Identification of CMTM6 and CMTM4 as PD-L1 protein regulators (original) (raw)

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Acknowledgements

We thank F. Scheeren, E. Stickel, V. Blomen, M. Brockmann and the other members of the Schumacher and Brummelkamp laboratories for discussions, J. Grabowska for technical assistance, K. Kemper and D. Peeper for sharing melanoma PDX models and the NKI- AVL flow facility, the NKI-AVL Core Facility Molecular Pathology & Biobanking (CFMPB) for supplying NKI-AVL Biobank material and/or laboratory support. This work was supported by The Queen Wilhelmina Cancer Research Award and European Research Council (ERC) Advanced Grant SENSIT (to T.N.M.S.), NWO Vici Grant (016.Vici.170.033), the Cancer Genomics Center (CGC.nl), and Ammodo KNAW Award 2015 for Biomedical Sciences (to T.R.B.), The Cancer Research Institute (CRI) Irvington Postdoctoral Fellowship (to C.S.), The Landsteiner Foundation for Blood Research, grant 1355 (to J.B.), Proteins@Work, a program of the Netherlands Proteomics Centre financed by NWO, the Netherlands Organisation for Scientific Research (to A.J.R.H.) and The Institute for Chemical Immunology, an NWO Gravitation project (to T.N.M.S. and A.J.R.H.).

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Author notes

1. Lucas T. Jae
   Present address: Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 25, 81377, Munich, Germany
2. Riccardo Mezzadra, Chong Sun and Lucas T. Jae: These authors contributed equally to this work.

Authors and Affiliations

1. Division of Molecular Oncology & Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
   Riccardo Mezzadra, Chong Sun, Raquel Gomez-Eerland, Meike E. W. Logtenberg, Maarten Slagter, Elisa A. Rozeman, Christian U. Blank & Ton N. M. Schumacher
2. Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
   Lucas T. Jae & Thijn R. Brummelkamp
3. Division of Tumor Biology & Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
   Evert de Vries, Yanling Xiao & Jannie Borst
4. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Padualaan 8, Utrecht, 3584 CH, The Netherlands
   Wei Wu & Albert J. R. Heck
5. Netherlands Proteomics Centre, Padualaan 8, Utrecht, 3584 CH, The Netherlands
   Wei Wu & Albert J. R. Heck
6. Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
   Maarten Slagter & Lodewyk F. A. Wessels
7. Division of Medical Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
   Elisa A. Rozeman & Christian U. Blank
8. Division of Pathology, Core Facility Molecular Pathology & Biobanking, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
   Ingrid Hofland & Annegien Broeks
9. Division of Pathology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
   Hugo M. Horlings
10. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, 1090, Austria
    Thijn R. Brummelkamp
11. Cancergenomics.nl, Amsterdam, Plesmanlaan 121, 1066 CX, The Netherlands
    Thijn R. Brummelkamp

Authors

1. Riccardo Mezzadra
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2. Chong Sun
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3. Lucas T. Jae
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4. Raquel Gomez-Eerland
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5. Evert de Vries
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6. Wei Wu
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7. Meike E. W. Logtenberg
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8. Maarten Slagter
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9. Elisa A. Rozeman
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10. Ingrid Hofland
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11. Annegien Broeks
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12. Hugo M. Horlings
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13. Lodewyk F. A. Wessels
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14. Christian U. Blank
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15. Yanling Xiao
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16. Albert J. R. Heck
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17. Jannie Borst
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18. Thijn R. Brummelkamp
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19. Ton N. M. Schumacher
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Contributions

R.M., C.S. and L.T.J. conceived the project, designed and performed experiments, interpreted data and co-wrote the paper. R.G.-E. designed, performed and interpreted functional assays. W.W. designed, performed and interpreted mass spectrometry analyses, A.J.R.H. designed and interpreted mass spectrometry analyses, E.d.V. designed, performed and interpreted immunoprecipitation experiments, Y.X. designed, performed and interpreted human dendritic cell experiments, M.E.W.L. performed and interpreted melanoma PDX experiments, M.S. performed bioinformatic analyses, L.F.A.W. supervised bioinformatic analysis, E.A.R. and I.H. identified samples and performed immunohistochemistry analyses, A.B. and H.M.H. supervised and scored immunohistochemistry analyses, C.U.B. provided and identified samples for immunohistochemistry analyses, J.B. designed and interpreted immunoprecipitation and human dendritic cell experiments, T.R.B. and T.N.M.S. designed experiments, interpreted data and co-wrote the manuscript.

