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Parameter IV Dose (1 Mg/Kg) Oral Dose (10 Mg/Kg) AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations Katharine Yen, Jeremy Travins, Fang Wang, Muriel D. David, Erin Artin, Kimberly Straley, Anil Padyana, Stefan Gross, Byron DeLaBarre, Erica Tobin, Yue Chen, Raj Nagaraja, Sung Choe, Lei Jin, Zenon Konteatis, Giovanni Cianchetta, Jeffrey O. Saunders, Francesco G. Salituro, Cyril Quivoron, Paule Opolon, Olivia Bawa, Véronique Saada, Angélo Paci, Sophie Broutin, Olivier Bernard, Stéphane de Botton, Benoît S. Marteyn, Monika Pilichowska, YingXia Xu, Cheng Fang, Fan Jiang, Wentao Wei, Shengfang Jin, Lee Silverman, Wei Liu, Hua Yang, Lenny Dang, Marion Dorsch, Virginie Penard-Lacronique, Scott A. Biller, Shin-San Michael Su SUPPLEMENTARY TABLES Supplementary Table 1. Drug metabolism and pharmacokinetic attributes of AG-221 Parameter IV dose (1 mg/kg) Oral dose (10 mg/kg) CL (l/h/kg) 0.83 ± 0.10 NA Vss (l/kg) 5.5 ± 1.6 NA AUC (h.ng/ml) 1,200 ± 130 5,000 ± 610 t1/2 (h) 5.4 ± 1.8 5.4 ± 1.2 F (%) NA 41 ± 5.0 IV and oral pharmacokinetic parameters of AG-221 in male Sprague-Dawley rats (mean ± s.d., n = 3). AUC, area under plasma concentration curve; CL, clearance; F, absolute oral bioavailability; IV, intravenous; NA, not applicable; t1/2, terminal half-life; Vss, volume of distribution at steady state. Page 1 of 12 Supplementary Table 2. Selectivity of AG-221 confirmed by testing against a panel of kinases Enzyme IC50 of AG-221 IC50 of reference Reference (nM) (nM) P13K >10,000 9.1 PI 103 JNK2 >10,000 5741.2 Staurosporine AKT1 >10,000 9.8 Staurosporine AKT2 >10,000 57.7 Staurosporine CDK2 >10,000 7.0 Staurosporine FLT3 >10,000 0.4 Staurosporine ABL >10,000 163.0 Staurosporine GSK3a >10,000 33.7 Staurosporine PKCa >10,000 3.4 Staurosporine JAK2 >10,000 0.7 Staurosporine cKIT >10,000 6.6 Staurosporine LCK >10,000 6.8 Staurosporine LYNa >10,000 6.8 Staurosporine SRC >10,000 11.8 Staurosporine ROCK2 >10,000 1.8 Staurosporine CHK1 >10,000 0.5 Staurosporine ALK >10,000 5.3 Staurosporine EGFR >10,000 45.8 Staurosporine FGR >10,000 2.2 Staurosporine Page 2 of 12 INSR >10,000 51.6 Staurosporine SYK >10,000 1.1 Staurosporine PDGFRa >10,000 0.7 Staurosporine PDGFRb >10,000 0.7 Staurosporine HER2 >10,000 476.8 Staurosporine AURB >10,000 2.8 Staurosporine Screening was carried out at concentrations up to 50 µM. Page 3 of 12 Supplementary Table 3. Clinical characteristics of patients with IDH2R140Q-mutated AML Patient Age Sex Disease FAB Karyotype IDH2 FLT3 NPM1 Other mutations VAF (y) status status status status AML-1 72 M Relapse M1 CN R140Q WT Mutant SRSF2 p.95-103del 55% NPM1 p.W288Cfs*12 50% IDH2 p.140Q 49% MPL p.W515L 41% RUNX1 p.L56Sa 44% AML-2 76 F Diagnosis M1 CN R140Q ITD Mutant NPM1 p.W288Cfs*12 