truncation) that can introduce artifacts. Using limited proteolysis (LiP) coupled to next-generation mass spectrometry we have developed a high-throughput, high-resolution approach (HR-LiP) that utilizes peptide-level resolution to characterize drug-protein interactions including for proteins that hindered by the previously mentioned limitations. Methods: To boost protein abundance in their native environment, proteins of interest were overexpressed in HEK293 cells using a simple plasmid construct. Cell lysates were incubated with the compounds of interest at increasing concentrations. Samples were then subjected to a limited digest using proteinase K and further processed for data independent acquisition (DIA)-MS analysis using trypsin. Data was analyzed using a directDIA workflow in Spectronaut. Results: Two well-characterized drug target proteins, bromodomain-containing protein 4 (BRD4) and epidermal growth factor receptor (EGFR), were selected for analysis. Using HR-LiP we identify the binding site of the BRD4 inhibitor JQ1 in the full-length protein, which is typically too large to be used directly in with conventional methods. Further, we map the intracellular binding location of both gefitinib and afatinib, two inhibitors of the membrane protein EGFR. Our data for both proteins are in good accordance with orthogonal data obtained by HDX-MS, NMR and X-ray studies. Conclusions: We demonstrate that HR-LiP can be used to dissect small molecule-protein binding events, including compound binding site prediction for protein targets classically considered to be difficult. Given its biological power, broad applicability and ease of implementation, we envision the use of HR-LiP as a routine approach for target validation and lead optimization in small molecule drug discovery pipelines. Citation Format: Nigel Beaton, Jagat Adhikari, Roland Bruderer, Ron Tomlinson, Yuehan Feng, Ivan Cornella-Taracido, Lukas Reiter. Prediction of small molecule-protein binding events for BRD4 and EGFR inhibitors using HR-LiP, a novel structural proteomics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2136.

Abstract 3271: BRD4 inhibition enhances the acalabrutinib tumor response by modulating PAX5 and interferon signaling in ABC-DLBCL

Abstract AZD5153 is a bivalent bromodomain and extraterminal (BET) inhibitor that simultaneously engages the two bromodomains of bromodomain-containing protein 4 (BRD4). Here, we initially tested AZD5153 in combination with acalabrutinib, a Bruton tyrosine kinase (BTK) inhibitor, in a panel of diffuse large B-cell lymphoma (DLBCL) cell lines. The ABC type DLBCL cells responded to the AZD5153 and acalabrutinib combination at lower drug concentrations compared to other DLBCL cell lines. Many of these lower concentrations were below the clinical equivalent dose for each drug. Similar results were achieved in DLBCL patient-derived xenografts, with most ABC-DLBCL tumors having more growth inhibition than the other B-cell lymphoma tumors after combination dosing at fixed concentrations. To delineate the mechanism of ABC-subtype preference for combination therapy, we performed RNA sequencing on ABC-DLBCL tumor samples treated with AZD5153, acalabrutinib, and the combination of AZD5153 and acalabrutinib. Pathway meta-analyses identified significant alterations of PAX5 signaling and interferon pathways in combination-treated tumors compared to monotherapy-treated samples. PAX5 is a key gene for B-cell activation and is associated with lymphoma pathogenesis. Protein levels of PAX5 and corresponding downstream transcription targets were also decreased by the combination of AZD5153 and acalabrutinib in ABC-DLBCL cell lines. In addition, STAT proteins related to PAX5 activation, the alternative RelB-NFκB pathway, and toll-like receptors in the interferon type I pathway were also modulated at the protein level by this drug combination in ABC-DLBCL cell lines. We hypothesized the AZD5153 and acalabrutinib combination induces changes to interferon signaling that impacts PAX5 activation. This mechanism is supported by evidence of AZD5153 and acalabrutinib combination-induced downregulation of interferons alpha and beta, as well as interferon gamma and IP-10 in vivo. In summary, our results demonstrate AZD5153 enhances the efficacy of acalabrutinib, and our in vitro data suggest adding AZD5153 to acalabrutinib concentrations much lower than the clinical equivalent dose results in a similar biomarker response. We show that this drug combination attenuates interferon signaling, which supports the mechanism of AZD5153 and acalabrutinib inhibiting PAX5 signaling in ABC-DLBCL. Citation Format: Derek B. Oien, Samanta Sharma, Ye Xu, Maureen Hattersley, Michelle DuPont, Kathleen Burke, Steven W. Criscione, Laura Prickett, Jingwen Zhang, Ho Man Chan, Lisa Drew, Yi Yao. BRD4 inhibition enhances the acalabrutinib tumor response by modulating PAX5 and interferon signaling in ABC-DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3271.

Abstract 5713: Alternative splicing of the chromatin modifier gene BAZ1A in colorectal cancer

Abstract Bromodomain Adjacent to Zinc finger Domain 1A (BAZ1A) is a non-catalytic subunit of the ATP-dependent chromatin assembly factor that regulates key cellular processes such as DNA replication, chromatin assembly, DNA repair, and transcriptional gene regulation (1). Defective DNA repair is a hallmark of cancer etiology, and BAZ1A is one of the deregulated proteins in colorectal cancer. The BAZ1A transcript exhibits full-length and truncated forms, generated via alternative splicing of exon-13. We observed that the histone deacetylase inhibitor sulforaphane (SFN) modulated alternative splicing of BAZ1A in human colon cancer cells by increasing the relative expression of the short vs. the long transcript. Cell Cycle and Apoptosis Regulator 2 (CCAR2) and Zinc finger protein 326 (ZIRD) are components of the DBIRD complex that integrates alternative splicing and the rate of transcript elongation (2). Knockdown of DBIRD members, including ZIRD and splicing factor hnRNPA1, downregulated expression of BAZ1A long form, whereas the BAZ1A short form was unaltered. Previous studies in SFN-treated colon cancer cells revealed that the BAZ1A bromodomain recognized a novel K97 acetylation site in CCAR2 (3). Co-immunoprecipitation analyses revealed that CCAR2 and BAZ1A interacted with each other in the presence and absence of SFN. On investigating the involvement of CCAR2 in BAZ1A alternative splicing, SFN treatment increased the expression of both BAZ1A long and short forms in CCAR2-/- HCT116 colon cancer cells. Although the total expression of BAZ1A was influenced by CCAR2 loss, alternative splicing of BAZ1A was independent of DBIRD complex-mediated regulation. Pursuing the role of p53 and p21 in BAZ1A alternative splicing, SFN treatment increased the expression of BAZ1A short form in p21-/- HCT116 cells, whereas this was impaired in p53-/- HCT116 cells. Overall, this investigation provided mechanistic insights into the regulation of alternative splicing in SFN-treated colon cancer cells, and BAZ1A functionalities that might be targeted via next-generation combinatorial epigenetic agents (4). Supported in part by NCI grant CA122959, by the John S. Dunn Foundation, and by a Chancellor’s Research Initiative.