Table S1 showing AML primary cell details for metabolomics

Supplementary Figure file showing Figures S1-S7

Abstract Isocitrate dehydrogenase 1 and 2 (IDH) are mutated in multiple cancers and drive production of (R)-2-hydroxyglutarate (2HG). We identified a lipid synthesis enzyme [acetyl CoA carboxylase 1 (ACC1)] as a synthetic lethal target in mutant IDH1 (mIDH1), but not mIDH2, cancers. Here, we analyzed the metabolome of primary acute myeloid leukemia (AML) blasts and identified an mIDH1-specific reduction in fatty acids. mIDH1 also induced a switch to b-oxidation indicating reprogramming of metabolism toward a reliance on fatty acids. Compared with mIDH2, mIDH1 AML displayed depletion of NADPH with defective reductive carboxylation that was not rescued by the mIDH1-specific inhibitor ivosidenib. In xenograft models, a lipid-free diet markedly slowed the growth of mIDH1 AML, but not healthy CD34+ hematopoietic stem/progenitor cells or mIDH2 AML. Genetic and pharmacologic targeting of ACC1 resulted in the growth inhibition of mIDH1 cancers not reversible by ivosidenib. Critically, the pharmacologic targeting of ACC1 improved the sensitivity of mIDH1 AML to venetoclax. Significance: Oncogenic mutations in both IDH1 and IDH2 produce 2-hydroxyglutarate and are generally considered equivalent in terms of pathogenesis and targeting. Using comprehensive metabolomic analysis, we demonstrate unexpected metabolic differences in fatty acid metabolism between mutant IDH1 and IDH2 in patient samples with targetable metabolic interventions. See related commentary by Robinson and Levine, p. 266. This article is highlighted in the In This Issue feature, p. 247

Supplementary Table from The Cell Type–Specific 5hmC Landscape and Dynamics of Healthy Human Hematopoiesis and TET2-Mutant Preleukemia

Abstract The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten–eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type–specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis. Significance: We show that 5-hydroxymethylation profiles are cell type–specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. TET2 loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of TET2 KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes. This article is highlighted in the In This Issue feature, p. 265

The interaction of germline variation and somatic cancer driver mutations is under-investigated. Here we describe the genomic mitochondrial landscape in adult acute myeloid leukaemia (AML) and show that rare variants affecting the nuclear- and mitochondrially-encoded complex I genes show near-mutual exclusivity with somatic driver mutations affecting isocitrate dehydrogenase 1 ( IDH1 ), but not IDH2 suggesting a unique epistatic relationship. Whereas AML cells with rare complex I variants or mutations in IDH1 or IDH2 all display attenuated mitochondrial respiration, heightened sensitivity to complex I inhibitors including the clinical-grade inhibitor, IACS-010759, is observed only for IDH1 -mutant AML. Furthermore, IDH1 mutant blasts that are resistant to the IDH1-mutant inhibitor, ivosidenib, retain sensitivity to complex I inhibition. We propose that the IDH1 mutation limits the flexibility for citrate utilization in the presence of impaired complex I activity to a degree that is not apparent in IDH2 mutant cells, exposing a mutation-specific metabolic vulnerability. This reduced metabolic plasticity explains the epistatic relationship between the germline complex I variants and oncogenic IDH1 mutation underscoring the utility of genomic data in revealing metabolic vulnerabilities with implications for therapy.

Cancer-related anemia is present in more than 60% of newly diagnosed cancer patients and is associated with substantial morbidity and high medical costs. Drugs that enhance erythropoiesis are urgently required to decrease transfusion rates and improve quality of life. Clinical studies have observed an unexpected improvement in hemoglobin and RBC transfusion-independence in patients with acute myeloid leukemia (AML) treated with the isocitrate dehydrogenase 2 (IDH2) mutant-specific inhibitor enasidenib, leading to improved quality of life without a reduction in AML disease burden. Here, we demonstrate that enasidenib enhanced human erythroid differentiation of hematopoietic progenitors. The phenomenon was not observed with other IDH1/2 inhibitors and occurred in IDH2-deficient CRISPR-engineered progenitors independently of D-2-hydroxyglutarate. The effect of enasidenib on hematopoietic progenitors was mediated by protoporphyrin accumulation, driving heme production and erythroid differentiation in committed CD71+ progenitors rather than hematopoietic stem cells. Our results position enasidenib as a promising therapeutic agent for improvement of anemia and provide the basis for a clinical trial using enasidenib to decrease transfusion dependence in a wide array of clinical contexts.

