Co-activation of Wnt/beta-catenin and c-MET induced inflamed hepatocellular carcinoma responsive to immune checkpoint therapy

Hepatocellular carcinoma (HCC), the major form of liver cancer, is often detected at advanced and unresectable stages, making it one of the deadliest cancers in adults. The front-line treatment for unresectable advanced HCC involves immune checkpoint therapy (ICT), which shows complete response only in 20-30% of patients. Mutational activation of Wnt/beta-catenin pathway due to gain-of-function mutations in beta-catenin gene ( CTNNB1 ) is a major oncogenic event driving hepatocarcinogenesis accounting for about a third of HCC. However, oncogenic forms of beta-catenin often cooperate with other oncogenes like c-MET, K-RAS, and AKT for tumor development and progression. For instance, activated beta-catenin in HCC can induce the expression of c-MYC, which facilitates metabolic adaptation of HCC cells and tumor associated macrophages (TAMs). Several clinical and preclinical studies have shown that HCC tumors with CTNNB1 mutation are often less inflamed with limited immune cell infiltration making the tumors “immune-cold” and resistant to ICT. On the other hand, mutational activation of the Wnt/beta-catenin also occurs in “immune-hot” HCC tumors suggesting that the tumor immune microenvironment (TIME) is co-regulated by other oncogenic events. Although, murine HCC tumors generated by hydrodynamic co-transfection of ΔN90-beta-cat (a constitutively active beta-catenin) and c-MYC are “immune-cold”, the events that cooperate with beta-catenin activation leading to “immune-hot” tumors are unknown. In this study, we used hydrodynamic tail vein injection (HTVI) of plasmids expressing ΔN90-beta-cat and c-MET to investigate their effect on HCC development, progression, and TIME. Immune-profiling of our ΔN90-beta-cat and c-MET tumor model showed increased infiltration of PD-1+ cytotoxic T cells and Th1 cells, both of which are crucial for anti-tumor response. Although T cells were present in the tumor microenvironment, the upregulation of PD-1 indicates an exhausted state of T cells, permitting HCC progression. Therefore, we hypothesized that checkpoint blockade reactivates the exhausted T cells leading to better anti-tumor response. Accordingly, HTVI generated HCC bearing FVB/NJ male mice treated with anti-PD1 antibody showed significant tumor regression/stabilization compared to those treated with isotype control antibody, as indicated by lower levels of plasma alpha-fetoprotein (AFP), a biomarker of HCC, along the treatment course as well as the terminal tumor size. Additionally, immune profiling of the anti-PD1 treated tumors show significantly elevated population of total and resident cytotoxic T cells compared to the isotype control group. Therefore, HCC tumors formed by combination of ΔN90-beta-cat and c-MET are “immune-hot” and respond to ICT. These findings further indicate that, co-activation of c-MET and beta-catenin could serve as biomarkers for HCC patient inclusion in ICT.