Erythroid Colonies Research Articles

A Clinical Study of OPN-305, a Toll-like Receptor 2 (TLR-2) Antibody, in Patients with Lower Risk Myelodysplastic Syndromes (MDS) That Have Received Prior Hypomethylating Agent (HMA) Therapy

▪INTRODUCTION: Alterations of innate immune signaling, including overexpression of TLR2, are common in MDS. Signaling mediated by this receptor results in activation of NF-kB and the histone demethylase JMJD3 (KDM6B) leading to expression of multiple cytokines that can result in inhibition of hematopoiesis. Significant TLR2 overexpression in MDS bone marrow CD34+ cells, especially after HMA therapy, has been reported (Wei, Leukemia 2013). OPN-305 is a fully humanized antagonistic IgG4 kappa monoclonal antibody to TLR2 which significantly increases the formation of erythroid colonies (CFU-E) in BM CD34+ cells isolated from pts with lower-risk MDS in vitro. This data suggested the potential therapeutic value of OPN-305 in patients (pts) with MDS.METHODS: We designed a phase I/II trial of OPN-305 for pts with Low or Int-1 risk MDS by IPSS after failure to prior therapy with a HMA. Pts with isolated del(5q) should have received therapy with lenalidomide. Pts with prior history of AML or alloSCT were excluded from the study. Because, OPN-305 had not been previously used in pts with hematological malignancies, the study had an initial phase of N=10 pts using OPN-305 at a dose of 5 mg/kg every 4 weeks for a maximum of 9 cycles. Therapy could be repeated as long as there was no excess toxicity or progression. If after 16 weeks of therapy, there was no response, azacitidine on a 3 day schedule, could be added to OPN-305. Responses were evaluated following the revised 2006 IWG criteria. This initial cohort allowed evaluation of toxicity, pharmacokinetic analysis, receptor occupancy, and sequential analysis of cytokine profile. Depending on results, dose escalation to 10 mg/kg monthly could be instituted in a second cohort of N=5 pts.RESULTS: At the time of this report, 15 pts have been enrolled, 11 at the initial 5 mg dose and 4 at 10 mg/kg. All pts were evaluable for toxicity and 12 (80%) were evaluable for response. Patient characteristics are shown in Table 1. Median age was 73 years (range 48-87). Three (20%) pts were classified as Low risk and 12 (80%) as Intermediate-1 risk by IPSS. Seven pts had normal karyotype, 2 del(5q), 2 trisomy 8, 1 del(20q), 1 monosomy Y and 2 other single or double abnormalities. Median number of prior therapies was 2 (range 1-4) with a median duration of prior therapies of 28 months (range 4-62) (Figure 1). A total of 13 (87%) pts were transfusion dependent of red cells prior to enrollment. Median follow up is 8 months (1-16). Median number of cycles administered is 5 (2-17) with 5 (33%) pts having received addition of azacitidine after 16 weeks of therapy. A total of 5 (42%) pts developed AEs related to OPN-305. All AEs were grade 1 with gastrointestinal disorders being the most frequent (33%). At this point, no significant drug related toxicity has been documented with no excess infectious complications. Overall response rate in the form of hematological improvement was 50% (6/12) with 2 (17%) pts achieving transfusion independence. A total of 5 (33%) pts remain on study with 3 pts continuing on therapy off study. Three (20%) pts were taken off study due to progression to AML and 4 (27%) due to no response. Half-lives of OPN-305 in serum were >200 h at 5 mg/kg and >300 h at 10 mg/kg (Figure 2A). There was a greater-than-dose proportional increase in mean OPN-305 exposure (AUC) between 5 and 10 mg/kg. PK profiles after repeated dosing at 5 mg/kg in N=2 subjects and pre-dose (trough) levels in other subjects indicated some variability in the potential for accumulation. TLR-2 receptor occupancy in blood PBMCs and bone marrow aspirates was essentially complete in virtually all samples taken after OPN-305 administration (Figure 2B). There is no evidence of treatment related anti-drug antibodies. Compared with baseline, no statistically significant dynamic changes of IL-23, IL-18, IFN-r, IL-10, IL-1β, IL-6, IL-12 (p40), IL-12 (p70) and IL-8 levels where observed after treatment, among responders or non-responders or based on OPN-305 dosing.CONCLUSIONS: Treatment with OPN-305 in pts with previously treated lower-risk MDS was well tolerated with no significant toxicities and 50% ORR. Correlative studies suggest adequate receptor occupancy with no correlation between responses and cytokine levels. Because no effect on cytokine profile, we plan to continue to escalate the dose of OPN-305 and investigate its activity profile in front line therapy. [Display omitted] [Display omitted] [Display omitted] DisclosuresDiNardo:Daiichi Sankyo: Research Funding; Agios: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

Quantification of IGF-1 Receptor May Be Useful in Diagnosing Polycythemia Vera-Suggestion to Be Added to Be One of the Minor Criterion.

Endogenous erythroid colony (EEC) formation is one of the minor criteria for diagnosing polycythemia vera (PV) according to 2008 WHO diagnostic criteria. But EEC requires bone marrow aspiration and sophisticated laboratory procedures; therefore, practically it is rarely used to diagnose PV. Insulin-like growth factor 1 receptor (IGF-1R) was found to be constitutively phosphorylated and was responsible for the EEC formation in PV; therefore, we measured IGF-1R levels in the peripheral blood of 26 PV patients and compared them with those of 33 patients with secondary polycythemia and 29 normal controls. Among the PV patients, 16 were treated with only phlebotomy, 9 received hydroxyurea, and 1 was treated with ruxolinitinib. We found that PV patients treated with only phlebotomy had significantly higher IGF-1R levels than did those PV patients treated with hydroxyurea or ruxolinitinib. None of the secondary PV patients or normal controls had elevated IGR-1R levels, while 14 of 16 (87%) PV patients had significantly elevated IGF-1R levels. The new 2016 WHO has eliminated EEC as a minor criterion for diagnosing PV, but there are still some cases that cannot be definitively diagnosed by the current criteria. Therefore, we suggest that quantifying the IGF-1R level in peripheral blood by flow cytometry to replace EEC as the minor criterion for diagnosing PV.

