SRC Activation Drives Acquired Pemetrexed Resistance in Non-Small Cell Lung Cancer and Is Targetable by Dasatinib

Pemetrexed, a standard antifolate chemotherapy for non-squamous non-small cell lung cancer (NSCLC), is often limited by acquired resistance. We investigated whether SRC kinase mediates pemetrexed resistance and whether SRC inhibition can suppress survival signaling in pemetrexed-resistant NSCLC cells.

Pemetrexed-resistant cell lines (PC9 R, NCI-H460 R, and NCI-H3122 R) were generated through chronic pemetrexed exposure. Protein phosphorylation profiling and Western blotting identified key signaling alterations. SRC was abrogated using transient and doxycycline-inducible CRISPR/Cas9 systems, and reciprocal siRNA knockdown of SRC and thymidylate synthase (TS) was performed. Three SRC inhibitors-dasatinib, bosutinib, and saracatinib-were assessed using cell viability, colony formation, apoptosis, and cell cycle assays.

All pemetrexed-resistant cell lines demonstrated elevated phosphorylated SRC (p-SRC Y416) and enhanced downstream signaling. SRC knockout reduced p-SRC Y416, p-STAT3, and p-FAK, while increasing markers of DNA damage (γ-H2AX) and apoptosis (cleaved caspase-3), impairing cell viability and clonogenicity. SRC knockdown reduced TS expression in two resistant models, whereas TS knockdown did not suppress p-SRC Y416. Among SRC inhibitors, dasatinib showed the most potent, dose-dependent suppression of SRC signaling. Dasatinib also induced G0/G1 arrest, DNA damage, and apoptosis, thereby reducing colony formation.

SRC activation is a critical mechanism of acquired pemetrexed resistance in NSCLC and may contribute to resistance through TS-associated survival signaling. SRC inhibition, particularly with dasatinib, disrupts pro-survival pathways and promotes DNA damage-mediated cell death in pemetrexed-resistant cells. These findings support further investigation of SRC inhibitors, alone or with pemetrexed, as a biomarker-guided therapeutic strategy for SRC-activated pemetrexed-resistant NSCLC.