ALBERT

Description: Activating mutations in the Vav guanine nucleotide exchange factor 1 (VAV1) gene are reported in various subtypes of mature T-cell neoplasms (TCN). However, oncogenic activities associated with VAV1 mutations in TCN remain unclear. To define them, we established transgenic mice expressing VAV1 mutants cloned from human TCN. Although we observed no tumors in these mice for up to a year, tumors did develop in comparably-aged mice on a p53-null background (p53-/- VAV1-Tg), and p53-/- VAV1-Tg mice died with shorter latencies than did p53-null (p53-/-) mice. Notably, various TCN with tendency of maturation developed in p53-/- VAV1-Tg mice, while p53-/- mice exhibited only immature TCN. Mature TCN in p53-/- VAV1-Tg mice mimicked human peripheral T-cell lymphoma (PTCL)-GATA3 and exhibited features of type2 T helper (TH2) cells. Phenotypes seen following transplantation of either p53-/- VAV1 or p53-/- tumor cells into nude mice were comparable, indicating cell-autonomous tumor-initiating capacity. Whole transcriptome analysis (WTA) showed enrichment of multiple Myc-related pathways in TCN from p53-/- VAV1-Tg mice relative to p53-/- or wild-type T cells. Remarkably, amplification of Myc locus were found recurrently in TCN of p53-/- VAV1-Tg mice. Finally, treatment of nude mice transplanted with p53-/- VAV1-Tg tumor cells with JQ1, a bromodomain inhibitor, which targets the Myc pathway, prolonged survival of mice. We conclude that VAV1 mutations function in malignant transformation of T cells in vivo and that VAV1-mutant expressing mice could provide an efficient tool for screening new therapeutic targets in TCN harboring these mutations. We conclude that VAV1 mutations function in malignant transformation of T cells in vivo and that VAV1-mutant expressing mice could provide an efficient tool for screening new therapeutic targets in TCN harboring these mutations.

Description: Background: Nestin-expressing cells (NeC) have been characterized to consist of hematopoietic stem cell (HSC) niche in the mouse bone marrow (BM). Decreases of BM NeC have been reported in myeloproliferative neoplasms (MPN) in humans and in the mouse model of MPN. These lines of information further emphasize the importance of the NeC for the maintenance of normal hematopiesis. Nevertheless, NeC appear to be heterogenous; nestin is reported to be expressed in multiple types of BM stromal cells distinct from each other, with regard to the anatomical localization and the cell-surface antigen expression pattern. One type is reported to be localized adjacent to sinusoids and another type surrounding arterioles. A subset of endothelial cells also appears to be a candidate of NeC in the BM. It is thus critical to define the identities of distinct subsets of BM NeC. Furthermore, each subset of NeC needs to be studied in the human BM from normal subjects and those with BM diseases to understand pathophysiologic significance of NeC in patients. Myelodysplastic syndromes (MDS) are a clonal disease characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. In this disease, anormalities of BM microenvironment have been repeatedly reported; however, consensus in detail has not been reached. Purpose: To define the identities of distinct subsets of NeC in the BM from normal human subjects and to explore their abnormalities in MDS. Methods:Formalin-fixed paraffin-embedded BM biopsy samples from lymphoma patients without BM involvement (designated normal) and from MDS patients were immunostained with antibodies against six markers: nestin, CD34, laminin, α-smooth muscle actin (αSMA), glial fibrillary acidic protein (GFAP), and neurofilament heavy chain (NFH). Immunohistochemistry (IHC) and immunofluorescence (IF) staining were performed. The microscopic analysis of IHC-stained samples involved 10 randomly selected fields of view at 400× magnification, where the numbers of NeC and CD34-positive spindle-shaped cells were counted for quantitative analysis, as well as the association of these two types of cells was evaluated. IF samples were analyzed by a confocal laser scanning microscope using 10 randomly selected fields of view at 63× magnification. Then, nestin-, GFAP-, and NFH-stained areas were measured using the confocal LAS AF software for quantitative analysis. Results:NeC were found at multiple locations in distinct contexts in the normal human BM. A majority of NeC were present in association with the arterior/arteriolar structures. These artery/arteriole-associated NeC were distributed at each of the three layers; the intimal layer inside the laminine-stained basement membrane, the tunica media epressing αSMA, and the adventitial layer outside the αSMA-stained structure. The NeC located at the intimal layer expressed the highest level of nestin. The NeC were present in a close proximity with the CD34-expressing endothelial cells, although whether the endothelial cells co-expressed nestin and CD34 was unclear. The NeC at the other layers showed relatively lower levels of nestin expression. We identified NeC which did not associate with the vascular structures, albeit at a low frequency and with weak nestin staining in the normal human BM. In MDS BM, there was a significant increase in the NeC that were unassociated with the vascular structures. A portion of these increased NeC co-expressed GFAP. These cells potentially represented Schwann cells, because some of them surrounded the NFH-stained structure. Consistent with this, GFAP- and NFH-stained areas were increases in the MDS BM, together with the nestin-stained areas when measured by the confocal LAS AF software. Discussion: Multiple subsets of NeC were identified in the normal human BM as well as in the MDS BM. It is yet elusive whether each subset of NeC has a HSC niche function. In MDS BM, there was an increase in a distinct subset of NeC. The origin of these cells was elusive, but the Shwann cells normally present along with the arterial/arteriolar structures could be a candidate, because in the normal BM, a portion of GFAP-expressing cells along with the vascular structures expressed nestin. It should be elucidated whether the increased sympathetic nervous structure is involved in the pathophysiology of MDS. Disclosures Obara: Alexion Pharmaceuticals: Honoraria, Research Funding.

