A Mouse Model of Hematopoietic Stem Cell Failure Associated with p53-Loss and Defective Fbw7-Dependent Cyclin E Regulation.

Abstract 2297

Recent findings have challenged the notion that increased proliferation of hematopoietic stem cells (HSCs) necessarily restricts their self-renewal capacity. We have studied the physiologic consequences to HSCs of ablating a key cell cycle regulatory mechanism, Fbw7-dependent cyclin E ubiquitination, using germline knock-in of a cyclin E^{T74A T393A} allele. Fbw7 is a tumor suppressor that regulates the abundance of several oncoprotein substrates by ubiquitin-mediated proteolysis, including cyclin E, Notch, and c-Myc. Cyclin E overexpression in vivo is associated with increased proliferation in some cellular contexts as well as a variety of deleterious consequences, including genomic instability, senescence, or apoptosis. In HSCs, Fbw7-loss has been shown to induce self-renewal and multi-lineage reconstitution defects, and the effect of Fbw7-loss in HSCs has been ascribed to dysregulated Myc and Notch expression. Using the cyclin E^{T74A T393A} mouse model, we tested the hypothesis that impaired Fbw7-mediated regulation of cyclin E, specifically, promotes HSC exhaustion due to loss of self-renewal capacity. We first examined bone marrow HSC counts and their cell cycle kinetics in cyclin E knock-in and wild-type control mice at steady state and following hematologic injury induced by 5-fluorouracil treatment. We found that cyclin E dysregulation reduces numbers of quiescent HSCs and increases cells in S/G2/M-phases, while decreasing total numbers of HSCs, phenotypes made more severe after recovery from hematologic stress. Using bromodeoxyuridine labeling studies, we found that excess cyclin E activity causes DNA hyper-replication in cyclin E^{T74A T393A} HSCs in a cell autonomous manner. By enumerating multi-potent progenitors (MPPs), we ruled out increased rate of transit from HSC-to-MPP as a cause of the apparent exhaustion of cyclin E knock-in HSCs. Thus, dysregulated cyclin E in HSCs promotes both increased proliferation and depletion of the HSC pool. Serial transplantation further revealed peripheral blood reconstitution defects associated with cyclin E^{T74A T393A} HSCs. Recently, we have found that p53 is activated by dysregulated cyclin E in hematopoietic cells in vivo, in association with phosphorylation of both p53 and Chk1 proteins, resembling a DNA damage-type response. Interestingly, p53-loss has been found to be associated with a gain of HSC self-renewal activity. We therefore hypothesized that p53-loss would rescue the self-renewal defect of cyclin E knock-in HSCs. Surprisingly, we discovered that cyclin E^{T74A T393A}; p53-null HSCs showed evidence of significantly worse self-renewal and peripheral reconstitution, compared to p53-null HSCs, defects that are more severe than those associated with impaired Fbw7-mediated cyclin E control in the setting of wild-type p53 (Chi-squared test, p<0.0001). Thus, our data are consistent with the concept that intact p53 function, in the setting of oncogenic insult, can preserve partial HSC self-renewal capacity, and its loss in vivo is detrimental to HSC viability when accompanied by defects in cell cycle control mechanisms.