Clonidine reduces norepinephrine and improves bone marrow function in a rodent model of lung contusion, hemorrhagic shock, and chronic stress

Propranolol has been shown previously to restore bone marrow function and improve anemia after lung contusion/hemorrhagic shock. We hypothesized that daily clonidine administration would inhibit central sympathetic outflow and restore bone marrow function in our rodent model of lung contusion/hemorrhagic shock with chronic stress. Male Sprague-Dawley rats underwent 6days of restraint stress after lung contusion/hemorrhagic shock during which the animals received clonidine (75μg/kg) after the restraint stress. On postinjury day 7, we assessed urine norepinephrine, blood hemoglobin, plasma granulocyte colony stimulating factor, and peripheral blood mobilization of hematopoietic progenitor cells, as well as bone marrow cellularity and erythroid progenitor cell growth. The addition of clonidine to lung contusion/hemorrhagic shock with chronic restraint stress significantly decreased urine norepinephrine levels, improved bone marrow cellularity, restored erythroid progenitor colony growth, and improved hemoglobin (14.1±0.6 vs 10.8±0.6g/dL). The addition of clonidine to lung contusion/hemorrhagic shock with chronic restraint stress significantly decreased hematopoietic progenitor cells mobilization and restored granulocyte colony stimulating factor levels. After lung contusion/hemorrhagic shock with chronic restraint stress, daily administration of clonidine restored bone marrow function and improved anemia. Alleviating chronic stress and decreasing norepinephrine is a key therapeutic target to improve bone marrow function after severe injury.

Colony-forming unit cell (CFU-C) assays at diagnosis: CFU-G/M cluster predicts overall survival in myelodysplastic syndrome patients independently of IPSS-R

BackgroundIn vitro colony-forming unit cell (CFU-C) assays are usually-used to detect the quantitative and qualitative features of haematopoietic stem cells (HSCs). We studies CFU-C assays in bone marrow samples from 365 consecutive subjects with newly-diagnosed myelodysplastic syndrome (MDS). Data were interrogated for associations with prognosis.MethodsCFU-C assays were performed according to the protocol of MethoCultTM H4435 Enriched. 365 consecutive newly-diagnosed, untreated subjects with MDS diagnosed from July, 2007 to April, 2014 were studied. All subjects were reclassified according to the 2008 WHO criteria. Subjects were observed for survival until July 31, 2015. Follow-up data were available for 289 (80%) subjects. Median follow-up of survivors was 22 months (range, 1-85) months. Erythroid and myeloid colonies were isolated from each subject with one cytogenetic abnormality such as del(5/5q), +8, del(7/7q) or del(20q). Cytogenetic abnormalities of each colony were analyzed by fluorescence in situ hybridization (FISH). SPSS 17.0 software was used to make statistical analysis.ResultsThe numbers of burst-forming units-erythroid (BFU-E), colony forming unit-erythroid (CFU-E) and colony forming unit-granulocytes/macrophages (CFU-G/M) were significantly lower than normals. A high ratio of cluster- to CFU-G/M was associated with poor-risk cytogenetics. In multivariable analyses a cluster- to CFU-G/M ratio >0.6 was an independent risk-factor for OS after adjusting for IPSS-R (HR 3.339, [95%CI 1.434-7.778]; P = 0.005) in very high-risk cohort.ConclusionThese data suggest abnormalities of proliferation and differentiation of erythroid and myeloid precursor cells in vitro parallel the ineffective hematopoiesis typical of MDS and may be useful in predicting outcomes of persons with higher-risk MDS.

Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia

Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous β-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the β-globin locus to mimic the naturally occurring Sicilian HPFH mutation. The efficiency of targeting deletion reached 31% in cells with the delivery of both upstream and downstream breakpoint guide RNA (gRNA)-guided Staphylococcus aureus Cas9 nuclease (SaCas9). The erythroid colonies differentiated from HSPCs with HPFH deletion showed significantly higher γ-globin gene expression compared with the colonies without deletion. By T7 endonuclease 1 assay, we did not detect any off-target effects in the colonies with deletion. We propose that this strategy of using nonhomologous end joining (NHEJ) to modify the genome may provide an efficient approach toward the development of a safe autologous transplantation for patients with homozygous β-thalassemia and SCD.

732. CRISPR/Cas9 Mediates Highly Efficient Gene Editing in Long-Term Engrafting Human Hematopoietic Stem/Progenitor Cells

Transplantation of genetically corrected autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for patients with inherited hematologic disease. Genome editing with CRISPR/Cas9 ribonucleoprotein (RNP) precisely modifies gene targets in human cells, including HSPCs. In order to translate this technology to a clinical setting, gene editing must be reproducible and efficient across multiple patient donors without compromising HSPC viability, multipotency, and long-term engraftment capability. To determine the reproducibility of Cas9 RNP mediated gene editing in HSPCs, human CD34+ cells obtained from 15 different patient donors (cord blood n=12, mobilized peripheral blood [mPB] n=3) were electroporated with S. pyogenes Cas9 RNP targeting the β-hemoglobin (HBB) or AAVS1 genetic locus. DNA sequence analysis of 15 separate experiments demonstrated that Cas9 supported up to 72% gene editing in cord blood CD34+ cells and up to 61% gene editing in adult mPB CD34+ cells (mean % editing by DNA sequencing: 57% ± 8%). Cas9 induced multiple modifications that comprise insertions and deletions at the HBB locus, and some of the lesions were repaired through an HDR repair mechanism that used the homologous sequences from the endogenous HBD gene as a template (Gene Conversion). Gene edited CD34+ cells retained viability and hematopoietic colony forming cell (CFC) activity ex vivo, with no significant differences between treatment groups or across donors. Indels were detected in one (BFU-E/GEMM: 50%-75%, CFU-GM/M: 50-66%) or both (BFU-E/GEMM: 12-38%, CFU-GM/M: 12-50%) alleles, at high frequencies in clonal erythroid and myeloid colonies (range of 63-100% of individual colonies assayed across experiments). To evaluate engraftment potential, both Cas9 modified (unsorted) and untreated control human CD34+ cells were transplanted into immunodeficient mice. Twelve weeks after transplantation, up to 34% human CD45+ cell engraftment was detected in the peripheral blood of recipients of Cas9 RNP treated CD34+ cells. There was no significant difference in the engraftment of Cas9 RNP treated and untreated control CD34+ cells which were detected in blood, spleen, and bone marrow. Human lymphoid, myeloid, and erythroid cells were detected in blood and hematopoietic organs with no difference in lineage distribution between cohorts. Analysis of the bone marrow from both groups revealed an average of 20% human CD45+ and 13% CD34+CD45+ cell engraftment long-term. Analysis of human gDNA revealed that up to 20% gene editing in the marrow of treated mice and edited cells were also detected in the blood and spleen. In summary, these data show that Cas9 RNP gene edited primary human CD34+ HSPCs retained the ability to engraft long-term and reconstitute hematopoiesis in vivo at levels higher than reported previously. These findings also show that electroporation of HSPCs with Cas9 RNP mediated highly reproducible gene editing levels across multiple donors and did not alter hematopoietic reconstitution or differentiation properties in comparison to untreated control CD34+ cells.

