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Acknowledgments We would like to thank the patients taking part in this study. Disclosures This study was funded by Agios Pharmaceuticals, Inc. CK, PAK, HM, LH, GC, MM, KS, M-HJ, and CB: Agios – employment and stockholder. RFG: Agios – advisory board and research funding. BG: Agios – advisory board. Editorial assistance was provided by Susanne Vidot, PhD, Excel Medical Affairs, Horsham, UK, and supported by Agios. References 1. Grace RF et al. Am J Hematol 2015;90:825-30. 2. Grace RF et al. Blood 2018;131:2183-92. 3. Kung C et al. 55th ASH Annual Meeting 2013: Abstr 2180. 4. Kung C et al. Blood 2017;130:1347-56. 5. Grace RF et al. 59th ASH Annual Meeting 2017: Poster 2194. 6. Kung C et al. 56th ASH Annual Meeting 2014: Abstr 4010. • Pyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by mutations in the PKLR gene, leading to a deficiency of the glycolytic enzyme red cell PK (PK-R) (Figure 1). 1,2 • AG-348 is an orally available small-molecule allosteric activator of PK-R that activates the wild type (WT) and a range of mutant PK-R enzymes associated with PK deficiency (Figure 2). 3,4 • Increased PK-R activity and ATP levels in patient red blood cells treated with AG-348 ex vivo may be linked to PKLR genotype and/or PK-R protein level (Figure 3). 4 • In a phase 2 clinical study of patients with PK deficiency (DRIVE PK; NCT02476916), 26 of 52 patients (50%) experienced a maximum Hb increase of >1.0 g/dL (mean maximum increase, 3.4 g/dL; range, 1.1–5.8 g/dL), including 25 of 42 patients (59.5%) with at least one missense mutation (Figure 4). 5 − In most cases, Hb increases were rapid and sustained, and seen across a wide dose range from 5 to 300 mg twice daily (BID) (Figure 5). − Hemolysis markers (reticulocytes, indirect bilirubin, haptoglobin) improved in patients who experienced a maximum Hb increase of >1.0 g/dL. − Hb increases were observed in patients with a variety of PKLR mutations, and increases were associated with the presence of at least one missense mutation. • Because PK deficiency is a genetically heterogeneous disease, with over 200 described mutations, we sought to understand in greater detail the molecular parameters associated with Hb increases in patients treated with AG-348. INTRODUCTION Scan code to receive PDF file of the poster or visit http://bit.ly/2zQfEaz Figure 1. Metabolic defects in PK deficiency 6 (A) The role of the PK-R enzyme in glycolysis. Defective glycolysis in PK-deficient red blood cells results in the accumulation of the upstream metabolites 2,3-DPG and PEP and the depletion of ATP and pyruvate, and decreased red blood cell lifespan. (B) Levels of 2,3-DPG and ATP in whole blood from healthy volunteers and patients with PK deficiency. PG = phosphoglycerate; DPG = diphosphoglycerate; mPK-R = mutated PK-R; PEP = phosphoenolpyruvate Glucose 1,3-DPG 3-PG PEP Pyruvate PK-R 2,3-DPG 2,3-DPG ATP Glucose 1,3-DPG 3-PG PEP Pyruvate mPK-R ATP Normal Chronic hemolysis © 2010 Mosby, Inc. © 2010 Mosby, Inc. 0 500 1000 WT PK deficiency WT PK deficiency [2,3-DPG] (μg/mL) 2,3-DPG 0 200 400 [ATP] (μg/mL) ATP Figure 2. AG-348 is an allosteric activator of PK-R 3,4 (A) Chemical structure of AG-348. (B) Recombinant WT PK-R enzyme activity was assessed after incubation with or without AG-348 (2 µM) in the presence of increasing concentrations of PEP. (C) Crystal structure of AG-348 bound to PK-R tetramer. (A) Genotype of patient samples. (B) PK-R activity and ATP levels in red blood cells from patients with PK deficiency; cells were incubated with AG-348 for 24 hr. (C) PK-R protein levels in red blood cells from healthy volunteers (indicated by WT) and patients with PK deficiency as measured by Meso Scale assay. DMSO = dimethyl sulfoxide Figure 4. Maximum Hb change by genotype in DRIVE PK patients 5 Figure 5. Hb change over time in DRIVE PK patients who had a maximum Hb increase of >1.0 g/dL 5 The majority of Hb increases were rapid and sustained. Median (range) days to the first Hb increase of >1.0 g/dL above baseline: 10 (7–187). The dose had to be held or reduced in nine patients owing to a rapid rise in Hb. Figure 6. An association between baseline PK-R protein level and maximum Hb change was observed in DRIVE PK patients RESULTS Figure 7. Distribution of mutations among 52 DRIVE PK patients Figure 8. Patients with an Hb increase of >1.0 g/dL have greater average PK-R protein levels PK-R protein levels in DRIVE PK patients (expressed as % of WT control sample) categorized by Hb change. Horizontal lines and percentage values indicate the mean, error bars show the standard deviation, and each symbol represents an individual patient. PK-R protein levels in patients with at least one missense mutation vs those with two non-missense change. Horizontal lines and percentage values indicate the mean, error bars show the standard deviation, and each symbol represents an individual patient. Figure 10. Patients with at least one R510Q or R479H missense mutation have lower PK-R protein levels than patients with other missense mutations PK-R protein levels in all patients with at least one missense mutation, stratified into those with at least one R510Q mutation, at least one R479H