Objective: PD-L1 and PD-L2 expression levels determine immune evasion and the therapeutic efficacy of immune checkpoint blockade. The factors that drive inducible PD-L1 expression have been extensively studied, but mechanisms that result in constitutive PD-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 was determined 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 this core DNA region but not to DNA surrounding PD-L1L2SE. Genetic knockout of ETV4 dramatically reduced the expressions of both PD-L1 and PD-L2. ETV4 transcription was dependent on ERK activation, and BRAF/TAK1-induced ERK activation was dependent on extracellular signaling from αvβ3 integrin, which profoundly affected ETV4 transcription and PD-L1/L2 expression. Genetic silencing or pharmacological inhibition of components of the PD-L1L2-SE-associated pathway rendered cancer cells susceptible to T cell-mediated killing. Conclusions: We identified a pathway originating from the extracellular matrix that signaled via integrin/BRAF/TAK1/ERK/ETV4 to PD-L1L2-SE to induce PD-L1-mediated immune evasion. These results provided new insights into PD-L1L2-SE activation and pathways associated with immune checkpoint regulation in cancer.

Panpan Ma;Xinxin Jin;Zhiwei Fan;Zhou Wang;Suhui Yue;Changyue Wu;Shiyin Chen;Yuanyuan Wu;Miaomiao Chen;Donghua Gu;Siliang Zhang;Renfang Mao;Yihui Fan

Laboratory of Medical Science,School of Medicine,Nantong University,Nantong 226001,China;Department of Pathogenic Biology,School of Medicine,Nantong University,Nantong 226001,China;Department of Clinical Laboratory,Yancheng No.1 People's Hospital,Yancheng 224005,China;School of Life Sciences,Nantong University,Nantong 226001,China;Department of Dermatology,Affiliated Hospital of Nantong University,Nantong University,Nantong 226001,China;The Department of Urology,the Second Affiliated Hospital of Nantong University,Nantong University,Nantong 226001,China;The Department of Radiotherapy Oncology,Harbin Medical University Cancer Hospital,Harbin 150086,China;Department of Pathophysiology,School of Medicine,Nantong University,Nantong 226001,China

癌症生物学与医学（英文版）

[1]Panpan Ma;Xinxin Jin;Zhiwei Fan;Zhou Wang;Suhui Yue;Changyue Wu;Shiyin Chen;Yuanyuan Wu;Miaomiao Chen;Donghua Gu;Siliang Zhang;Renfang Mao;Yihui Fan-.Super-enhancer receives signals from the extracellular matrix to induce PD-L1-mediated immune evasion via integrin/ BRAF/TAK1/ERK/ETV4 signaling)[J].癌症生物学与医学（英文版）,2022(05):669-684

L1L2,L1L2SE,signaled

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