Academic literature on the topic 'EGFR-BRD4'

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults with a dismal 15-month median survival. Standard therapy consisting of surgical resection, radiation, and temozolomide has been unsuccessful in meaningfully extending survival and preventing recurrence; thus, novel therapeutics are urgently needed. One proposed targeted treatment strategy for GBM involves using small molecule inhibitors against common genetic mutations. Epidermal growth factor receptor (EGFR) is the most commonly overexpressed oncogene in GBM (~56%). While EGFR tyrosine kinase inhibitors (TKI) have shown promise in other cancers, GBM clinical trials with EGFR TKI have failed. One reason for this failure is the development of adaptive therapeutic resistance. Understanding the mechanisms behind drug resistance is essential for the development of novel, effective therapeutics for GBM. To better understand adaptive resistance in GBM, we utilized two genetically engineered mouse astrocyte lines harboring common GBM mutations: Cdkn2a-/-, EGFRvIII (CEv3) and Cdkn2a-/-, Pten-/-, EGFRvIII (CEV3P). CDKN2A and PTEN are commonly deleted or otherwise inactivated tumor suppressor genes in GBM while the vIII variant of EGFR is the single most common oncogene mutation, making it an attractive therapeutic target. Cell lines CEv3 and CEv3P are both sensitive to neratinib, an irreversible second-generation EGFR TKI, at IC50 of 0.24μM and 0.13µM, respectively. To better understand adaptive response to neratinib treatment, we profiled the transcriptome with RNA sequencing at 0, 4, 24, and 48 hours. Our data shows that kinome rewiring is detectable after just 4 hours of treatment and sustained through 48 hours, with differential expression of 70% or more of the expressed kinome. We propose that differentially expressed kinases in response to neratinib can potentially activate alternative signaling pathways that bypass EGFR inhibition, which ultimately confers resistance to EGFR targeted therapy. Furthermore, we hypothesize that the epigenome is directly responsible for this adaptive kinome response through BRD4 dependent enhancer remodeling. Because dual therapy against EGFR and BRD4 has shown promising results in other cancers, targeting the epigenome through BRD4 represents a potential combination therapy with EGFR TKI in GBM. To profile BRD4-associated epigenomic changes, we used Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to interrogate several regulatory marks (H3K4me1, K3K4me3, H3K27ac) in addition to BRD4. We seek to integrate RNA sequencing and CUT&RUN data to determine if kinases differentially expressed following neratinib treatment correlate with epigenetic marks for their respective enhancer(s). This work will provide insight into the adaptive resistance mechanism of EGFR driven GBM. Citation Format: Benjamin Lin, Julia Ziebro, Kasey R. Skinner, Abigail Shelton, Erin Smithberger, Ryan Bash, Frank B. Furnari, Ryan Miller. Elucidating the transcriptomic response to EGFR-targeted therapy in EGFR-driven glioblastoma [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 1125.

232 Background: BRD4 plays an important role in transcription, DNA repair and drug resistance. High expression and polymorphisms of BRD4 regulating pathways were reported to be related to worse prognosis in colorectal cancer. Therefore, we hypothesized that genetic variants in BRD4 regulating pathway may predict first-line treatment outcome in mCRC pts. Methods: The impact on outcome of 22 SNPs in 7 genes involved in BRD4 regulating pathway (BRD4, SIPA1, MYC, 53BP1, H2AX, BATF, CD47) was analyzed through the OncoArray, a customized array manufactured by Illumina, on genomic DNA from blood samples of pts enrolled in 2 randomized trials. MAVERICC FOLFIRI/bevacizumab (bev) arm served as discovery cohort (N = 107), FIRE3 FOLFIRI/bev arm as validation (N = 107) and FOLFIRI/cetuximab (cet) arm as control (N = 129). Results: In the discovery cohort, right(R)-sided pts with BRD4 rs4808272 any G allele (N = 46) showed significantly shorter PFS (9.5 vs 18 m) compared to carriers of A/A (N = 21) in both uni- and multi-variable analysis ( p < .01); R-sided pts carrying any T allele of BATF rs7161377 (N = 50) showed longer PFS (12.3 vs 6.8 m) compared to carriers of C/C (N = 14) in univariate analysis ( p < .05) and had a strong trend in multivariable analysis ( p = .06). These findings were all validated in R-sided pts in FIRE3 bev arm (BRD4 rs4808272, PFS 9.8 vs 18.7 m; BATF rs7161377, PFS 15.1 vs 4.2 m) in uni- (both p < .01) and multi-variable ( p = .08 and p < .05 respectively) analysis. No significant association was observed in the control arm. Interestingly, pts carrying CD47 rs3206652 any C allele (N = 13) only showed significant longer PFS (9.0 vs 3.0 m, univariable p < .01 and multivariable p = .07) in the R-sided pts of FIRE3 cet cohort, but no association was observed in the bev-based treatment. Conclusions: Our study demonstrates for the first time that BRD4 and BATF polymorphisms may predict outcomes of bev-based treatment in R-sided mCRC pts; Meanwhile CD47 polymorphism could predict outcomes of cet-based treatment in R-sided mCRC pts. This finding supports a possible role of BRD4 regulating pathway in contributing to resistance to anti-VEGF/EGFR treatment.

