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Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm immunophenotypically resembling regulatory T cells, associated with human T-cell leukemia virus type-1. Here, we performed whole-genome sequencing (WGS) of 150 ATL cases to reveal the overarching landscape of genetic alterations in ATL. We discovered frequent (33%) loss-of-function alterations preferentially targeting the CIC long isoform, which were overlooked by previous exome-centric studies of various cancer types. Long but not short isoform–specific inactivation of Cic selectively increased CD4+CD25+Foxp3+ T cells in vivo. We also found recurrent (13%) 3′-truncations of REL, which induce transcriptional upregulation and generate gain-of-function proteins. More importantly, REL truncations are also common in diffuse large B-cell lymphoma, especially in germinal center B-cell–like subtype (12%). In the non-coding genome, we identified recurrent mutations in regulatory elements, particularly splice sites, of several driver genes. In addition, we characterized the different mutational processes operative in clustered hypermutation sites within and outside immunoglobulin/T-cell receptor genes and identified the mutational enrichment at the binding sites of host and viral transcription factors, suggesting their activities in ATL. By combining the analyses for coding and noncoding mutations, structural variations, and copy number alterations, we discovered 56 recurrently altered driver genes, including 11 novel ones. Finally, ATL cases were classified into 2 molecular groups with distinct clinical and genetic characteristics based on the driver alteration profile. Our findings not only help to improve diagnostic and therapeutic strategies in ATL, but also provide insights into T-cell biology and have implications for genome-wide cancer driver discovery.

Mutations in JAK2, myeloproliferative leukemia virus (MPL), or calreticulin (CALR) occur in hematopoietic stem cells (HSCs) and are detected in more than 80% of patients with myeloproliferative neoplasms (MPNs). They are thought to play a driver role in MPN pathogenesis via autosomal activation of the JAK-STAT signaling cascade. Mutant CALR binds to MPL, activates downstream MPL signaling cascades, and induces essential thrombocythemia in mice. However, embryonic lethality of Calr-deficient mice precludes determination of a role for CALR in hematopoiesis. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr-deficient mice. CALR deficiency had little effect on the leukocyte count, hemoglobin levels, or platelet count in peripheral blood. However, Calr-deficient mice showed some hematopoietic properties of MPN, including decreased erythropoiesis and increased myeloid progenitor cells in the bone marrow and extramedullary hematopoiesis in the spleen. Transplantation experiments revealed that Calr haploinsufficiency promoted the self-renewal capacity of HSCs. We generated CALRdel52 mutant transgenic mice with Calr haploinsufficiency as a model that mimics human MPN patients and found that Calr haploinsufficiency restored the self-renewal capacity of HSCs damaged by CALR mutations. Only recipient mice transplanted with Lineage-Sca1+c-kit+ cells harboring both CALR mutation and Calr haploinsufficiency developed MPN in competitive conditions, showing that CALR haploinsufficiency was necessary for the onset of CALR-mutated MPNs.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma that develops in about 5% of human T-cell leukemia/lymphoma virus 1 (HTLV-1) carriers. In addition to viral oncogenes, namely tax and HTLV-1 bZIP factor (HBZ), gene mutations, highly enriched for T-cell receptor (TCR)-NF-kB signaling, should be involved in the development of ATL. Among gene mutations, mutation in CARD11, a cytoplasmic scaffolding protein required for TCR-induced NF-kB activation, was detected in 24% of ATL patients. Here we generated a mouse model for conditional expression of a human ATL-derived CARD11(E626K) gain-of-function mutant, and demonstrated CARD11 activation induced oligoclonal expansion of T-cell and infiltration to many organs. We also showed that expression of HBZ accelerated mutant CARD11-induced lymphoproliferative diseases. We introduced a human Card11(E626K) into the mouse genome at the ROSA26 locus. After crossing with CD4-Cre Tg, CARD11(E626K)CD4-Cre mice was obtained. In CD4+ cells from CARD11(E626K)CD4-Cre, the amount of cleaved BCL-10 and NF-kBp65 increased compared with those in WT CD4+cells, confirming the activation of NF-kB. About half of CARD11(E626K)CD4-Cre mice died on or after 6 months after birth. At 6 months, leukocytosis was observed in CARD11(E626K)CD4-Cre, and accordingly the number of CD4+ cells cells was about 1.43 times greater than those in WT mice. The most affected organ in CARD11(E626K)CD4-Cre mice was lung. Alveolar septum was thickened by infiltrated cells at 6 months, and worsened subsequently. CD3+ T-cell accumulated around capillary blood vessels, and had high proliferation indices (>50%), as assayed by Ki-67 staining. CARD11(E626K)CD4-Cre mice developed lymphadenopathy (4/8 mice (50%) at 6M and 4/6 mice (66.7%) at 12M). Normal lymph node (LN) architecture was barely preserved, and medullary sinus was expanded with CD3+ T-cell. Some of them were positive for FoxP3, and had moderate proliferation indices (25%-50%). Among CD4+ T-cell, the proportion of naive T-cell (Tn) decreased, and that of effector/memory T-cell (Tem) and regulatory T-cell (Treg) increased compared to WT LNs. The proportion of Treg in CD4+ T-cell from LN of 6M- and 12M-old CARD11(E626K)CD4-Cre mice was 25% and 40%, respectively, which values were much larger than the normal range as 10-15%. We next examined the clonality of CD4+ cells in spleen and swollen LNs from CARD11(E626K)CD4-Cre mice. The clonality of the TCR repertoires of 20 individual Vb gene famines from Vb1-20 was assessed by a PCR. The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 5 splenic CD4+ cells, and 1 of 2 LN CD4+ cells from