Background. Despite the progress made in identifying key molecules that coordinate self-renewal, proliferation and differentiation during adult hematopoiesis, little is known about the physiologic and molecular regulation of these processes during bone marrow (BM) homeostasis and during its adaptive response to stress. Notch signaling plays an essential role in the regulation of cell fate decisions in normal hematopoiesis, favoring stem cell self-renewal and inhibiting differentiation of human bone marrow progenitors by promoting their expansion. Notch signaling has also been shown to participate in leukemogenesis. Preliminary results. Our previous studies showed that Notch activation inhibits differentiation of BM progenitors, preserving them in a more immature phenotype, and induces perturbation of their cell cycle by shortening their G1 phase and promoting premature entry into S-phase. We have recently identified the molecular mechanism that mediates this effect. We discovered that Notch1 activation induces the transcriptional activation of SKP2, the F-box subunit of the SCFskp2 complex that targets the cell cycle inhibitors p27Kip1 and p21Cip1 for degradation, promoting their downregulation and the entry into S-phase. Of note, SKP2 overexpression, is frequently altered in leukemias and lymphoma. To address the physiological relevance of these results we investigated the BM response during inflammatory stress. We found that cytokines released during inflammation, such as LPS and TNF1, induce upregulation of Notch signaling in BM cells and result in 4-5 fold increase in stem cells and progenitors. Hypothesis. Based on these results, we propose a model in which BM microenvironmental cues signal to hematopoietic cells and their cell cycle machinery via Notch signaling. We hypothesize that the SKP2/p27Kip1 pathway is critical for Notch-mediated stem cells and progenitor expansion, in particular during conditions of stress, and that it may be involved in Notch-induced leukemogenesis.
Aims and Strategy. To test this hypothesis we propose to: (1) Determine the role of the Notch/SKP2/CKIs pathway in the expansion of BM stem and progenitor cells by using mice deficient for SKP2 or Notch signaling (RBP-J conditional knock out) in combination with transplantation models;(2) Define whether SKP2 is critical for Notch-mediated effects on stem/progenitor expansion and Notch-induced leukemogenesis by analyzing the in vivo effects of Notch iperactivation on the hematopoietic compartment in the absence of SKP2;(3) Determine the role of different Notch ligands and of intensity of Notch signaling in the recruitment of the SKP2/CKIs pathway. This question will be addressed by stimulating hematopoietic cells through co-culture with feeders overexpressing different Notch ligands and with increasing doses of recombinant Delta4 ligand. Relevance. We believe that these experiments will yield insight into the molecular mechanisms underlying the physiologic regulation of bone marrow homeostasis and the process of leukemogenesis and that they will contribute to the development of novel therapeutic strategies in hematopoietic disorders. PROJECT NARRATIVE: The present proposal aims at defining the mechanisms that regulate blood cell production by the bone marrow in normal conditions and in conditions of stress, such as inflammation, infections or bone marrow transplantation. In particular, we are focused on understanding the role of the Notch receptors in the maintenance and expansion of bone marrow hematopoietic progenitors and stem cells. A better comprehension of the events that coordinate cell proliferation and differentiation during normal hematopoiesis is crucial for the understanding of the mechanisms of leukemogenesis and, therefore, is essential for the development of novel therapeutic strategies. The long term goal of this project is to evaluate the role of the Notch signaling pathway both in normal bone marrow reconstitution and in leukemias and to identify novel targets of therapeutic intervention.
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