Leukemia cell infiltration causes defective erythropoiesis partially through MIP-1α/CCL3.

Leukemia often results in severe anemia, which may significantly contribute to patient mortality and morbidity. However, the mechanisms underlying defective erythropoiesis in leukemia have not been fully elucidated. In this study, we demonstrated that insufficient erythropoiesis in an immunocompetent acute myeloid leukemia (AML) murine model was due to reduced proliferation of megakaryocyte erythroid progenitors and increased apoptosis of erythroblasts. Colony-forming cell assays indicated that the leukemic bone marrow (BM) plasma inhibited erythroid colony formation, whereas they had no inhibitory effect on other types of colonies. Cytokine array analysis demonstrated that the chemokine CCL3 was elevated in the plasma of AML mice and patients. CCL3 inhibited erythroid differentiation of hematopoietic stem cells, common myeloid progenitors and especially megakaryocytic-erythroid progenitors. Administration of the CCR1 antagonist partially recovered the yield of erythroid colonies in the presence of CCL3 or leukemic BM plasma. Mechanistically, we observed an increase of p38 phosphorylation and subsequent downregulation of GATA1 after CCL3 treatment. Furthermore, knockdown of CCL3 attenuated leukemic progression and alleviated anemia. Therefore, our results demonstrate that elevated CCL3 in the leukemic environment suppresses erythropoiesis via CCR1-p38 activation, suggesting a novel mechanism for the erythroid defects observed in leukemia.

Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9High Committed Megakaryocytic Progenitors.

This study aimed at reinvestigating the controversial contribution of Notch signaling to megakaryocytic lineage development. For that purpose, we combined colony assays and single cells progeny analyses of purified megakaryocyte-erythroid progenitors (MEP) after short-term cultures on recombinant Notch ligand rDLL1. We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage. Interestingly, we also identified a CD9High MEP subset that spontaneously generated almost exclusively megakaryocytic progeny mainly composed of single megakaryocytes. We showed that Notch activation decreased the extent of polyploidization and maturation of megakaryocytes, increased the size of megakaryocytic colonies and surprisingly restored the generation of erythroid and mixed colonies by this CD9High MEP subset. Importantly, the size increase of megakaryocytic colonies occurred at the expense of the production of single megakaryocytes and the restoration of colonies of alternative lineages occurred at the expense of the whole megakaryocytic progeny. Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages. This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells.

In Vivo Deletion of the Cebpa +37 kb Enhancer Markedly Reduces Cebpa mRNA in Myeloid Progenitors but Not in Non-Hematopoietic Tissues to Impair Granulopoiesis.

The murine Cebpa gene contains a +37 kb, evolutionarily conserved 440 bp enhancer that directs high-level expression to myeloid progenitors in transgenic mice. The enhancer is bound and activated by Runx1, Scl, GATA2, C/EBPα, c-Myb, Pu.1, and additional Ets factors in myeloid cells. CRISPR/Cas9-mediated replacement of the wild-type enhancer with a variant mutant in its seven Ets sites leads to 20-fold reduction of Cebpa mRNA in the 32Dcl3 myeloid cell line. To determine the effect of deleting the enhancer in vivo, we now characterize C57BL/6 mice in which loxP sites flank a 688 bp DNA segment containing the enhancer. CMV-Cre mediated germline deletion resulted in diminution of the expected number of viable Enh(f/f);CMV-Cre offspring, with 28-fold reduction in marrow Cebpa mRNA but normal levels in liver, lung, adipose, intestine, muscle, and kidney. Cre-transduction of lineage-negative marrow cells in vitro reduced Cebpa mRNA 12-fold, with impairment of granulocytic maturation, morphologic blast accumulation, and IL-3 dependent myeloid colony replating for >12 generations. Exposure of Enh(f/f);Mx1-Cre mice to pIpC led to 14-fold reduction of Cebpa mRNA in GMP or CMP, 30-fold reduction in LSK, and <2-fold reduction in the LSK/SLAM subset. FACS analysis of marrow from these mice revealed 10-fold reduced neutrophils, 3-fold decreased GMP, and 3-fold increased LSK cells. Progenitor cell cycle progression was mildly impaired. Granulocyte and B lymphoid colony forming units were reduced while monocytic and erythroid colonies were increased, with reduced Pu.1 and Gfi1 and increased Egr1 and Klf4 in GMP. Finally, competitive transplantation indicated preservation of functional long-term hematopoietic stem cells upon enhancer deletion and confirmed marrow-intrinsic impairment of granulopoiesis and B cell generation with LSK and monocyte lineage expansion. These findings demonstrate a critical role for the +37 kb Cebpa enhancer for hematopoietic-specific Cebpa expression, with enhancer deletion leading to impaired myelopoiesis and potentially preleukemic progenitor expansion.

