Abstract: Introduction: Hematopoietic stem/progenitor cell differentiation is blocked in acute myeloid leukemia (AML) resulting in cytopenias and high risk of death. Most patients with AML become resistant to treatment due to lack of effective cytotoxic and differentiation fostering compounds. High expression of MN1 confers poor prognosis to AML patients and induces resistance to cytarabine and all-trans-retinoic acid (ATRA) induced differentiation. We thus set out to identify compounds which could potentially overcome the differentiation block in AML. Methods: Based on the above concepts and in an effort to identify novel compounds which are potent inducers of differentiation and apoptosis in AML, high-throughput drug screening was employed in the MN1 leukemic model. A total of 3580 bioactive compounds were tested in duplicate at a concentration of 2.5 µM using alamar blue fluorescence as readout. As MN1 cells are resistant to ATRA (at 1µM and even 10µM ATRA), the drug screening was performed in the presence of a clinically relevant dose of ATRA (1 µM) to identify compounds that concurrently act with the cytotoxic and/or differentiating effects of ATRA. To determine whether a compound was effective as monotherapy or if it synergized with ATRA, we also performed a validation phase study in which the IC50 of each candidate compound was tested alone and in combination with ATRA. Fifty-four inhibitors were chosen from the primary screen for further validation based on presumed mechanism of action and novelty. The shortlisted compound pyrimethamine (PMT) was validated for its differentiation and apoptosis promoting effects in various murine and human AML models. Results: Our high-throughput drug screening identified 117 compounds, which reduced MN1 leukemic cell proliferation by more than 80% above the ATRA-treated control in both replicates (inhibitors), 8 borderline inhibitors (one replicate with more than 80% inhibition and one with 74 to 80% inhibition), and 35 outliers, which inhibited cell proliferation by 80% or more in only one replicate. The biologic processes most frequently targeted by the 117 inhibitors were DNA replication (n=26), microtubule assembly (n=12), NF-kB pathway (n=8), dihydrofolate reductase (DHFR, n=3) and heat shock protein 90 (HSP90). Dihydrofolate reductase inhibitors, pyrimethamine and amethopterin/methotrexate emerged as top hits from the screening and preliminary validation studies. Validation studies identified the antifolate pyrimethamine (PMT) that potently induced apoptosis and differentiation in several murine and human leukemic cell lines when administered as a single agent. The cytotoxic effects of pyrimethamine were reversed by addition of an excess of folic acid whereas induction of myeloid differentiation at higher concentrations of pyrimethamine was not mediated through DHFR inhibition. We further evaluated the effect of pyrimethamine in an in vivo xenograft mouse model by subcutaneously inducing tumors with HL60 and THP1 cell lines. Oral pyrimethamine treatment significantly reduced tumor volumes after 14, 19 and 24 days post-transplantation and at death compared to solvent treated mice (P 〈 0.01). The effect of pyrimethamine was further assessed in primary human AML cells and normal CD34+ cells by CFC assays. Colony numbers from primary AML cells, but not normal CD34+ bone marrow cells, were significantly reduced by pyrimethamine as compared to solvent control. Thus, our study identifies pyrimethamine as a candidate drug that is a potent and specific inducer of apoptosis and differentiation with the property of specifically targeting leukemic cells. Conclusion: Our high-throughput drug screening identified pyrimethamine as a potent and specific antileukemic compound and reinforces targeting of folate metabolism as a treatment strategy in acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

Abstract: Vaso-Occlusive Crises (VOCs) in patients with SCD cause acute and chronic morbidity including disabling pain, hospitalizations, missed school and work, end-organ damage, and early mortality. VOC prophylaxis, while beneficial, does not address the disabling pain and morbidity of breakthrough VOC events that still occur. E-selectin upregulation on vascular endothelium leads to leukocyte trapping, activation and aggregation and is a critical driver of acute VOC (Morikis et al, Blood 2017). Rivipansel, a pan-selectin inhibitor with potent activity against E-selectin, prevents interaction between leukocytes and vascular endothelium (Morikis et al, Blood 2017) and increases blood flow by reducing cell-cell aggregates and vascular occlusion in a SCD mouse model (Chang et al, Blood 2010). A phase 2 study of rivipansel in VOC demonstrated shorter hospital stays and reduced opioid use (Telen et al Blood, 2015). The RESET trial (NCT02187003) was a phase 3, randomized, double-blind, placebo-controlled, study of the efficacy and safety of rivipansel for VOC requiring hospitalization. Three hundred forty-five patients (204 patients ≥18 and 141 patients 6-17 years of age) were randomized to an intravenous rivipansel loading dose, followed by up to 14 additional doses Q 12 hr, or placebo, in addition to standard care. Overall, 320 were treated: 162 with rivipansel, 158 with placebo. Pre-treatment variables were well balanced, including concomitant hydroxyurea use, genotype, chronic opioid use, gender, and country. The primary endpoint was time to readiness for discharge (TTRFD), and key secondary endpoints were time to discharge (TTD), time to discontinuation of IV opioids (TTDIVO), and cumulative IV opioid use (CIVO). Hazard ratios or ratios of medians, and confidence intervals were calculated for each. Both study arms had comparable baseline soluble E-selectin (sE-sel) levels and inflammatory/coagulation biomarkers. Median sE-sel in the rivipansel group decreased by 59% from baseline after a loading dose while increasing by 9% in the placebo group. Although the RESET study did not show statistically significant improvements in outcomes for the total population, additional analysis demonstrated that rivipansel treatment within 26.4 hr of pain onset (earliest quartile of duration of VOC until treatment) reduced median TTRFD by 56.3 hrs (from 122.0 to 65.7 hrs), reduced median TTD by 41.5 hrs (from 112.8 to 71.3 hrs), and reduced median TTDIVO by 50.5 hrs (from 104.0 to 53.5 hrs), compared to placebo. There was also a consistent trend for lower hazard ratios for rivipansel treatment earlier during VOC (up to ~36 hr from onset) for all endpoints (Figure 1). Pediatric subjects (6-17 yrs, n = 141) were 41% of patients treated in the RESET trial (71 in rivipansel arm, 70 in placebo arm). As in the overall population the observed benefit with rivipansel in pediatric subjects depended on duration of VOC prior to treatment. Children 6-17 yrs of age treated with rivipansel within 30 hrs of onset of VOC experienced reduction in median TTRFD by 29.3 hrs (from 94.1 to 64.8 hrs), reduction in median TTD by 23.2 hrs (from 92.8 to 69.6 hrs), and reduction in median TTDIVO by 15.4 hrs (from 68.9 to 53.5 hrs). Early treatment with rivipansel decreased median TTRFD by more than one day and led to more children ready for discharge by 24, 48, and 72 hrs, compared to patients receiving placebo (Table 1). Additionally, pain scale assessments (VAS and Faces scale) showed a substantial reduction in the time to first clinically meaningful reduction in pain (data not shown). Thus, the hazard ratios for the efficacy endpoints favored early rivipansel treatment over placebo in the overall population and the pediatric population (Table 2). Summary: Rivipansel administered early in VOC results in clinically meaningful benefit for adults and children with SCD, shortening IV opioid use and hospital stay. Biomarker data confirm on-target effect, suggesting that the diminishing effect of later rivipansel treatment results from downstream pathophysiology. These findings suggest the utility of early treatment to shorten or interrupt acute VOCs, analogous to thrombolysis for heart attack or stroke. This could change the VOC treatment paradigm from deferral of hospitalization to one of early intervention to reduce length of hospitalization and IV opioid requirement, relieve VOC symptoms, and possibly mitigate end-organ damage from tissue ischemia. Disclosures Dampier: Merck: Research Funding; Hudson Publishing Company: Consultancy, Research Funding; CLS Behring: Consultancy, Other: DSMB Chair; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Micelle Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclerion: Consultancy, Other: DSMB Chair; Novartis: Research Funding. Telen:GlycoMimetics Inc.: Consultancy; Forma Therapeutics: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wun:GlycoMimetics, Inc.: Consultancy; Pfizer, Inc.: Other: Steering Committee for clinical study, Research Funding. Smith:Incyte: Other: Investigator; Pfizer: Consultancy; Ironwood: Consultancy; Novo Nordisk: Consultancy; Imara: Research Funding; Shire: Research Funding; NHLBI: Research Funding; Patient-Centered Outcomes Research Institute: Other: Investigator, Research Funding; Health Resources and Services Administration: Other: Investigator, Research Funding; Shire, Inc.: Other: Investigator, Research Funding; Global Blood Therapeutics, Inc.: Consultancy, Research Funding; Novartis, Inc.: Consultancy, Other: Investigator, Research Funding; Emmaeus Pharmaceuticals, Inc.: Consultancy; GlycoMimetics, Inc.: Consultancy. Brown:Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Forma Therapeutics, Inc,: Research Funding; Imara, Inc.: Consultancy, Research Funding; Novartis, Inc.: Consultancy, Research Funding. Desai:Pfizer, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; GBT, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ironwood Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Rockpointe Continuing Medical Education Company: Consultancy. El Rassi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclerion: Research Funding; Pfizer: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bluebird bio: Membership on an entity's Board of Directors or advisory committees. Kanter:Wells Fargo: Honoraria; Medscape: Honoraria; Guidepoint Global: Honoraria; NHLBI Sickle Cell Advisory Board: Membership on an entity's Board of Directors or advisory committees; SCDAA Medical and Research Advisory Board: Membership on an entity's Board of Directors or advisory committees; AGIOS: Membership on an entity's Board of Directors or advisory committees; BEAM: Membership on an entity's Board of Directors or advisory committees; Jeffries: Honoraria; GLG: Honoraria; Sanofi: Consultancy; bluebird bio, inc: Consultancy, Honoraria; Novartis: Consultancy; Cowen: Honoraria. Pastore:Pfizer: Honoraria. Rothman:Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Agios Pharmaceuticals: Honoraria, Research Funding. Readett:Pfizer, Inc.: Current Employment. Lozier:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Thackray:GlycoMimetics, Inc.: Current Employment. Hassell:Pfizer, Inc.: Other: RESET Study Steering Committee; Pfizer, Inc.: Research Funding.

