CIP2A recruits SLX4-MUS81-XPF in mitosis and protects against replication stress.

DNA replication stress can generate mitotic defects because incompletely replicated chromosomes or unresolved replication intermediates tether sister chromatids and hinder their segregation. We and others recently uncovered a mitotic role for the oncoprotein CIP2A, which promotes chromosome stability and is essential in homologous recombination-deficient (HRD) cells. However, how CIP2A safeguards mitotic genome integrity remains unclear. Here, we investigate the role of CIP2A in mitotic responses to replication stress. We show that replication stress induces a strong increase in CIP2A foci during mitosis, highlighting its involvement in processing under-replicated DNA. In wild-type cells, CIP2A is required for efficient recruitment of the scaffold SLX4 and the nucleases MUS81 and XPF to sites of under-replicated DNA. CIP2A loss disrupts this recruitment and leads to increased anaphase lagging chromosomes and micronuclei formation. CIP2A also contributes to mitotic DNA synthesis (MiDAS), although this varies across cell lines, indicating that MiDAS and SMX complex recruitment are not strictly coupled. Together, our findings identify CIP2A as a regulator of mitotic processing of under-replicated DNA and provide a framework for understanding context-dependent vulnerabilities in cancer cells.