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

癌症干细胞在多种癌症类型中仍然处于最高层次。在急性髓系白血病（AML）中，白血病干细胞（LSC）的特征相当明确，被认为是治疗结果的主要决定因素。LSC特征是表观遗传学嵌入的，因为LSC和散装细胞通常在单个患者中具有相同的突变。内在治疗抗性似乎与LSC特征相关。白血病干细胞具有特殊的特性，包括无限的自我更新潜力，分化阻滞和重新填充宿主的能力。此外，对各种药物的内在抗性与LSC的特征有关。然而，仍然不清楚LSC活性如何决定其本身的治疗抗性，或者治疗抗性是否代表LSC中过度表达的一种细胞特征。因此，本项目的主要目的是确定LSC功能和治疗抗性是否错综复杂地联系在一起，或者是否可以将干性和治疗抗性分开。为此，我们将利用遗传性白血病小鼠模型，其影响抑制性组蛋白修饰H3 K27 me 3。在这个模型中，我们将使用来自表达CAS9的C57 BL/6小鼠的造血细胞的逆转录病毒转导来耗尽已知影响干细胞的EZH 2细胞。除了经验证的靶向EZH 2的向导RNA之外，逆转录病毒构建体还将含有三种经常描述的白血病癌基因之一，MLL-AF 9，AML 1-ETO 9a或MYC，以及随机条形码，其将在以后用于鉴定单个克隆。这将产生各种各样的单个致白血病克隆，其在逆转录病毒整合位点、等位基因命中的数量以及其NHEJ修复缺陷的确切性质方面将有所不同。随后，我们将在不同的体内试验（有限稀释试验、二次或连续移植）中分析白血病发生、白血病进展和LSC功能，以及每种癌基因在体外和体内对阿糖胞苷的治疗耐药性。将使用1000个单个克隆的合并液进行这些试验，随后可通过对其各自的条形码进行测序对这些克隆的干性和阿糖胞苷耐药性进行排序。将对最佳候选物的整合位点进行遗传表征，以发现受影响基因的协同效应。将在人类原发性白血病样本中分析鉴定的靶基因和途径以进行功能验证，并在临床试验患者队列中分析其与临床特征和患者结局的相关性。最终，这些分析将有助于确定LSC在干性和治疗抗性方面的功能是否可以区分。这些发现将揭示不同的LSC功能，并可能有助于设计新的治疗策略，以克服治疗耐药性。

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.