43% IDH2 p.140Q 48% CBLB p.T81I 55% FLT3p.E611delinsDKWEF 35% PRENLE AML-3 73 F Diagnosis M1 CN R140Q WT Mutant DNMT3A p.R749C 51% IDH2 p.140Q 50% NPM1 p.W288Cfs*12 46% SRSF2 p.P95H 50% Page 4 of 12 AML-4 59 M Relapse M5 CN R140Q ITD Mutant DNMT3AR882H, CEBPA insertion AML-5 61 M Relapse M2 Trisomy 9 R140Q I867M WT IDH2 p.R140Q 54% Monosomy 7 JAK2 p.V617F 37% BCOR p.M1043Ifs 88% SRSF2 p.P95H 49% RUNX1 p.R166* 96% AML-6 62 M Relapse M1 Trisomy 10 R140Q WT WT IDH2 p.R140Q 50% Other mutations not determined AML-7 80 F Relapse M2 Trisomy 11 R172K D835A - IDH2 p.R172K 45% DNMT3A p.R749C 48% DNMT3A p.W860R 44% NRAS p.A59D 14% FLT3 p.D835A 13% BCOR p.R1341W 11% AML-8 73 F Relapse M2 CN R172K WT WT IDH2 p.R172K 62% Page 5 of 12 DNMT3A p.R736C 52% AML-9 84 M Relapse M2 CN R172K - - IDH2 p.R172K 56% U2AF1 p.S34F 57% AML-10 45 F Diagnosis M1 CN WT D835 Mutant NPM1 p.W288Cfs*12 51% TET2 p.Q1191* 50% TET2 p.V1064Qfs*3 40% AML-11 69 F Refractory M2 CN WT WT WT Not determined AML-12 51 M Diagnosis M1 CN WT ITD WT FLT3 ITD p.Y599_ 92% E608dupYDLKWEFPRE NPM1 p.W288Cfs*12 50% DNMT3A p.R882H 49% aThe somatic or germline origin of this mutation is controversial in the literature. BCOR, BCL6 corepressor; CBLB, Cbl proto-oncogene B; CEBPA, CCAAT/enhancer-binding protein ; CN, cytogenetically normal; DNMT3A, DNA methyltransferase 3A; FAB, French–American–British classification; FLT3, fms-related tyrosine kinase 3; ITD, internal tandem duplication; JAK2, Janus kinase 2; MPL, myeloproliferative leukemia proto-oncogene; NPM1, nucleophosmin; NRAS, neuroblastoma RAS viral oncogene homolog; RUNX1, runt-related transcription factor 1; SRSF2, serine/arginine-rich splicing factor 2; TET2, Tet methylcytosine dioxygenase 2; U2AF1, U2 small nuclear RNA auxiliary factor 1; VAF, variant allele frequency. Page 6 of 12 Supplementary Table 4. Treated NSG mice (AML-1, AML-2, AML-3) engrafted with human IDH2R140Q mononuclear cells display stable levels of AG-221 in serum AG-221 concentration in serum (ng/ml) Pre-dose Day 7 Day 14 Day 21 Day 28 Day 38 Vehicle < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ AG-221 < LOQ 6,976 ± 8,223 ± 7,540 ± 6,877 ± 8,454 ± 1,409 1,402 1,017 1,323 1,178 AG-221 was detected through liquid chromatography-mass spectrometry in serum following treatment at 30 mg/kg twice daily. Data are expressed as mean ± s.d. of values obtained from all three models combined. LOQ, limit of quantification; NSG, NOD scid interleukin-2Rγ–/–. Page 7 of 12 Supplementary Table 5. AG-221 inhibits 2HG production in models AML-1, AML-2, and AML-3 A Pre dose Day 7 Day 14 Day 21 Day 28 Day 38 2HG serum concentration (ng/ml) Vehicle 456 ± 165 261 ± 128 346 ± 154 307 ± 137 406 ± 146 502 ± 284 AG-221 337 ± 139 248 ± 138 144 ± 18 180 ± 72 162 ± 54 137 ± 40 2HG reduction by – 