CARD11(E626K)CD4-Cre mice. To assess the effect of HBZ constant expression on CARD11(E626K)CD4-Cre mice, we generated HBZ Tg, in which HBZ cDNA was expressed under the CD4 promoter. Similar to the previous report (by Satou et al.), our HBZ Tg showed increased number of Tem, destroyed architecture of lung such as thickened alveolar septum by infiltrated cells and decreased alveolar space by edema, and the lymphadenopathy after 12M (66.7%). We then crossed CARD11(E626K)CD4-Creand HBZ Tg, and obtained the compound mice. The compound mice caused more aggressive lymphoproliferative diseases compared with CARD11(E626K)CD4-Cre mice. Most of compound mice died within 8M. At 6M, architecture of lung, kidney, spleen, and LN was destroyed. In lung, alveolar space of lung was scarcely observed caused of T-cell invasion. Alveolar septum was thickened with infiltrated cells, and CD3+ cells accumulated around capillary blood vessel. Some of them were positive for FoxP3, and indicated moderate proliferation indices (25-50%). T-cell invasion was also observed in kidney. Lymphadenopathy was detected in 6 of 9 (66.7%) with completely destroyed architecture, increment of the proportion of Fox3+ cells, and moderate proliferation indices (25-50%). The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 4 splenic CD4+ cells, and 2 of 2 LNs CD4+ cells from compound mice. These results suggest that CARD11 mutant-induced NFkB activation and constant HBZ expression may have similar effects, such as T-cell infiltration into organs and LN adenopathy, and that the simultaneous occurrence of both may have additive effects. Disclosures Sugiyama: Chordia Therapeutics, Japan.: Current Employment. Morishita:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Shimoda:Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Celgene: Honoraria; Shire plc: Honoraria; Novartis: Honoraria, Research Funding; Takeda Pharmaceutical Company: Honoraria; Bristol-Myers Squibb: Honoraria.

Acquired mutations of JAK2 and TET2 are frequent in myeloproliferative neoplasms (MPNs). We examined the individual and cooperative effects of these mutations on MPN development. Recipients of JAK2V617F cells developed primary myelofibrosis-like features; the addition of loss of TET2 worsened this JAK2V617F-induced disease, causing prolonged leukocytosis, splenomegaly, extramedullary hematopoiesis, and modestly shorter survival. Double-mutant (JAK2V617F plus loss of TET2) myeloid cells were more likely to be in a proliferative state than JAK2V617F single-mutant myeloid cells. In a serial competitive transplantation assay, JAK2V617F cells resulted in decreased chimerism in the second recipients, which did not develop MPNs. In marked contrast, cooperation between JAK2V617F and loss of TET2 developed and maintained MPNs in the second recipients by compensating for impaired hematopoietic stem cell (HSC) functioning. In-vitro sequential colony formation assays also supported the observation that JAK2V617F did not maintain HSC functioning over the long-term, but concurrent loss of TET2 mutation restored it. Transcriptional profiling revealed that loss of TET2 affected the expression of many HSC signature genes. We conclude that loss of TET2 has two different roles in MPNs: disease accelerator and disease initiator and sustainer in combination with JAK2V617F.

Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008). First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice. Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis. In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

Mutations in the Calreticulin (CALR) gene were identified in cases of myeloproriferative neoplasms (MPNs), and various functions of the CALR mutant protein are being elucidated. On the other hand, few data are available on the role of CALR in the hematopoietic system. The knockout (KO) mice of Calr are impaired in expression of transcription factors necessary for cardiac development and are embryonic lethal. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr KO mice. Mice carrying floxed allele targeted exons 4-7 of Calr (Calrf/+ mice) and Calr heterozygous KO mice (Calr+/− mice) (Tokuhiro et al., Sci Rep 2015) were crossed with Mx1-Cre transgenic mice and obtained mice with three genotypes; Mx1-cre;Calr+/+, Mx1-cre;Calr+/−, and Mx1-cre;Calrf/−. Floxed alleles were then deleted by intraperitoneal injection with polyinosinic: polycytidilic acid. No differences were found in the peripheral blood (PB) leukocyte count, hemoglobin levels, or platelet count among the three genotypes of mice. The proportions of Mac1+ or Gr1+ myeloid lineage cells, B220+ B cells, and CD3+ T cells among the three groups were comparable. In the bone marrow (BM), cell pellet from Mx1-cre;Calrf/− mice appeared anemic and the proportion of CD71+/Ter119+ erythroid cells and the number of CFU-E were significantly lower in Mx1-cre;Calrf/− BM cells compared to the other two genotypes of BM cells. On the other hand, no difference was found in other mature cells such as myeloid, T, B, or CD41+ megakaryocytes (Mks) in BM. As for HSCs and progenitors, Mx1-cre;Calrf/− mice exhibited a higher proportion of MPP and GMP compared to the other two genotypes of mice. We found no difference in other progenitor compartments including long- and short-term HSCs among the three groups. In contrast to the minor effect on BM and PB cells, The spleen weight in Mx1-cre;Calrf/− mice was about 2-fold heavier than that in Mx1-cre;Calr+/+ mice. In spleens from Mx1-cre;Calrf/− mice, the border between the white and red pulp was obscured. Mks and maturing myeloid cells had markedly infiltrated into the red pulp. In FACS analysis, mature myeloid cells, erythroid cells, and Mks were significantly increased in spleens from Mx1-cre;Calrf/− mice compared to those from the other two genotypes of mice. HSCs and most types of myeloid progenitors were also increased in the spleen. Hematopoiesis in the spleen may compensate for the reduced erythropoiesis in the BM, as no anemia was seen in Mx1-cre;Calrf/− mice. No onset of