47-Year-Old Man With Pruritus

47-year-old man with a medical history of headaches and seasonal allergies presented to the primary care clinic in late fall for evaluation of diffuse pruritus of 6 weeks’ duration that had occurred after initiating a new diet. The diet mostly restricted foods like dairy, soy, nuts, grains, and processed foods, but he stated that he had substantially increased his consumption of coconut and various spices including turmeric. The itching was present over most of his body but most prominent on the inner thighs and most notable after a hot shower. He provided picturesfromrecent episodes thatillustrated linear excoriations on the thighs. When he showered in cooler water, the diffuse pruritus was still present but minimally improved compared with a hot shower. He had been using the same body soap for years but tried a new soap without relief from the itching. In addition, he changed laundry detergent from one he had used for years with no relief. He had no occupational or habitual exposures that are suspected to contribute to pruritus. Overthe-counter hydrocortisone cream applied to his thighs provided minimal relief. His weight had been stable, and he reported no constitutional symptoms. On physical examination, the patient was a

The induction and cryopreservation of erythroid progenitor cells derived from umbilical cord blood mononuclear cells

目的探索含多种细胞因子无血清培养基诱导脐血单个核细胞体外分化为红系祖细胞效率及红系祖细胞的保存方法。方法利用羟乙基淀粉沉降脐血中红细胞，人淋巴细胞分离液（Ficoll）分离单个核细胞，采用含FMS样酪氨酸激酶3配体、干细胞生长因子、胰岛素样生长因子1、重组人红细胞生成素的无血清培养基进行体外诱导脐血单个核细胞向红系祖细胞分化，并将诱导的红系祖细胞用不同的冻存液进行冷冻保存，观察红系祖细胞诱导、分化能力和红系祖细胞冷冻保存效果。结果随着诱导时间延长，细胞总数明显增多，培养14 d红系祖细胞扩增约110倍，存活率为（88.92±0.95）%，红系祖细胞特异性标志CD71+细胞占（86.77±9.11）%，CD71 +/CD235a+细胞占（64.47±16.67）%。诱导10 d多为中幼红细胞，可见血红蛋白表达的阳性细胞；诱导14 d开始出现晚幼红细胞，细胞沉淀呈红色。诱导7 d红系集落数为326.00±97.96，高于诱导前（61.60±20.03）。10％二甲基亚砜（DMSO）+2％人血清白蛋白保存细胞复苏后红系祖细胞存活率、回收率分别为（90.32±1.80）%、（93.66±1.87）%，将50％自体脐血浆与10% DMSO、2％人血清白蛋白联用可获得更好的保护效果。结论该无血清培养基可高效诱导、扩增红系祖细胞，10% DMSO+2％人血清白蛋白+50％自体脐血血浆可很好保存红系祖细胞。

Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts.

Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30–40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts.

Lineage-Specific Aberrant mRNA Splicing By U2AF1 Mutation Alters Erythroid and Granulomonocytic Differentiation

Splicing factor genes are the most common targets of somatic mutations in myelodysplastic syndromes (MDS). The splicing factor U2AF1 is an auxiliary factor that forms a heterodimer for the recognition of the 3′ splice site during pre-mRNA splicing. Heterozygous mutations of U2AF1 occur in ~10% of MDS patients and are predominantly located at S34 and Q157 within the zinc fingers domains. Recently an inducible transgenic mouse model expressing mutant U2AF1 S34F demonstrated altered hematopoiesis and aberrant pre-mRNA splicing in hematopoietic progenitor cells.MDS are clonal stem-cell disorders characterized by ineffective hematopoiesis in one or more myeloid lineages of the bone marrow. To investigate the effects of U2AF1 S34F mutation on hematopoiesis, U2AF1 S34F mutant (S34F) and U2AF1 wild type (WT) were overexpressed in human bone marrow CD34+ progenitor cells by retroviral transduction and the cells were differentiated along erythroid and granulomonocytic lineages. S34F erythroblasts exhibited impaired erythroid differentiation compared to WT and empty vector (EV) controls. A significant increase in CD71-CD235a- non-erythroid cells (p≤0.02, n=7) followed by a significant decrease in CD71+CD235a+ (p≤0.002, n=7) and CD71-CD235a+ (p=0.005, n=7) erythroid cells was observed in S34F erythroblasts from day 11 to 14 using flow cytometry, when compared to WT and EV controls. Moreover, S34F inhibited formation and hemoglobinization of BFU-E colonies from bone marrow CD34+ cells in colony forming cell (CFC) assays compared to WT (p=0.002, n=7) and EV (p=0.0006, n=7) controls. S34F erythroblasts also exhibited impaired cell growth and increased apoptosis (Annexin V+) compared to WT (p<0.05, n=6-8) and EV (p≤0.01, n=6-8) controls. Thus, the S34F mutation results in impaired erythropoiesis.S34F perturbed the granulomonocytic lineage by skewing differentiation of myeloid cells towards granulocytes. A reduction in the CD11b+ population was observed in S34F myeloid cells compared to WT (p≤0.001, n=9) and EV (p≤0.001, n=9) controls from day 11 to 14. An increase in granulocytes (CD15+, p≤0.001, n=5) followed by a concomitant decrease in monocytes (CD14+,p=0.026, n=5) was also observed in S34F myeloid cells on day 20 compared to WT and EV controls. Morphological analysis of myeloid cells confirmed a reduction in monocytes caused by an expansion of granulocyte eosinophils. Moreover, S34F bone marrow CD34+ cells produced a significantly higher number of CFU-G (p=0.035, n=5) with a decrease in the number of CFU-M (p≤0.03, n=5) in myeloid CFC assays compared to WT (p≤0.01, n=7) and EV (p≤0.01, n=7) controls. S34F myeloid cells exhibited impaired cell growth associated with G2/M cell cycle arrest compared to WT (p=0.0003, n=6) and EV (p=0.0002, n=6) controls.To investigate aberrant splicing events, we performed RNA sequencing on individual erythroid (BFU-E) and granulomonocytic (CFU-G and CFU-M) colonies formed by S34F, WT and EV transduced bone marrow CD34+ cells (n=3 each). By comparison with WT and EV colonies of the same lineage, we observed that S34F differentially alters the splicing pattern in different lineages. We have observed aberrant splicing of many genes, including BCOR and H2AFY, two genes previously shown to be aberrantly spliced in common myeloid progenitors from a U2AF1 S34F mouse model. The transcriptional co-repressor BCOR is commonly mutated in MDS/AML. Alternative 3' splice site usage in BCOR, resulting in reduced expression of its long isoform, was observed in S34F granulomonocytic colonies, but not in S34F erythroid colonies. In contrast, reduced expression of isoform 1.1 of H2AFY (a member of H2A histone family), due to mutually exclusive exons, was observed in both S34F erythroid and granulomonocytic colonies. Deregulation in isoform expression levels in BCOR and H2AFY was validated by isoform-specific qRT-PCR in S34F transduced cells compared to WT (p≤0.015, n=5) and EV (p≤0.045, n=5) controls. We are currently introducing these isoform imbalances into bone marrow CD34+ cells as they differentiate towards the erythroid and granulomonocytic lineages to elucidate the lineage-specific effect of S34F.Our results indicate that U2AF1 S34F mutant alters erythroid and granulomonocytic differentiation by inducing lineage-specific aberrant splicing patterns, providing new insights into the molecular pathogenesis of U2AF1 mutant MDS. DisclosuresNo relevant conflicts of interest to declare.

