LAUSR

Background: VAV1 is known as an important mediator of T-cell receptor (TCR) signaling through its guanine exchange factor (GEF)-dependent and independent functions. Recent studies identified activating VAV1 mutations in several types of T-cell malignancies including peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), ALK-negative anaplastic large cell lymphoma (ALCL), and adult T-cell lymphoma/leukemia (ATLL). However, the functions of VAV1 mutations in T-cell malignancies have not been clarified. Objective: We aim to identify the oncogenic signaling of VAV1 mutations in T cells using genetically engineered mice. Methods: Human VAV1 mutant (p.165_174del) (VAV1-Del) and VAV1-STAP2 fusion cDNAs, identified in our PTCL cohort (Fujisawa, Leukemia 2018) were cloned into a VA vector under the CD2 promoter. The vectors were injected into eggs to generate VAV1-Del and VAV1-STAP2 transgenic mice. The mice were further crossed with p53-/- mice to generate p53-/- x VAV1-Del or p53-/- x VAV1-STAP2 mice. Cell surface markers of tumor cells were analyzed by flowcytometry. Cell suspension of tumors were cultured, and were intraperitoneally injected into BALBc/nu mice to examine the cell-autonomous proliferative activity in vitro and tumor-initiating capacity in vivo, respectively. RNA sequencing was performed to clarify the downstream signaling of VAV1 mutations. Results: The p53-/- mice expressing VAV1 mutants showed significantly poorer overall survival (OS) compared to p53-/- mice (p53-/- x VAV1-Del, median 16.6 weeks; p53-/- x VAV1-STAP2, median 18.6 weeks; vs p53-/-, median 33.7 weeks: p<0.001), while mice with VAV1 expression in the wild-type (WT) background as well as WT mice remained alive during the observation period (>50 weeks). p53-/- x VAV1-Del and p53-/- x VAV1-STAP2 mice developed either T-cell lymphoblastic leukemias (LBL) infiltrating into thymus, lung, spleen, and liver, or mature T-cell lymphomas (Lym) into lymph nodes, spleen, and liver. In contrast, p53-/- mice developed only T-LBL at thymus. Flow cytometric analysis showed that most of T-LBL cells developed in p53-/- mice with VAV1 mutants were CD8+ single positive (SP), while those in p53-/- mice were either CD4+CD8+ (double positive, DP) or CD8+ SP. Lym cells in p53-/- mice with VAV1 mutants were either CD4+ SP or CD4-CD8- (double negative, DN) (in p53-/- x VAV1-Del mice, 5/9 CD8+ SP T-LBL, 1/9 DP T-LBL , 2/9 CD4+ SP Lym , and 1/9 DN Lym; in p53-/- x VAV1-STAP2 mice, 9/13 CD8+ SP T-LBL, 1/13 CD4+ SP Lym , and 3/13 DN Lym; p53-/- mice, 3/7 CD8+ SP T-LBL and 4/7 DP T-LBL). T-LBL with or without VAV1 mutants were immortalized in vitro over 4 weeks without any cytokines, while Lym with VAV1 mutations could not be maintained in vitro. The BALBc/nu mice transplanted with cell suspension of either T-LBL or Lym with VAV1 mutants were succumbed to death around 10 or 15 weeks, respectively. All the tumor cells developed in transplanted mice showed the similar immunophenotype to those of donor cells. Gene set enrichment analysis (GSEA) following RNA sequencing showed that G2M check point, E2F targets, mitotic spindle, PI3K/Akt/mTOR signaling, and hedgehog signaling were enriched in CD8+ SP T-LBL with VAV1 mutants compared with DP T-LBL in p53-/- mice, while E2F targets, MYC targets, G2M checkpoint, Oxidative phosphorylation, and MTORC1 signaling were upregulated in CD4+ SP Lym with VAV1 mutants in comparison with WT CD4+ spleen cells. We previously reported that TCR and Tfh pathways were enriched in TET2-/-/RHOA G17V mice through activation of VAV1 (Tran, ASH 2018). Curiously, neither of them were enriched in CD4+ SP Lym with VAV1 mutants, while TCR pathway was enriched in T-LBL with VAV1 mutants. Cell viability assay using panels over 1400 drugs showed that PI3K/Akt/mTOR pathway inhibitors and cell-cycle inhibitors effectively suppressed the cell growth of T-LBL with VAV1 mutants in vitro. Conclusions: Expression of VAV1 mutants promoted the development of T-cell malignancies in mice. Our mouse models may provide the efficient tools to screen new therapeutic targets in T-cell malignancies with VAV1 mutations. Ohshima: NEC Corp.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; SRL, Inc.: Consultancy. Ogawa:Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership.