ALBERT

Description: Abstract 794FN2 LBH589 is a novel pan-deacetylase inhibitor (DACi) that has demonstrated clinical activity in phase I/II studies in patients with a variety of hematologic malignancies. Our group has previously presented preliminary results of a phase I study of LBH589 in patients with myelofibrosis (MF) (Mascarenhas et al, ASH 2009, a308) while a phase II trial using higher doses of LBH589 has also been reported (DeAngelo et al, ASH 2010,a630). Both studies identified reversible thrombocytopenia as the DLT and reported evidence of clinical responses. The final results of our phase I study and the effects of extended treatment with LBH589 are reported here. We enrolled 18 patients at 3 dose levels. Fifty-five percent of these patients had PMF, 28% Post-PV MF and 17% Post ET MF; all were intermediate/high risk based on Lille classification. Twenty-five mg PO TIW was determined to be the recommended phase II dose. All patients experienced resolution of their systemic symptoms and 10/11 patients with baseline palpable splenomegaly, who were evaluable after 1 month of therapy, had a median reduction of 30%, range 0–100%. Five patients entered into an extension phase of the trial and received 〉 6 months of therapy with a mean dose of 20mg PO TIW at time of optimal response (Table 1). Of these patients, 2 were initially enrolled in the 20 mg PO TIW cohort, 1 in the 30 mg PO TIW cohort and 2 in the 25 mg PO TIW cohort. Both patients at the lowest dose achieved clinical improvement (CI) by IWG-MRT response criteria at 6 months as did one patient at the 25 mg dose. The remaining 2 patients had SD at 30 and 25 mg. A mean reduction in palpable splenomegaly at 3 and 6 months of 55% and 83%, respectively, was observed in this group. Two of these patients had marked and durable improvement in anemia (patients 1 and 4). Patient 4 achieved a near CR at 16 months with resolution of palpable splenomegaly, elimination of peripheral blood dacrocytes and leukoerythroblastosis, a 4g/dL increase in hemoglobin, improvement in overall marrow cellularity and megakaryocyte atypia with an increase in erythroid precursors and a significant reduction of reticulin/collagen fibrosis. Patient 1 was heavily transfusion dependent requiring RBC transfusions weekly to maintain a mean hemoglobin of 6.5g/dL and after 6 months on LBH589 achieved 〉50% reduction in transfusion dependence maintaining a mean hemoglobin of 9g/dL. Patient 2 had resolution of palpable splenomegaly and leukoerythroblastosis by cycle 6 and the bone marrow at cycle 26 was characterized by a reduction in marrow fibrosis from grade 4 to 1. A phase II study is ongoing, 14 patients are currently enrolled, with a planned goal of 22 patients. Pharmacokinetic and pharmacodynamic studies as well as cytokine profiling of the phase I patients are being analyzed and will be presented at the meeting. We conclude that low doses of LBH589 delivered for greater than 6 months in patients with MF are capable of ameliorating symptoms, improving clinical features and reversing pathologic marrow changes. Disclosures: Off Label Use: Oral histone deacetylase inhibitor that targets epigenetic changes in malignant myelofibrosis cells with an goal to modify the disease process.

