Inhibition of CMTM4 Sensitizes Cholangiocarcinoma and Hepatocellular Carcinoma to T Cell-Mediated Antitumor Immunity Through PD-L1

Abstract

Liver cancers consist primarily of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Immune checkpoint inhibitors have emerged as promising therapeutic agents against liver cancers. Programmed cell death protein 1 (PD-1) is an immunoinhibitory receptor present on T cells that 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, which enhances their antitumor activity. 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/PD-L1 treatment. Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 6 (CMTM6) has been recently identified as a major regulator of PD-L1. Another member in the CMTM family, CKLF-like MARVEL transmembrane domain-containing 4 (CMTM4), has been shown to compensate for the effects of CMTM6 when CMTM6 is lost. Interestingly, we found that CMTM4 is the major regulator of PD-L1 in the context of liver cancer. Up-regulated CMTM4 in patients with HCC and ICC is associated with poor patient survival, potentially due to its function in stabilizing PD-L1 expression, hence facilitating escape from T cell-mediated cytotoxicity. We confirmed the role of CMTM4 as a positive regulator of PD-L1 in multiple HCC and ICC cell lines and demonstrated that CMTM4 stabilizes PD-L1 through posttranslational mechanisms. In vivo, suppression of Cmtm4 inhibited HCC growth and increased CD8⁺ T-cell infiltration in immunocompetent mice. Furthermore, we found that depletion of CMTM4 sensitized HCC tumor to anti-PD-L1 treatment compared with control. This suggests that CMTM4 expression level could be a predictive marker for patient response to anti-PD-L1 treatment, and CMTM4 depletion can potentially be used to enhance the clinical benefits of anti-PD-L1 immunotherapy in patients with liver cancer.

FIG. 1

CMTM4 is up‐regulated in HCC and ICC and is associated with poor prognosis. CMTM6 (A) and CMTM4 (B) mRNA expression in 49 pairs of HCC (left) or 9 pairs of ICC (right) are shown with their corresponding NT liver tissues from TCGA database. (C,D) Waterfall plot showing CMTM4 overexpression by at least two‐fold in 55% and 63% of patients with HCC from TCGA database (C) and the University of Hong Kong, Queen Mary Hospital (HKU‐QMH) (D), respectively. (E) CMTM4 mRNA expression in 75 cases of paired HCC and NT tissues from HKU‐QMH patients. (F) Representative images of IHC staining of CMTM4 in two pairs of human HCC and NT tissues. (G) Kaplan‐Meier curves showed that high CMTM4 mRNA expression is associated with poor disease‐free (left) and overall survival (right) in patients with HCC from TCGA. (H) Correlation of CMTM4 expression with degrees of cellular differentiation in HKU‐QMH HCC patients. (I) Correlation of CMTM4 mRNA expression with CMTM4 copy‐number alterations in HCC tissues from TCGA. (J) CMTM4 copy‐number analysis in genomic DNA extracted from paired HCC and NT tissues from 18 HKU‐QMH patients with HCC. (A,B,E) Lines indicate median. (H‐J) Error bars indicate mean ± SEM. **P < 0.01, ***P < 0.001. (A,B,E) Wilcoxon signed‐rank test. (H,I) Student t test. (G) Log rank test. Abbreviation: T, tumor.

FIG. 2

CMTM4 stabilized PD‐L1 surface expression in HCC and ICC cell lines. (A,C) CMTM4 knockdown efficiency in CLC5 and RBE cells confirmed by western blot. (B,D) Relative median fluorescence intensity (MFI) of surface PD‐L1 (left) and representative histograms (right) from flow cytometry analysis showing PD‐L1 expression in −shCMTM4 cells compared to −NTC cells, with or without IFN‐γ stimulation. (E) Cmtm4 knockout efficiency in mouse syngeneic HCC cell line Hepa1‐6 transduced with Cas9. (F) Relative MFI (left) and representative histograms (right) of PD‐L1 surface expression in Hepa1‐6‐Cas9 −sgCmtm4 cells compared with −EV cells. (See Supporting Figs. S1 and S2 for the full data set.) Cells were pretreated with 25 ng/mL human or mouse IFN‐γ for 24 hours before analysis. Error bars indicate mean ± SD. **P < 0.01, ***P < 0.001. (B,D,F) Student t test.

