Статті в журналах з теми "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.

e21008 Background: NUT midline carcinoma (NMC) is an aggressive squamous cell carcinoma molecularly defined by a chromosomal rearrangement of nuclear protein in testis (NUTM1) with bromodomain-containing protein 3 or 4 (BRD3/4). While NMCs are characterized by this rare canonical gene rearrangement little is known about the transcriptome and proteosome of this rare disease. As such, we set out to comprehensively characterize five NMC cases in which we attained targeted DNA sequencing, full-transcriptome RNA sequencing, and targeted proteomics. We further examine and integrate these results in order to better understand the relationship between gene expression and protein abundance within the context of NMC. Methods: All cases were analyzed for genomic and transcriptomic alterations against a custom panel via the Tempus xT tissue biopsy assay (DNA sequencing of 648 genes in tumor and matched normal samples at 500x depth and full-transcriptome RNA sequencing) for germline and/or somatic mutations. The xT assay detects single nucleotide variants, specific insertion/deletions, amplifications and gene fusions, as well as tumor mutational burden (TMB) and microsatellite instability (MSI) status. Proteomic data were obtained utilizing digital spatial profiling through Nanostring immune, MAPK and PI3/AKT, and pan tumor nCounter GeoMix panels. Results: Clinical characteristics, histology, and genomic/proteomic alterations for 5 NMC cases are presented. Cases were defined by pathological assessment and the identification of the canonical NUTM1 fusion, further broken down by fusion partner with three patients having NUTM1-BRD4 fusions, one NUT-BRD3, and one NUT-ZMYND8. TMBs ranged for 0.8-.6 mutations/megabases (n=5). All patients were MSI stable (5/5). Of three patients with available PD-L1 IHC result, one had elevated PD-L1 tumor staining at 70%. Results will be presented from full-transcriptome RNA expression analysis indicating overexpression of BRAF, MYC, mTOR, and EGFR, among others. Targeted proteomics were performed to assess relative abundance at the protein level (results to be presented). Clinical follow up for the five patients revealed that two have survived beyond 7 months. A lung primary patient treated with surgical resection and post op radiation (XRT) is NED at 63 months. A sinus primary patient is NED at 16 months after a partial response (PR) to taxotere/5FU/Cisplatin followed by resection and XRT/cis platin. One patient had a brief PR from ifosphamide/etoposide/vorinostat. One patient's tumor grew through XRT/cisplatin. Conclusions: Multi-omic analysis has the potential to further elucidate the mechanisms of tumor growth in NMC and identify new targets for the treatment of this aggressive and poor prognosis disease.