Description: Abstract 4968 Elucidation of the molecular aberrations underlying the development of myeloproliferative neoplasms (MPNs) has progressed rapidly during the previous years. In addition to the JAK2V617F mutation, which is found in over 90% of patients with polycythemia vera (PV) and in around 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF), several novel markers have recently been described. These include mutations in the Ten-Eleven-Translocation-2 (TET-2) gene, as well as overexpression of the hematopoietic transcription factor nuclear factor erythroid-2 (NF-E2). While the individual frequency of these abnormalities is well known, so far, studies that evaluate the relationship between these markers and their correlation with hematological parameters have not been conducted. The International Myeloproliferative Research Consortium (MPD-RC) has instituted a Tissue Bank (MPD-RC Trial 106) allowing the collection of MPN patient samples from member institutions in the US and Europe. Using this resource, we have investigated the relationship among the following molecular markers and hematological parameters in a series of 66 MPN patients: TET-2 mRNA expression, JAK2V617F allele burden, NF-E2 mRNA expression, granulocyte clonality (female patients) as well as hematocrit, hemoglobin concentration, platelet numbers and leukocyte counts. Our crossectional cohort included 37 PV patients, 14 ET, 4 PMF and one post PV MF patient. Sixtyeight percent of the patients were treated, medication including hydroxyurea, anagrelide, ASA, interferon and one patient treated with imatinib mesylate. We acknowledge that treatment may affect the molecular markers being investigated. In addition, the desired effect of treatment on hematological parameters may not be paralleled by a similar effect on molecular markers. Therefore, investigation of treated patients may not reveal biological relationships present in untreated diseases states. We thus tested the effect of cytoreductive treatment on the expression of molecular markers by comparing treated and untreated patients in our cohort. The JAK2V617F allele burden was significantly lower in treated patients than in untreated patients (p = 0.008). The same difference was noted when PV patients were analyzed alone (p = 0.006) In contrast, neither TET-2 nor NF-E2 mRNA expression were affected by treatment. In the 66 MPN patients evaluated, a significant inverse Spearman correlation of -0.25 was noted between NF-E2 mRNA expression and hemoglobin concentration (p = 0.05). This relationship was more pronounced when PV patients were analyzed alone (Spearman's r = -0.41, p = 0.01). In addition, a correlation of 0.51 between JAK2V617F allele burden and NF-E2 mRNA expression was noted (p = 0.0007). Occurrence of the recently discovered TET-2 gene mutations, which are present in around 15% of MPN patients, was measured indirectly by determining TET-2 mRNA expression. None of the other markers and parameters assessed was significantly correlated with the amount of TET-2 mRNA expressed. We report a previously unrecognized inverse relationship between NF-E2 mRNA expression and hemoglobin concentration. These data complement several recent findings in MPN patients. While NF-E2 is overexpressed in granulocytes of all three MPN subtypes, PV, ET and PMF, its overexpression in CD34+ hematopoietic stem cells has so far only been observed in PMF. We have recently shown that ex vivo overexpression of NF-E2 in CD34+ hematopoietic stem cells drastically reduces erythroid colony formation. It was previously noted that mean JAK2V617F allele burdens in PMF patients are higher than in PV or ET. Here, we confirm the previously noted positive correlation between JAK2V617F allele burden and NF-E2 mRNA expression. Our data therefore suggest that high levels of JAK2V617F in PMF patients directly or indirectly augment NF-E2 overexpression, which may contribute to the anemia of Primary Myelofibrosis. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1907 Poster Board I-930 Primary myelofibrosis (PMF) is characterized by abnormalities of megakaryocyte maturation, bone marrow fibrosis, increased angiogenesis, bone formation and extramedullary hematopoiesis. Patients are often anemic and thrombocytopenic and, as patients with other myeloproliferative disorders, have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles (PMP) and/or to pathological platelet-neutrophil interactions. The megakaryocytes of these patients, in fact, express normal levels of P-selectin (P-sel, an adhesion receptor that mediates interaction with neutrophils) that remains abnormally localized to the demarcation membrane system (DMS) rather than being assembled into the a-granules. Mice carrying the hypomorphic Gata1low mutation (deletion of the regulatory sequences that drive Gata1 expression in megakaryocytes) express the same megakaryocyte abnormalities presented by PMF patients, including abnormal P-sel localization to the DMS and develop with age myelofibrosis, a disease that closely resembles PMF. Whether these mice would also develop thrombosis has not been investigated as yet. The aim of this study was to determine whether Gata1low mice would develop thrombosis with age and, in this case, the role played by P-sel in the development of the trait. To this aim, Gata1low mice were crossed with P-selnull mice (kindly provided by dr. P. Frenette) according to standard genetic protocols and Gata1lowP-selWT, Gata1lowP-selnull and Gata1WTP-selnull or Gata1WTP-selWT (as controls) littermates obtained. The hematocrit (Hct), blood platelet counts (ptl) and PMP counts were compared among the four experimental groups (Table 1).Table IHct, ptl and PMP in the blood of mice carrying the Gata1low and/or P-selnull mutation.*PMP were defined by FACS as anti-CD61pos events against the events of constant numbers of microbeads added to platelet poor plasma produced by serial centrifugation steps as calibrators. There were no significant differences among the four groups of mice with respect of Hct (Kruskal-Wallis nonparametric analysis of variance, 2-sided p=0.89) although there were differences among these groups with respect to platelet levels (Kruskal-Wallis 2-sided, p= 0.002). In particular, Gata1WT mice had significantly greater platelet levels than Gata1low mice regardless of P-sel (Wilcoxon rank test, p ≤0.0001, no adjustment for multiple comparisons). PMP were found to be reduced in Gata1low mice compared to Gata1WT littermates (Table I). Gata1WTP-selnull and Gata1low/P-selnull littermates had also reduced numbers of PMP. P-sel was only expressed by PMP from Gata1WTP-selWT mice. On the other hand, not only adult and old Gata1lowP-selWT mice but also Gata1WT/P-selnull and Gata1low/P-selnull littermates had bleeding times longer (