FIG. 3

CMTM4 stabilized PD‐L1 through posttranslational mechanisms. (A‐C) PD‐L1 mRNA expressions measured by quantitative RT‐PCR in control and CMTM4‐knockdown or CMTM4‐knockout CLC5, RBE, and Hepa1‐6‐Cas9 cells with or without IFN‐γ stimulation (24 hours). CLC5 (D) and RBE (E) control and CMTM4‐knockdown cells were stimulated with IFN‐γ (24 hours). A total of 30 μM Dyngo4a (clathrin‐dependent endocytosis inhibitor) and/or 8 μM MG132 (proteasome inhibitor) were added into culture medium 24 hours and 6 hours before flow cytometry analysis, respectively. Relative MFI (left) and representative histograms (right) of surface PD‐L1 level. Error bars indicate mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (A‐C) Student t test. Abbreviations: D, Dyngo4a; M, MG132; n.s., not significant.

FIG. 4

Inhibition of Cmtm4‐suppressed tumor growth in mouse by increasing T‐cell recruitment. (A) 3.5 × 10⁶ of control (EV) or Cmtm4‐knockdown (shCmtm4‐1, shCmtm4‐2) Hepa1‐6 cells were orthotopically implanted into the liver of wild‐type mice. Tumors were harvested 12 days following implantation and dissociated for immune profiling. (B) Picture (left) and volume (right) of orthotopically implanted tumors. (C) Flow cytometry analysis of CD8⁺ T cells in the tumors. Quantification of the percentage of CD8⁺ T cells over total tumor‐infiltrating leukocytes (left) and representative contour plots showing the gating of CD8⁺ T cells (right). (D) HDTV injection model for the generation of HCC tumor. Genome editing plasmids were injected into mice through lateral veins. Large volume of plasmid solution was delivered into heart within 6‐8 seconds, causing transient cardiac arrest and retrograde flow of plasmids into the mouse liver. Plasmids entered hepatocytes by high pressure created by HDTV injection. Tumors developed in the liver spontaneously, and mice were killed 6 weeks after HDTV injection. (E) Picture (left) and mass (right) of control (EV) or Cmtm4‐knockout (sgCmtm4‐1, sgCmtm4‐2) liver tumors. (F) CD4 and CD8 were stained by IHC in formalin‐fixed paraffin‐embedded HDTV tumor specimens. Representative pictures of CD4 and CD8 staining in EV and sgCmtm4 tumors (left) and quantification of CD4⁺ and CD8⁺ T cells in the tumors (right). Cells with positive staining at three random regions per sample were counted. (B,C,E) Lines indicate mean. (F) Lines indicate mean, and error bars indicate SD. *P < 0.05, **P < 0.01. (B,C,E,F) Student t test. Abbreviations: FSC‐A, forward scatter; n.s., not significant.

FIG. 5

Inhibition of CMTM4 sensitized HCC toward PD‐L1 blockade in mouse. (A) Scheme of experimental design. A total of 3 × 10⁶ of Hepa1‐6 EV and Cmtm4‐knockdown (shCmtm4‐1) cells were subcutaneously implanted into the right and left sides of the lateral flank of wild‐type mice, respectively. Four doses of anti‐PD‐L1 mAb or saline control were administered by intraperitoneal injection during weeks 2‐3 (10 mg/kg/dose). Control group, n = 7; anti‐PD‐L1, group n = 12. (B) Growth curves of individual tumors, measured from the commencement of anti‐PD‐L1 treatment onward. (C) Schematic diagram explaining the therapeutic effect of CMTM4 inhibition in anti‐PD‐L1 immunotherapy. In CMTM4^hi tumor cells, anti‐PD‐L1 monoclonal antibodies fail to occupy all PD‐L1 binding sites (left), whereas with lowered PD‐L1 surface expression in CMTM4^lo tumor cells, anti‐PD‐L1 mAb is able to completely abrogate PD‐1/PD‐L1 signaling between T cells and tumor cells and hence fully unleash T cell–mediated antitumor immunity (right). Abbreviations: Ctrl, saline control; EE, early endosome; RE, recycling endosome.

FIG. 6

Schematic diagram of the regulation of PD‐1/PD‐L1 signaling by CMTM4 in liver cancer. CMTM4 physically interacts with PD‐L1 in tumor cells to promote and sustain PD‐L1 expression at the plasma membrane by shuttling PD‐L1 to recycling endosomes, preventing it from being degraded by the lysosome or proteasome. As a result, CMTM4 potentiates the binding of PD‐L1 on tumor cells to PD‐1 on T cells, inhibiting T‐cell activation and facilitating immune escape. Inhibition of proteasomal activity by MG132 and inhibition of clathrin‐dependent endocytosis by Dyngo4a restored PD‐L1 surface expression after CMTM4 inhibition. Abbreviations: EE, early endosome; RE, recycling endosome; MHC‐Ag, MHC class I conjugated with tumor antigen; TCR, T‐cell receptor; U, ubiquitin.