Focal Adhesion Kinase-Dependent Reprogramming of IFN-γ Signaling through PYK2 Coinhibition Sensitizes Melanoma to Immune Checkpoint Blockade.

Immune checkpoint blockade has transformed melanoma therapy but is frequently limited by acquired resistance. Focal adhesion kinase (FAK) and its homolog PYK2 are nonreceptor tyrosine kinases that coordinate cell-matrix adhesion and cytoskeletal signaling. In this study, we define the distinct roles of FAK (as the principal scaffold) and PYK2 (in a supportive capacity) in IFN-γ-mediated immune checkpoint blockade resistance. Label-free phosphoproteomic profiling revealed that IFN-γ stimulation drives extensive phosphorylation across pathways governing cytoskeletal remodeling, transcriptional regulation, mRNA splicing, and ribosomal RNA biogenesis. Selective FAK inhibition markedly suppressed IFN-γ-induced signal transducer and activator of transcription 1 and PD-L1 upregulation, and the addition of PYK2 blockade achieved maximal suppression, culminating in enhanced CD8 T-cell-mediated tumor cytotoxicity. In an anti-PD-1-resistant murine melanoma model, high tumoral FAK expression correlated with treatment failure. Analysis of The Cancer Genome Atlas-Skin Cutaneous Melanoma cohort showed that FAK levels associate with immunosuppressive gene signatures, whereas in an independent clinical dataset (GSE91061), patients exhibiting post-treatment downregulation of FAK experienced improved outcomes. Single-cell RNA sequencing distinguished tumor cell-intrinsic FAK expression from PYK2 enrichment in immune subsets. Together, these data position FAK as the dominant driver of IFN-γ-dependent resistance, with PYK2 playing a subsidiary role, and suggest that selective FAK targeting-potentially combined with limited PYK2 inhibition-may overcome immune checkpoint blockade resistance in melanoma.