Abstract: A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite ( R )-2-hydroxyglutarate (2HG). We developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia cells in vitro. These data provide proof-of-concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.

Abstract: Squalene epoxidase (SQLE), also known as squalene monooxygenase, catalyzes the stereospecific conversion of squalene to 2,3( S )-oxidosqualene, a key step in cholesterol biosynthesis. SQLE inhibition is targeted for the treatment of hypercholesteremia, cancer, and fungal infections. However, lack of structure-function understanding has hindered further progression of its inhibitors. We have determined the first three-dimensional high-resolution crystal structures of human SQLE catalytic domain with small molecule inhibitors (2.3 Å and 2.5 Å). Comparison with its unliganded state (3.0 Å) reveals conformational rearrangements upon inhibitor binding, thus allowing deeper interpretation of known structure-activity relationships. We use the human SQLE structure to further understand the specificity of terbinafine, an approved agent targeting fungal SQLE, and to provide the structural insights into terbinafine-resistant mutants encountered in the clinic. Collectively, these findings elucidate the structural basis for the specificity of the epoxidation reaction catalyzed by SQLE and enable further rational development of next-generation inhibitors.

Abstract: Introduction: Somatic mutations in the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 are found in multiple solid and hematologic tumors and drive tumorigenesis via production of the oncometabolite D-2-hydroxyglutarate (2-HG). The mutant IDH1 (mIDH1) inhibitor ivosidenib is approved in the US for the treatment of adults with mIDH1 newly diagnosed acute myeloid leukemia (AML) in patients ≥75 years old or with comorbidities that preclude induction chemotherapy, and mIDH1 relapsed or refractory (R/R) AML. In patients with R/R AML who were enrolled in a phase 1 study (NCT02074839), ivosidenib was associated with an overall response rate of 42%, a complete remission (CR) rate of 24%, and a CR plus CR with partial hematologic response (CR+CRh) rate of 32%, with durable responses and additional clinical benefits observed (Pollyea et al. J Clin Oncol 2018 Abs). Baseline co-occurring mutations in receptor tyrosine kinase (RTK) pathway genes were shown to be associated with primary resistance to ivosidenib (DiNardo et al. N Engl J Med 2018). Case studies of secondary resistance to ivosidenib monotherapy have revealed examples of 2-HG restoring mechanisms at relapse, including IDH2 isoform switching and the emergence of mIDH1-S280F (Harding et al. Cancer Discov 2018; Intlekofer et al. Nature 2018). Aim: To characterize the mechanisms of response and relapse to ivosidenib monotherapy via a comprehensive genomic analysis of samples from a large cohort of patients with mIDH1 R/R AML enrolled in the pivotal phase 1 study whose starting dose was 500 mg daily (approved dose). Methods: Baseline and longitudinal co-occurring mutation profiling was performed on whole bone marrow, bone marrow mononuclear cell, and peripheral blood mononuclear cell samples by next generation sequencing (NGS; detection sensitivity 1-5%). 3D modelling of second-site IDH1 mutations was performed with an ivosidenib analog using Molecular Operating Environment 10.0 software. Enzymatic assays were performed with mIDH1 as previously reported. The clinical response data cut-off was Nov 10, 2018. Results: In 167 patients with baseline NGS data from whole bone marrow, the genes with most frequent co-occurring mutations were DNMT3A (35%), NPM1 (26%), SRSF2 (24%), ASXL1 (18%), RUNX1 (18%), NRAS (14%), and TP53 (13%). RTK pathway mutations, and mutations in the individual genes NRAS and PTPN11, were significantly associated with a lack of CR or CRh response to ivosidenib (p & lt;0.001 [RTK], p=0.004 [NRAS] , and p=0.016 [PTPN11], Fisher's exact test). Investigation of clonal hierarchy at baseline showed that neither subclonal nor clonal status of mIDH1 was associated with clinical response (p=0.4, Fisher's exact test). To