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

项目摘要/摘要 耐药性是胶质母细胞瘤的一个主要问题，胶质母细胞瘤是最常见和最具侵袭性的 成人的恶性脑瘤。通过异常激活的激酶增殖和存活的GBM细胞 信号通路被认为可以通过以下途径避免治疗性激酶抑制的有害影响 替代信号和代谢途径的利用。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 到目前为止，EGFR的改变和该途径的抑制剂(EGFRi)在很大程度上是无效的。尽管 了解EGFRi在其他癌症(尤其是非小细胞肺癌)中的耐药性方面取得了相当大的进展 肿瘤)，GBM中的EGFRi耐药机制仍知之甚少。为了确定新的机制， EGFR激酶抑制剂在GBM中的耐药性，我已经进行了基因组规模的成簇规则间隔 在几种EGFR突变的GBM细胞系中筛选短回文重复序列(CRISPR)文库 以及PAN-ErbB抑制剂neratinib的缺失。氨基酸传感通路中多个成员的缺失 (如GCN2、GCN1L1)与奈拉替尼耐药相关。我还观察到， 急性EGFR抑制诱导氨基酸感觉通路(例如，EIF2a和ATF4的磷酸化 表达)。基于这些结果，我假设EGFR抑制会导致一种氨基酸状态。 整合应激反应(ISR)的饥饿和激活，这可以通过GCN2或GCN2或GCN2的缺失来绕过 ISR途径中的其他相关基因。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 在遗传特征的、患者来源的人类GBM模型中的意义。我的总体目标是进一步 了解EGFR抑制的分子和细胞后果，目标是设计更多 EGFR改变的基底膜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.