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Introduction Essential thrombocythemia (ET) and polycythemia vera (PV) patients suffer from various symptoms that worsen quality of life (QOL), yet serial data on symptom changes resulting from therapy are sparse in the literature. Patient questionnaires from 2 large multicenter trials (MPN-RC 111, 112) were used to assess change in symptom burden and QOL over 12 months and impact of baseline symptom burden on subsequent change in ET / PV patients on hydroxyurea (HU) or pegylated interferon alfa-2a (PEG). Methods Trials MPN-RC 111 was a single-arm, open-label, phase II trial evaluating response to PEG in high-risk ET / PV patients with HU resistance/intolerance or splanchnic vein thrombosis (SVT; NCT01259817). MPN-RC 112 was a randomized, open-label, phase III trial comparing response to PEG versus HU in cytoreductive therapy naïve high-risk ET / PV patients diagnosed < 5 years ago (NCT01258856). Measures Patients reported disease-related symptoms via the validated Myeloproliferative Neoplasms Symptom Assessment Form (MPN-SAF), QOL via the European Organisation for the Research and Treatment of Cancer Core QOL Questionnaire (EORTC QLQ-C30), and (if applicable) PEG-related symptoms (flu-like symptoms, injection site irritation, blurry vision, vision change, flushing) at baseline, 3, 6, 9, and 12 months. Analysis Mixed models assessed mean changes from baseline in the MPN-SAF Total Symptom Score (TSS), MPN-SAF items, QOL, and PEG-related symptoms in MPN-RC 111, 112 PEG, and 112 HU patients. Mixed models also assessed the impact of baseline symptom burden (high [TSS ≥ 20] versus low) on subsequent change in PEG (MPN-RC 111 and 112) and HU patients. Results Patients Of the 135 enrolled MPN-RC 111 patients, 20 with SVT and 1 with no questionnaires were excluded. Of the remaining 114, 64 (56%) / 50 (44%) had ET / PV. Patients were 51% / 48% female. Median age was 65 / 64 years, and median time since diagnosis was 38 / 55 months. 31% / 22% had prior thrombosis, and 19% / 56% had splenomegaly. Of the 168 enrolled MPN-RC 112 patients (82 PEG, 86 HU), 2 with no questionnaires were excluded. Of the remaining 166, 79 (48%) / 87 (52%) had ET / PV. Patients were 50% / 33% female. Median age was 60 / 62 years, and median time since diagnosis was 3 / 3 months. 25% / 29% had prior thrombosis, and 11% / 37% had splenomegaly. Symptoms Questionnaire completion rates ranged from 90 - 99%, 87 - 100%, and 75 - 96% for on-treatment MPN-RC 111, 112 PEG, and 112 HU patients. At baseline, TSS (0 [absent] - 100 [worst imaginable]) and QOL (0 [very poor] - 100 [excellent]) means (SDs) were 19.5 (18.4) and 71.6 (20.1) for MPN-RC 111, 17.0 (13.6) and 67.9 (24.3) for MPN-RC 112 PEG, and 14.6 (11.4) and 73.8 (18.8) for MPN-RC 112 HU patients. On average, MPN-RC 111 patients had significant improvement of TSS, fatigue, abdominal pain, abdominal discomfort, dizziness, numbness, night sweats, and fever; MPN-RC 112 PEG patients had significant worsening of fever; and MPN-RC 112 HU patients had significant worsening of inactivity (no mean changes indicating improvement were observed). PEG patients had significant worsening of PEG-related symptoms. The greatest improvements occurred in the 46 (40%), 27 (33%), and 23 (28%) MPN-RC 111, 112 PEG, and 112 HU patients with high baseline symptom burden. On average, PEG patients with high baseline symptom burden had significant improvement of TSS, fatigue, early satiety, abdominal pain, abdominal discomfort, inactivity, headache, concentration, dizziness, numbness, insomnia, cough, night sweats, itching, bone pain, fever, weight loss, and QOL, while those with low baseline symptom burden had significant worsening of TSS, early satiety, headache, itching, and bone pain. On average, HU patients with high baseline symptom burden had significant improvement of TSS, early satiety, abdominal discomfort, headache, dizziness, numbness, insomnia, itching, and weight loss, while those with low baseline symptom burden had significant worsening of TSS, early satiety, abdominal discomfort, inactivity, concentration, and sexual desire/function (Figures 1 and 2). Conclusions Although no statistical comparisons were made across trials, overall improvements were seen in MPN-RC 111 but not 112. Patients with high baseline symptom burden experienced the greatest improvements in symptom burden and QOL during treatment with PEG or HU, which may explain the improvements seen in the more advanced patients in MPN-RC 111 compared to 112. Disclosures Mascarenhas: Celgene, Prelude, Galecto, Promedior, Geron, Constellation, and Incyte: Consultancy; Incyte, Kartos, Roche, Promedior, Merck, Merus, Arog, CTI Biopharma, Janssen, and PharmaEssentia: Other: Research funding (institution). Yacoub:Dynavax: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Cara Therapeutics: Current equity holder in publicly-traded company; Hylapharm: Current equity holder in private company; Incyte: Speakers Bureau; Agios: Honoraria, Speakers Bureau; Novartis: Speakers Bureau; Roche: Other: Support of parent study and funding of editorial support. Hoffman:Protagonist: Consultancy; Forbius: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding. Silver:PharmaEssentia: Speakers Bureau. Mesa:Bristol Myers Squibb: Research Funding; Incyte: Research Funding; AbbVie: Research Funding; Samus Therapeutics: Research Funding; Genentech: Research Funding; CTI BioPharma: Research Funding; Promedior: Research Funding; Sierra Oncology: Consultancy; LaJolla Pharmaceutical Company: Consultancy; Novartis: Consultancy.

The goal of therapy for essential thrombocythemia (ET) and polycythemia vera (PV) patients is to reduce thrombotic events by normalizing blood counts. Hydroxyurea (HU) and interferon-α (IFN-α) are the most frequently used cytoreductive options for ET and PV patients at high-risk for vascular complications. Myeloproliferative Disorders Research Consortium 112 was an investigator-initiated, phase 3 trial comparing HU to pegylated IFN-α (PEG) in treatment naïve, high-risk ET/PV patients. The primary endpoint was complete response (CR) rate at 12 months. A total of 168 patients were treated for a median of 81.0 weeks. CR for HU was 37% and 35% for PEG (p=0.80) at 12 months. At 24/36 months, CR was 20%/17% for HU and 29%/33% for PEG. PEG led to a greater reduction in JAK2V617F at 24 months, but histopathologic responses were more frequent with HU. Thrombotic events and disease progression were infrequent in both arms, while grade 3/4 adverse events were more frequent with PEG (46% vs. 28%). At 12 months of treatment there was no significant difference in CR rates between HU and PEG. This study indicates that PEG and HU are both effective treatments for PV and ET. With longer treatment PEG was more effective in normalizing blood counts and reducing driver mutation burden, while HU produced more histopathologic responses. Despite these differences, both agents did not differ in limiting thrombotic events and disease progression in high-risk ET/PV patients. (Funded by the National Cancer Institute, 5P01CA108671-09; clinicaltrials.gov number (NCT01259856). [Abstract copyright: Copyright © 2022 American Society of Hematology.]

Polycythemia and pulmonary hypertension are 2 human diseases for which better therapies are needed. Upregulation of hypoxia-inducible factor-2α (HIF-2α) and its target genes, erythropoietin (EPO) and endothelin-1, causes polycythemia and pulmonary hypertension in patients with Chuvash polycythemia who are homozygous for the R200W mutation in the von Hippel Lindau (VHL) gene and in a murine mouse model of Chuvash polycythemia that bears the same homozygous Vhl(R200W) mutation. Moreover, the aged Vhl(R200W) mice developed pulmonary fibrosis, most likely due to the increased expression of Cxcl-12, another Hif-2α target. Patients with mutations in iron regulatory protein 1 (IRP1) also develop polycythemia, and Irp1-knockout (Irp1-KO) mice exhibit polycythemia, pulmonary hypertension, and cardiac fibrosis attributable to translational derepression of Hif-2α, and the resultant high expression of the Hif-2α targets EPO, endothelin-1, and Cxcl-12. In this study, we inactivated Hif-2α with the second-generation allosteric HIF-2α inhibitor MK-6482 in Vhl(R200W), Irp1-KO, and double-mutant Vhl(R200W);Irp1-KO mice. MK-6482 treatment decreased EPO production and reversed polycythemia in all 3 mouse models. Drug treatment also decreased right ventricular pressure and mitigated pulmonary hypertension in Vhl(R200W), Irp1-KO, and Vhl(R200W);Irp1-KO mice to near normal wild-type levels and normalized the movement of the cardiac interventricular septum in Vhl(R200W)mice. MK-6482 treatment reduced the increased expression of Cxcl-12, which, in association with CXCR4, mediates fibrocyte influx into the lungs, potentially causing pulmonary fibrosis. Our results suggest that oral intake of MK-6482 could represent a new approach to treatment of patients with polycythemia, pulmonary hypertension, pulmonary fibrosis, and complications caused by elevated expression of HIF-2α.

The patients with mantle cell lymphoma (MCL) have translocation t(11;14) associated with cyclin D1 overexpression. We observed that iron (an essential cofactor of dioxygenases including prolyl hydroxylases [PHDs]) depletion by deferoxamine blocked MCL cells’ proliferation, increased expression of DNA damage marker γH2AX, induced cell cycle arrest and decreased cyclin D1 level. Treatment of MCL cell lines with dimethyloxalylglycine, which blocks dioxygenases involving PHDs by competing with their substrate 2‐oxoglutarate, leads to their decreased proliferation and the decrease of cyclin D1 level. We then postulated that loss of EGLN2/PHD1 in MCL cells may lead to down‐regulation of cyclin D1 by blocking the degradation of FOXO3A, a cyclin D1 suppressor. However, the CRISPR/Cas9‐based loss‐of‐function of EGLN2/PHD1 did not affect cyclin D1 expression and the loss of FOXO3A did not restore cyclin D1 levels after iron chelation. These data suggest that expression of cyclin D1 in MCL is not controlled by ENGL2/PHD1‐FOXO3A pathway and that chelation‐ and 2‐oxoglutarate competition‐mediated down‐regulation of cyclin D1 in MCL cells is driven by yet unknown mechanism involving iron‐ and 2‐oxoglutarate‐dependent dioxygenases other than PHD1. These data support further exploration of the use of iron chelation and 2‐oxoglutarate‐dependent dioxygenase inhibitors as a novel therapy of MCL.

Exposure to hypoxia elicits a myriad of adaptive responses that are mediated by hypoxia inducible factors (HIFs). An essential physiologic response to hypoxia is increase of production of erythrocytes necessary to maintain adequate oxygen delivery to the tissues; it has also been reported that the survival of erythrocyte may increase. Congenital defects of upregulation of the hypoxia-sensing pathway result in inappropriately elevated erythrocyte concentration. Evolutionarily, after many generations of living at high altitude, high altitude dwellers such as Tibetans, Andeans, and Ethiopians have selected genomic signatures, some in HIF pathway, enabling them to prosper in high altitude hypoxic environment. However, the entire diverse complex of molecular mechanisms of high-altitude adaptation is still largely unknown.

Normal human red blood cells have an average life span of about 120 days in the circulation after which they are engulfed by macrophages. This is an extremely efficient process as macrophages phagocytose about 5 million erythrocytes every second without any significant release of hemoglobin in the circulation. Despite large number of investigations, the precise molecular mechanism by which macrophages recognize senescent red blood cells for clearance remains elusive. Red cells undergo several physicochemical changes as they age in the circulation. Several of these changes have been proposed as a recognition tag for macrophages. Most prevalent hypotheses for red cell clearance mechanism(s) are expression of neoantigens on red cell surface, exposure phosphatidylserine and decreased deformability. While there is some correlation between these changes with aging their causal role for red cell clearance has not been established. Despite plethora of investigations, we still have incomplete understanding of the molecular details of red cell clearance. In this review, we have reviewed the recent data on clearance of senescent red cells. We anticipate recent progresses in in vivo red cell labeling and the explosion of modern proteomic techniques will, in near future, facilitate our understanding of red cell senescence and their destruction.

Clonality studies can establish the single-cell origin of tumors and thus differentiate clonal malignant and premalignant processes from reactive polyclonal processes. Detection of clonal cells may be based on direct tracking of cell lineage-specific sequences or disease-specific somatic mutations identifying the clonal population. Historically, clonal hematopoiesis was defined using the principle of X-chromosome inactivation based on observation that in circulating clonal cells, only one of the active chromosomes was expressed. In myeloproliferative neoplasms (MPNs) virtually all circulating erythrocytes, platelets, and granulocytes are products of single mutated stem cells that preferentially differentiate into the myeloid rather than lymphoid lineage. Thus, clonal differentiated myeloid cells co-exist in circulation with polyclonal long-lived T lymphocytes that originated before the MPN-initiating somatic clonal event. Chronic lymphocytic leukemia (CLL) starts in a differentiating B cell, but other lymphoid lineages and myeloid cells remain polyclonal. Normal T and B cells co-exist with the CLL clone, but are diluted by the massively expanded CLL population, which outnumbers the residual normal cells. Clonal hematopoiesis of undetermined potential (CHIP) has been identified by whole-genome sequencing of healthy individuals. These clones contain a specific somatic mutation previously considered to be disease defining but are detected in only a small proportion of circulating leukocytes, and there is no obvious suppression of normal hematopoietic stem cells. However, more studies are needed to properly define these clones, their persistence or disappearance, and their relative propensity for transforming into leukemias, myeloproliferative neoplasms, or other clonal hematological malignancies.

In North America, jaundiced neonates are not usually tested for G6PD deficiency if the family is of European ancestry. However, we describe such a family where ≥35 males have had severe (Class I) G6PD deficiency. Many of the jaundiced neonates did not have this diagnosis considered, at least three of whom developed bilirubin neurotoxicity. Over seven generations 35 affected males were identified. Three developed signs of kernicterus spectrum disorder; three had exchange transfusions for hyperbilirubinemia; and nine received one or more blood transfusions during childhood.

