Global proteome and phosphoproteome alterations in 3rd generation EGFR TKI resistance reveal drug targets to circumvent resistance

Lung cancer continues to be the leading cause of cancer mortality worldwide. The treatment of lung cancer patients harboring mutant EGFR with orally administered EGFR TKIs has been a paradigm shift. Osimertinib and rociletinib are two 3rd generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance to it. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here, we have characterized the proteome and phosphoproteome of a series of isogenic EGFR mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs. To our knowledge, this is the most comprehensive proteomic dataset resource to date to investigate 3rd generation EGFR TKI resistance in lung adenocarcinoma. We have interrogated this unbiased global quantitative proteomic and phosphoproteomic dataset to uncover alterations in signaling pathways, and to reveal a proteomic signature of EMT and kinases / phosphatases with altered protein expression and phosphorylation in the TKI resistant cells. We validated the significant role of SHP2 in the activation of RAS/MAPK and PI3K/AKT signaling pathways. Furthermore, we performed anticorrelation analyses of this phosphoproteomic dataset with the published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures (LINCS) program to predict drugs with the potential to overcome EGFR TKI resistance. We identified that dactolisib, a PI3K/mTOR inhibitor, in combination with osimertinib, may overcome osimertinib resistance both in vitro and in vivo. Introduction Lung cancer continues to be the leading cause of cancer mortality in the world (1). Many lung adenocarcinoma patients with activating epidermal growth factor receptor (EGFR) mutations initially respond dramaticlly to the first- or second-generation EGFR tyrosine kinase inhibitors (TKIs). However, they eventually develop resistance. The most common mechanism of acquired resistance is the EGFR T790M gatekeeper site residue mutation (2). Osimertinib, a third generation irreversible EGFR TKI has been approved by the FDA to treat patients harboring the EGFR T790M mutation who have developed resistance to first- and second- generation EGFR TKIs (3). Recently, osimertinib was also approved for the front-line treatment of patients harboring EGFR mutations (4). Rociletinib is another irreversible inhibitor targeting the EGFR T790M mutation, which has minimal activity against wild-type EGFR. Both drugs have therapeutic benefits and have demonstrated activity in tumors with T790M-mediated resistance to other EGFR tyrosine kinase inhibitors (5, 6). Further development of rociletinib was ceased in 2016 due to less than expected efficacy, poor brain penetration leading to tumor progression in brain tissues and off-target effects on IGFR activation leading to hyperglycemia (7, 8). Although 3rd-generation TKIs provide clinical benefit to most patients with EGFR mutations, some patients, demonstrating primary resistance, still do not respond to these inhibitors. Complete responses are rare, and all patients eventually develop resistance, suggesting primary and acquired resistance mechanisms decrease the efficacy of the drugs (9, 10). Genomic approaches have been used primarily to interrogate osimertinib resistance mechanisms (9, 11-15). Several mechanisms of osimertinib resistance have been identified (16), including novel second site EGFR mutations, activated bypass pathways such as MET amplification, HER2 amplification, RAS mutations, BRAF mutations, PIK3CA mutations, and novel fusion events (17). However, the resistance mechanism is complex and still not fully understood. Previously, we have used SILAC-based quantitative phosphoproteomics to identify the global dynamic modification which occur upon treatment of TKI-sensitive and -resistant lung adenocarcinoma cells with the 1st and 2nd generation EGFR TKIs, erlotinib and afatinib. Utilizing this strategy, we identified the targets of mutant EGFR signaling pathways responsible for TKI resistance, and possible off-target effects of the drugs (18, 19). In this study, we employed SILAC-based quantitative mass spectrometry to characterize alterations in the proteome and phosphoproteome which occur upon acquired resistance and sought to identify novel mechanisms of resistance to the third generation EGFR TKIs, osimertinib and rociletinib. To our knowledge, this is the most comprehensive 3rd generation EGFR TKI resistant proteome and phosphoproteome analysis resource available to date.

