Deciphering Effects of Idh1-R132H Mutations on the Regulation of Hematopoietic Differentiation

The hematopoietic system is a highly versatile regenerative tissue, in which hematopoietic stem cells drive the life-long production of multiple mature blood cell types. During hematopoietic differentiation, the regulation of genome-wide epigenetic patterns of histone modification or DNA methylation marks is an essential process orchestrating cell identities, lineage decisions and developmental cell fates. In acute myeloid leukemia, mutations frequently affect direct and indirect epigenetic regulators and modifiers such as isocitrate dehydrogenase 1 (IDH1) or DNA methyltransferase 3 alpha (DNMT3A), and result in disturbed epigenetic landscapes and differentiation patterns. Here, IDH1 mutations promote oncogenic transformation through the de novo production of the metabolite D2-hydroxyglutarate, which induces a genome and epigenome instability by inhibiting multiple histone and DNA demethylases. Yet, molecular details of how IDH1 mutations alter characteristics of individual hematopoietic cell types remain poorly understood. In the course of this thesis, combinatorial mouse models carrying specific Idh1-R132H and DNMT3A-R882H mutations, which frequently co-occur in acute myeloid leukemia patients, were extensively characterized. By integrating phenotypic readouts in combination with latest advances in high-throughput single-cell RNA-sequencing approaches, cooperativity and impact of these mutations on individual cell types of the hematopoietic system were delineated and gene regulatory networks which are altered upon the expression of an Idh1-R132H or a DNMT3A-R882H mutation were identified. At a phenotypic level, neither an Idh1-R132H mutation alone nor in combination with a DNMT3A-R882H mutation resulted in the development of myeloid malignancies, suggesting a restricted oncogenic potential of these mutations and additional intrinsic or extrinsic factors to be required for further malignant transformation. However, Idh1-R132H mutated hematopoietic stem cells displayed increased engraftment and reconstitution potential during serial transplantations and featured aberrant expression of genes associated with DNA damage and DNA repair. Furthermore, both Idh1-R132H single-mutant and Idh1-R132H DNMT3A-R882H double-mutant mice displayed aberrant differentiation patterns predominantly affecting the myelo-monocytic lineage, culminating in a favored monocytic cell fate and increased monocyte and monocyte progenitor counts in the bone marrow. By employing a multi-layered single-cell transcriptome analysis of nearly all cell types within the hematopoietic compartment, differentiation trajectories from hematopoietic stem cells towards mature differentiated cells were reconstructed and underlying molecular defects characterized. Pseudotime-inferred myeloid lineage trajectories revealed an aberrant lineage specification in particular for Idh1-R132H DNMT3A-R882H double-mutated myeloid progenitor cells, resembling a differentiation arrest at the stage of common myeloid progenitors and an ineffective hematopoietic differentiation as seen in myelodysplastic syndromes. At the molecular level, this aberrant population was characterized by an altered metabolic signature and elevated Myc signaling, which is involved in the regulation of terminal myeloid differentiation. Importantly, we could correlate this transcriptome-defined population to a surface marker-defined population, allowing the prospective isolation of these cells for further investigation. Independent of a DNMT3A-R882H mutation, the expression of an Idh1-R132H mutation resulted in the deregulation of several key regulatory factors which either orchestrate monocyte and macrophage development or their activation upon inflammatory stimuli. In line with this, monocyte progenitor cells displayed elevated interferon signaling levels, suggesting that a proinflammatory environment is a common characteristic of an Idh1-R132H mutated hematopoietic compartment and could contribute to leukemic transformation upon additional events. In summary, the experimental framework presented in this thesis enhanced our understanding of how IDH1-R132H mutations alone or in combination with a DNMT3A-R882H mutation in patients synergistically drive leukemia initiation and progression. The identified molecular characteristics will be of benefit in designing treatment strategies for patients carrying IDH1-R132H and DNMT3A-R882H mutations and can be used as a resource when studying these mutations in the context of altered physiological conditions and upon additional extrinsic stimuli