Drug-specific resistance mechanisms to RET inhibitors in RET fusion-positive NSCLC

Purpose: Ret proto-oncogene (RET) fusions are highly heterogeneous oncogenic drivers occurring in approximately 1-2% of non-small cell lung cancer (NSCLC). While selpercatinib and pralsetinib have dramatically improved clinical outcomes of patients, acquired resistance inevitably develops and effective therapeutic strategies following resistance remain limited. Understanding RET-dependent resistance mechanisms is critical for improving clinical outcomes for this patient population. Experimental Design: A rapid lentivirus-based mutagenesis approach, LentiMutate, was employed to identify potential secondary RET mutations conferring resistance to selective RET inhibitors (SRIs). Candidate resistance mutations were validated using Ba/F3 cell models. Biochemical and structural analysis were conducted to investigate the underlying mechanisms. Candidate mutations were evaluated against next-generation SRIs. Clinical responses after SRIs progression were retrospectively analyzed in patients with RET fusion-positive NSCLC using the MD Anderson real-world cohort. Results: We identified RET secondary mutations that induced SRIs resistance in a drug-specific manner. V804E/M mutations conferred resistance to selpercatinib while L730I/V and A883V mediated resistance to pralsetinib, with A883V representing a novel resistance mutation identified in this study. G810C/D/S, Y806N and E732K mutations induced resistance to both drugs. Molecular modeling suggested that both drug structure and fusion partner-mediated dimerization impacted differential sensitivity of secondary mutations. In a retrospective clinical cohort, one patient with RET fusion-positive NSCLC who acquired G810X mutations at progression on selpercatinib subsequently achieved a durable response to pralsetinib. Conclusions: Our findings indicate that secondary RET mutations can impart drug-specific effects on resistance to SRIs. These results underscore the need for precision therapy based on the specific resistance mutations present.