leukemia or myelofibrosis was observed, and no difference in survival was seen among the three groups following observation for 2 years. As CALR plays a role as a chaperone in the endoplasmic reticulum (ER) during protein synthesis, we searched for proteins with expression that was reduced by Calr deficiency. The quantitative differences of proteins in Mac1+/Gr1+ BM cells between Mx1-cre;Calrf/− and Mx1-cre;Calr+/+ mice were compared using proteomics analysis by 2DICAL, a shotgun proteomics analysis system (Ono et al. Cellular Proteomics 2006). We found that only a few proteins, including myeloperoxidase and CALR itself, were reduced, and conversely, many proteins were increased by the absence of CALR. List of differentially upregulated proteins higher than those in WT samples were analyzed using Metascape (http://metascape.org) to determine enriched pathways. The most enriched cluster was "protein processing in the ER", and 19 out of 76 upregulated (>1.8-fold) proteins were included in this cluster. In real-time PCR analysis, we confirmed that ER chaperon genes (BiP, Grp94, Hsp40, Pdia3, Pdia4, Pdia6) and genes involved in ER-related degradation (Trap, Bap31, Derl1, p97) were significantly upregulated in Mx1-cre;Calrf/− myeloid cells. These observations suggested that chaperone dysfunction due to Calr deficiency is compensated by upregulation of unfolded protein response (UPR) pathway. In summary, Calr deficiency induced erythroid hypoplasia in the BM, and induced splenomegaly and extramedullary hematopoiesis. This suggests that the amount of WT CALR expression remaining in CALR mutant cells may modify the phenotype of MPN patients. Absence of myeloperoxidase protein and upregulation of UPR pathway in myeloid cells from Calr KO mice indicates that CALR plays a significant role as a chaperone also in hematopoiesis and that CALR deficient cells are exposed to ER stress. Disclosures No relevant conflicts of interest to declare.

CALR exon9 frameshift mutations function as driver mutations in essential thrombocythemia (ET) and primary myelofibrosis patients with non-mutated JAK2 or MPL. The mutations augment signal transducer and activator of transcription activity in the presence of MPL, induce increased cell proliferation and growth factor independence in cell lines, and cause ET-like myeloproliferative neoplasms (MPN) in mice. In tumor cells bearing the CALR mutation, mutant CALR protein expression occurs while wild-type (WT) CALR expression is decreased by half. Although the biological activity of mutant CALR has been elucidated in detail, the significance of CALR haploinsufficiency is unclear. The purpose of this study was therefore to clarify the influence of CALR haploinsufficiency on hematopoiesis in normal and CALR-mutated MPN in mice. First, we analyzed the effect of CALR haploinsufficiency on hematopoiesis using CALR heterozygous knockout (CALR-hKO) mice (Tokuhiro et al. Sci Rep. 2015). Blood cell counts, liver and spleen weight, histology, cell fraction in bone marrow (BM) and spleen, and survival of CALR-hKO mice were all equivalent to that observed in WT mice. In the analysis of progenitor cells by fluorescence-activated cell sorting and colony formation assays, no difference was observed in the amount of progenitor cells and in colony replating capacity between WT mice and CALR-hKO mice. Interestingly, in competitive serial transplantation experiments using whole BM cells in primary and secondary transplanted mice, CALR-hKO cells showed higher levels of chimerism than WT cells. Next, the effect of CALR haploinsufficiency on hematopoiesis in mutant CALR-del52 overexpressing mice was analyzed. We compared three groups of mice, WT mice, CALR-del52 transgenic (TG) mice (Shide et al. Leukemia 2017) and CALR-del52 TG/CALR-hKO double-mutant (TG-hKO) mice. Both TG mice and TG-hKO mice developed ET-like MPN. Compared to WT mice, increases in megakaryocytes, platelets and hematopoietic progenitor cells (including HSCs) were observed in these mice. However, no differences were observed between TG mice and TG-hKO mice. Finally, 4000 LSK cells sorted from WT mice, TG mice, and TG-hKO mice (B6-Ly5.2) and B6-Ly5.1 competitor cells (1 × 106 WT BM cells) were mixed and injected into lethally irradiated B6-Ly5.1 recipient mice, and the percent chimerism of donor cells was followed for 1 year after transplantation. From 12 weeks after transplantation, the chimerism of TG cells was significantly lower than that of control WT cells, suggesting that the CALR mutation has a negative influence on clonal expansion of HSCs. The recipient mice transplanted with TG LSK cells showed very mild thrombocythemia. On the other hand, chimerism in TG-hKO cells was significantly higher than that in control WT cells up to 12 weeks after transplantation. Chimerism at 20 weeks after transplantation was equivalent to that in control WT cells, and the recipient mice transplanted with TG-hKO LSK cells showed severe thrombocythemia. These findings show that CALR haploinsufficiency compensates for HSCs functioning, which has been impaired by the CALR mutation, enhancing the ability of HSCs to develop ET. Mice experiments have showed that HSCs with the JAK2V617F mutation are fragile, and it is considered that mutations in DNMT3A or TET2 often occurred prior to mutations in JAK2 to compensate for the defect in self renewal capacity. Conversely, since the CALR mutation is not typically preceded by any mutations, it is considered that the CALR mutation may not require any preceding mutations. Our results showed that, like JAK2 V617F mutants, overexpression of the CALR-del52 mutant impairs HSC functioning. Furthermore, we found that CALR haploinsufficiency restores the functions of impaired HSCs to the same extent as that found in WT HSCs. When an HSC acquires the CALR mutation, "defect" and "recovery" states thus occur simultaneously in the cell. This finding may explain why mutant CALR clones expand without needing to undergo any preceding mutation. Disclosures No relevant conflicts of interest to declare.

Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell lymphoma that is caused by HTLV-1. The prognosis of acute and lymphomatous variants of ATLL is poor, ranging from 2 weeks to >1 year. Compared to other types of malignant lymphomas, the organ infiltration is frequently observed in ATLL (Yamada et al. Leuk Lymphoma 1997). We previously reported the landscape of genetic mutations in ATLL, and showed that various mutations occurred in the TCR-NFκB pathway in more than 90% of ATLL cases (Kataoka et al. Nat Genet 2015). These somatic mutations are thought to develop ATLL in combination with viral genes such as HTLV-1 bZIP factor (HBZ). Among them, mutations in TET2, an epigenetic regulator, was observed in about 10% of ATLL cases. Higher frequencies inTET2 mutation was reported in other types of peripheral T-cell lymphoma (PTCL); it was observed in about 80% of angioimmunoblastic T-cell lymphoma (AITL) and in about 50% of PTCL, not otherwise specified. In PTCL, it has been reported that additional mutations in lymphoid progenitors derived from TET2 mutated hematopoietic stem cells cause increased cell proliferation and anti-apoptosis, leading to the disease progression. In ATLL, the role of TET2 mutation in disease progression is still unknown. In this study, we investigated the role of TET2 mutation in ATLL using mouse model and acute and lymphomatous variant ATLL cohort. Materials and methods: As an animal model of HTLV-1 infection or ATLL, transgenic mice expressing HBZ under the control of the mouse CD4 promoter (HBZ-Tg) were generated with C57BL/6 background. Heterozygous TET2 knock-down mice (TET2KD) were generated with C57BL/6 background by gene trapping (Tang et al. Transgenic Res 2008; Shide et al. Leukemia 2012). HBZ-Tg/TET2KD compound mice (double mutant) were generated by crossing them. HBZ-Tg, TET2KD, and double mutant mice were investigated by cell counts, organ weight, FACS analysis, pathological analysis, and survival analysis. The relationship between the TET2 mutation status and the clinical feature was investigated using our acute and lymphomatous variant ATLL cohort (n=115). Result: At 12 months, compared to wild type mouse (WT), sporadic splenomegaly and lymphadenopathy were observed in HBZ-Tg. No significant increase was observed in peripheral blood (PB) leukocyte and mononuclear cell (MNC) of BM and spleen, but an increase was observed in the estimated whole body MNC (Femur x 100/6 + spleen) (WT vs. HBZ-Tg; estimated whole body MNC (x106 cells/body), 416±162 vs. 621±147, p=0.01). In FACS analysis, the frequency of CD4+ T-cell was increased in PB, spleen, and BM (WT vs. HBZ-Tg; PB-CD4+ T-cell%, 4.9±0.9 vs. 28.2±22.8, p Next, to elucidate the role of TET2 mutation in ATLL, the double mutant was analyzed. At six months, compared to HBZ-Tg, no increase was observed in the number of PB leukocyte, spleen-MNC, and BM-MNC, and also in the frequency and the number of CD4+ T-cells in PB, spleen and BM. However, in pathological and survival analysis, the double mutant showed severe cell infiltration in lung and liver and demonstrated inferior OS (median OS (month), 11.1 vs. 6.0, p Conclusion: In both mice model and human cohort, TET2 mutation exacerbated organ infiltration of ATLL cells. Disclosures No relevant conflicts of interest to declare.