characterize mechanisms of secondary resistance to ivosidenib, longitudinal NGS profiling was performed for 129 patients, 74 of whom had data at both baseline and relapse or disease progression. Mutations not detected at baseline but identified at the time of relapse/progression were categorized into canonical biological pathways. Mutations in RTK pathway genes were identified at relapse/progression in 20 of 74 (27%) patients, with IDH1/2 mutations detected at a comparable rate (17 [23%] ; Table). IDH2-R140Q mutations were newly detected during treatment in 9 of 74 patients (12%). IDH1 second-site mutations emerged in 10 of 74 patients (14%), including previously unreported mutations at 5 amino acid positions. Detection of these IDH1 second-site mutations was concurrent with an increase in plasma 2-HG. Five patients acquired both RTK pathway mutations and 2-HG restoring mutations (IDH1 and/or IDH2) at relapse/progression. All identified second-site IDH1 mutations were clustered into two sub-regions and were predicted by 3D modeling to impact ivosidenib and/or co-factor (NADPH) binding either directly or indirectly. Biochemical characterization of two of these mutations confirmed loss of ivosidenib inhibitory potency. Conclusions: Relapse to ivosidenib monotherapy is mediated by diverse mechanisms that include emergent mutations, most frequently in RTK pathway genes and IDH1/IDH2. The observation of frequent 2-HG restoration at relapse underscores the key role of 2-HG production in mIDH AML disease. These results are important for the rational design of combination treatment strategies in mIDH1 AML, and to understand whether these patterns are replicated in ongoing combination trials. Disclosures Choe: Agios: Employment, Equity Ownership. Wang:Agios: Employment, Equity Ownership. DiNardo:syros: Honoraria; abbvie: Consultancy, Honoraria; medimmune: Honoraria; celgene: Consultancy, Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; agios: Consultancy, Honoraria; jazz: Honoraria; daiichi sankyo: Honoraria. Stein:PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees. De Botton:Agios: Consultancy, Research Funding; Celgene: Consultancy, Speakers Bureau; Pierre Fabre: Consultancy; Servier: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Forma: Consultancy, Research Funding; Syros: Consultancy; Bayer: Consultancy; AbbVie: Consultancy; Daiichi Sankyo: Consultancy; Astellas: Consultancy. Fathi:Amphivena, Kite, Jazz, NewLink Genetics,: Honoraria; Agios, Astellas, Celgene, Daiichi Sankyo, Novartis, Takeda, Amphivena, Kite, Forty Seven,Trovagene, NewLink genetics, Jazz, Abbvie, and PTC Therapeutics: Consultancy. Tallman:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; UpToDate: Patents & Royalties; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; UpToDate: Patents & Royalties; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Cellerant: Research Funding; Cellerant: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Biosight: Research Funding; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; UpToDate: Patents & Royalties; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Cellerant: Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; UpToDate: Patents & Royalties; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties. Kantarjian:Daiichi-Sankyo: Research Funding; Novartis: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Jazz Pharma: Research Funding; Takeda: Honoraria; Immunogen: Research Funding; Ariad: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; BMS: Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Agios: Honoraria, Research Funding. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Novartis, Agios, Arog: Research Funding; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: . Quek:Celgene: Research Funding, Speakers Bureau; Agios: Research Funding. Konteatis:Agios: Employment, Equity Ownership. Dang:Agios: Employment, Equity Ownership. Zhang:Agios: Employment, Equity Ownership; Agios: Employment, Equity Ownership. Liu:Agios: Employment, Equity Ownership. Attar:Aprea Therapeutics: Employment; Agios: Employment, Equity Ownership. Wu:Agios: Employment, Equity Ownership.

