APC/C prevents non-canonical order of cyclin/CDK activity to maintain CDK4/6 inhibitor-induced arrest

Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase APC/C (anaphase promoting complex/cyclosome), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear if APC/C maintains all types of arrest. Here by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological CDK4/6 inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves cyclin-dependent kinases acting in an atypical order to inactivate RB-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability. Significance Appropriate stable cell cycle arrest is critical to prevent cancer. However, it is not well-understood how cells maintain arrest. It is known that arrest requires repressing proliferation-stimulating genes, but the role of targeted protein degradation is unclear. This work demonstrates that continuous degradation of cyclin A through the action of the anaphase promoting complex/cyclosome (APC/C) is required to maintain arrest induced by a cancer drug that blocks cell cycle kinase enzymes. APC/C activity is required to prevent cell cycle re-entry. Impaired APC/C activity causes arrest bypass, inefficient DNA replication, and ultimately long-term proliferation defects. These results suggest that the activity level of the APC/C in tumors may profoundly influence the response to drugs that target cell cycle kinases. ### Competing Interest Statement The authors have declared no competing interest.