Regulation of the guanine nucleotide exchange factor DOCK1 through interaction of leukemic cells and the bone marrow niche: pathway analysis and investigation of functional aspects in acute myeloid leukemia

通过白血病细胞和骨髓生态位的相互作用调节鸟嘌呤核苷酸交换因子 DOCK1:急性髓性白血病功能方面的途径分析和研究

基本信息

项目摘要

Leukemic stem cells (LSC) give rise to the mass of leukemic blasts in acute myeloid leukemia (AML). LSCs reside within the so called stem cell niche comprising of endothelial cells, osteoblasts and other stromal cells. LSCs gain resistance against chemotherapy through interactions with stromal niche cells therefore being responsible for the high relapse rates of AML patients. Details of the complex interactions between LSCs and stromal niche cells need to be clarified.We performed co-culture experiments of freshly isolated human leukemic blasts with osteoblasts as well as endothelial cells in order to determine genes involved in the interactions of leukemic and stromal cells. Subsequent gene expression analysis identified the guanine nucleotide exchange factor DOCK1 as one of the genes being upregulated in co-cultures of AML and stromal cells. The induction of DOCK1 expression upon co-culture with stromal cells could be verified using several AML cell lines as well as primary AML blasts. Analysis of a mRNA expression data base of about 300 AML patients being treated by stardard chemotherapy revealed a prognostic impact of DOCK1 as AML patients with high DOCK1 expression had a significantly poorer overall survival compared to patients with low DOCK1 expression.The blockade of DOCK1 using the specific inhibitor CPYPP resulted in reduced proliferation of AML cell lines as well as primary AML cells in vitro. Therefore DOCK1 inhibition might represent a novel therapeutic target for AML treatment.In our planned project, we would like to investigate the DOCK1 signalling cascade in AML cells in detail. Furthermore, the therapeutic potential of DOCK1 inhibition should be evaluated. One part of the project will focus on the phosphorylation status of DOCK1 after treatment with the DOCK1 inhibitor CPYPP using mass spectrometry (SILAC marking, DOCK1 immunoprecipitation, LC-MS/MS analysis). Furthermore, mass spectrometric analysis will be used to identify DOCK1 binding proteins in order to gain more insight into the DOCK1 signalling cascade in AML.In another part of the project, the potential of DOCK1 as therapeutic target in AML will be evaluated using functional assays such as Rac-1 activity, proliferation or colony-formation assays. DOCK1 activity will be blocked using the inhibitor CPYPP. Furthermore in AML cell lines with endogenous DOCK1 expression, shRNA approaches will be used to investigate the biological functions of DOCK1. Moreover, a mouse model with parallel transplantation of CRISPR/Cas9-generated DOCK1 knockout vs. wildtype AML cells is planned in order to investigate the role of DOCK1 within the bone marrow niche. Subsequent immunohistochemical analyses of the murine bone marrow will provide more information about the relevance of DOCK1 expression within the bone marrow niche.
白血病干细胞(LSC)在急性髓细胞白血病(AML)中产生大量白血病原始细胞。LSC位于所谓的干细胞龛内,包括内皮细胞、成骨细胞和其他基质细胞。LSC通过与基质小生境细胞的相互作用获得对化疗的抗性,因此是AML患者的高复发率的原因。LSC和基质细胞之间复杂的相互作用的细节需要澄清,我们进行了共培养实验新鲜分离的人白血病细胞与成骨细胞以及内皮细胞,以确定参与白血病和基质细胞的相互作用的基因。随后的基因表达分析鉴定了鸟嘌呤核苷酸交换因子DOCK 1作为AML和基质细胞共培养物中上调的基因之一。与基质细胞共培养后DOCK 1表达的诱导可以使用几种AML细胞系以及原代AML母细胞来验证。对约300名正在接受标准化疗的AML患者的mRNA表达数据库的分析揭示了DOCK 1的预后影响,因为具有高DOCK 1表达的AML患者与具有低DOCK 1表达的患者相比具有显著较差的总体存活率。使用特异性抑制剂CPYPP阻断DOCK 1导致AML细胞系以及体外原代AML细胞的增殖减少。因此,DOCK 1抑制可能代表AML治疗的一个新的治疗靶点。在我们计划的项目中,我们希望详细研究AML细胞中的DOCK 1信号级联。此外,应评估DOCK 1抑制的治疗潜力。该项目的一部分将集中在使用质谱法(SILAC标记,DOCK 1免疫沉淀,LC-MS/MS分析)使用DOCK 1抑制剂CPYPP治疗后DOCK 1的磷酸化状态。此外,质谱分析将用于识别DOCK 1结合蛋白,以获得更多的了解DOCK 1信号级联在AML中。在该项目的另一部分,DOCK 1作为AML治疗靶点的潜力将使用功能测定,如Rac-1活性,增殖或集落形成测定进行评估。DOCK 1活性将使用抑制剂CPYPP阻断。此外,在具有内源性DOCK 1表达的AML细胞系中,shRNA方法将用于研究DOCK 1的生物学功能。此外,计划平行移植CRISPR/Cas9产生的DOCK 1敲除与野生型AML细胞的小鼠模型,以研究DOCK 1在骨髓小生境中的作用。随后对小鼠骨髓的免疫组化分析将提供更多关于骨髓龛内DOCK 1表达相关性的信息。

项目成果

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