Dissecting EGFR Inhibitor Resistance in Glioblastoma through genome-wide CRISPR screening

项目总结/摘要 耐药性是胶质母细胞瘤（GBM）中的一个主要问题， 成人恶性脑肿瘤通过激酶的异常激活增殖和存活的GBM细胞 信号传导途径被认为通过以下途径避免了治疗性激酶抑制的有害作用 利用替代信号和代谢途径。表皮生长因子受体（EGFR） 代表了一个令人信服的例子来剖析这个问题，因为至少40%的人类GBM具有一个 迄今为止，EGFR改变和该途径的抑制剂（EGFRi）在很大程度上无效。尽管 在了解其他癌症（最值得注意的是，非小细胞肺癌）中的EGFR i抗性方面取得了相当大的进展。 癌症），GBM中的EGFR i抗性机制仍然知之甚少。为了确定新的机制， 在GBM中EGFR激酶抑制剂耐药，我进行了基因组规模的定期间隔聚类， 短回文重复序列（CRISPR）文库筛选在存在EGFR的情况下在几种EGFR突变GBM细胞系中进行。 以及不存在泛ErbB抑制剂来那替尼。氨基酸传感途径多个成员的丢失 (e.g., GCN 2、GCN 1 L1）与这些细胞系中的来那替尼耐药性相关。我还观察到， 急性EGFR抑制诱导氨基酸感应途径（例如，EIF 2a和ATF 4的磷酸化 表达式）。基于这些结果，我假设EGFR抑制诱导了一种氨基酸的状态， 饥饿和整合应激反应（ISR）的激活，这可以通过GCN 2的丢失或 ISR通路中的其他相关基因。我建议阐明EGFR抑制剂的这种新机制 通过一系列生化和遗传实验，进一步探索其更广泛的生物学特性， 在遗传表征的、患者来源的人GBM模型中具有显著性。我的总体目标是进一步 了解EGFR抑制的分子和细胞后果，目的是设计更多 EGFR改变的GBM患者的有效治疗策略。

PROJECT SUMMARY/ABSTRACT Drug resistance represents a major problem in glioblastoma (GBM), the most common and aggressive malignant brain tumor in adults. GBM cells that proliferate and survive through aberrant activation of kinase signaling pathways are believed to avoid the deleterious effects of therapeutic kinase inhibition through utilization of alternative signaling and metabolic pathways. The epidermal growth factor receptor (EGFR) represents a compelling example to dissect this question because at least 40% of human GBMs harbor an EGFR alteration, and inhibitors of this pathway (EGFRi) have been largely ineffective thus far. Despite considerable progress in understanding EGFRi resistance in other cancers (most notably, non-small cell lung cancer), mechanisms of EGFRi resistance in GBM remain poorly understood. To identify novel mechanisms of EGFR kinase inhibitor resistance in GBM, I have performed genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) library screens in several EGFR-mutant GBM cell lines in the presence and absence of the pan-ErbB inhibitor neratinib. Loss of multiple members of the amino-acid sensing pathway (e.g., GCN2, GCN1L1) were associated with neratinib resistance in these cell lines. I have also observed that acute EGFR inhibition induces the amino acid sensing pathway (e.g., phosphorylation of EIF2a and ATF4 expression). Based on these results, I hypothesize that EGFR inhibition induces a state of amino acid starvation and activation of the integrated stress response (ISR) which can be bypassed by loss of GCN2 or other related genes in the ISR pathway. I propose to elucidate this novel mechanism of EGFR inhibitor resistance through a series of biochemical and genetic experiments and further explore its broader biological significance in genetically characterized, patient-derived human GBM models. My overall goal is to further understand the molecular and cellular consequences of EGFR inhibition, with the goal of designing more effective therapeutic strategies for patients with EGFR-altered GBM.