Abstract: BACKGROUND Pyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by deficiency of the glycolytic enzyme red cell PK (PK-R). AG-348 is an orally available, small molecule, allosteric activator of PK-R that activates wild-type and a range of mutated PK-R enzymes in vitro, and increases PK-R activity and restores adenosine triphosphate (ATP) levels in red blood cells from patients with PK deficiency ex vivo. AIMS To describe data from the ongoing DRIVE PK study (NCT02476916), an open-label, dose-ranging trial of AG-348 in transfusion-independent adults with PK deficiency. Early data from this study have been reported (Grace et al. EHA 2016, S466). METHODS After providing informed consent, patients are randomized to AG-348 50 mg or 300 mg orally twice daily (BID) for 6 months. Transfusion independence is defined as no more than 3 units of red blood cells transfused in the 12 months preceding first dose and no transfusions in the 4 months preceding first dose. Patients are followed weekly for Weeks 1-3, then every 3 weeks until Week 12, and then monthly until Week 24. Sex hormone levels and iron status are evaluated at Baseline, Week 12 and End of Study. RESULTS As of the data cutoff date of June 20, 2016, 25 patients have been enrolled and treated for ≥3 weeks. Adverse events (AEs) were mild to moderate. The most frequent AEs were nausea (n=11/25), headache, and insomnia (each n=8/25). One serious AE (Grade 2 osteoporosis) has been reported. Grade 3 AEs were hypertension (n=1), hypertriglyceridemia (n=1), insomnia (n=2), and anemia (n=1). One patient was discontinued from treatment because anemia worsened. There were no Grade 4 AEs and no deaths. Three patients had dose reductions because of headache, nausea, or insomnia. Serum levels of sex steroids were measured at baseline, 12 and 24 weeks; increases in free and total testosterone, and decreases in estradiol and estrone, indicate aromatase inhibition by AG-348, consistent with previously reported results. In 25 patients with ≥3 weeks on treatment, 13 patients (52%) showed a hemoglobin (Hgb) increase 〉 1.0 g/dL (range: 1.2-4.9 g/dL; median: 3.40 g/dL). Three patients had dose reductions because of Hgb values exceeding the protocol-mandated maximum. Hgb responses were typically observed within 2 weeks of treatment and were stable with continued treatment. Two patients had dose increases because of insufficient response to 50 mg. Maximal change in Hgb from baseline according to genotype is shown in Figure 1. Of the 25 enrolled patients with ≥3 weeks on treatment, 6 (24%) had two non-missense mutations and none of these had an Hgb response 〉 1.0 g/dL. Of the 19 patients with at least one missense mutation, 13 (68%) had an Hgb increase exceeding 1.0 g/dL. CONCLUSION AG-348 is a novel, first-in-class, PK-R activator in clinical testing as a disease-altering therapy to improve anemia in patients with PK deficiency. The ongoing DRIVE PK study has now enrolled over 25 patients, and data from additional patients will be available at the time of presentation. Daily dosing with AG-348 is well tolerated and has demonstrated clinically relevant durable increases in Hgb in the majority of the patients with at least 1 missense mutation. Out of the 19 patients with at least one missense mutation, 13 had an Hgb increase of 〉 1.0 g/dL. These data highlight the potential of AG-348 as the first disease-altering treatment for patients with PK deficiency. Figure 1 Maximum hemoglobin (Hgb) increase by genotype Figure 1. Maximum hemoglobin (Hgb) increase by genotype Disclosures Grace: Agios Pharmaceuticals: Other: Scientific Advisor, Research Funding. Rose:Novartis: Honoraria; Celgene: Honoraria; Genzyme: Honoraria. Layton:Novartis: Other: Advisory board meeting; Alexion: Other: Advisory board meeting; GSK: Other: Advisory board meeting; Agios: Speakers Bureau. Yaish:Baxalta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Consultancy; Octapharma: Consultancy. Barcellini:Agios: Consultancy. Kuo:Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Apotex: Other: Unrestricted education grant; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Silver:Agios Pharmaceuticals, Inc.: Consultancy. Merica:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kung:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Cohen:Agios Pharmaceuticals, Inc.: Consultancy. Yang:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Hixon:Agios