Herein a novel series of histone deacetylases (HDACs) and epidermal growth factor receptor (EGFR) dual inhibitors were designed and synthesized based on the structure of the approved EGFR inhibitor osimertinib (AZD9291). Among them, four compounds 5D, 5E, 9D and 9E exhibited more potent total HDAC inhibition than the approved HDAC inhibitor SAHA. However, these compounds only showed moderate to low inhibitory potency towards EGFR with compounds 5E and 9E possessing IC50 values against EGFRWT and EGFRT790M in the micromolar range. 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay revealed the potent antiproliferative activities of compounds 5D, 5E, 9D and 9E, among which 9E was even more potent against HeLa, MDA-MB-231, MDA-MB-468, HT-29 and KG-1 cell lines than SAHA and AZD9291. Further selectivity profile of 9E showed that this compound was not active against other 13 cancer-related kinases and two epigenetic targets lysine specific demethylase 1 (LSD1) and bromodomain-containing protein 4 (BRD4). These results support further structural modification of 9E to improve its EGFR inhibitory activity, which will lead to more potent and balanced HDAC and EGFR dual inhibitors as anticancer agents.

234 Background: The PI3K/Akt pathway is frequently activated in aggressive and resistant prostate cancer. Here we detail our pre-clinical evaluation of AZD8186, a novel β and δ selective PI3K small molecule inhibitor. Further, we investigate how increased transcription of the myc oncogene may represent a mechanism of resistance to monotherapy PI3K/Akt inhibition. Therefore, we further evaluated co-targeting strategies against both the PI3K/Akt pathway and the epigenetic reader protein BRD4. Methods: Human prostate cancer cell lines LNCaP and 22RV1 were tested for sensitivity to AZD8186 in vitro. Caspase-3 activity and flow cytometry were used to assess apoptosis. Western blotting and RT-qPCR were used to measure AR and myc pathway genes and protein expression. Castrate resistant LNCaP xenografts were treated with 10mg/kg and 25mg/kg doses of AZD8186 given orally 4 days on, 3 off. Results: In vitro results demonstrated sensitivity to AZD8186 with decreases in cell proliferation and increases in apoptosis. In vivo results demonstrated a dose-dependent decrease in tumor growth velocity with AZD8186 with on-target decreases in pAkt was observed in xenograft tumour samples. Further, increases in myc protein and mRNA levels were seen in xenograft samples treated with AZD8186 compared to control. Downstream increases in EGFR and IGF-IR mRNA transcripts induced by AZD8186 were also seen in vitro and in vivo. Increases in myc, EGFR and IGF-IR was also seen in cells and tumours treated with an Akt inhibitor. Addition of the BDR4 inhibitor JQ1 decreased the increase of myc induced by AZD8186 and also partially abrogated the increases in EGFR, IGFR. Greater suppression of PSA expression was seen with the combination of AZD8186 and JQ1 compared to AZD8186 and enzalutamide. Similar results were seen in the 22RV1 cell line. Conclusions: Inhibition of PI3K with AZD8186 inhibits growth of PTEN-negative LNCaP cells. However, feedback activation of myc and AR pathways occurs. We demonstrate BRD4 inhibition using JQ1 which co-targets both myc and AR feedback pathways as a rationale combination strategy with PI3K inhibition. This strategy warrants further investigation in prostate cancers with an activated PI3K/Akt pathway.