Background: Pacritinib, a novel JAK2/IRAK1 inhibitor, demonstrated clinically significant activity in spleen volume and symptom reduction in patients with advanced cytopenic myelofibrosis (MF), including those with severe thrombocytopenia (platelet [PLT] count Methods: Patients with baseline PLT Results: A total of 71 patients were analyzed as the pooled pacritinib 200 mg BID group (n=47 in PERSIST-2; n=24 in PAC203) and 42 patients in the BAT group. In addition to having severe thrombocytopenia, patients in the pooled pacritinib group had significant anemia, with median hemoglobin 8.6 g/dL, and 34% required red blood cell transfusion at baseline (Table 1). Sustained dose intensity was observed for the pacritinib group, with median dose of 400 mg/day in PERSIST-2 and 396 mg/day in PAC203. In the BAT subgroup from PERSIST-2, among the patients who received ruxolitinib the median post-titration dose was 10 mg/day. A total of 44% of patients treated with pacritinib and 21% treated with BAT had drug exposure ≥6 months. All-grade treatment-emergent adverse events (TEAEs) and those leading to study drug discontinuation were observed at similar rates in the pooled pacritinib and BAT groups. The patients in the pooled pacritinib group had a higher incidence of grade ≥3 and treatment-emergent serious AEs compared to those on BAT, which included patients on supportive care strategies. In the pooled pacritinib group, all-grade TEAEs were mostly driven by thrombocytopenia (32%) and gastrointestinal events, which included low grade nausea (30%) and diarrhea ([41%], which was manageable with over-the-counter antidiarrheals and resolved without leading to drug discontinuation). Rates of other commonly reported AEs in both trials were lower in the pooled pacritinib group compared to BAT, including epistaxis and peripheral edema. Fewer patients experienced fatal TEAEs in the pooled pacritinib group (Table 2). The incidence of serious and grade ≥3 bleeding AE was lower with pacritinib 200 mg BID in PAC203 than those reported with pacritinib 200 mg BID or BAT in PERSIST-2, likely attributable to the additional safety measures in PAC203 (Table 2). Incidence of cardiac events of any grade and those grade ≥3 were lower in the pooled pacritinib group compared to BAT. No patients in the pooled pacritinib group and 2 in the BAT group (1 fatal) had a MACE event (Table 2). Conclusion: In this analysis of patients with cytopenic MF who have severe thrombocytopenia, the safety profile of pacritinib 200 mg BID was comparable to BAT. This analysis suggests that pacritinib 200 mg BID may represent the first fully-dosed therapeutic option for this challenging patient population. Figure 1 Figure 1. Disclosures Mascarenhas: Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merus: Research Funding; Geron: Consultancy, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Constellation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prelude: Consultancy; Geron: Consultancy; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Galecto: Consultancy; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Forbius: Research Funding; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech/Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Consultancy, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Harrison: Constellation Pharmaceuticals: Research Funding; Sierra Oncology: Honoraria; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau. Gerds: Constellation: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; AbbVie: Consultancy; Sierra Oncology: Consultancy; CTI BioPharma: Research Funding; PharmaEssentia Corporation: Consultancy; Novartis: Consultancy. Rampal: Incyte: Consultancy, Research Funding; BMS/Celgene: Consultancy; Stemline: Consultancy, Research Funding; Kartos: Consultancy; Constellation: Research Funding; Jazz Pharmaceuticals: Consultancy; Blueprint: Consultancy; CTI: Consultancy; Abbvie: Consultancy; Novartis: Consultancy; Pharmaessentia: Consultancy; Sierra Oncology: Consultancy; Memorial Sloan Kettering: Current Employment; Disc Medicine: Consultancy. Buckley: CTI Biopharm: Current Employment. Craig: CTI BioPharma: Current Employment. Smith: CTI: Current Employment. Volpone: CTI: Current Employment. Roman-Torres: CTI Biopharm: Current Employment. Mesa: Samus: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; Incyte Corporation: Consultancy, Research Funding; Pharma: Consultancy; CTI: Research Funding; CTI: Research Funding; Sierra Oncology: Consultancy, Research Funding; Abbvie: Research Funding; Promedior: Research Funding; AOP: Consultancy; La Jolla Pharma: Consultancy; Genentech: Research Funding; Novartis: Consultancy; Gilead: Research Funding; Celgene: Research Funding.

Background: Selinexor is an oral, small molecule, selective inhibitor of nuclear export (SINE) compound that specifically blocks the karyopherin protein exportin 1 (XPO1, CRM1). In an shRNA library screen, we discovered that the survival of JAK2V167F mutant HEL cells is dependent on XPO1-mediated nuclear-cytoplasmic transport. Selinexor selectively suppressed primary myelofibrosis (MF) cells as compared with normal progenitor cells and induced hematologic responses in an MPN mouse model. Methods: An open label, prospective, investigator-initiated single center study is ongoing in adults with primary or secondary MF with resistance or intolerance to JAK inhibitor (JAKi) therapy with platelets > 30 K/μL and neutrophils > 500/μL. Selinexor was given orally once a week. Spleen volume was assessed by MRI at week 12 and week 24. The study was amended to include additional MRIs every 12 weeks in the year 1 and 24 weeks in the year 2. Primary end point is spleen response, defined as ≥ 35% spleen volume reduction (SVR) by MRI or CT, where applicable) at week 24. Bone marrow was evaluated at baseline and at week 24. The projected sample size of 24 will provide 83% power to reject a response rate of 15% and allow for up to a 25% dropout rate. We provide an interim report after completing 50% enrollment. Results: Between May 2019 and February 2021, 12 patients (pts) were enrolled. JAK2, CALR and MPL mutations were present in 7 (58.3%), 4 (33.3%) and 1 (8.3%) pts respectively. Eight pts (66.6%) had at least one high molecular risk mutation at baseline (Table 1). Median duration of prior JAKi therapy was 22 months (0.5 to 96 months) and 11 out of 12 were refractory to ruxolitinib at study enrollment. Median baseline spleen volume was 1454 cm 3(range 835 to 5792). Selinexor starting dose was 80 mg weekly in the first 6 pts and 60 mg for subsequent pts. At data cutoff, median duration of selinexor therapy was 36 weeks (range 11-114 weeks). One pt was not response evaluable and died due to liver abscess at week 12 (unrelated). One pt discontinued selinexor at week 18 due to grade 3 fatigue and was not evaluable for the primary end point. Of the 11 pts who had week 12 MRI or CT, 6 showed ≥ 10% SVR, 3 showed ≥ 25% SVR and 1 pt had early progression (Figure 1). At week 24, 5/9 (56%) pts had ≥ 25% SVR and 2/9 (22%) had ≥ 35% SVR (Figure 1). In 9 pts who had ≥24 weeks of selinexor, SVR ≥ 25% and ≥ 35% occurred at any point during study treatment in 4 (44%) and 3 (33%) pts, respectively. Two pts were red cell transfusion dependent at baseline; 1 became transfusion independent after 36 weeks of treatment, has not required transfusion for 49 weeks and remains on study treatment to date (114 weeks). Six pts (50%) discontinued selinexor. Reasons for treatment discontinuation are death in 1 pt, progressive disease in 1 pt, alternative treatment in 2 pts, and toxicity in 2 pts. Ten pts required dose reduction due to fatigue (1pt), anemia (1 pt), thrombocytopenia (2 pts), abdominal pain (1pt) and weight loss (5 pts). The most common treatment related adverse event was weight loss (grade 2 in 4 pts and grade 3 in 1 pt). This was manageable with treatment interruption and dose reduction, except in one pt who discontinued selinexor. As yet no changes in reticulin fibrosis MF grade were observed among 9 patients who received at least 24 weeks of treatment. Conclusions: Once weekly, oral selinexor showed single agent activity with sustained spleen responses in pts with JAKi refractory MF. Long-term administration of selinexor was well tolerated over time in MF pts. Correlatives studies including circulating inflammatory cytokine levels and mutant allele burden, as well as clonality studies by X-chromosome inactivation studies in woman, are underway and will be presented. Figure 1 Figure 1. Disclosures Tantravahi: BMS: Research Funding; Novartis: Research Funding; CTI BioPharma: Research Funding; Abbvie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Consultancy, Honoraria, Research Funding. Patel: Stemline: Research Funding; Genentech: Research Funding; Roche: Research Funding. Chamoun: Karyopharm Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Shah: Karyopharm: Current Employment. George: Celgene: Consultancy; Bristol Meyers Squibb: Consultancy; Incyte Corporation: Consultancy; Blueprint Medicines: Consultancy. Deininger: Fusion Pharma, Medscape, DisperSol: Consultancy; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Novartis: Consultancy, Research Funding; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Research Funding.

We previously created and characterized a mouse model of congenital erythrocytosis with low erythropoietin (EPO) levels from a gain-of-function mutation of the human erythropoietin receptor gene (mtHEPOR) (Divoky et al. PNAS. 2001; 98:986; Divoky et al. JMM Berl. 2016; 94:597). These mice develop fetal erythrocytosis, followed by transient amelioration of erythrocytosis in perinatal life, and reappearance at 3-6 weeks of age. Similarly, erythrocytosis is observed in heterozygous mtHEPOR patients postnatally but not at birth. We previously reported dynamic changes of the erythron with iron homeostasis during ontogenesis in these mice (Kralova et al. Blood 2017; 130: 170). We observed that while perinatal mtHEPOR mice exhibit relative iron deficiency, aged mice had iron overload. Here, we evaluated developmentally-determined factors associated with hyperactivation of EPOR signaling which could cause a transition from iron deficiency (neonates) to hyperferremia and increased iron deposition (aged mice). To assess the consequences of different levels of EPOR-JAK2-STAT5 signaling, we studied hetero- and homozygous mtHEPOR mice that differ in their severity of erythrocytosis. We found that prenatally and perinatally, mtHEPOR hetero- and homozygous mice have increased erythroferrone (Erfe) transcripts and reduced hepcidin, consistent with the known inverse correlation between Erfe and hepcidin and in accordance with increased numbers of immature erythroid progenitors in the fetal hepatic circulation. At birth, previously normal Epo expression decreased and remained low in adulthood. Iron deficiency, observed in mtHEPOR hetero- and homozygotes at postnatal day 7, was likely related to increased iron consumption by augmented erythropoiesis at this stage. Postnatally, hepcidin levels increased in mutant mice, accompanied by low Erfe induction and iron accumulation in the liver and spleen as reflected by the upregulation of hepatic Bmp6 expression in mature adult (aged ~6.5 months) and old (~16 months) mtHEPOR homozygotes. We hypothesized that this could be a consequence of diminished iron consumption due to a progressive decline of erythropoiesis in mtHEPOR mice, possibly mediated by premature aging of erythroid progenitors with cell-autonomously increased proliferative history and/or increased inflammation. Indeed, young mutant erythrocytes had decreased erythrocyte survival and expression of a senescent marker CD47, an inhibitor of erythrocytes' phagocytosis. Additionally, a progressive decline in the percentage of Ter119-positive bone marrow cells and immature erythroblasts was observed in mtHEPOR hetero- and homozygotes with aging. Clonogenic assays of old mice revealed suppression of early (BFU-E) and late (CFU-E) erythroid progenitors and myeloid bias of hematopoiesis, paralleled by the up-regulation of PU.1 expression, elevation of platelet counts, and an increase in megakaryocytes chiefly in the bone marrow of mtHEPOR homozygotes. Serum levels of inflammatory cytokines did not indicate systemic inflammation; however, induced transcripts of IL-6, Inf-γ, Tgf-β, and Tnf-α, mainly in mtHEPOR homozygotes showed local bone marrow inflammatory stress. These data indicate progressive attenuation of erythroid drive in mtHEPOR homozygotes, and less so in mtHEPOR heterozygotes, paralleled by a decline in hematocrit levels with aging. In response to attenuated erythropoietic activity, iron consumption was reduced in mtHEPOR mice, leading to iron accumulation in the liver and spleen accompanied by markedly increased hepcidin synthesis. Our data suggest that even in the absence of systemic inflammation, albeit with possible paracrine inflammatory signals, known to affect bone marrow remodeling and hematopoietic aging, life-lasting prolonged activation of EPOR-JAK2-STAT5 signaling promoted exhaustion of erythroid progenitors and resulted in an age-related decline of accelerated erythropoiesis in this mouse model of congenital erythrocytosis with human gain-of-function EPOR. Grant support: Czech grant agencies projects GA17-05988S, NV19-07-00412 and LTAUSA17142, Palacky University project IGA_LF_2021_004. Disclosures No relevant conflicts of interest to declare.

Reticulocytosis in sickle cell disease (SCD) is driven by tissue hypoxia from hemolytic anemia and vascular occlusion. Gene expression changes caused by hypoxia and other factors during reticulocytosis may impact SCD outcomes. We detected 1226 differentially expressed genes in SCD reticulocyte transcriptome compared to normal Black controls. To assess the role of hypoxia-mediating HIFs from other regulation of changes of the SCD reticulocyte transcriptome, we compared differential expression in SCD to that in Chuvash erythrocytosis (CE), a disorder characterized by constitutive upregulation of HIFs in normoxia. Of the SCD differentially expressed genes, 28% were shared between CE and SCD and thus classified as HIF-mediated. The HIF-mediated changes were generally in genes promoting erythroid maturation. We found that genes encoding the response to endoplasmic reticulum stress generally lacked HIF mediation. We then investigated the clinical correlation of erythroid gene expression for the 1226 differentially expressed genes detected in SCD reticulocytes, using clinical measures and gene expression data previously profiled in peripheral blood mononuclear cells (PBMCs) of 157 SCD patients at the University of Illinois at Chicago (UIC). Normal PBMCs contain only a small number of erythroid progenitors, but in SCD or CE PBMCs the erythroid transcriptome is enriched due to elevated circulating erythroid progenitors from heightened erythropoiesis (PMID: 32399971). We applied deconvolution analysis to assess the clinical correlation of erythroid gene expression, using a 16-gene expression signature of erythroid progenitors previously identified in SCD PBMCs. Deconvolution analysis uses the proportion of cell/tissue or specific marker genes (here the erythroid specific 16-gene signature) to dissect gene expression variation in biological samples with cell/tissue type heterogeneity. We correlated, in the 157 UIC patients, erythroid gene expression with i) degree of anemia as indicated by hemoglobin concentration, ii) vaso-occlusive severe pain episodes per year, and iii) degree of hemolysis measured by a hemolysis index. The analysis identified 231 genes associated with at least one of the complications. Increased expression of 40 erythroid specific genes, including 15 HIF-mediated genes, was associated with all three complications. These 40 genes are all upregulated in SCD reticulocytes and correlated with low hemoglobin concentration, frequent severe pain episodes, and high hemolysis index, suggesting that these manifestations may share a relationship to stress erythropoiesis-driven transcriptional activity. Expression quantitative trait loci (eQTL) contain genetic polymorphisms that associate with gene expression level, which can be viewed as a natural experiment to investigate the causal relations between gene expression change and phenotypic outcomes. To assess the causal effect of erythroid gene expression, we tested association between erythroid eQTL and the clinical manifestations in 906 SCD patients from the Walk-PHaSST and PUSH cohorts. We first mapped erythroid eQTL in the 157 UIC patients, who were previously genotyped by array, applying deconvolution algorithm on the same PBMC data for the 1226 differential genes in SCD reticulocytes, and detected 54 distinct eQTL for 30 genes at 5% false discovery rate. After adjusting for multiple comparisons, we found that the C allele of rs16911905, located in the β-globin cluster and associated with increased erythroid expression of HBD (encodes δ-globin of hemoglobin A 2), significantly correlated with lower hemoglobin concentration (β=-0.064, 95% CI -0.092 - -0.036, P=6.7×10 -6). The C allele was also associated with higher hemolytic rate (P=0.031), less frequent pain episodes (P=0.045), and increased erythroid expression of HBB here encoding sickle β-globin (P=5.1x10 -5). The association of the C allele with lower hemoglobin concentration was then validated in 242 patients from the UIC cohort (β=-0.071, 95% CI -0.13 - -0.011, P=0.023), as was the trend of association with higher hemolytic rate (P=0.0031) and less pain episodes (P=0.034). Our findings reveal HIF- and non-HIF-mediated genes in SCD stress erythropoiesis, and identify novel clinical associations for a HBD eQTL. Our study highlights the correlation of altered erythroid gene expression with SCD hemolytic and vaso-occlusive manifestations. Disclosures Saraf: Global Blood Therapeutics: 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; Pfizer: Research Funding. Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy.

