The transmembrane protein CMTM6 promotes plasma membrane expression of the immune checkpoint protein PD-L1, a key suppressor of anti-tumor immunity. Targeting CMTM6 has been proposed as a strategy to enhance tumor cell killing by reducing PD-L1 surface expression. In accord, ablation of CMTM6 in mouse cancer models was shown to efficiently suppress tumor growth, but unexpectedly in a manner partially independent of PD-L1, suggesting that CMTM6 may regulate additional proteins involved in anti-tumor immunity. Using mass spectrometry, we discovered that mouse CMTM6 strongly associates with the cell death receptor FAS and negatively regulates its surface expression in mice. Deletion of CMTM6 increases FAS plasma membrane localization and sensitizes murine cells to FAS ligand-induced cytotoxicity. However, the interaction between CMTM6 and FAS is absent in human cells due to the difference in three amino acids at the boundary of the FAS extracellular and transmembrane domains. Altogether, our findings urge caution when translating promising data regarding the targeting of CMTM6 from mouse cancer models to potential human therapies.

Department of Immunobiology University of Lausanne Epalinges Switzerland

Faculty of Science Charles University Prague Czech Republic

Laboratory of Adaptive Immunity Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Laboratory of Immunity and Cell Communication Division BIOCEV 1st Faculty of Medicine Charles University Vestec Czech Republic

Zobrazit více v PubMed

Adachi M, Suematsu S, Kondo T, Ogasawara J, Tanaka T, Yoshida N, Nagata S (1995) Targeted mutation in the Fas gene causes hyperplasia in peripheral lymphoid organs and liver. Nat Genet 11:294–300 PubMed DOI

Barnhart BC, Legembre P, Pietras E, Bubici C, Franzoso G, Peter ME (2004) CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells. EMBO J 23:3175–3185 PubMed DOI PMC

Burr ML, Sparbier CE, Chan YC, Williamson JC, Woods K, Beavis PA, Lam EYN, Henderson MA, Bell CC, Stolzenburg S et al (2017) CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature 549:101–105 PubMed DOI PMC

Chrifi I, Louzao-Martinez L, Brandt MM, van Dijk CGM, Burgisser PE, Zhu C, Kros JM, Verhaar MC, Duncker DJ, Cheng C (2019) CMTM4 regulates angiogenesis by promoting cell surface recycling of VE-cadherin to endothelial adherens junctions. Angiogenesis 22:75–93 PubMed DOI

Consortium I (2015) INFRAFRONTIER—providing mutant mouse resources as research tools for the international scientific community. Nucleic Acids Res 43:D1171–D1175 DOI

Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteom 13:2513–2526 DOI

Cullen SP, Henry CM, Kearney CJ, Logue SE, Feoktistova M, Tynan GA, Lavelle EC, Leverkus M, Martin SJ (2013) Fas/CD95-induced chemokines can serve as “find-me” signals for apoptotic cells. Mol Cell 49:1034–1048 PubMed DOI

Dai X, Bu X, Gao Y, Guo J, Hu J, Jiang C, Zhang Z, Xu K, Duan J, He S et al (2021) Energy status dictates PD-L1 protein abundance and anti-tumor immunity to enable checkpoint blockade. Mol Cell 81:2317–2331.e2316 PubMed DOI PMC

Davidovich P, Higgins CA, Najda Z, Longley DB, Martin SJ (2023) cFLIP(L) acts as a suppressor of TRAIL- and Fas-initiated inflammation by inhibiting assembly of caspase-8/FADD/RIPK1 NF-kappaB-activating complexes. Cell Rep 42:113476 PubMed DOI

Ho P, Melms JC, Rogava M, Frangieh CJ, Pozniak J, Shah SB, Walsh Z, Kyrysyuk O, Amin AD, Caprio L et al (2023) The CD58-CD2 axis is co-regulated with PD-L1 via CMTM6 and shapes anti-tumor immunity. Cancer Cell 41:1207–1221.e1212 PubMed DOI PMC

Holler N, Tardivel A, Kovacsovics-Bankowski M, Hertig S, Gaide O, Martinon F, Tinel A, Deperthes D, Calderara S, Schulthess T et al (2003) Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex. Mol Cell Biol 23:1428–1440 PubMed DOI PMC

Karray S, Kress C, Cuvellier S, Hue-Beauvais C, Damotte D, Babinet C, Levi-Strauss M (2004) Complete loss of Fas ligand gene causes massive lymphoproliferation and early death, indicating a residual activity of gld allele. J Immunol 172:2118–2125 PubMed DOI

Knizkova D, Pribikova M, Draberova H, Semberova T, Trivic T, Synackova A, Ujevic A, Stefanovic J, Drobek A, Huranova M et al (2022) CMTM4 is a subunit of the IL-17 receptor and mediates autoimmune pathology. Nat Immunol 23:1644–1652 PubMed DOI PMC

Kural MH, Djakbarova U, Cakir B, Tanaka Y, Chan ET, Arteaga Muniz VI, Madraki Y, Qian H, Park J, Sewanan LR et al (2024) Mechano-inhibition of endocytosis sensitizes cancer cells to Fas-induced Apoptosis. Cell Death Dis 15:440 PubMed DOI PMC

Labun K, Montague TG, Krause M, Torres Cleuren YN, Tjeldnes H, Valen E (2019) CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res 47:W171–W174 PubMed DOI PMC

Lee KH, Feig C, Tchikov V, Schickel R, Hallas C, Schutze S, Peter ME, Chan AC (2006) The role of receptor internalization in CD95 signaling. EMBO J 25:1009–1023 PubMed DOI PMC

