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.
CAM Kinase IV Regulates Lineage Commitment and Survival of Erythroid Progenitors in a Non-Cell–Autonomous Manner
