Identification of Transcriptional Regulators of Immune Evasion across Cancers: An Alternative Immunotherapeutic Strategy for Cholangiocarcinoma

Background: Immune evasion in cancer is a multifaceted process that synchronizes pro-tumoral immune infiltration, immunosuppressive inflammation, and inhibitory immune checkpoint expression (IC). Recent developments have immunotherapies such as immune checkpoint blockade (ICB) and chimeric antigen receptor T-cell (CAR-T) therapy effectively impede this process but benefit a limited patient cohort. This investigation introduces a systemic immunotherapeutic strategy by inhibition of master-regulators of immune evasion (MR-IE). Methods: We utilized the TCGA PanCancer Atlas transcriptomic data to subset and stratify cancer samples based on IC expression levels and CIBERSORT to estimate immune cell infiltration. Differential gene expression analysis was conducted to unravel pathways associated with the immune evasion. Transcription Factor Enrichment Analysis and Survival Analysis was conducted to identify and rank candidate MR-IE per cancer type. The top-ranking candidate MR-IE of cholangiocarcinoma (CCA) was validated for its regulation of ICs using quantitative PCR and western blots. Orbitrap Proteomics was employed to assess changes in the immune-related proteome of CCA cells after MR-IE is inhibited or knocked-down. Lastly, CCA cell viability and immune-mediated cell death was assessed after MR-IE inhibition in combination with fourth-generation anti-folate receptor alpha (FRɑ) CAR-T cell therapy. Results: This workflow ranked and identified candidate MR-IE for 33 tumor types. MYC was identified as the high-ranking candidate MR-IE for CCA. MYC modulated PD-L1 expression adn key markers of inflammation and IC expression in CCA cells. Moreover, MYC inhibition was able to potentiate CAR-T mediated cell death in CCA cells. Conclusions: Cumulatively, these results offer a candidate list of MR-IE immunotherapeutic targets per cancer type that can be further explored and validated. This study also highlights the promise of MR-IE inhibition as a novel potent immunotherapeutic strategy for the treatment of CCA.