Description: Myelofibrosis (MF) is characterized by anemia, thrombocytopenia and marrow fibrosis. Malignant rather than normal hematopoietic progenitor cells (HPC) preferentially proliferate. A form of MF can also occur in patients with a prior history of PV or ET which is referred to as PV or ET related MF (PV-MF and ET-MF). In MF, excessive proliferation of marrow stromal cells occurs in response to cytokines elaborated by the malignant clone. Previously, we found that lipocalin-2 (LCN2) levels were elevated in patient plasmas with MPN, and a greatest degree of elevation was observed in the PMF plasma and PMF conditioned medium. We also found that LCN2 increased PMF HPC proliferation and increased numbers of JAK2V617F homozygous HPCs, but depressed normal BM HPC (Lu. et al, 2013 ASH meeting). In this study, a series of experiments were performed to further explore the effects of LCN2 on MF pathogenesis. ELISA data showed that plasma LCN2 levels not only were higher in all MF patients, plasma LCN2 levels in PV-MF as well as ET- MF were significantly greater than that in PV or ET plasma (p=0.0002 and p=0.0175, respectively). Importantly, in MPN patients, a non-parametric spearman correlation coefficient analysis suggested an inverse relationship between the patients’ hemoglobin levels and LCN2 levels (coefficient=-0.35, p

Description: Abstract 754 Introduction: The identification of mutations in JAK2 in myeloproliferative neoplasms (MPN) has led to the hypothesis that tyrosine kinase inhibitors will have therapeutic benefit. CEP701 (lestaurtinib, Cephalon Oncology), an orally available multikinase inhibitor, has been tested extensively in acute myeloid leukemia (AML) as a FLT3 inhibitor. CEP701 also inhibits JAK2 with an in vitro IC50 of 1 nM, and selectively inhibits primary cells from patients with MPN. Limitations of the drug in AML studies (doses ranging from 60 to 100 mg BID) have included gastrointestinal toxicity, low free drug levels related to in vivo plasma protein binding, and, as a consequence, incomplete target inhibition. Further, a maximum tolerated dose (MTD) for CEP701 has not yet been reached. Methods: The Myeloproliferative Disorders Research Consortium (MPD-RC) is conducting a multicenter, open label dose escalation phase I study of a new, capsule formulation of CEP701 in subjects with myelofibrosis (primary or evolved from polycythemia vera or essential thrombocytosis) who have the JAK2 V617F mutation. Patients are entered into cohorts in a classic 3+3 design with CEP701 administered orally at BID dosing of 80mg, 100mg, 120mg, 140mg, and 160mg for a minimum of 28 consecutive days. In the absence of a dose limiting toxicity, patients may continue treatment for 6 months, with the option to extend if responding. Results: Four of five dose levels from 80 mg – 160 mg PO BID have been tested. 80 mg and 100 mg were tested using the older liquid formulation; subsequent cohorts (100 mg – 160 mg) have or will be treated with the capsule formulation. No MTD has been reached at doses up to 140 mg; the final dosing cohort is accruing at the time of this submission (full data will be available from the phase I for reporting at the time of the meeting). Of 19 patients enrolled, 5 (26%) are females, 68% have primary myelofibrosis and the remaining patients have post-PV or post-ET myelofibrosis; 26% have had a prior thrombotic event and 47% a prior hemorrhagic event. At baseline 12 of 19 patients had at least 1 adverse prognostic factor by the Lille classification system. 9 patients (47%) of 19 available had a normal karyotype at baseline; the remaining had at least one cytogenetic abnormality. 7 of 19 patients continue to receive study drug with one subject treated for more than 1 year (range 1 to 54+ weeks). There were 2 drug-attributable grade 3/4 non-hematologic toxicities: 2 subjects receiving the 100 mg liquid formulation experienced grade 3 diarrhea. Diarrhea was the most common grade ≥2 adverse event, affecting 5 of the 7 subjects treated with the liquid formulation and 2 of 12 subjects treated with the capsule formulation (both at the 140 mg dose level). Nausea was also common, with 2/7 (liquid) and 2/12 (capsule) patients experiencing grade 2 toxicity. There was one death in a patient at the 100mg (liquid) cohort, who died from hepatic failure following surgery in the setting of cirrhosis, deemed to be unrelated to CEP701. All patients had splenomegaly at baseline with spleen sizes ranging from 2 cm – 30 cm below the left costal margin (mean of 16.7. s.d., 7.6). Spleen size reduced markedly from baseline through 20 weeks (linear mixed models, p 120 mg has reached 12 weeks of treatment. The median baseline JAK2 T% (mutant) in peripheral blood granulocytes was 66.4% (range 16.2 – 99.6%). In the 5 patients with measurements at baseline and at either 12 or 24 weeks on study (last on study visit), JAK2 T% was markedly decreased (one sample Wilcoxon rank sum test, p – 0.06, 2-sided), with a median of 49.6% (range 34-96) at last on study observation; the median change from baseline to their last study observation was 8.6% (range 0.2 – 40.3). Taken together, these data demonstrate that CEP701 administered as a capsule formulation is safe at dose levels higher than those previously tested. CEP701 treatment decreases JAK2 V617F allelic burden and decreases spleen size, suggesting both biologic and clinical activity. Disclosures: Carroll: Sanofi Aventis Corp: Research Funding; Cephalon Oncoloy: Consultancy.

