Abstract: 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 〈 0.05; spleen CD4+ T-cell%, 10.3±3.1 vs. 18.2±3.3, P 〈 0.01; BM-CD4+ T-cell%, 1.2±0.6 vs. 2.5±1.1, p 〈 0.05), and the estimated whole body CD4+ T-cell count was also increased (WT vs. HBZ-Tg; CD4+ T-cells (x106 cells/body) 13.6±7.6 vs. 41.9±24.4, p 〈 0.01). In the survival analysis, compared to WT, the shortened overall survival (OS) was observed in HBZ-Tg (median survival time (MST, month), unreached vs. 11.1, p 〈 0.01). In pathological analysis, HBZ-Tg showed increased leukocyte infiltration to various organs such as lung and liver, and the infiltrated cells were mainly composed of T-cells. In the lung, in addition to the cell infiltration, alveolar edema was observed, which was presumed to be the main cause of death. 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 〈 0.05). Further, the double mutant showed increased frequency of CD103 (integrin alpha E), an adhesion molecule, expressing cells (CD4+CD103+% in spleen; 6.7±1.0 vs. 10.9±1.9, p 〈 0.05). In the acute and lymphomatous variant ATLL cohort analysis, genetic and clinical investigation revealed that organ infiltration detectable by imaging studies was frequently observed in TET2 mutated patients (WT-Pt (n=100) vs. TET2 mutated-Pt (n=15); extra nodular lesion, 78/100 vs. 14/15). Conclusion: In both mice model and human cohort, TET2 mutation exacerbated organ infiltration of ATLL cells. Disclosures No relevant conflicts of interest to declare.

