APC/C^Cdh1 As a Mediator Of Hematopoietic Differentiation, Self-Renewal and Malignant Transformation

Cdh1 is an important activator of the anaphase-promoting complex/cyclosome (APC/C) and may play a major role in both the stabilization of G1-phase and the induction of cell cycle arrest and differentiation. Our work focuses on the function of APC/C^Cdh1in hematopoietic stem cells (HSCs) with regard to their potential for differentiation, self-renewal and malignant transformation.

Physiological expression levels of Cdh1 were studied among different human hematopoietic lineages (defined by staining for the cell surface markers CD11b, CD41a, CD34, CD3 and CD19) obtained from bone marrow (BM) of healthy donors and mobilized peripheral blood (PB). Next we established a strong lentiviral Cdh1 knock down (kd) in human CD34+ cells and performed colony forming cell (CFC) assays and replating assays. We also analyzed Cdh1-protein levels in 30 samples of BM or PB of patients first diagnosed with acute myeloid leukemia (AML). Finally experiments to further look into possible mechanisms of Cdh1 regulation during leukemogenesis were carried out. On the transcriptional level we reanalyzed published microarray data from CD34+-AML blasts and normal CD34+ cells (Leukemia 2011;25:1825-1833). On the post-transcriptional level we tested the hypothesis of Cdh1 degradation mediated by the ubiquitin-ligase SCF by expressing a dominant negative mutant of the core SCF subunit Cullin-1 (delta-Cul1) in the AML cell lines Kasumi-1 and HL-60.

Western blot analysis of physiological Cdh1-distribution among the variable human hematopoietic lineages showed significant differences in Cdh1 protein levels. We saw diminishing levels of Cdh1 from HSCs to mature lymphoid and myeloid cells, suggesting that Cdh1 may be important to induce differentiation but dispensable for maintaining the differentiated state.

In the Cdh1-kd-CFC assays a significant decrease of total colony numbers, CFU-Gs, CFU-GMs and BFU-Es >50% was observed. At morphological examination and FACS analysis these colonies proved to be more immature than the control colonies. Thus, depletion of Cdh1 in HSC hinders normal differentiation into the myeloid and erythroid lineage both by decreasing the number of mature lineage progenitors and by delaying individual cell maturation. Upon replating, we noticed a significant increase in the number of secondary colonies, with a doubling of total colony numbers, when using Cdh1 deficient HSC. This result indicates an advantage for self-renewal over differentiation in these cells, which seems to correlate with the intensity of the Cdh1-kd.

Examination of Cdh1 protein levels in AML blasts revealed that basically all AML samples showed a strong down-regulation of Cdh1 protein levels compared to normal CD34+ cells, which may be a contributing factor to the differentiation block in leukemogenesis. Indeed, if we performed knockdown of Cdh1 in the HL-60 leukemia cell line they were in a less differentiated state as judged by CD11b expression. The evaluation of microarray data, in order to further address the mechanism of Cdh1 down-regulation in AML blasts, showed that Cdh1 transcription levels were not significantly different in CD34+ AML cells compared to normal CD34+ cells. This would be consistent with a posttranscriptional cause of decreased Cdh1-protein levels in AML blasts. Our ongoing work indicates SCF-dependent degradation of Cdh1, since inhibition of the SCF function (by expression of a dominant-negative form of the SCF subunit Cullin-1 (delta-Cul1)) in AML cell lines leads to a strong upregulation of Cdh1.

Our data establish Cdh1 as an important cell cycle regulator in the regulation of differentiation and self-renewal in HSCs. Its posttranscriptional downregulation by the SCF ubiquitin ligase may contribute to leukemogenesis.

No relevant conflicts of interest to declare.