Abstract
Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development1,2,3,4,5,6. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma6,7,8,9,10. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3′ region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3′-untranslated region (UTR). Disruption of the Pd-l1 3′-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3′-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.
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Data deposits
Sequencing data have been deposited in the European Genome-phenome Archive (EGA) under accession EGAS00001001296 (https://www.ebi.ac.uk/ega/studies/EGAS00001001296).
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Acknowledgements
This work was supported by Grant-in-Aid from the Japan Agency for Medical Research and Development (Practical Research for Innovative Cancer Control (15Ack0106014h0002) and Medical Research and Development Programs Focused on Technology Transfer (15im0210102h0001)), Grant-in-Aid for Scientific Research (KAKENHI 22134006, 15H05909, 25250020), and National Cancer Center Research and Development Funds (26-A-6). We thank M. Sago, M. Nakamura and S. Baba for technical assistance, and R. Velaga for English editing. The supercomputing resources were provided by the Human Genome Center, the Institute of Medical Science, the University of Tokyo. This research also used computational resources of the K computer provided by the RIKEN Advanced Institute for Computational Science through the HPCI System Research project (hp140230, hp160219, and hp150232). The results shown here are partly based on data generated by the TCGA Research Network (http://cancergenome.nih.gov/).
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K.Kataoka, Y.S., H.T., K.C., S.I., and S.Miyano performed sequencing data analyses. H.S., T.Y., Y.Totoki, H.N., N.H. and T.Shibata assisted sequencing data analyses. K.Kataoka, Y.N., Y.W., N.K., K.Y., M.S. and K.Kashiwase performed sequencing experiments. K.Kataoka, S.N., T.M., K.M., N.M., H.K., and Y.A. performed functional assays. S.Mizuno. and S.T. designed sgRNAs. Y.Takeda, M.M., and T.Seya performed in vivo experiments. S.S. and K.T. performed IHC assay. A.K., H.I., Y.I., W.M., K.Shide, Y.K., T.H., T.K., K.I., A.T.-K., Y.M., and K.Shimoda collected specimens. K.Kataoka, Y.S., and S.O. generated figures and tables and wrote the manuscript. S.O. led the entire project. All authors participated in discussions and interpretation of the data and results.
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Reviewer information Nature thanks J. Cools, M. Meyerson and A. Ribas for their contribution to the peer review of this work
Extended data figures and tables
Extended Data Figure 1 PD-L1 SVs affecting 3′-UTR in ATL.
a, Diagram showing numbers of ATL samples investigated by WGS/RNA-seq. b, Genome-wide distribution of SV (without deletion) breakpoints in 49 ATL samples, showing a prominent peak at the PD-L1 locus. c, Summary of different types of SVs commonly affecting 3′ region of the PD-L1 gene are shown by indicated colours. d, 3′-truncated PD-L1 mRNA transcripts observed in ATL cases. RNA-seq data are visualized by IGV for ATL samples with or without PD-L1 SVs. Aberrant PD-L1 transcripts are shown in red.
Extended Data Figure 2 Fusion genes involving PD-L1 and non-coding sequences identified in ATL.
a, ATL020 fused sequence: the sequences derived from PD-L1 exons 1–6 and INSL6 intron 1 are marked in yellow and green, respectively. The non-template sequence is marked in blue, and stop codon (UAG), poly-A signal (AAUAAA) and poly-A in red. The putative translated region is underlined. b, Genomic structure of the rearranged PD-L1 locus and transcription in two representative cases (ATL012 and ATL079) with 3′-UTR-truncated PD-L1 transcripts, in which PD-L1 ORF is terminated before exon 6 or 7, and merged into an intergenic sequence. Breakpoints (blue dotted lines) are shown with accompanying copy number alterations. c, Structure and breakpoint sequence of PD-L1 fusion transcripts (top) with Sanger sequencing chromatogram (bottom). d, Length of abnormal PD-L1 transcripts identified in ATL samples with PD-L1 SVs, compared with wild-type PD-L1. e, Genomic and transcript sequences from the PD-L1 locus containing 327 bp inversion within the last exon identified in case ATL017. Aberrant PD-L1 transcripts have a putative poly-A signal sequence in the inverted region, followed by poly-A tract.
Extended Data Figure 3 Elevated PD-L1 mRNA expression in ATL according to PD-L1 SV state.
Diagonal plots between PD-L1 exon 3 expression (RPKM) and its relative value to that of 3′-UTR (exon 3 to 3′-UTR ratio) for 43 ATL cases. SV (+) cases are indicated by corresponding colours to each SV type.
Extended Data Figure 4 PD-L1 fusion proteins identified in ATL.
a, Amino acid sequence alignment of wild-type and truncated PD-L1 proteins. Transmembrane domain is shaded in blue. Conserved regions are shown in red. b, Western blot analysis with antibodies against the N-terminal and C-terminal domains of PD-L1 in PC-9 cells transduced with indicated PD-L1 constructs. c, d, Flow cytometry plots for PD-L1 surface expression (c) and PD-1 Ig binding (d) in PC-9 cells transduced with indicated PD-L1 constructs. e, Western blot of PD-L1 SV(+) cases harbouring intact or truncated ORFs, compared with SV(−) cases using antibodies specifically detecting N-terminal and C-terminal domains of PD-L1. b–e, Representative of three independent experiments.
Extended Data Figure 5 Flow chart for detecting abnormal PD-L1 transcripts in the TCGA cohort.