Polycythemia vera (PV) is characterized by elevated red cell mass due to gain-of-function mutations of the tyrosine kinase JAK2 gene. Major causes of morbidity and mortality in PV are venous and arterial thromboses. Risk factors of thrombosis in PV are age > 60 years, previous thrombosis history, and leukocytosis due to increased granulocytes (PMID: 31865003). High hematocrit has been considered as a causative factor of thrombosis due to high viscosity; however, this has been challenged. Phlebotomy to maintain hematocrit below 45% is performed by most hematologists to prevent thrombosis. However, in our prospective controlled study, we reported that phlebotomies, but not high hematocrit, are associated with increased risk of thrombosis in Chuvash erythrocytosis (CE). CE is due to a hypomorphic mutation of the Von Hippel-Lindau (VHL) gene, a negative regulator of hypoxia inducible factors (HIFs) (PMID 289208), leading to augmented HIFs. We hypothesize that repeated phlebotomies may increase the risk of thrombosis in PV. We reported that HIFs, and some HIFs-regulated thrombotic genes, are upregulated in PV in both granulocytes and platelets, and even more in PV with a thrombosis history (PMID: 32203583). PV patients with mutated JAK2 have lower ferritin, serum iron, transferrin saturation, and increased soluble transferrin receptor levels compared to JAK2-wild type erythrocytosis (PMID: 30042411), indicating that PV patients with JAK2 mutation are more likely to be iron deficiency (ID). Repeated phlebotomies further augment ID, which increases the level of HIF-1 and HIF-2 by inhibiting the principal negative regulator of HIFs, prolyl hydroxylase domain 2 (PHD2) enzyme. PHD2 requires iron as a co-factor (PMID: 18066546). This led us to hypothesize that further increase of HIFs due to phlebotomy-induced ID increases expression of HIF-regulated prothrombotic genes, leading to increased risk of thrombosis in PV. We studied 17 PV with ID, 32 PV without ID, and 19 healthy control and measured their prothrombotic genes' transcript levels in the granulocytes. Of 17 PVs with ID, 9 (56.3 %) had thrombosis while 14/32 PVs without ID (43.8 %) had thrombosis history. We then measured transcript levels of prothrombotic genes, such as F3 (tissue factor), SELP (P-selectin), THBS1 (thrombospondin-1), SERPINE1(plasminogen activator inhibitor-1) and HIF-target genes VEGFA and SLC2A1 (glucose transporter-1). The transcripts of these genes were upregulated in PV with and without ID compared to the controls. However, PV with ID had higher transcripts of these genes compared to PV without ID, except SERPINE1 (Figure 1). These prothrombotic gene transcripts were inversely correlated with ferritin levels and positively correlated with HIF target genes. Thus, ID in PV further augmented HIFs activity and induced expression of prothrombotic genes. In ongoing studies of PV patients serving as their own controls, PV subjects with ID are treated with hydroxyurea to prevent rebound erythrocytosis. The hydroxyurea treatment alone did not normalize high transcripts of prothrombotic and HIF target genes. However, transcripts of these prothrombotic and HIF-target genes decreased after iron correction while their JAK2V617F allele burden did not change. We then measured transcript levels of these prothrombotic genes in the patients with germline mutations augmenting HIF activity including loss of function VHL, gain-of-function EPAS1 (HIF2a), and loss-of-function EGLN1 (PHD2). These patients have high transcript levels of prothrombotic genes due to high HIF activity although they don`t have ID. Transcripts of F3, SELP, VEGFA, and SLC2A1 of the patients with HIF-pathway mutations were increased compared to the controls and compared to PV without ID but similar to the levels in PV with ID. On the other hand, transcript levels of THBS1 and SERPINE1 in the patients with HIF-pathway mutations were higher than the levels seen in the patients with PV with ID. These data indicate that ID in PV augments HIF activity similar, although not identical, to the patients with germline HIF-pathway mutations and may facilitate propensity to thromboses. These data suggest that therapeutic phlebotomy to control high hematocrit in PV and other erythrocytoses has the potential to increase thrombosis risk. In our ongoing studies, transcript levels of these prothrombotic genes are being correlated with their functions and protein levels. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked inherited enzymopathic disorder that may lead to transfusion-requiring acute hemolytic anemia (AHA) triggered by fava beans ingestion, infection or some drugs. The gene encoding for G6PD carries a large number of genetic variants that have varying pathogenicity. We reported on three G6PD variants in the Gaza Strip Palestinian population with differing clinical impacts and frequencies: G6PD Mediterraneanc.563T, African G6PD A−c.202A/c.376G, and G6PD Cairoc.404C. We also identified a novel G6PD missense (Ser179Asn) mutation c.536G > A “G6PD Gaza”. In this work we explore the effect of these four genetic variants on the structural and substrate (NADP+ and G6P) binding characteristics of the G6PD enzyme using the Monte Carlo (MC) flexible docking and molecular dynamics (MD) simulation approaches. We report that G6PD A−c.202A/c.376G, G6PD Mediterraneanc.563T, G6PD Cairoc.404C and G6PD Gazac.536A mutations cause significant structural changes in G6PD enzyme to induce conformational instability leading to the loss of binding of one or both substrates and are causative of G6PD deficiency.

Polycythemia vera (PV) and essential thrombocythemia (ET) patients have a higher risk of arterial and venous thrombosis than healthy individuals; thromboses are their principal cause of morbidity and mortality. We reported increased transcription of prothrombotic and inflammatory genes in granulocytes and platelets of PV and ET. There were differences in the expression of prothrombotic genes between platelets and granulocytes, suggesting that these cells have cell-specific contributions to thrombosis in PV and ET. Some of these prothrombotic genes are regulated by hypoxia inducible factors (HIFs) (PMID: 32203583). However, the molecular mechanism of thrombosis in PV and ET remains unknown. KLF2 (Kruppel like factor 2) is a transcription factor regulating primitive erythropoiesis and inflammation. Knockdown of KLF2 in cultured endothelial cells increases prothrombotic gene expression and reduces blood clotting time and flow rates (PMID: 15718498). Targeted deletion of KLF2 in neutrophils increases thrombosis by inducing the expression and activity of tissue factor (Blood, 2018, 132:75). To study the role of KLF2 in PV and ET thrombosis, we measured KLF2 mRNA in granulocytes from 53 PV and ET patients (25 with a history of thrombosis) and in platelets from 40 patients (21 with a history of thrombosis). We also measured KLF2 mRNA in granulocytes from 38 controls and platelets from 18 controls. Althrough the role of KLF2 in thrombosis has been studied in neutrophils, we also tested KLF2 mRNA in platelets since we previously observed a different pattern of expression of prothrombotic genes between granulocytes and platelets in PV and ET. We found lower KLF2 mRNA in both granulocytes and platelets of PV and ET patients compared to the controls (Fig. 1A and B). Compared to patients without thrombosis, those with thrombosis had lower KLF2 mRNA in platelets but not granulocytes. KLF2 mRNA in these cells correlated inversely with JAK2V617F allele burden in granulocytes (Fig. 1C and D). We then measured mRNA of prothrombotic genes: F3 (tissue factor), SELP (P-selectin), IRAK1 (interleukin 1 receptor associated kinase 1), IL1RAP (interleukin 1 receptor accessory protein), VEGFA (vascular endothelial growth factor-A), THSB1 (thrombospondin 1), SERPINE1 (encoding plasminogen activator inhibitor 1 [PAI-1]). The mRNA levels of these prothrombotic genes correlated inversely with KLF2 mRNA in platelets while SELP and THSB1 transcripts correlated inversely with KLF2 mRNA in granulocytes. KLF2 and HIFs are reported to interact (PMID: 19491109, PMID: 21565532). In order to elucidate the regulatory machanism of KLF2 in thrombosis, we measured KLF2 mRNA in patients with two inherited disorders of hypoxia sensing characterized by thrombosis: 1) Chuvash erythrocytosis (CE) due to homozygous mutation of VHLR200W (13 patients) and 2) erythrocytosis due to gain-of-function mutation of HIF-2a (two patients with HIF2AM535V and two patients HIF2AE548K). KLF2 mRNA levels did not differ in granulocytes and platelets between these patients and controls. However, two CE patients and two patients with HIF2AM535V with a history of thrombosis had lower KLF2 mRNA levels compared to patients without thrombosis (ASH this meeting, 2020 Song J). In conclusion, we report here that KLF2 transcripts are down regulated in both granulocytes and platelets from PV and ET patients and they correlate inversely with the transcripts of prothrombotic genes and JAK2V617F allelic burden, suggesting that KLF2 might be a negative regulator of thrombotic gene expression in PV and ET. Here we did not detect any changes of KLF2 transcripts in congential disorders with elevated HIFs. However, two CE patients and 2 patients with HIF2AM535V with thrombosis had less KLF2 expression compared to those without thrombosis. These results suggest that, by inference from findings in congenital disorders with elevated HIFs, KLF2 in PV and ET granulocytes and platelets may be regulated in a HIF-independent manner but that thrombosis may be regulated in a HIF-dependent manner. Thus, KLF2 may be a novel therapeutic target to prevent thrombosis in PV and ET, but confirmation by further studies is needed. The upstream regulation of KLF2 in PV and ET granulocytes and platelets needs to be elucidated. *PT &JTP contributed equally Disclosures Gangaraju: Sanofi Genzyme, Consultant for Cold Agglutinin Disease: Consultancy. Gordeuk:CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Imara: Research Funding; Ironwood: Research Funding; Novartis: Consultancy.

Background: There are limited therapeutic options for patients (pts) with myelofibrosis (MF) who lose response to ruxolitinib (Rux). The combination of navitoclax (Nav) plus Rux (NCT03222609) has been shown to induce clinically meaningful spleen volume (SV) responses, improvement in Total Symptom Score (TSS), and reduction in bone marrow fibrosis (BMF) grading in pts with MF who no longer benefit from Rux. At diagnosis, pts with primary MF who have high-molecular-risk (HMR) mutations, defined as mutations in ASXL1, SRSF2, EZH2, U2AF1, and IDH1/2, have shorter overall survival and/or increased risk of leukemic transformation. In addition to mutations in HMR genes, the total number of all genes mutated (regardless of whether they are HMR genes) also correlates with reduced SV responses, treatment (Tx) duration, and overall survival. This phase 2 study explored whether the presence of HMR, or the total number of genes mutated at study entry, correlated with clinical outcomes (SV reduction ≥35% [SVR35], reductions in TSS and BMF) and reductions in driver gene (JAK2 p.V617F and mutated CALR) variant allele frequency (VAF) following Nav plus Rux. As pts with MF have demonstrated a markedly abnormal cytokine profile at baseline, the ability of Nav plus Rux to mediate known MF inflammatory cytokines was also assessed. Methods: MF pts with Rux failure who had received ≥12 weeks of continuous Rux and had persistent splenomegaly that required a new Tx were enrolled. Pts continued Rux, and Nav was started at 50 mg QD with stepwise escalation to 300 mg on the basis of tolerability. Study endpoints included SVR35 (by MRI), change in TSS (MF Symptom Assessment Form version 4.0) at week (Wk) 24, and change in BMF (locally assessed). At baseline and Wk 24, mutational analyses including VAF measurement were performed in peripheral blood by next-generation sequencing with the 54-gene Focus::Myeloid™ panel (3% limit of detection). At baseline, Wk 12 and 24, levels of inflammatory cytokines were measured in plasma with the 133-analyte ExplorerMAP™ panel. Results: As of February 28, 2020, 34 pts with MF had received ≥1 dose of Nav plus Rux. At study entry, 33 pts were evaluable for biomarker analysis. JAK2 was mutated in 26/33 (79%) pts and 7/33 (21%) pts were positive for mutated CALR. Median VAFs for JAK2- and CALR-mutated pts were 88% and 39%, respectively. Baseline mutational analysis revealed the presence of HMR genes in 19/33 (58%) pts; of these, 8/19 (42%) had ≥2 HMR genes mutated. Mutation rates for ASXL1, SRSF2, EZH2, U2AF1, and IDH1 were 13/19 (68%), 7/19 (37%), 4/19 (21%), 2/19 (10%), and 1/19 (5%), respectively (Figure 1). At baseline, the median number of all genes mutated was 3, and 17/33 (52%) pts harbored ≥3 mutations. At Wk 24, among evaluable pts, 9/34 (27%) achieved SVR35, 6/20 (30%) reached ≥50% reduction in TSS (TSS50), 7/34 (21%) had -1/-2 grade improvement in BMF, and 12/26 (46%) had >10% driver gene VAF reductions. Pts who achieved either SVR35, TSS50, BMF improvement, or VAF reduction were assessed and the distribution of pts with or without HMR mutations, and with ≥3 or Further analyses revealed a direct correlation between changes from baseline in known MF-associated cytokines and SV changes (Figure 2A and 2B) (ie, beta-2 microglobulin [B2M; Wk 12 and 24], tumor necrosis factor receptor 2 [TNFR2; Wk 12], tissue inhibitor of metalloproteinases 1 [TIMP-1; Wk 12], and vascular cell adhesion molecule 1 [VCAM-1; Wk 12]). The Wk 24 TNFR2, TIMP-1, and VCAM-1 analyses will be available for presentation. These 4 cytokines have also been shown historically to correlate with TSS improvement. Conclusions: Pts with MF previously treated with Rux who then receive Nav plus Rux in combination achieved clinically meaningful SVR, TSS improvement, reduction in BMF, and driver gene VAF reductions independent of HMR mutations and the total number of genes mutated. Ongoing cytokine analyses suggest that the combination of Nav plus Rux may have a role in modulating key cytokines implicated in TSS improvement in pts with MF with suboptimal response to Rux alone. Disclosures Pemmaraju: MustangBio: Honoraria; Pacylex Pharmaceuticals: Consultancy; Samus Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Cellectis: Research Funding; Plexxikon: Research Funding; Affymetrix: Other: Grant Support, Research Funding; Novartis: Honoraria, Research Funding; Roche Diagnostics: Honoraria; Blueprint Medicines: Honoraria; Stemline Therapeutics: Honoraria, Research Funding; Celgene: Honoraria; AbbVie: Honoraria, Research Funding; Incyte Corporation: Honoraria; DAVA Oncology: Honoraria; SagerStrong Foundation: Other: Grant Support; LFB Biotechnologies: Honoraria. Garcia:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Eli Lily: Research Funding. Potluri:AbbVie: Current Employment, Other: may hold stock or stock options. Holes:AbbVie Inc.: Current Employment, Current equity holder in publicly-traded company. Harb:AbbVie: Current Employment, Other: may hold stock or stock options. Jung:AbbVie Inc.: Current Employment, Current equity holder in publicly-traded company. Hutti:AbbVie Inc.: Current Employment, Other: may hold stock or stock options. Verstovsek:Blueprint Medicines Corp: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Roche: Research Funding; AstraZeneca: Research Funding; ItalPharma: Research Funding; Incyte Corporation: Consultancy, Research Funding; CTI Biopharma Corp: Research Funding; Protagonist Therapeutics: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Consultancy, Research Funding; PharmaEssentia: Research Funding. Harrison:CTI Biopharma Corp: Honoraria, Speakers Bureau; Janssen: Speakers Bureau; Gilead Sciences: Honoraria, Speakers Bureau; Incyte Corporation: Speakers Bureau; Sierra Oncology: Honoraria; Shire: Honoraria, Speakers Bureau; AOP Orphan Pharmaceuticals: Honoraria; Roche: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Promedior: Honoraria.

Thrombosis is the major cause of morbidity and mortality in Chuvash erythrocytosis (CE), caused by a hypomorphic R200W mutation of the Von Hippel-Lindau (VHL) gene, a negative regulator of hypoxia inducible factors (HIFs). This mutation augments HIF activity even in normoxia. High hematocrit associated with hyperviscosity has been considered a risk factor for thrombosis suggesting that maintaining normal hematocrit may prevent thrombosis. However, our prospective controlled study reported that phlebotomies, but not the high hematocrit, are associated with increased risk of thrombosis (PMID 289208) by a mechanism that remains to be elucidated. Phlebotomies lead to iron deficiency. We explored the potential role of iron deficiency as a cause of thrombosis. Iron deficiency induces HIF activity by inhibiting prolyl hydroxylase domain 2 (PHD2), an inhibitor of HIFs that requires iron as a co-factor (PMID 18066546). The expression of certain prothrombotic genes regulated by HIFs is modestly increased in CE mononuclear cells (PMID 23993337). We hypothesized that further augmentation of already high HIF activity by iron deficiency might further increase expression of prothrombotic genes leading to increased risk of thrombosis in phlebotomized subjects. We reported that, in polycythemia vera (PV) and essential thrombocythemia (ET), the pattern of increase in prothrombotic and inflammatory gene expression differs between granulocytes and platelets (PMID: 32203583), suggesting cell-specific contributions to thrombosis in these disorders. We analyzed the whole transcriptome of the platelets of 10 CE patients, 6 with iron deficiency (ferritin 1). Dysregulated genes as analyzed by Ingenuity Pathway Analysis (Qiagen) were associated with platelet binding, hemostasis and thrombus signaling and decreased bleeding time. To explore our hypothesis further, we quantitated the mRNA of these HIF-regulated prothrombotic genes: THBS1 (thrombospondin 1), SERPINE1 (plasminogen activator inhibitor-1 [PAI-1]), ITGA2B (integrin alpha-IIb), PTGS2 (prostaglandin-endoperoxide synthase 2), SELP (P-selectin), PDGFA (platelet derived growth factor subunit A), and ITGB3 (integrin beta-3). We analyzed granulocytes from 16 CE subjects (8 iron deficient) and platelets from 12 CE subjects (7 iron deficient). In platelets, THBS1, SELP, SERPINE1, and PDGFA mRNA levels were higher in iron deficient CE subjects than those with normal ferritin (p=0.015-0.088). In all CE subjects, the mRNA levels of these four genes correlated inversely with ferritin (figure 1). PTGS2 (known to be down regulated in thrombosis) was down regulated in iron deficient CE patients and correlated positively with ferritin. ITGB3 and ITGA2B mRNA levels were not different between the two groups. In granulocytes, SELP mRNA was augmented in CE patients with iron deficiency and both SELP and ITGB3 mRNA levels correlated inversely with ferritin. We did not find a difference in expression of KLF2, a regulator of thrombotic genes, in iron deficient versus iron sufficient CE patients; see this meeting Song J et al. We then tested our hypothesis of augmentation of thrombosis risk by iron deficiency in granulocytes from 50 PV and ET patients (9 with iron deficiency) and platelets from 41 patients (5 with iron deficiency). In granulocytes, THBS1, SELP, and IRAK1 (Interleukin 1 Receptor Associated Kinase 1) mRNA levels were higher in patients with iron deficiency, and IRAK1, THBS1, and SERPINE1 mRNA levels correlated inversely with ferritin. In platelets, THBS1, and SERPINE1 mRNA were higher in patients with iron deficiency and SELP, THBS1, and SERPINE1 mRNA levels correlated inversely with ferritin. JAK2V617F allele burden also correlated inversely with ferritin. Our study demonstrates that iron deficiency is associated with increased expression of HIF-regulated prothrombotic genes in CE platelets and granulocytes in a pattern that differs between these two cell types. We also report increased expression of prothrombotic genes in PV and ET patients with iron deficiency. These results underline the potential danger of phlebotomies in attempts to control high hematocrit. We caution against indiscriminate use of therapeutic phlebotomy for treatment of patients with PV and other erythrocytoses. VG &JTP contributed equally Figure Disclosures Gordeuk: Ironwood: Research Funding; Novartis: Consultancy; CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Imara: Research Funding.

