Unique vulnerability of RAC1-mutant melanoma to combined inhibition of CDK9 and immune checkpoints

RAC1^P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1^P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1^P29S-harboring melanomas. To investigate the RAC1^P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1^P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1^P29S-mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1^P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1^P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.