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.

(1) Background: Despite the evidence that ferroptosis is involved in myocardial ischemia-reperfusion (MIR), the critical regulator of ferroptosis in MIR remains unclear. (2) Methods: We included three GEO datasets and a set of ferroptosis-related genes with 259 genes. Following the identification of the differentially expressed ferroptosis-related genes (DEFRGs) and hub genes, we performed the functional annotation, protein–protein interaction network, and immune infiltration analysis. The GSE168610 dataset, a cell model, and an animal model were then used to verify key genes. (3) Results: We identified 17 DEFRGs and 9 hub genes in the MIR samples compared to the control. Heme oxygenase 1 (Hmox1), activating transcription factor 3 (Atf3), epidermal growth factor receptor (Egfr), and X-box binding protein 1 (Xbp1) were significantly upregulated in response to ischemic and hypoxic stimuli. In contrast, glutathione peroxidase 4 (Gpx4) and vascular endothelial growth factor A (Vegfa) were consistently decreased in either the oxygen and glucose deprivation/reoxygenation cell or the MIR mouse model. (4) Conclusions: This study emphasized the relevance of ferroptosis in MIR. It has been successfully demonstrated that nine ferroptosis-related genes (Hmox1, Atf3, Egfr, Gpx4, Cd44, Vegfa, asparagine synthetase (Asns), Xbp1, and bromodomain containing 4 (Brd4)) are involved in the process. Additional studies are needed to explore potential therapeutic targets for MIR.

In the design of antineoplastic drugs, quinazolinone derivatives are often used as small molecule inhibitors for kinases or receptor kinases, such as the EGFR tyrosine kinase inhibitor gefitinib, p38MAP kinase inhibitor DQO-501, and BRD4 protein inhibitor PFI-1. A novel and convenient approach for the solid-phase synthesis of dihydroquinazoline-2(1H)-one derivatives was proposed and 19 different compounds were synthesized. Cytotoxicity tests showed that most of the target compounds had anti-proliferative activity against HepG-2, A2780 and MDA-MB-231 cell lines. Among them, compounds CA1-e and CA1-g had the most potent effect on A2780 cells, with IC50 values of 22.76 and 22.94 μM, respectively. In addition, in an antioxidant assay, the IC50 of CA1-7 was 57.99 μM. According to bioinformatics prediction, ERBB2, SRC, TNF receptor, and AKT1 were predicted to be the key targets and play an essential role in cancer treatment. ADMET prediction suggested 14 of the 19 compounds had good pharmacological properties, i.e., these compounds displayed clinical potential. The correct structure of the final compounds was confirmed based on LC/MS, 1H NMR, and 13C NMR.

Abstract Glioblastoma (GBM) is the most common and malignant adult brain tumor. Despite years of research, few advancements have been made in its management. One promising avenue of research has been treatment with BRD4 inhibitors, which decrease oncogene expression in GBM cells. However, resistance to these inhibitors is rapidly acquired. Kinome reprogramming is thought to underlie this resistance, suggesting a need for combination therapy with kinase inhibitors. The goal of this study is to determine whether transcriptomic and kinomic profiling of GBM tumors will identify synergistic drug pairs for GBM treatment. We profiled the active kinome on a set of three newly-diagnosed GBM patient-derived xenograft (PDX) tumors and three recurrent tumors using quantitative SILAC mass spectrometry. Additionally, kinome reprogramming following BET inhibition was profiled in vitro using the BET inhibitor JQ1. Kinome activity was uploaded into our novel computational platform, SynergySeq, to assess the synergistic potential of kinase inhibitors with JQ1. Additionally, single cell RNA-sequencing of a GBM tumor was used to determine the cell populations affected by each inhibitor. To quantify synergy in response to combination therapy, cells were treated with a combination matrix of JQ1 and a kinase inhibitor in variable concentrations and cell death was quantified via ATP levels. Our results showed that newly-diagnosed tumors were predicted to be sensitive to combined BET inhibition with Bcr/Abl, EGFR, and FGFR kinase inhibitors. Recurrent tumors were sensitive to combined Bcr/Abl and FGFR inhibitors but were not sensitive to EGFR inhibitors. We screened inhibitors in vitro and found a synergistic effect for the combination of JQ1 and TAS120, a pan-FGFR inhibitor. These data suggest that clinically a brain penetrant BET inhibitor should be effective in combination with a brain penetrant FGFR inhibitor. Importantly, our computational platform is a novel informatics-based approach for targeted therapy in a patient-specific and disease-specific manner.

