Abstract 1111: MYC promotes tyrosine kinase inhibitor resistance in ROS1 fusion positive lung cancers

Abstract Targeted therapy of ROS1 fusion-driven non-small cell lung cancer (NSCLC) with the tyrosine kinase inhibitor (TKI), crizotinib, has achieved notable clinical success. Despite these promising results, resistance to therapy poses a significant challenge. 47-92% of crizotinib-resistant tumors have a wild-type ROS1 kinase domain suggesting that concurrent aberrations in bypass signaling pathways contribute to sustained cancer growth even in the presence of ROS1 inhibition. Bypass resistance pathways are not well-characterized in ROS1 fusion-driven cancers; hence there is a dearth of secondary therapeutic strategies. To identify recurrent genomic alterations that co-occur with ROS1 fusions and could potentially impinge on sensitivity to crizotinib and other emerging ROS1 TKIs, we interrogated MSK-IMPACT datasets and found that 10% of patients with ROS1-fusion have concurrent MYC copy number amplification (CNA). We hypothesized that c-Myc overexpression compensates for the loss of ROS1 signaling due to ROS1 TKI treatment. Here we dissected the role of c-Myc overexpression in diminishing ROS1 TKI sensitivity using two models: 1) a MYC-amplified and crizotinib-resistant patient-derived ROS1-fusion NSCLC cell line (LUAD0006) and 2) a CRISPR-Cas9 engineered human bronchial epithelial EZR-ROS1 cell line (HBEC-ER) exhibiting sensitivity to ROS1 TKI. We validated LUAD0006 ROS1 TKI-resistance using cell viability assays and confirmed MYC CNA and protein overexpression with fluorescence in situ hybridization and immunoblotting, respectively. Live cell imaging assays revealed that ROS1-TKI or c-Myc knockdown via shRNA alone were insufficient to inhibit LUAD0006 cell growth; however combined ablation of ROS1 kinase activity and c-Myc expression significantly suppressed cell proliferation. Gain of function experiments using HBEC-ER revealed that these cells acquire functional resistance to multiple ROS1 TKIs upon expression of a degradation-resistant c-Myc T58A mutant. Notably, immunoblotting confirmed 50-fold overexpression of c-Myc T58A relative to endogenous c-Myc in unmodified cells. We also performed RNA sequencing and reverse-phase protein array (RPPA) analysis of LUAD0006 cells treated with or without crizotinib and found that targets related to cell cycle, apoptosis, and metabolism were differentially expressed. Finally, we found that CDK4/6 and BET bromodomain inhibitors re-sensitized LUAD0006 cells to crizotinib, demonstrating the importance of c-Myc to the survival of these TKI-resistant cells. Taken together, we demonstrate that CNA or post-translational stabilization that leads to c-Myc overexpression confers resistance to crizotinib and other emerging ROS1 TKIs. These patients will likely benefit from a combination therapeutic strategy that targets both ROS1 and c-Myc. Citation Format: Sudarshan R. Iyer, Igor Odintsov, Adam J. Schoenfeld, Hiroki Sato, Evan Siau, Alexander Drilon, Gregory J. Riely, Romel Somwar, Marc Ladanyi, Monika A. Davare. MYC promotes tyrosine kinase inhibitor resistance in ROS1 fusion positive lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1111.