The Leukemic Isocitrate Dehydrogenase (IDH) 1/2 Mutations Impair Myeloid and Erythroid Cell Differentiation of Primary Human Hematopoietic Stem and Progenitor Cells (HSPCs)

Abstract

How hematopoietic stem and progenitor cell (HSPC) fate decisions are affected by genetic alterations acquired during AML leukemogenesis is poorly understood and mainly explored in animal models. Here, we study isocitrate dehydrogenase (IDH) gene mutations in the human model of HSPC and discuss the available literature on this topic. IDH1/2 mutations occur in ~20% of AML cases, are recognized among the mutations earliest acquired during leukemogenesis, and are targets of specific inhibitors (ivosidenib and enasidenib, respectively). In order to investigate the direct effects of these mutations on HSPCs, we expressed IDH1-R132H or IDH2-R140Q mutants into human CD34+ healthy donor cells via lentiviral transduction and analyzed the colony-forming unit (CFU) ability. CFU ability was dramatically compromised with a complete trilineage block of differentiation. Strikingly, the block was reversed by specific inhibitors, confirming that it was a specific effect induced by the mutants. In line with this observation, the CD34+ leukemic precursors isolated from a patient with IDH2-mutated AML at baseline and during enasidenib treatment showed progressive and marked improvements in their fitness over time, in terms of CFU ability and propensity to differentiate. They attained clonal trilinear reconstitution of hematopoiesis and complete hematological remission.

Keywords: IDH1/2 mutation; acute myeloid leukemia; human HSPC modeling.

Figure 1

Experimental workflow.

Figure 2

IDH1-R132H and IDH2-R140Q mutations drive a block of CFU ability in human CD34+ HSPC, which is released by specific inhibitors. The CFU assay of transduced hCD34+ cells with an empty vector, IDH1 wild-type, or IDH1-R132H (n = 7), ±AG-120 inhibitor (n = 3 of 7) (A); empty vector, IDH2 wild-type, or IDH2-R140 (n = 6), ±AG-221 inhibitor (n = 4 of 6) (B). Graphs on the left represent the percentage of total colonies that rose after 14 days. Panels on the right are representative images of wells (top row) and types of colonies (bottom row) obtained using the STEMvision™. Colony marker application: red circles identify BFU-E-derived colonies; yellow circles identify CFU-G/M/GM-derived colonies; blue circles identify CFU-GEMM-derived colonies; and orange circles identify CFU-E-derived colonies (one-way ANOVA analyses **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 3

The block of differentiation induced in human CD34+ by either IDH1 (A) or IDH2 (B) mutations and the release by the specific inhibitors affect all lineages: erythroid (left), myeloid (middle), or GEMM (right) precursors (one-way ANOVA analyses **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 4

Enasidenib treatment induces the progressive improvement of CFU ability in primary CD34+ cells carrying the IDH2-R140Q mutation. Experimental scheme (left) and stacked columns chart (right) of colonies that rose after 14 days from CD34+ cells isolated from the patient’s BM before the start of treatment (Day 0) and at I, II, and IV cycles of enasidenib therapy (Days 28, 56, and 112, respectively). The table in the middle shows IDH2-R140Q VAF obtained by targeted NGS on DNA extracted from either bulk BM or isolated cell subpopulations at cycle IV/Day 112 of treatment.