Pharmaceuticals, Inc.: Equity Ownership, Other: Employment (former); KSQ Therapeutics: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Dang:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yuan:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Barbier:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Glader:Agios Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Abstract: Pyruvate kinase deficiency (PKD) is an autosomal recessive enzymopathy that is the most common cause of hereditary nonspherocytic hemolytic anemia (HNSHA). PKD is a rare disease characterized by a life-long chronic hemolysis with severe co-morbidities. It is hypothesized that insufficient energy production to maintain red cell membrane homeostasis promotes the chronic hemolysis. Treatment is generally palliative, focusing on the resultant anemia, and there are no approved drugs that directly target mutated pyruvate kinase. Here, we describe the mechanism of action and cellular effects of AG-348, an allosteric activator of the red cell isoform of pyruvate kinase (PKR). Hundreds of mutant alleles of PKR have been identified and are known to have deleterious effects on catalytic activity, protein stability, or protein expression. We demonstrate that AG-348 can potently activate a spectrum of recombinantly expressed PKR mutant proteins, including mutations that span distinct subdomains of the enzyme. The R532W mutation is quite sensitive to AG-348 modulation, with over 4-fold activation of the enzyme activity, even as the mutation renders PKR insensitive to stimulation by its endogenous allosteric regulator fructose 1,6-bisphosphate (FBP) (Figure A). Crystallographic analysis reveals that very few mutations associated with PKD occur within the AG-348 binding pocket, accounting for its broad activity. The binding of AG-348 attenuates the thermostability defect of several mutant alleles of PKR, including the commonly observed R510Q mutant that has a half-life of ∼2% of the half-life of wild-type PKR when incubated at 53°C. Pre-incubation of the R510Q protein with AG-348 restores the half-life to ∼70% that of the wild-type enzyme (Figure B). PKD red cells are characterized by changes in metabolism associated with defective glycolysis, including a build-up of the PKR substrate phosphenolpyruvate (PEP) and deficiency in the PKR product adenosine triphosphate (ATP). PKD red cells from several patients with distinct compound heterozygous PKR mutations exposed to AG-348 had increased PKR enzyme activity (up to 4-fold over control) and showed consistent dose and time-dependent metabolic responses (Figure C), including sharp reductions in PEP (up to 70% compared to control) and increases in ATP levels (up to 100% over control). These responses were observed in patient samples harboring PKR mutations that we had studied biochemically (including R486W and R510Q), but also in an instance where the mutation had not previously been biochemically characterized (A495V). In these ex-vivo settings, ATP levels in AG-348 treated cells can reach levels that are typical of normal, non-PKD red cells. These data support the hypothesis that drug intervention with AG-348 may restore glycolytic pathway activity and normalize red cell metabolism in vivo. This therapeutic approach may be an effective way to correct the underlying pathology of PKD and, importantly, provide clinical benefit to patients. Disclosures: Kung: Agios Pharmaceuticals: Employment, Equity Ownership. Hixon:Agios Pharmaceuticals: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals: Employment, Equity Ownership. Histen:Agios Pharmaceuticals: Employment, Equity Ownership. Hill:Agios Pharmaceuticals: Employment, Equity Ownership. Si:Agios Pharmaceuticals: Employment, Equity Ownership. Kernytsky:Agios Pharmaceuticals: Employment, Equity Ownership. Chen:Agios Pharmaceuticals: Employment, Equity Ownership. DeLaBarre:Agios Pharmaceuticals: Employment, Equity Ownership. Clasquin:Agios Pharmaceuticals: Employment, Equity Ownership. Ho:Agios Pharmaceuticals: Employment, Equity Ownership. Salituro:Agios Pharmaceuticals: Employment, Equity Ownership. Popovici-Muller:Agios Pharmaceuticals: Employment, Equity Ownership. Agresta:Agios Pharmaceuticals: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals: Employment, Equity Ownership. Dang:Agios Pharmaceuticals: Employment, Equity Ownership.

