Progression through mitosis is balanced by the timely regulation of phosphorylation and dephosphorylation events ensuring the correct segregation of chromosomes before cytokinesis. This balance is regulated by the opposing actions of CDK1 and PP2A, as well as the Greatwall kinase/MASTL. MASTL is commonly overexpressed in cancer, which makes it a potential therapeutic anticancer target. Loss of Mastl induces multiple chromosomal errors that lead to the accumulation of micronuclei and multilobulated cells in mitosis. Our analyses revealed that loss of Mastl leads to chromosome breaks and abnormalities impairing correct segregation. Phospho-proteomic data for Mastl knockout cells revealed alterations in proteins implicated in multiple processes during mitosis including double-strand DNA damage repair. In silico prediction of the kinases with affected activity unveiled NEK2 to be regulated in the absence of Mastl. We uncovered that, RAD51AP1, involved in regulation of homologous recombination, is phosphorylated by NEK2 and CDK1 but also efficiently dephosphorylated by PP2A/B55. Our results suggest that MastlKO disturbs the equilibrium of the mitotic phosphoproteome that leads to the disruption of DNA damage repair and triggers an accumulation of chromosome breaks even in noncancerous cells.

Cell cycle regulation, especially faithful DNA replication and mitosis, are crucial to maintain genome stability. Cyclin-dependent kinase (CDK)/cyclin complexes drive most processes in cellular proliferation. In response to DNA damage, cell cycle surveillance mechanisms enable normal cells to arrest and undergo repair processes. Perturbations in genomic stability can lead to tumor development and suggest that cell cycle regulators could be effective targets in anticancer therapy. However, many clinical trials ended in failure due to off-target effects of the inhibitors used. Here, we investigate in vivo the importance of WEE1- and MYT1-dependent inhibitory phosphorylation of mammalian CDK1. We generated Cdk1AF knockin mice, in which two inhibitory phosphorylation sites are replaced by the non-phosphorylatable amino acids T14A/Y15F. We uncovered that monoallelic expression of CDK1AF is early embryonic lethal in mice and induces S phase arrest accompanied by γH2AX and DNA damage checkpoint activation in mouse embryonic fibroblasts (MEFs). The chromosomal fragmentation in Cdk1AF MEFs does not rely on CDK2 and is partly caused by premature activation of MUS81-SLX4 structure-specific endonuclease complexes, as well as untimely onset of chromosome condensation followed by nuclear lamina disassembly. We provide evidence that tumor development in liver expressing CDK1AF is inhibited. Interestingly, the regulatory mechanisms that impede cell proliferation in CDK1AF expressing cells differ partially from the actions of the WEE1 inhibitor, MK-1775, with p53 expression determining the sensitivity of cells to the drug response. Thus, our work highlights the importance of improved therapeutic strategies for patients with various cancer types and may explain why some patients respond better to WEE1 inhibitors.