Targeting of Hepatic Stellate Cell PD-L1 to Inhibit Tumor Microenvironment and Cholangiocarcinoma
Cholangiocarcinoma (CCA) is a lethal type of liver cancer. Therapies targeting the immune-regulating molecules PD-L1 and PD-1 are under clinical investigation as treatments for CCA, but how PD-L1 affects the development of CCA and the microenvironment surrounding it are not completely understood.
CCA tumors are surrounded by myofibroblasts, a type of cell that is mainly derived from liver resident cells called hepatic stellate cells (HSCs). The myofibroblasts contribute to cholangiocarcinoma growth, metastasis, immune suppression and resistance to chemotherapy. The conversion of HSCs into myofibroblasts is driven by transforming growth factor beta (TGFβ).
In the work proposed here through the Developmental Research Program in the Mayo Clinic Hepatobiliary SPORE, we are focusing on the PD-L1 that is produced in HSCs and are studying how PD-L1 regulates the conversion of HSCs into myofibroblasts that support CCA. We are also implanting cholangiocarcinoma tumors into mice that are genetically engineered to have no PD-L1 in the HSCs to test whether the lack of PD-L1 in HSCs suppresses the conversion of HSCs into myofibroblasts and the development of cholangiocarcinoma and allows chemotherapy to work better for CCA in mice.
Cholangiocarcinoma is a lethal hepatic malignancy originating from the biliary epithelium. CCA is often diagnosed at an advanced stage, when it is incurable.The first-line treatment for advanced cholangiocarcinoma is a gemcitabine-cisplatin combination. The median overall survival rate is less than 12 months, suggesting a critical need to understand the mechanisms of cholangiocarcinoma in order to develop novel therapies.
PD-L1/PD-1 blockade is currently under investigation for CCA in clinical trials, but monotherapy with anti-PD-1 (pembrolizumab) has been disappointing, with only a 5.8% overall response rate, indicating that more mechanistic studies are needed to understand the role of the PD-L1/PD-1 axis in the development of cholangiocarcinoma and its microenvironment. CCA is a desmoplastic tumor containing abundant myofibroblasts that are mainly derived from hepatic stellate cells through a transdifferentiation or activation process mediated by TGFβ. Because the role of PD-L1 in myofibroblastic activation of HSCs remains undetermined, we have focused on the function of PD-L1 in HSCs.
Our preliminary data show that HSCs are a source of PD-L1 and that PD-L1 is required for HSC activation and promotion of CCA growth in a subcutaneous CCA implantation mouse model. Mechanistically, PD-L1 protects the mRNA of TGFβ receptor I (TβRI) from degradation in HSCs. Because N6-methyladenosine (m6A), an epitranscriptomic modification, directs degradation of mRNAs in eukaryotic cells, we are testing the central hypothesis of this proposal: That PD-L1 suppresses m6A modifications on TβRI mRNA, thereby promoting TβRI and activation of HSCs into CCA-promoting myofibroblasts. The m6A modification is induced by an N6-adenosine-methyltransferase complex, METTL3/14, and is erased by an m6A-specific demethylase, ALKBH5.
In Aim 1, we are using in vitro experiments to study whether PD-L1 competes with METTL3/14 for TβRI mRNA binding, thereby suppressing m6A modifications of TβRI mRNA and its degradation. In addition, we are testing whether ALKBH5 removes m6A modifications from TβRI mRNA and rescues the TβRI mRNA level that is reduced in PD-L1-deficient cells.
In Aim 2, we are using cre/loxP recombination technology, orthotopic CCA implantation and in vivo tumor imaging to test whether an HSC-specific PD-L1 knockout suppresses HSC activation and cholangiocarcinoma, and potentiates gemcitabine-cisplatin chemotherapy for CCA in mice.
Because the clinical trials of anti-PD-L1/PD-1 monotherapy or combination therapy for CCA launched without mechanistic and preclinical studies, this proposal is highly relevant in this regard because it lays the groundwork for the clinical investigations aimed at improving the treatment outcomes of patients with cholangiocarcinoma.