Corresponding authors

Correspondence toThijn R. Brummelkamp or Ton N. M. Schumacher.

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Competing interests

R.M., C.S., L.T.J., T.R.B. and T.N.M.S. are inventors on a patent application covering the use of CMTM6, CMTM4 and STUB1 as therapeutic and diagnostic targets. T.R.B. is co-founder and shareholder of Scenic Biotech. The other authors declare no competing interests

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Reviewer Information Nature thanks S. Ogawa, A. Ribas and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 PD-L1 is regulated by IFNγ and by the 3′ UTR in HAP1 cells.

a, Flow cytometry analysis of PD-L1 expression in untreated HAP1 cells and HAP1 cells treated with the indicated concentrations of IFNγ. An IFNγ concentration of 0.5 ng ml−1 was chosen for the subsequent genetic screen, to allow identification of gene integrations that either enhance or suppress PD-L1 expression. Data are representative of three independent experiments. b, Schematic representation of the PD-L1 gene and of the gene trap insertions observed in HAP1 cells sorted on the basis of either low or high PD-L1 expression. Note the bias towards integrations within introns 5 and 6 in the PD-L1 gene in PD-L1high cells relative to PD-L1low cells, consistent with the structural variants beyond exon 4 of PD-L1 that have been shown to result in increased PD-L1 expression in a subset of adult T-cell leukaemia, diffuse large B-cell lymphoma and stomach cancers11. c, Screen data as depicted in Fig. 1, but now with PD-L1 (CD274) data plotted when either including (CD274) or excluding (CD274*) integrations downstream of exon 5 (Refseq identifier NM_014143.3). MI, mutation index.

Extended Data Figure 2 RNA expression of CMTM6 in human cancers and correlation with PD-L1 mRNA levels.

Pearson’s correlation coefficients (r) are shown along with associated unadjusted P values. Because randomly selected genes are on average also weakly positively correlated (not shown), empirical P values, which represent one minus the quantile of the _CMTM6_- and _CD274_-expression correlation coefficient among a reference distribution composed of correlation coefficients between CMTM6 and randomly selected genes, are also depicted. Empirical P values smaller than 0.5 denote a stronger correlation between CMTM6 and CD274 than the median observed correlation in the reference distribution. TPM, transcript per million. ACC, adrenocortical carcinoma; BLCA, urothelial bladder carcinoma; BRCA, breast cancer; CESC, cervical squamous cell carcinoma; CHOL, cholangiocarcinoma; COAD, colorectal adenocarcinoma; DLBC, diffuse large B-cell lymphoma; ESCA, oesophageal cancer; GBM, glioblastoma multiforme; HNSC, head and neck squamous cancer; KICH, chromophobe renal cell carcinoma; KIRC, clear cell kidney carcinoma; KIRP, papillary kidney carcinoma; LAML, acute myeloid leukaemia; LGG, lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic ductal adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SKCM, cutaneous melanoma;, STAD, stomach cancer; TGCT, testicular germ cell cancer; THCA, papillary thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.

