MASTL Kinase Mediates the DNA Replication Stress Response Through PP2A/B55 Phosphatase Modulation

MASTL kinase promotes mitosis by inhibiting PP2A-B55 through ENSA/ARPP19 phosphorylation. Our analyses of cancer datasets support the involvement and diagnostic/prognostic potential of the MASTL-ENSA-PP2A/B55 module. In head and neck and other cancers, increased MASTL/ENSA or reduced B55 is associated with resistance to multiple chemotherapeutic agents, including anti-replication drugs. Experimentally, MASTL enhances cellular tolerance by promoting replication fork progression under stress, supporting fork restart, and promoting new origin firing. These effects require PP2A/B55 suppression, whereas B55 upregulation reduces stalled fork recovery and new origin firing in fiber assays, consistent with increased sensitivity to replication stress. Phosphoproteomics identifies replication factors, including RPA, MCM, and DNA polymerases, as potential downstream targets. RPA32 functions as a key effector, with its phospho-deficient mutant showing impaired replication dynamics. Furthermore, replication stress reduces RPA-associated phosphatase activity, and induces a MASTL-dependent ENSA-PP2A interaction during S-phase. Collectively, our findings indicate that the mitotic MASTL-ENSA-PP2A/B55 pathway is partially activated in response to replication stress, promoting the replication stress response, and conferring therapeutic resistance.