In total, 10,210 tumour samples in 33 tumour types, for which RNA-seq data were available in TCGA, were assessed by exon-level expression quantification and fusion detection by STAR algorithm. Viral integration within or near the PD-L1 gene was also searched in this cohort. After manual review by IGV, a total of 32 cases with aberrant PD-L1 transcription were identified.
Extended Data Figure 6 3′-truncated PD-L1 mRNA transcripts in the TCGA cohort.
RNA-seq data are visualized by IGV for TCGA samples with abnormal PD-L1 transcription. Aberrant PD-L1 transcripts were shown in red.
Extended Data Figure 7 Aberrant transcription affecting PD-L1 3′-UTR and associated genomic alterations identified in multiple cancers.
a, Genomic structure of the rearranged PD-L1 locus and transcription in FA-A4XK-01 (DLBC), BP-4983-01 (KIRC), L5-L4OE-01 (ESCA), and F5-6814-01 (READ), showing loss of PD-L1 3′-UTR transcription and fusion transcripts between PD-L1 and intronic or intergenic segments. Breakpoints (blue dotted lines) are shown with accompanying copy number alterations. Del, deletion. b, PD-L1 DNA copy number versus JAK2 mRNA expression across 48 DLBC (left), 415 STAD (middle), and 43 ATL (right) samples. SV(+) samples (red) and those with 9p24.1 copy number gains involving both JAK2 and PD-L1 genes (orange) are indicated. P values for the effects of PD-L1 SVs and copy number on JAK2 expression (GLM) are shown. c, Genomic structure of the rearranged PD-L1 locus and transcription in two cases with viral integrations around the PD-L1 gene; a STAD case (FP-7998-01) with an EBV integration (top) and an HNSC case (CV-5443-01), showing HPV16 integration, which was described previously19, and premature termination of PD-L1 transcripts within intron 4 (bottom).
Extended Data Figure 8 Induction of Pd-l1 3′-UTR deletions and inversions in mouse cell lines using the CRISPR-Cas9 system.
a, Positions of targeting sgRNAs used for CRISPR-Cas9-mediated disruption of Pd-l1 3′-UTR are indicated by arrows. b, Pd-l1 surface expression in EG7-OVA cells transfected with Cas9 and no, single, or pairwise sgRNAs. Representative of three independent experiments. c, d, PCR detection of the Pd-l1 3′-UTR deletion (c) or inversion (d) breakpoint junction from EG7-OVA, P815, and B16-F10 cells in which Cas9 was expressed without (parental) or with no sgRNA (mock), or a pair of Pd-l1 sgRNAs. e, Sequence chromatogram of the detected Pd-l1 3′-UTR deletions from sgPd-l1-transfected EG7-OVA, P815, and B16-F10 cells. f, g, Pd-l1 exon 4 mRNA expression (RPKM) was calculated from the RNA-seq data for EG7-OVA, P815, and B16-F10 cells in which Cas9 was expressed without (parental) or with no sgRNA (mock), or a pair of Pd-l1 sgRNAs (f). RNA-seq reads within the Pd-l1 gene were visualized by IGV (g).
Extended Data Figure 9 Induction of PD-L1 3′-UTR deletions and inversions in human cell lines using the CRISPR-Cas9 system.
a, PCR detection of the PD-L1 3′-UTR deletion breakpoint junction from T2 cells in which Cas9 was expressed without (parental) or with no sgRNA (mock), or a pair of PD-L1 sgRNAs. b, Sequence chromatogram of the detected PD-L1 3′-UTR deletions from sgPD-L1-transfected HEK293T and T2 cells. c, PCR detection of the PD-L1 3′-UTR inversion breakpoint junction from HEK293T, T2, and PC-9 cells in which Cas9 was expressed without (parental) or with no sgRNA (mock), or a pair of PD-L1 sgRNAs. d, Sequence chromatogram of the detected PD-L1 3′-UTR inversions from sgPD-L1-transfected HEK293T, T2, and PC-9 cells. e, Visualization of RNA-seq reads within the PD-L1 gene for T2 and PC-9 cells in which Cas9 was expressed without (parental) or with no sgRNA (mock), or a pair of PD-L1 sgRNAs. f, Flow cytometric analysis of PD-L1 surface expression in parental or sgPD-L1-transfected PC-9 cells stimulated with IFN-γ (100 or 300 U ml−1) for 48 h. Representative of three independent experiments.
Extended Data Figure 10 Tumour-intrinsic Pd-l1 activation by 3′-UTR loss suppresses CD8+ cytotoxic T lymphocyte recruitment within the tumour microenvironment.
a, Strategy for evaluating the effect of Pd-l1 3′-UTR disruption on anti-tumour immunity. b, Representative immunofluorescence images (from experiments in Fig. 4c) of CD8 (green) and DAPI (purple) staining in mock- and sgPd-l1-transfected EG7-OVA tumours treated with PBS or poly(I:C). c, Flow cytometric analysis showing frequency of CD8+ T cells infiltrating into mock- and sgPd-l1-transfected EG7-OVA tumours treated with PBS or poly(I:C) (n = 6 per group; Welch’s t-test). Data represent mean ± s.e.m. d, Flow cytometric analysis showing frequency of CD8+ T cells infiltrating into sgPd-l1-transfected, poly(I:C)-treated EG7-OVA tumours treated with isotype control or anti-Pd-l1 antibody (n = 7 per group; Welch’s t-test). Data represent mean ± s.e.m.
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Kataoka, K., Shiraishi, Y., Takeda, Y. et al. Aberrant PD-L1 expression through 3′-UTR disruption in multiple cancers. Nature 534, 402–406 (2016). https://doi.org/10.1038/nature18294
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DOI: https://doi.org/10.1038/nature18294
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