Extended Data Figure 3 Regulation of PD-L1 by CMTM6 in different tumour types.

a, c, e, o, q, Flow cytometry analysis of PD-L1 expression in WM2664 melanoma cells (a), COLO679 melanoma cells (c), DLD1 colorectal cancer cells (e), H2122 non-small-cell lung cancer cells (o) and three short-term cultures of melanoma xenografts (q). Cells are independently transduced with two vectors expressing different CMTM6 shRNAs and are compared with cells transduced with the control vector. b, d, f, p, Western blot analysis of CMTM6 and PD-L1 expression in WM2664 melanoma cells (b), COLO679 melanoma cells (d), DLD1 colorectal cancer cells (f), H2122 non-small-cell lung cancer cells (p). Cells are independently transduced with two vectors expressing different CMTM6 shRNAs and are compared with cells transduced with the control vector. g–j, l, m, Flow cytometry analysis of PD-L1 expression upon staining with anti-PD-L1 antibody or with PD-1–Fc in 8505C thyroid cancer cells (g, h), RKO colorectal cancer cells (i, j) and H2030 non-small-cell lung cancer cells (l, m). k, n, Western blot analysis of CMTM6 and PD-L1 expression in RKO colorectal cancer cells (k) and H2030 non-small cell lung cancer cells (n). Cells are independently transduced with two vectors expressing different CMTM6 shRNAs and are compared with cells transduced with the control vector. In all cases, cells treated with IFNγ (25 ng ml−1) or left untreated were compared. g, The graph shown is identical to Fig. 2f, shown again here to facilitate comparison. Data are representative of three (c–n), two (a, b, o, p) or one (q) independent experiments and were analysed by unpaired _t_-test (a, c, e, g–m, o, q). Data are mean ± s.d. of triplicates (a, c, e, g–m, o, q). ***P < 0.001. Ctrl, control; MFI, median fluorescence intensity. CRC, colorectal cancer; NSCLC, non-small-cell lung cancer; PDX, patient-derived xenograft.

Extended Data Figure 4 CMTM4 and CMTM6, but no other CMTM family members, are regulators of PD-L1.

a, Validation of CMTM6 and CMTM4 downregulation by western blot analysis of cells shown in Fig. 3b. b, c, Ectopic expression of CMTM4 restores IFNγ-induced PD-L1 expression in CMTM6-deficient cells. Two clones of CMTM6-deficient A375 cells (CMTM6 KO #6 and CMTM6 KO #12) were transduced with retroviral vectors encoding CMTM4 (CMTM4 OE) or CMTM6 (CMTM6 OE) individually. After blasticidin selection, cells were cultured in the absence (untreated) or presence of 25 ng ml−1 IFNγ for 72 h before flow cytometry (b) and western blot (c) analysis. Untransduced A375 parental cells served as controls. d, e, Western blot analysis of expression of the indicated CMTM family members, as determined by staining with an anti-Flag antibody. Two exposures of the same gel are shown. Expression of CMTM2 and CMTM8 is not detected and CMTM5 expression is low compared to that of other CMTM family members. f, Phylogenetic analysis of the CMTM family by CLUSTALW. CMTM6 and CMTM4 form the two most closely related members. In view of the lack of detectable expression/low expression observed for CMTM2, CMTM8 and CMTM5, an effect of these CMTM members on PD-L1 protein fate cannot be excluded. However, the observation that CMTM family members 7 and 3, which are more closely related to CMTM4 and CMTM6, do not influence PD-L1 expression makes this unlikely. g, Results of the flow-cytometry-based screen as shown in Fig. 1a, with the position of all CMTM family members indicated. Data are representative of two independent experiments (a–e). Data are mean ± s.d. of triplicates (b). KO, knockout; MFI, median fluorescence intensity; MI, mutation index; OE, overexpression.

