Abstract 5833: Genomic mechanisms of RET inhibitor resistance in RET-fusion positive NSCLC
Authors: Liu Ximeng , Nilsson Monique , Takehara Tomohiro , He Junqin , Huo Xiaofang , Kumar Ashwani , Heymach John , Kittler Ralf 
Published: March 2024

Abstract
RET fusions are a driver oncogene detected in 1-2% of non-small cell lung cancer (NSCLC) cases. Currently, two selective RET inhibitors (RETi), selpercatinib and pralsetinib have received FDA approval for RET fusion-positive NSCLC. However, RET fusions are highly heterogeneous and the impact of specific fusion partners and breakpoints on RETi sensitivity is not well understood. Moreover, most patients eventually develop resistance to RETi, and secondary RET mutations are thought to be a key mechanism of acquired resistance. Thus, we aimed to identify RET-dependent mechanism of RETi resistance in effort to improve treatment options and clinical outcomes. We engineered a panel of Ba/F3 cells expressing various RET fusion partners and breakpoints observed in clinical cases of NSCLC and evaluated their drug sensitivity profile against a panel of RETi including selpercatinib, pralsetinib, TPX-0046, lovatinib, zeteletinib and ponatinib. We determined that individual RET fusions impart distinct sensitivities to RETi. For example, cells expressing KIF-RET (K22, R12) were sensitive to selpercatinib and pralsetinib but less sensitive to ponatinib, whereas cells expressing KIF-RET (K15, R12) were highly sensitive to selpercatinib, pralsetinib, and ponatinib. Moreover, using RET-fusion positive NSCLCs, we observed that the RETi sensitivity profile of human cell lines was similar to that observed using Ba/F3 models. To identify potential resistance mutations, we utilized the LentiMutate approach. In KIF5B-RET (K15, R12) expressing cells, we identified G810S as being associated with resistance to both selpercatinib and pralsetinib, a finding consistent with previous reports and clinical observations. In addition, we identified Y806H/N, V804M/E, and M918T as being associtated with selpercatinib-resistance and L730I/V, E732K, and Y806H/N as being associated with pralsetinib-resistance. We next constructed Ba/F3 cells expressing KIF5B-RET in combination with each secondary mutation and evaluated RETi sensitivity. G810S and Y806N conferred resistance to selpercatinib and pralsetinib, as well as to RET-targeting multi-kinase inhibitors. V804M/E mutations conferred resistance to selpercatinib but not pralsetinib, whereas L730I/V and E732K mutations conferred resistance to pralsetinib but not selpercatinib, demonstrating RETi-specific resistance mutations. Cells expressing G810S secondary mutations were resistant to selpercatinib and pralsetinib, but sensitive to zeteletinib. Collectively, we show that individual RET fusion variants have distinct drug sensitivity profiles and that secondary resistance mutations are non-overlapping between RETi. The comprehensive characterization of RET-dependent mechanisms of resistance to RETi may provide therapeutic guidance for treating RET-fusion driven NSCLC and provide structural insights that can guide the development of new therapeutic regimens.
Citation Format: Ximeng Liu, Monique Nilsson, Tomohiro Takehara, Junqin He, Xiaofang Huo, Ashwani Kumar, John Heymach, Ralf Kittler. Genomic mechanisms of RET inhibitor resistance in RET-fusion positive NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5833.