Abstract: Background. Sickle cell disease (SCD) is a devastating disorder initiated by polymerization of the deoxy-hemoglobin S (HbS) to form fibers that distort (sickle) erythrocytes. Increased intracellular 2,3- diphosphoglycerate (2,3-DPG) stabilizes fibers and promotes sickling, while decreased intracellular adenosine triphosphate (ATP) levels can lead to hemolysis. Mitapivat (AG-348) is an oral, small molecule, allosteric activator capable of activating both mutant and wild type red cell pyruvate kinase (PKR), thus decreasing 2,3-DPG and increasing ATP levels in red cells and potentially acting as an anti-sickling agent. The safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of multiple ascending doses of mitapivat were assessed in subjects with SCD in a Phase I study. Methods. Subjects aged ≥ 18 years with a confirmed diagnosis of SCD (HbSS), adequate organ function, baseline Hb ≥ 7 g/dL, and no transfusions or erythropoietin therapy in the prior 3 months were eligible. Concomitant stable hydroxyurea (HU) and/or L-Glutamine therapy was permitted on study. Subjects received either 3 or 4 ascending dose levels of mitapivat (5 mg BID, 20 mg BID, 50 mg BID, 100 mg BID) for 2 weeks' duration each, followed by a 12-15-day drug taper. The primary endpoint was safety and tolerability as assessed by frequency and severity of adverse events (AEs) and laboratory parameters. Secondary endpoints included changes in hematological parameters, 2,3-DPG and ATP levels, and markers of Hb S polymerization (p50 and t50). Results. Nine subjects have been enrolled as of July 2020. One patient discontinued treatment early and was lost to follow-up. Results pertain to the 8 subjects who completed study: mean age was 43.6 years (range 34-55 years), 5 were male, and 7 were stabilized on HU. The initial 6 subjects escalated to a maximum dose of 50 mg BID, and the subsequent 2 subjects escalated up to a 100 mg BID dose following a protocol amendment. All 8 subjects reported AEs; the most common treatment related AEs were hypertension (n=3, Grades 1-3), insomnia (n=3, Grade 2), and asymptomatic heart rate increase on vital signs (n=3, Grade 1). 5 serious AEs (SAEs) were reported in 4 subjects, with only 1 SAE possibly attributable to study drug - a vaso-occlusive crisis (VOC) during drug taper, after which the protocol was amended to increase the length of taper from 9 to 12 days for the 50 mg BID dose and 15 days for the 100 mg BID dose. No VOCs have occurred during core period or on the prolonged drug taper. The SAEs not attributable to study drug included a VOC at the end of study, VOC-related transaminitis (Grade 2), a non-VOC-related pain episode, and a pre-existing pulmonary embolism discovered shortly after study drug initiation. Table 1 details the PD and clinical laboratory measures. Mean 2,3-DPG levels decreased and ATP levels increased in a dose-dependent manner. ATP levels remained elevated immediately following drug taper in 5/8 subjects. Both ATP and 2,3-DPG levels returned to near baseline by 4 weeks after drug taper. Decreases in p50 and increases in t50 at various dose levels support an anti-sickling effect of mitapivat in a subset of subjects, though responses were highly variable. Mitapivat increased Hb levels in a dose-dependent manner with a concomitant decrease in hemolytic markers. Mean change in Hb from baseline for all 8 subjects at the 50 mg BID dose level was 1.2 g/dL (range -0.3-2.7 g/dL), followed by near return to mean Hb baseline after drug taper. Hemolytic markers similarly returned to near baseline after drug taper. 3/8 and 5/8 subjects achieved a Hb increase of ≥ 1 g/dL at the 20 and 50 mg BID dose levels, respectively. A small dose-dependent increase in mean corpuscular volume (MCV) was also observed, suggesting a potential alternative mechanism of mitapivat, acting through the ATP-dependent Gardos channel to increase red cell hydration, decrease hemolysis, and improve red cell survival in SCD. Conclusion. Mitapivat demonstrated an acceptable safety profile across the tested dose levels in 8 subjects with SCD. Analyses of data show promising evidence of efficacy in terms of Hb increase from baseline with concomitant decreases in hemolytic markers. The accompanying changes in metabolites and sickling studies are consistent with the proposed mechanism of the drug. The study is ongoing with a planned sample size of 15 subjects completing 6-8 weeks of treatment. Additional data including PK will be presented. Disclosures Iyer: Novartis: Current equity holder in publicly-traded company; Agios Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Mangus:Agios Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Kung:Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dang:Agios Pharmaceuticals Inc.: Current Employment, Current equity holder in publicly-traded company. Kosinski:Agios Pharmaceuticals Inc: Current Employment, Current equity holder in publicly-traded company. Hawkins:Infinity Pharmaceuticals: Current equity holder in publicly-traded company; Bristol-Myers Squibb: Current equity holder in publicly-traded company; Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company; Jazz Pharmaceuticals: Current equity holder in publicly-traded company.