mutation, or neither. Horizontal lines and percentage values indicate the mean, error bars show the standard deviation, and each symbol represents an individual patient. SUMMARY AND CONCLUSIONS • A statistically significant correlation was observed between baseline PK-R protein level and Hb increases in patients with PK deficiency treated with AG-348. • This correlation is evidence that AG-348 is working via its proposed mechanism of action of stimulating the residual activity of the mutant enzyme. • Although neither genotype nor PK-R protein level could predict Hb increases with absolute precision, some trends were observed: − Patients with two non-missense mutations had lower protein levels than those with at least one missense mutation. − Patients with R479H or R510Q mutations had lower protein levels than patients with other missense mutations. • These preliminary findings will be examined further in the ongoing phase 3 studies of AG-348 (NCT03548220 and NCT03559699). N N O H N S O O N AG-348 (yellow) binds at the PK-R dimer-dimer interface, away from the active site and the most common mutations Active PK-R is a tetramer; mutations (green) decrease the catalytic activity –0.2 0.2 0.4 PK-R WT PK-R WT + AG-348 0.6 PEP (mM) PK-R activity (μmol/sec/g) 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 AG-348 binding enhances the affinity of PK-R for its substrate PEP –1 0 1 2 3 4 5 6 Patient Missense/missense Missense/non-missense Non-missense/non-missense Max Hb change from baseline (g/dL) 4 6 8 10 12 14 16 18 Study day Hb (g/dL) 300 mg BID 50 mg BID 25 mg BID <25 mg BID 1 22 43 64 85 113 141 169 200 mg BID 100 mg BID 0 50 100 150 Baseline PK-R protein level (% of WT control) Max Hb change (g/dL) –1 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 100 120 110 130 140 Patient Baseline PK-R protein level (% of WT control) Max Hb change >1.0 g/dL Max Hb change ≤1.0 g/dL –50 0 50 100 150 4% 38% Two non-missense At least one missense Baseline PK-R protein level (% of WT control) R510Q R479H Other missense –50 0 50 100 150 Baseline PK-R protein level (% of WT control) 18% 19% 59% (A) Correlation plot between maximum Hb change observed in DRIVE PK patients and normalized PK-R protein level (r 2 = 0.39, p<0.0001). Dots represent individual patients. (B) PK-R protein levels in DRIVE PK patients categorized by maximum Hb change. OBJECTIVE • To analyze the relationship between Hb increase and patient genotype, biochemical response to AG-348 treatment, and baseline PK-R protein level. B A A A B B C Patient Mutations (nucleotide) Mutations (protein) A 1529 G>A/1532 G>A R510Q/G511R B 1456 C>T/1168 G>A R486W/D390N C 1529 G>A/721 G>T R510Q/E241stop D 1483 G>A/721 G>T A495V/E241stop E 1456 C>T/1022 G>A R486W/G341D F 1529 G>A/1241 C>G R510Q/P414R G 401T>A/1487 T>G V134D/V496G WT A B D E F G Patient sample PK-R protein level (arbitrary units/ μg lysate) 0 300 200 100 400 0 2 1 3 0.0001 0.01 1 100 [AG-348] (μM) PK-R activity (fold of DMSO control) 0.0 2.0 1.5 1.0 0.5 2.5 0.0001 0.01 Patient B Patient E Patient F 1 100 [AG-348] (μM) ATP (fold of DMSO control) PK-R activity ATP A B C METHODS • Whole blood samples were collected from patients with PK deficiency enrolled in the phase 2 DRIVE PK study. • Patient genotypes were determined by Centogene AG (http://www.centogene.com). • Levels of PK-R protein were quantitated using a Meso Scale assay as described previously (antibodies from Abcam, Cambridge, UK [ab89071] and Aviva Systems Biology, London, UK [OAGA00912]). 4 − The signal was normalized to a reference control sample from a subject without PK deficiency. − For PK-R protein-level testing, the sample was obtained on Day 0 prior to the initiation of AG-348 treatment, except in a single patient for whom the sample from Day 15 was used. • Patient consent was received for all testing procedures. R510Q R479H E241stop R486W A495V T384M V134D T408I G165V N393S 0 5 10 15 Number of patients (at least one copy of mutation) 10 mutations observed in at least 2 patients Max Hb change >1.0 g/dL Max Hb change ≤1.0 g/dL 106 mutations 52 patients 47 unique 19 non-missense 28 missense Max Hb change >1.0 g/dL Max Hb change ≤1.0 g/dL –50 0 50 100 150 Baseline PK-R protein level (% of WT control) 52% 10% Figure 9. Patients with two non-missense mutations have significantly lower PK-R protein levels Genotype PK-R protein level Figure 3. Ex vivo response may be linked to genotype and/or PK-R protein level 4 These are the treatment doses that each patient received for the longest duration during the core period 3621 Genotype-response correlation in DRIVE PK, a phase 2 study of AG-348 in patients with pyruvate kinase deficiency Charles Kung 1 , Penelope A Kosinski 1 , Heidi Mangus 1 , Lei Hua 1 , Gary Connor 1 , Michelle Mobilia 1 , Karen Sullivan 1 , Marie-Hélène Jouvin 1 , Rachael F Grace 2 , Bertil Glader 3 , Chris Bowden 1 1 Agios Pharmaceuticals, Inc., Cambridge, MA, USA; 2 Dana-Farber Boston Children’s Cancer and Blood Disorders Center, Boston, MA, USA; 3 Stanford University School of Medicine, Palo Alto, CA, USA Presented at the 60th American Society of Hematology (ASH) Annual Meeting, December 1–4, 2018, San Diego, CA, USA
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