Receptor tyrosine kinase (RTK)-targeted therapies are invariably limited by drug resistance. A major mechanism of acquired resistance involves “bypass” switching to alternative pathways driven by non-targeted RTKs that reactivate proliferation. These pathways are often centered around Erk/Akt signaling; however, a more fundamental understanding of network-level bypass resistance is required to better inform the design of effective combination therapies. Overexpression of the RTK AXL is frequently observed in bypass resistant tumors which, in addition to cell survival, promotes the development of further malignant phenotypes such as epithelial-to-mesenchymal transition and immunosuppression. Nonetheless, the molecular pathways engaged to enact these phenotypic outcomes still remain elusive. Here, we sought to analyze the global downstream signaling changes of lung cancer cells (PC9) when switching to
AXL and other RTKs upon the use of an EGFR inhibitor erlotinib. Using k-means clustering and partial least square regression (PLSR), LC-MS/MS-generated phosphorylation measurements across the different bypass conditions predict the cell viability and enable systematic identification
of the key signaling molecules and pathways involved. Consistent with previous findings, we observed a strong correlation between cell viability and pro-mitotic kinases such as MAPK1 & 3, Grb2, and AXL, and an inverse correlation with pro-apoptotic signaling molecules such as RIPK1 and cytoskeletal-remodeling and migration-related proteins Filamin-A, Annexin A2, and Paxillin. Our future directions will focus on reimplementing this analysis using a panel of established mutant cell lines in which each AXL intracellular tyrosine is mutated to phenylalanine. This will enable us to attribute variation in bypass resistance capacity to individual phosphorylation events. Our study underscores the relevance of investigating non-genomic variation to effectively identify key signaling molecules driving bypass resistance and concomitant phenotypes in cancer.

Authors: Marc Creixell^1, Jacqueline Gerritsen², Song Yi Bae¹, Forest M. White² and Aaron S. Meyer^1
1Department of Bioengineering, Jonsson Comprehensive Cancer Center, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; ²Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Center for Precision Cancer Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139, USA