Abstract: Objective  To investigate the role and molecular mechanisms of Rac GTPase-activating protein 1 (RACGAP1) in resistance to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, and to determine whether targeting RACGAP1 can synergistically enhance the efficacy of CDK4/6 inhibitors and reverse drug resistance phenotypes, thereby providing a novel therapeutic target strategy for overcoming resistance in breast cancer and other malignancies.  Methods  A Ribociclib-resistant MCF-7 sub-line was established by long-term exposure. Whole-cell pellets were subjected to RNA-seq to identify differentially expressed transcripts between parental and resistant cells. Genes consistently up-regulated across multiple CDK4/6 inhibitor models were prioritized. Public breast cancer data from The Cancer Genome Atlas (TCGA) were interrogated using the R package clusterProfiler to perform gene set enrichment analysis (GSEA) and map downstream pathways.  Results  Both RACGAP1 was markedly elevated at the mRNA and protein levels in resistant cells and in TCGA tumors, and their high expression correlated with poor response to Ribociclib. Genetic knockdown of RACGAP1 restored sensitivity to CDK4/6 inhibitors and synergistically inducing apoptosis and suppressing clonogenicity.  Conclusion  Elevated RACGAP1 drive CDK4/6 inhibitor resistance by modulating cell-cycle progression and microtubule dynamics. Targeting these genes re-sensitizes resistant breast cancer cells, offering a clinically implementable strategy to overcome CDK4/6 inhibitor resistance.

Key words: Rac GTPase-activating protein 1, cyclin-dependent kinase 4/6 inhibitors, resistance, Ribociclib, breast cancer, cell cycle