Calreticulin (CALR) exon 9 mutations were reported in about two-thirds of JAK2 or MPL mutation negative ET and PMF patients. The mutations cause frameshifts that result in proteins with novel C-terminus.Retrovirus-mediated gene transfer into cell lines and mouse bone marrow (BM) cells is a common technique, but the expression level is very high compared to the physiological expression.We investigated the effects of physiological expression of mutant CALR using CRISPR/Cas9 gene editing techniques for cell lines, and as for the mouse model, we generated a transgenic mice (TG) expressing human CALR del52 mutant. We used two human cell lines expressing MPL: human acute megakaryoblastic leukemia cell line CMK11-5 which expressed endogenous MPL, and F-36P-MPL cell line which was generated by introducing MPL to GM-CSF-dependent erythroleukemia cell line F-36P. Plasmids coexpressing hCas9 and single-guide RNA were prepared by ligating oligonucleotides (5'-CACCGACAAGAAACGCAAAGAGGAGG-3', 5'-AAACCCTCCTCTTTGCGTTTCTTGTC-3') for the target sequence of human CALR exon 9 into pX330. The plasmids were introduced with a electroporator to each of the cell lines. After limiting dilution cloning, we identified cell lines which have indel mutation at the target site. We produced two types of CMK11-5 subline knocked in a CALR mutation, namely CALR del25 CMK cells and CALR del25/del17 CMK cells, respectively. The former lacks 25 bases in one CALR allele, causing a frameshift that results in a protein resembling human CALR mutant, while the latter lacks an additional 17 bases in another allele in CALR exon 9 and induces a frameshift that causes a deletion in CALR exon 9. Both kinds of CALR mutant CMK11-5 cells showed increased cell proliferation compared to WT cells. We also produced one type of F-36P-MPL subline, CALR del1/ins1 F-36P-MPL cells which had 1 base deletion in one CALR allele resembling human mutation and 1 base insertion in another allele. Though the growth of this subline in the presence of GM-CSF was comparable to WT cells, it showed GM-CSF independent autonomous cell growth. We generated TG mice expressing human CALR del52 mutant driven by the murine H2Kb promoter. We compared the expression level of human CALR mRNA in TG BM cells with the expression of endogenous WT CALR in human cell lines (CMK11-5, F-36P-MPL, CHRF288) using Rn18s as an endogenous control. The expression of human CALR in TG BM was approximately 0.6 times that of endogenous WT CALR in human cell lines, and the physiological expression level was obtained. They exhibited thrombocytosis, with platelet (PLT) counts as high as 2,000 x 109/L. Leukocyte number and the proportion of granulocytes and T and B lymphocytes, were comparable to WT mice. CALR mutation had no impact on Hb level or spleen weight. There was a striking difference in the number of megakaryocytes (Mgks), which was 2-fold higher in BM from TG mice than in WT mice. The TG Mgks were also more mature, with larger diameter, and contained higher number of alpha-granules compared to WT cells. In one year of observation, there is no fibrosis in BM. These observations showed that TG developed human ET-like disease. The survival of TG mice was comparable to that of WT mice. The disease phenotype was transplantable into WT recipient mice. To characterize in detail the impact of MPNs induced by the CALR del52 mutant, we evaluated the frequencies of HSCs and progenitors in BM. The frequency of both LT-HSC and ST-HSC in BM was higher inTG mice compared to WT mice. The frequencies of progenitors (CMP, MEP, and MKP) were also greater in BM from TG mice than from WT mice. However, BM cells did not have enhanced replating capacity. We next examined whether or not ruxolitinib (RUX) treatment ameliorated thrombocytosis in TG mice. Either 90 mg/kg bid of RUX or vehicle was administrated to TG mice for 4 weeks.TG mice treated with vehicle showed a mean 16% increase in PLT count during the treatment period, probably due to the disease progression. RUX treatment attenuated the increase in the number of PLTs in TG mice by a mean of 22%, but the overall count was still higher than that in WT mice. BM sections showed that RUX reduced the Mgks number in TG. In summary, physiological expression of CALR mutant increases cell growth and cytokine independency in human cell lines expressing MPL, and develops ET in mice. RUX therapy attenuated the increased numbers of peripheral blood PLTs and BM Mgks, and ameliorated CALR mutation-induced ET. Disclosures No relevant conflicts of interest to declare.

Introduction Adult T-cell leukemia/lymphoma (ATLL) is an aggressive peripheral T cell neoplasm that is resistant to conventional chemotherapy and carries a poor prognosis. The effect of mogamulizumab, an immunoglobulin (Ig) G1 monoclonal antibody targeting CCR4 for ATLL cells, was reported in a previous phase 2 study in which mogamulizumab monotherapy was evaluated in relapsed ATLL patients. The overall response rate (ORR), median progression-free survival (PFS) and median overall survival (OS) were 50%, 5.2 and 13.7 months, respectively. It was not stated whether these values were derived in the real world or in clinical practice. Here we evaluate the clinical impact of mogamulizumab treatment in CCR-4-positive aggressive ATLL patients in clinical practice. Patients and methods We retrospectively analyzed 101 CCR-4-positive ATLL patients who received at least one cycle of mogamulizumab infusion between March, 2012 and April, 2016 in 7 facilities in Miyazaki prefecture, an HTLV-1 endemic area in Southwestern Japan. The ORR, PFS, OS and adverse effects (AEs) were evaluated. We next compared OS in patients with at least one course of mogamulizumab therapy with that in historical control patients without mogamulizumab therapy. Results Of the 101 patients, 92 were evaluable for treatment response, survival and AEs. The median age was 70 years old (range; 45 to 90), and 52 patients (51%) were more than 70 years old. According to Shimoyama's criteria, 66 patients were classified as acute type, 32 as lymphoma type, and 3 as chronic type. All 3 chronic-type ATLL patients had at least one unfavorable risk factor. Of the 101 patients, 96 had refractory or relapsed ATLL when mogamulizumab treatment was started, and the prior treatments consisted of VCAP-AMP-VECP, CHOP, DeVIC or CHASE therapy, with an average of 2 courses. In the 5 remaining cases, mogamulizumab was administered as the initial therapy for ATLL. Mogamulizumab was administered as monotherapy in 87 cases (86%), and as combination therapy with other drugs in 14 cases (14%). The ORR was 37%, including a complete remission rate of 19%. The median PFS and OS were 1.8 and 4.2 months, respectively. Among the 101 patients treated with mogamulizumab, only 26 (26%) fulfilled the inclusion criteria of the phase 2 clinical study. Among patients who met those inclusion criteria, the median PFS and OS were 6.0 and 8.4 months, respectively. The use of mogamulizumab improved OS in clinical practice. The median OS of patients receiving mogamulizumab therapy was 12 months, whereas that of patients who did not receive mogamulizumab in the historical cohort was 8.4 months. Hematologic toxicity and skin rash were the most common AEs, and both were manageable Conclusion Mogamulizumab therapy showed clinically meaningful activity in ATLL patients, with an acceptable toxicity in clinical practice. Disclosures No relevant conflicts of interest to declare.

