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.
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.
Asterisk with author names denotes non-ASH members.