It is now important to conduct clinical studies to evaluate effects of dasatinib in AITL patients

Oct 25, 2024 PI-PLC

It is now important to conduct clinical studies to evaluate effects of dasatinib in AITL patients. 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 mutations and a new translocation, mutation-negative samples (8.2%), whereas none of the 41 mutation-positive samples exhibited mutations. Augmentation of 174Tyr phosphorylation was also demonstrated in Rabbit Polyclonal to ZADH1 VAV1CSTAP2. Dasatinib, a multikinase inhibitor, efficiently blocked the accelerated VAV1 phosphorylation and the associating TCR signaling by both G17V RHOA and VAV1CSTAP2 expression. Phospho-VAV1 staining was demonstrated in the clinical specimens harboring G17V and mutations at a higher frequency than those without. Our findings indicate that the G17V RHOACVAV1 axis may provide a new therapeutic target in AITL. Introduction Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of peripheral T-cell lymphoma.1 AITL patients display generalized lymphadenopathy and immune system-mediated manifestations including high fever, skin rash, polyarthritis, hemolytic anemia and hypergammaglobulinemia.2 We and others previously conducted gene mutational profiling of AITL samples and observed mutations converting glycine to valine at amino acid 17 (the G17V mutation) in up to 70% of AITL.3, 4, 5 Genes encoding the epigenetic regulators and are also frequently mutated in AITL.3, 4, 6, 7, 8, 9, 10 RHOA is a small GTPase that cycles between guanosine diphosphate (GDP)-bound inactive and guanosine-triphosphate (GTP)-bound active forms. Guanine nucleotide exchange factors (GEFs) activate RHOA by replacing GDP with GTP. Physiologically, RHOA mediates migration and polarity of T cells.11, 12 RHOA also functions in thymocyte development13, 14 and activation VRT-1353385 of pre-T-cell receptor (pre-TCR) signaling in thymocytes.13, 15 Glycine at RHOA residue 17 is located at a critical position for GTP binding. G17V RHOA protein is considered to be a loss-of-function mutant, as G17V RHOA does not bind Rhotekin, a molecule with high affinity for the GTP-bound form.3, 4, 5 Nonetheless, the impact of G17V RHOA expression on AITL remains unclear. The VAV1 protein mediates a signaling cascade triggered by the TCR engagement partly through GEF activity,16 whereas GEF-independent VAV1 functions are also reported.17, 18 In the latter, VAV1 functions as an adaptor in a protein complex that promotes phosphorylation of phospholipase C-1 (PLC1).18, 19 PLC1 phosphorylation induces its own enzymatic activity to upregulate the second messengers diacylglycerol and inositol 1,4,5-triphosphate, in turn promoting calciumCcalmodulin signaling and enhancing nuclear factor of VRT-1353385 activated T cells (NFAT) transcription.20 VAV1 also functions in extracellular signal-regulated kinase, c-Jun N-terminal kinase and nuclear factor-B pathways,21 and its activation is tightly regulated by multilayered autoinhibition by interaction of its Dbl-homology (DH) domain with both acidic (167C178)22 and C-terminal Src homology 2 (SH2)/SH3 domains. TCR engagement initially triggers the phosphorylation of Tyr142 and Tyr160 of VAV1 protein, destabilizing modulatory contacts and facilitating recruitment of the Src kinases LCK and FYN by providing a docking site for their SH2 domains. Thereafter, VAV1 Tyr174 is phosphorylated,23 relieving core inhibitory interactions with the acidic and DH domains, resulting in the activation of downstream effectors. Conversion of Tyr174 to either Phe17 or Asp24 or physiologic phosphorylation of the wild-type Tyr174 residue reportedly activates VAV1 signaling. In addition, deletion of the VAV1 C terminus enhances its signaling.25 Here we used mass spectrometry and immunoprecipitation to show that the G17V RHOA protein specifically binds to VAV1 protein. Upon TCR stimulation, VAV1 binding to G17V RHOA accelerated VAV1 phosphorylation and the eventual downstream signaling cascade. Materials and methods Patients and samples Samples were obtained from patients with approval of local ethics committees in all participating institutes. Informed consent was obtained from all living subjects. Cells Jurkat cells inducibly expressing the wild-type (WT) and G17V RHOA VRT-1353385 mutant complementary DNA (cDNA) and mock-transduced cells have been previously described.3 We newly established VAV1CSTAP2-expressing Jurkat cells with a method similar to the previous one.3 SU9T01 cells inducibly expressing WT or VRT-1353385 G17V RHOA cDNA and mock-transduced cells were also established previously. Jurkat cells and SU9T01 cells were cultured at 37?C in RPMI-1640 Medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal calf serum and 1% penicillin streptomycin. The 293T cells were cultured at 37?C in Dulbeccos modified Eagles medium (Sigma-Aldrich) supplemented with 10% fetal calf serum and 1% penicillin streptomycin. Other experimental methods DNA extractions, targeted sequencing of and genes, antibodies, inhibitors, mRNA sequencing, plasmids, TCR stimulation, immunoprecipitation, mass spectrometry, NFAT reporter assay, interleukin-2 (IL-2) expression assay and immunohistochemistry are described in Supplementary Information. Results Identification of G17V RHOA-specific binding proteins in Jurkat cells To identify specific binding partners of G17V RHOA, we performed immunoprecipitation analysis in Jurkat cells overexpressing Flag-tagged WT RHOA (JurkatWTRHOA) or the G17V RHOA mutant (JurkatG17V). Specifically, we used ANTI-FLAG Magnetic beads in either line, with or without TCR stimulation (Supplementary Figure 1). By targeted pull-down followed by mass spectrometry, we identified G17V RHOA-specific binding proteins: 44 proteins were identified specifically using G17V bait,.