Investigating the role of the polarity regulator Scribble in maintenance and polarity formation of hematopoietic stem cells.

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Evolutionary conserved regulators of stem cell properties as well as RNA-/DNA-binding proteins have been shown to influence hematopoietic stem cell-polarity and -division. Recently, we were able to characterize a polarity regulator and member of the Scribble polarity complex (lethal giant larvae homolog 1: Llgl1) and provided evidence for its role in regulation of HSC biology. Loss of Llgl1 resulted in competitive advantage of hematopoietic stem cells (HSC) and Llgl1 KO HSC revealed a higher degree of stress-resistance. Despite an increase in cell cycle, Llgl1 KO HSC did not exhaust. Moreover, low expression of Llgl1 in primary patient blasts was associated with dismal prognosis and decreased overall survival of patients with acute myeloid leukemia. In the proposed study we aim to investigate the impact of Scribble polarity complex on hematopoietic stem cells. We will characterize the functional relevance using a conditional knockout mouse model for the central complex member Scribble. The influence of its genetic inactivation in steady state hematopoiesis will be investigated as well as the impact on stem cell biology by serial bone marrow transplantation into recipient hosts. Cell cycle analysis will complement the functional characterization of hematopoietic stem cells. In a second step, we aim to clarify the role of both Scribble complex members (Llgl1 and Scribble) in regulation of HSC polarity and bone marrow-niche interaction. To address these questions we will use time-lapse microscopy, analysis of triggered and spontaneous cell polarization on fibronectin coated surfaces as well as in-vivo 2D microscopy of the bone marrow. Intracellular interactions of Scribble complex members will be analyzed by global gene-expression profiling (on a transcriptional level using Scribble knockout versus wildtype HSC) and by mass-spectrometry (for protein-protein interactions). The proposed experiments will allow functional and structural characterization of Scribble polarity complex in hematopoietic stem cells. This will deepen our knowledge on regulation of stem cell biology and -fate through the polarity machinery. We and others already provided first evidence for a role for polarity complex members in pre-leukemic transformation of hematopoietic stem cells and their prognostic relevance. This may lead to direct therapeutic implications for targeting leukemic stem cells through interference with the polarity network.