The V617F mutation in the JH2 domain of Janus kinase 2 (JAK2) is an oncogenic driver in several myeloproliferative neoplasms (MPNs), including essential thrombocythemia, myelofibrosis, and polycythemia vera (PV). Other mutations in JAK2 have been identified in MPNs, most notably exon 12 mutations in PV. Here, we describe a novel recurrent mutation characterized by a common 4-amino-acid deletion and variable 1-amino-acid insertion (Leu583-Ala586DelInsSer/Gln/Pro) within the JH2 domain of JAK2. All 4 affected patients had eosinophilia, and both patients with Leu583-Ala586DelInsSer fulfilled diagnostic criteria of both PV and chronic eosinophilic leukemia (CEL). Computational and functional studies revealed that Leu583-Ala586DelInsSer (herein referred to as JAK2ex13InDel) deregulates JAK2 through a mechanism similar to JAK2V617F, activates signal transducer and activator of transcription 5 and extracellular signal-regulated kinase, and transforms parental Ba/F3 cells to growth factor independence. In contrast to JAK2V617F, JAK2ex13InDel does not require an exogenous homodimeric type 1 cytokine receptor to transform Ba/F3 cells and is capable of activating β common chain family cytokine receptor (interleukin-3 receptor [IL-3R], IL-5R, and granulocyte-macrophage colony stimulating factor receptor) signaling in the absence of ligand, with the maximum effect observed for IL-5R, consistent with the clinical phenotype of eosinophilia. Recognizing this new PV/CEL-overlap MPN has significant clinical implications, as both PV and CEL patients are at high risk for thrombosis, and concomitant cytoreduction of red cells, neutrophils, and eosinophils may be required for prevention of thromboembolic events. Targeted next-generation sequencing for genes recurrently mutated in myeloid malignancies in patients with unexplained eosinophilia may reveal additional cases of Leu583-Ala586DelInsSer/Gln/Pro, allowing for complete characterization of this unique MPN.

We studied 10 affected and 11 non affected relatives of a five generation kindred with autosomal dominant familial erythrocytosis. We have excluded other known inherited forms of erythrocytosis. i.e., mutations of globin, the 2,3 DPG generating PBGM gene causing increased Hb/O2 affinity (low p50), gain-of-function mutations of erythropoietin receptor (EPOR), germ-line JAK2 mutations, and hypoxia inducible factor 2A (HIF2-A(EPAS1)),PHD2(EGLN1), and VHL mutations associated with augmented oxygen-sensing pathway. Those affected family members had moderately increased erythropoietin (EPO) levels, no splenomegaly, normal leukocyte and platelet numbers and normal p50 (presented at this mtg, Blood. 2003;102,162b). We sequenced whole exomes and adjacent portions of introns of two affected individuals and found a novel heterozygous 5`UTR EPO variant with change -136 nt upstream from the ATG EPO initiation site (NG_021471 -136 G>A). This variant segregated with the erythrocytosis phenotype in 15 relatives examined: the 7 affected subjects were heterozygous for this variant and the 8 unaffected were negative, suggesting its causative role in erythrocytosis (presented at this mtg, Blood. 2013;122,950). Other authors (NEJM 2018; 378:924) reported a variant of autosomal dominant familial erythrocytosis with a different EPO mutation: a single-nucleotide deletion (c.32delG) in exon 2 of the EPO gene causing a frameshift and alternative EPO mRNA transcripts, leading to increased production of functional EPO protein with shortened signal peptide and a novel N-terminus as cause of their familial erythrocytosis. In order to characterize function of our 5`UTR EPO variant, we introduced it into the EPO producing human hepatoma cell line Hep3B using CRISPR/Cas9 editing system by homologous recombination with single-stranded donor oligonucleotides. The targeted cells were sorted in 96 well plates (20 cells per well) and then each well tested for presence of -136 G>A variant by allele-specific PCR. We identified 3 heterozygous Hep3B for EPO-136 G>A; the second round of targeting generated homozygous Hep3B clones. The EPO mRNA of homozygous recombinants was greatly increased and detected even in normoxia, unlike non-edited Hep3B cells. No alternative EPO mRNA transcripts were detected in the engineered and non-edited Hep3B cells. To emulate human phenotype, the supernatants of cultured three heterozygousEPO-136 G>A Hep3B single-cell derived clones and controls in normoxic and hypoxic conditions were used to detect production of EPO. The hypoxic treatment increased ~2x the production of EPO from edited clones compared to non-edited Hep3B cells. The secreted EPO from heterozygousEPO-136 G>A Hep3B clones supported growth of EPO-dependent BaF3-EPOR cells more than supernatants from non-edited Hep3B cells. We then measured EPO transcript levels in Hep3B with EPO-136 G>A and parental Hep3B cell lines in normoxia and hypoxia. The hypoxia increased the relative expression of EPO-136 G>A allele in all three targeted heterozygousEPO-136 G>A Hep3B cell lines. The EPO gene promoter was largely unmethylated in both wild and mutated clones. To evaluate activity of this mutant EPO promoter, we sub-cloned wild and mutated 5`UTR EPO sequence upstream of luciferase reporter gene and transfected them into two EPO producing cell lines - Hep3B, Hep2G. The mutant significantly increased activity of the reporter. To study the interaction of EPO-136 G>A mutant promoter with HIF2 (principal transcription factor regulating EPO), we co-transfected EPO-luc reporter with HIF2-A expression plasmid. The activity of reporter with mutated EPO-136 G>A was further increased in these cells with augmented HIF2 levels. Indeed, alignment tools predicted the EPO-136 G>A genomic region as putative HIF2 binding site. This suggests that mutated 5`UTR of EPO augments interaction with HIF2, leading to increase production of EPO. Chromatin immunoprecipitation experiments are ongoing to model the transcriptional regulatory network accounting for augmented transcriptional regulation of this 5`UTR EPO gene variant. Here we report a novel mechanism of inherited erythrocytosis caused by increased transcription of mutated 5`UTR of EPO. Supported by Czech HRC, grant NV19-07-00412 and Ministry of Education, grant LTAUSA17142 and University of Utah. Disclosures No relevant conflicts of interest to declare.

Background:Chuvash erythrocytosis (CE), an inherited autosomal recessive disease endemic to Russia's mid-Volga River region, is caused by a germlineVHLC598T mutation (encoding VHLR200W) that alters oxygen sensing (PMID: 12415268). VHLR200W protein displays impaired degradation of hypoxia inducible transcription factor (HIF)-α subunits leading to increased HIF-1 and HIF-2 in normoxia and increased transcription of many HIF-regulated genes including erythropoietin (PMID: 14726398). CE patients have increased risk of venous and arterial thromboses, which are the major cause of morbidity and mortality (PMID:28104701). Thrombosis occurs despite lower blood pressure, body mass index and white blood cells compared to controls and is not related to the elevation in hematocrit but is increased in patients treated with phlebotomy therapy (PMID: 31289208). We have shown by microarray analysis of CE peripheral blood mononuclear cells modestly increased expression of several HIF-regulated pro-thrombotic genes at false discovery rate Methods:EightVHLC598T homozygotes and 6VHLwild type controls from Cheboksary, Chuvashia, Russia were studied as outpatients under basal circumstances. Blood was collected from these participants by venipuncture into vacutainer tubes containing 0.129 M sodium citrate. Immediately after venipuncture, platelet free plasma (PFP) was prepared by two rounds of centrifugation at 2,500 × g for 15 minutes at room temperature and EVs were isolated from PFP by centrifugation at 20,000 g for 15 minutes at 4 ºC. EV-TF was measured in duplicate by a two-stage Factor Xa generation assay with and without anti-TF antibody using Innovin (Siemens Healthcare Diagnostics) as a standard (PMID: 30656275). We also isolated granulocyte mRNA from 3 of the patients and 2 of the controls at a different time point and measured F3 transcripts by RT-qPCR. Results:We detected EV-TF in 4 of 8 CE patients but in no wild type controls (one-sided P = 0.043). The range of 0.45 to 1.25 pg/ml is similar to the range recently reported in US patients with cancer (PMID: 32548563). MCHC tended to be lower among 4 CE patients with detectable EV-TF than 4 without (one-sided P = 0.12), but this was not the case for serum ferritin. We also detectedF3transcripts in granulocytes, and these levels were higher in the 3 patients than the 2 controls that we analyzed (one-sided P = 0.12). Furthermore,F3mRNA correlated with plasma EV-TF in these five subjects (Spearman rho = 0.71, one-sided P < 0.05). As previously reported, CE patients had higher hemoglobin, hematocrit and red blood cells and lower platelets compared to controls (PMID: 14726398). They also had lower values for serum ferritin and mean corpuscular hemoglobin concentration (MCHC) suggesting iron deficiency, likely induced by phlebotomy therapy or by the presence of gastritis, which is increased in CE. Discussion:The presence of EV-TF in the plasma of 4 out of 8 CE patients and none of six controls, and increasedF3transcripts in CE granulocytes, may point to a potential thrombogenic role, although none of the CE patients in this study had a history of thrombosis. Further studies on larger numbers of patients are warranted to confirm these findings and to clarify the potential role of EV-TF in thrombosis in CE. *NM, JTP & VRG contributed equally Figure Disclosures Gordeuk: CSL Behring:Consultancy, Research Funding;Ironwood:Research Funding;Imara:Research Funding;Global Blood Therapeutics:Consultancy, Research Funding;Novartis:Consultancy.

Acquired uniparental disomy (aUPD) is a common and recurrent molecular event in human cancers that leads to homozygosity for tumor suppressor genes as well as oncogenes, while retaining the diploid chromosomal complement. Because of the lack of copy number change, aUPD is undetectable by comparative genome hybridization, so the magnitude of this genetic change was underappreciated in the past. 9p aUPD was first described in 2002 in patients with polycythemia vera (PV). Since then, systematic application of genomewide single-nucleotide polymorphism arrays has indicated that 9p aUPD is the most common chromosomal aberration in myeloproliferative neoplasms (MPNs), contributing to discovery of the PV-defining mutation JAK2V617F21. It was also found in other myeloid and lymphoid malignancies, though at a relatively lower frequency. By leading to JAK2V617F 23 homozygosity, 9p aUPD plays a causal role in the development of PV and is also associated with less favorable clinical outcomes. It is also possible that new targets other than JAK2V617F 25 are present within 9p aUPD that may contribute to diversity of PV outcome and phenotype. This review summarizes recent discoveries on 9p aUPD in hematologic malignancies and discusses possible underlying mechanisms and potential roles of 9p aUPD in the pathogenesis of PV, the relationship between 9p aUPD and JAK2V617F29, and possible new cancer-related targets within the 9p aUPD region.

Obstructive sleep apnea (OSA) is characterized by chronic intermittent hypoxia (CIH) caused by recurrent episodes of upper airway obstruction during sleep. Chronic hypoxia increases red blood cell volume (RBCV) and hemoglobin concentration (Hb) in order to improve tissue oxygen delivery. However, the prevalence of increased Hb in OSA was only 1.7% (9 out of 527 patients) in our prior study (Gangaraju et al Blood 2016 128:2444). The reasons for this relative lack of discernable erythrocytosis is not yet fully understood. Further, the studies of possible changes in RBC and plasma volume in OSA are lacking. We collected blood samples from OSA patients before and after 3 months of continuous positive airway pressure (CPAP) treatment. We estimated erythropoietic activity by measuring erythropoietin (EPO) and reticulocyte count. EPO levels and reticulocytes were higher in OSA patients compared to controls; CPAP treatment normalized these levels. Time spent below sPO2 89% correlated with EPO levels (r=0.3434 and p=0.0403) indicating that episodes of hypoxia augmented erythropoiesis. However, Hb remained normal, leading us to hypothesize that augmented erythropoietic stimulus in OSA is counterbalanced by either 1) increase of hemolysis or 2) erythropoietic resistance to EPO. Hemolysis was detected in some but not all OSA patients by end-tidal carbon monoxide (ETCO), a product of heme catabolism; however, the instrument's limited sensitivity precluded detection of mild hemolysis. We previously showed that hypoxia-increased RBCV is transiently overcorrected upon rapid return to normoxia by preferential destruction of young red cells -neocytolysis. Neocytolysis is caused by excessive accumulation of reactive oxygen species (ROS) from increased retention of mitochondria in reticulocytes and decreased catalase in young RBCs (1). In our OSA subjects, we found that mitochondrial mass and mitochondrial ROS were higher in reticulocytes. Further, ROS were higher in B- cells, T-cells, monocytes, and granulocytes compared to controls. After CPAP treatment, these values normalized and catalase mRNA and enzyme activity also increased. These results support neocytolysis as one of the factors that prevent the increase of Hb in OSA. OSA also induces inflammation, a factor known to inhibit erythropoiesis, in part by induction of hepcidin which inhibits release of iron from macrophages. IL-6 and TNF transcript levels in granulocytes were higher in OSA compared to controls and normalized after CPAP treatment. Hepcidin levels were higher in OSA compared to controls. Hepcidin levels were correlated with inflammatory markers, IL-6 levels were inversely correlated with EPO levels, and iron and transferrin saturation levels were lower in OSA, inversely correlating with elevated hepcidin. The normal Hb does not rule out the presence of polycythemia, as increased RBCV could be offset by concomitant increase in plasma volume resulting in a normal Hb concentration. This was recently demonstrated in Tibetan Sherpas living in hypoxic high altitude who have normal Hb but increased RBCV and plasma volume (PV) (2). One can determine RBCV and PV by inhaling known amount of CO and measuring increments of carboxyhemoglobin together with Hb and hematocrit. We recently obtained the required instrumentation (OpCO, DETALO INSTRUMENTS, Denmark), and are currently correlating our clinical and biochemical measurement with OSA subjects' RBCV and PV to either confirm presence of hypoxia-induced polycythemia or conclusively rule it out. We conclude that the absence of elevated Hb in OSA is attributable not only to neocytolysis but also to the suppression of erythropoiesis by inflammation. 1) Song J, Yoon D, Christensen RD., Horvathova M,Thiagarajan P, and Prchal JT. HIF mediated increased ROS from reduced mitophagy and decreased catalase causes neocytolysis. J Mol. Med., (Berl). 2015 Aug., PMID: 26017143. 2) Stembridge M et all. The overlooked significance of plasma volume for successful adaptation to high altitude in Sherpa and Andean natives. Proc Natl Acad Sci U S A.2019 Jul 29. [Epub ahead of print]. Disclosures Lundby: Detalo: Other: CEO and founder. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership.

