Super-enhancer receives signals from the extracellular matrix to induce PD-L1-mediated immune evasion via integrin/BRAF/TAK1/ERK/ETV4 signaling

Abstract

Objective: PD-L1 and PD-L2 expression levels determine immune evasion and the therapeutic efficacy of immune checkpointblockade. The factors that drive inducible PD-L1 expression have been extensively studied, but mechanisms that result in constitutivePD-L1 expression in cancer cells are largely unknown. Methods: DNA elements were deleted in cells by CRISPR/Cas9-mediated knockout. Protein function was inhibited by chemical inhibitors.Protein levels were examined by Western blot, mRNA levels were examined by real-time RT-PCR, and surface protein expression wasdetermined by cellular immunofluorescence and flow cytometry. Immune evasion was examined by in vitro T cell-mediated killing. Results: We determined the core regions (chr9: 5, 496, 378–5, 499, 663) of a previously identified PD-L1L2-super-enhancer (SE).Through systematic analysis, we found that the E26 transformation-specific (ETS) variant transcription factor (ETV4) bound to thiscore DNA region but not to DNA surrounding PD-L1L2SE. Genetic knockout of ETV4 dramatically reduced the expressions of bothPD-L1 and PD-L2. ETV4 transcription was dependent on ERK activation, and BRAF/TAK1-induced ERK activation was dependenton extracellular signaling from αvβ3 integrin, which profoundly affected ETV4 transcription and PD-L1/L2 expression. Geneticsilencing or pharmacological inhibition of components of the PD-L1L2-SE-associated pathway rendered cancer cells susceptible toT cell-mediated killing. Conclusions: We identified a pathway originating from the extracellular matrix that signaled via integrin/BRAF/TAK1/ERK/ETV4to PD-L1L2-SE to induce PD-L1-mediated immune evasion. These results provided new insights into PD-L1L2-SE activation andpathways associated with immune checkpoint regulation in cancer.