Infectious diseases account for a large proportion of morbidity and mortality worldwide. The major global efforts lie in effectively enhancing the health span of infected individuals and more importantly, curbing infection onset and spread. A multitude of host- and pathogen-derived factors contribute towards coining the outcome of infections. In this regard, the virulence of major successful infectious agents is believed to be determined by their prowess to swivel host immune system to their own benefit. The pathogens effectuate such immune subversions by modulating various host signalling pathways and reprogramming host cellular homeostatic processes. Thus, understanding the transactions occurring at the host-pathogen interface becomes an asset for the development of rational diagnostic, preventative and therapeutic interventions for infectious diseases. The current study focuses on the implications of Epidermal Growth Factor Receptor (EGFR) signalling during selected bacterial infections. Conventionally associated with cancers of various origins; accumulating evidences indicate aberrant activation of EGFR (Receptor Tyrosine Kinase, RTK) pathway during infection scenarios and Pattern Recognition Receptor (PRR) engagements. For instance, EGFR has been reported to be deregulated during infections with Helicobacter pylori, Shigella flexneri, Mycobacterium tuberculosis and Toxoplasma sp.; however, there is a dearth of information relating to the molecular mechanisms governed by EGFR during such infections. In the purview of the stated literature, the specific contributions of EGFR in the pathogenesis of Mycobacterium tuberculosis (Mtb) and Shigella flexneri (S. flexneri) was conceived. Infection with both Mtb and S. flexneri activated signalling through EGFR pathway. During Mtb infection, EGFR was intricately involved in regulating key processes that allow pathogen survival. EGFR was found to employ a specific reader of epigenetic marks, Bromodomain-containing protein 4 (BRD4), to alter gene expression and assist Mtb pathogenesis by regulating the phenomenon of angiogenesis, i.e. blood vessel formation (Chapter 3). Further, the receptor-epigenetic reader pair was also found to orchestrate Mtb-induced accumulation of lipid droplets and inhibition of autophagy (Chapter 4). In studies with the gram-negative bacterium S. felxneri, it was found that EGFR regulated the expression of anti-inflammatory indoleamine 2, 3 dioxygenase (IDO), which contributed significantly to host immune homeostasis during S. flexneri infection (Chapter 5). Mtb afflicts over one-fourth of the world population and poses a global health concern. The successful co-evolution of the pathogen with its host, along with the emergence of its drug resistant variants presses on the need for unravelling critical host factors that aid in its persistence. Within the host, alveolar macrophages form the primary site of infection for the pathogen and are extensively modulated by Mtb to generate a secure and nutrient-rich niche. In this scheme of events, infection drives the formation of a highly organized immune structure - the granuloma, consisting of concentric whorls of distinct immune cells, with infected macrophages residing in the core, surrounded by neutrophils, T lymphocytes and B lymphocytes. With such an organization, it has been perceived that one of the major constraints to effective antibiotic therapy for tuberculosis (TB) would stem from the lack of vascular perfusion to the bacteria-populated core of the granuloma. Therefore, granuloma-associated host angiogenic modulation stands critical for Mtb survival, dissemination and evasion from antibiotics as well as host immune effectors. Existing literature suggests the upregulation of angiogenic markers in mycobacteria-infected host cells. Macrophages contained in the hypoxic microenvironment of the granuloma are reported to form the major source of the potent angiogenic factor, VEGF-A. Interestingly, it has been observed that the extent of expression of VEGF-A is strongly correlated with the virulence of Mtb. Apart from the mice and zebrafish model for the study of Mtb-associated angiogenesis, another study has recently reported the potential efficacy of anti-VEGF therapy as an adjunct for TB control in rabbit model. Further, the presence of angiogenic properties