In primary myelofibrosis patients, somatic mutations such as JAK2V617F(JAKVF) and MPLW515 that activate JAK-STAT signaling are often seen. Small-molecule JAK2 inhibitors are effective for organomegaly and constitutional symptoms, but the drugs have little effect on BM fibrosis. To clarify the mechanism by which MPN cells with JAK2 mutations cause BM fibrosis, we compared the gene expression patterns of Lin−Sca1+ BM cells in JAK2VF transgenic mice (JAK2VF-TG), which develop myelofibrosis (MF), with that in WT mice. We found that TGFb1 and HOXB4, the target genes of transcription factor USF1 were highly expressed. TGFβ1, which is secreted by hematopoietic cells, is essential for fibrotic development in a murine model of MF (Chagraoui et al. Blood 2002), and increased expression of HOXB4 enhances human megakaryocytic development (Zhong et al. BBRC 2010). To investigate the mechanism of the high expression of these genes downstream of JAK2 signaling, USF1 and a cytokine receptor gene (MPL, EPOR or CSF3R) were co-transfected into 293T cells along with either a TGF-β1/HOXB4 promoter-driven or a STAT5 response element-driven luciferase reporter. Stimulation of MPL with TPO enhanced USF1 transcriptional activity about 3 fold, but stimulation of EPOR with EPO or of CSF3R with G-CSF did not change this activity. However, stimulation with any of the 3 types of cytokines enhanced STAT5 transcriptional activity. JAK2VF upregulated USF1 and STAT5 much more highly than JAK2WT without TPO stimulation. This USF1 upregulation specifically to TPO/MPL signaling was suppressed by a dominant negative mutant of USF1, JAK2 inhibitors (AG490, NS-018) or MEK inhibitors (U0126, PD325901). Inhibition of PI3K or p38MAPK did not affect the USF1 activation. Co-treatment with JAK2 and MEK inhibitors showed a synergistic effect in blocking both USF1 upregulation and STAT5 activation induced by JAK2VF. Next, we tested the MEK inhibitor, PD325901, in combination with the JAK2 inhibitor, NS-018, in the JAK2VF-TG mice. After disease was established 12 weeks after birth, JAK2VF-TG mice were divided into the following 4 groups: vehicle control; PD325901 monotherapy; NS-018 monotherapy; and combined therapy. PD325901 (5 mg/kg) and NS-018 (50 mg/kg) were orally administered once and twice daily, respectively. After 12 weeks of treatment, we evaluated the effect on BM fibrosis. The grading of MF in each group (n = 5-6) was as follows: vehicle control (MF-0: 0/6, MF-1 or 2: 6/6); PD325901 monotherapy (MF-0: 4/5, MF-1 or 2: 1/5); NS-018 monotherapy (MF-0: 0/6, MF-1 or 2: 6/6); and combined therapy (MF-0: 3/6, MF-1 or 2: 3/6). In the 2 groups treated with PD325901, 50~80% of mice showed MF-0. In contrast, in vehicle-treated or NS-018 monotherapy groups, all mice showed MF-1 or 2. Consistent with the MF grading, BM cellularity was significantly increased in the PD325901 monotherapy or combined therapy groups compared with the vehicle-treated group. A significant reduction was seen in the plasma TGFβ1 concentration in the PD325901 monotherapy and combined therapy groups compared with the vehicle-treated group (9.7 ng/ml, 8.1 ng/ml vs. 18.2 ng/ml, respectively). The TGFβ1 concentration in the extracellular fluid of BM (Wagner et al blood 2007) was also significantly reduced (5.6 ng/ml, 6.8 ng/ml vs. 9.1 ng/ml, respectively). BM cellularity and the TGFβ1 concentration in the NS-018 monotherapy group were comparable to those in the vehicle-treated group. Interestingly, megakaryocytes in the PD325901 monotherapy and combined therapy groups were decreased in number and were smaller than those in the vehicle-treated or NS-018 monotherapy groups. Regarding the effect on splenomegaly, spleen weight was significantly reduced in the NS-018 monotherapy and combined therapy groups compared with the vehicle-treated group (0.83 g, 0.69 g vs. 1.18 g, respectively). PD325901 monotherapy had little effect on splenomegaly. It is known that MEK-ERK1/2 pathway is critical in normal megakaryocyte development. In vitro data suggest that JAK2VF activates this pathway downstream of MPL and may contribute to TGFβ1 overproduction and dysmegakaryopoiesis, causing BM fibrosis via transcriptional enhancement of USF1. In vivo data suggest that MEK inhibition has the potential to improve dysmegakaryopoiesis and BM fibrosis. The combined therapy of JAK2 inhibitors with MEK inhibitors might be a promising therapy for improving both splenomegaly and BM fibrosis. Disclosures No relevant conflicts of interest to declare.

