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Abstract

In acute myeloid leukemia (AML), initiation of tumorigenesis via multiple oncogenic mutations occurs throughout various stages of hematopoiesis that disrupt the corresponding transcriptomic and epigenetic profiles. The cancer cells that emerge are referred to as blasts and share biological features from these disease-specific alterations and patterns associated with differentiation and the tumor cell of origin. The resulting blasts show a large inter- and intra-tumor heterogeneity within molecularly defined AML subgroups that are highly relevant for risk stratification and personalized treatment strategies. Sequencing methods that analyze the transcriptome (scRNA-seq) and epigenome (scATAC-seq) are ideally suited to resolve tumor cell heterogeneity as well as non-malignant cell types in the microenvironment. Additionally, scATAC-seq allows to map the binding of transcription factors (TF) and infer cell-specific regulatory networks. Here, I dissected inter- and intra-tumor heterogeneity in patients with different genetic aberrations representing major subgroups in AML, namely MLL fusions, IDH mutated, and FLT3-ITD rearranged AMLs. I established and adjusted the experimental and bioinformatic procedures to generate reproducible and scalable data by scRNA/ATAC-seq of peripheral blood and bone marrow biopsies from AML patients. I could demonstrate that leukemic cells could be successfully distinguished from the microenvironment based on marker gene annotation from the human cell atlas and ploidy inference. Furthermore, I used the experimental and data analysis framework to analyze specific molecular features of the three AML subgroups. First, I characterized changes in the transcriptome and classified developmental stages of leukemic cells carrying MLL-EDC4 fusions along the hematopoietic stem cell to the myeloid trajectory compared to other MLL fusions. Cell type prediction revealed extensive malignant cell diversity and a phenotype skewed towards stem- and progenitor-like populations in MLL-EDC4 leukemic cells. To further elucidate transcriptomic properties of MLL-EDC4 cells, TF activity was inferred. The results agreed with differential gene expression highlighting many TFs that play a critical role in hematopoiesis, endothelial-to-hematopoietic transition, or leukemic stem cell activation. Second, I developed an approach to resolve the subclone- specific response during FLT3 inhibition with midostaurin. Analysis from scRNA and scATAC V showed different FLT3 activity/chromatin signatures within clusters of leukemic cells in the relapse that could be explained by midostaurin resistance and the emergence of distinct subclones as detected by scDNA-seq. Third, I characterized how the chromatin accessibility landscape was influenced by IDH1 mutated cells treated ex vivo with targeted therapy compared to IDH1 wild-type cells. Treatment with the IDH1 inhibitor revealed a partially reversible pattern of accessibility while other mutation-induced epigenetic modifications could not be reverted. The scRNA-seq data acquired for the three different AML subgroups were then exploited to perform a cell type prediction analysis. The relative abundance of different malignant cell types discovered varied amongst tumors, with some having just two identities and others having a wide range of malignant cells. MLL fusions, except for MLL-EDC4, generally conferred a more differentiated phenotype predominantly consisting of monocytes/macrophage CD14-like and promonocyte-like cells. Both tumor entities harboring FLT3-ITDs or IDH1 mutations showed a more complex composition of cell types along the myeloid differentiation trajectory than MLL fusions. The composition of cell types was generally more skewed to early progenitors at the point of diagnosis when compared to their matching relapse sample. This indicates a partial differentiation of AML cells that treatment might induce. In summary, this thesis provides novel insights into the tumorigenesis process in AML by using a systematic and functional analysis approach of the transcriptome and open chromatin in single cells for three major genetically defined AML subgroups. A better comprehension of cellular hierarchies, epigenetic effects, clonal evolution, and their impact on gene regulation might help to understand disease progression, stratify patient risk, and help to improve the treatment of hematopoietic malignancies in the future.