Abstract Background: The treatment of HNSCC in Taiwan is still very challenging and might be related to betel-nuts use. Betel nut chewing might contribute to (1)strong inflammation, angiogenesis, and invasion ; (2)easy recurrence; (3)refractory to traditional therapies. In our previous research, we found betel-nuts exposed HNSCC cell line, TW2.6, might reflect treatment refractoriness of betel-nuts related HNSCC in Taiwan with high PDL1, defective p53 mutation, p16 loss, and BCL2 overexpression. PI3K/mTOR dual inhibition, PI3Kalpha inhibitor, AKT inhibitor, FGFR inhibitor, ALK/IGF1R inhibitor, CDK4/6 inhibitor, BCl2 inhibitor, WEE1 inhibitor, ATR inhibitor, DNA-PK inhibitor, AT2AR inhibitor, Mcl-1 inhibitor, MEK1/2 inhibitor, EZH2 inhibitor, HDAC inhibitor, CDK9 inhibitor, DNMT3 inhibitor, BRD4/BET inhibitor, JAK2 inhibitor, CXCR4 inhibitor, FAK inhibitor, BTK inhibitor, eribulin, & VEGFR2/PDGFR/FGFR or VEGFR2/c-MET/Axl triple blockage might be effective on TW2.6 and reverse treatment refractoriness, maybe through the inhibition of mesenchymal transformation, pRB, & PI3K/AKT/mTOR signaling and the modulation of stemness & PD1/PDL1 pathway. Besides, polo like kinase inhibition seemed a good radiosensitizer for TW2.6. Methods: We try to prove the difference of TW2.6 reflecting clinical characteristics of HNSCC in Taiwan and design effective treatment combinations. Other HNSCC cell lines will also be evaluated for their genomic signatures. All cell lines will be tried to be categorized as TCGA subtypes for the reference of future drug combinations. Results: Conclusions: Further NGS analysis may translate these HNSCC cell lines to represent TCGA subtypes for the reference of future drug combinations, esp. immunotherapy, basic/translational research, and animal models. LRP1B will be a potential ICIs efficacy biomarker in HNSCC. Specific Tx for TW2.6 & others are listed above. Cell lines SCC25 KB SAS CAL27 FaDu SCC15 SCC9 SCC4 TW2.6 Differ- entiation Well Poor Poor Poor Poor Well Well Well Well, but rapidly replicated, with high hyper-diploidy & complex rearrangements HPV status HPV 16/18 HPV18 - - HPV 16/18 - - HPV 6/11 - EGFR status Medium Low High High Medium High Low Medium to high Unknown Docetaxel sensitivity +++ +++ ++ ++ + ++ to +++ ++ - + Cisplatin sensitivity +++ ++ +++ ++ ++ + - to + - - to + 5-FU sensitivity +++ + ++ +++ ++ - + to ++ - - to + Afatinib sensitivity +++ - to + - + ++ ++ to +++ +++ +++ - Polo-like kinase Inhibitor sensitivity +++ +++ ++ ++ + ++ to +++ - to + - - to + VEGFR2 Inhibitor sensitivity - - - - ++ +++ - - ++ PI3K/mTOR inhibitor All cell lines sensitive CDK4/6 Inhibitor response +++ - to + +++ ++ to +++ + ++++ + ++ ++ to +++ Western blots Weak p-AKT & VEGF-A, mild PDL1 and BMI-1, Gli-1(+) Weak p-AKT, mild PDL1 and strong VEGF-A & BMI-1, p16(+) Moderate p-AKT & BMI-1, high PDL1, mild VEGF-A High p-AKT & VEGF-A, mild PDL1 & BMI-1 High VEGF-A, moderate p-AKT & PDL1, weak BMI-1, Gli-1(+) Weak p-AKT & VEGF-A, mild PDL1 & BMI-1 Weak p-AKT, VEGF-A, & BMI-1, moderate PDL1 Moderate p-AKT & VEGF-A, strong BMI-1, mild PDL1, Gli-1(+) High p-AKT, PDL1, & VEGF-A and moderate BMI-1 NGS CCND1 gain, CDKN2A deletion, FRG1 mutation, HGF mutation, p53 mutation, ATR mutation, SMO mutation, RUNX1T1 mutation STK11 mutation, PDGFRA mutation, IGF1 mutation, BCOR mutation, EGFR mutation, NOTCH1 mutation, MET mutation, IKZF1 mutation, NFKB1 mutation, DPYD mutation, FGFR4 mutation, BRCA1 mutation, MSH2 mutation, DNMT3A mutation KRAS mutation, MDM2 mutation, TMB-H, AXIN1 