Description: Abstract 1750 The Myeloproliferative Disorder-Research Consortium (MPD-RC) designed the first US prospective phase II study of allogeneic hematopoietic stem cell transplantation (HSCT) in patients with primary myelofibrosis (PMF) or MF secondary to essential thrombocythemia (ET-MF) or polycythemia vera (PV-MF). Between May 2007 and March 2011, 66 patients were entered into the MPD-RC 101 study. Thirty-two patients received an allogeneic HSCT from a related and 34 patients from an unrelated donor. In the two groups diagnoses were: PMF: 44%, 74%; ET-MF: 47%, 12% and PV-MF: 9%, 15%, respectively. A reduced intensity regimen with fludarabine/melphalan (FluMel) ± ATG was used. Of 66 patients, 63 were at intermediate/high risk according to Lille score system and 3 patients were at low risk but had thrombocytopenia. Recipients of related and unrelated HSCT were comparable with respect to age (median: 54 vs 55 years), gender (M/F 19/13 vs 19/15), Lille score intermediate (63% vs 68%) or high risk (28% vs 32%) and time from diagnosis to transplant (median, range: 16, 1–247 vs 20, 2–341 months). At the time of transplant, 66% and 82% of patients in the related and unrelated cohorts had splenomegaly and 19% and 15% had previous splenectomy. Jak-2 V617F mutation was present in 38% and 50% and an abnormal karyotype was detected in 56% and 59% of patients with known status, respectively. Bone marrow stem cells were utilized in 19% of related and 9% of unrelated transplants, while in the remaining cases patients received peripheral blood stem cell (PBSC) grafts. Donor compatibility was assessed by molecular typing for HLA A, B, C, DRB1 and DQB1 antigens; inclusion criteria allowed enrollment with maximum 1 antigen mismatched donor. Thirty of 32 transplants (94%) in the related cohort and 25/36 (74%) in the unrelated cohort were fully matched. In transplants from related donor, engraftment of neutrophils and platelets occurred in 31/32 patients, One patient in this group experienced a secondary graft failure. In contrast, among patients in the unrelated group only 26/34 (76%) patients engrafted and of these 4 had a secondary graft failure post transplant. Median time to neutrophils 〉 0.5 × 109/L and platelets 〉20 × 109/L engraftment was: day 22 and day 28 in the related, and day 18 and day 28 in the unrelated cohort, respectively. Acute GVHD grade II-IV was observed in 37% related (grade III-IV: 12%) and in 42% unrelated transplants (grade III-IV: 21%). Based on the International Working Group criteria, in patients who were followed for at least 6 months there were 7 CR, 8 PR, and 11 CI among the 28 patients in the related group, and 5 CR, 1 PR, and 5 CI among the 16 patients in the unrelated group. After a median follow-up for survivors of 24 months, 25 patients (78%) in the related group are alive, 6 patients (18%) died for causes related to transplant (GVHD n=3, cardiac toxicity n=1, renal failure n=1, secondary cancer n=1) and 1 (3%) for progression of disease. In the unrelated group, 15 patients (44%) are alive at 12 months follow-up for survivors, 18 patients (53%) died for causes related to transplant and 1 patient (3%) due to relapse. Median survival time has not been reached in the related transplant group and is 7 months in the unrelated group (hazard ratio 4.2, 95% CI: 1.7–10.1, p

Description: Key Points A high survival rate was seen in primary or secondary MF patients transplanted from matched related donors using the FluMel regimen. FluMel plus ATG in HSCT from unrelated donors for MF patients is associated with an increased risk of graft failure.