More Efficient Generation of β-Globin-Expressing Erythroid Cells Using Stromal Cell-Derived Induced Pluripotent Stem Cells

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. Using traditional embryoid body methods, iPS cell-derived erythroid cells predominantly produce ε-globin and γ-globin without β-globin expression. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with β-globin production, which is associated with emergence of CD34+ hematopoietic stem/progenitor cells (HSPCs) (2014 ASH). In the current study, we extend this work to evaluate erythroid cell generation using iPS cell lines generated from various sources including patients with sickle cell disease (SCD).To test our two hypotheses; (1) erythroid progenitor (EP)-derived iPS cells more efficiently differentiate to erythroid cells, and (2) stromal cell (ST)-derived iPS cells more efficiently emerge hemangioblast-like immature HSPCs which results in greater erythroid cell generation, we generated several clones of iPS cells which were derived from (1) EPs (6 clones) which were differentiated from peripheral blood mononuclear cells and (2) bone marrow STs (5 clones) in SCD patients. Transgene-free iPS cells were generated and characterized according to Merling et al. (Blood. 2013). These iPS cells and controls (2 clones of fibroblast (FB)-derived iPS cells and H1 ES cells) were used to generate ES/iPS sacs for 15 days. After a 2 day culture of ES/iPS sac-derived spherical cells on OP9 feeder cells, the suspension cells were differentiated into erythroid cells using human erythroid massive amplification culture for 13 days (Blood cells Mol Dis. 2002).Following ES/iPS sac generation, 3.5-4.8 fold greater amounts of CD34+CD45+ HSPCs emerged in both EP- and ST-derived iPS sacs, compared to FB-derived iPS sacs (p<0.01). After an additional 2 weeks of erythroid differentiation, we observed 4.5-8.7 fold greater amounts of GPA+ erythroid cells from both EP- and SC-derived iPS sacs, compared to FB-derived iPS sacs (p<0.01). Interestingly, ST-derived iPS sacs resulted in 1.4-2.0 fold greater amounts of CD34+CD45+ HSPCs and GPA+ erythroid cells (p<0.01), compared to EP-derived iPS sacs. Higher β-globin expression (21.5±4.3%) was observed by RT-qPCR in erythroid cells from ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (4.4±2.5% and 8.3±4.2%, respectively, p<0.01), which was comparable to ES sacs (23.3%). Sickle hemoglobin was detected by hemoglobin electrophoresis. The ES/iPS sac-derived erythroid cell generation was more strongly affected by cell sources (5-6 fold larger SD) than variations among iPS cell clones. These data demonstrate that ST-derived iPS sacs allow more efficient erythroid cell generation with higher β-globin production, compared to EP- and FB-derived iPS sacs.We hypothesized that ST-derived iPS sacs contain greater amounts of immature HSPCs (including hemogenic endothelium) and immature EPs (including megakaryoerythroid progenitors), since more expansion of ST-derived cells was observed during the late phase of erythroid differentiation, compared to EP- and FB-derived cells. We evaluated hemogenic endothelium markers at day 15, and observed 7.7 fold greater amounts of VEGFR+GPA- cells (p<0.01) and 1.3-1.4 fold greater amounts of CD31+CD34+ cells in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (not detectable VEGFR+GPA- cells in EP-derived iPS sacs). Before erythroid differentiation, 3.2-16.4 fold greater amounts of GPA+CD41a+ megakaryoerythroid progenitors were observed in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (p<0.05). In colony forming unit assays, 1.8-5.0 fold greater amounts of myeloid and erythroid colonies were observed in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (p<0.01). These data suggest that ST-derived iPS sacs more efficiently produce immature HSPCs and immature EPs, which may result in more efficient generation of erythroid cells with β-globin production.In summary, we demonstrated that human ST-derived iPS sacs allow for more efficient erythroid cell generation with higher β-globin production, which could be caused by heightened emergence of hemogenic endothelium in ST-derived iPS sacs. Our findings should be important for in in vitro iPS cell-derived erythroid cell generation with high β-globin expression. DisclosuresWinkler:Novartis: Research Funding; GSK: Research Funding.

A Novel Erythroid Regulator MLF1IP Promotes Proliferation in Mice By Regulating Cell Cycle

Purpose: MLF1IP is a novel constitutive kinetochore component essential in physiological progress of cell cycle. Mouse homologue appears to be lineage-specifically expressed, but little is known about how it functions in hematopoiesis and whether it is involved in hematopoietic disorders. The goal of the study was to confirm biological function of MLF1IP in human K562 erythroleukemic cells, analyzed hematological abnormalities in MLF1IP transgenic mice and potential mechanisms underlying these abnormalities, and evaluated MLF1IP expression in erythroid colonies from PV and MDS.Methods: MLF1IP was down-regulated by lentivirus in K562 cell line. The proliferation of K562 cells was analyzed by cell colony formation assay. VP16 was used to induce apoptosis of K562 cells. The entire coding region of human MLF1IP was cloned into the HS21/45-vav plasmid to generate transgenic mice, which were crossed with wild-type C57BL/6J mice, negative siblings of transgenic mice were used as controls. Genotypes of the transgenic mice were identified by PCR of tail genomic DNA with primers specific for transgenic MLF1IP sequence. Relative quantitative real-time PCR was used to detect mRNA level in BM cells. Western blot were carried out to check the expression of cell cycle associated proteins. Flow cytometry analyses were used to assess cell cycles and proportions of different stages of erythroblast populations. Absolute quantitative real-time PCR were used to examine MLF1IP expression in colonies from healthy donors and subjects.Results: MLF1IP knock-down K562 cells are arrested in G2/M and have reduced colony-forming activity. MLF1IP knockdown had no detectable effect on apoptosis in K562 cells, while significantly enhanced etoposide-induced apoptosis was seen in MLF1IP-knockdown K562 cells at 48 h after treatment compared with that in same treated control cells. MLF1IP transgenic mice show increased hemoglobin, with normal levels of platelets and neutrophils. Erythroid progenitors and early-stage erythroblasts in bone marrow are markedly increased. These mice have increased cyclin D2, reduced p27 and p21 and increased cycling, suggesting that enhanced MLF1IP expression leads to erythroid expansion via accelerating cell cycle progression in bone marrow cells. Interestingly, MLF1IP expression in colony forming unit-erythropoiesis (CFU-E) is increased in polycythemia vera (PV) and decreased in low-risk myelodysplastic syndrome (MDS), consistent with erythropoiesis characteristics of these disorders.Conclusion: Our data demonstrate that MLF1IP expression promotes erythroid proliferation via regulating cell cycle status of erythroid cells, implicating abnormal MLF1IP levels in erythropoiesis disorders. Correction of MLF1IP expression could be a novel therapy target in diverse dysfunctional erythropoiesis conditions. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Analysis of Linsitinib for the Treatment of Polycythemia Vera