loss, RAD51D mutation, NOTCH1/2 mutation, ERBB4 mutation, PALB2 mutation, p53 mutation, POLE mutation, CASP8 mutation, BRCA2 mutation, RNF43 mutation, LRP1B mutation, MET mutation CDKN2A deletion, EGFR amplification, SMAD4 mutation, TMB-H, LRP1B mutation, APC mutation, CASP8 mutation, CREBBP mutation, PIK3CG mutation, NRAS mutation, ABL1 mutation, FGF23 mutation, HGF mutation, ATRX mutation, p53 mutation, ERBB2 mutation, ROS1 mutation, EP300 mutation, NRAS mutation, CDKN1A mutation, KDM6A mutation, FLT4 mutation CCND1 gain, CDKN2A deletion, FLCN mutation, TMB-H, LRP1B mutation, SMAD4 loss, SF3B1 mutation, FAT1 mutation, VHL mutation, NOTCH3 mutation, EPHA5 mutation, p53 mutation, ERCC2 mutation CCND1 gain, EGFR amplification, SMO mutation, ATR mutation, FAT1 loss, NTRK1 mutation, KMT2D mutation, p53 mutation, NOTCH3 mutation CDKN2A deletion, AXIN2 amplification, SMAD3 loss, HRAS mutation, ATR mutation, NF1 mutation. IGF1R mutation, FLCN mutation, KEAP1 mutation, ASXL1 mutation, PMS2 mutation CCND1 gain, NF1 loss, LRP1B mutation, NSD1 mutation, KMT2D mutation, p53 mutation, EPHA2 mutation FAT1 loss, CCND3/FGF10 amplification, PIK3CA H1047R mutation, STK11 mutation, RICTOR/FLCN amplification, VEGF-A amplification , TSC2 mutation, EPHB1 mutation, MAP2K4 mutation, KDM5A mutation, PDGFRB mutation, SETD2 mutation, RPTOR mutation, APC mutation, DDR2 mutation, ATM mutation, MDM2 mutation, p53 mutation, CDK12 mutation, HRAS mutation, MYC mutation, CDK8 mutation, ARID1B loss Outcomes Best; like TCGA CL (HPV+) subtype Like TCGA basal subtype, but responded to particular treatments each Basal Basal Like TCGA mesen-chymal subtype (HPV+) Like TCGA CL(HPV-) subtype, different characters between these 3 cell lines CL(HPV-) subtype CL(HPV-) subtype Worse; like TCGA EMT subtype (HPV-) Potential treatments All sensitive maybe; Hedgehog inhibitor , HGF/c-MET inhibitor, and I/O could be tried (1) Taxane, cisplatin, PLKi (2) mTORi (3) IGF1Ri, METi, PDGFRi, FGFRi (4) Epigenetics (5) I/O (1) Taxane, cisplatin, 5-FU, PLKi (2) CDK4/6i (3) I/O (4) DDRi (5) KRASi, METi, HERi (6) p53 reactivator and MDM2/Mcl-1 inhibitor (1) Taxane, cisplatin, 5-FU, PLKi (2) CDK4/6i (3) Mild EGFRi response (4) I/O (5) NRASi, FGFRi, HGF/c-METi/ROS1i/HERi (6) p53 reactivator/DDRi/Epigenetics (1) Cisplatin, 5-FU (2) EGFRi and VEGFR2i (3) Weak to PLKi & CDK4/6i (4) I/O (5) mTORi (6) Ephi (7) DDR/Epigenetics (8) p53 reactivator (9) HIFi (1) Taxane and PLKi (2) EGFRi, VEGFR2i, CDK4/6i (3) NTRKi (4) Hedgehog inhibitor (5) DDRi, epigenetics,& p53 reactivator (1) Taxane &5-FU (2) EGFRi (3) HRASi (4) DDRi/Epigenetics (5) I/O (6) IGF1Ri (7) mTORi (1) EGFRi (2) CDK4/6i (3) I/O (4) Epigenetics (5) Ephi (6) p53 reactivator (1) CDK4/6 inhibitor (2) Multi-targeted VEGFR TKI (3) PI3K/AKT/mTOR inhibitor (4) ICIs combination (5) p53 reactivator/DDR interventions/Epigenetics (6) Dasatinib, HRASi, EphB1/B4 interventions Citation Format: Jo-Pai Chen Chen, Jui-Ying Chang, Zhong-Zhe Lin, Ruey-Long Hong. Different treatment response in several head and neck squamous cell carcinoma(HNSCC) cell lines reflecting underlying genomic & molecular signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1411.

Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that selectively inhibits BCL-2 causing cell death. First generation BET inhibitor ABBV-075 and Venetoclax were recently shown to be synergistic in AML cell lines (Bui MH,Cancer Res 2017). ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia efficacy of the concomitant BCL-2 blockade by venetoclax and of BDII inhibition with ABBV-744 in primary AML samples. Anti-leukemia activity of venetoclax and ABBV-744 was examined in 21 primary AML samples with diverse genomic alterations. The combination significantly enhanced cell death (57.0 ± 6.3%) compared to the single agent treatment (43.9 ± 5.7% in ABT-199 10 nM group, p&lt;0.001 and 23.8 ± 2.9% in ABBV-744 20nM group, p&lt;0.001, Fig.1A). ABBV-744 reduced viable cell numbers in the majority of AML cases (31.7 ± 5.2%) and the cell growth suppression was more profound in the combination group (77.2 ± 6.3 %, p&lt;0.001, Fig.1B). In two AML primary samples tested, combination treatment of ABBV-744 and ABT-199 induced apoptosis with caspase-3 activation and PARP cleavage through regulation of the key proteins regulating survival and proliferation pathways (e.g. (BCL-2, BCL-XL, MCL-1, c-Myc)). To identify biomarkers of response to therapy, we performed the baseline transcriptome analysis of AML cells used for in vitro response assessment (n=25) by RNA-sequencing (RNA-seq), and correlated baseline gene expression levels with in vitro response to therapy. Based on response to venetoclax or combination, samples were divided into 3 groups: sensitive to venetoclax (n=10), samples with no response to single agent or combination ("low apoptosis", n=7) and samples resistant to venetoclax as a single agent but responsive to venetoclax/ ABBV-744 combination ("synergy", n=4). AML samples sensitive to venetoclax and venetoclax/ABBV-744 combination were characterized by high level of BCL2 and lower levels of MCL1 and BCL2L1 transcripts, consistent with known inability of venetoclax to inhibit MCL-1 and BCL2L1 (Fig.1C).The resistant samples additionally expressed higher levels of anti-apoptotic genes such as GADD45, BCL2L10, PMAIP1. AML cells that showed synergy between venetoclax/ABBV-744 expressed low levels of AR, IL1R1 genes and had high CCND1 expression. The gene expression analysis indicated that the genes differentially expressed in the synergy vs. low apoptosis samples overlap with the genes inhibited by dual BCL-2/BCL-XL inhibitor ABT-737. To test the efficacy of this regimen in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, EGFR, IDH1, NPM1, TET2 mutations in NSG mice. Upon engraftment, mice were randomized to receive vehicle; single agent venetoclax at 50 mg/kg; ABBV-744 at 9.4 mg/kg; or venetoclax plus ABBV-744 for 21 days. After 21 days of therapy, flow cytometry data demonstrated significantly reduced leukemia burden in venetoclax treated group (9.5% ± 1.7%) but not in ABBV-744 group (22.3% ± 5.8%) compared to controls (30.8% ± 3.9%), with lowest tumor burden in the combination group (5.0% ± 0.8%, p&lt;0.01) (Fig. 1E). Combination of ABBV-744 and venetoclax treatment delayed AML progression and extended the survival compared to the untreated mice (median survival, 193 days vs 99 days, p&lt;0.001) (Fig. 1D). No significant impact on mice' weight was noted, and no clinical signs of toxicity recorded over the course of therapy. In summary, combinatorial blockade of BDII bromodomain and of BCL-2 anti-apoptotic pathway facilitates apoptotic cell death, suppresses proliferation in the majority of primary AML cells and produces anti-AML activity in AML PDX models in vivo at tolerable doses of both agents. This combination is currently undergoing testing in a Phase I clinical trial in AML (NCT03360006). Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. Lin:AbbVie: Employment. Uziel:AbbVie: Employment, Other: stock or other options. Lu:AbbVie: Employment. Zhang:AbbVie: Employment. Huang:AbbVie: Employment. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Shen:AbbVie: Employment. Konopleva:Astra Zeneca: Research Funding; Ablynx: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy, Honoraria; Ascentage: Research Funding; Agios: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Genentech: Honoraria, Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria.