Introduction JAK2V617F (JAK2VF) is the most frequent mutation in myeloproliferative neoplasms (MPN), and its role has been demonstrated in mouse models. Actually, JAK2VF transgenic (JAK2VF Tg) mice generated by us induce lethal MPN (Shide et al. Leukemia 2008). Recently, mutations of epigenetic regulator such as TET2 are also frequently identified in MPN, and several TET2 knock out or knock down (TET2KD) mouse models are generated. We previously analyzed TET2KD mice (Ayu17-449) (Shide et al. Leukemia 2012). TET2KD fetal liver (FL) or bone marrow (BM) cells showed a growth advantage over Wt BM cells, with increased self-renewal capacity of hematopoietic stem cells; however TET2KD mice didn’t develop MPN, and its role in MPN remained unclear. To explore the role of TET2 deficiency in MPN harboring JAK2VF, we examined the cooperative effect, using these mutant mice. Materials and methods (1) Mice and collection of test cells. JAK2VF Tg mice (C57BL/6, Ly5.2) and TET2KD mice (Ayu17-449, C57BL/6, Ly5.2) were used. We crossed them, and collected JAK2Wt-TET2Wt (Wt-Wt), JAK2Wt-TET2KD (Wt-KD), JAK2VF-TET2Wt (VF-Wt), and JAK2VF-TET2KD (VF-KD) FL cells. (2) Non-competitive repopulation assay (NCRA). FL cells (Ly5.2, 1x106 cells) were transplanted into lethally irradiated recipients (Ly5.1) without competitor cells. Recipients were analyzed by complete blood counts, flow cytometry, colony-forming assay, colony-replating assay, pathology at 20-28 weeks post-transplantation, and overall survival. (3) Competitive repopulation assay (CRA) and serial BM transplantation (sBMT). FL cells (Ly5.2, 1x106 cells) were transplanted into lethally irradiated recipients (Ly5.1) with competitor Wt BM cells (Ly5.1, 5x106 cells), and sBMT was performed by 1x106 BM cells of the recipients at every 12 weeks post-transplantation. Recipients which were not selected as the donors were analyzed. (4) Analyses of adult mutant mice. Mice were bred in BDF1 background and analyzed at 20 or more weeks of age, as well as the recipients in NCRA. (5) Statistical analysis. Results were presented as means±S.D. Two-tailed Student’s t-test and log-rank test were used. Result In NCRA, both recipients transplanted with VF-Wt cells and VF-KD cells developed MPN with increase in WBC and Plt, decrease in Hb, fibrosis in BM and spleen, and extramedullary hematopoiesis (EMH) of lung and liver; and the latter developed more severe MPN and died earlier: VF-Wt (n=10) vs. VF-KD (n=10); WBC (x104/µl), 4.2±1.6 vs. 7.3±3.3 (p Conclusion TET2 deficiency increases severity of MPN harboring JAK2VF. TET2 deficiency enhances disease initiating potential of JAK2VF-MPN stem cells. TET2 deficiency is considered to be critical for both onset and progression of MPN harboring JAK2V617F. Disclosures: No relevant conflicts of interest to declare.

Background Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T cell neoplasm caused by human T-cell lymphotropic virus type I with very poor prognosis. The relationship between chemotherapy dose intensity and clinical outcome of ATL in clinical practice remains unclear. Patients and methods To elucidate the clinical characteristics and outcome of ATL patients, we retrospectively analyzed 118 patients diagnosed with ATL at 7 institutes in Miyazaki Prefecture, Japan from 2010 to 2012. There were 67 males and 51 females. The median age of the patients was 70 years (range 44–92). Subtypes included acute- (n=85) and lymphoma-type (n=33). One hundred one patients were treated with one of the below combination chemotherapy: (1) VCAP-AMP-VECP (LSG15); (2) CHOP (including pirarubicin (THP)-COP); and (3) non-LSG15, non-CHOP regimen. The prognostic value of the recently proposed prognostic index for ATL, namely ATL-PI (Katsuya et al. J Clin Oncol. 2012), was evaluated in this cohort. Relative dose intensity (RDI) during the first 12 weeks of therapy was calculated based on the standard regimen. Average RDI (ARDI) for LSG15 and CHOP was calculated. Results The median survival time (MST), 1- and 2-years overall survival (OS) rates of the entire cohort were 8.5 months, 35.3% and 23.0%, respectively. MSTs of patients less than 70 years and patients 70 years or older were 11.8 and 5.7 months, respectively (p=0.03). MSTs among all age groups for acute- and lymphoma- type were 8.3 and 10.0 months, respectively (p=0.445). Although ATL-PI could efficiently discriminate high-risk patients, it failed to separate the intermediate- and low-risk patients in this cohort. As almost all patients in this cohort were stage III or IV, ATL-PI was modified to exclude the Ann Arbor stage from variables. This modified ATL-PI could stratify our cohort into three distinct risk groups. MSTs were 3.9, 10.9, and 18.1 months for patients in high, intermediate, and low risk groups, respectively (P < 0.01). Among this cohort, 38 patients have received LSG15 and 47 patients received CHOP. Variables known to affect outcome were similar in both groups, except the age. MSTs were 11.5 and 8.1 months for patients treated with LSG15 and CHOP, respectively (p=0.206). As the median age of CHOP group is about 10 years greater than that of LSG15 group, we examined the MST in each therapy group according to the age. The MSTs for less than 70 years old were 11.7 and 21.9 months for patients treated with LSG15 and CHOP, respectively (p=0.311). Similarly, the MSTs for 70 years or older were 8.6 and 5.5 months for patients treated with LSG15 and CHOP, respectively (p=0.142). In practice, we conclude that CHOP is still a standard therapy for ATL. We next examined the RDI in each therapy groups and its relationship with OS. During the first 12 weeks, 73.7% of patients treated with LSG15 received ≥50% of planned DI, whereas 50.0% of patients treated with CHOP received ≥50% of planned DI. MSTs were significantly longer in patients treated with higher ARDI (≥50%) than that in patients with lower ARDI ( Conclusion Our modified ATL-PI excluding the Ann Arbor stage from variables in original ATL-PI may have prognostic value for patients with ATL. In the daily practice in Japan, no superiority of LSG15 compared to CHOP could be demonstrated both in young and elderly patient groups. Reduced ARDI in the first line chemotherapy for ATL is pervasive. Considering the significantly poor outcome of patients treated with ARDI less than 50%, changes in therapy with alternative strategy such as applying mogamulizumab, relatively early in the course of treatment, may improve outcome. Disclosures: No relevant conflicts of interest to declare.

