Tyrosine phosphoproteomics identified both co-drivers and co-targeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer RUNNING TITLE: Phosphoproteomics of EGFR-TKI resistance

Purpose : Irreversible EGFR-tyrosine kinase inhibitors (TKIs) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gate-keeper mutations in non-small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M. Experimental Design: We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI-resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass-spectrometry-based identification/quantification. Profiles of erlotinib perturbations were examined. Results: We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src-family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK-inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced anti-tumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib. Conclusions: Our results identified both co-drivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in NSCLC patients with acquired T790M. a the molecular between EGFR-TKI-sensitive and -resistant pairs. The driving force this the limited efficacy of irreversible EGFR-TKIs in targeting T790M, as shown in both preclinical and clinical studies (8, 9, 12, 14-19), and the ability of other tyrosine kinases, especially RTKs, to limit EGFR-TKI efficacy. Through a systematic interrogation of pTyr peptides and proteins using LC-MS/MS, we identified both RTKs and non-RTKs able to be recruited to confer erlotinib sensitivity. In PC9GR cells, we identified higher levels of pTyr peptides corresponding to MET signaling, including more MET, ROR1, and Gab1/2 proteins. Interestingly, this coordinated activation of MET signaling was not secondary to MET gene amplification, as our FISH results revealed no amplification of the MET gene. The results with the basal phosphoproteome corresponded to HGF’s strong effects in protecting both PC9 and PC9GR cells from erlotinib or afatinib, respectively. Additionally, these results suggest a form of “lineage” addiction, whereby resistant cells with T790M can carry forward RTKs that can cooperate to drive resistance. Importantly, these results suggest that interrogating protein activation status or network signaling may highlight proteins that play a role in protecting cells against EGFR-TKI, especially when in a microenvironment rich with cognate growth factor ligands. Despite observing more AXL pTyr peptides in PC9GR cells, we demonstrated no ability of AXL pathway activation by Gas6 ligand to drive resistance to either erlotinib or afatinib. not clear, our of of pTyr peptides. adaptive resistance to targeted agents and could in some cases with factors, such as growth factors, to limit TKI efficacy. Collectively, these results suggest that cell intrinsic (receptors, signaling proteins) and extrinsic (ligands) factors can collaborate to drive resistance to kinase inhibitors in a systems-level manner. Our phosphoproteomics analyses in PC9 and PC9GR cells demonstrated that SFKs are also critical as an EGFR-independent co-signal in NSCLC cells with T790M. These results were enabled by tyrosine phosphorylation profiling combined with analysis of proteins based on known protein-protein interactions. We validated the inferences derived from the phosphoproteomics by showing that afatinib combined with dasatinib resulted in anti-tumor activity regarding cell proliferation and apoptosis in and HCC827-T790M cells (each harboring T790M). These results appear to be generalized to additional T790M EGFR-TKIs, as dasatinib demonstrated similar combination effects with the T790M-selective EGFR-TKI WZ4002 (40). We did not observe combination effects with afatinib plus dasatinib compared to either agent alone in cells with TKI-sensitive EGFR mutation only (PC9, HCC4006, and HCC827 cells) or wild-type EGFR (H460, A549, and H1299 cells). The enhanced apoptosis with combined afatinib and dasatinib in the cells with T790M translated into improved in vivo effects on tumor growth in PC9GR cells. Collectively, our results suggest that dasatinib can be generally used as a combination therapy with irreversible or T790M-selective EGFR-TKIs for NSCLC patients results of cell models. In addition, our mass spectrometry data from tumor samples with TKI-sensitive EGFR mutation demonstrated a high degree of overlap with results from cell models, thereby validating the overall approach. These results from tumor samples suggest that our results from lung cancer cell line models are applicable to translate in to the clinic. Our previous chemical and phosphoproteomic characterization identified nearly 40 different kinase targets of dasatinib and showed that SRC, FYN, and EGFR are relevant targets for dasatinib action in NSCLC (32). Our recent phase I/II study showed that dasatinib combined with erlotinib is tolerable, with 63% of advanced NSCLC patients showing disease control, including two having partial response and one having bone response (48). Another group also showed that dasatinib combined with erlotinib is safe and feasible in NSCLC Based on these clinical studies along with the experiments reported here, dasatinib has potential clinical activity in NSCLC treatment, but this is limited to combinations with T790M-targeted agents and in genotype-specific patients. This will be formally tested in a phase I trial of afatinib and dasatinib (NCT01999985). Our results also highlight the ability of phosphoproteomics to identify other important mediators of drug sensitivity, and examination of these proteins may be important in clinical studies of T790M-targeting agents.