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

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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.