56P - SLC34A2-ROS1 L2026M+G2032R confers resistance to ROS1 tyrosine kinase inhibitors in Ba/F3 cells through a reduced ATP binding pocket volume

Background

The ROS1 G2032R mutation is the most common on-target resistance mutation in crizotinib treated ROS1-positive lung cancer patients. The aim of our study was to investigate resistance mechanisms in SCL34A2-ROS1^G2032R positive Ba/F3 cells against second line treatment with lorlatinib.

Methods

Ba/F3 SLC34A2-ROS1^G2032R cells were subjected to N-ethyl-N-nitrosourea (ENU) mutagenesis and clones were selected upon treatment with 1000 nM lorlatinib for 4 weeks. Resistant clones were analyzed for presence of on-target resistant mutations using Sanger sequencing. In addition, we generated subclones expressing SLC34A2-ROS1^{L2026M+G2032R} and SLC34A2-ROS1^L2026M in Ba/F3 cells. Sensitivity to ROS1 TKIs was determined by measuring cell viability and ROS1 phosphorylation. Molecular Dynamic simulations of the ATP binding pocket were performed for all ROS1 variants.

Results

The ENU-screen of 41 lorlatinib resistant clones revealed one with a mutation in the kinase domain: L2026M. Cell viability assays of the ENU-induced resistant cell line and the Ba/F3 cells transfected with the mutant SCL34A2-ROS1 fusion gene constructs revealed a decreased sensitivity of SLC34A2-ROS1^{L2026M+G2032R} cells for lorlatinib, crizotinib, entrectinib and repotrectinib compared to the single mutants. Consistent with these findings, we observed phosphorylation of ROS1 fusion protein in the double mutant cells which was not inhibited upon treatment with ROS1 TKIs. The single mutant cells showed as expected a clear reduction in phosphorylated ROS1 fusion protein . Molecular modeling to unravel the effect of the mutations demonstrated that the volume of the ATP-binding pocket was reduced in single and double mutants compared to wild type. The double L2026M+G2032R mutant displayed the smallest pocket.

Conclusions

We identified a novel on-target mutation after inducing lorlatinib resistance in SLC34A2-ROS1^G2032R Ba/F3 cells. This SLC34A2-ROS1^{L2026M+G2032R} cell line was also resistant to crizotinib, entrectinib and repotrectinib. The resistance can be explained by a smaller ATP binding pocket in the mutated ROS1 fusion protein preventing effective binding of the investigated TKIs.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

The Netherlands Organization for Scientific Research (NWO) Doctoral Grant for Teachers to C. Dijkhuizen Applied Research SIA (regieorgaan SIA) HBO-Postdoc Grant to M. Smelt.

Disclosure

A.J. Van Der Wekken: Financial Interests, Institutional, Funding: AstraZeneca, Boehringer Ingelheim, Pfizer, Roche, Takeda; Non-Financial Interests, Institutional, Advisory Board: AstraZeneca, Lilly, Roche, Takeda; Non-Financial Interests, Institutional, Speaker, Consultant, Advisor: AstraZeneca, BMS, Lilly, Pfizer, Roche; Financial Interests, Institutional, Other, involved in Clinical Studies: AstraZeneca, Amgen, Blueprint Medicin, Nuvalent, Novartis, Merck, Pfizer, Roche, Takeda. All other authors have declared no conflicts of interest.