Crosstalk between leukemic blasts and the BM microenvironment contribute to leukemic transformation

项目摘要 急性髓性白血病（AML）是一种血液和骨髓的克隆性、高度异质性恶性肿瘤， 预后不良。沿着白血病细胞自身的基因突变，骨髓中的 微环境（BMM）也在白血病转化、治疗抗性和 复发因此，要开发有效的AML疗法，关键是要了解内在的遗传改变是如何影响AML的。 白血病原始细胞与BMM合作以促进白血病发生。我们发现分泌的蛋白 上皮生长因子样7（EGFL 7）在一个大的AML患者队列中高度表达， 预后不佳。我们进一步证明：1）EGFL 7蛋白增加胚细胞生长，并影响 白血病干细胞（LSC）功能; 2）白血病骨髓间充质干细胞（MSC）表达和 与正常MSC相比，以增加的水平分泌EGFL 7; 3）正常MSC表现出增加的生长， 应答重组EGFL 7蛋白的存活率和4）Egfl 7在AML母细胞中增加，但在AML前 来自原代MII PTD; Flt 3 ITD鼠AML模型的白血病造血干细胞和祖细胞（HSPC）。 基于我们的工作，我们假设白血病细胞和骨髓小生境细胞的EGFL 7异常表达， 有助于白血病发生，并且是AML患者治疗的相关靶标。我们提案的目的是 为了充分表征EGFL 7对髓系白血病发生的表型贡献，剖析其分子生物学特性， 机制，并确定靶向EGFL 7在AML中的治疗益处。我们计划实现 通过以下具体目标（SA）实现这些目标：SA#1：证明EGFL 7在以下方面的重要性： 白血病细胞改变BM微环境以促进白血病发生。在这里，我们假设EGFL 7 促进白血病细胞和导致疾病的BM微环境之间的正反馈回路 启动和进展。我们将通过一系列体外和体内实验来验证这一假设 在三个任务中。在任务1中，我们将研究EGFL 7在BMM中对AML启动的贡献 使用我们的Mll PTD; Flt 3 ITD和Egfl 7 fl/fl条件性敲除小鼠模型。对于任务2，我们将确定如何 EGFL 7表达的改变影响骨髓间充质干细胞的白血病发生。任务3我们将描述 MSC中Egfl 7依赖性信号传导途径的分子机制; SA#2：研究抗白血病 单克隆EGFL 7阻断抗体（Parsatuzumab）与FLT 3抑制剂组合的活性 （Gilteritinib），使用AML的患者来源的异种移植物（PDX）鼠模型和原代遗传鼠模型。 在这里，我们建议使用AML细胞系、原代AML样品和细胞培养物进行体外和体内临床前研究。 和两个FLT 3突变AML的小鼠模型，以评估靶向自分泌和旁分泌的疗效。 EGFL 7在AML中的作用总的来说，该提案旨在定义EGFL 7在 BMM和白血病细胞，并生成临床前数据以支持抗EGFL 7的临床开发 单克隆抗体与FLT 3抑制剂组合用于FLT 3突变的AML。

PROJECT SUMMARY Acute myeloid leukemia (AML) is a clonal, highly heterogeneous malignancy of the blood and bone marrow with poor prognosis. Along with genetic mutations within the leukemic cells themselves, alterations in the BM microenvironment (BMM) also play an important role in leukemia transformation, therapy resistance, and relapse. Thus, to develop effective AML therapies, it is critical to understand how intrinsic genetic alterations in the leukemic blasts cooperate with the BMM to facilitate leukemogenesis. We identified that the secreted protein Epithelial Growth Factor Like 7 (EGFL7) is highly expressed in a large cohort of AML patients and is associated with poor prognosis. We further demonstrate that: 1) EGFL7 protein increases blast cell growth and impacts on leukemic stem cell (LSC) function; 2) leukemic bone marrow mesenchymal stromal cells (MSCs) express and secrete EGFL7 at increased levels compared to normal MSCs ; 3) normal MSCs exhibit increased growth and survival in response to a recombinant EGFL7 protein and 4) Egfl7 is increased in AML blasts, but not in pre- leukemic hematopoietic stem and progenitor cells (HSPCs) from a primary Mll PTD; Flt3 ITD murine AML model. Based on our work, we hypothesize that aberrant EGFL7 expression by leukemic cells and BM niche cells contribute to leukemogenesis and is a relevant target for treatment for AML patients. The goal of our proposal is to fully characterize the phenotypic contributions of EGFL7 to myeloid leukemogenesis, dissect its molecular mechanism(s), and determine the therapeutic benefit of targeting EGFL7 in AML. We are planning to achieve these goals through the following specific aims (SA): SA#1: To demonstrate the importance of EGFL7 in leukemic cells to alter the BM microenvironment to promote leukemogenesis. Here, we hypothesize that EGFL7 promotes a positive feedback loop between leukemic cells and the BM microenvironment contributing to disease initiation and progression. We will test this hypothesis with a series of in vitro and in vivo experiments organized in three tasks. In Task 1 we will examine the contribution of EGFL7 in the BMM towards the initiation of AML using our Mll PTD; Flt3 ITD and Egfl7fl/fl conditional knock-out mouse models. For Task 2 we will determine how alterations in the expression of EGFL7 effects MSCs in leukemogenesis. Task 3 we will characterize the molecular mechanisms of Egfl7-dependent signaling pathways in MSCs; SA#2: To investigate anti-leukemic activity of a monoclonal EGFL7 blocking antibody (Parsatuzumab) in combination with a FLT3 inhibitor (Gilteritinib), using patient derived xenograft (PDX) murine models and primary genetic murine models of AML. Here, we propose to conduct in vitro and in vivo preclinical studies using AML cell lines, primary AML samples and two murine models of FLT3 mutated AML to evaluate the efficacy of targeting the autocrine and paracrine action of EGFL7 in AML. Overall, this proposal seeks to define the role of EGFL7 in the cross-talk between the BMM and leukemic cells and to generate preclinical data to support clinical development of anti-EGFL7 monoclonal antibodies in combination with FLT3 inhibitors for FLT3 mutated AML.