Extended Data Figure 5 CMTM6 downregulation does not affect MHC class I and PD-L2 cell-surface levels or PD-L1 mRNA levels and regulates PD-L1 stability after egress from the endoplasmic reticulum.

a–c, Flow cytometry analysis of MHC class I and PD-L2 expression in the panel of cell lines tested in Fig. 2 and Extended Data Fig. 3 (a), and in bone marrow progenitor-derived dendritic cells (b, c) in which cells transduced with the control vector are compared with cells independently transduced with two vectors expressing different shRNA directed against CMTM6. For cell lines, cells treated with IFNγ (as indicated in the other figure legends) or left untreated were compared, for bone marrow progenitor-derived dendritic cells, cells treated with 500 ng ml−1 LPS or left untreated were compared. d, qPCR analyses were performed to quantify relative mRNA levels of PD-L1 in the indicated tumour cell lines. e, Quantification of the experiment shown in Fig. 4c. f, Immunoprecipitates of the samples used in Fig. 4c. Samples were mock-treated, treated with EndoH or with PNGaseF to examine the kinetics of protein maturation. No difference in maturation kinetics was observed between CMTM6-overexpressing and CMTM6-deficient cells. Pulse–chase experiments were performed three times, once comparing CMTM6-overexpressing and CMTM6-deficient cells (a) and twice comparing wild-type and CMTM6-deficient cells. Other data are representative of at least two independent experiments. BM, bone marrow; DC, dendritic cell; EndoH, endoglycosidase H; KO, knockout; MFI, median fluorescence intensity; OE, overexpression; PNGaseF, peptide-N-glycosidase F.

Extended Data Figure 6 Specificity and membrane localization of CMTM6, and co-localization with PD-L1 in human tumours.

a, Membrane-fractionated proteome of 8505C and RKO cells. CMTM6 and PD-L1 were detected by LC–MS/MS predominantly from the plasma membrane fractions. Label-free quantification (LFQ) performed by intensity-based normalization of four fractions together across different cell lines is depicted. b, A375 parental cells, CMTM6-knockout or CMTM6-overexpressing cells were fixed and formalin embedded, and stained for CMTM6 with a monoclonal antibody (clone RCT6) generated against a peptide from the C-terminal domain of CMTM6. Analysis shows a mainly membranous stain, as indicated by the arrowheads. c, Sequential slides from lymph node and subcutaneous metastases from three melanoma patients were stained for PD-L1 (left) or CMTM6 (right), showing frequent localization of PD-L1 within CMTM6+ areas. In total, samples from nine melanoma patients and five PD-L1+ lung cancer samples were analysed. KO, knockout; OE, overexpression.

Extended Data Figure 7 Interactions between CMTM6, PD-L1 and CMTM4, and effect of CMTM6 on PD-L1 stability.

a, A375 parental cells, CMTM6-deficient cells, PD-L1-deficient cells and cells ectopically expressing CMTM6 or PD-L1, were cultured in the absence or presence of 25 ng ml−1 IFNγ for 48 h before preparation of cell lysates. Immunoprecipitation was performed using a CMTM6-specific antibody immobilized on protein-A-coated beads. Immunoprecipitates and whole cell lysate were subjected to SDS–PAGE and immunoblotted for CMTM6 and PD-L1. Two exposures of the same western blot are shown. Arrows indicate PD-L1 bands. b, Parental and CMTM6-deficient 8505C cells were cultured in the absence or presence of 50 ng ml−1 IFNγ for 72 h before preparation of cell lysates. Immunoprecipitation was performed using CMTM6- or PD-L1-specific antibodies immobilized on protein-A-coated beads. Immunoprecipitates and whole cell lysates were subjected to SDS–PAGE and immunoblotted for CMTM6 and PD-L1. Two exposures of the same western blot are shown. Normal IgG served as a control. Arrows indicate PD-L1 bands. c, d, Parental and CMTM6-knockout RKO cells (c) and 8505C cells (d) were lysed and immunoprecipitation was performed using antibodies immobilized on protein-G-coated beads as indicated. Immunoprecipitates and whole-cell lysates were subjected to SDS–PAGE, and western blot analysis of CMTM4 and PD-L1 expession was carried out. Two exposures of the same western blots are shown. Arrows indicate CMTM4 (c) or PD-L1 (d). Data are representative of three independent experiments. KO, knockout; OE, overexpression.

