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.

BCL6, a master transcription factor for differentiation of follicular helper T (TFH) cells, is highly expressed in angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphomas (PTCL) containing tumor cells with TFH features. TET2, encoding an epigenetic regulator, is frequently mutated in AITL/PTCL. We previously reported that Tet2 knockdown mice developed T-cell lymphomas with TFH features. Hypermethylation of the Bcl6 locus followed by BCL6 upregulation was thought to be the key event for lymphoma development in mice. The mechanisms by which BCL6 expression is upregulated in human AITL/PTCL, however, have not been elucidated. Here, we investigated the impact of TET2 mutations on methylation of BCL6 locus in human AITL/PTCL samples. Hypermethylation of the BCL6 locus was more frequent in PTCL samples than B-cell lymphoma samples (PTCL vs B-cell lymphomas: 9/42 vs 0/35). PTCL samples with TET2 mutations were more frequently hypermethylated than those without TET2 mutations (PTCL with TET2 mutations vs without mutations: 6/22 vs 0/17). BCL6 expression in hypermethylated samples was higher than that in low methylated samples. Deregulated BCL6 expression caused by hypermethylation and TET2 mutations may result in skewed TFH differentiation and eventually contribute to AITL/PTCL development in patients, as well as lymphoma development in Tet2 knockdown mice.

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.

functional annotation was done by gene set enrichment analysis (GSEA).Results: Genes downregulated after ETS1 silencing by siRNAs in 5 ABC DLBCL cell lines (TMD8, HBL-1, U2932, OCI-Ly10, SU-DHL-2) belonged to the signaling of BCR (NES 2.45 FDR <0.001), CD40 (NES 2.67 FDR <0.001), and NFKB/TNFA (NES 2.03 FDR 0.01), as well as to inflammatory response (NES 2.14 FDR 0.005) and differentiation (NES 2.5 FDR <0.001). Three candidate genes were selected for further studies according to the following educated guess criteria: i) potential biologic role; and ii) presence of binding sites for ETS factors in their promoter regions. HCST, RGS1 and FAIM3 were confirmed to be downregulated, by qPCR, also after silencing with shRNA. Their expression in clinical specimens was confirmed in two large series of 181 and 233 DLBCL cases, respectively. FAIM3 expression was higher in ABC DLBCL cases than germinal center type (GCB) DLBCL in both series (P < 0.0001), and a similar pattern was confirmed at protein level in cell lines derived from ABC (n = 6) or GCB DLBCL (n = 8). Conclusions: NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.