Abstract: Background: Sickle cell disease (SCD) is a monogenetic red blood disorder that is characterized by hemolytic anemia and vaso-occlusive crises. Among the many factors that contribute to disease pathophysiology is stiffening and sickling of red blood cells (RBC), which is the direct result of the formation of abnormal hemoglobin S. Sickling is one of the core factors that cause vaso-occlusion and sickling is modulated by glycolytic intermediates such as 2,3-diphosphoglycerate (2,3-DPG) and ATP. Previously we showed that red blood cell pyruvate kinase (PKR), the key regulatory enzyme of glycolysis, is impaired in SCD and that ex vivo treatment with mitapivat, an allosteric activator of PKR, increased enzymatic activity and thermostability, reduced 2,3-DPG levels, decreased p50, and subsequently reduced sickling (Rab et al, Blood 2021). Currently, mitapivat is in phase 1 and phase 2 trials for SCD (#NCT04000165 and EudraCT#2019-003438). Aims: Recently, AG-946, a next-generation activator of PKR has been developed. Here we investigate the pharmacodynamic effects of AG-946 in ex vivo treatment of RBC from SCD patients in comparison with mitapivat. Methods: Buffy coat depleted whole blood obtained from five patients with SCD was incubated for 20-24 hrs in absence or presence of mitapivat (100 mM) or AG-946 (1 mM, 5 mM, 50 mM). After ex vivo treatment, enzymatic activities of PKR and PK-thermostability was measured. Glycolytic intermediates ATP and 2,3-DPG were measured using LC-MS/MS. Hemoglobin oxygen affinity (p50) was measured with the Hemox Analyzer. RBC sickling was analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior by oxygen gradient ektacytometry. Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO 2 at which RBCs start to sickle during deoxygenation under shear stress. Results: PKR activity was increased compared to vehicle (DMSO) to a similar extent in presence of both mitapivat and AG-946 (Figure 1A). In addition, PKR thermostability was significantly increased compared to vehicle (mean 22%, SD 6%) in samples treated with mitapivat 100 mM (mean 78%, SD 11%), as well as AG-946 5 mM (mean 66%, SD 23%), and AG-946 50 mM (mean 95%, SD 17%, Figure 1B). The glycolytic intermediate 2,3-DPG decreased after incubation with both mitapivat and AG-946 (Figure 1C), which was further illustrated by the improved ATP/2,3-DPG ratio (Figure 1D). In line with these latter results p50 decreased significantly after incubation with mitapivat 100 mM (mean 95%, SD 2%), as well as AG-946 1 mM (mean 96%, SD 2%), AG-946 5 mM (mean 94%, SD 2%), and AG-946 50 mM (mean 95%, SD 3%, Figure 1E). The improved metabolic status and p50 was accompanied by a decreased PoS compared to vehicle in RBCs treated with mitapivat or AG-946, indicating reduced RBC sickling tendency in vitro (Figure 1F). Conclusion: Ex vivo treatment of SCD RBCs with the next-generation PKR activator AG-946 activates and stabilizes PK, decreases 2,3-DPG levels, improves the ATP/2,3-DPG ratio, improves p50 and lowers the PoS. These beneficial effects are similar to ex vivo treatment with mitapivat but, importantly, are obtained at much lower concentrations. Therefore, AG-946 may be a potent activator of PKR in SCD. Taken together, these results are the first in an ex vivo model to demonstrate that the next-generation PK activator AG-946 has a similar favorable pharmacodynamic profile to mitapivat with enhanced PK-stabilizing properties and, hence, represents a potential novel therapeutic option in addition to mitapivat for the treatment of SCD and other hemolytic anemias. Figure 1 Figure 1. Disclosures Rab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Van Dijk: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Kosinski: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kung: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Van Beers: Pfizer: Research Funding; RR Mechatronics: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dang: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.