AXL tyrosine kinase inhibition restores anti-PD-1 efficacy in melanoma through tumor-associated macrophage—dependent mechanisms 2255689

Abstract Introduction Immune checkpoint blockade (ICB) has transformed melanoma care, yet resistance is common and often driven by tumor-associated macrophages (TAMs). AXL, a Gas6-activated tyrosine kinase, is classically a tumor-intrinsic driver of invasion but is predominantly expressed by TAMs in melanoma, suggesting a role in regulating the tumor-immune microenvironment. We tested if AXL inhibition could reprogram macrophage function, disrupt PD-1:PD-L1 interactions, and restore responsiveness to PD-1 blockade. Methods AXL expression was analyzed using TCGA, serum ELISA, and single-cell RNA-seq datasets. ICB-resistant melanoma models (Yumm1.7, B16F10) were treated with warfarin or bemcentinib (AXL inhibitors) alone or with anti-PD-1, with or without macrophage depletion. PD-1:PD-L1 interactions were quantified by immune Förster resonance energy transfer (iFRET). In vitro, polarized macrophages were analyzed for AXL-dependent efferocytosis, T cell crosstalk, and cytokine secretion. Results AXL was enriched in TAMs across cohorts, with soluble AXL highest in stage IV disease. AXL inhibition reduced tumor burden and synergized with anti-PD-1, efficacy was lost after CSF1R depletion and enhanced by F4/80 depletion. iFRET showed restoration of PD-1:PD-L1 blockade despite unchanged PD-L1 expression. In vitro, AXL function was context-dependent: M1-like macrophages upregulated AXL with immunostimulatory outputs, while M2-like macrophages became more AXL-driven under anti-PD-1. Combination therapy reprogrammed the secretome toward a Th1/Th17, chemokine-rich milieu ↑CXCL9/10, IL-12, IL-23; ↓IL-10, MCP-1). Conclusion AXL is a dominant TAM-driven regulator of ICB resistance in melanoma. AXL inhibition restores functional PD-1:PD-L1 blockade and rebalances macrophage signaling toward immunostimulation in a microenvironment-specific manner. These findings support AXL as both a biomarker and therapeutic target to overcome ICB resistance, with implications for tailoring macrophage-targeted strategies in refractory melanoma. Funding Source N/A Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)