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Abstract

The CKLF-like MARVEL transmembrane proteins CMTM4 and CMTM6 stabilize PD-L1 and have been implicated in tumor immune evasion, yet their PD-L1-independent functions and in vivo relevance remain unknown. My thesis integrates functional assays, multi-omics profiling, and mouse liver cancer models to dissect shared and distinct roles of CMTM4/6 and to evaluate their therapeutic potential. In melanoma cells, overexpression of CMTM4 or CMTM6 did not alter global transcriptional programs, consistent with a PD-L1 “ceiling” whereby endogenous levels are sufficient for maximal immune resistance. By contrast, CRISPR knockouts produced broad and partially overlapping transcriptomic shifts with convergent enrichment of antigen processing/presentation, alongside paralog-specific signatures: CMTM4KO engaged cell-cycle/chromatin axes, whereas CMTM6KO activated p53 and PI3K–AKT pathways. Membrane proteomics revealed that and suppressed mitochondrial metabolism/oxidative phosphorylation from CMTM6KO. During antigen-specific T cell co-culture, tumor transcription was dominated by interferon-driven programs; CMTM4KO subtly attenuated the induction of selected interferon-stimulated genes (ISGs) (e.g., CXCL10/11), whereas CMTM6KO showed minimal transcriptional divergence, pointing to a predominant protein-level mode of action for CMTM6. In hydrodynamic tail-vein injection (HTVi) liver cancer models, CMTM6 loss conferred context-dependent benefit: no survival extension in rapidly progressive KRASG12D/TP53KO, but significant prolongation in more indolent myr-AKT/NICD cholangiocarcinoma and c-MYC/TP53KO hepatocellular carcinoma. Importantly, hepatocyte-specific CMTM6 deletion (AAV8-TBG-Cre) unmasked a robust, CD8⁺ T cell–dependent antitumor phenotype even where global knockout failed, characterized by higher intratumoral CD8⁺ density, preserved progenitor potenGal, increased tissue residency, and enhanced type I effector function (IFNγ, TNFα, granzyme B). CD8 depletion abrogated protection. In mice, the absence of a CD58 ortholog focuses the mechanism on PD-L1 destabilization as the principal driver. Across matched experiments, CMTM4 exerted smaller and less consistent in vivo effects. Collectively, these data establish CMTM6 as the dominant, tumor-intrinsic enabler of immune escape in liver cancer and show that hepatocyte-restricted inhibition can convert “cold” tumors into an inflamed, CD8-centered state when disease kinetics permit. The work outlines testable paths to translate CMTM6 suppression, prioritizing tumor-restricted delivery and mechanistic dissection of PD-L1–independent facets, while indicating scenarios where dual CMTM4/6 targeting may be warranted.