Long Y, Chen R, Yu X, Tong Y, Peng X, Li F, Hu C, Sun J, Gong L (2023) Suppression of tumor or host intrinsic CMTM6 drives antitumor cytotoxicity in a PD-L1-independent manner. Cancer Immunol Res 11:241–260 PubMed DOI

Magri Z, Jetton D, Muendlein HI, Connolly WM, Russell H, Smirnova I, Sharma S, Bunnell S, Poltorak A (2024) CD14 is a decision-maker between Fas-mediated death and inflammation. Cell Rep 43:114685 PubMed DOI PMC

Masuda T, Tomita M, Ishihama Y (2008) Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res 7:731–740 PubMed DOI

Mellacheruvu, Wright D, Couzens AL Z, Lambert JP, St-Denis NA, Li T, Miteva YV, Hauri S, Sardiu ME, Low TY et al (2013) The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods 10:730–736 PubMed DOI PMC

Mezzadra R, Sun C, Jae LT, Gomez-Eerland R, de Vries E, Wu W, Logtenberg MEW, Slagter M, Rozeman EA, Hofland I et al (2017) Identification of CMTM6 and CMTM4 as PD-L1 protein regulators. Nature 549:106–110 PubMed DOI PMC

Miao B, Hu Z, Mezzadra R, Hoeijmakers L, Fauster A, Du S, Yang Z, Sator-Schmitt M, Engel H, Li X et al (2023) CMTM6 shapes antitumor T cell response through modulating protein expression of CD58 and PD-L1. Cancer Cell 41:1817–1828.e1819 PubMed DOI PMC

Morad G, Helmink BA, Sharma P, Wargo JA (2021) Hallmarks of response, resistance, and toxicity to immune checkpoint blockade. Cell 184:5309–5337 PubMed DOI PMC

Moudra A, Niederlova V, Novotny J, Schmiedova L, Kubovciak J, Matejkova T, Drobek A, Pribikova M, Stopkova R, Cizkova D et al (2021) Phenotypic and clonal stability of antigen-inexperienced memory-like T cells across the genetic background, hygienic status, and aging. J Immunol 206:2109–2121 PubMed DOI

Nagata S (1997) Apoptosis by death factor. Cell 88:355–365 PubMed DOI

Ni Q, Li G, Chen Y, Bao C, Wang T, Li Y, Ruan X, Wang H, Sun W (2024) LECs regulate neutrophil clearance through IL-17RC/CMTM4/NF-kappaB axis at sites of inflammation or infection. Mucosal Immunol 17:723–738 PubMed DOI

Perez-Riverol Y, Bandla C, Kundu DJ, Kamatchinathan S, Bai J, Hewapathirana S, John NS, Prakash A, Walzer M, Wang S et al (2025) The PRIDE database at 20 years: 2025 update. Nucleic Acids Res 53:D543–D553 PubMed DOI

Peter ME, Hadji A, Murmann AE, Brockway S, Putzbach W, Pattanayak A, Ceppi P (2015) The role of CD95 and CD95 ligand in cancer. Cell Death Differ 22:549–559 PubMed DOI PMC

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308 PubMed DOI PMC

Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906 PubMed DOI

Rieux-Laucat F, Magerus-Chatinet A, Neven B (2018) The autoimmune lymphoproliferative syndrome with defective FAS or FAS-ligand functions. J Clin Immunol 38:558–568 PubMed DOI

Risso V, Lafont E, Le Gallo M (2022) Therapeutic approaches targeting CD95L/CD95 signaling in cancer and autoimmune diseases. Cell Death Dis 13:248 PubMed DOI PMC

Rudra M, Chatterjee B, Bahadur M (2016) Phylogenetic relationship and time of divergence of Mus terricolor with reference to other Mus species. J Genet 95:399–409 PubMed DOI

Sharma S, Carmona A, Skowronek A, Yu F, Collins MO, Naik S, Murzeau CM, Tseng PL, Erdmann KS (2019) Apoptotic signalling targets the post-endocytic sorting machinery of the death receptor Fas/CD95. Nat Commun 10:3105 PubMed DOI PMC

Siegmund D, Lang I, Wajant H (2017) Cell death-independent activities of the death receptors CD95, TRAILR1, and TRAILR2. FEBS J 284:1131–1159 PubMed DOI

Susa KJ, Kruse AC, Blacklow SC (2024) Tetraspanins: structure, dynamics, and principles of partner-protein recognition. Trends Cell Biol 34:509–522 PubMed DOI

Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland NG, Suda T, Nagata S (1994) Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76:969–976 PubMed DOI

Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13:731–740 PubMed DOI

Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson A, Kampf C, Sjostedt E, Asplund A et al (2015) Proteomics. Tissue-based map of the human proteome. Science 347:1260419 PubMed DOI

van Deventer S, Arp AB, van Spriel AB (2021) Dynamic plasma membrane organization: a complex symphony. Trends Cell Biol 31:119–129 PubMed DOI

Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S (1992) Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314–317 PubMed DOI

Xu Y, Kang K, Coakley BA, Eisenstein S, Parveen A, Mai S, Wang YS, Zheng J, Boral D, Mai J et al (2025) Modulation of tumor inflammatory signaling and drug sensitivity by CMTM4. EMBO J 44:1866–1883 PubMed DOI PMC

Zhang Y, Liu Q, Yang S, Liao Q (2021) CD58 Immunobiology at a glance. Front Immunol 12:705260 PubMed DOI PMC

Zheng L, Fisher G, Miller RE, Peschon J, Lynch DH, Lenardo MJ (1995) Induction of apoptosis in mature T cells by tumour necrosis factor. Nature 377:348–351 PubMed DOI