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

项目总结 急性髓系白血病(AML)是一种克隆性、高度异质性的血液和骨髓恶性肿瘤。 预后不良。随着白血病细胞本身的基因突变，骨髓的变化 微环境(BMM)在白血病的转化、治疗耐药和 旧病复发。因此，要开发有效的急性髓细胞白血病治疗方法，关键是要了解内在的基因改变是如何在 白血病母细胞与骨髓基质细胞协同促进白血病的发生。我们鉴定出分泌的蛋白质 表皮生长因子样蛋白7(EGFL7)在大量AML患者中高表达，并与 预后不良。我们进一步证明：1)EGFL7蛋白可促进BLCs的生长并影响其功能。 白血病干细胞(LSC)功能；2)白血病骨髓间充质基质细胞(MSCs)表达和 与正常的MSCs相比，分泌EGFL7的水平增加；3)正常的MSCs生长和 在急性髓系白血病细胞中，重组EGFL7蛋白和Egfl7反应后的存活率增加，但在Pre. 白血病造血干细胞和祖细胞(HSPC)来自原发MLL PTD；Flt3 ITD小鼠AML模型。 根据我们的工作，我们假设白血病细胞和骨髓细胞巢细胞异常表达EGFL7 有助于白血病的发生，也是AML患者治疗的相关靶点。我们提案的目标是 为了充分表征EGFL7在髓系白血病发生中的表型作用，解剖其分子 机制(S)，并确定靶向EGFl7在AML的治疗益处。我们计划实现 这些目标通过以下具体目标(SA)实现：SA#1：证明EGFL7在 白血病细胞改变骨髓微环境，促进白血病发生。在这里，我们假设EGFL7 促进白血病细胞和导致疾病的骨髓微环境之间的正反馈循环 开始和进展。我们将通过一系列体外和体内实验来验证这一假设 分三项任务。在任务1中，我们将研究BMM中的EGFL7对AML启动的贡献 使用我们的MLL PTD；Flt3 ITD和Egfl7fl/fl条件性基因敲除小鼠模型。对于任务2，我们将确定如何 EGFL7表达的改变影响骨髓间充质干细胞在白血病发生中的作用任务3我们将描述 MSCs中Egfl7依赖的信号通路的分子机制；SA#2：研究抗白血病 EGFL7单抗与Flt3抑制剂联合应用的活性研究 (Gilteritinib)，使用患者来源的异种移植(PDX)小鼠模型和AML的原始遗传小鼠模型。 在这里，我们建议使用AML细胞系、原代AML样本进行体外和体内的临床前研究 以及两种Flt3突变的AML小鼠模型以评估靶向自分泌和旁分泌的疗效 EGFL7在急性髓系白血病中的作用总体而言，这项建议旨在定义EGFL7在 BMM和白血病细胞，并产生临床前数据以支持抗EGFL7的临床开发 单抗联合Flt3抑制剂治疗Flt3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.