Abstract: Abstract 845 Background: Transfusion therapy can effectively treat many complications associated with sickle cell disease (SCD), but iron overload will develop without iron chelation therapy. Despite long-term transfusion requirements, long-term data for iron chelation in SCD are limited. The oral iron chelator, deferasirox, effectively reduced iron burden in SCD patients aged ≥2 years during 1 year of treatment (Vichinsky et al. Br J Haematol 2007). This 5-year follow-up is the first report of long-term deferasirox treatment in SCD patients. Methods: Eligible patients that completed the 1-year core study (randomized to deferasirox [deferasirox cohort] or deferoxamine [crossover cohort] ) entered the 4- year extension. All received deferasirox while continuing their regular transfusion regimen. Deferasirox dose in the extension was initially based on serum ferritin (SF) trends in the core study (deferasirox cohort) or transfusion requirements (crossover cohort). Dose adjustments were based on monthly SF and safety assessments (investigator-reported adverse events [AEs] and centrally processed lab parameters). Growth and sexual development (Tanner staging) were assessed annually. Results: Of the patients in the deferasirox (n=132) and crossover (n=53) cohorts that received ’1 deferasirox dose during the core or extension, 43 (33%) and 19 (36%) patients, respectively, completed the extension. Reasons for discontinuation included withdrawal of consent (n=44, 23.8%), loss to follow-up (n=17, 9.2%) and AEs (n=14, 7.6%). Three deaths occurred, all in the extension (intraventricular hemorrhage, n=2; intracranial bleed post liver transplantation, n=1); none considered by investigators to be deferasirox-related. Mean dose during the study was 18.7 ± 6.5 and 21.2 ± 5.3 mg/kg/day in the deferasirox and crossover cohorts, respectively. Investigator-assessed drug-related AEs (≥5% overall) included nausea (14.6%), diarrhea (10.8%), increased blood creatinine (5.9%) and vomiting (5.4%). Generally, these AEs were manageable and transient, and decreased in frequency over time. Serious AEs were reported in 70.8% of patients overall and were mostly related to the underlying disease. Serious investigator-assessed drug-related AEs were reported in 8 patients (6.1%) in the deferasirox cohort and 1 patient (1.9%) in the crossover cohort. In the deferasirox and crossover cohorts respectively, 9 (6.8%) patients and 1 (1.9%) patient had 2 consecutive serum creatinine level increases 〉 33% above baseline and 〉 upper limit of normal (ULN). Median creatinine clearance remained stable within normal range throughout the study. One patient from each cohort had alanine aminotransferase (ALT) 〉 10 × ULN on 2 consecutive visits; both had ALT values ≤ULN at the start of deferasirox treatment. In 37 patients with data available before and after dose increases to ≥30 mg/kg/day, no clinically relevant differences were observed in AE profile or laboratory parameters before and after dose increase. Deferasirox had no adverse effect on pediatric growth and adolescent sexual development. Overall, median SF levels in patients who received deferasirox treatment for ≥4 years decreased significantly from 3410 to 3108 ng/mL at end of study (median absolute change, –591 ng/mL, P=0.027; n=67). Decreases in SF were more pronounced when mean deferasirox dose increased to 〉 20 mg/kg/day (Figure 1). In the deferasirox cohort, the median absolute change in SF levels from start of deferasirox to end-of-study was greater in patients aged ≥16 than 2– 〈 16 years (–476 vs –156 ng/mL) reflecting low 1st-year deferasirox doses and conservative dose escalation in pediatric patients. Conclusions: This is the first study to report that deferasirox can significantly decrease SF levels over the long-term in SCD patients without evidence of renal toxicity. Treatment for up to 5 years was associated with a clinically manageable safety profile, without progressive decreases in median creatinine clearance, which remained within normal range. The modest, but statistically significant, decrease in SF is most likely due to under-dosing in the first 2 years and variability in SF related to disease factors. Patients titrated to an appropriate dose had a clinically relevant decrease in SF, highlighting the need to adjust dose, usually to 〉 20 mg/kg/day, to achieve negative iron balance. Disclosures: Vichinsky: Novartis: Consultancy, Research Funding, Speakers Bureau; Apotex: Consultancy, Research Funding; Hemaquest: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bernaudin:Novartis: Investigator for SCD deferasirox (Exjade). Forni:Novartis: Research Funding. Gardner:Novartis: Research Funding. Hassell:Novartis: Research Funding. Heeney:Novartis: Research Funding. Kutlar:Novartis: Research Funding. Lane:Novartis: Research Funding. Mathias:Novartis: Research Funding. Porter:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Tebbi:Novartis: Speakers Bureau. Wilson:Novartis: Honoraria, Research Funding, Speakers Bureau. Griffel:Novartis: Employment. Deng:Novartis: Employment. Giannone:Novartis: Employment. Coates:Novartis: Research Funding, Speakers Bureau.