Somatic G17V RHOA mutations were found in 50–70% of angioimmunoblastic T-cell lymphoma (AITL). The mutant RHOA lacks GTP binding capacity, suggesting defects in the classical RHOA signaling. Here, we discovered the novel function of the G17V RHOA: VAV1 was identified as a G17V RHOA-specific binding partner via high-throughput screening. We found that binding of G17V RHOA to VAV1 augmented its adaptor function through phosphorylation of 174Tyr, resulting in acceleration of T-cell receptor (TCR) signaling. Enrichment of cytokine and chemokine-related pathways was also evident by the expression of G17V RHOA. We further identified VAV1 mutations and a new translocation, VAV1–STAP2, in seven of the 85 RHOA mutation-negative samples (8.2%), whereas none of the 41 RHOA mutation-positive samples exhibited VAV1 mutations. Augmentation of 174Tyr phosphorylation was also demonstrated in VAV1–STAP2. Dasatinib, a multikinase inhibitor, efficiently blocked the accelerated VAV1 phosphorylation and the associating TCR signaling by both G17V RHOA and VAV1–STAP2 expression. Phospho-VAV1 staining was demonstrated in the clinical specimens harboring G17V RHOA and VAV1 mutations at a higher frequency than those without. Our findings indicate that the G17V RHOA–VAV1 axis may provide a new therapeutic target in AITL.
Recent genetic studies identified that the disease-specific G17V RHOA mutation, together with mutations in TET2, DNMT3A, and IDH2, is a hallmark of angioimmunoblastic T cell lymphomas (AITL). The diagnostic value of these mutations is now being investigated. Circulating tumor DNAs (ctDNAs) may offer a non-invasive testing for diagnosis and disease monitoring of cancers. To investigate whether these mutations are useful markers for ctDNAs in AITL and its related lymphomas, we performed targeted sequencing for TET2, RHOA, DNMT3A, and IDH2 in paired tumors and cell-free DNAs from 14 patients at diagnosis. Eighty-three percent of mutations detected in tumors were also observed in cell-free DNAs. During the disease course, mutations were detectable in cell-free DNAs in a refractory case, while they disappeared in a chemosensitive case. These data suggest that the disease-specific gene mutations serve as sensitive indicators for ctDNAs and may also be applicable for non-invasive monitoring of minimal residual diseases in AITL.
Angioimmunoblastic T‐cell lymphoma (AITL) is a subtype of nodal peripheral T‐cell lymphoma (PTCL). Somatic RHOA mutations, most frequently found at the hotspot site c.50G > T, p.Gly17Val (G17V RHOA mutation) are a genetic hallmark of AITL. Detection of the G17V RHOA mutations assists prompt and appropriate diagnosis of AITL. However, an optimal detection method for the G17V RHOA mutation remains to be elucidated. We compared the sensitivity and concordance of next‐generation sequencing (NGS), droplet digital PCR (ddPCR) and peptide nucleic acid‐locked nucleic acid (PNA‐LNA) clamp method for detecting the G17V RHOA mutation. G17V RHOA mutations were identified in 27 of 67 (40.3%) PTCL samples using NGS. ddPCR and PNA‐LNA clamp method both detected G17V mutations in 4 samples in addition to those detected with NGS (31 of 67, 46.3%). Additionally, variant allele frequencies with ddPCR and those with NGS showed high concordance (P < .001). Three other RHOA mutations involving the p.Gly17 position (c.[49G > T;50G > T], p.Gly17Leu in PTCL198; c.[50G > T;51A > C], p.Gly17Val in PTCL216; and c.50G > A, p.Gly17Glu in PTCL223) were detected using NGS. These sequence changes could not appropriately be detected using the ddPCR assay and the PNA‐LNA clamp method although both indicated that the samples might have mutations. In total, 34 out of 67 PTCL samples (50.7%) had RHOA mutations at the p.Gly17 position. In conclusion, our results suggested that a combination of ddPCR/PNA‐LNA clamp methods and NGS are best method to assist the diagnosis of AITL by detecting RHOA mutations at the p.Gly17 position.