in the serum of active TB patients and the abundance of VEGF-A during TB infection necessitates the exploration of the consequences and the molecular mechanisms governing the process. In this regard, we found that EGFR signalling is a significant regulator of Mtb-induced angiogenic effects. EGFR was found to mediate this differential regulation through BRD4, in conjunction with KLF5, a transcription factor belonging to the Kruppel-like family. Interestingly, both these members have earlier been associated with VEGF-A expression. Also literature evidences suggest the contribution of distinct bromodomain proteins during bacterial infections and LPS treatment. Finally, through the use of pharmacological inhibitors of EGFR and BRD4, in an established model of TB, we demonstrate the ability of these small molecule inhibitors in potentiating anti-TB effects. The different phenomena exploited by Mtb to accentuate its survival within the host includes alteration of nutrient sources such as lipids; skewing of immune responses such as T cell functions and MHC recognition; and perturbation of bacterial elimination processes such as autophagy and apoptosis. EGFR was earlier reported as a potential therapeutic target for TB infection. Here, we additionally find the implication of EGFR signalling dependent BRD4 in differentially modulating two key cellular events during Mtb infection; i.e. lipid accumulation and autophagy. Mtb-induced lipid droplets (LDs) are suggested to act as source of nutrients and a secure niche for the bacterium; and autophagy (as a bacterial clearance mechanism) is suppressed by the pathogen. We determine the interplay of these phenomena and underscore their relevance in Mtb pathogenesis. The intricate regulatory circuits of these Mtb survival events form essential targets for effective TB therapy. Several studies propose key signalling intermediates/ transcription factors in the regulation of Mtb-driven immune evasion. In the current study, we uncover that inhibition of EGFR signalling dependent BRD4 compromises the ability of Mtb to induce lipid droplet formation. We also observe the induction of host autophagic process in Mtb-infected cells treated with the concerned pharmacological inhibitors. Corroborating these observations, in vitro CFU enumeration revealed compromised mycobacterial survival upon perturbation of EGFR signalling dependent BRD4; validating their possible implication in Mtb pathogenesis. In another facet, we determined the implication of EGFR in S. flexneri infection. Recognition of S. flexneri by PRRs triggers a gamut of immune responses, which majorly culminate in hyper-inflammation. Resolution of such overt inflammation is crucial for host survival. Several anti-inflammatory factors such as RUNX3, NRF2 and ERBIN have been reported to provide homeostasis in a context-dependent manner. Using in vitro and in vivo models of S. flexneri infection, we find that S. flexneri leads to the expression of the immune homeostat indoleamine 2, 3 dioxygenase 1 (IDO1). We observed a critical role of EGFR-driven signalling cascade in governing IDO1 production. Further insights into the functional significance of IDO1 revealed that the loss of IDO1 function in vivo leads to exacerbated Shigellosis. We delineated that the observed effect is correlated with the skewed inflammatory mediators, with excessive pro-inflammatory cytokines (IL-1β, IL-12, IL-17) and compromised expression of the anti-inflammatory arm (IL-10, Arginase1, IL-4, TGF-β). Interestingly, administration of recombinant EGF in IDO1 inhibited mice restored the cytokine balance and consequently ameliorated exacerbated Shigellosis and bacterial growth. This study focused on the homeostatic effect of IDO1 and uncovered the possible contribution of EGFR in conserving host inflammatory milieu. Together, we unravel the potential implications of EGFR in discrete bacterial infections. We find that pathogen-specific activation of EGFR pathway may have diverse manifestations during distinct infections. Here, we observed that whereas on one hand EGFR assists mycobacterial pathogenesis; it protects the host from exacerbated Shigellosis. These mechanisms shed light on the conundrum of events that determine infection outcome and also project the relevance of calibrated host responses to specific immune insults.