Targeting the Anaphase Promoting Complex/Cyclosome (APC/C) in Multiple Myeloma

The discovery of novel agents such as the proteasome inhibitor bortezomib has significantly increased the survival of multiple myeloma (MM) patients. However MM remains an incurable disease mainly due to relapse, associated with significant resistance to therapy including bortezomib. Therefore further investigation to elucidate the disease and the mechanisms leading to drug resistance is necessary.

The success of bortezomib highlights the importance of the ubiquitin-proteasomal system (UPS) in MM. The UPS regulates protein turnover and plays a key role is several cellular processes such as apoptosis, cell cycle progression, cell proliferation and DNA replication. The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase protein complex involved in controlling cell cycle progression. The regulation of APC/C is dependent on 2 co-activators: Cdc20 and Cdh1. The APC^Cdc20 complex is controlling the metaphase to anaphase transition in mitosis, while APC^Cdh1 controls mitotic exit and early G1 phase. During metaphase, the activity of APC^Cdc20 is inhibited by the spindle assembly checkpoint. When all kinetochores are properly attached, the spindle assembly checkpoint is silenced and APC^Cdc20 becomes activated. When APC^Cdc20is active, cell cycle proteins are targeted for degradation by the proteasome such as securin and cyclin A and B leading to mitotic exit. Recent studies described that spindle assembly checkpoint is defective in MM cells and that patient samples after chemotherapy and at relapse displayed an increased chromosomal instability signature including Cdc20.

The aim of our study is to elucidate the importance and therapeutic potential of APC/C and its co-activators Cdc20 and Cdh1 in MM. Analysis of gene expression in the data of Zhan et al. (Blood 108, 2020-8, 2006) revealed that the co-activator Cdc20 was higher expressed in certain MM sub-groups (PR, MS, CD1, MF) compared to healthy bone marrow plasma cells. Moreover, high Cdc20 expression is correlated with poor prognosis. Cdh1 on the other hand was significantly lower expressed in all MM sub-groups compared to healthy bone marrow plasma cells. Interestingly, lower Cdh1 expression is correlated with poor prognosis. Next, we analyzed whether blocking APC/C would affect MM cells. For this study the pro-drug of TAME (tosyl-L-arginine methyl ester) that has been described as an inhibitor of the APC/C, was used. When the human myeloma cell lines LP-1 and RPMI-8226 were treated with proTAME, an accumulation of the APC^Cdc20 substrate cyclin B1 was seen already after 6 hours. However the levels of Skp2, an APC^Cdh1 substrate, were not affected by proTAME treatment. This suggests that proTAME inhibits the APC^Cdc20 complex but not the APC^Cdh1complex. We morphologically assessed the effect on number of metaphases on May-Grünwald Giemsa stained cytospins. ProTAME clearly induced an accumulation of LP-1 and RPMI-8226 cells in metaphase. Since a metaphase arrest can lead to cell death, we investigated the effect of proTAME on the viability and apoptosis. A significant dose-dependent decrease in viability and increase in apoptosis was observed after treatment with proTAME of human myeloma cell lines and primary MM cells purified from human and 5T33MM diseased mice. In contrast, other cells from the bone marrow microenvironment were not affected upon proTAME treatment. The induction of apoptosis was accompanied with caspase 3, 8, 9 and PARP cleavage. Western Blot analysis also showed phosphorylation of H2AX suggesting DNA damage upon proTAME treatment. Previous studies showed that MM is a heterogeneous disease consisting of a bulk CD138+ population and a minor CD138- population which is less sensitivity to drugs such as bortezomib. Interestingly, treatment of CD138+/- 5T33MM cells with proTAME demonstrated an equal targeting of both populations.

From these results we can conclude that overexpression of Cdc20 by MM cells is correlated with a bad prognosis. Inhibition of APC^Cdc20 results in a metaphase arrest in MM cells which is associated with reduced viability and induction of apoptosis. Moreover, APC/C inhibition equally targets CD138+ and the more resistant CD138- 5T33MM cells. This study suggests that APC/C and its co-activator Cdc20 could be a new and promising target in MM.