Abstract
Natural killer/T-cell lymphoma (NKTCL) is a malignant proliferation of CD56+ and cytoCD3+ lymphocytes with aggressive clinical course, which is prevalent in Asian and South American populations1. The molecular pathogenesis of NKTCL has largely remained elusive. We identified somatic gene mutations in 25 people with NKTCL by whole-exome sequencing and confirmed them in an extended validation group of 80 people by targeted sequencing. Recurrent mutations were most frequently located in the RNA helicase gene DDX3X (21/105 subjects, 20.0%), tumor suppressors (TP53 and MGA), JAK-STAT-pathway molecules (STAT3 and STAT5B) and epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3). As compared to wild-type protein, DDX3X mutants exhibited decreased RNA-unwinding activity, loss of suppressive effects on cell-cycle progression in NK cells and transcriptional activation of NF-κB and MAPK pathways. Clinically, patients with DDX3X mutations presented a poor prognosis. Our work thus contributes to the understanding of the disease mechanism of NKTCL.
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Acknowledgements
This work was supported by the Chinese National Key Basic Research Project 973 (2013CB966800 to S.-J.C.); the Chinese Ministry of Health (201202003 to S.-J.C.); the Mega-projects of Scientific Research for the 12th Five-Year Plan (2013ZX09303302 to S.-J.C.); the State Key Laboratories Project of Excellence (81123005 to S.-J.C.); the National Natural Science Foundation of China (81325003 and 81172254 to W.-L.Z.); the Shanghai Commission of Science and Technology (11JC1407300 to W.-L.Z.); the Program of Shanghai Subject Chief Scientist (13XD1402700 to W.-L.Z.); the Doctoral Innovation Fund Projects from SJTU School of Medicine (BXJ201312 to Z.-X.Y.); the Samuel Waxman Cancer Research Foundation Co-PI Program; and the Multi-center Hematology-Oncology Protocols Evaluation System (M-HOPES). We are grateful to Y. Zhao, J. Xu and F. Yang for sample preparation, E.-D. Wang for experimental guidance and X.-C. Fei, L.-L. Wu and X.-Q. Li for pathological analysis.
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L.J., Z.-X.Y., Y.-Y.X., L.W., Z.Z. and W.X. performed experiments. Z.-H.G., Z.-G.Z. and J.-Y.H. were responsible for bioinformatics investigation. X.Z. and Y.S. gathered detailed clinical information for the study and carried out clinical analysis. C.-M.P., J.-Y.S., L.X., Y.L. and J.L. carried out the exome sequencing and participated in the validation experiments. Y.H. and C.-P.C. gave technical support. Y.D., J.-F.Z., J.-D.H., J.-F.W., Y.-H.L., L.-H.Y., F.Z., J.-M.W., Z.W., Z.-G.P., F.-Y.C., Z.-M.S., H.D., J.-M.S., J.H. and J.-S.Y. participated in the preparation of biological samples. G.M. carried out the structural analysis. S.-J.C., Z.C. and W.-L.Z. conceived the study, directed and supervised research and wrote the manuscript.
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Supplementary Figure 1 Correlation of nonsilent somatic mutations with the age and the disease stage of 25 people with NKTCL, subjected to whole-exome sequencing.
(a) Distribution of non-silent somatic mutations according to the age of the patients. P value was calculated by two-sided F-test (n = 25 subjects). (b) Distribution of non-silent somatic mutations according to the disease stage of the patients. P value was calculated by two-sided Student’s t test (mean ± s.d.; n = 25 subjects).
Supplementary Figure 3 Somatic copy-number alterations (CNAs) and uniparental disomies (UPDs) in 25 people with NKTCL, subjected to whole-exome sequencing.
(a) Types and genomic distribution of somatic copy number alterations (CNAs) and uniparental disomies (UPDs) in each patient. (b) Types and genomic distribution of somatic CNAs and UPDs according to the chromosomes.
