Abstract
The programmed cell death protein 1 (PD-1) / programmed death-ligand 1 (PD-L1) axis represents a cornerstone of cancer immunotherapy, yet the dynamic shuttling of PD-L1 between endosomal recycling and lysosomal degradation routes limits durable responses. Using a CRISPR screen targeting glycosphingolipid metabolism, we identify transmembrane 9 superfamily member 2 (TM9SF2) as a key regulator of PD-L1 levels. TM9SF2 orchestrates a dual mechanism: it recruits phosphoglycerate kinase 1 (PGK1) to promote PD-L1 recycling to the plasma membrane while dismantling the huntingtin-interacting protein 1-related protein (HIP1R)-mediated lysosomal degradation pathway. Genetic or pharmacological disruption of the TM9SF2-PGK1 complex depletes PD-L1 levels and boosts antitumor immunity. Further, the endogenous ceramide species Cer(d18:1/26:0) destabilizes this complex, triggering PD-L1 lysosomal destruction and potentiating antitumor immunity. These findings delineate a ceramide-gated sorting mechanism within the endosomal network, revealing a druggable metabolic switch to disrupt immune evasion and amplify checkpoint blockade efficacy.
Similar content being viewed by others
Data availability
The lung adenocarcinoma data (TCGA-LUAD) used in this study are publicly available in the Database of Genotypes and Phenotypes (dbGaP) under accession number phs000178 (https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000178.v11.p8). The acute myeloid leukemia data (TARGET-AML) used in this study are publicly available in the dbGaP under accession number phs000218 (https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000218.v26.p8). The genomic and proteomic data generated in this study are publicly available as follows: The mass spectrometry proteomics data of shTM9SF2 and shNC H460 cells have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD061780. The raw data and search results of sgNTC and sgPGK1 H460 cells have been uploaded to the iProX database under the accession number IPX0011331001. The raw data and search results of IP-MS analysis with FLAG-tagged TM9SF2 have been uploaded to the iProX database under the accession number IPX0015889001. The lipidomics data generated in this study have been deposited in the MetaboLights database under accession code MTBLS13838. The sequencing data from the focused CRISPR screen have been deposited to the NCBI GEO database under accession number GSE320531. All additional data are available from the corresponding author (tingdong2025@pumc.edu.cn) upon request. Source data are provided with this paper.
References
Sanmamed, M. F. & Chen, L. A paradigm shift in cancer immunotherapy: from enhancement to normalization. Cell 175, 313–326 (2018).
Kim, T. K., Vandsemb, E. N., Herbst, R. S. & Chen, L. Adaptive immune resistance at the tumour site: mechanisms and therapeutic opportunities. Nat. Rev. Drug Discov. 21, 529–540 (2022).
Kubli, S. P., Berger, T., Araujo, D. V., Siu, L. L. & Mak, T. W. Beyond immune checkpoint blockade: emerging immunological strategies. Nat. Rev. Drug Discov. 20, 899–919 (2021).
Bonaventura, P. et al. Cold tumors: a therapeutic challenge for immunotherapy. Front. Immunol. 10, 168 (2019).
Sharma, P., Hu-Lieskovan, S., Wargo, J. A. & Ribas, A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell 168, 707–723 (2017).
Choi, S. H. et al. DRG2 is required for surface localization of PD-L1 and the efficacy of anti-PD-1 therapy. Cell Death Discov. 10, 260 (2024).
Pang, K. et al. Research progress of therapeutic effects and drug resistance of immunotherapy based on PD-1/PD-L1 blockade. Drug Resist. Updates 66, 100907 (2023).
Schoenfeld, A. J. & Hellmann, M. D. Acquired resistance to immune checkpoint inhibitors. Cancer cell 37, 443–455 (2020).
Ribas, A. & Wolchok, J. D. Cancer immunotherapy using checkpoint blockade. Science 359, 1350–1355 (2018).
Sharma, P. et al. Immune checkpoint therapy-current perspectives and future directions. Cell 186, 1652–1669 (2023).
Li, C. W. et al. Eradication of triple-negative breast cancer cells by targeting glycosylated PD-L1. Cancer Cell 33, 187–201.e110 (2018).
Burr, M. L. et al. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature 549, 101–105 (2017).
Ren, Y. et al. TRAPPC4 regulates the intracellular trafficking of PD-L1 and antitumor immunity. Nat. Commun. 12, 5405 (2021).
Yu, X. et al. PD-L1 translocation to the plasma membrane enables tumor immune evasion through MIB2 ubiquitination. J. Clin. Investig. 133, https://doi.org/10.1172/jci160456 (2023).
