Computational-experimental repurposing reveals synergistic sorafenib/hydroxychloroquine response in KRAS-mutant breast cancer.

Abstract

BACKGROUND: KRAS mutations are approximately 25% of human cancers, with particularly high incidence in breast and lung cancers, by constitutively triggering MAPK/ERK and PI3K/AKT pathways that advance proliferation, survival, and autophagy-mediated resistance. Targeting these pathways with kinase inhibitors upregulates autophagy, thereby diminishing therapeutic efficacy. Thus, combining kinase inhibitors with autophagy inhibitors offers a rational strategy to enhance antitumor benefits in KRAS-mutant malignancies. FDA-approved Sorafenib (multi-kinase inhibitor targeting Raf) and Hydroxychloroquine (autophagy inhibitor) show promise for repurposing, as Sorafenib induces autophagy leading to resistance, warranting combination testing in KRAS-mutant models. This study integrates computational modeling and in vitro assays to evaluate their synergistic potential in MDA-MB-231 breast and A549 lung cancer cells. METHODS: Raf-Sorafenib stability was evaluated using RMSD, RMSF, Rg, hydrogen bonds, contact frequency, and MM/GBSA for binding free energy in molecular dynamics simulations (100 ns, GROMACS with CHARMM36 force field). MTT assays were used for cytotoxicity on MDA-MB-231, A549, and normal gingival fibroblasts (48 h treatment); Chou-Talalay Combination Index and Dose Reduction Index were used for synergy; Annexin V/PI flow cytometry was performed for apoptosis; PI staining was used for cell cycle; and ANOVA/Tukey’s test (GraphPad Prism, p < 0.05) was conducted for statistics. RESULTS: Sorafenib bound Raf stably (RMSD ~ 0.25 nm protein/0.15 nm ligand, G_bind − 49.90 ± 2.69 kcal/mol), with persistent interactions (3–4 H-bonds, key residues VAL471, LEU513). IC50 values: Sorafenib 9.4 µM (MDA-MB-231), 12 µM (A549), 23.1 µM (fibroblasts); HQ 23.6/62.4/86.2 µM; SN:2HQ ratio showed synergy in MDA-MB-231 (CI = 0.32, DRI 9.36 Sorafenib/4.68 HQ at Fa = 0.5) but antagonism in A549 (CI > 1). Combination enhanced late apoptosis/necrosis (49.41%) in MDA-MB-231 with minimal normal cell cycle disruption. CONCLUSIONS: Sorafenib-HQ combination offers potent, context-specific synergy for KRAS-mutant breast cancer via Raf inhibition and autophagy blockade, enabling dose reductions and apoptosis enhancement. Tumor-type dependence (synergy vs. antagonism) highlights need for patient stratification; findings support repurposing with limited normal cell impact.