Chromosome Instability Underlies Stem Cell Dysfunction and Neoplasia In Widespread Hematologic Disease Induced By Deregulated Cyclin E

The Fbw7 ubiquitin ligase controls the expression of a number of oncoprotein substrates including cyclin E, Notch, c-Jun, and c-Myc. Using a knock-in mouse model (cyclin E^T74AT393A), in which mutations were introduced into the cyclin E1 allele (Ccne1) to disrupt Fbw7-mediated ubiquitination specifically, we previously found that cyclin E dysregulation in vivo induces anemia with defects in erythroid differentiation and morphologic dysplasia of erythroid progenitors. We also found that cyclin E^T74AT393A mice have fewer hematopoietic stem cells (HSCs) during steady-state hematopoiesis compared to wild-type counterparts. We performed serial transplantation experiments to assay comprehensively the self-renewal and multi-lineage reconstitution capacities of cyclin E^T74AT393A HSCs. Contrary to our expectations, cyclin E^T74AT393A HSC self-renewal appears normal after three rounds of serial transplantation; however, we identified defects in their multi-lineage reconstitution function. In cyclin E^{T74A T393A} bone marrow erythroid cells, induction of a p53-dependent DNA damage response pathway appears to promote compensated erythropoiesis. In cyclin E^{T74A T393A} HSCs, we similarly observed induced expression of canonical p53 target genes. We studied the effect of p53-loss on cyclin E^{T74A T393A} HSCs and found that p53-null; cyclin E^{T74A T393A} HSCs exhibit defects in both self-renewal and multi-lineage reconstitution. By enumerating chromosomes in metaphase spreads, we found p53-null; cyclin E^{T74A T393A} hematopoietic stem and progenitor cells (HSPCs) demonstrate significant chromosomal instability (CIN). Importantly, we can recapitulate the self-renewal defects and CIN of cyclin E^{T74A T393A} HSPCs with intact p53 by treating recipient animals with a single dose of 5-fluorouracil (5-FU). Thus, chromosomal stability is a key determinant for the maintenance of HSC self-renewal, and hematologic stress appears to unmask the potential for impaired Fbw7-dependent cyclin E ubiquitination to engender CIN in the presence of intact p53. Moreover, CIN is a characteristic feature of fatal T-cell malignancies that ultimately develop in recipients of cyclin E^{T74A T393A}; p53-null HSCs. In pre-malignant thymocytes isolated from recipients of cyclin E^{T74A T393A}; p53-null HSCs, aneuploidy is associated with the marked potentiation of cyclin E kinase activity in these cells by p53-loss. In malignant thymocytes, comparative genome hybridization analysis demonstrates clonal CIN associated with deregulated cyclin E expression combined with p53-loss. In toto, our data demonstrate the functional importance of cyclin E regulation by the Fbw7 ubiquitin ligase to the hematopoietic system and highlight CIN as a key mechanism underlying HSC dysfunction and malignancy induced by deregulated cyclin E in vivo.