Yi, M., Niu, M., Xu, L., Luo, S. & Wu, K. Regulation of PD-L1 expression in the tumor microenvironment. J. Hematol. Oncol. 14, 10 (2021).
Ye, Z. et al. Manipulation of PD-L1 Endosomal Trafficking Promotes Anticancer Immunity. Adv. Sci. 10, e2206411 (2023).
Guo, H. et al. Insights into the role of derailed endocytic trafficking pathway in cancer: from the perspective of cancer hallmarks. Pharmacol. Res. 201, 107084 (2024).
Lemma, E. Y., Letian, A., Altorki, N. K. & McGraw, T. E. Regulation of PD-L1 trafficking from synthesis to degradation. Cancer Immunol. Res. 11, 866–874 (2023).
Wang, H. et al. HIP1R targets PD-L1 to lysosomal degradation to alter T cell-mediated cytotoxicity. Nat. Chem. Biol. 15, 42–50 (2019).
Xu, X. et al. Hsc70 promotes anti-tumor immunity by targeting PD-L1 for lysosomal degradation. Nat. Commun. 15, 4237 (2024).
Dong, T. et al. Targeting VPS18 hampers retromer trafficking of PD-L1 and augments immunotherapy. Sci. Adv. 10, eadp4917 (2024).
Contreras, F. X. et al. Molecular recognition of a single sphingolipid species by a protein’s transmembrane domain. Nature 481, 525–529 (2012).
Breslow, D. K. & Weissman, J. S. Membranes in balance: mechanisms of sphingolipid homeostasis. Mol. cell 40, 267–279 (2010).
Bieberich, E. Sphingolipids and lipid rafts: novel concepts and methods of analysis. Chem. Phys. lipids 216, 114–131 (2018).
Hannun, Y. A. & Obeid, L. M. Sphingolipids and their metabolism in physiology and disease. Nat. Rev. Mol. Cell Biol. 19, 175–191 (2018).
Lee, M., Lee, S. Y. & Bae, Y. S. Functional roles of sphingolipids in immunity and their implication in disease. Exp. Mol. Med. 55, 1110–1130 (2023).
Chluba-de Tapia, J., de Tapia, M., Jäggin, V. & Eberle, A. N. Cloning of a human multispanning membrane protein cDNA: evidence for a new protein family. Gene 197, 195–204 (1997).
Schimmöller, F., Díaz, E., Mühlbauer, B. & Pfeffer, S. R. Characterization of a 76 kDa endosomal, multispanning membrane protein that is highly conserved throughout evolution. Gene 216, 311–318 (1998).
Lozupone, F. et al. The human homologue of Dictyostelium discoideum phg1A is expressed by human metastatic melanoma cells. EMBO Rep. 10, 1348–1354 (2009).
Hoekstra, M. E. et al. Distinct spatiotemporal dynamics of CD8(+) T cell-derived cytokines in the tumor microenvironment. Cancer Cell 42, 157–-167.e159 (2024).
Aktas, E., Kucuksezer, U. C., Bilgic, S., Erten, G. & Deniz, G. Relationship between CD107a expression and cytotoxic activity. Cell. Immunol. 254, 149–154 (2009).
Qiu, A. et al. Phosphoglycerate Kinase 1: An effective therapeutic target in cancer. Front. Biosci. 29, 92 (2024).
Liu, H. et al. PRMT1-mediated PGK1 arginine methylation promotes colorectal cancer glycolysis and tumorigenesis. Cell Death Dis. 15, 170 (2024).
Chu, S. L. et al. Phosphoglycerate kinase 1 acts as a cargo adaptor to promote EGFR transport to the lysosome. Nat. Commun. 15, 1021 (2024).
Liao, L. et al. A potent PGK1 antagonist reveals PGK1 regulates the production of IL-1β and IL-6. Acta Pharm. Sin. B 12, 4180–4192 (2022).
Singla, A. et al. Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling. Nat. Commun. 10, 4271 (2019).
Yamaji, T. et al. A CRISPR screen identifies LAPTM4A and TM9SF proteins as glycolipid-regulating factors. iScience 11, 409–424 (2019).
He, Y. et al. PGK1-mediated cancer progression and drug resistance. Am. J. Cancer Res. 9, 2280–2302 (2019).
Alizadeh, J. et al. Ceramides and ceramide synthases in cancer: Focus on apoptosis and autophagy. Eur. J. Cell Biol. 102, 151337 (2023).
