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 ET74A 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 ET74A 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 ET74A 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 ET74A 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 ET74A 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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.