49.3 >99.9 >99.9 >99.9 >99.9 AG-221 (%)a B 2HG concentration at Day 38, ng/106 cells Bone marrow Spleen Vehicle 299 ± 15 203 ± 25 AG-221 BLQ BLQ AG-221 lowers 2-HG levels in (A) serum and (B) tissues of NSG mice engrafted with primary human IDH2R140Q-mutant acute myeloid leukemia (AML-1, AML-2, AML-3), as measured by liquid chromatography-mass spectrometry. Data are expressed as mean ± s.d. of values obtained from all three models combined. aPlasma concentrations of 2HG in naïve CB17-SCID mice (234 ng/ml) were considered as background levels for the calculation of 2HG suppression. BLQ, below the limit of quantitation (1 ng/ml); IDH, NSG, NOD scid interleukin-2Rγ–/–. Page 8 of 12 Supplementary Table 6. Summary of pharmacokinetics/pharmacodynamics in primary human acute myeloid leukemia xenograft model (AML-4) Group AG-221 concentration 2HG concentration (ng/mL or ng/g) (ng/ml or ng/g) (% 2HG suppression) Blood Spleen Blood Spleen Bone marrow Vehicle NA NA 5,087 ± 1,534,503 ± 4,137 ± 780 2,000 731,453 AG-221 5 mg/kg 1,472 ± 380 3,018 ± 700 1,485 ± 286 34,462 ± 32,509 276 (n = 2)a (89.7) (97.8) (93.3) AG-221 15 mg/kg 4,957 ± 9,378 ± 1,398 ± 166 3,650 ± 1,360 BLQ 1,136 4,736 (91.9) (99.8) AG-221 45 mg/kg 6,808 ± 17,480 ± 1,331 ± 142 2,714 ± 767 BLQ 1,384 3,397 (93.6) (99.9) Naïve mice BLQ BLQ 1,074 ± 103 811 ± 260 BLQ Mean ± s.d. concentrations of AG-221 and 2HG in blood, spleen, and bone marrow of mice 8 hours after the last twice-daily oral dose (108 days). a2HG levels were BLQ in seven of nine animals. BLQ, below limit of quantitation; NA, not applicable. Page 9 of 12 Supplementary Table 7. Summary of data collection and refinement statistics Structure IDH2R140Q·KG IDH2R140Q·AG-221 PDB ID 5I95 5I96 Data collectiona Resolution (Å) 29.83–1.54 (1.595–1.54) 48.4–1.55 (1.605–1.55) Space group C2221 P212121 Unit cell a, b, c (Å) 67.18, 154.88, 93.57 58.37, 120.35, 124.73 Unit cell α, β, γ (°) 90, 90, 90 90, 90, 90 Unique reflections 71,924 (7,103) 121,518 (11,825) Rmeas 0.078 (0.534) 0.059 (0.576) <I/σI> 19.5 (2.4) 24.3 (2.9) Completeness (%) 99.5 (98.3) 95.0 (94.1) Redundancy 4.8 (4.3) 4.9 (4.6) Refinement Rwork/Rfree 0.142/0.174 0.153/0.194 Number of non-hydrogen 4,134 8,157 atoms Protein 3,334 6,976 Ligands 88 (1 KG, 1 NADPH) 200 (1 AG-221, 2 NADPH) Water 589 976 Average B-factor (Å2) 18.40 24.7 Protein 14.6 23.6 Ligand 19.10 21.9 Solvent 33.70 32.6 Page 10 of 12 RMS bonds (Å) 0.027 0.032 RMS angles (degree) 2.51 2.95 Ramachandran statistics: 97, 3, 0 96, 3.9, 0.1 Most favored, allowed, outlier regions (%) aValues in parentheses are for the highest-resolution shell. Page 11 of 12 Supplementary Table 8. Percentage of human chimerism in peripheral blood in models AML-1 and AML-2 Page 12 of 12 .
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