Tanaka, A. et al. Genome-wide screening uncovers the significance of N-Sulfation of heparan sulfate as a host cell factor for chikungunya virus infection. J. Virol. 91, https://doi.org/10.1128/jvi.00432-17 (2017).
Li, Q. et al. LINC01232 exerts oncogenic activities in pancreatic adenocarcinoma via regulation of TM9SF2. Cell Death Dis. 10, 698 (2019).
Clark, C. R. et al. Transposon mutagenesis screen in mice identifies TM9SF2 as a novel colorectal cancer oncogene. Sci. Rep. 8, 15327 (2018).
Fu, Q. & Yu, Z. Phosphoglycerate kinase 1 (PGK1) in cancer: a promising target for diagnosis and therapy. Life Sci. 256, 117863 (2020).
Nie, H. et al. O-GlcNAcylation of PGK1 coordinates glycolysis and TCA cycle to promote tumor growth. Nat. Commun. 11, 36 (2020).
Fougère, L., Mongrand, S. & Boutté, Y. The function of sphingolipids in membrane trafficking and cell signaling in plants, in comparison with yeast and animal cells. Biochim. et. Biophys. Acta Mol. Cell Biol. Lipids 1869, 159463 (2024).
Hanada, K., Kumagai, K., Tomishige, N. & Kawano, M. CERT and intracellular trafficking of ceramide. Biochim. et. Biophys. Acta 1771, 644–653 (2007).
Morad, S. A. & Cabot, M. C. Ceramide-orchestrated signalling in cancer cells. Nat. Rev. Cancer 13, 51–65 (2013).
Ogretmen, B. & Hannun, Y. A. Biologically active sphingolipids in cancer pathogenesis and treatment. Nat. Rev. Cancer 4, 604–616 (2004).
Finicle, B. T. et al. Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6. J. Cell Sci. 131, https://doi.org/10.1242/jcs.213314 (2018).
Hanada, K. et al. Molecular machinery for non-vesicular trafficking of ceramide. Nature 426, 803–809 (2003).
Turpin-Nolan, S. M. & Brüning, J. C. The role of ceramides in metabolic disorders: when size and localization matters. Nat. Rev. Endocrinol. 16, 224–233 (2020).
Cong, L. & Zhang, F. Genome engineering using CRISPR-Cas9 system. Methods Mol. Biol. 1239, 197–217 (2015).
York, A. G. et al. IL-10 constrains sphingolipid metabolism to limit inflammation. Nature 627, 628–635 (2024).
Chen, X. et al. A membrane-associated MHC-I inhibitory axis for cancer immune evasion. Cell 186, 3903–3920.e3921 (2023).
Acknowledgements
This work was supported by the National Key R&D Project on Biomedical Frontiers (2025YFC3409705) to T.D., Beijing Nova Program (2025-20250484836) to T.D., Shandong Provincial Natural Science Foundation of China (ZR2024MB138) to T.D., National Natural Science Foundation of China (82474005 to Y.Z., 82373957 to W.Z.), Central Public-interest Scientific Institution Basal Research Fund (2025-RC350-01, 2025-JKCS-28) to T.D., National Key R&D Program of China (2023YFC2706100) to W.Z. and Y.Z., Taishan Scholar Program of Shandong Province (tstp20230660) to W.Z.
Author information
Authors and Affiliations
Contributions
Conceptualization: T.D.; bioinformatics, data analyses and visualization: F.Y. and M.W.; AML syngeneic mouse models: F.Y., Y.Z., C.H., and A.N.; solid tumor syngeneic mouse models: F.Y., M.W. and C.H.; tumor dissection: W.L., A.D., Z.D., S.Z., and L.S.; mechanistic studies: F.Y. and M.W.; clinical sample collection: W.J.; project administration: D.T., W.Z., and Y.Z.; data curation: T.D., M.W., Z.W., X.Z., and M.L.; funding acquisition: T.D., W.Z., and Y.Z.; writing—original draft: T.D. and M.W.; Writing—review & editing: T.D., M.W., F.Y., B.G., Z.W., X.Z., and Y.Z. All authors reviewed the results and approved the final version of the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks Yeonseok Chung, Xiaolong Liu, Besim Ogretmen and the other anonymous reviewers for their contribution to the peer review of this work. [A peer review file is available.]
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Zheng, Y., Yang, F., Wang, M. et al. Ceramide disrupts TM9SF2-PGK1 axis to redirect PD-L1 trafficking and enhance antitumor immunity. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70764-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-70764-x