AbstractEGFR upregulation is an established biomarker of treatment resistance and aggressiveness in head and neck cancers (HNSCC). EGFR-targeted therapies have shown benefits for HPV-negative HNSCC; surprisingly, inhibiting EGFR in HPV-associated HNSCC led to inferior therapeutic outcomes suggesting opposing roles for EGFR in the two HNSCC subtypes. The current study aimed to understand the link between EGFR and HPV-infected HNSCC particularly the regulation of HPV oncoproteins E6 and E7. We demonstrate that EGFR overexpression suppresses cellular proliferation and increases radiosensitivity of HPV-positive HNSCC cell lines. EGFR overexpression inhibited protein expression of BRD4, a known cellular transcriptional regulator of HPV E6/E7 expression and DNA damage repair facilitator. Inhibition of EGFR by cetuximab restored the expression of BRD4 leading to increased HPV E6 and E7 transcription. Concordantly, pharmacological inhibition of BRD4 led to suppression of HPV E6 and E7 transcription, delayed cellular proliferation and sensitised HPV-positive HNSCC cells to ionising radiation. This effect was shown to be mediated through EGFR-induced upregulation of microRNA-9-5p and consequent silencing of its target BRD4 at protein translational level, repressing HPV E6 and E7 transcription and restoring p53 tumour suppressor functions. These results suggest a novel mechanism for EGFR inhibition of HPV E6/E7 oncoprotein expression through an epigenetic pathway, independent of MAPK, but mediated through microRNA-9-5p/BRD4 regulation. Therefore, targeting EGFR may not be the best course of therapy for certain cancer types including HPV-positive HNSCC, while targeting specific signalling pathways such as BRD4 could provide a better and potentially new treatment to improve HNSCC therapeutic outcome.

AbstractThird-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs), including osimertinib, an irreversible EGFR-TKI, are important treatments for non-small cell lung cancer with EGFR-TKI sensitizing or EGFR T790M resistance mutations. While patients treated with osimertinib show clinical benefit, disease progression and drug resistance are common. Emergence of de novo acquired resistance from a drug tolerant persister (DTP) cell population is one mechanism proposed to explain progression on osimertinib and other targeted cancer therapies. Here we profiled osimertinib DTPs using RNA-seq and ATAC-seq to characterize the features of these cells and performed drug screens to identify therapeutic vulnerabilities. We identified several vulnerabilities in osimertinib DTPs that were common across models, including sensitivity to MEK, AURKB, BRD4, and TEAD inhibition. We linked several of these vulnerabilities to gene regulatory changes, for example, TEAD vulnerability was consistent with evidence of Hippo pathway turning off in osimertinib DTPs. Last, we used genetic approaches using siRNA knockdown or CRISPR knockout to validate AURKB, BRD4, and TEAD as the direct targets responsible for the vulnerabilities observed in the drug screen.

AbstractN6-methyladenosine (m6A) is methylation that occurs in the N6-position of adenosine, which is the most prevalent internal modification on eukaryotic mRNA. Accumulating evidence suggests that m6A modulates gene expression, thereby regulating cellular processes ranging from cell self-renewal, differentiation, invasion and apoptosis. M6A is installed by m6A methyltransferases, removed by m6A demethylases and recognized by reader proteins, which regulate of RNA metabolism including translation, splicing, export, degradation and microRNA processing. Alteration of m6A levels participates in cancer pathogenesis and development via regulating expression of tumor-related genes like BRD4, MYC, SOCS2 and EGFR. In this review, we elaborate on recent advances in research of m6A enzymes. We also highlight the underlying mechanism of m6A in cancer pathogenesis and progression. Finally, we review corresponding potential targets in cancer therapy.