Description: Key Points Abnormal signatures in TGF-β1 signaling gene expression were identified in spleen and marrow from the Gata1low model of MF. These signatures include abnormalities in individual gene (Id2, Stat1, mTOR) in spleen and of gene pathways (Smads and BMPs) in marrow.

Description: Introduction HU is the treatment of choice for patients (pts) with high risk ET/PV, however, PEG has been proposed as an alternative option due to its proposed potential to modify disease course. An interim analysis of MPN-RC 112 (Blood 2016 128:479;) did not reveal a difference in PR/CR rates between HU and PEG therapy after 12 months in the first evaluable 75 pts treated. Here we present the results and long-term follow-up of all pts participating in this pivotal study [NCT01259856]. Methods MPN-RC 112 was a randomized, open label, phase 3 clinical trial comparing HU and PEG in pts with high risk ET/PV. Pts were treated for up to 12 months to achieve PR or CR (ELN/IWG-MRT response criteria). Pts who achieved a PR/CR continued therapy for up to a maximum of 6 years. Minimum follow up was 1 year from the time the last pt was randomized. The primary objective was to compare the CR rate following HU vs. PEG at 12 months with 3 month confirmation. Secondary objectives included a comparison of toxicity and tolerability; PR rates; incidence of specific pre-defined toxicities and tolerance to therapy; impact of therapy on key biomarkers; survival and incidence of myelodysplastic syndrome, myelofibrosis, or leukemic transformation; and incidence of major cardiovascular events. Bone marrow pathologic responses were evaluated by central blinded expert review at baseline, 12, 24 months and end of study. Results The study accrued 168 pts; 86 were randomized to HU and 82 to PEG. A summary of pt baseline characteristics by treatment arm is shown in Table 1 and were well balanced between the treatment arms except for median age which was higher in the HU arm (p=0.02). Median duration of follow up was 89.9 weeks (range, 0 to 292.3) and the median treatment duration was 86.0 weeks (range, 0 to 287.3). At 12 months, the overall response rate (ORR= PR+CR) was 69.8% and 78% for HU and PEG, respectively (p=0.22). Figure 1 shows the distribution of responses stratified by disease type. At 24 months, 59 pts were on treatment with an ORR of 22/25 (88%) for HU and 31/34 (91%) for PEG. When considering all 106 pts who were eligible to receive treatment for 24 months (due to study closure), the ORR was 22/54 (40.7%, PR: 11 (20.4%), CR: 11 (20.4%)) for HU and 31/52 (59.6%, (PR: 15 (28.9%), CR: 16 (30.8%)) for PEG, p=0.04. Best ORR at any time on study was seen in 70.9% and 81.7% of HU and PEG treated pts, respectively, p=0.10. The median maximum change from baseline spleen volume was -6% (-100.0 - +53.8) in 112 evaluable pts and was similar between arms, p=0.99. Bone marrow morphologic responses are shown in Table 2 and the best response (CR) seen at any time on study for ET treated with HU was 52% (12/23) vs PEG 32% (8/25) and for PV treated with HU 19% (6/31) vs PEG 6% (2/34). Cytogenetic analyses at diagnosis were available in 86% (144/168). An abnormal karyotype was seen in 15% (22/144). Five PV and one ET pt lost their chromosomal abnormalities: 3 after one year (HU=1, PEG=2) and three after two years of therapy (HU=2, PEG=1). AE information is available for 162 pts (HU: 80; PEG: 82) (Table 3). Six pts randomized to HU never received treatment due to study withdrawal prior to initiation of treatment. Sixty pts had a grade 3 or higher event [HU: 22 (27.5%) and PEG: 38 (46.3%)]. 28 PV pts had a grade 3/4 [HU: 10 (24.4%) and PEG: 18 (41.9%)]. Four pts had a grade 4/5 event [HU: 3 (7.3%) and PEG: 1 (2.3%)]. 32 ET pts had a grade 3/4 [HU:12 (30.8%) and PEG:20 (51.3%)]. Two pts had a grade 4/5 event [HU:1 (2.6%), PEG:1 (2.6%)]. Additional outcomes of interest on study include one death attributed to new diagnosis of lung cancer (HU:1), progression to myelofibrosis (HU:1), vertebral artery occlusion (HU:1), and cerebral vascular accident (PEG:1). Reasons for study discontinuation are shown in Table 4. The effect of therapy on symptom burden and quality of life will be presented in a companion abstract (Mesa et al). The impact of mutation status on therapeutic outcome as well as the molecular responses will be presented at the meeting. Conclusion The final analysis of MPN-RC 112 revealed that the CR rates in pts