Even after the discovery of JAK2V617F in majority of Polycythemia Vera (PV) patients and the generation of JAK2 inhibitors, the treatment of PV remains to be improved. An increased understanding of aberrant signaling in PV cells may yield novel drug targets for this patient population. The formation of erythroid colonies in the absence of exogenous erythropoietin (Epo), so called Epo-independent erythroid colonies (EECs), is a pathognomonic hallmark of PV. Over 20 years ago, Axelrad and colleagues proposed that EEC formation is not due to Epo-independence. Rather, they postulated that PV cells are hypersensitive to Insulin Growth Factor-1 (IGF-1). Subsequently, increased IGF-1 receptor (IGF-1R) phosphorylation was demonstrated in PV MNCs. In addition, serum concentrations of IGF-1 binding protein (IGF-1BP) are significantly elevated in PV patients. It has been shown that IGF-1 cooperates with JAK2V617F to increase JAK/STAT signaling, elevating constitutive phosphorylation of STAT factors and thereby augmenting aberrant proliferation. We therefore hypothesized that inhibition of IGF-1 signaling would ameliorate the PV phenotype, even in the presence of JAK2V617F.The dual IGF-1R and insulin receptor (IR) kinase inhibitor linsitinib (formerly called OSI-906) was used to investigate the contribution of the IGF-1 pathway to PV pathophysiology. In methylcellulose colony assays, linsitinib significantly reduced the EEC formation from PV MNCs. In contrast, in the presence of EPO, colony formation from either PV or healthy control MNCs was not affected. These data suggest that linsitinib specifically inhibits growth of the PV clone. Linsitinib likewise significantly reduced the cytokine independent proliferation of Ba/F3 cells expressing JAK2V617F but did not affect proliferation of Ba/F3 cells expressing either JAKWT or JAK2V617F in the presence of cytokine (mIL3). These data again argue that IGF-1 signalling is required for aberrant, cytokine independent growth. In addition to reducing proliferation, Linsitinib also induced apoptosis in IL3-independent JAK2V617F transduced Ba/F3 cells. Using the JAK2V617F bone marrow transplant (BMT) model, we investigated the effect of linsitinib on the PV phenotype in vivo. Linsitinib treatment significantly reduced the elevated RBC count, hematocrit and hemoglobin in this PV mouse model. In addition, the white blood cell count was decreased.The data presented here provide a pre-clinical rationale for expanding the investigation of linsitinib as well as similar IGF-1 pathway inhibitors for the treatment of PV. The effect of combining JAK2 and IGF-1 inhibition may prove especially promising in this patient population and is currently being investigated. DisclosuresNo relevant conflicts of interest to declare.

