Abstract 1865: Phosphoproteomic analyses identify EGFR as a critical cooperative kinase for ALK
Authors: Chen Nan , Le Anh T. , Doak Andrea E. , Zhang Guolin , Fang Bin , Haura Eric B. , Doebele Robert C. 
Published: September 2020

ALK fusion oncogenes occur in ~5% of human lung adenocarcinomas. ALK tyrosine kinase inhibitors (TKIs) improve patient outcomes, but rarely achieve a complete tumor response and all patients ultimately relapse. In order to minimize or eliminate residual disease, it is crucial to understand the mechanisms that allow a survival niche for cancer cells under oncogene inhibition. Our lab has shown that EGFR signaling can mediate responses to fusion kinase inhibitors. However, the detailed signaling mechanism of EGFR and ALK in cancer cells is still not fully understood. In order to interrogate the contribution of EGFR to downstream signaling under ALK inhibition, we conducted phosphoproteomics for two ALK-driven cell lines, DFCI032 and H3122, using the ALK inhibitor crizotinib or in combination with the EGFR inhibitor gefitinib. Kinase-Substrate Enrichment Analysis (KSEA) and Phosphoproteomics Dissection Using Networks (PHOTON) analysis were used to identify whether a signaling node was functionally regulated based on substrate phosphorylation or on protein-protein interaction partner phosphorylation, respectively. Strikingly, both methods showed that EGFR was functionally downregulated by ALK inhibition. ALK was also considered functionally more downregulated under EGFR/ALK dual inhibition compared to ALK inhibition alone by KSEA analysis. These data suggest ALK and EGFR engage in overlapping signaling pathways. We speculated that based on the signaling patterns shared by EGFR and ALK, EGFR can be preferably selected by cancer cells to maintain critical downstream signaling mediated by ALK so upfront inhibition of EGFR may prevent ALK TKI resistance. Indeed, co-treatment with the pan-HER inhibitor afatinib in combination with crizotinib or certinib eliminated residual colonies in 2-week clonogenic assay and prevented resistance emergence in 6 weeks monitored by live cell imaging. To investigate the resistance mechanism induced by dual ALK/EGFR inhibition, we generated resistant cells by exposing H3122 to a fixed high dose of crizotinib alone (H3122-CR) or crizotinib plus afatinib (H3122-CAR). Resistance to combination therapy was once again delayed in generating H3122-CAR cells, which took ~6 months as compared to ~3 months for H3122-CR cells (n=2 for CAR and n=3 for CR cell lines). Interestingly, FAK phosphorylation was increased in H3122-CAR cells compared to H3122-CR cells (and parental cells). Although FAK inhibitors modestly re-sensitize H3122-CAR cells to ALK inhibition, the distinct signaling alterations between H3122-CAR and H3122-CR cells suggest that that cancer cells are forced to reprogram signaling under dual ALK/EGFR inhibition compared to ALK inhibition alone. Collectively, EGFR and ALK dual inhibition can significantly delay or prevent the emergence of ALK TKI resistance by suppressing critical overlapping signaling pathways and promotes signaling reprogramming in cancer cells.

Citation Format: Nan Chen, Anh T. Le, Andrea E. Doak, Guolin Zhang, Bin Fang, Eric B. Haura, Robert C. Doebele. Phosphoproteomic analyses identify EGFR as a critical cooperative kinase for ALK [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1865.