Dual targeting of <scp>RET</scp> and <scp>SRC</scp> synergizes in <scp> <i>RET</i> </scp> fusion‐positive cancer cells

Rearranged during transfection (RET) fusions drive subsets of non-small cell lung cancer (NSCLC) and papillary thyroid carcinoma (PTC). Despite new selective RET tyrosine kinase inhibitors (TKIs), resistance usually occurs and is often driven by RET-independent bypass mechanisms. Previous studies have implied crosstalk between RET and proto-oncogene tyrosine-protein kinase SRC, but the anticancer effects of targeting SRC combined with selective RET TKIs, and the underlying molecular mechanisms involved, are not fully understood. Our results show that the multitargeted SRC TKI dasatinib significantly enhanced the efficacy of RET TKIs in RET fusion-positive (RET⁺) NSCLC and PTC cells. Genetic rescue experiments validated that the combination effects between RET TKIs and dasatinib were indeed SRC-dependent. Phosphoproteomics analysis and validation using selective inhibitors and small interfering RNAs (siRNAs) determined that synergy was primarily mediated by suppression of downstream serine/threonine-protein kinase PAK signaling, with contributions from AKT and ribosomal protein S6. Importantly, synergy was also observed with eCF506 (NXP900), a next-generation clinical SRC inhibitor. Finally, both SRC TKIs restored sensitivity in selpercatinib-resistant RET⁺ PTC cells. These results elucidate RET and SRC signaling crosstalk in RET⁺ NSCLC and PTC, suggesting that co-inhibiting SRC has clinical potential in TKI-naïve and -resistant RET⁺ cancers.