Hypoxia alters cellular metabolism and chemosensory endocrine regulation. Hypoxia increases production of erythrocytes in order to improve oxygen delivery to tissues. Organisms respond differentially to duration and patterns of hypoxia. Acute hypoxia stabilizes transcription factors' a-subunits of hypoxia inducible factors (HIFs; HIF-1a and HIF-2a) while in chronic hypoxia HIF-2a continues to be augmented, but HIF-1a decreases (1, 2). There are two clinical patterns of hypoxia: chronic sustained hypoxia (CSH) and chronic intermittent hypoxia (CIH). CSH is present in people living at high altitudes, patients with respiratory diseases, and Eisenmenger complex. CIH is characterized by repeated series of hypoxia followed by normoxia exposure and best exemplified by obstructive sleep apnea (OSA). The hematological responses to CSH and CIH has not been compared and studied in detail. We studied 2 separate hypoxic mouse cohorts: 1) CSH (12% O2 for 24 hours/day for 33 days), and 2) CIH emulating human OSA (8% O2 for 30 sec followed by 21% O2 for 90 sec; 8 hours/day for 33 days) and nonhypoxic controls. Hematocrits (Hct) were higher in both CSH and CIH compared to controls. In CSH, Hct increased by 22.0% (p Hepcidin (encoded by Hamp) is the principal regulator of iron metabolism; its expression is decreased by hypoxia, iron deficiency, and augmented erythropoiesis. It suppresses erythropoiesis by inhibiting release iron from macrophages. We measured Hamp transcript in the liver. Despite presence of hypoxia, both CSH and CIH had higher Hamp transcripts. These results are consistent with co-existent suppression of erythropoiesis in both CSH and CIH, but more so in CSH. Since inflammation induces hepcidin expression, we measured inflammatory marker transcripts in granulocytes and found increased Il-6 transcripts in both CSH and CIH; Il-1a levels increased only in CSH. As Epo transcripts were decreased in the kidney, we also measured inflammatory markers` transcripts in kidney. The Ccl2, Il-10 and Il-6 transcripts were higher in both CSH and CIH. These results suggest that increased inflammation in CSH and CIH suppresses erythropoiesis via increase of hepcidin. We also measured reactive oxygen species (ROS) in blood cells. The amount of mitochondrial ROS and the number of mitochondrial ROS positive reticulocytes increased in CSH and CIH and correlated with mitochondrial mass. We previously showed that excessive ROS associated with increased mitochondrial mass is due to reduced Bnip3L (the mediator of mitophagy) and decreased catalase upon normoxic return from CSH (2). Bnip3L and Cat (encodes catalase) were also decreased in both CSH and CIH in reticulocytes, which correlated with their increased mitochondrial mass and ROS. We conclude that in CIH there is an increased hematocrit in mouse OSA model which contrasts with human OSA where Hct is not increased. Our data support that the increased Hct occurs in CSH and mouse CIH at the early stages of hypoxia, but that further increases of Hct is prevented by inflammation and hepcidin. The high Hct is further contributed by recent demonstration of prolonged mouse erythrocyte survival in hypoxia (4). Further, in this OSA mouse model increased ROS is restricted to reticulocytes but not present in leukocytes, unlike in human OSA (3). This demonstrates some limitation of using mouse as a model for human OSA, but also some similarities. Excessive ROS in reticulocytes due to increased mitochondrial mass and decreased catalase (due to a decrease of HIF-1a) may explain some features of pathophysiology of CSH and CIH (2). 1. Uchida et al, JBC 2004 2. Song et al, JMM 2015 3. Song et al," Normal Hemoglobin Concentrations in OSA", abstract this meeting 4. Song et al, High Alt Med Biol. 2019 Disclosures No relevant conflicts of interest to declare.

Background: Myelofibrosis (MF) is associated with bone marrow fibrosis (BMF), splenomegaly, a high symptom burden, and poor prognosis; the JAK/STAT pathway is the central pathway implicated in its pathogenesis. Ruxolitinib, a JAK1/2 inhibitor and the only FDA-approved pharmacotherapy for treatment (Tx) of MF patients (pts), improves splenomegaly, but is unable to control all clinical manifestations of disease. Navitoclax is an orally bioavailable, novel small-molecule that targets and binds with high affinity to multiple antiapoptotic B-cell lymphoma 2 (BCL2) family proteins, including BCL-XL, BCL2, and BCL-W. Preclinical studies have demonstrated cytotoxic activity of navitoclax against myeloproliferative neoplasm-derived cell lines. Herein, the results of a phase 2 study (NCT03222609) evaluating the combination of navitoclax with ruxolitinib in pts with MF are reported. Methods: This phase 2 single-arm, multicenter, open-label study assessed the efficacy and safety of navitoclax combined with ruxolitinib in pts with MF. Eligible pts (≥18 yr, diagnosis of primary MF, post-essential thrombocythemia [PET]-MF, or post-polycythemia vera [PPV]-MF, ECOG 0-2, receiving at least 12 wk of continuous ruxolitinib therapy prior to study Tx initiation) received a starting dose of 50 mg navitoclax once-daily combined with the current stable dose of ruxolitinib (≥10 mg BID). Weekly intra-patient dose-escalation of navitoclax was allowed to a maximum daily dose of 300 mg based on tolerability and platelet count. Tx continued until the end of clinical benefit, unacceptable toxicity, or discontinuation. The primary efficacy endpoint was percentage reduction in splenic volume from baseline. Secondary endpoints included effect on total symptom score (TSS), overall response rate, rate of anemia response, improvement in BMF, and safety profile. Results: As of May 1, 2019, 34 pts (primary MF, n=16; PET-MF, n=5; PPV-MF, n=13) had received ≥1 dose of navitoclax in combination with ruxolitinib. Median age was 68 yr (range 42-86), 68% were male, and 9 pts (26%) had ≥3 prior lines of MF therapy. The median duration of prior ruxolitinib exposure was 745 days (range 134-4549). Of the 34 pts enrolled, 27 (79%) had JAK2 and 7 pts (21%) had CALR mutations. There were no pts enrolled with triple-negative MF. Of 33 pts with available baseline testing, 17 (52%) had high molecular risk, defined by mutations within ASXL1, EZH2, IDH1/2, SRSF2, or U2AF1. The mean baseline platelet count was 231 x 109/L (range 99-706); mean baseline Hgb was 10.8 g/dL; 19 (56%) pts had elevated WBC at baseline (>1.5× ULN). Maximal navitoclax dose of 300 mg was achieved in 23 pts (68%). Of the 25 (74%) pts that enrolled on ruxolitinib doses >10 mg BID, 22 (88%) subsequently had the dose of ruxolitinib reduced to 10 mg BID. At the time of this analysis, 24 pts were evaluable for efficacy, with 20 pts completing ≥24 wk on study and 4 pts with Tx discontinuations prior to 24 wk. At wk 24, 7 of 24 pts (29%) achieved a spleen volume reduction of ≥35% (SVR35) from baseline by MRI as determined by prespecified central review; the median TSS was 7.4 (range 0-23), a 20% improvement from baseline. A SVR35 at any time on study was achieved in 10 pts (42%). Reductions in driver mutation allelic burden of >5% were observed in 10 (42%) pts; 6 pts (25%) had BMF improvement of ≥1 grade. One pt (4%) had an anemia response; the mean Hgb at wk 24 was slightly improved over baseline at 11.3 g/dL. Of the 19 pts with elevated baseline WBC, 16 (84%) reduced to within normal limits during Tx, with a median WBC reduction of 17.7 × 109/L. All pts experienced a Tx-emergent adverse event (TEAE); most common (≥20%) were thrombocytopenia (82%), diarrhea (62%), fatigue (53%), anemia (27%), nausea (27%), contusion (24%), and vomiting (21%). Grade ≥3 TEAEs occurred in 26 pts (77%); most common were thrombocytopenia (n=15, 44%; Grade 4 n=1, 3%) and anemia (n=8, 24%; no Grade 4). Five pts (15%) experienced serious AEs that resolved including anemia, upper abdominal pain, vomiting, chest pain, increased C-reactive protein, and abnormal liver function test (3% each). There were no significant episodes of bleeding and no TEAE-related deaths. Conclusions: Navitoclax in combination with ruxolitinib was well tolerated with clinically meaningful spleen responses, allelic burden reductions, TSS improvements, and encouraging improvements in BMF in pts with MF who have received prior Tx with ruxolitinib. Disclosures Harrison: CTI: Speakers Bureau; Promedior: Honoraria; Roche: Honoraria; Celgene: Honoraria, Speakers Bureau; Gilead: Speakers Bureau; Shire: Speakers Bureau; Incyte: Speakers Bureau; Sierra Oncology: Honoraria; Janssen: Speakers Bureau. Garcia:Genentech: Research Funding; Abbvie: Research Funding. Mesa:CTI: Research Funding; Galena Biopharma: Consultancy; Samus: Research Funding; Genotech: Research Funding; AbbVie: Research Funding; NS Pharma: Research Funding; Baxalta: Consultancy; LaJolla: Consultancy; Shire: Honoraria; PharmaEssentia: Research Funding; Genentech: Consultancy; Celgene Corporation: Research Funding; AOP Orphan Pharmaceuticals: Honoraria, Other: travel, accommodations, expenses; Promedior: Research Funding; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses; Incyte: Other: travel, accommodations, expenses, Research Funding; Gilead Sciences: Research Funding; Pfizer: Research Funding; Sierra Oncology: Consultancy. Somervaille:Novartis: Consultancy. Komrokji:pfizer: Consultancy; celgene: Consultancy; DSI: Consultancy; Incyte: Consultancy; Agios: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau. Pemmaraju:sagerstrong: Research Funding; celgene: Consultancy, Honoraria; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; samus: Research Funding; novartis: Consultancy, Research Funding; plexxikon: Research Funding; incyte: Consultancy, Research Funding; abbvie: Consultancy, Honoraria, Research Funding; mustangbio: Consultancy, Research Funding; cellectis: Research Funding; affymetrix: Research Funding. Papadantonakis:Agios: Consultancy, Honoraria. Foran:Agios: Honoraria, Research Funding. O'Connell:Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Shionogi: Membership on an entity's Board of Directors or advisory committees. Holes:AbbVie Inc: Employment, Other: Stock/stock options. Jia:AbbVie: Employment, Other: Stock/stock options. Harb:AbbVie Inc: Employment, Other: Stock/stock options. Hutti:AbbVie: Employment, Other: Stock/stock options.

Hydroxyurea is usually the first line treatment for high-risk polycythemia vera (PV) and essential thrombocythemia (ET), but interferon is an equally effective treatment. However, unlike hydroxyurea, several studies have shown higher rate of molecular response with pegylated interferon-alpha (PegINFa) and tolerable toxicity. We had reported earlier that in some female patients, polyclonal hematopoiesis is restored (Liu E et al, Blood, 2003) after interferon therapy. However, the mechanism of action of interferon on rescue of normal dormant hematopoiesis in PV and ET is not fully understood. Presence of immune response against PV & ET clone is long speculated and we had previously screened human testis cDNA library in plasma from a PV subject who converted to polyclonal hematopoiesis after interferon therapy, where presence of immune response were noted. We analyzed 48 patients diagnosed with PV and ET per WHO 2012 criteria who were treated with PegINFa at our institution. The majority of patients receiving PegINFa - 28 (58%) were second-line treatments after receiving hydroxyurea. The overall hematological response rate for PegINFa treated patients was 46 (95.8%), but 14 (29%) had to discontinue due to various toxicities. Of the 48 patients, 31 had evaluable JAK2V617F allelic burden at least at 2 time points, and the overall molecular response rate was 45% (14 of 31) with a median time of 24 months. In 4 (12.9%) patients, JAK2V617F allelic burden decreased to In this pilot study, using plasma samples from 7 PV patients collected before and after treatment with PegINFa, we screened for antibody responses to a panel of 16 commercially available and 11 custom generated recombinant tumor-associated antigens (TAA) by enzyme-linked immunosorbent assay (ELISA) as previously described (Luetkens T et al., Leukemia Research 2010). In brief, we coated 96-well MaxiSorp (Nunc) plates over night at 4°C with 5mg/ml recombinant protein, blocked plates in 4% non-fat milk PBS (M-PBS) and incubated antigens with patient sera diluted 1:100 in M-PBS for 2h at room temperature. Bound antibodies were detected using a secondary anti-human immunoglobulin G antibody linked to alkaline phosphatase (Southern Biotech) followed by development with p-nitrophenyl phosphate. In patients #1, #2 and #3, who showed a decrease in JAK2V617F allelic burden after PegINFa treatment, we observed antibody responses against antigens MAGEB1, MAGEH1, MAGEA1, and DKK1 before and after PegINFa treatment. In patients #4, #5 and #7, JAK2V617F allelic burden increased after PegINFa treatment, and we did not detect substantial immune responses against any antigens in our panel. In patient #6, no immune response was seen against any antigen on the panel even though there was a decrease in JAK2V617F allele burden. These preliminary data suggest that PegINFa can induce deep molecular responses in a subset of PV and ET patients, and is associated with presence of immune responses against tumor antigens. We speculate that PegINFa may augment the efficacy of pre-existing TAA-specific immune responses against PV and ET clone. We are in the process of validating these findings in a larger cohort of PV and ET patients treated with PegINFa. Disclosures No relevant conflicts of interest to declare.

Sickle cell anemia (SCA) is associated with an increased hypoxic response from anemia and vaso-occlusion-impaired tissue perfusion. The effects of hypoxia are mediated by hypoxia transcription factors (HIFs). Chuvash erythrocytosis (CE) is an inherited condition due to homozygosity for the missense mutation in VHL gene (VHLR200W) that impairs interactions of VHL with HIF-α subunits, thereby augmenting transcription of HIF-regulated genes. CE and SCA share increased expression of erythropoietin (EPO) and other HIF target genes. As HIF-regulation of transcription is tissue and differentiation-stage specific, in this study we used reticulocytes, which are easily accessible and purified peripheral blood erythroid cells. We compared the transcriptomes of SCA and CE reticulocytes to differentiate HIF-mediated dysregulation from non-hypoxic dysregulation of SCA transcripts. Our study revealed reticulocyte gene expression changes that are common to both diseases as well as SCA-specific changes. The reticulocytes were purified from 5 HbSS and 5 age- and gender-matched HbAA African-American individuals, and from 17 VHLR200W homozygotes from Chuvashia and 13 wild type Chuvash individuals. Total RNA was depleted of ribosomal RNA and globin transcripts, and reverse transcribed. Strand-specific libraries were constructed for 100 or 125 bp paired-end sequencing to 30-45 million reads using Illumina HiSeq 2500 or 4000 platform. The sequencing data were mapped to human reference genome version GRCh37 using the splice-aware aligner STAR and analyzed using DESeq2. In separate analyses of the two diseases, we identified 1435 genes differentially expressed in SCA among 6965 analyzed genes, 848 increased and 587 decreased in SCA relative to control individuals. We also identified 1498 genes differentially expressed in CE among 8989 analyzed genes, 862 increased and 636 decreased in CE relative to control individuals. Across all analyzed genes, there was a moderate correlation (r=0.30) of expression changes between the two diseases. Among genes differentially expressed, 258 up-regulated and 155 down-regulated genes overlapped between the two diseases, representing a 1.4-fold enrichment. In a combined analysis of the two diseases, we identified 1228 genes among 6924 analyzed genes that shared altered regulation in both diseases. The 693 genes increased in both diseases were enriched (adjusted P < 0.05) in multiple metabolic, inflammatory, and oxidative pathways. The 535 genes decreased in both diseases were enriched in a cell cycle pathway. Among the commonly increased genes, the expression level of ERFE encoding erythroferrone was increased by 9.4-fold in CE and by 4.3-fold in SCA, suggesting markedly altered iron regulation in CE and SCA. This would be expected as both SCA and CE share upregulated erythropoiesis that is associated with augmented erythroferrone. Among the commonly decreased genes, RPL3L encoding Ribosomal Protein L3 Like was decreased by 68% in CE and by 93% in SCA. These common expression changes reflect hypoxic regulation related to chronic anemia in SCA. To assess disease-specific gene expression change, we tested the disease (SCA versus CE) by genotype (mutation versus wildtype) interaction effect. We identified 822 genes that showed disease-specific expression changes among 6924 analyzed genes. Of these disease-specific genes, 304 were increased and 153 decreased in SCA (adjusted P 0.05). For example, SLC16A1, encoding Solute Carrier Family 16 Member 1 and associated with an erythrocyte lactate transporter defect, was increased in SCA by 11-fold whereas PPBP encoding Pro-Platelet Basic Protein was decreased in SCA by 92%; both genes showing no change in CE. The 304 genes with SCA-specific increased expression were enriched in "Thyroid hormone signaling" (5.1 fold) and "Glioma" (7.0-fold) pathways. The 153 genes with SCA-specific decreased expression were enriched in "Ribosome" (12-fold) pathway. Among the disease-specific genes, only 34 increased and 28 decreased in CE but not in SCA. Our study demonstrates high HIF transcriptional activity in both CE and SCA reticulocytes but also reveals hypoxia-independent gene expression changes in SCA reticulocytes. These results suggest that HIF might be a therapeutic target of SCA. These data also shed light on the different molecular mechanisms underlying SCA complications. Disclosures Gordeuk: Pfizer: Research Funding; Modus Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding.

