APOBEC3B (A3B) is aberrantly overexpressed in a subset of breast cancers, where it associates with advanced disease, poor prognosis, and treatment resistance, yet the causes of A3B dysregulation in breast cancer remain unclear. Here, A3B mRNA and protein expression levels were quantified in different cell lines and breast tumors and related to cell cycle markers using RT-qPCR and multiplex immunofluorescence imaging. The inducibility of A3B expression during the cell cycle was additionally addressed after cell cycle synchronization with multiple methods. First, we found that A3B protein levels within cell lines and tumors are heterogeneous and associate strongly with the proliferation marker Cyclin B1 characteristic of the G₂/M phase of the cell cycle. Second, in multiple breast cancer cell lines with high A3B, expression levels were observed to oscillate throughout the cell cycle and again associate with Cyclin B1. Third, induction of A3B expression is potently repressed throughout G₀/early G₁, likely by RB/E2F pathway effector proteins. Fourth, in cells with low A3B, induction of A3B through the PKC/ncNF-κB pathway occurs predominantly in actively proliferating cells and is largely absent in cells arrested in G₀. Altogether, these results support a model in which dysregulated A3B overexpression in breast cancer is the cumulative result of proliferation-associated relief from repression with concomitant pathway activation during the G₂/M phase of the cell cycle.

APOBEC3B (A3B) is a single-stranded DNA cytosine deaminase overexpressed in cancer, where it causes genomic DNA damage and mutations associated with tumor evolution. Enforced A3B overexpression triggers a dependency on the replication-stress response in different cellular models. However, whether endogenous A3B in cancer cells might yield a similar vulnerability is unclear. Here, we investigate how endogenous A3B expression and catalytic activity affect sensitivity to CHK1 inhibition, using two cancer cell lines, JHOC5 and U2OS. A3B-expressing cancer cells are sensitive to two chemically distinct CHK1 inhibitors, GDC-0575 and Prexasertib. CHK1 inhibitor sensitivity is reduced by A3B CRISPR knockout and restored by re-expressing wildtype A3B in knockout cells. Moreover, an endogenous A3B-E255A catalytic mutant generated by homology-directed repair phenocopies the reduced CHK1 inhibitor sensitivity of A3B -null cells, demonstrating a DNA deamination-dependent mechanism. CHK1 inhibition induces replication-associated DNA damage and cell-cycle perturbation dependent on A3B expression. As a result, A3B-expressing cells accumulate more pan-nuclear γH2AX, aberrant DNA-content profiles, and an expanded EdU-negative S-phase population, which are hallmarks of stalled DNA replication. In comparison, A3B -null and A3B-E255A cells retain defined cell-cycle distributions and are less sensitive to CHK1 inhibition. Together, these findings identify endogenous A3B-catalyzed deamination as a therapeutically actionable source of replication-associated DNA damage that renders tumor cells selectively dependent on CHK1 function. Statement of significance: APOBEC3B causes mutations in cancer cells and simultaneously imposes DNA replication stress. This combines to sensitize tumor cells to chemical inhibitors of the DNA damage response kinase CHK1.