肺癌仍是全球范围内癌症相关死亡的首要病因。针对携带表皮生长因子受体（EGFR）突变的肺癌患者，口服EGFR酪氨酸激酶抑制剂（TKIs）的治疗方案堪称治疗范式的革新。奥希替尼（Osimertinib）与罗西替尼（rociletinib）是两款靶向EGFR T790M突变的第三代不可逆EGFR TKIs。奥希替尼凭借更高的疗效、更低的不良反应以及更强的血脑屏障穿透性，目前已成为EGFR突变患者的标准治疗方案。遗憾的是，所有患者最终都会对其产生耐药性。既往研究多采用基因组学方法探究其耐药机制。本研究针对一系列对上述药物敏感或耐药的同基因EGFR突变肺腺癌细胞系，对其蛋白质组与磷酸化蛋白质组进行了系统表征。据我们所知，这是目前为止用于探究肺腺癌中第三代EGFR TKI耐药机制的最全面的蛋白质组学数据集资源。我们通过分析该无偏倚的全局定量蛋白质组与磷酸化蛋白质组数据集，揭示了信号通路的异常改变，并鉴定出TKI耐药细胞中上皮间质转化（EMT）相关蛋白质组特征以及蛋白表达与磷酸化水平发生异常的激酶/磷酸酶。我们验证了SHP2在RAS/MAPK与PI3K/AKT信号通路激活中的关键作用。此外，我们将该磷酸化蛋白质组数据集与已发表的、来自整合网络细胞信号库（Library of Integrated Network-Based Cellular Signatures, LINCS）项目的药物诱导P100磷酸化蛋白质组数据集进行反相关分析，以预测有望克服EGFR TKI耐药的药物。我们发现，PI3K/mTOR抑制剂达科替尼（dactolisib）与奥希替尼联合使用，可在体内外模型中克服奥希替尼耐药。

引言
肺癌仍是全球范围内癌症相关死亡的首要病因[1]。众多携带激活型表皮生长因子受体（EGFR）突变的肺腺癌患者，最初对第一代或第二代EGFR酪氨酸激酶抑制剂（TKIs）可产生显著应答。但这类患者最终会产生耐药性。最常见的获得性耐药机制为EGFR T790M守门位点突变[2]。第三代不可逆EGFR TKI奥希替尼已获美国食品药品监督管理局（Food and Drug Administration, FDA）批准，用于治疗对第一代、第二代EGFR TKIs产生耐药的EGFR T790M突变患者[3]。近期，奥希替尼还被批准用于EGFR突变患者的一线治疗[4]。罗西替尼（rociletinib）是另一款靶向EGFR T790M突变的不可逆抑制剂，对野生型EGFR的活性极低。两款药物均具有治疗获益，且在因T790M介导而对其他EGFR TKIs产生耐药的肿瘤中展现出抗肿瘤活性[5,6]。由于疗效未达预期、血脑屏障穿透性差导致脑组织肿瘤进展，以及对IGFR激活的脱靶效应引发高血糖，罗西替尼的开发于2016年终止[7,8]。

尽管第三代EGFR TKIs为多数EGFR突变患者带来临床获益，但仍有部分表现为原发性耐药的患者无法从这类治疗中获益。完全应答十分罕见，且所有患者最终都会产生耐药，这表明原发性与获得性耐药机制会降低药物疗效[9,10]。既往研究多采用基因组学方法探究奥希替尼的耐药机制[9,11-15]。目前已鉴定出多种奥希替尼耐药机制[16]，包括新型EGFR第二位点突变、激活的旁路通路（如MET扩增、HER2扩增、RAS突变、BRAF突变、PIK3CA突变）以及新型融合事件[17]。但耐药机制十分复杂，目前尚未完全阐明。

此前，我们采用基于稳定同位素标记氨基酸细胞培养（Stable Isotope Labeling with Amino acids in Cell culture, SILAC）的定量磷酸化蛋白质组学技术，分析了TKI敏感与耐药肺腺癌细胞经第一代、第二代EGFR TKIs厄洛替尼与阿法替尼处理后的全局动态修饰变化。通过该策略，我们鉴定出了与TKI耐药相关的突变EGFR信号通路靶点，以及药物可能存在的脱靶效应[18,19]。本研究采用基于SILAC的定量质谱技术，对获得性耐药过程中蛋白质组与磷酸化蛋白质组的改变进行系统表征，以期鉴定出第三代EGFR TKIs（奥希替尼与罗西替尼）耐药的新型机制。据我们所知，这是目前为止最为全面的第三代EGFR TKI耐药蛋白质组与磷酸化蛋白质组分析资源。