Background Interferons are recognized as active agents in the treatment of patients with high risk essential thrombocythemia (ET) or polycythemia vera (PV), both in the upfront setting as well as beyond. Several trials have shown high rates of hematologic and molecular responses with the use of interferons, however, data on direct comparison of interferon activity in patients with early disease in comparison to patients refractory or resistant to prior therapies, such as hydroxyurea (HU) are lacking. We conducted a controlled analysis of the activity of pegylated interferon alfa-2a (PEG) in two prospective parallel clinical trials conducted in these two unique patient populations. Methods The MPD-RC 111 (NCT01259817) was an international, multicenter, phase 2 open-label clinical trial that evaluated PEG therapy in patients with high risk PV and high-risk ET who were either refractory or intolerant (R/I) to HU by modified ELN criteria. The MPD-RC 112 trial (NCT01258856) enrolled patients with high risk ET/PV who were treatment-naïve (TN) (HU Results Patients ET: 39 TN and 65 R/I ET patients were available for this analysis. Median disease duration was 2.9 months in TN and 37.3 months in R/I patients. Baseline characteristics and demographics were similar in the two cohorts except lower baseline hemoglobin level in RI patients. (Table1A) PV: 43 TN and 50 R/I PV patients were included. Median disease duration was 2.5 months in TN and 54.8 months in R/I patients. Baseline characteristics only differed by lower frequency of phlebotomy rate in R/I patients. (Table1B) Baseline symptoms scores and quality of life were similar in TN and RI groups (Table 2) Response ET: CR/PR/ORR at 12 months were observed in 43.1%/26.2%/69.2% in R/I ET patients and in 43.6%/25.6%/69.2% in TN ET patients (p=0.99 for ORR). (Table 3, Figure 1) PV: CR/PR/ORR at 12 months were observed in 22%/38%/60% in R/I PV patients, and in 27.9%/58.1%/86% in TN PV patients (p=0.005 for ORR). (Table 3, Figure 1) Safety PEG was equally well tolerated throughout both treatment groups with treatment discontinuation due to adverse events occurring in 14.6% in TN patients and 13.9% in R/I patients. The mean (SD) dose of PEG was 102.7 (52.3) mcg in R/I ET patients and 128.7mcg (46.4) in R/I PV patients. For TN patients, mean dose was 85.7mcg (59.7) in ET and 93.5 mcg (44.0) in PV. Adverse events were consistent with historic reports of PEG use and the distribution of events was similar in R/I and TN patients. (Table 4) Conclusion This intention to treat response analysis included TN and R/I ET and PV patients with balanced baseline characteristics who received prospective therapy with PEG. Patients with ET had a higher overall response rate at 12 months that was equivalent in patients who were treatment-naïve and in patients who were intolerant or refractory to HU. By contrast, patients with PV who were treatment-naïve had a higher ORR than patients those intolerant or refractory to HU. We conclude that treatment with PEG is an effective therapeutic option both treatment naïve PV and ET as well as those previously treated with HU, however PEG as a second line agent is especially effective in ET patients. Disclosures Yacoub: Hylapharm: Equity Ownership; Agios: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Seattle Genetics: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau; Ardelyx: Equity Ownership; Cara: Equity Ownership; Dynavax: Equity Ownership. Mascarenhas:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding. Mesa:AbbVie: Research Funding; Samus: Research Funding; Incyte: Research Funding; Sierra Onc: Consultancy; Genotech: Research Funding; Promedior: Research Funding; Novartis: Consultancy; Celgene: Research Funding; CTI Biopharma: Research Funding; La Jolla Pharma: Consultancy. Rampal:Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy; Constellation, Incyte, and Stemline Therapeutics: Research Funding. Silver:PharmEssentia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. McMullin:Daiko Sanyo: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Speakers Bureau; Italopharma: Membership on an entity's Board of Directors or advisory committees. Ewing:Novartis: Honoraria, Other: Meeting attendance sponsorship ; Bristol Myers-Squibb: Other: Meeting attendance sponsorship . O'Connell:Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Shionogi: Membership on an entity's Board of Directors or advisory committees. Mead:Bristol Myers-Squibb: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel/accommodation expenses, Research Funding, Speakers Bureau; CTI: Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Pfizer: Consultancy. De Stefano:Alexion: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau. Baer:Astellas: Research Funding; Al Therapeutics: Research Funding; Abbvie: Research Funding; Incyte: Research Funding; Forma: Research Funding; Kite: Research Funding; Takeda: Research Funding. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kremyanskaya:La Jolla: Consultancy; Incyte, Celgene, Constellation, Protagonist.: Research Funding. Hexner:novartis: Research Funding. Rambaldi:Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, travel support. Ritchie:Genentech: Other: Advisory board; Tolero: Other: Advisory board; agios: Other: Advisory board; Pfizer: Other: Advisory board, travel support; Celgene: Other: Advisory board; Jazz Pharmaceuticals: Research Funding; Celgene, Novartis: Other: travel support; AStella, Bristol-Myers Squibb, Novartis, NS Pharma, Pfizer: Research Funding; Ariad, Celgene, Incyte, Novartis: Speakers Bureau; Celgene, Incyte, Novartis, Pfizer: Consultancy. Kiladjian:Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding; Celgene: Consultancy. Harrison:Promedior: Honoraria; Incyte: Speakers Bureau; Sierra Oncology: Honoraria; Celgene: Honoraria, Speakers Bureau; Janssen: Speakers Bureau; CTI: Speakers Bureau; AOP: Honoraria; Shire: Speakers Bureau; Roche: Honoraria; Gilead: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Hoffman:Merus: Research Funding. OffLabel Disclosure: Pegylated Interferon Alfa-2a for in Patients with Polycythemia Vera or Essential Thrombocythemia

In this issue of Blood, Pilli and colleagues1 report that the essential antithrombotic factor protein S is inversely regulated by the transcription factor hypoxia inducible factor 1 (HIF1). This is an important contribution to our understanding of the molecular basis of the augmentation of thrombosis by hypoxia.

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors.

In the first days of life, low grade jaundice is essentially universal. The source of the elevated bilirubin level giving rise to "physiological jaundice of the newborn" is only partly known. We hypothesized that it is, at least in part, the result of active and specific hemolysis involving a physiological mechanism to lower the high fetal hematocrit, appropriate for the relatively low oxygen environment in utero, to a lower level appropriate for the state of oxygen abundance after birth. We tested this by quantifying end tidal carbon monoxide (ETCO) as a marker of the rate of heme metabolism to bilirubin. We found that ETCO values of 20 neonates and children with known hemolytic disorders were higher than 20 age-matched healthy controls (p

Anemia is common in older persons and is associated with substantial morbidity and mortality. One third of anemic older adults have unexplained anemia of the elderly (UAE). We carried out a randomized, wait list control trial in outpatients with UAE and serum ferritin levels between 20 and 200ng/mL. Intravenous iron sucrose was given as a 200-mg weekly dose for 5weeks either immediately after enrollment (immediate intervention group) or following a 12-week wait list period (wait list control group). The primary outcome measure was changed in 6-minute walk test (6MWT) distances from baseline to 12weeks between the two groups. Hematologic, physical, cognitive, and quality of life parameters were also assessed. The study was terminated early after 19 subjects enrolled. The distance walked in the 6MWT increased a mean 8.05±55.48m in the immediate intervention group and decreased a mean 11.45±49.46m in the wait list control group (p=0.443). The hemoglobin increased a mean 0.39±0.46g/dL in the immediate intervention group and declined a mean 0.39±0.85g/dL in the wait list control group (p=0.026). Thus, a subgroup of adults with UAE may respond to intravenous iron. Enrollment of subjects into this type of study remains challenging.

Overexpression of transcription factors runt-related transcription factor 1 (RUNX1) and nuclear factor, erythroid-derived 2 (NF-E2) was reported in granulocytes of patients with polycythemia vera and other myeloproliferative neoplasms (MPNs). Further, a transgenic mouse overexpressing the NF-E2 transgene was reported to be a model of MPN. We hypothesized that increased transcripts of RUNX1 and NF-E2 might characterize other polycythemic states with primary polycythemic features, that is, those with exaggerated erythropoiesis due to augmented erythropoietin (EPO) sensitivity. We tested the expression of RUNX1 and NF-E2 in polycythemic patients of diverse phenotypes and molecular causes. We report that RUNX1 and NF-E2 overexpression is not specific for MPN; these transcripts were also significantly elevated in polycythemias with augmented hypoxia-inducible factor activity whose erythroid progenitors were hypersensitive to EPO. RUNX1 and NF-E2 overexpression was not detected in patients with EPO receptor (EPOR) gain-of-function, suggesting distinct mechanisms by which erythroid progenitors in polycythemias with defects of hypoxia sensing and EPOR mutations exert their EPO hypersensitivity.

In congenital Chuvash polycythemia (CP), VHL(R200W) homozygosity leads to elevated hypoxia inducible factor (HIF) levels at normoxia. CP is often treated by phlebotomy resulting in iron deficiency, permitting us to examine the separate and synergistic effects of iron deficiency and HIF signaling on gene expression. We compared peripheral blood mononuclear cell gene expression profiles of eight VHL(R200W) homozygotes with 17 wildtype individuals with normal iron status and found 812 up-regulated and 2120 down-regulated genes at false discovery rate of 0.05. Among differential genes we identified three major gene regulation modules involving induction of innate immune responses, alteration of carbohydrate and lipid metabolism, and down-regulation of cell proliferation, stress-induced apoptosis and T-cell activation. These observations suggest molecular mechanisms for previous observations in CP of lower blood sugar without increased insulin and low oncogenic potential. Studies including 16 additional VHL(R200W) homozygotes with low ferritin indicated that iron deficiency enhanced the induction effect of VHL(R200W) for 50 genes including hemoglobin synthesis loci but suppressed the effect for 107 genes enriched for HIF-2 targets. This pattern is consistent with potentiation of HIF-1α protein stability by iron deficiency but a trend for down-regulation of HIF-2α translation by iron deficiency overriding an increase in HIF-2α protein stability.

Evolutionary adaptations to high altitude in Tibetans, Ethiopians, and Andean populations of South America have shown that Tibetans and Ethiopians have normal hemoglobin %, while most of Aymara and Quechua of the Andean highlands are polycythemic. Whole genome sequencing (WGS) in Quechua identified enriched SENP1 and ANP32D genes correlating with polycythemia (Zhou et al, Am J Hum Genet. 2013 Sep 5; 93(3): 452-462) but these genes were neither enriched nor segregated with polycythemia in Aymara. Instead, we identified that genes enriched in Aymara are related to regulation of cardiovascular development in high-altitude adapted Andeans, BRINP3, NOS2, and TBX5 (Crawford et al, Am J Hum Genet. 2017 Nov 2;101(5):752-767). To further search for Aymara propensity to polycythemia, we analyzed transcriptomes from Aymara and Europeans living in La Paz, Bolivia (3,639-4,150m) from limited amount of peripheral blood reticulocytes, platelets and granulocytes, but only granulocyte RNA was adequate for unbiased whole transcriptome analyses. In Aymaras, 2,585 genes were upregulated and 365 genes were downregulated (Adjp2.0). Many of these modulated genes are involved in inflammatory pathways including B-cell activation (FDR=0.005) and NF-κB signaling pathway (FDR=0.011). We then analyzed differential exon usage in the transcriptome and identified 2,475 genes with alternative splicing events, comprising 1,568 exon skipping, 485 intron retention, 175 alternative 3' splice sites, 144 alternative 5' splice sites, and 902 mutually exclusive exons. These alternative spliced genes were also overrepresented in inflammatory pathways (TNF receptor, IL-1 and IL-23 mediated signaling, and NF-κB signaling). Notably we detected the previously unreported NFKB1 alternate transcripts skipping exon 4 or 5, which lead to the out-of-framed NFKB1 mRNA, generating the truncated nonfunctional NF-κB protein (Figure). Inflammation is a potent suppressor of erythropoiesis and the NF-κB is transcriptional regulator of plethora of inflammatory genes. Further, NF-κB also interacts with erythropoiesis-regulators, hypoxia-inducible factors (HIFs). By the integrative analysis of the Aymara transcriptome and WGS, we identified 46 NFKB1 splicing quantitative trait loci (sQTLs). Among these 46 sQTLs, five single nucleotide polymorphisms (SNP) were in high linkage disequilibrium, and two (rs230511 and rs230504) were more enriched in Aymara (allele frequency: 0.878) (Figure) and within a genomic region where Andeans are genetically differentiated from lowland Native Americans (peak FST = 0.37, peak PBSn1 = 0.31). These sQTLs rs230511 and rs230504 were corelated with two functionally important exon skipping (exon 4 and 5) in NFKB1 as described above. Furthermore, these two SNPs were correlated with higher hemoglobin levels and lower leukocytes; the wild-type NFKB1 transcript inversely correlated with hemoglobin%. We report Aymara have differentially expressed and alternatively spliced transcripts of genes modulating inflammation, particularly NFKB1. This Aymara enriched NFKB1 haplotype variant stands out as a major cause of Aymara adaptation to high altitude, as this truncated nonfunctional NF-κB variant peptide correlates with higher hemoglobin, lower leukocytes and suppresses inflammation. These data indicate that NFKB1 SNPs enriched in Aymara are associated with alternative spliced NFKB1 transcripts which contribute to polycythemia in Aymara. Further evaluation of NF-κB and HIFs' transcriptional activity and their correlation with inflammatory makers, hepcidin and erythroferrone in Aymara and Europeans living at the same high altitude is under way. JS and SH contributed equally to this work. YL and JTP act as equivalent co-senior authors. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Introduction: JAK2 V617Fmutation is present in majority of polycythemia vera (PV) and in 50-60% of patients with primary myelofibrosis (MF). JAK1/2 inhibitor ruxolitinib (RUX) is approved for treatment of both PV and intermediate/high risk MF. Two independent randomized studies showed the benefit of RUX in MF with significant reduction in spleen volume and improvement in constitutional symptoms compared to best supportive care. Clonal evolution may occur in PV and MF upon acquisition of new mutations and transformation to acute myeloid leukemia. Rarely, patients with JAK2V617Fpositive PV or MF have also concomitant BCR-ABL1 fusion gene with phenotype of chronic myelogenous leukemia (CML). Imatinib, a BCR-ABL1 inhibitor, induces long-term cytogenetic and molecular remissions in CML. Single agent imatinib has shown no clinical benefit in MF patients despite in vitro efficacy (Gaikwad et al Exp. Hematol. 2007). We describe here a patient with JAK2V617F positive post-PV MF who later developed concomitant BCR-ABL+ CML. Splenomegaly was initially resolved by RUX but returned coincidently with the diagnosis of CML and was again normalized by imatinib alone. Massive splenomegaly reappeared along with elevated hematocrit on imatinib and was resolved by the addition of low dose RUX. Case report: A 71-year old woman initially presented in September 2011 with isolated erythrocytosis and splenomegaly. A diagnosis of PV was established based on the presence of the JAK2V617Fmutation and hypercellular marrow. Bone marrow karyotype was normal (Table 1). The patient was randomized to pegylated interferon arm in the MPD Consortium study. A complete hematologic response was achieved after nearly 2 years on maximal dose 180 mcg weekly. In early 2015, she developed constitutional symptoms, and progressive splenomegaly. Progression to post-PV MF was confirmed on bone marrow evaluation. The patient was taken off study and started on RUX 20 mg BID. She developed severe anemia 4 months later requiring dose reduction to 10 mg BID. The anemia markedly improved, while JAK2V617F allele burden remained high at 88% with normal cytogenetics. In December 2017, the patient developed anemia, fatigue and rapid regrowth of spleen. Marrow karyotype showed Philadelphia (Ph) chromosome in 25% of the cells analyzed, PCR was positive for BCR-ABL1 p210, and JAK2V617F allele burden was reduced to 56.4%. In January 2018, she started imatinib 400 mg daily and RUX was discontinued due to toxicity concerns. In April 2018, her hematocrit increased and massive splenomegaly returned. Restart of initial RUX 20 mg BID was followed by severe pancytopenia which normalized when RUX was reduced to 5 mg BID with continuing imatinib regimen. After 8 weeks, splenomegaly completely resolved (Figure 1). A complete cytogenetic response and major molecular response were achieved after 3 months and 6 months of combination therapy respectively. Methods: Mononuclear cells from peripheral blood collected in June 2018 were plated in methylcellulose without erythropoietin or other cytokines. After 14 days, colonies were plucked, with ½ of each colony used for JAK2V617F allele-specific PCR and ½ prepared as cytospins for BCR-ABL fluorescent in situ hybridization (FISH) with the Vysis LSI BCR/ABL Dual Color, Dual Fusion Translocation Probe from Abbott Laboratories. Results: Analysis of 3-6 EPO-independent colonies of each CFU-E, G, M, GM, GEMM, BFU-E showed no BCR-ABL1 positive cells, while a single CFU-GEMM colony was positive and also homozygous for JAK2V617F. Frozen marrow cells from the time of the original diagnosis are being analyzed. Conclusion: The recurrence of splenomegaly in this patient following single agent RUX indicates emergence of novel BCR-ABL1+ clone. While single agent imatinib temporarily normalized the splenomegaly and leukocytosis for several months, MF phenotype reoccurred. Splenomegaly resolved completely in 8 weeks with the addition of very low dose RUX to standard dose imatinib, providing the first clinical observation of in vivo synergism of RUX and imatinib. We are investigating serial samples during her course, which will be presented at the meeting. The analysis of a single clone demonstrate that BCR-ABL1 mutation did not occur in previously normal dormant stem cell but in JAK2V617F positive progenitors and represent in this pts a novel subclone of previous PV/MF stem cell. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

