Mutant Idh1 Blocks Neutrophil Maturation By Repressing Myeloid Progenitor Programs

Mutations of Isocitrate dehydrogenase 1 (IDH1) are found in several malignancies, including myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Despite rarely being detected in clonal hematopoiesis, IDH1 mutations are considered to be early pathogenetic events. IDH1 mutations are always heterozygous and result in neomorphic enzymatic activity leading to the production of the oncometabolite D-2-hydroxyglutarate (D2HG). D2HG inhibits α-ketoglutarate-dependent dioxygenases resulting in DNA and histone hypermethylation. However, to date the intricate molecular alterations resulting from IDH1 mutations and how they contribute to malignant transformation are only poorly understood.The aim of this study was to investigate the cellular and molecular aberrations elicited by mutant Idh1 in hematopoiesis to better understand the mechanisms driving IDH1-mutant (mut) hematopoietic neoplasms.We established a conditional Idh1-R132H mouse model in which Idh1-R132H and YFP-reporter expression are induced in hematopoietic stem and progenitor cells (HSPC) using a tamoxifen-inducible Cre driven by the Scl enhancer (Scl-CreERT). Lineage^-/YFP⁺ cells were transplanted into lethally irradiated mice. Blood and bone marrow cells from fully chimeric Idh1-wildtype (WT) and Idh1-R132H mice were subjected to scRNA-seq, and ex vivo/in vivo assays. The Idh1-R132H mutation was also expressed in 32D cells using lentiviral transduction. Publicly available DNA methylation data from AML patients were collected from TCGA (LAML), BEAT-AML and from Schmutz et al. (Clin Epigenetics 2023). Peripheral blood counts from MDS and AML patients were obtained from patient registries.

Analysis of scRNA-seq data of normal human and murine bone marrow revealed dynamic regulation of IDH1/Idh1 expression in hematopoiesis with peak expression in myeloid progenitor (MP) cells. In contrast, expression of IDH2/Idh2 was observed uniformly across all hematopoietic stem and progenitor cell types. In Idh1-R132H mice, this upregulation strongly correlated with a significant expansion of MPs (CD55^- CMPs and Ly6C⁺ GMPs) and a loss of neutrophils. Adoptive transfer confirmed that Idh1-R132H CD55^- CMPs produce fewer neutrophils than their WT counterparts, suggesting that Idh1-R132H MPs exhibit a cell-intrinsic defect in granulopoiesis. This differentiation defect was reproduced in 32D cells expressing Idh1-R132H. Treatment of Idh1-R132H CD55^- CMPs with a mutant-specific IDH1-inhibitor virtually reversed the differentiation defect in an ex vivo assay. Further, scRNA-seq revealed a depletion of late neutrophil progenitors in Idh1-R132H mice and reduced expression of the granulocyte transcription factor Cebpe, which was again validated in Idh1-mut 32D cells. Therefore, 32D cells were modified with a tamoxifen-inducible Cebpe constract (Theilgaard-Mönch et al., 2022). Tamoxifen treatment and with that translocation of CEBPE to the nucleus was sufficient to overcome the differentiation block of Idh1-mut cells.

To test the relevance of our findings for AML patients, we compared DNA methylation data from IDH1-mut AML samples with data from normal human hematopoietic cell types. This revealed epigenetic scars of granulocytic lineage commitment in 9/16 IDH1-mut AML samples which were absent in IDH2-mut AML samples. This suggested that a granulocytic differentiation defect might contribute to leukemogenesis in IDH1-mut AML. In line with this we found that MDS and AML patients with IDH1-mut presented significantly lower neutrophil counts as compared to IDH2-mut and IDH1/2-WT patients. In contrast, no other cell lineage presented a significant change between IDH1- and IDH2-mut patients.

In the present study, we identified cellular defects in MPs of Idh1-mut mice, which are mediated by a physiologic upregulation of Idh1 expression in MPs. This results in downregulation of Cebpe in neutrophil progenitors and impaired granulopoiesis. Re-introducing CEBPE activity is sufficient to overcome the differentiation block. Identification of granulocytic epigenetic scars in IDH1-mut AML samples and neutropenia in IDH1-mut MDS and AML patients suggest that neutrophilic differentiation defects might play a role in leukemogenesis in humans. In summary, our molecular and cellular data provide an explanation for the preferential association of IDH1 mutations with myeloid neoplasms.

Bullinger:Hexal: Consultancy, Honoraria; Menarini: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Bayer: Research Funding; Sanofi: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria. Cross:Novartis: Research Funding; Curis: Research Funding. Platzbecker:Amgen: Consultancy, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; MDS Foundation: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Research Funding; Curis: Consultancy, Honoraria, Research Funding; Geron: Consultancy; Janssen: Consultancy, Honoraria, Research Funding; Merck: Research Funding; Novartis: Consultancy, Research Funding. Kneisel:SERVIER Germany GmbH: Current Employment. Germing:Abbvie: Research Funding; BMS: Honoraria; BMS: Research Funding; Novatis: Honoraria; JAZZ: Research Funding. Hoermann:MLL Munich Leukemia Laboratory: Current Employment. Lipka:Infectopharm GmbH: Honoraria; Illumina Inc: Honoraria.