The role of CKLF-Like MARVEL transmembrane domain-containing 4 in tumour immunology of liver cancer

Abstract

Liver cancers consist mainly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). In recent years, immune checkpoint inhibitors have emerged as a potential therapy against liver cancers. Programmed cell death protein 1 (PD-1) is an immunoinhibitory receptor present on T cells which interacts with its ligand programmed death-ligand 1 (PD-L1) found on cancer cells. Blocking PD-1/PD-L1 binding improves T cell survival, proliferation and cytotoxicity. Nivolumab (anti-PD-1) has been recently approved by the FDA for treatment in HCC. However, many clinical trials demonstrated that only a subset of HCC patients responds to anti-PD-1 treatment. Better understanding of the molecular mechanisms governing PD-1/PD-L1 response is essential to the development of predictive markers and therapeutic combinations that could improve the efficiency of anti-PD-1/anti-PD-L1 treatment. CKLF Like MARVEL Transmembrane Domain-Containing 6 (CMTM6) has been recently identified as a major post-translational regulator of PD-L1. Another member of the CMTM family, CMTM4, has been shown to compensate for the effects of CMTM6 only when CMTM6 is lost. Interestingly, we found that CMTM4 is the major regulator of PD-L1 in the context of liver cancer. CMTM4 is upregulated in HCC and ICC patients and high expression of CMTM4 is associated with low patient survival, potentially due to its function in stabilizing PD-L1 expression hence facilitating escape from T cell-mediated cytotoxicity. Using flow cytometry, we found that PD-L1 surface expression is significantly downregulated after the knockdown of CMTM4 in multiple HCC and ICC cell lines, confirming the role of CMTM4 as a positive regulator of PD-L1 in liver cancer. In vivo, knockdown of Cmtm4 suppressed tumour growth and increased CD4+ T cells infiltration in orthotopic model in immunocompetent mice. We hypothesized that the inhibition of CMTM4 coupled with anti-PD-1/PD-L1 immunotherapy can further activate T cells in tumour microenvironment and expedite anti-tumour immune response. Using syngeneic mouse liver cancer model, we found that after the depletion of CMTM4, HCC was more sensitive to anti-PD-1/PD-L1 treatment compared to control. This suggests that CMTM4 expression level could be a predictive marker for anti-PD-1/PD-L1 treatment and CMTM4 depletion can potentially be employed to enhance the clinical benefits of anti-PD-1/PD-L1 immunotherapy in liver cancer patients.