Thrombosis is a major cause of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET). The mechanistic basis of thrombosis in PV/ET, however, is unknown. To better understand the pathophysiology of thrombosis in PV and ET, we first studied transcript levels of selected thrombotic, inflammatory and hypoxia- inducible factor (HIF) pathway genes in granulocytes and platelets of PV and ET patients with and without thrombosis. Genes selected for the study included: tissue factor (F3); P-selectin (SELP); serpin peptidase inhibitor clade E member 1 (SERPINE1, encoding plasminogen activator inhibitor I, PAI1); thrombospondin 1 (THBS1); interleukin 1 receptor associated kinase 1 (IRAK1); interleukin 1 receptor accessory protein (IL1RAP) and HIF-regulated genes: vascular endothelial growth factor A (VEGFA) and solute carrier family 2 (SLC2A1, encoding glucose transporter 1). We have previously reported at this meeting that PV and ET patients with a history of thrombosis had higher transcripts of F3, SERPINE1, IL1RAP, VEGFA and SLC2A1 compared to those without thrombosis. We also performed unbiased total RNA sequencing of platelets and granulocytes (Gangaraju R et al Blood, 2016;128:3143). Tissue factor (TF) is the principal initiator of coagulation in vivo. The presence of TF transcript in leukocytes and platelets may or may not reflect the translated protein level. Furthermore, TF functional activity is modulated by encryption. Therefore, we proceeded to evaluate the functional activity of microvesicle-associated TF (MVTF) in the plasma of 10 ET and 33 PV patients considered to have high thrombotic risk (Tefferi A, Barbui T Am J Hematol. 2017;92(1):94-108). TF activity was measured in MVs collected by centrifugation of patient plasma to 20,200 xg by a two-step FXa generation assay with and without an inhibitory TF antibody to determine the contribution of TF to FXa generation (Owens 3d et al. Circ Res. 2011;108(10):1284-97). We found significantly increased levels of MVTF activity in PV and ET compared to normal controls (Figure 1). However, MVTF levels in PV and ET patients with and without thrombosis were comparable (Figure 1). In the vasculature, leukocytes can synthesize TF (upon stimulation) and it has been shown that monocytes, not neutrophils, are the principal source of TF under normal conditions (Osterud B, Thromb Res. 2010;25 Suppl 1:S31-4). MPN granulocytes, in contrast to normal granulocytes, had increased levels of TF transcripts, a novel and important finding of as yet undetermined significance (Figure 2). Since TF synthesis is regulated by hypoxia-inducing factor-1 (HIF-1) (Rolfs et al. J Biol Chem. 1997;272(32):20055-62), we also examined MVTF activity in the plasma of Chuvash polycythemia (CP) patients. These patients have a germline VHLC598T mutation in the negative regulator of HIFs, the von Hippel Lindau gene (VHLC598T), and as a result they have increased levels of HIF-1, HIF-2 and transcripts of a vast array of HIF-regulated genes. CP subjects have an even higher propensity for arterial and venous thrombosis than PV. As predicted, some CP plasmas also demonstrated elevated levels of MVTF activity (Figure 1). In conclusion, hypoxia-induced increased levels of plasma MVTF activity may play a role in the increased thrombotic risk of PV and ET. TF joins TSP-1 (Sergeueva et al. Haematologica. 2017;102(5):e166-e169) and protein S (Pilly et al. Blood 2018;132(4):452-455) as a potential HIF-regulated mechanism of thrombotic risk in patients with PV/ET and CP. Granulocytes may also be a source of hypoxia-induced TF in these patients. The hypoxia-mediated upregulation of thrombotic risk is further underscored by the observation that PV patients living in moderate hypoxia at Salt Lake City have a higher risk of thrombosis than those living at sea level (Baltimore MD) in multivariate analysis (Zangari et al, Blood Coagul Fibrinolysis 2013; 24(3):311-316). The data described here may facilitate identification of novel targets and their therapies including the use of HIF-1 inhibitors such as digoxin to prevent thrombosis in these patients (Zhang et al. PNAS 2008;105(50):19579-86). Disclosures Key: UniQure BV: Research Funding.

Tibetans have been living at altitudes over 3500 m for ~20,000 years and have developed unique beneficial evolutionary genetic adaptions (PMID:28448578). Our previous study identified selected genetic haplotypes in two genes, EPAS1 (encoding hypoxia-inducible factor 2-alpha [HIF2-a], a transcription factor that mediates the hypoxic response), and EGLN1 (encoding prolyl hydroxylase 2 [PHD2], a principal negative regulator of HIF stability (PMID:25129147). The presence of these two haplotypes correlates with lower hemoglobin levels in Tibetans compared to other highlanders. However, the entire diverse complex of molecular mechanisms of high altitude adaptation is still largely unknown and our study showed that neither EPAS1 nor EGLN1 variants fully explain the mechanism of protection from polycythemia in Tibetans in high altitude (PMID:28233034). We found an enriched haplotype in the PKLR gene (encoding pyruvate kinase [PK] expressed only in liver and red blood cells). The PK enzyme is in the terminal portion of the glycolytic pathway, and its decreased activity leads to accumulation of proximal glycolytic intermediates, including 2,3-diphosphoglycerate (DPG) which shifts the hemoglobin dissociation curve to right (high p50) and increases oxygen release to tissues from a unit of hemoglobin. We hypothesized that Tibetan enriched PKLR variants might improve oxygen delivery to tissues and help explain the protection from polycythemia at high altitude. Genomic analyses revealed that this PKLR haplotype is enriched in Tibetans but is not unique to Tibetans. It has the highest frequency in Tibetans (89%), with a lower prevalence in Chinese and Mongolians (~77%), Kyrgyz (~60 %), Aymara (~44 %), and Colombians (~30 %) and a much lower frequency in Caucasians (11%), perhaps explaining the heterogeneity of responses to hypoxia within and among these populations. Our study of reticulocyte RNA showed that transcript levels of PKLR progressively decrease with increasing altitude in controls and even more in Tibetans with the Tibetan evolutionary selected PKLR haplotype. Tibetans with the PKLR haplotype (heterozygotes and homozygotes) have lower PKLR transcript levels than wild type Tibetans. Because of the paucity of wildtype PKLR haplotype in Tibetans and the challenges of acquiring Tibetan samples in high altitude in China, we collected samples from 125 m (Cheboksary, Chuvashia); 800 m (Bishkek, Kyrgyzstan) and 2640 m (Bogota, Colombia). PK activity, PKLR transcript levels, and ATP decreased at 2640 m compared to 800 m, while p50 increased at 2640 m. PKLR transcript levels correlated with PK activity and ATP and inversely correlated with p50. PK activities also correlated with PKLR transcript levels and ATP and inversely correlated with p50. At 2640 m, PK activity was lower and p50 levels were higher in those with the enriched PKLR haplotype. These results demonstrate that increasing altitude decreases PK activity, resulting in increasing p50 providing a molecular basis for the previously reported improvement of oxygen delivery at high altitude (PMID:17394415). To study the roles of HIFs in the regulation of PKLR gene expression, we also collected samples from Chuvash polycythemia (CP) homozygotes and Chuvash controls. CP homozygotes have a mutation in the VHL gene, a negative regulator of HIFs, that results in augmented HIF levels. CP homozygotes had lower PKLR mRNA in reticulocytes, PK activity, and PKR protein levels in red blood cells compared to controls, while their 2,3 DPG levels were higher. These data confirm that PKLR expression levels are negatively regulated by HIFs. Our findings demonstrate that individuals in high altitudes have lower PKLR transcript levels and PK activity, resulting in high 2,3DPG and p50 which shifts the hemoglobin dissociation curve to right. This decreases affinity of hemoglobin for oxygen, which improves tissue oxygen delivery and as such is another mechanism in Tibetans that protects from high altitude polycythemia. We also demonstrate that HIFs negatively regulate PKLR expression, leading to better oxygen release from hemoglobin at high altitude. Disclosures Kosinski: Agios: Employment, Equity Ownership. Kung:Agios: Employment, Equity Ownership. van Wijk:RR Mechatronics: Research Funding; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative neoplasms (MPN) associated with morbidity and mortality resulting from thrombosis or transformation to myelofibrosis and/or secondary acute leukemia. The acquired somatic mutation JAK2V617Fhas been observed in 98% of PV patients, and around 50% of ET patients. Interferon is known to be an effective treatment for chronic MPNs, but unlike hydroxyurea (HU, Hydrea®), it has been shown to target the malignant clone by decreasing JAK2V617Fallelic burden, and in some patients restoring polyclonal hematopoiesis. (Liu et al, Blood. 2003;101:3294-3301). The covalent binding of polyethylene glycol to the interferon molecule, through pegylation, prolongs the half-life by lowering the clearance of interferon, thereby extending the duration of its therapeutic effects, permitting less frequent administration and fewer side-effects. Currently, there are two commercially available pegylated interferons in the U.S., peginterferon alfa-2b (PEG-Intron®, Merck) and peginterferon alfa-2a (Pegasys®, PEG, Roche), both with heterogeneous pegylation with multiple pegylation sites. Ropeginterferon-alfa 2b/P1101 (ROPEG) is a novel longer acting mono-pegylated recombinant proline-interferon alfa-2b which has shown good efficacy in PV and has successfully completed a phase III trial in PV patients comparing it against HU (PROUD-PV study) in Europe (ASH2016 Abstract;Blood. 2016;128:475). Earlier, we had enrolled 53 high risk PV and ET patients to the Myeloproliferative Disorders Research Consortium (MPD-RC) trials #111 (refractory to HU) and #112 (randomized to HU vs PEG)(www.clinicaltrials.gov; NCT01259856 and NCT01259817). After the completion of these trials, PEG was no longer supplied by the study and not adequately covered by insurance for many of our patients. Therefore, ROPEG was procured under an FDA-approved expanded access program and used as a substitute for those controlled on PEG, and these patients are being transitioned from PEG to ROPEG according to the dosing strategy shown in Table 1. Seven patients have been switched onto ROPEG as of writing this abstract (Table 2). After a mean follow-up of roughly 8 weeks (range: 2 - 10 weeks), all have maintained response to therapy and none have had a thrombotic event or new or increased interferon-related side effects thus far. Patient 002 experienced elevated AST/ALT while on PEG, which normalized after four q2 week doses of ROPEG (8 weeks of therapy). Patients 003 and 004 who had persistent fatigue on PEG, saw significant improvement of their symptoms after switching to ROPEG. In patient 005 who has ET, bone marrow cytogenetics at entry to PEG had a paracentric inversion of the long arm of chromosome 3, namely 3q21.3q26.2 (RPN1/MECOM, inv3; diagnostic of AML per WHO criteria), in 16/20 (80%) metaphases. Over the 4 ensuing years while on PEG, this cytogenetic abnormality progressively decreased. FISH assay was set up 2 years ago wherein 86/200 cells (46%) were positive with lowest achieved level of 36/200 cells (18%) without any evidence of emerging AML. Upon transition to ROPEG (2 fortnightly injections of 50 mcg), this response was maintained (23/200 cells scored (11.5%). Of the 3 patients who were JAK2V617Fpositive, one patient (patient 003) showed a decrease in the JAK2V617Fallelic burden (37% to 18%) after two months of ROPEG despite decreasing the dose to 50 mcg (from 100 mcg) 2 weeks after the first injection, while the other two patients had no statistically significant change in their JAK2V617Fallelic burden. Clonality will be followed in female subjects to see if clonal hematopoiesis is suppressed and normalizes with long-term administration of ROPEG. Our early experience suggests ROPEG is a safe and effective alternative to PEG permitting less frequent administration for patients with high risk PV and ET. Notable observations include normalization of hepatic transaminases previously induced by PEG, maintenance or even suppression of one highly deleterious cytogenetic abnormality, maintenance of ET & PV remission, and amelioration of interferon side-effects with lower equivalent doses. In the future, we intend to increase the pool of patients by designing a protocol for newly diagnosed high risk ET and PV with randomization to HU versus ROPEG. Disclosures No relevant conflicts of interest to declare.

