Abstract 1958: EGFR and mTOR signaling facilitate RET-independent resistance to selective RET-TKIs

Abstract RET fusions are an oncogenic driver mutation occurring in 1-2% non-small cell lung cancer (NSCLC). Currently, RET-selective tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib are approved for this patient population, demonstrating favorable antitumor activity and safety profiles in advanced RET fusion-positive NSCLC. However, tumors eventually become refractory to selective RET inhibitor monotherapy. We sought to identify signaling pathways which mediate RET-independent acquired resistance to RET inhibitors and effective therapeutic combinations to overcome resistance. Using LC-2/ad (CCDC6-RET) NSCLC cells, we assessed the expression and activation of critical signaling pathways at a proteomic level following RET knockdown using reverse-phase protein array. RET knockdown resulted in rapidly increased adaptive upregulation of EGFR, HER2, MET, mTOR and MEK pathway signaling; observations were confirmed by Western Blotting. To determine whether EGFR or MET signaling facilitate RET inhibitor resistance, LC-2/ad and NCCE-TH1101 (KIF5B-RET) NSCLC cells were treated with increasing concentrations of selpercatinib or pralsetinib with or without ligands for these receptors, EGF or HGF respectively, and after five days cell viability was measured by CellTiter Glo. EGFR and MET signaling promoted RET TKI resistance in both cell lines. Next, to evaluate whether blockade of EGFR or MET signaling could enhance the activity of RET inhibitors, we treated LC-2/ad and NCCE-TH1101 cells with RET TKIs alone or in combination with the EGFR TKI erlotinib or poziotinib, or the MET inhibitor SU11274. We observed an additive effect with EGFR but not MET inhibitors when combined with RET TKIs. Western blot analysis revealed that EGFR signaling could reactivate mTOR and MAPK signaling after RET TKI treatment. Therefore, we next tested whether inhibition of these downstream pathways might enhance RET TKI sensitivity. We observed that treatment of LC-2/ad and NCCE-TH1101 cells with an mTOR inhibitor (everolimus) or a MEK inhibitor (trametinib) in combination with RET TKIs increased the anti-tumor activity or RET inhibitors. Finally, we established cells with acquired resistance to selpercatinib and pralsetinib. RET TKI resistant cells did not harbor secondary RET fusion mutations but underwent epithelial to mesenchymal transition and exhibited pan-RET inhibitor resistance. Moreover, RET TKI resistant cells showed increased cell surface expression of EGFR and MET by flow cytometry. RET TKI resistant cells were sensitive to mTOR or MEK inhibitors in combination with RET TKIs. Collectively, our findings indicate that in RET-fusion positive NSCLC tumor cells, activation of bypass pathways including the EGFR, MAPK, mTOR pathways may facilitate RET inhibitor resistance and that blockade of bypass pathways may enhance the efficacy of selective RET inhibitors and overcome acquired RET TKI resistance. Citation Format: Tomohiro Takehara, Monique B. Nilsson, Ximeng Liu, Hibiki Udagawa, Naoto Morikawa, Alissa Poteete, Junqin He, Xiaoxing Yu, Thiru Arumugam, Koichi Goto, John V. Heymach. EGFR and mTOR signaling facilitate RET-independent resistance to selective RET-TKIs 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 (6Suppl): Abstract nr 1958.