Acute Myeloid Leukemia (AML) remains one of the most difficult cancers to treat, with a 30% 2-year survival rate. High-throughput sequencing of AML patients has identified mutations, including FLT3, IDH1, and IDH2, for which targeted therapies have been developed. Enasidenib is an FDA-approved, first-in-class agent that preferentially inhibits IDH2-mutant activity and reduces levels of the oncometabolite 2-HG, inducing differentiation of IDH2-mutated blasts. Interestingly, greater than 50% of enasidenib-treated patients who had no objective clinical response still demonstrated improvement in their peripheral blood counts and reached RBC transfusion independence. The mechanism underlying this phenomenon is unknown but is of great clinical relevance given the high transfusion dependence and anemia-associated complications universally associated with AML. Thus, we sought to investigate how enasidenib drives normal hematopoiesis to improve quality of life and reduce morbidity in AML patients. In this study, we demonstrate that enasidenib enhances erythropoiesis from normal CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from cord blood (CB) and bone marrow. Enasidenib doubled the proportion and total number of mature CD71+/GPA+ erythroblasts after 8 days of culture with EPO, SCF, and IL-3. In the presence of EPO, enasidenib induced a gene signature characteristic of maturing erythrocytes, with increased expression of GATA1 (1.3 fold), EPOR (2 fold), and KLF1 (1.4 fold), and decreased PU.1 (0.5 fold) and GATA2 (0.7 fold). Enasidenib-treated progenitor cells further demonstrated increased hemoglobin production (1.9 fold) and morphologic characteristics of increased erythroid maturation. Next, we sought to determine if enasidenib augments erythroid differentiation through IDH2 and IDH2-dependent pathways. First, we found that other IDH inhibitors (AG-120, AGI-6780, and AG-881) did not increase erythropoiesis at doses ranging from 1-10μM. As expected for normal HSPCs, 2-HG was not present at detectable levels in either the DMSO or enasidenib-treated conditions, and addition of 2-HG (50, 200μM) did not affect the ability of enasidenib to increase the proportion of CD71+GPA+ cells. Because it is possible that enasidenib acts through inhibition of wild-type IDH2, we generated CRISPR-Cas9 engineered IDH2 knockout (KO) CD34+ cells and treated them with enasidenib. Similar to wildtype cells, IDH2 KO CB CD34+ cells demonstrated a 3.4-fold increase in %CD71+GPA+ erythroid cells. Thus, enasidenib augments erythropoiesis independently of both mutant and wildtype IDH2 pathways. We then investigated the progenitor population that enasidenib acts on to drive erythroid maturation. Enasidenib did not increase the number of BFU-E or CFU-E colonies or the proportion of BFU-E (IL3R-CD34+CD36-) and CFU-E (IL3R-CD34-CD36+) progenitors in colony forming or liquid culture assays, respectively, leading us to conclude that enasidenib acts on more mature erythroid progenitors. Indeed, treating sorted mature CD71+ erythroid progenitors with enasidenib increased %CD71+GPA+ cells compared to DMSO control, whereas enasidenib treatment of CD71- early erythroid progenitors showed no effect. These observations provide evidence that enasidenib acts on CD71+ erythroid progenitors to increase late-stage erythroid differentiation. Given that CD71 allows for iron uptake into erythroid progenitors, we hypothesized that enasidenib modulates the heme biosynthesis pathway. Enasidenib inhibited the ABCG2 transporter, which effluxes protoporphyrin IX (PPIX), the direct precursor to heme, from the mitochondria and cytosol. Inhibition of ABCG2 by enasidenib could lead to PPIX accumulation within the cell, driving increased heme synthesis. To investigate this hypothesis, we treated cells with 20μM Ko143, a potent ABCG2 inhibitor, and observed a similar increase in %CD71+GPA+ cells as seen with enasidenib. Measurement of PPIX autofluorescence by flow cytometry and microscopy revealed an increase of PPIX in enasidenib-treated cells by 1.2-fold. Together, our data suggests that enasidenib drives maturation of CD71+ erythroid precursors independently of wildtype or mutant IDH2. Our results position enasidenib as a promising therapy to stimulate erythropoiesis and provide the basis for a clinical trial using enasidenib to improve anemia in a wide array of clinical contexts. Disclosures Majeti: Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy.

This study explores cytomorphologic features and their predictive role for early identification of acute myeloid leukemia (AML) with morphological distinctive recurrent cytogenetic abnormalities (RCA): t(15;17), t(8;21) and inv(16)/t(16;16).We retrospectively evaluated 396 de novo AML cases, diagnosed and treated at single institution, between 2013-2017. Specific cytomorphologic features suggesting distinctive AML-RCA were revealed at diagnosis in 62 (15.65%) patients, including AML with t(15;17) in 41 (66.13%), t(8;21) in 13 (20.97%) and inv(16)/t(16;16) in 8 (12.90%). Final diagnoses of AML-RCA according to WHO integrated diagnostic criteria were established in 66 (16.66%) cases, including AML with t(15;17) 40 (60.60%), t(8;21) 17 (25.76%), and inv(16)/t(16;16) 9 (13.64%).Discordance between cytomorphological and other integrated criteria was detected as missed/wrong-call in 0/1 for t(15;17), 6/2 for t(8;21) and 2/1 for inv(16)/t(16;16).The cytomorphological accuracy was 97.56% (40/41) for t(15;17), 57.89% (11/19) for t(8;21) and 70% (7/10) for inv (16)/t(16;16).Positive/negative predictive values of cytomorphological evaluation were: 97.56%/100% for t(15;17); 84.62%/88.68% for t(8;21); 87.50%/96.65% for inv(16)/t(16;16). Sensitivity/specificity were: 100%/96.15% for t(15;17); 64.10%/95.92% for t(8;21); 77.78%/98.25% for inv(16)/t(16;16).We confirmed that morphology is still a highly relevant evaluation method in diagnosing several common AML-RCAs before completing cytogenetic and molecular studies, enabling early detection, particularly of AML with t(15;17).