Role of Cks1 for l-dependent and -indepentend cell cycle regulation, survival and quiescence in normal and leukemic hematopoiesis

The cyclin-dependent kinase inhibitors (CKI) p27Kip1 and p21Cip1 have been shown to regulate the size, quality and repopulation efficiency of the hematopoietic stem/ progenitor cell (HSC/ HPC) pool. Both p27Kip1 and p21Cip1 are targets of the E3 ubiquitin ligase SCFSkp2/Cks1 that controls their protein levels to regulate G1-S transition, S phase progression and G2-M transition. In the absence of the essential SCFSkp2 component Cyclin-dependent kinase subunit 1 (Cks1) p27Kip1 and p21Cip1 protein accumulate. Preliminary results indicate that Cks1 is highly expressed in early HSC and that loss of Cks1 results in Cks1 in normal and malignant hematopoiesis a depletion of the HSC/HPC pool. Furthermore, our preliminary data indicate that Cks1 protects early hematopoietic cells from apoptosis. To test the hypothesis that Cks1, either CKI-dependent via SCFSkp2/Cks1, or independent of CKI, regulates HSC homeostasis, we will generate mutant mice that lack Cks1 and p27Kip1 (Cks1-/-;p27-/-), and mice that lack Cks1 and p21Cip1 (Cks1-/-;p21-/-), as well as required compound knock-out mice. Normal and challenged hematopoiesis will be evaluated. Bone marrow from these compound mice and from control mice will be infected with retrovirus encoding Bcr- Abl and then transplanted into wild type recipient mice to induce a myeloproliferative disorder/CML-like disease and the stem cell pool will be analyzed. Our hypothesis is that Cks1 regulates the normal and malignant hematopoietic stem cell pool. The proposed experiments will allow addressing the role and mechanism of Cks1 in steady state and challenged hematopoiesis with regard to proliferation, self renewal, survival, and differentiation.

We identified Cks1 as an essential regulator of HSC fate operating upstream of the CKI p27, p21 and p57, and the Rb family member p130. Cks1 loss prominently affects the most immature CD150+ LSK HSC compartment by inhibiting exit from quiescence and cell cycling, and protects more mature HPC from apoptosis. Our analyses revealed that p27 is an essential downstream target of the SCFSkp2-Cks1 complex in the regulation of early hematopoiesis. Most likely all SCFSkp2/Cks1 CKI targets and p130 are involved in the HSC/HPC control. Only genetic in vivo studies involving composite gene kockout mice would allow deciphering this complex network. In CML, Cks1 is overexpressed. Cks1 is a mediator of Bcr-Ablinduced clonogenic activity, where Bcr-Abl expression also involves high Myc levels. Therefore Cks1 is proposed to be a central intermediate for normal and oncogene-induced hematopoietic cell cycle regulation. Cks1 thus represents a therapeutic target in various hematologic malignancies.