PB1691 CDC20 IS A NOVEL POTENTIAL THERAPEUTIC TARGET IN ACUTE MYELOID LEUKEMIA
Authors: Bruno S. , di Rorà A. Ghelli Luserna , Soverini S. , Martinelli G. , Simonetti G. 
Published: June 2019

Background:

Cell‐division cycle protein 20 (Cdc20) is one activator of the anaphase‐promoting complex (APC) during mitosis. APC‐Cdc20 has been characterized to play a role in cell cycle progression through ubiquitination and proteolysis of several cell cycle regulatory targets, including securin and cyclin B1 (Holland. SEB Exp Biol Ser. 2008). Many sudies have shown that CDC20 may function as oncoprotein to promote cancer development and progression (Smolders. Expert Opin Ther Targets, 2011). We previously have shown that Cdc20 is overexpressed in aneuploid acute myeloid leukemia (AML) patients compared with euploid ones (Simonetti, Padella. Cancer, 2019). We hypothesize that CDC20 upregulation might favor the survival and division of AML cells.

Aims:

This study aims to clarify the consequences of Cdc20 inhibition on AML cell lines.

Methods:

Intracellular staining of Cdc20 was performed on primary AML sample at diagnosis on CD34+ cells. AML cell lines were treated for 48 h with two different APC‐Cdc20 inhibitors: Apcin and proTAME. Cell viability, apoptosis and cell cycle were evaluated by RalTime‐Glo assay, Annexin‐V and PI staining, respectively. Cell cycle synchronization was obtained through 72 h of serum starvation for G0/G1 phase, double thymidine block (3 μM) for S phase, and single thymidine block (1 μM) followed by nocodazole (50ng/mL) for G2/M phase. PI staining was performed to evaluate the synchronization level. Western blot analysis was performed to quantify the protein level of Cdc20 and its target.

Results:

CDC20 is expressed at variable levels both in proliferating and resting CD34+ stem‐progenitor cells and in blast cells isolated from bone marrow or peripheral blood of AML patients. Conversely, synchronization of AML cell lines in each of the three phases of cell cycle showed preferential expression of Cdc20 in G2/M phase, with very low levels in G0/G1 and S phases.To evaluate the effect of Cdc20 functional inhibition we treated two different AML cell lines, KG‐1 (TP53‐mutated) and MV‐4‐11 (t(4;11) and FLT3‐ITD), with Apcin or proTAME as single agent. KG‐1 being most sensitive with IC50 values of 37.6 μM for Apcin, and 5.16 μM for proTAME after 48 h of treatment. Whereas MV‐4‐11 showed an IC50 of 180 μM for Apcin and 11.6 μM for proTAME. Annexin V/PI staining performed after treatment with different concentration of two inhibitors (50‐100 μM for Apcin, and 5‐10‐20 μM for proTAME) showed a reduction of cell viability in a dosage‐dependent manner. In particular, the percentage of apoptotic cells was 45.7% and 66.8% in KG‐1 after 48 h of treatment with 50 and 100 μM of Apcin, and 15%, 16.8% and 23.5% after treatment with 5, 10 and 20 μM of proTAME. At the same condition of treatment MV‐4‐11 showed low level of apoptotic cells, with values of 4.6% and 5.6% after Apcin treatment, and 5%, 6% and 6.9% after proTAME treatment. Moreover we observed that both molecules caused a significant arrest in G2/M phase of the cell cycle only in KG‐1 cells, in line with APC‐Cdc20 inhibition effect. WB analysis showed a reduction of Cdc20 protein level, especially in KG‐1, without stabilization of securin and cyclin B1 which results down‐regulated in both cell lines after treatment.

Summary/Conclusion:

Our data showed that Cdc20 is expressed at different levels in AML patients’ blast and in AML cell lines. The different effect of Cdc20 functional inhibition on two tested AML cell lines suggests a potential mechanism of action related to a genetic‐driven control of cell cycle progression. Further investigations are on the way to better clarify the potential therapeutic role of Cdc20 in AML.