hDLG Associated Protein 1 (DLGAP1) Supports Mpl-Driven Cell Proliferation and Differentiation Through Its Centrosomal Function,

Abstract 3402

The Mpl proto-oncogene functions as a basic factor in megakaryocytic development and platelet production as well as contributing to hematopoietic stem cell (HSC) homeostasis and self-renewal. Several mutations in Mpl have been shown to be associated with the myeloproliferative neoplasms. We used retroviral insertional mutagenesis to screen for factors that might give a proliferative and/or survival advantage for cells dependent on Mpl signaling. We developed a retroviral construct expressing a dimerizable form of Mpl that can interrupt genes through insertion, and can activate adjacent genes with its long terminal repeats. We transduced the human leukemia cell line K562 with this construct and blocked the endogenous transforming BCR-ABL kinase using Imatinib. Cells dependent on Mpl signaling were selected by the addition of the dimerizing drug, AP20187. In the absence of Mpl signaling the cells underwent erythroid differentiation and died. Cells that acquired a proliferative advantage and were dependent on Mpl function were expanded. Cloning of retroviral integration sites (RIS) from the selected populations allowed the identification of recurrent RIS that co-localized in the genome. We have identified 668 RIS from 36 independent transductions with a presumed ∼ 1.9 × 10⁶ independent insertions in the initial non-selected, cell population. Among these, 203 RIS represented independent insertion events. Three independent RIS were located in the fourth intron of the Discs large homolog-associated protein 1 (DLGAP1). This protein is a member of the Discs-large/Scribble/Lethal Giant Larvae pathway. DLGAP1, in cooperation with DLG1 and CDC42, has recently been shown to control centrosome positioning and cell polarity in astrocytes (Manneville J-B et al. 2010, JBC, v.191, no 3, pp. 585–598). We investigated the role of DLGAP1 in MPL signaling.

Overexpression of full length DLGAP1 had minimal effect on plain K562 cells, but it significantly slowed down the proliferation of K562 cells switched to Mpl signaling. The identified insertion sites would predict the increased expression of an amino-end truncated isoform. When this truncated form was overexpressed in K562 cells and Mpl dependent K562 cells, the cells proliferated at an increased rate. Immunofluorescent studies revealed that the full length DLGAP1 colocalized with major centrosomal markers including gamma-tubulin, PCM1, and APC in K562, HEL, UT7/TPO and Mo7e cell lines. We did not observe colocalization with centrosomes when the truncated isoform of DLGAP1 was overexpressed. Immunoflourescent microscopy revealed that endogenous DLGAP1 in myeloid cell lines was localized in the centriolar satellites, a cellular structure essential for centrosome integrity and function. The immunoflourescent pattern using an anti DLGAP1 antibody showed cell cycle dependent assembly and disassembly. Protein sequence analyses of DLGAP1 indicated consensus sites for presumptive Jak2 phosphorylation as well as consensus sites for SRC tyrosine kinases. To test the possible influence of the two types of kinases on DLGAP1 we treated K562, HEL and UT7/TPO cell lines with an inhibitor of Jak2 (AG490) and independently with an inhibitor of SRC (SU6656). In each case the centrosomal staining of DLGAP1 was diminished with minimal effect on the cytoplasmic fraction of DLGAP1. Interestingly, treatment with SU6656 of the K562 cells switched to Mpl signaling and overexpressing full length GFP-DLGAP1 fusion resulted in a significant increase in the number of polyploid cells. The changes correlated with upregulation of the megakaryocytic marker CD41a and downregulation of the erythroid marker Glycophorin A as assessed by FACS analyses. As centrosomal aberrations are frequent markers of the myeloproliferative neoplasms, we intend to study the role of DLGAP1 in these bone marrow disorders.