Identifying and Exploiting Combinatorial Synthetic Lethality by Characterizing Adaptive Kinome Rewiring of EGFRvIII-driven Glioblastoma
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ABSTRACT:
PROVIDER: PRJNA1257934 | ENA |
REPOSITORIES: ENA
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Project description:GBM is an aggressive primary malignant brain tumor that has a poor prognosis. Molecular characterization of GBM has shown that EGFR mutations are present in over 50% of tumors. However, EGFR inhibitors have not shown clinical efficacy in contrast to other EGFR-driven neoplasms due to the unique EGFR biology found in GBM. We believe that upfront combinatorial therapy with EGFR inhibitors can overcome these challenges. To identify combinatorial drug targets, we temporally characterized drug induced EGFRvIII kinome rewiring in a cell line with an isogenic genetically engineered GBM EGFRvIII kinome. Kinomic characterization show that kinome rewiring has both shared and unique kinases after acquired resistance develops despite a common genetic origin. Additionally, we noted that the kinases altered in the acute setting are distinct from those in acquired resistance. By identifying these differential temporal kinomic vulnerabilities throughout the drug response process, we generated a kinase signature associated with inhibition of EGFR. Further molecular interrogation of these signature genes reveals that drug treatment induces an unexpected increase in Cdk6 protein but not mRNA despite a decrease in proliferation measured by both live cell counts and transcriptomics. Survival experiments with two cohorts of orthotopic allografts show that upfront combination inhibition of Cdk6, with abemaciclib, and EGFR, with neratinib, significantly prolonged median survival compared to neratinib alone. Our findings suggest that identifying and inhibiting targets with synthetic lethality in the upfront combinatorial setting is a viable approach for precision oncology and may help provide an avenue to overcome the resistance mechanisms that contributed to the failures of EGFR as a molecular target in GBM.
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Project description:Next generation sequencing platforms were used to identify STAT3 targets in the background of EGFRvIII expresssion mRNA of 3 EGFRvIII positive brain tumor stem cell lines (BTSC #68, 73, and 90) were compared to an EGFRvIII negative line (#41) to identify EGFRvIII-regulated targets in human BTSC. EGFRvIII-dependent targets in mouse astrocytes were identified by mRNA-seq analyses of EGFRvIII- or MSCV-expressing astrocytes. STAT3-differentially regulated genes in EGFRvIII expressing mouse astrocytes were obtained by subjecting EGFRvIII,STAT3f/f and EGFRvIII, STAT3-/- astrocytes to mRNA-Seq analyses. Sites of STAT3 occupancy in EGFRvIII expressing astrocytes were identified by ChIP-Seq using a STAT3 antibody or IgG control. We identified OSMR as a direct target of STAT3 in both EGFRvIII-expressing human BTSC and mouse astrocytes. Therefore to identify OSMR-regulated genes, we used a lentiviral mediated RNAi system to knockdown OSMR in EGFRvIII-expressing astrocytes. OSMR-dependent differentially expressed genes were obtained by comparison of OSMR knockdown (KD1 and KD2) astrocytes to control groups (ShRNA control and Vector control)
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Project description:EGFRvIII is the most common deletion mutant of EGFR in human cancer and its levels are highly correlated with poor prognosis in GBM. The deletion of exons 2-7 removes most of the extracellular ligand binding domain, so it is unable to bind EGF or other EGFR-binding ligands. Nevertheless, the mutant receptor is constitutively phosphorylated, and is capable of activating downstream signaling pathways at a low level. To comprehensively identify the downstream signaling consequences of the EGFRvIII, we incorporated phosphoproteomic, transcription profiling and DNase-Seq data from U87MG glioblastoma cells expressing titrated levels of this mutant receptor. Total RNA were extracted from U87MG cells engineered to expressed different levels of EGFRvIII: medium (U87M; 1.5 million copies of EGFRvIII receptor per cell), high (U87H; 2 million copies per cell), super-high (U87SH; 2.5 million copies per cell), and kinase-dead EGFRvIII (U87DK; 2 million copies of kinase dead EGFRvIII per cell). RNA was hybridized to Affymetrix microarrays.
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Project description:We found that U87 cells expressing EGFR and EGFRvIII upregulated a large group of cytokines. The upregulation of many of these cytokines was dependent on KRAS.
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Project description:Next generation sequencing platforms were used to identify STAT3 targets in the background of EGFRvIII expresssion
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Project description:Co-amplification of EGFR and EGFRvIII, a tumor-specific truncation mutant of EGFR, represent hallmark genetic lesions in glioblastoma. We report that EGFR and EGFRvIII stimulate the innate immune defense receptor Toll-like Receptor 2 (TLR2); and that knockdown of TLR2 led to a dramatic survival advantage in glioblastoma xenografts. EGFR and EGFRvIII activated TLR2 in a ligand-independent manner, promoting tumor growth and immune evasion. We show that EGFR and EGFRvIII cooperate to activate the Rho-associated protein kinase ROCK2, modulating malignant progression both by activating TLR2 and WNT signaling, and through remodeling the tumor microenvironment.
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Project description:The effect of CD151 expression onto the kinome of Jurkat T cells was assessed using kinome analysis. CD151 was expressed in Jurkat T cells by retroviral transduction based on a pMSCV vector. Entrez Gene: 977 UniProtKB: P48509 Jurkat T cells were transduced with the MSCV-CD151 vector and successfully transduced cells were selected using puromycin. For the kinome array experiments 3 independent samples of Jurkat cells and three independent samples of J-CD151 cells were collected. To minimize unspecific background signals, lysates from Jurkat and J-CD151 T cells harvested at different growth stages, which were then pooled to provide one sample prior to loading on the Kinexus antibody microarrays.
2017-09-30 | E-MTAB-5978 | biostudies-arrayexpress
Project description:EGFRvIII is the most common deletion mutant of EGFR in human cancer and its levels are highly correlated with poor prognosis in GBM. The deletion of exons 2-7 removes most of the extracellular ligand binding domain, so it is unable to bind EGF or other EGFR-binding ligands. Nevertheless, the mutant receptor is constitutively phosphorylated, and is capable of activating downstream signaling pathways at a low level. To comprehensively identify the downstream signaling consequences of the EGFRvIII, we incorporated phosphoproteomic, transcription profiling and DNase-Seq data from U87MG glioblastoma cells expressing titrated levels of this mutant receptor.
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