Abstract 1102: Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC

Abstract ALK/ROS1 TKIs have significantly improved outcomes for patients with ALK/ROS1+ lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately 1/3 of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in most patients remains unknown/poorly characterized. To model and characterize acquired resistance to crizotinib, we used ROS1+ and ALK+ primary, patient tumor-derived non-small cell lung cancer (NSCLC) cell lines to generate acquired resistance models to the ALK/ROS1 inhibitor crizotinib. We used several techniques to probe the mechanisms driving resistance: DNA and RNA sequencing (seq), fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. The crizotinib-resistant line initially driven by a TPM3-ROS1 fusion (CUTO28-CR) displayed exquisite sensitivity to Src family kinase (SFK) TKIs in proliferation assays, which was modestly improved by addition of ERBB TKI afatinib. Bulk RNA seq revealed upregulation of SFKs Fyn and Src transcripts (141- and 2.31-fold, respectively). Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells vs parental (EGFR:CEP7 ratio 2.1 vs 1.0), and RNA seq revealed upregulation of EGFR and HER2 transcripts (1.79- and 2.45-fold, respectively). Western blot analysis confirmed upregulation of Fyn at the protein level. CUTO28-CR cells treated with a SFK TKI revealed dose-dependent deactivation of Akt signaling, and addition of afatinib caused inhibition of MAPK signaling. Stimulation with epidermal growth factor (EGF) enhanced MAPK signaling in CUTO28-CR cells more than in parental cells, consistent with EGFR/HER2 dependence. Because of a noted propensity for the EML4-ALK line (H3122) to upregulate ERBB kinase activity under sustained ALK TKI treatment, we maintained these cells in low dose (25nM) afatinib while driving crizotinib resistance to identify additional bypass signaling pathways. These H3122-CAR cells were partially resensitized to crizotinib with the addition of SFK TKIs, and the addition of afatinib with SFK TKIs further resensitized these resistant cells to crizotinib. Bulk RNA seq of H3122-CAR cells revealed upregulation of Fyn and Src (2.64- and 3.63-fold, respectively), and western blot analysis confirmed upregulation at the protein level. In conclusion, we demonstrated increased SFK (particularly Fyn and/or Src) expression and/or activation is a mechanism utilized to drive bypass signaling, in cooperation with ERBB family kinases, to circumvent treatment with ALK/ROS1 TKI crizotinib. To our knowledge, this is the first report of SFK-driven bypass signaling as a mechanism of acquired resistance to an ALK/ROS1 TKI in NSCLC. The upregulation of Src and/or Fyn in NSCLC biopsies may guide the exploration of combination SFK TKIs with ALK/ROS1 TKIs. Citation Format: Logan C. Tyler, Nan Chen, Anh T. Le, Hala Nijmeh, Liming Bao, Robert C. Doebele. Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1102.