Colony-Forming Unit Cell (CFU-C) Assays in Myelodysplastic Syndrome

Introduction: The myelodysplastic syndromes (MDS) are a group of clonal diseases derived from hematopoietic stem cells (HSC). Colony-forming unit cell (CFU-C) assay is an effective method to study the number and the function of HSC in vitro. In this study, we focus on the characteristics and the prognostic value of CFU-C in patients with MDS.Patients and Method: CFU-C assays were performed according to the protocol of MethoCultTM H4435 Enriched (STEMCELL Technologies). A colony was defined as an aggregate of >40 cells. Clusters consisted of 4 to 40 cells. 560 consecutive newly-diagnosed, untreated subjects with MDS diagnosed from March, 2001 to April, 2013 were studied. All subjects were reclassified according to the 2008 WHO criteria. 535 subjects with evaluable cytogenetics were classified using the International Prognostic Scoring System (IPSS) and the revised International Prognostic Scoring System (IPSS-R) criteria. Follow-up data were available for 470£¨84%£©subjects. Median follow-up of survivors was 26 months (range, 1-170) months. Subjects receiving an allotransplants were censored in survival analyses. Erythroid and myeloid colonies were isolated from each subject with one cytogenetic abnormality such as del(5/5q-) or +8. Cytogenetic abnormalities of each colony were analyzed by fluorescence in situ hybridization (FISH). SPSS 17.0 software was used to make statistical analysis.Results: Frequencies of burst-forming units-erythroid (BFU-E), colony forming unit-erythroid (CFU-E) and colony forming unit-granulocytes/macrophages (CFU-G/M) were significantly lower than normals (P<0.05) (Table 1). Subjects classified as lower risk in IPSS and IPSS-R had significantly higher numbers of BFU-E and CFU-E (P<0.05) but similar numbers CFU-G/M and clusters-G/M compared with higher risk subjects (Table 2). In 11 subjects with del(-5/5q-) or +8 identified by G- and/or R-banding, both normal and abnormal CFU-Cs were identified in 8 subjects studied by FISH. A high ratio of cluster- to CFU-G/M (>0.6) was associated with poor-risk cytogenetics (Table 2) and with worse overall survival in univariable (Figure 1, P=0.001) and multivariable analyses (HR 1.748, [1.01-3.0]; P=0.046) after adjusting for IPSS.Conclusions: These data suggest abnormalities of proliferation and differentiation of erythroid and myeloid precursor cells in vitro parallel the ineffective hematopoiesis typical of MDS and may be useful in predicting outcomes of patients with MDS.Table 1CFU-C in MDS subtypesNBFU-ECFU-ECFU-G/MNRatio of cluster- to CFU-G/MRA218 (0-44)40 (0-134)14 (0-127)160.25 (0.40-1.00)RT418 (4-55)75 (60-90)130 (18-70)122RARS2712 (0-33)35 (1-140)12 (0-70)1100.45 (0.17-0.80)RCMD27510 (0-80)33 (0-178)14 (0-100)1260.35 (0-0.83)RAEB111210 (0-258)32 (0-312)14 (0-89)530.47 (0-1.00)RAEB21039 (0-46)25 (0-120)13 (0-72)420.37 (0-1.00)MDS-U154 (0-58)25 (0-161)10 (0-43)32Del(5q)32 (2-4)15 (0-20)5 (5-41)1121No significant difference compared with normals.2Too few cases to analyze.Table 2Associations between CFU-C and clinical and laboratory variablesNBFU-EPCFU-EPCFU-G/MPNumberRatio of cluster- to CFU-GMPIPSS0.0640.0060.3610.089Low3013 (0-44)60 (0-169)19 (0-45)100.44 (0.24-0.70)Int-136110 (0-258)33 (0-312)14 (0-127)1500.33 (0-1.00)Int-21159 (0-61)30 (0-137)14 (0-72)520.45 (0-1.00)High297 (0-34)21 (0-93)12 (0-67)120.44 (0-1.00)IPSS-R0.0030.0030.1250.209Very low716 (9-25)30 (15-120)18 (5-33)20.29 (0.10-0.49)Low13014 (0-80)42 (0-178)17 (0-70)480.31 (0-0.77)Intermediate17310 (0-66)34 (0-161)13 (0-127)810.37 (0-1.00)High1399 (0-259)29 (0-312)11 (0-89)510.33 (0-1.00)Very high868 (0-61)25 (0-137)14 (0-91)420.47 (0-1.00)Cytogenetics (IPSS)0.8670.0550.2900.007Good32710 (0-258)36 (0-312)15 (0-89)1330.33 (0-1.00)Intermediate13310 (0-69)30 (0-162)12 (0-127)630.45 (0-1.00)Poor7510 (0-61)25 (0-137)14 (0-91)280.42 (0-1.00)Cytogenetics (IPSS-R)0.9900.0900.6760.022Very good711 (4-20)48 (1-110)14 (8-28)20.49 (0.43-0.56)Good32410 (0-258)35 (0-312)15 (0-89)1320.33 (0-1.00)Intermediate12910 (0-69)30 (0-162)12 (0-127)620.45 (0-1.00)Poor2710 (0-61)35 (0-137)16 (0-48)80.36 (0.15-1.00)Very poor4811 (0-42)22 (0-120)14 (0-91)200.53 (0-1.00) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MTH1 Inhibitor-Induced Cytotoxicity in Acute Myeloid Leukemia

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive leukemia with 5-year overall survival of 20-25%. The major reason for treatment failure in AML is resistance to chemotherapy. Thus, there is an urgent need for identification of novel therapeutic agents for AML.Neoplastic cells, including AML, have dysfunctional redox regulation that results in increased reactive oxygen species (ROS). Accumulation of ROS leads to oxidation of free and incorporated nucleotides, leading to DNA damage and cell death. MTH1 is a nudix family hydrolase that sanitizes the oxidized nucleotide pool to prevent incorporation of these damaged bases in the DNA. MTH1 is thought to be non-essential for normal cells but crucial for neoplastic cells in order to avoid incorporation of oxidized dNTPs into DNA, thereby evading DNA damage and cell death. Whether MTH1 inhibitors have any activity against AML is not known.METHODS: Neoplastic myeloid cell lines HL-60, HEL, K562, KG1A, ML1, MV-4-11, SET2, and U937 were treated with varying concentrations of TH588 for a total of 48 hours. In experiments using the pan-caspase inhibitor Q-VD-OPh (Qvd), cells were pre-treated with 5µM Qvd for 1 hour followed by TH588. Cells were washed and stained with annexin, propidium iodide (PI), or MitoTracker (Life Technologies, Carlsbad, CA) for flow cytometry. To evaluate the potential impact of MTH1 inhibition on chemorefractory AML, HL-60/VCR cells were treated with vehicle control or TH588 in culture medium with or without vincristine (1µg/ml). Percentage apoptosis was calculated by normalizing to vehicle only control.With IRB approval, bone marrow aspirate samples were obtained from patients with untreated AML or healthy controls. Mononuclear cells were analyzed using colony-forming unit (CFU) assays. The total number of erythroid (CFU-E) and myeloid (CFU-G, CFU-GM) colonies containing ≥50 cells were read on day 14 and reported as percentage colonies compared to vehicle control.RESULTS: TH588 induced dose-dependent cell death in each of the neoplastic cell lines tested except HEL. In particular, treatment with TH588 resulted in a dose-dependent increase in the number of cells undergoing apoptosis as indicated by annexin V and/or PI staining (IC50 3.1-21.3µM, Figure 1). Pre-treatment with Qvd significantly inhibited TH588-induced cell death in all the cell lines studied except KG1A and SET2, suggesting a caspase-dependent mechanism of cell death. In further studies, cells treated with TH588 exhibited decreased MitoTracker staining; and Qvd pretreatment increased the number of MitoTrackerLow cells at the same time apoptotic cells decreased, suggesting that mitochondrial damage is upstream of caspase activation in TH588-induced apoptosis. Treatment with TH588 not only induced apoptosis in HL-60/VCR cells, but also facilitated further apoptosis in cells co-treated with vincristine and TH588 (Figure 2). Treatment with TH588 also diminished colony formation in a primary AML sample (IC50 6µM, Figure 3). Analysis of additional primary AML samples is ongoing.DISCUSSION: Our results show that the MTH1 inhibitor TH588 induces apoptosis in most neoplastic myeloid cells. MTH1 causes mitochondrial damage that, in turn, leads to caspase-dependent apoptosis in these cells. In HL-60/VCR cells representing chemorefractory phenotype, TH588 induces apoptosis as a single agent and resensitizes cells to vincristine. Moreover, TH588 significantly diminished colony formation in primary AML ex vivo. Further preclinical and possible clinical study of this class of agent appears warranted. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HH-002 Effectively Suppresses Myeloproliferative Neoplasm Phenotypes in JAK2V617F Transgenic Mice