Description: Background: Bone marrow (BM) fibrosis is common in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN), its pathogenesis remains unclear. It is also unknown whether and how the pathophysiology of BM fibrosis in MDS and MPN are similar or different, while it is anticipated that altered BM microenvironment plays a key role. Methods: To visualize various BM microenvironmental cells in MDS or MPN patients as compared to those in control subjects, we utilized multicolor immunostaining using a tyramide signal amplification system and a fluorescence spectrum analyzer. Paraffin-embedded BM biopsy samples from 47 patients with MDS (26 without and 21 with fibrosis), 10 with primary myelofibrosis or myelofibrosis secondary to essential thrombocythemia (MPN-F), and 17 with non-Hodgkin lymphoma without BM involvement (as control) were immunostained with antibodies for mesenchymal/neuronal cell markers. Gene mutations were analyzed by targeted sequencing. Results: In control samples, C-X-C motif ligand 12 (CXCL12) was stained mainly in cells at perivascular areas and the periphery of a minor proportion of adipocytes. These patterns did not differ in MDS without fibrosis from control. However, CXCL12-stained non-adipose cells were significantly increased in MDS with fibrosis (MDS-F) [median (range), 5190 (2117-14299) μm2/mm2 as compared to 628 (464-1331) μm2/mm2 in control; p=0.00453]. These cells tended to be increased in also MPN-F though statistical significance was not reached [6096 (1098-24458) μm2/mm2; p=0.06304]. The localization of these cells was not confined to the perivascular areas in both diseases. There were no apparent differences in the degree or the pattern of staining for CXCL12 between MDS-F and MPN-F. We also focused on Schwann cells and Nestin-expressing stromal cells because these cells have been independently reported to function as an important hematopoietic stem cell niche. Neurofilament heavy chain (NFH) or neuron-specific class III beta-tubulin-positive nerve fibers (Tuj1) surrounded by glial fibrillary acidic protein (GFAP)-positive Schwann cells were rarely observed in control samples. This observation was true of MDS without fibrosis. However, GFAP-positive Schwann cells were significantly increased in MDS-F and MPN-F [median (range), 39457 (1325-60654) um2/mm2 and 75951 (19879-326216) um2/mm2, as compared to 6258 (1325-14842) um2/mm2 in control; p=0.0017 and 0.0034, respectively]. When the section was stained for Nestin, Schwann cells in MPN-F did not show positive signals except for one case. In contrast, Nestin was frequently stained in the GFAP-positive Schwann cells of MDS-F (9 of 21 MDS-F, 42.9%); particularly 5 of 7 cases (71.4%) in which fibrosis was judged as severe (MF-3), showed positive signals. One exceptional case of MPN-F that showed Nestin-positivity in increased Schwann cells was a triple-negative case (no mutations in JAK2, CALR, or MPL); instead, mutations were found in NRAS, TET2, BCOR, and SMC1A, indicating that this case may have a feature of MDS. Conclusion: Abnormal increases in CXCL12-expressing mesenchymal cells and nerve fiber-surrounding Schwann cells were visualized by multicolor fluorescence in MDS-F and MPN-F. Schwann cells in MDS-F and MPN-F showed a remarkable difference in the expression of Nestin. It is to be elucidated whether the increase in the Schwann cells has a role in the pathogenesis/pathophysiology, and if so, whether those Schwann cells with distinct phenotypes have differential roles in these two disease conditions. Disclosures Ogawa: ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; RegCell Corporation: Equity Ownership; Qiagen Corporation: Patents & Royalties; Asahi Genomics: Equity Ownership.