Supplementary Figure 4 Functional categories of somatic mutations in 25 people with NKTCL, subjected to whole-exome sequencing.
(a) Gene ontology analysis of 795 somatic mutations expected to alter the function or structure of the encoded protein. Adjusted P value < 0.01 and False Discover Rate < 0.1 (Online Methods ). (b) Functional categories of 26 candidate gene mutations according to 6q21 deletions.
Supplementary Figure 5 Relationship between mutations of DDX3X, tumor suppressors, JAK-STAT-pathway molecules and epigenetic modifiers in NKTCL.
(a) Distribution of mutations in DDX3X, tumor suppressors (TP53 and MGA), JAK-STAT pathway (STAT3 and STAT5B) and epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3) in NKTCL. (b) Comparison of the allele frequencies of selected mutations in samples harboring mutations in DDX3X and tumor suppressors. (c) Comparison of the allele frequencies of selected mutations in samples harboring mutations in DDX3X and JAK-STAT pathway molecules. (d) Comparison of the allele frequencies of selected mutations in samples harboring mutations in and DDX3X and epigenetic modifiers. Each axis showed the frequencies of the mutant alleles. When a single gene had 2-Hit mutation, the frequencies of major alleles were indicated. Data were analyzed statistically by two-sided Wilcoxon rank-sum test.
Supplementary Figure 6 Cellular localizations of DDX3X and EBV-associated RNA or protein in EBV-infected natural killer cell line KAI3.
(a) Cellular localizations of DDX3X and EBV-Encoded RNA (EBER). (b) Cellular localizations of DDX3X and latent membrane protein 1 (LMP-1). Images were visualized by confocal laser scanning microscopy. Scale bar, 5 μm.
Supplementary Figure 7 Apoptosis assay of DDX3X wild-type natural killer cell line KAI3 transfected with empty vector (vector), wild-type or DDX3X mutants (E348K and A404P).
P values were calculated using two-sided Student’s t test (mean ± s.d.; n = 3 per group).
Supplementary Figure 8 shRNA-mediated knockdown of DDX3X enhanced the growth of DDX3X wild-type natural killer cell line KAI3.
(a) Flow cytometric plots showing the percentage of GFP+ KAI3 cells 3 days (considered as day 0 for cell growth evaluation) and 15 days (considered day 12 for cell growth evaluation) post transduction of cells with scrambled shRNA (Scrambled shRNA) or shRNA targeting DDX3X (DDX3X shRNA1). Cells were switched to natural killer culture medium with reduced IL-2 (25 U/mL). (b) Quantification of the percentages of GFP+ KAI3 cells transfected with scrambled shRNA or DDX3X shRNA1 at regular time intervals, as normalized to the levels at day 0. **: P < 0.01 as compared to the scrambled shRNA. P values were calculated using two-sided Student’s t test (mean ± s.d.; n = 3 per group). (c) Western blot images of DDX3X levels in shRNA-transfected, GFP+ sorted cells post transduction with the scrambled shRNA or DDX3X shRNA1. (d) Flow cytometric plots showing the percentage of GFP+ KAI3 cells 3 days (considered as day 0 for cell growth evaluation) and 15 days (considered day 12 for cell growth evaluation) post transduction of cells with scrambled shRNA (Scrambled shRNA) or shRNA targeting DDX3X (DDX3X shRNA2). Cells were switched to natural killer culture medium with reduced IL-2 (25 U/mL). (e) Quantification of the percentages of GFP+ KAI3 cells transfected with scrambled shRNA or DDX3X shRNA2 at regular time intervals, as normalized to the levels at day 0. **: P < 0.01; ***: P < 0.001, as compared to the scrambled shRNA. P values were calculated using two-sided Student’s t test (mean ± s.d.; n = 3 per group). (f) Western blot images of DDX3X levels in shRNA-transfected, GFP+ sorted cells post transduction with the scrambled shRNA or DDX3X shRNA2.