Background: Angioimmunoblastic T cell lymphoma (AITL) is a subset of peripheral T cell lymphomas (PTCLs). AITL has very specific clinical features, including high fever, skin rush, and autoimmune-like manifestations. The molecular pathogenesis of AITL is poorly understood despite the recent progress in genetics of this disease. We and others previously identified disease specific ras homolog family member A (RHOA) mutations together with muations in epigenetic regulators, tet methylcytosine dioxygenase 2 (TET2) and DNA methyltransferase 3 alpha (DNMT3A) mutations, and isocitrate dehydrogenase (NADP(+)) 2, mitochondrial (IDH2) in AITL. Thesemutations were also frequent in PTCL, not otherwise specified (PTCL-NOS) having features of AITL. RHOA, a small GTPase is predominantly activated by guanine nucleotide exchange factors (GEFs). VAV1encodes a GEF, serving as an important mediator of T-cell receptor signaling pathway. Phosphorylation of VAV1 occurs within seconds in response to antigen stimulation of the T-cell receptors by Syk and Src-family tyrosine kinases and initiate downstream signaling. Objective: We aim to identify novel disease specific gene mutations in AITL besides RHOA. Methods: We performed RNA sequencing of 9 PTCL samples, including 6 AITL and 3 PTCL-NOS. Targeted deep sequencing of VAV1 was performed for 139 PTCL samples, including 93 AITL and 46 PTCL-NOS, 48 of which have RHOAmutations. VAV1 wild-type (WT), VAV1-STAP2, and VAV1 mutant (p.173_177del, p.165_174del, and p.Pro615Leu) cDNA was subcloned into pEF vector. Nuclear factor of activated T cell (NFAT) activity in response to CD3 stimulation was examined in Jurkat cells transiently transfected with a reporter vector containing NFAT response element (NFAT-RE) together with VAV1 WT and mutant cDNAs. The levels of interleukin-2 (IL-2) in response to CD3 stimulation were examined for the supernatant of Jurkat cells inducibly expressing VAV1 WT or VAV1-STAP2 cDNAs. Rac1 activation was examined in NIH3T3 cells transiently transfected with VAV1WT and mutant cDNAs. Results: RNA sequencing identified a fusion gene involving VAV1 and STAP2 in an AITL sample without RHOA mutations. Moreover, targeted sequencing of VAV1 identified 2 in-flame deletion mutations in an acidic region (c.C518_529del:p.173_177del and c.C494_520del:p.165_174del) in AITL samples and 2 missense mutations in a zinc finger and SH3-SH2-SH3 module (c.G1668C:p.Glu556Asp and c.C1844T:p.Pro615Leu) in PTCL-NOS and AITL samples, respectively. All of these VAV1 mutations were found in the samples without RHOA mutations. The phosphorylation of VAV1 at Tyr 174 was enhanced in Jurkat cells expressing VAV1-STAP2 cDNA than those with VAV1 WT cDNA or mock. The phosphorylation was efficiently blocked by Src inhibitors, PP2 and dasatinib. Transient transduction of VAV1-STAP2, VAV1 p.173_177del, p.165_174del, and p.Pro615Leu cDNA in Jurkat cells resulted in enhanced NFAT activity. Moreover, the aberrant reporter activity was blocked by Src-family kinase inhibitors. Jurkat cells inducibly expressing VAV1-STAP2 cDNA showed higher IL-2 secretion than those with mock or VAV1 WT. RAC1 activation was higher in NIH3T3 cells transfected with VAV1 p.173_177del and p.165_174del mutant cDNA than those with VAV1 p.Pro615Leu mutant and VAV1-STAP2 cDNA. Conclusions: Recurrent VAV1 abnormalities were found in AITL and PTCL-NOS samples. Our data suggest that the VAV1 mutations may contribute to its clinical features and the VAV1 mutants can be a new therapeutic target. Disclosures Izutsu: Abbvie: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Janssen Pharmaceutical K.K.: Honoraria; Eisai: Honoraria; Kyowa Hakko Kirin: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria; Mundipharma KK: Research Funding. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.
We isolated and characterized Ralstonia solanacearum-responsive genes (RsRGs) related to the hypersensitive response (HR) by virus-induced gene silencing using Nicotiana benthamiana and the potato virus X vector system. We focused on RsRGM10, because induction of HR was delayed in RsRGM10-silenced plants challenged with incompatible R. solanacearum 8107, which induces the HR in, N. benthamiana. e amino acid sequence deduced from the full-length RsRGM10 cDNA showed approximately 90% identity with rolB overexpressed 1 (ROX1) from N. tabacum, and contained an asparagine-rich sequence. We designated this protein NbARP (N. benthamiana asparagine-rich protein). Expression of HRrelated hin1 and onset of the oxidative burst were delayed in NbARP-silenced plants, and induction of myelin basic kinase activity was reduced. Growth of R. solanacearum 8107 was accelerated in NbARP-silenced plants. Induction of the HR by Pseudomonas cichorii and Pseudomonas syringae pv. syringae was also delayed in NbARP-silenced plants. Silencing of NbARP reduced induction of HR-cell death by Agrobacterium tumefaciens-mediated transient expression of HR elicitors and a constitutively active form of mitogen activated protein kinase kinase. e population of R. solanacearum 8107 was decreased in NbARP-overexpressing plants. ese results suggest that NbARP is closely related to the HR.
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