Description: 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: p50 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. Disclosures 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.

Description: [Background] Nestin-expressing cells (NeC) have been characterized as one of many types of bone marrow (BM) microenvironmental cells, including endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, etc. Recent studies have gradually provided information about anatomy and functions of each of these cells. Nevertheless, subcellular signaling and transcriptional regulations in individual miciroenvironmental cells have poorly been demonstrated. On a different line of studies, it has been suggested that NOTCH signaling in BM microenvironmental cells affects hematopoiesis; despite this, information is limited whether NOTCH signaling plays a role in NeC. It is of note that nestin was originally identified in neural stem cells (NSC), that NOTCH signaling is known to play a pivotal role in the NSC, and that the BM NeC could be derived from neuroectoderm. This potential linkage urged us to investigate whether and how downregulation of NOTCH signaling in BM NeC affects hematopoiesis. [Method] Mice with an rbpj-flox allele were crossed with those with a CreERT2/GFP (Green Fluorescent Protein) transgene under the nestin promoter. At 8-12 weeks, tamoxifen was intraperitoneally injected for 4-12 weeks to delete the rbpj gene only in NeC (rbpj cKO mice). In this experimental system, GFP is expected to be expressed as a surrogate marker for the rbpj gene deletion, by the deletion of stop codon inserted at 5' to the cDNA of GFP. Then, transplantation assays were performed using rbpj-null BM cells as a donor to reconstitute hematopoiesis in the wild-type mice, or using rbpj-null mice as recipients to see reconstitution of hematopoiesis from wild-type BM cells. The effect of splenectomy was investigated in the untransplanted and transplanted conditions. The littermate rbpjnull/wt orrbpjwt/wtmice were used as controls. [Results] GFP was detected in 0.1-0.5% of flow cytometry (FCM)-sorted CD45(-) cells only after tamoxifen injection. Deletion of rbpj was specifically confirmed in GFP-positive BM and spleen cells. Tamoxifen induced mild splenomegaly in rbpj cKO mice compared with littermate control mice. Enlarged spleen showed preserved follicular architecture but increased CD71(+)Ter119(+) mature erythroid cells in the red pulp. In contrast, BM of rbpj cKO mice bearing mild splenomegaly demonstrated marked decrease in the CD71(+)Ter119(+) mature erythroid cells without obvious anemia. There was a substantial animal-to-animal variation in the phenotypes; however, the strength of phenotypes was correlated with the frequency of GFP-positive cells in the BM, suggesting that the phenotypic variation was a result of the efficiency of rbpj gene deletion. We hypothesized that the rbpj cKO mice would develop anemia if spenectomized, because extramedullary erythropoiesis in spleen might compensate the defective BM erythropoiesis. However, tamoxifen did not cause significant anemia in splenectomized rbpj cKO mice. In these mice, reduction of the CD71(+)Ter119(+) mature erythroid cells in BM was significantly milder than nonsplenectomized mice. In transplantation analysis, the recipient rbpj cKO mice transplanted with BM cells from wild-type mice showed a reduction in CD71(+)Ter119(+) mature erythroid cells and mild splenomegaly, as were seen in rbpj cKO mice without transplantation. The phenotypes were again erased by the splenectomy. The recipient wild-type mice transplanted with BM cells from rbpjcKO mice did not show any phenotypes. [Discussion] Rbpj cKO in NeC induced impaired erythroid differentiation in BM together with mild splenomegaly. We confirmed that these phenotypes were caused by rbpj cKO in BM NeC by transplantation experiments. Surprisingly, such BM erythropoiesis impairment was reversed by splenectomy. This unexpected finding uncovered the presence of a previously unidentified balance controller between BM and spleen erythropoiesis. Hematopoietic stem cell-autonomous NOTCH signaling has been shown to be dispensable for adult murine hematopoiesis; however, NOTCH signaling in BM-NeC is responsible for control of balance of erythropoiesis at the BM and the spleen. Disclosures Obara: Alexion Pharmaceuticals: Honoraria, Research Funding.