Abstract: Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy with a dismal prognosis, caused by HTLV-1. Although our previous study, mainly using whole-exome sequencing and SNP array karyotyping, discovered many driver mutations and copy number alterations (CNAs), the whole-genome landscape of ATL still remains elusive. To this end, we have performed high-depth whole-genome sequencing (WGS) of 155 ATL cases with a median sequencing depth of 96-fold for tumors. Among them, 75 cases were also analyzed by RNA sequencing (RNA-seq). In total, we detected 1,952,490 single nucleotide variants (SNVs) and 159,141 insertion-deletions (4.0 SNVs and 0.3 indels/Mb/case), 10,279 SVs (66.3 SVs/case), and 3,975 CN altered segments (25.7 segments/case). Using several driver discovery algorithms (dNdScv, MutSig2CV, and DriverPower), we identified 47 significantly mutated genes, 19 of which were mutated in more than 10% of cases. These included several novel mutations, such as those affecting XPO1 (7.1%), ZNF292 (6.5%), and ITGB1 (5.2%). Using GISTIC2.0, we identified 13 significant CNAs, such as IRF4 amplifications and CDKN2A deletions, consistent with previous SNP array data. To detect significantly recurrent SVs, we calculated SV breakpoint frequency and identified 13 genes affected by SVs, including the previously identified genes (such as CARD11, CD274, and TP73). In addition, we investigated recurrent mutations in non-coding elements by DriverPower and LARVA and discovered 12 recurrently mutated elements. Among them, the most frequent were splice site mutations, including those of HLA-A and HLA-B, most of which caused loss of function as revealed by RNA-seq. By contrast, we found recurrent mutations in TP73 splice site, which induced skipping of exons 2 and 3, generating a dominant-negative variant similar to their SVs. In addition, recurrent non-coding elements contained several novel regions, such as 3´-untranslated region (UTR) of NFKBIZ and 5´- UTR of TMSB4X. Altogether, a total of 56 genes were recurrently altered. The median number of driver alterations was eight per case, and at least one driver alteration was found in 149 cases (96.1%). Among 56 driver genes, 40 (71.4%) genes were affected by more than one alteration class. Some drivers, such as CDKN2A, IKZF2, and CD274, were affected almost exclusively by CNAs and/or SVs, while showing quite high alteration frequencies (11.6-29.0%). These observations suggest that WGS presented a substantially different overview of driver alterations from our previous study. The overall numbers of mutations and SVs were linked to these driver alterations, suggesting their etiology. In particular, inactivation of EP300 and immune-related molecules, such as HLA-A, HLA-B, and CD58, were associated with an increased number of mutations and SVs, especially deletions and tandem duplications. By contrast, cases with TP53-altered cases harbored more inversions and translocations. These results emphasize a pivotal role of immune evasion for acquiring genetic alterations to drive ATL progression. To define molecular subgroups in ATL, we integrated the 56 identified genetic drivers using non-negative matrix factorization clustering and identified two robust subgroups with discrete clinical and genetic characteristics. Group 1 was enriched with alterations affecting distal components of T-cell receptor (TCR)/NF-κB signaling (such as CARD11, PRKCB, and IRF4) and immune-related molecules (HLA-A, HLA-B, and CD58), whereas proximal regulators of TCR/NF-κB signaling (PLCG1, VAV1, and CD28) and a JAK/STAT signaling molecule (STAT3) were more frequently altered in group 2. In addition, group 1 cases had a larger number of mutations, SVs, and CNAs than group 2 cases. Clinically, most cases with lymphoma subtype were classified into group 1, whereas group 2 mainly consisted of cases with leukemic subtypes. Moreover, group1 cases showed a worse overall survival than group 2, independently of clinical subtype. These results suggest the biological and clinical relevance of the molecular classification of ATL. In summary, our WGS analysis not only identifies novel somatic alterations but also extends the overview of ATL genome. We also propose a new molecular classification of ATL, with its clinical relevance, which can lead to the future improvement of patient management. Disclosures Kogure: Takeda Pharmaceutical Company Limited.: Honoraria. Nosaka:Kyowa Kirin Co.Ltd: Honoraria; Chugai pharmaceutical Co. Ltd: Honoraria; Novartis international AG: Honoraria; Celgene K.K: Honoraria; Eisai Co., Ltd: Honoraria; Merck Sharp & Dohme K.K.: Honoraria; Bristol-Myer Squibb: Honoraria. Imaizumi:Kyowa Kirin Co. Ltd.: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Eisai: Honoraria. Utsunomiya:Kyowa Kirin: Honoraria; Celgene: Honoraria. Shah:Celgene: Research Funding; BMS: Research Funding; Physicians Education Resource: Honoraria. Janakiram:Takeda, Fate, Nektar: Research Funding. Ramos:NIH: Research Funding. Takaori-Kondo:Astellas Pharma: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Thyas Co. Ltd.: Research Funding; Takeda: Research Funding; CHUGAI: Research Funding; Eisai: Research Funding; Nippon Shinyaku: Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; OHARA Pharmaceutical: Research Funding; Sanofi: Research Funding; Novartis Pharma: Honoraria; MSD: Honoraria. Miyazaki:Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Celgene: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Otsuka Pharmaceutical: Honoraria; Novartis Pharma KK: Honoraria; Astellas Pharma Inc.: Honoraria. Matsuoka:Chugai Pharmaceutical Co. Ltd: Research Funding; Bristol-Myers Squibb: Research Funding; Kyowa Kirin Co. Ltd.: Research Funding. Ishitsuka:Takeda: Other: Personal fees, Research Funding; mundiharma: Other: Personal fees; Taiho Pharmaceuticals: Other: Personal fees, Research Funding; Janssen Pharmaceuticals: Other: Personal fees; Novartis: Other: Personal fees; Pfizer: Other: Personal fees; Astellas Pharma: Other, Research Funding; Genzyme: Other; Sumitomo Dainippon Pharma: Other, Research Funding; Eisai: Other, Research Funding; Mochida: Other, Research Funding; Shire: Other; Otsuka Pharmaceutical: Other; Ono Pharmaceutical: Other, Research Funding; Teijin Pharma: Research Funding; MSD: Research Funding; Asahi kasei: Research Funding; Eli Lilly: Research Funding; Daiichi Sankyo: Other; Huya Japan: Other; Celgene: Other: Personal Fees; Kyowa Hakko Kirin: Other: Personal fees, Research Funding; BMS: Other: Personal fees; Chugai Pharmaceutical: Other: Personal fees, Research Funding. Ogawa:Asahi Genomics Co., Ltd.: Current equity holder in private company; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding. Shimoda:Takeda Pharmaceutical Company: Honoraria; Bristol-Myers Squibb: Honoraria; Shire plc: Honoraria; Celgene: Honoraria; 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; Novartis: Honoraria, Research Funding. Kataoka:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Genomics: Current equity holder in private company.

Abstract: The prognosis of aggressive adult T-cell leukemia/lymphoma (ATL) is poor, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment. In order to identify favorable prognostic patients after intensive chemotherapy, and who therefore might not require upfront allo-HSCT, we aimed to improve risk stratification of aggressive ATL patients aged 〈 70 years. The clinical risk factors and genetic mutations were incorporated into risk modeling for overall survival (OS). We generated the m7-ATLPI, a clinicogenetic risk model for OS, that included the ATL prognostic index (PI) (ATL-PI) risk category, and non-silent mutations in seven genes, namely TP53, IRF4, RHOA, PRKCB, CARD11, CCR7, and GATA3. In the training cohort of 99 patients, the m7-ATLPI identified a low-, intermediate-, and highrisk group with 2-year OS of 100%, 43%, and 19%, respectively (hazard ratio [HR] =5.46; P 〈 0.0001). The m7-ATLPI achieved superior risk stratification compared to the current ATL-PI (C-index 0.92 vs. 0.85, respectively). In the validation cohort of 84 patients, the m7-ATLPI defined low-, intermediate-, and high-risk groups with a 2-year OS of 81%, 30%, and 0%, respectively (HR=2.33; P=0.0094), and the model again outperformed the ATL-PI (C-index 0.72 vs. 0.70, respectively). The simplified m7-ATLPI, which is easier to use in clinical practice, achieved superior risk stratification compared to the ATLPI, as did the original m7-ATLPI; the simplified version was calculated by summing the following: high-risk ATL-PI category (+10), low-risk ATL-PI category (−4), and non-silent mutations in TP53 (+4), IRF4 (+3), RHOA (+1), PRKCB (+1), CARD11 (+0.5), CCR7 (−2), and GATA3 (−3).

