The molecular mechanisms underlying angioimmunoblastic T cell lymphoma (AITL), a common type of mature T cell lymphoma of poor prognosis, are largely unknown. Here we report a frequent somatic mutation in RHOA (encoding p.Gly17Val) using exome and transcriptome sequencing of samples from individuals with AITL. Further examination of the RHOA mutation encoding p.Gly17Val in 239 lymphoma samples showed that the mutation was specific to T cell lymphoma and was absent from B cell lymphoma. We demonstrate that the RHOA mutation encoding p.Gly17Val, which was found in 53.3% (24 of 45) of the AITL cases examined, is oncogenic in nature using multiple molecular assays. Molecular modeling and docking simulations provided a structural basis for the loss of GTPase activity in the RHOA Gly17Val mutant. Our experimental data and modeling results suggest that the RHOA mutation encoding p.Gly17Val is a driver mutation in AITL. On the basis of these data and through integrated pathway analysis, we build a comprehensive signaling network for AITL oncogenesis.
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We appreciate helpful discussion and comments from P.J. Park (Harvard Medical School). This work was supported by grants from the Samsung Biomedical Research Institute (SP2-B2-04 to Y.H.K., W.S.K. and H.Y.Y. and GE1-B2-071 to H.Y.Y.), the Samsung Cancer Research Institute (cancer genomics project SCRI-12-02), the National Research Foundation of Korea (NRF-2012M3A9D1054744 and NRF-2012M3A9B9036673 to S.L. and NRF-2011-0019745), the GIST (Gwangju Institute of Science and Technology) Systems Biology Infrastructure Establishment Grant through ERCSB (S.L. and J.K.) and the Ewha Global Top5 Grant of Ewha Womans University.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Classical morphology with effacement of normal architecture and marked vascular proliferation associated with aggregates of atypical lymphoid cells (H&E staining). (b) The majority of atypical cells express CD3. (c) CD21 immunostaining showing hyperplastic follicular dendritic cell meshwork. (d) A portion of tumor cells express CD10. (e) Some tumor cells express CXCL13. (f) PD-1 is expressed in some neoplastic cells.
Frozen section of the tumor sample showing hypercellular lymphoid tissue without necrosis (H&E staining).
Patient ID with RHOA G17V mutation is indicated by red text and arrows. Representative traces of wild-type and G17V mutant RHOA are shown in the top left panel. The horizontal red arrow shows the direction of genomic DNA sequencing. The mutant sequence shows a clear heterozygous mutation. Traces from tumor samples obtained from 45 AITL, 20 NK/T cell and 13 PTCL-NOS patients are shown in the rectangles. Patients 5, 6, 9, 10, 29, 30 and 43 in the AITL group showed the mutant as the dominant peak. The other 17 AITL patients showed the heterozygous mutation clearly. All three NK/T cell patients with somatic mutation showed the heterozygous genotype. Only one PTCL-NOS patient showed a heterozygous mutation. Sequencing trace chromatograms are representative of at least three independent experiments.
In vitro proliferation assays in (a) the SUP-T1 cell line and (b) the MOLT-4 cell line. Cells expressing inactive RHOA (G17V and T19N) displayed enhanced cell proliferation. P < 0.01 compared with the cells expressing WT. Each condition with three replicates was repeated three times and expressed as the ± s.d.
Supplementary Figure 6 Inhibition of the RHOA-ROCK pathway downregulates AKT phosphorylation and promotes cell proliferation.
Inhibition of the RHOA-ROCK pathway downregulates AKT phosphorylation and promotes cell proliferation. (a) After transfection of Jurkat cells with control siRNA or RHOA siRNA, AKT phosphorylation levels and cell proliferation were assessed. Cell lysates were prepared 48 h after transfection and processed for immunoblotting with the indicated antibodies. The extent of RHOA depletion was determined by immunoblotting with anti-RHOA antibodies. As a loading control, a-tubulin was used. Cells transfected with siRNA were incubated for 48 h, and relative cell proliferation was determined using a cell counting kit (CCK-8). Five replicates of each condition were repeated three times; the data are expressed as mean ± s.d. (b) After treatment with the ROCK inhibitor, Y-27632 (30 μM), AKT phosphorylation levels and cell proliferation were measured. Cell lysates were prepared 48 h after treatment and processed for immunoblotting with the indicated antibodies. Cells treated with Y-27632 were incubated for 48 h, and relative cell proliferation was determined using a cell counting kit (CCK-8). Five replicates of each condition were repeated three times, and the data are expressed as mean ± s.d.
Structural models of wild-type and mutant RHOA mutant proteins. Homology modeling was based on the PDB structure of 3LXR. GTP substrate and its ribose moiety are indicated in yellow and pink, respectively. The glycine and valine residues are shown in green and red, respectively.
Heat map from hierarchical clustering of differentially expressed genes. AITL patients from our study (PAT1–PAT9) and GSE6338 (AITL1–AITL6) were included. Normal control samples are indicated as CD4+. Gene clusters from hierarchical classification were subjected to the DAVID web server for Gene Ontology analysis, and the most enriched term for each cluster was determined using the q value from the FDR test.
Supplementary Figure 9 Statistically significant KEGG pathways from GAGE gene set enrichment analysis.
Pathways are grouped into functional categories. Up- and downregulated pathways are indicated by pink and green backgrounds, respectively. Numbers of SNVs, indels, CNVs and DEGs indicate the number of genes affected in each pathway.
(a) Patients with wild-type RHOA (PAT1 and PAT3). (b) Patients with G17V RHOA (PAT2 and PAT4–PAT9).
Supplementary Tables 1–3, 5–7 and 9, and Supplementary Figures 1–10 (PDF 2830 kb)
Full list of somatic SNVs. (XLS 294 kb)
Full list of differentially expressed genes. (XLS 979 kb)
List of genes affected by copy number variations. (XLS 3064 kb)
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Yoo, H., Sung, M., Lee, S. et al. A recurrent inactivating mutation in RHOA GTPase in angioimmunoblastic T cell lymphoma. Nat Genet 46, 371–375 (2014) doi:10.1038/ng.2916
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