Introduction Acquired severe aplastic anemia (SAA) is a low frequency hematologic disease associated with significant morbidity and mortality. The destruction by immunological mechanisms of hematopoietic progenitor cells appears to play a fundamental role in the pathophysiology of the disease, therefore, immunosuppressive treatment (IST) is one of the pillars of management. In Colombia and Latin-America there is lack of information about the characteristics of patients with SAA and the effectiveness of IST and other treatments. The main objective of this study was to assess the outcomes of a group of patients treated in different centers in Colombia during the last five years. Materials and methods This is a retrospective observational study, carried out within 6 institutions of 3 Colombian cities (Bogota, Medellín, Bucaramanga) of patients treated from 2013 to 2018. An anonymized database was constructed. All patients diagnosed with SAA and treated with IST as first line of therapy were included. Results We analyzed the data of 37 patients; 22 of them were women. The average age was 37 years old (with a range of 7-68 years). The median time since the onset of the symptoms to the time of diagnosis was 88 days (with a range of 7-655 days). Clastogenic effect of diepoxybutane (DEB) tests were available only in 9 patients (7 of which were positive); Paroxysmal Nocturnal Hemoglobinuria (PNH) clone was detected in 9 of the 27 tested, all of which were under 5%. Mean leukocytes, hemoglobin concentration, and platelets at the moment of diagnosis were 3.090, 8.5 and 22.000. Bacterial infection was the most important clinical manifestation at diagnosis (in 16 of the 37 patients); 19 out of 37 patients had more than 5 red blood cell transfusions before the first treatment. As first line therapy, 25 patients received IST (most by antithymocyte globulin (ATG) of which 10 were treated with rabbit ATG, 12 with horse ATG and 3 combined with Eltrombopag); 4 patients had cyclosporin and prednisone, 2 had allogeneic transplant and 6 received other therapies. Those treated with IST patients, 8 had a complete response (CR) (32%), 6 had partial response (PR) (24%), 8 had no response (32%). 5 received a second course of IST and 3 had CR. 3 patients were lost in follow up. At a mean follow up of 18.7 months (1.7-65), 44% were alive in CR, 26% in PR, 9% were in relapse and 21% died. Causes of death were SAA related infection in 5 patients, transplant related in 1 and non-disease related in 1. Conclusions This is the first multicenter report of SAA treatment in Colombia, it includes children and adults, resulting in a very heterogeneous population. This preliminary information shows that there is a significant delay in the diagnosis and initiation of treatment in patients, most patients have more than 5 transfusions before treatment. This could, in part, explain the apparent low response rates to IST in comparison to that of what have been reported in literature. A larger registry of hematologic pathologies is required so we can confirm these findings, start proposing strategies applicable in our country to achieve better outcomes in these patients and planning clinical trials to answer questions relevant to our population. Disclosures No relevant conflicts of interest to declare.

Background: Patients (pts) with polycythemia vera (PV) and essential thrombocythemia (ET) suffer from difficult MPN associated symptoms which impact quality of life (QoL). The impact of initial cytoreduction on ET/PV symptoms, resulting toxicity and QoL have not been prospectively studied. Methods: MPD-RC 112 trial (NCT01258856) randomized 168 therapy-naive (hydroxyurea [HU] Results: Patients: Of 168 randomized pts (86 HU, 82 PEG), 164 (98%) completed the survey package at baseline and 151 (HU 83%; PEG 98%) completed at least 1 survey during treatment. Median age was 61 (range 18-87) with 70 (42%) females; 81 (48%) / 87 (52%) with ET / PV; 46 (27%) had a history of a thrombosis. 119 (71%) received prior HU (up to 3 months). Baseline characteristics were balanced between arms with the exception of age (median HU 63 vs. PEG 60 yrs; p=0.02). Baseline Symptom Burden /QoL: Mean MPN-SAF Total Symptom Score (TSS), scale 0 [absent]-100 [worst imaginable] was 15.8 (SD 12.6; range 0-62.0) with means of 15.1 (SD 12.6) / 16.5 (SD 12.6) for ET / PV which were slightly lower than reported means of a previous cohort receiving any line of treatment (Scherber RM, JCO 2012). Median number of moderate (defined as score >=3) symptoms in ET patients was 3.0 and PV 4.0. Most common moderate symptoms were fatigue (65.9%), insomnia (37.2%), numbness (33.5%) , itching (29.9%), dizziness (27.4%), early satiety (26.8%) , headache (23.2%), sad mood (23.2%), inactivity (22%), concentration (20.7%). 50.6% of patients had one or more symptom with a score >=6. Mean QLQ-C30 global health status/QoL (GHS/QoL) was 70.8 (SD 21.9) - comparable to a general population (mean 71.2, SD 22.4) and better than a general cancer population (mean 61.3, SD 24.2; QLQ-C30 Reference Manual 2008). At baseline, TSS, symptoms, and QoL were similar between treatment arms. In patients with TSS >20 at baseline (highly symptomatic, n=50; ET:20, PV:30), median TSS was 28.0 (range 20.0 to 62.0); the highest scored symptom was fatigue (median=7.0), itching (median=4.5) and insomnia (median=4.0). Impact of Therapy on Symptom Burden/QoL: On HU, pts experienced worsening QoL (physical, cognitive functioning, HRQoL) and some persistent or transient worsened symptoms (inactivity, concentration (p 20, n=50), improvements in weight loss, abdominal discomfort, fatigue, itching, fever, early satiety (12 m.) were observed in pts on HU. For PEG, in highly symptomatic pts, improvements were observed for overall TSS score, fatigue, inactivity, night sweats, and itching (with similar toxicities to all PEG patients). ELN Response and Symptom Burden/QoL: Among the 133 pts with 12-mo symptom data, complete hematologic response (CHR) rate was 43%. Inactivity and sad mood were worse in those patient achieving a CHR (inactivity: CHR worsening 0.68 vs PHR/NR better 0.26; p=0.03; sad mood: CHR worsening 0.67 vs PHR/NR better 0.67, p=0.002)). These latter changes were not related to higher doses utilized (in either arm), suggesting obtaining CHR may have negative effects on patient symptoms. Conclusions:In pts with a significant baseline MPN symptom burden, cytoreductive therapy has both a beneficial impact on baseline MPN symptom burden but also leads to therapy associated toxicity, although different patterns of efficacy and toxicity between HU and PEG. Achievement of a CHR in PV/ET may be associated with higher rates of drug related toxicities, and the value of achieving CHR may need further validation. Disclosures Mesa: Incyte Corporation: Research Funding; CTI Biopharma: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Novartis: Consultancy; Celgene: Research Funding; UT Health San Antonio - Mays Cancer Center: Employment; Genentech: Research Funding; Pfizer: Research Funding. Mascarenhas:Merck: Research Funding; CTI Biopharma: 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; Roche: Research Funding; Promedior: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Janssen: Research Funding. Rambaldi:Omeros: Consultancy; Roche: Consultancy; Celgene: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Italfarmaco: Consultancy; Amgen Inc.: Consultancy. Yacoub:Novartis: Honoraria, Speakers Bureau; Cara Therapeutics: Equity Ownership; Ardelyx, INC.: Equity Ownership; Inycte: Honoraria, Speakers Bureau; Seattle Genetics: Honoraria, Speakers Bureau; Dynavax: Equity Ownership. Harrison:Gilead: Honoraria, Speakers Bureau; CTI BioPharma: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, 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:Elstar: Research Funding; Celgene: Research Funding; ARIAD: Consultancy; Cell Therapeutics: Consultancy; Evotek: Research Funding; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy. Kessler:Sangamo: Research Funding; Octapharma: 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; Novo Nordisk: Honoraria, Research Funding; Dimension Advisory boards: Membership on an entity's Board of Directors or advisory committees; Bayer: 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; Genentech: Research Funding; Biomarin: Research Funding. Kremyanskaya:Incyte: Research Funding. Rampal:Incyte: Honoraria, Research Funding; Constellation: Research Funding; Jazz: Consultancy, Honoraria; Stemline: Research Funding; Celgene: Honoraria. Hoffman:Incyte: Research Funding; Janssen: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Summer Road: Research Funding. Dueck:Pfizer: Honoraria; Bayer: Employment; Phytogine: Employment.

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.

Obstructive sleep apnea (OSA), characterized by intermittent hypoxia, causes cardiovascular, metabolic, neurocognitive and cancer complications. Hypoxia expands the red cell mass by stimulating erythropoietin (EPO) production; yet in our analysis of 527 OSA patients, It is also well-known that OSA induces systemic inflammation markers including C-reactive protein, IL-6, TNF-α, IL-8, and NF-κb. Inflammation participates in the control of the number of RBCs by inducing hepcidin, the principal regulator of iron metabolism. Increased hepcidin suppresses erythropoiesis by inhibiting iron release from macrophages. Based on this evidence, we also hypothesized that the absence of polycythemia in OSA might be caused by an independent contribution of inflammation-mediated suppression of erythropoiesis. We studied OSA patients before and after treatment with continuous positive airway pressure (CPAP). Increased erythropoiesis was evidenced by increased EPO and reticulocytosis. EPO levels correlated with time spent below sPO2 89 %, indicating that severe OSA patients had more augmented erythropoiesis. However, hematocrit levels were normal. Hemolysis was detected in some but not all OSA patients by end tidal carbon monoxide (a product from heme catabolism). After CPAP treatment, these changes diminished but hematocrits did not change. Conditions favoring neocytolysis were confirmed by increased ROS from expanded reticulocytes' mitochondria which correlated with time spent below sPO2 89 %. Downregulated catalase resulting from increased miR-21 was also detected. Also these changes normalized with CPAP. These results indicate that hemolysis of hypoxia-born RBCs prevents OSA patients from becoming polycythemic. Increased ROS was not only found in reticulocytes but also in leukocytes; these also normalized with CPAP. Expression of inflammatory markers (NFKB1, TNF, and IL6) in granulocytes was higher in OSA compared to controls and normalized by CPAP; these levels correlated with apnea-hypopnea Index (AHI). OSA patients had higher hepcidin levels, correlating with inflammatory marker levels and inversely correlated with EPO. Iron and transferrin saturation levels were lower in OSA compared to controls, inversely correlating with high hepcidin levels. These data indicated that besides neocytolysis, coexistent suppression of erythropoiesis by inflammation contributed to the lack of polycythemia in OSA. In OSA, inflammation mediated increase of ROS in leukocytes is a known causative factor of cardiovascular disease. We now report increase of both ROS and inflammatory markers in leukocytes. We conclude that the absence of polycythemia in OSA is the result of hemolysis via neocytolysis and inflammation-mediated suppression of erythropoiesis. Increased ROS in blood cells and systemic inflammation from OSA-constitute mechanisms likely contributing to the pathophysiology of OSA. Disclosures Ganz: Vifor: Consultancy; Ablynx: Consultancy; Keryx Pharma: Consultancy, Research Funding; Silarus Pharma: Consultancy, Equity Ownership; La Jolla Pharma: Consultancy, Patents & Royalties: Patent licensed to La Jolla Pharma by UCLA; Akebia: Consultancy, Research Funding; Intrinsic LifeScience: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy.

Germline von Hippel-Lindau (VHL) gene mutations underlie dominantly inherited familial VHL tumor syndrome comprising a predisposition for renal cell carcinoma, pheochromocytoma/paraganglioma, cerebral hemangioblastoma, and endolymphatic sac tumors. However, recessively inherited congenital polycythemia, exemplified by Chuvash polycythemia, has been associated with 2 separate 3' VHL gene mutations in exon 3. It was proposed that different positions of loss-of-function VHL mutations are associated with VHL syndrome cancer predisposition and only C-terminal domain-encoding VHL mutations would cause polycythemia. However, now we describe a new homozygous VHL exon 2 mutation of the VHL gene:(c.413CT):P138L, which is associated in the affected homozygote with congenital polycythemia but not in her, or her-heterozygous relatives, with cancer or other VHL syndrome tumors. We show that VHL(P138L) has perturbed interaction with hypoxia-inducible transcription factor (HIF)1α. Further, VHL(P138L) protein has decreased stability in vitro. Similarly to what was reported in Chuvash polycythemia and some other instances of HIFs upregulation, VHL(P138L) erythroid progenitors are hypersensitive to erythropoietin. Interestingly, the level of RUNX1/AML1 and NF-E2 transcripts that are specifically upregulated in acquired polycythemia vera were also upregulated in VHL(P138L) granulocytes.

We report a series of neonates who developed a total serum bilirubin (TSB) > 20 mg/dL during a recent ten-year period in a multihospital healthcare system. The incidence of a TSB > 20 mg/dL fell after instituting a pre-hospital discharge bilirubin screening program in 2003/2004 (91.3 cases/10,000 births before vs. 72.4/10,000 after), but the incidence has subsequently remained unchanged. No specific cause for the hyperbilirubinemia was identified in 66% of ( n = 32) cases with a TSB > 30 mg/dL or in 76% of ( n = 112) cases with a TSB 25.0–29.9 mg/dL. We hypothesized that hemolysis was a common contributing mechanism, but our review of hospital records indicated that in most instances these infants were not evaluated sufficiently to test this hypothesis. Records review showed maternal and neonatal blood types and direct antiglobulin testing were performed in > 95% cases, but rarely were other tests for hemolysis obtained. In the ten-year period reviewed there were zero instances where erythrocyte morphology from a blood film examination or Heinz body evaluation by a pediatric hematologist or pathologist were performed. In 3% of cases pyruvate kinase was tested, 3% were evaluated by hemoglobin electrophoresis, 3% had a haptoglobin measurement, and 16% were tested for G6PD deficiency. Thus, determining the cause for hyperbilirubinemia in neonates remains a problem at Intermountain Healthcare and, we submit, elsewhere. As a result, the majority of infants with a TSB > 25 mg/dL have no specific causation identified. We speculate that most of these cases involve hemolysis and that the etiology could be identified if searched for more systematically. With this in mind, we propose a “consistent approach” to evaluating the cause(s) of hyperbilirubinemia among neonates with a TSB > 25 mg/dL.

Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting more than 500 million people worldwide, is one of the most common of inherited disorders. There are 186 G6PD mutations published, with mutational clustering within defined ethnic/racial groups. However comprehensive molecular characterization of ethnically associated G6PD mutants and their clinical implications are lacking. Design and methods Eighty unrelated Palestinian children hospitalized for hemolysis were studied. G6PD activity was determined by quantitative spectrophotometry and G6PD mutations were analyzed by sequencing of gDNA. Results 65 of 80 children (81%) had G6PD deficiency, accounting for most of the hemolytic disease in this age group. G6PD Mediterraneanc.563T, African G6PD A-c.202A/c.376G, and G6PD Cairoc.404C were common with relative allele frequencies of 0.33 [1], 0.26, and 0.18 respectively. Two other variants were discovered, G6PD Beverly Hillsc.1160A mutation, and a novel G6PD missense mutation c.536G > A (Ser179Asn), designated G6PD “Gaza”. Three samples exhibited enzyme deficiency without detectable exonic or exon/intron boundary mutations. Conclusion G6PD deficiency accounts for the majority of diagnoses for hemolysis in Palestinian children (81%), providing support for newborn G6PD deficiency screening programs. We report unanticipated molecular heterogeneity of G6PD variants among Gaza Strip Palestinians greater than reported in neighboring Arab populations. We report a high proportion of affected children with G6PD Cairo, which was observed previously in only a single Egyptian, and a novel mutation G6PD “Gaza”.

During erythropoiesis, erythropoietin stimulates induction of erythroid transcription factors that activate expression of erythroid genes including the erythropoietin receptor (EPO-R) that results in increased sensitivity to erythropoietin. DNA binding of the basic helix-loop-helix transcription factor, TAL1/SCL, is required for normal erythropoiesis. A link between elevated TAL1 and excessive erythrocytosis is suggested by erythroid progenitor cells from a patient that exhibits unusually high sensitivity to erythropoietin with concomitantly elevated TAL1 and EPO-R expression. We found that TAL1 regulates EPO-R expression mediated via three conserved E-box binding motifs (CAGCTG) in the EPO-R 5' untranslated transcribed region. TAL1 increases association of the GATA-1·TAL1·LMO2·LDB1 transcription activation complex to the region that includes the transcription start site and the 5' GATA and 3' E-box motifs flanking the EPO-R transcription start site suggesting that TAL1 promotes accessibility of this region. Nucleosome shifting has been demonstrated to facilitate TAL1 but not GATA-1 binding to regulate target gene expression. Accordingly, we observed that with induced expression of EPO-R in hemotopoietic progenitor cells, nucleosome phasing shifts to increase the linker region containing the EPO-R transcription start site and TAL1 binds to the flanking 5' GATA and 3' E-box regions of the promoter. These data suggest that TAL1 binds to the EPO-R promoter to activate EPO-R expression and provides a potential link to elevated EPO-R expression leading to hypersensitivity to erythropoietin and the resultant excessive erythrocytosis.