Abstract 2471 Loss-of-fuction mutations of Ten-Eleven-Translocation-2 (TET2) was found in a variety of myeloid malignancies. But little is known about the function of TET2 in normal hematopoiesis and in the pathogenesis of myeloid malignancies. To study the function of TET2 in hematopoiesis in vivo, we conducted detailed analyses on hematopoiesis of TET2 gene trap mice (Tang et al. 2008), in which gene trap vector was inserted into exon 2 of TET2. Homozygous (trap/trap) mice were born at the proportion with Mendelian expectations, but died at a high rate by 2 days after birth. The fraction of trap/trap mice at P0 and P3 was 33% and 12%, respectively. TET2 mRNA expression level in fetal liver cells (FLs) at embryonic day 14.5 (E14.5) was approximately 60% in trap/wt mice and 20% in trap/trap mice compared to wild-type (WT) mice. Homozygous TET2 gene trap mice revealed to be TET2 low expression mice (TET2low). In E14.5 TET2low FLs, cellularity was comparable to WT FLs. The proportion of lineage-marker−Mac-1+Sca-1+ cells containing hematopoietic stem cells (HSCs) and multipotent progenitors was higher in TET2low FLs compared to WT FLs (0.43±0.05% vs 0.18±0.01%, p We next transplanted FLs from WT or TET2low mice into lethally irradiated recipient mice, and the hematological parameters were examined. Twenty weeks after transplantation, there were no significant differences in blood counts between two groups. The difference in peripheral blood (PB) was observed in the proportion of Gr-1+/Mac1+ cells and monocytes. The proportion of Gr-1+/Mac1+ cells and monocytes of WBCs in recipient mice transplanted with WT FLs was 20.7±1.6% and 11±1%, respectively, and that in recipient mice transplanted with TET2low FLs was 29.7±3.4% and 16±6%, respectively (p Recipient mice transplanted with TET2low FLs showed splenomegaly and extramedullary hematopoiesis. The spleen weights in recipient mice transplanted with WT FLs and TET2low FLs was 0.076±0.003g and 0.092±0.005g, respectively (p=0.02). The proportion of LSK, CD41+, and CD71+TER119− cells was higher in recipient mice transplanted with TET2low FLs compared to those with WT FLs. The number of CFU-GM and CFU-MEP was also higher in TET2low group. To determine whether TET2 low expression affects the HSC function in vivo, we performed a competitive repopulation assay by transplanting 1 × 106 CD45.2+ FLs from WT or TET2low mice along with 1 × 106 CD45.1+ WT BM cells (BMs), into lethally irradiated CD45.1+ recipient mice (1st recipient mice). After 12 weeks, 1 × 106 BMs from 1st recipient mice were serially transplanted into irradiated recipient mice (2nd recipient mice). The contribution of CD45.2+ cells in total and differential lineage of WBC was analyzed. The proportion of CD45.2+ in total WBC from 1st recipient mice transplanted with WT and TET2low FLs was 75±2.6% and 88±1.1%, respectively. In the 2nd recipient mice, the contribution of TET2 low cells was still high as 89±1.1%, although that from WT cells decreased to 54±4.5%. In both myeloid and lymphoid lineages, the contribution of TET2low cells was higher compared to that of WT cells. The proportion of CD45.2+ cells in Gr-1+/Mac1+ cells, B220+ cells, and CD4+/CD8+ cells in 2nd recipient mice transplanted with WT BMs was 49±6.5%, 72±4.7%, and 41±3.3%, respectively, and that in 2nd recipient mice transplanted with TWT2low BMs was 92±0.6%, 97±0.7%, and 64±2.7%, respectively. These data indicate that (1) TET2 is essential for survival in mice, (2) the decreased TET2 expression resulted in the enlargement of HSCs, and cell-autonomous growth and competitive advantage in hematopoietic cells, (3) the decreased TET2 expression altered the cell differentiation skewing toward myeloid/monocytic lineages. TET2 controls the hematopoietic homeostasis. Disclosures: No relevant conflicts of interest to declare.