Extended Data Figure 8 Aspects of PD-L1 regulation by CMTM6 and STUB1.

a, V5-tagged PD-L1 was introduced into parental, CMTM6-overexpressing and CMTM6-deficient A375 cells. Cell lysates were denatured and then subjected to immunoprecipitation with anti-V5 antibody immobilized on protein-G-coated beads. Immunoprecipitates were then analysed by immunoblotting with an anti-V5 antibody as a control for the experiments shown in Fig. 4e. b, c, Results of the FACS-based genetic screens in CMTM6-expressing and CMTM6-deficient HAP1 cells as shown in Fig. 1a (b) and in Fig. 3a (c), with the position of STUB1 indicated. d, Relative expression of PD-L1, PD-L2 and the indicated PD-L1–PD-L2 chimaeric proteins in CMTM6 knockdown A375 cells compared to matched control. Chimaeras were detected with an anti-PD-L1 or an anti-PD-L2 antibody. e, Schematic overview of the chimaeric proteins analysed. f, g, HEK293T human embryonic kidney cells were co-transfected with a vector encoding either PD-L1, PD-L2 or the indicated chimaeric protein, together with a vector encoding CMTM6. Cell lysates were denatured and subjected to immunoprecipitation with anti-Flag antibody immobilized on protein-G-coated beads. Lysates and immunoprecipitates were then analysed by immunoblotting with the indicated antibodies. Data are representative of three (a, d), one (f) or two (g) independent experiments. Data represent mean ± s.d. of triplicates. EC, extracellular; IC, intracellular; KO, knockout; MFI, median fluorescence intensity; OE, overexpression; TM, transmembrane.

Extended Data Figure 9 Orientation mapping of CMTM6.

a, Predicted domain topology of CMTM6 according to TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). b, c, A375 cells were transduced with C- or N-terminal haemagglutinin (HA)-epitope-tagged CMTM6. HA-epitope tag staining was performed in both live cells and fixed and permeabilized cells followed by flow cytometry analysis and quantified in c. Data represent mean ± s.d. of triplicates. MFI, median fluorescence intensity.

Extended Data Figure 10 Selectivity of CMTM6 and CMTM6 loss alleviates PD-L1-mediated T-cell suppression.

a, Comparative membrane-fractionated mass spectrometry of CMTM6-proficient or -deficient RKO cells. Four wild-type and four CMTM6-knockout RKO clones were analysed by LC–MS/MS and differential protein abundance is shown in a volcano plot. b, Proteins found up- or downregulated upon CMTM6 removal in both 8505C and RKO cells. c, d, Flow cytometry (c) and western blot (d) analysis of CMTM6 and PD-L1 expression in parental A375 or CMTM6-deficient A375 clones in which PD-L1 is ectopically expressed by lentiviral transduction. e, Primary human T cells were transduced with the MART-1-specific 1D3 TCR31 and PD-1. Transduced T cells were co-cultured with unloaded or MART-I peptide-loaded PD-L1-overexpressing A375 cells (Parental + PD-L1 OE), parental A375 cells (Parental) or CMTM6-deficient A375 cells that overexpressed PD-L1 (CMTM6 KO + PD-L1 OE). IL-2 production in T cells that expressed high (PD-1high), intermediate (PD-1inter) or low (PD-1low) levels of PD-1 were analysed by flow cytometry. Untransduced A375 cells (Parental) served as controls. Data are representative of three independent experiments and were analysed by unpaired _t_-test (c). Data represent mean ± s.d. of triplicates. **P < 0.01; ***P < 0.001; NS, not significant. KO, knockout; OE, overexpression; PM, plasma membrane; TM, transmembrane; WT, wild type.

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Mezzadra, R., Sun, C., Jae, L. et al. Identification of CMTM6 and CMTM4 as PD-L1 protein regulators.Nature 549, 106–110 (2017). https://doi.org/10.1038/nature23669

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• Received: 13 December 2016
• Accepted: 25 July 2017
• Published: 16 August 2017
• Issue Date: 07 September 2017
• DOI: https://doi.org/10.1038/nature23669