Downregulation of the Cell-Cycle Regulating Ubiquitin-Ligase APC/CCdh1 May Contribute to the Differentiation Block of AML1/Eto Positive AML.
Authors: Hein Manuel , Schnerch Dominik , Schmidts Andrea , Felthaus Julia , Wider Dagmar , Ihorst Gabriele , Engelhardt Monika , Waesch Ralph M. 
Published: October 2019

Abstract
Abstract 5045

Introduction
The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase regulating cell cycle progression by targeting various cell cycle regulators for proteasomal degradation. It is activated by the adaptor proteins Cdc20 in mitosis and by Cdh1 in late mitosis and G1/G0. Thereby, Cdh1 establishes a stable G1 phase enabling the cell to either exit the cell cycle and differentiate or to prepare for a new round of cell division. It has also been shown that Cdh1 plays a role in the differentiation of various cell types, such as neurons, myocytes, hepatocytes and lens epithelial cells.

Methods and Results
We have examined the regulation of Cdh1 in several acute myeloid leukemia (AML) cell lines. We found that in the AML1/Eto positive leukemia cell lines SKNO-1 and Kasumi-1, Cdh1 protein and RNA levels are lower than in AML1/Eto negative cell lines KG-1 and HL-60. In addition, Cdh1 protein level in an AML1/Eto positive primary blast sample was lower than in AML1/Eto negative patient samples. The translocation t(8;21) is one of the most frequent chromosomal rearrangement in AML and results in an AML1/Eto fusion protein, which can act as a transcriptional repressor. Thus, our results are consistent with AML1/Eto mediated downregulation of Cdh1. To evaluate the potential role of APC/CCdh1 in myeloid differentiation, we established a stable Cdh1 knockdown (kd) in the AML1/Eto negative HL60 cell line with high Cdh1 expression by lentiviral vector mediated RNA interference. HL60 cells harbouring either a Cdh1 shRNA or a control shRNA against GFP were established simultaneously. We used PMA at concentrations of 0.5, 1, 2 and 50 nM to differentiate these cells into CD11b positive macrophage-like cells over 48h. Protein isolation and analysis of CD11b expression by flow cytometry were performed at 0, 6h, 12h, 24h and 48h to examine differentiation kinetics. Cdh1 and target proteins with a potential role in cell cycle arrest and differentiation, such as Skp2 (an activator of the SCF-ubiquitin ligase targeting p21 and p27) and ID2 (inhibitor of differentiation 2), were analyzed by Western blotting. We observed that kd of Cdh1 in HL60 cells resulted in 10% to 20% lower CD11b expression at any time, when PMA was used at concentrations 0, 0.5, 1nM over 48h. ID2 and Skp2 were stabilized in these Cdh1 kd cells compared to the control correlating with the less differentiated state. In addition, HL60 cells with a stable Skp2 kd showed a higher CD11b expression indicating a more differentiated status compared to the control.

Conclusion
This is the first report that indicates a role for APC/CCdh1 in the differentiation of myeloid cells with SCFSkp2 being one of the relevant targets. Downregulation of Cdh1 may contribute to the differentiation block of AML1/Eto postive AML.

Disclosures
No relevant conflicts of interest to declare.