Ph-negative myeloproliferative neoplasms (MPNs) represent a group of conditions characterized by chronic increases in some or all of the blood cells (platelets, white blood cells, and red blood cells). A major molecular defect in MPNs is JAK2V617F, a gain-of-function mutant form of tyrosine kinase JAK2 found in the majority of MPN patients. In earlier studies, we generated JAK2V617F transgenic mice by using the vav promoter which drives gene expression in all hematopoietic cells. These mice develop MPN-like phenotypes in a transgene dose-dependent manner. The objective of this study is to use this animal MPN model to test the efficacy of HH-002, a derivative of natural plant alkaloid homoharringtonine.Treatment of JAK2V617F transgenic mice with a daily dose of 1 mg/kg HH-002 through subcutaneous injection reduced blood cell counts to the normal range or slightly below the normal level. HH-002 causes a preferential reduction of myeloid cells in JAK2V617F transgenic mice since percentages of lymphocytes (CD3e+ T cells and CD19+ B cells) were increased (despite somewhat decreases in absolute numbers) while the percentage of Gr-1+/CD11b+ granulocytes sharply declined. HH-002 also effectively reduced the spleen size of JAK2V617F transgenic mice and prevented development of myelofibrosis. With a JAK2V617F bone marrow transplant mouse model, HH-002 showed a similar effect. Although it did not increase the ratio of JAK2V617F-negative cells to JAK2V617F-positive, it stopped further expansion of JAK2V617F-containing malignant cells in JAK2V617F bone marrow recipient mice. In vitro experiments with primary hematopoietic cells demonstrated that HH-002 potently inhibited formation of erythroid and myeloid colonies with an IC50 value of 1-3 nM. However, it does not show a preferential inhibition of JAK2V617F-containing cells. This is not unexpected since homoharringtonine is known to inhibit protein synthesis. Taken together, HH-002 is a promising candidate for development of therapeutic drugs to treat MPNs and related diseases. Combination drug therapies with JAK2 inhibitors need to be explored. DisclosuresHallenbeck:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Employment, Equity Ownership. Rong:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Employment, Equity Ownership. Zhao:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Research Funding.

Critical Role of SHP2 in the Initiation and Maintenance of Myeloproliferative Neoplasms Evoked By JAK2V617F

The JAK2V617F mutation has been found in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of the JAK2 tyrosine kinase and its downstream signaling pathways. JAK2 inhibitor therapy can reduce splenomegaly and constitutional symptoms but is not sufficient to cure or produce complete remission in patients with MPNs. Identification of critical signaling pathways/molecules required for the initiation and maintenance of MPNs will facilitate the development of more effective targeted therapies.To determine the role of JAK2V617F mutation in MPNs, we previously generated a conditional Jak2V617F knock-in mouse. We observed that expression of heterozygous Jak2V617F in mice hematopoietic compartments is sufficient to induce a PV-like MPN, whereas homozygous Jak2V617F expression accelerates the development of myelofibrosis. We have found that SHP2, a protein tyrosine phosphatase that positively regulates hematopoietic signaling, is constitutively phosphorylated in mouse and human hematopoietic cells expressing JAK2V617F. However, the contribution of SHP2 in the pathogenesis of MPNs induced by JAK2V617F remains elusive. Here, we sought to determine the role of SHP2 in JAK2V617F-evoked MPNs using conditional SHP2 knockout (SHP2 floxed) and Jak2V617F knock-in mice and MxCre line.Whereas expression of heterozygous Jak2V617F induced a PV-like MPN characterized by increase in red blood cells (RBC), white blood cells (WBC), neutrophils and platelets in the peripheral blood and splenomegaly, deletion of SHP2 normalized the blood parameters and spleen size in Jak2V617F knock-in mice. Flow cytometric analysis showed that deletion of SHP2 inhibited the expansion of erythroid, megakaryocytic, and granulocytic precursors in the bone marrow (BM) and spleens of Jak2V617F mice. Deletion of SHP2 also inhibited the expansion of hematopoietic stem cells and megakaryocyte-erythroid progenitors in the BM and spleens of Jak2V617F knock-in mice. Furthermore, deletion of SHP2 markedly inhibited the erythropoietin (Epo)-independent CFU-E colonies in the BM and spleens of Jak2V617F mice.In order to determine whether the effects of SHP2 deletion in Jak2V617F mice were cell autonomous, BM cells from uninduced control, MxCre;V617F/+ and MxCre;V617F/+;SHP2fl/fl mice were transplanted into lethally irradiated syngeneic recipient mice. Four weeks after transplantation, mice were injected with pI-pC to induce deletion of SHP2 and expression of Jak2V617F simultaneously. Transplanted animals receiving MxCre;V617F/+ BM developed a PV-like disease within 6 weeks after pI-pC induction. However, recipients of MxCre;V617F/+;SHP2fl/fl BM failed to induce the PV disease due to deletion of SHP2. Together, these data suggest that SHP2 is required for the initiation of PV-like MPN mediated by Jak2V617F.To determine if SHP2 is required for the maintenance of MPN evoked by Jak2V617F, uninduced wild type or MxCre;SHP2fl/fl mice BM cells were transduced with retroviruses expressing Jak2V617F and transplanted into lethally irradiated syngeneic recipient animals. Five weeks after transplantation, we assessed the peripheral blood counts to confirm the development of MPN disease. Transplanted animals receiving Jak2V617F-transduced wild type or MxCre;SHP2fl/fl mice BM were then divided into two groups- one group was injected with pI-pC (to induce deletion of SHP2 after establishment of the MPN disease); another group (control group) was injected with saline. We observed that deletion of SHP2 by pI-pC induction significantly inhibited the RBC, hematocrit, WBC and platelet counts in the peripheral blood and reduced the spleen size in transplanted animals expressing Jak2V617F. Moreover, deletion of SHP2 markedly inhibited the Epo-independent erythroid colonies in the BM and spleens of transplanted animals expressing Jak2V617F. Thus, SHP2 is not only required for the initiation of MPN but also required for the maintenance of MPN mediated by Jak2V617F. We have also observed that deletion of SHP2 blocks the development of myelofibrosis in Jak2V617F knock-in mice. In conclusion, our results suggest that SHP2 plays a critical role in the initiation and maintenance of MPNs evoked by Jak2V617F. DisclosuresNo relevant conflicts of interest to declare.