The relevance of EZH2 dependent Stemness mechanisms for Therapy Response and Resistance in Acute Myeloid Leukemia

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Cancer stem cells remain at the top of the hierarchy in multiple cancer types. In Acute Myeloid Leukemia (AML), the leukemic stem cells (LSCs) are rather well characterized and are thought to constitute a major determinant of therapy outcome. LSC features are epigenetically embedded since LSC and bulk cells usually harbor the same mutations within a single patient. Intrinsic therapy resistance appears to be coupled to LSC features. Leukemic stem cells harbor specific traits including unlimited self-renewal potential, a differentiation block and the ability to repopulate the host. Also, intrinsic resistance towards various drugs relates to features of LSCs. However, it remains unknown how LSC activity determines therapy resistance in itself or whether therapy resistance represents one cellular feature that is just overrepresented in LSC. Accordingly, the main aim of this project is to identify whether LSC functions and therapy resistance are intricately linked or whether stemness and therapy resistance can be separated. For this purpose, we will utilize a genetic leukemia mouse model, which affects the repressive histone modifications H3K27me3. In this model, we are going to deplete cells of EZH2, which is known to influence stemness, using retroviral transduction of hematopoietic cells derived from CAS9-expressing C57BL/6 mice. In addition to a validated guide-RNA targeting EZH2, the retroviral constructs will also contain one of three frequently described leukemic oncogenes, MLL-AF9, AML1-ETO9a or MYC, as well as a randomized barcode, which will be used later to identify individual clones. This will generate a large variety of individual leukemogenic clones, which will vary in their retroviral integration site, the number of alleles hit as well as the exact nature of their NHEJ repair defects. Subsequently, we are going to analyze leukemogenesis, progression of leukemia and LSC functions in different assays in vivo (limiting dilution assays, secondary or serial transplantation) as well as therapy resistance against cytarabine in vitro and in vivo for each oncogene. These assays will be performed using a pool of 1000 individual clones whose stemness and cytarabine resistance can be ranked afterwards by sequencing of their respective barcodes. Top candidates will be genetically characterized for integration sites in order to discover synergistic effects of affected genes. Identified target genes and pathways are going to be analyzed in human primary leukemia samples for functional verification and in clinical trial patient cohorts for association with clinical features and patient outcome. Ultimately, these analyses will help to determine whether LSC functions in terms of stemness and therapy resistance can be distinguished. These findings will shed light on distinct LSC functions and may help to devise novel therapeutic strategies to overcome therapy resistance.