Abstract 5351: LTK mutations responsible for resistance to lorlatinib in non-small cell lung cancer harboring CLIP1-LTK fusion
Authors: Mori Shunta , Izumi Hiroki , Liu Jie , Tanaka Kosuke , Kumagai Shogo , Hayashida Takuma , Kagawa Yosuke , Shibata Yuji , Matumoto Shingo , Goto Koichi , Kobayashi Susumu 
Published: June 2022

Background: CLIP1-LTK was recently discovered as a novel oncogenic gene fusion in non-small cell lung cancer (NSCLC). Lorlatinib, an ALK/ROS1 inhibitor, can inhibit CLIP1-LTK constitutive kinase activity, and showed dramatic and promising efficacy in a patient with NSCLC harboring CLIP1-LTK fusion. However, acquired resistance to lorlatinib will be inevitably developed, especially by mutations in LTK gene, as 50-60% of oncogenic driver-positive NSCLC develop resistance to kinase inhibitors by acquired mutations in the target gene. The aim of this study is to identify the LTK mutations responsible for resistance to lorlatinib in CLIP1-LTK fusion positive NSCLC, and to explore compounds that can overcome resistance.

Methods: As LTK and ALK share nearly 80% protein sequence identity in their respective kinase domains, we established Ba/F3 cells expressing CLIP1-LTK (Ba/F3-CLIP1-LTK) with six different LTK mutations (I565N, F568C, L590M, L592F, G596R, and G663A), all of which are analogous to reported ALK mutations responsible for resistance to lorlatinib. Ba/F3 cells expressing CLIP1-LTK with these different LTK mutations, as well as CLIP1-LTK-wild type (WT) were treated with several concentrations of lorlatinib. Then, the efficacy of other compounds including crizotinib, alectinib, ceritinib, brigatinib, entrectinib, giltertinib, repotorectinib was assessed to detect which compounds can overcome lorlatinib resistance.

Results: All LTK mutations tested showed resistance to lorlatinib, with IC50s of lorlatinib ranging 2.7 to 31.3 nM, which were higher than that in Ba/F3-CLIP-LTK-WT (1.0 nM) in cell viability assay. An IC50 of giltertinib in Ba/F3-CLIP1-LTK-F568C was lower than that of lorlatinib (0.9 vs 2.7 nM). Moreover, an IC50 of giltertinib in Ba/F3-CLIP1-LTK-G663A was lower than that of lorlatinib (2.7 vs 19.4 nM). Ba/F3-CLIP1-LTK-L592F was rather sensitive to crizotinib compared with Ba/F3-CLIP1-LTK-WT (IC50, &lt;0.1 vs 16.5 nM). Similarly, Ba/F3-CLIP1-LTK-G596R was similar to repotorectinib compared with WT (IC50, 10.4 vs 11.3 nM). The results of western blotting assay evaluating phosphorylation of CLIP1-LTK were in accordance with that obtained cell viability assay.

Conclusion: We identified that several LTK mutations, analogous to ALK resistant mutations, were responsible for lorlatinib resistance, some of which can be overcome by existing compounds. Further validation and exploration are warranted to establish resistance mechanism-based precision medicine following lorlatinib treatment in CLIP1-LTK fusion-positive NSCLC.

Citation Format: Shunta Mori, Hiroki Izumi, Jie Liu, Kosuke Tanaka, Shogo Kumagai, Takuma Hayashida, Yosuke Kagawa, Yuji Shibata, Shingo Matumoto, Koichi Goto, Susumu Kobayashi. LTK mutations responsible for resistance to lorlatinib in non-small cell lung cancer harboring CLIP1-LTK fusion [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 5351.