Supplementary Figure 9 Gene-expression profiles of NF-κB and MAPK pathways in NKTCL.
(a) Significantly upregulated and downregulated genes of NF-κB pathway in DDX3X-mutated tumors, as compared to those in DDX3X-wild-type (wt) tumors of NKTCL. (b) Significantly upregulated and downregulated genes of MAPK pathway in DDX3X-mutated tumors, as compared to those in DDX3X-wild-type (wt) tumors of NKTCL.
Supplementary Figure 10 Kaplan-Meier analysis of the survival of people with NKTCL, excluding extranasal cases or stage III and stage IV cases.
(a) Overall survival (OS) and progression-free survival (PFS) of NKTCL patients excluding extranasal cases according to mutation status of DDX3X and TP53 (upper panel) and according to risk stratification combining International Prognostic Index (IPI) and mutation status of DDX3X and TP53 (lower panel). (b) OS and PFS of NKTCL patients excluding stage III–IV cases according to mutation status of DDX3X and TP53 (upper panel) and according to risk stratification combining IPI and mutation status of DDX3X and TP53 (lower panel). Risk stratification combining IPI and mutation status of DDX3X and TP53: Group 1: IPI0–1 and wtDDX3X/wtTP53; Group 2: IPI0–1 and mutDDX3X/mutTP53 or IPI2–5 and wtDDX3X/wtTP53; Group 3: IPI2–5 and mutDDX3X/mutTP53. wt: wild-type, individuals without DDX3X or TP53 mutations; mut: mutation, individuals with DDX3X or TP53 mutations. One individual with both DDX3X and TP53 mutations was grouped into two cohorts with mutations.
Supplementary Figure 11 Variant allele frequency of single-nucleotide variations in 25 people with NKTCL, subjected to whole-exome sequencing.
In each plot, the density curve depicted the clustered variant allele frequency (VAF) to determine the number of clusters.
Supplementary Figure 13 Representative GeneScanning results of TRB or TRG rearrangement in NKTCL.
(a) TCRB rearrangement negative. (b) TCRG rearrangement negative. (c) TCRG rearrangement positive. The control gene peaks were shown at 101, 200, 300 and 395nt.
Supplementary Figure 14 Representative GeneScanning results of TRD rearrangement in NKTCL.
(a) TCRD rearrangement negative. (b) TCRD rearrangement positive. The control gene peaks were shown at 101, 200, 300 and 395nt.
Supplementary Figure 15 Positive controls of immunohistochemistry of human anti-RelB antibody and human anti-p-ERK antibody.
(a) The positive control of immunohistochemistry of human colon cancer tissue using anti-RelB antibody (Proteintech, 25027-1-AP). (b) The positive control of immunohistochemistry of human lung cancer tissue using anti-p-ERK antibody (Cell Signaling, 4370S). Scale bar, 20 μm.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–15 and Supplementary Tables 4 and 6–12 (PDF 2826 kb)
Supplementary Table 1
Clinicopathological characteristics of 105 subjects with NKTCL (XLSX 48 kb)
Supplementary Table 2
Somatic nonsilent mutations identified in 25 subjects with NKTCL, subjected to whole-exome sequencing (XLSX 149 kb)
Supplementary Table 3
Somatic copy-number alterations and uniparental disomies in 25 subjects with NKTCL, subjected to whole-exome sequencing (XLSX 21 kb)
Supplementary Table 5
Mutation screening in test and validation cohorts of NKTCL (n = 105) (XLSX 22 kb)
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Jiang, L., Gu, ZH., Yan, ZX. et al. Exome sequencing identifies somatic mutations of DDX3X in natural killer/T-cell lymphoma. Nat Genet 47, 1061–1066 (2015). https://doi.org/10.1038/ng.3358
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DOI: https://doi.org/10.1038/ng.3358
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