Abstract: 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.

Abstract: The efficacy of mogamulizumab in adult T‐cell leukemia/lymphoma ( ATLL ) was reported in a previous phase 2 study. Compared with patients in clinical trials, however, most patients in real‐life settings have demonstrated worse outcomes. Method We retrospectively analyzed 96 patients with relapsed/refractory ATLL who received mogamulizumab treatment. Results Relapsed/refractory ATLL patients with a median age of 70 years received a median of five courses of mogamulizumab. Hematologic toxicity and skin rash were the most common adverse events, and both were manageable. Of 96 patients, 87 were evaluable for efficacy. The overall response rate was 36%, and the median progression‐free survival ( PFS ) and overall survival ( OS ) from the start of mogamulizumab therapy were 1.8 and 4.0 months, respectively. Of the original 96 patients, only 25 fulfilled the inclusion criteria of the phase 2 study. Those who met the criteria demonstrated longer median PFS and OS durations of 2.7 and 8.5 months, respectively. The median OS from diagnosis in relapsed/refractory ATLL patients receiving mogamulizumab was 12 months, longer than the 5.8 months in a historical cohort without mogamulizumab. Conclusion In clinical practice, mogamulizumab exhibited antitumor activity in patients with relapsed/refractory ATLL , with an acceptable toxicity profile. Mogamulizumab therapy improved the OS of ATLL patients.

Abstract: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by clonal myeloproliferation, progressive bone marrow (BM) fibrosis, splenomegaly, and anemia. BM fibrosis was previously thought to be a reactive phenomenon induced by mesenchymal stromal cells that are stimulated by the overproduction of cytokines such as transforming growth factor (TGF)-β1. However, the involvement of neoplastic fibrocytes in BM fibrosis was recently reported. In this study, we showed that the vast majority of collagen- and fibronectin-producing cells in the BM and spleens of Jak2V617F-induced myelofibrosis (MF) mice were fibrocytes derived from neoplastic hematopoietic cells. Neoplastic monocyte depletion eliminated collagen- and fibronectin-producing fibrocytes in BM and spleen, and ameliorated most characteristic MF features in Jak2V617F transgenic mice, including BM fibrosis, anemia, and splenomegaly, while had little effect on the elevated numbers of megakaryocytes and stem cells in BM, and leukothrombocytosis in peripheral blood. TGF-β1, which was produced by hematopoietic cells including fibrocytes, promoted the differentiation of neoplastic monocytes to fibrocytes, and elevated plasma TGF-β1 levels were normalized by monocyte depletion. Collectively, our data suggest that neoplastic fibrocytes are the major contributor to BM fibrosis in PMF, and TGF-β1 is required for their differentiation.

Abstract: 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.

Abstract: 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 〈 0.05); peripheral blood (PB) myeloid cells (%), 59.6±9.7 vs. 71.9±8.2 (p 〈 0.05); liver weight (g), 1.15±0.22 vs. 1.48±0.22 (p 〈 0.01); spleen weight (g), 0.26±0.11 vs. 0.52±0.19 (p 〈 0.01): VF-Wt (n=36) vs. VF-KD (n=30); mean survival time (weeks), 36 vs. 39 (p 〈 0.05). In colony-forming assay, number of CFU-GM was more increased in VF-KD cells than VF-Wt cells: VF-Wt (n=9) vs. VF-KD (n=9); colonies/2x104 BM cells, 107±37 vs. 157±46 (p 〈 0.05). In colony-replating assay, VF-Wt BM cells lost replating capacity by 3rd to 5th passage; VF-KD BM cells retained replating capacity beyond 5th passage: VF-Wt (n=9) vs. VF-KD (n=6); number of colonies in 4th passage, 5.9±6.8 vs. 896±613 (p 〈 0.01). In CRA, all recipients transplanted with VF-Wt cells (n=9) or VF-KD cells (n=9) showed ≥ 70% test cell-derived PB chimerism, and developed MPN with fibrosis and EMH at 12 weeks. In 2nd BMT, 4/9 recipients transplanted with VF-Wt cells showed ≥ 35% PB chimerism at 12 weeks. Six recipients were analyzed at 12-16weeks, and no one (0/6) showed pathological findings of MPN. Whereas, 7/9 recipients transplanted with VF-KD cells showed ≥ 35% PB chimerism. Five recipients were analyzed, and 3/5 developed MPN with fibrosis and EMH: VF-Wt (n=6) vs. VF-KD (n=5); liver weight (g), 1.00±0.12 vs. 1.39±0.15 (p 〈 0.002); spleen weight (g), 0.069±0.019 vs. 0.20±0.097 (p 〈 0.05). In analyses of adult mutant mice, both VF-Wt mice and VF-KD mice developed MPN, and disease severities or colony-replating capacities are similar tendencies as those in transplantation model. 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.