Abstract A98: An in vitro mutagenesis screen identifies L1951R and G2032R as drug-resistant mutants of CD74-ROS1.

Abstract

Abstract NSCLC can be divided into a series of genomically-defined subsets, each generally containing a distinct molecular driver. The development of drugs that specifically target drivers such as mutant EGFR (erlotinib) and ALK fusions (crizotinib) has proven to be an effective therapeutic strategy. Despite these treatment advances, drug resistance inevitably occurs via a variety of mechanisms. Drug resistant point mutations of the driver oncogene are a common occurrence, with gatekeeper mutants often being problematic. ROS1 is rearranged in approximately 1.5% of NSCLC patients and is sensitive to crizotinib. Since ALK and ROS1 are structurally related, we generated Ba/F3 cells lines expressing CD74-ROS1 and tested their sensitivity to key ALK- targeted inhibitors currently in clinical testing. Crizotinib, AP26113, LDK378, and ASP3026 inhibited the viability of CD74-ROS1 expressing cells with IC50s significantly below their clinically-achievable plasma concentrations while CH5424802 was inactive thus highlighting that co-optimization of ROS1 activity is not a universal property of all ALK inhibitors. To investigate potential drug resistant mutant liabilities associated with each inhibitor, we performed a large scale mutagenesis screen, coupled with next generation sequencing, in Ba/F3 CD74-ROS1 cells exposed to increasing concentrations of each inhibitor. From our screens we identified a number of candidate mutants spanning the kinase domain of ROS1. L1951R and G2032R were the most frequent mutants in each screen, but mutation of the ROS1 gatekeeper residue (L2026) was not observed. To confirm the degree of resistance imparted by each mutant, we engineered CD74-ROS1 Ba/F3 cells to express the key ROS1 mutants identified and determined their sensitivities to crizotinib, AP26113, LDK378 and ASP3026. In addition to their frequent occurrence in our screens, L1951R and G2032R also conferred the highest degree of resistance (>50-fold) to each inhibitor. A ROS1 structural homology model indicates that L1951 and G2032 form part of the ATP-binding pocket of ROS1, and mutation of either residue to the larger amino acid arginine negatively impacts inhibitor activity without affecting the ability of CD74-ROS1 to transform cells. Interestingly, the analogous G2032R mutant in ALK (G1202R) has been observed as a clinical resistance mechanism induced by crizotinib, but remains sensitive to AP26113 (IC50 379 nM) in preclinical studies. In conclusion, we have demonstrated that crizotinib, AP26113, LDK378, and ASP3026 inhibit ROS1 activity. However, L1951R and G2032R ROS1 mutants are particularly resistant to each inhibitor. These preclinical data are supported by the recent identification of a G2032R mutant in a ROS1 patient who acquired resistance to crizotinib treatment, suggesting that generation of a ROS1-targeted compound that potently inhibits L1951R and G2032R mutants will ultimately be needed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A98. Citation Format: Rana Anjum, Sadanand Vodala, Anna Kohlmann, Victor M. Rivera, Andrew P. Garner. An in vitro mutagenesis screen identifies L1951R and G2032R as drug-resistant mutants of CD74-ROS1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A98.