with high risk ET/PV treated with PEG and HU at 12 and 24 months were similar. PEG was associated with a higher rate of grade 3/4 toxicity. Each drug appeared equally capable of modifying the natural history of high risk ET/PV based upon their effects on spleen size, karyotypic abnormalities, histopathological parameters and the low incidence of thrombotic complications and disease evolution in both arms. Disclosures Mascarenhas: Novartis: Research Funding; Promedior: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Merck: Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding. Rambaldi:Amgen Inc.: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Italfarmaco: Consultancy; Omeros: Consultancy; Roche: Consultancy; Celgene: Consultancy. Harrison:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; CTI BioPharma: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Gilead: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau. Kiladjian:AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Mead:Bristol-Myers Squibb: Consultancy; Elstar: Research Funding; Celgene: Research Funding; ARIAD: Consultancy; Evotek: Research Funding; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Cell Therapeutics: Consultancy. Kessler:Genentech: Research Funding; Sangamo: Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biomarin: Research Funding; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; DSMB: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Research Funding; Dimension Advisory boards: Membership on an entity's Board of Directors or advisory committees. Kremyanskaya:Incyte: Research Funding. Rampal:Stemline: Research Funding; Incyte: Honoraria, Research Funding; Constellation: Research Funding; Jazz: Consultancy, Honoraria; Celgene: Honoraria. Mesa:CTI Biopharma: Research Funding; Novartis: Consultancy; Genentech: Research Funding; Promedior: Research Funding; UT Health San Antonio - Mays Cancer Center: Employment; Pfizer: Research Funding; Celgene: Research Funding; Incyte Corporation: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding. Dueck:Pfizer: Honoraria; Bayer: Employment; Phytogine: Employment. Hoffman:Incyte: Research Funding; Formation Biologics: Research Funding; Merus: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.

Description: Abstract 964 The JAK2 V617F mutation is primarily associated with three chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) but how a single mutation could be responsible for three different disorders is still unresolved. A gene dosage effect was proposed based on the MPD phenotypes in mice with differential expression of a JAK2 V617F transgene, where low expression correlated with an ET phenotype and high expression with a PV phenotype. However, quantitative studies of JAK2 V617F expression in humans revealed significant overlap between PV and ET. Since JAK2 is the cognate tyrosine kinase for the erythropoietin (EPO) and thrombopoietin (TPO) receptors, and JAK2 V617F is expressed in pluripotent hematopoietic stem cells, PV is the ultimate clinical phenotype of the mutation. Furthermore, TPO but not EPO promotes the survival and proliferation of pluripotent hematopoietic stem cells, suggesting that the TPO receptor (Mpl) is essential not only for generating thrombocytosis, but also the stem cell expansion that is characteristic of PV. To examine the role of Mpl in the genesis of the JAK2 V617F MPD phenotype, we manipulated the MPL genotype in a transgenic mouse expressing 13 copies of JAK2 V617F (V617Ftg) (Blood 111:5109, 2009) by breeding these mice with MPL knockout mice (Science265:1445, 1994), which are hematologically normal except for profound thrombocytopenia, to create three genotypes: V617Ftg/MPL wild type (wt); V617Ftg/MPL heterozygote (het), and V617Ftg/MPL knockout (ko). We compared the blood counts, spleen weights, plasma TPO levels, and bone marrow and spleen histology of these three genotypes with each other and with MPL wt, MPL het and MPL ko mice over a 33 week period. Crossbreeding gave the expected genotypes, JAK2 V617F transgene expression was stable in all groups, platelet Mpl expression by immunoblotting correlated with MPL genotype, there was no unexpected mortality, and body weights were not different for any of the genotypes during the observation period. As expected, in V617Ftg/MPL wt mice there was a robust and persistent thrombocytosis (2087 +/− 641 × 106/μL vs 1005 +/− 176 × 106/μL, p