MARCKS mediates the AXL/PD-L1 signaling axis and contributes to an immunosuppressive environment in lung cancer

Abstract

Abstract The advent of immunotherapy with immune checkpoint blockade (ICB) has led to significant advances in lung cancer management; unfortunately, many issues remain to be resolved, especially regarding a low objective response rate. Through integrated large-scale omics data and biomarkers from published immune ICB trials, we identified the PIP2-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), and the AXL receptor as top-ranked therapeutic targets in mediating immunotherapy resistance. Both immunofluorescence and immunoprecipitation data demonstrated that MARCKS forms a molecular complex with AXL in aggressive lung cancer cells. In a screening of 200 patients using immunohistochemistry staining, we observed a positive correlation between AXL phosphorylation and MARCKS expression in advanced lung cancer. MARCKS knockdown abated the activity of AXL and its downstream pathways including PI3K/AKT and STAT3/Src signaling. Data from our cytokine arrays showed upregulation of anti-tumoral cytokines including GM-CSF and CXCL10 in MARCKS-knockout cells. We further discovered that MARCKS acts in accordance with PD-L1 to modulate T cell activity and killing. Upon co-culture with MARCKS-expressing lung cancer cells, cytotoxic T cells expressed higher PD-1 levels on cell surfaces, whereas T cell killing activity was potentiated in the MARCKS-knockout group. In mouse xenograft models, mice bearing MARCKS-deficient tumors displayed increased sensitivity to PD-L1 treatment. Our data suggest an oncogenic role of the AXL-MARCKS axis in modulating the tumor immune microenvironment. Therapeutic targeting of MARCKS may warm up an immunologically “cold” microenvironment, sensitizing tumors to immunotherapy. This work was supported by grants funded by the Department of Defense Kidney Cancer Research Program (KCRP) W81XWH1910831 (log# KC180170) and the California UCOP Tobacco-Related Disease Research Program (TRDRP) T29IR0704.