Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.

Cells undergo tens of thousands of DNA-damaging events each day. Defects in repairing double-stranded breaks (DSBs) can lead to genomic instability, contributing to cancer, genetic disorders, immunological diseases, and developmental defects. Cohesin, a multi-subunit protein complex, plays a crucial role in both chromosome organization and DNA repair by creating architectural loops through chromatin extrusion. However, the mechanisms by which cohesin regulates these distinct processes are not fully understood. In this study, we identify two separate roles for cohesin in DNA repair within mammalian cells. First, cohesin serves as an intrinsic architectural factor that normally prevents interactions between damaged chromatin. Second, cohesin has an architecture-independent role triggered by ATM phosphorylation of SMC1, which enhances the efficiency of repair. Our findings suggest that these two functions work together to reduce the occurrence of translocations and deletions associated with non-homologous end joining, thereby maintaining genomic stability.

DNA synthesis in metazoans initiates within a select group of replication origins (baseline origins), whereas other (dormant) origins do not initiate replication despite recruiting apparently indistinguishable pre-replication complexes. Dormant origins are activated as backups when DNA synthesis stalls, allowing for complete genome duplication, yet it is unclear how cells selectively differentiate between baseline and dormant origins. We report here that during unperturbed cell proliferation, dormant origins selectively bind phosphorylated RecQL4 (pRecQL4), a member of the RecQ helicase family mutated in Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. Origin-bound pRecQL4 prevents the binding of an essential replication initiation complex, MTBP-TICRR/TRESLIN, to dormant origins, thus restricting replication initiation to baseline origins. When cells encounter replication stress, pRecQL4 is required for the dissociation of the MTBP-TICRR/TRESLIN complex from chromatin, which, in turn, facilitates the subsequent redistribution of MTBP-TICRR/TRESLIN to both baseline and dormant origins and allows recovery from replication inhibition. Thus, the interactions between the MTBP-TICRR/TRESLIN complex and pRecQL4 at replication origins are critical for replication origin choice and facilitate recovery from replication stress.

Triple-negative breast cancer (TNBC) is clinically aggressive and relatively unresponsive to current therapies. Therefore, the development of new anticancer agents is needed to satisfy clinical needs. Oxyphenisatin acetate (Acetalax), which had been used as a laxative, has recently been reported to have anticancer activity in murine models. In this study, we demonstrate that Acetalax and its diphenolic laxative structural analogue bisacodyl (Dulcolax) exhibit potent antiproliferative activity in TNBC cell lines and cause oncosis, a nonapoptotic cell death characterized by cellular and nuclear swelling and cell membrane blebbing, leading to mitochondrial dysfunction, ATP depletion, and enhanced immune and inflammatory responses. Mechanistically, we provide evidence that transient receptor potential melastatin member 4 (TRPM4) is poisoned by Acetalax and bisacodyl in MDA-MB468, BT549, and HS578T TNBC cells. MDA-MB231 and MDA-MB436 TNBC cells without endogenous TRPM4 expression as well as TRPM4-knockout TNBC cells were found to be Acetalax- and bisacodyl-resistant. Conversely, ectopic expression of TRPM4 sensitized MDA-MB231 and MDA-MB436 cells to Acetalax. TRPM4 was also lost in cells with acquired Acetalax resistance. Moreover, TRPM4 is rapidly degraded by the ubiquitin–proteasome system upon acute exposure to Acetalax and bisacodyl. Together, these results demonstrate that TRPM4 is a previously unknown target of Acetalax and bisacodyl and that TRPM4 expression in cancer cells is a predictor of Acetalax and bisacodyl efficacy and could be used for the clinical development of these drugs as anticancer agents. Significance: Acetalax and bisacodyl kill cancer cells by causing oncosis following poisoning of the plasma membrane sodium transporter TRPM4 and represent a new therapeutic approach for TNBC.