Targeting PLCG1 in AML1-ETO-positive acute myeloid leukemia (AML)

急性髓性白血病（AML）是一种克隆性造血疾病，其特征是未成熟和增殖性母细胞在骨髓、外周血和其他器官中积累。AML的分子异质性仍然是一个主要的治疗挑战。不同的AML基因组图谱有助于疾病生物学并转化为不同的结果。这种分子和临床异质性也可能取决于不同AML亚型的细胞起源。另一方面，特定异常的存在会产生次要依赖和漏洞。在第一个资助期内，我们的目标是确定融合癌基因诱导的急性髓性白血病（AML）的新分子靶点。我们进行了高分辨率蛋白质组学分析。在AML1-ETO （AE）驱动的AML中，我们发现了磷脂酶C （PLC）信号的解除管制。值得注意的是，在具有平衡易位t的AML中发现了最高的表达水平（8;21），这导致了AML1-ETO的致癌融合。我们发现PLCgamma1 （PLCG1）是AE融合蛋白的特异性靶点，该蛋白在AE与基因间调控DNA元件结合后被诱导。在新建立的条件小鼠模型和人类AML中，PLCG1基因失活抑制了AML1-ETO依赖的自我更新程序、白血病增殖和白血病维持。相比之下，PLCG1对于正常的造血干细胞和祖细胞功能是必不可少的。这些发现被扩展到AML1-ETO AML细胞中钙信号的药理学扰动并得到证实，表明PLCG1途径是AML1-ETO1白血病干细胞的重要治疗靶点。虽然PLCG1在AE-AML中的上游调控已被表征，但其下游调控和机制功能尚不清楚。根据我们提出的工作计划，我们将确定相关的效应物，明确PLCG1在AE-AML中的依赖关系，并旨在揭示PLCG1的机制作用，以促进PLCG1在AE-AML中的靶向治疗。使用crispr介导的筛选，我们已经确定了PLCG1的相关结构域，指示可靶向区域。使用全局磷酸化蛋白质组分析和功能基因组编辑方法，我们将定义PLCG1在AML1-ETO （AE）驱动的AML中的效应物和依赖性。利用第一期资助期建立的条件小鼠模型，我们将进行拯救实验、相关PLCG1结构域的遗传扰动和相互作用组分析，以揭示PLCG1下游在AML1-ETO AML中的靶向分子机制。

Acute myeloid leukemia (AML) is a clonal hematopoietic disorder characterized by the accumulation of immature and proliferating blasts in the bone marrow, peripheral blood and other organs. Molecular heterogeneity of AML remains a major therapeutic challenge. Different genomic AML profiles contribute to disease biology and translate to variable outcomes. This molecular and clinical heterogeneity may also depend on the cellular origin of the different AML subtypes. On the other hand, presence of specific aberrations can create secondary dependencies and vulnerabilities. Within the first funding period we aimed to identify novel molecular targets for fusion oncogene–induced acute myeloid leukemia (AML). We performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. Of note, highest expression levels were found in AML harboring the balanced translocation t(8;21), which gives rise to the oncogenic fusion AML1-ETO. We identified PLCgamma1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in a newly developed conditional mouse model and in human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of calcium-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO1 leukemic stem cells. While the upstream regulation of PLCG1 in AE-AML has been characterized, its downstream regulation and mechanistic function remains elusive. According to our proposed working program we will identify relevant effectors and specify dependencies of PLCG1 in AE-AML and aim to uncover mechanistic roles of PLCG1 to facilitate targeting of PLCG1 in AE-AML. Using CRISPR-mediated screens, we have identified relevant domains of PLCG1 indicating targetable regions. Using global phospho-proteome analysis and functional genome editing approaches we will define effectors and dependencies of PLCG1 in AML1-ETO (AE) driven AML. Using the conditional mouse model created in the first funding period, we will conduct rescue experiments, genetic perturbation of relevant PLCG1 domains and interactome analyses to uncover targetable molecular mechanisms downstream of PLCG1 in AML1-ETO AML.