Microarray expression data of NCI-H1975 cells treated with osimertinib, alpelisib, or combination of these in the presence or absence of L-histidinol.. Microarray expression data of NCI-H1975 cells treated with osimertinib, alpelisib, or combination of these in the presence or absence of L-histidinol.

Oncogenic signals often activate abnormal proliferation, while simultaneously activate stress-adaptive mechanisms such as the integrated stress response (ISR) to ensure rapid growth under intrinsic and extrinsic stress conditions. In this study, we investigated the involvement of EGFR-PI3K pathway in the regulation of ISR in EGFR-mutant NSCLC cell lines under amino acid deprivation. We found that the third generation EGFR inhibitor osiemrtinib suppressed induction of activation transcription factor 4 (ATF4), the key ISR effector, in EGFR mutant cells, while the effect was to a less extent in cells harboring PIK3CA-co-alteration. PI3K inhibitors including P110a-specific inhibitor alpelicib markedly suppress ATF4 induction in PIK3CA-mutant cell lines. To further explore the role of EGFR-PI3K, transcriptome analysis was performed in EGFR- and PIK3CA-mutated NCI-H1975 cells treated with osimertinib, alpelisib, or combination of these in the absence or presence of histidyl-tRNA inhibitor L-histidinol (His), mimicking amino acid deprived conditions. Among His-induced genes, either osimertinib or alpelisib partially, but the combination dramatically suppressed a cluster of genes targeted by ATF4. Furthermore, combination of osimertinib and alpelisib increased apoptotic cells under amino acid deprived conditions. These results indicate that oncogenic EGFR-PI3K pathway contributes to cellular adaptation to stress conditions through ATF4. We used microarrays to identify genes whose expression is up- or down-regulated by inhibition of EGFR, PI3K, or both under amino acid deprivation. Overall design: NCI-H1975 cells were treated with DMSO, osimertinib (10 nM), alpelisib (1 µM), or a combination of these in the presence or absence of L-histidinol (2 mM) for 24 hours.

致癌信号通常会激活异常增殖，同时还会激活整合应激反应（integrated stress response, ISR）等应激适应机制，以确保细胞在内在和外在应激条件下能够快速生长。本研究探究了EGFR-PI3K通路在氨基酸剥夺条件下对EGFR突变非小细胞肺癌细胞系中整合应激反应的调控作用。我们发现，第三代EGFR抑制剂奥希替尼（osimertinib）可抑制EGFR突变细胞中关键ISR效应分子激活转录因子4（activation transcription factor 4, ATF4）的诱导表达，而该效应在携带PIK3CA共突变的细胞中有所减弱。包括P110α特异性抑制剂阿培利司（alpelisib）在内的PI3K抑制剂，可显著抑制PIK3CA突变细胞系中ATF4的诱导表达。为进一步探究EGFR-PI3K通路的作用，我们对经奥希替尼、阿培利司或二者联合处理，且在存在或不存在组氨酰-tRNA抑制剂L-组氨醇（L-histidinol, His，模拟氨基酸剥夺条件）的EGFR及PIK3CA突变NCI-H1975细胞中开展了转录组分析。在L-组氨醇诱导的基因中，奥希替尼或阿培利司单独处理仅能部分抑制ATF4靶向基因簇的表达，而二者联合处理则可显著抑制该基因簇的表达。此外，在氨基酸剥夺条件下，奥希替尼与阿培利司联合处理可增加凋亡细胞比例。上述结果表明，致癌性EGFR-PI3K通路可通过ATF4介导细胞对应激条件的适应性调控。我们采用基因芯片（microarrays）鉴定了在氨基酸剥夺条件下，经EGFR、PI3K单靶点或双靶点抑制后表达上调或下调的基因。实验整体设计：将NCI-H1975细胞在存在或不存在2 mM L-组氨醇的条件下，分别用二甲基亚砜（DMSO）、10 nM奥希替尼、1 µM阿培利司或二者联合处理24小时。