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WEE1 kinase phosphorylates CDK1 and CDK2 to regulate origin firing and mitotic entry. Inhibition of WEE1 has become an attractive target for cancer therapy due to the simultaneous induction of replication stress and inhibition of the G2/M checkpoint. WEE1 inhibition in cancer cells with high levels of replication stress results in induction of replication catastrophe and mitotic catastrophe. To increase potential as a single agent chemotherapeutic, a better understanding of genetic alterations that impact cellular responses to WEE1 inhibition is warranted. Here, we investigate the impact of loss of the helicase, FBH1, on the cellular response to WEE1 inhibition. FBH1-deficient cells have a reduction in ssDNA and double strand break signaling indicating FBH1 is required for induction of replication stress response in cells treated with WEE1 inhibitors. Despite the defect in the replication stress response, FBH1-deficiency sensitizes cells to WEE1 inhibition by increasing mitotic catastrophe. We propose loss of FBH1 is resulting in replication-associated damage that requires the WEE1-dependent G2 checkpoint for repair.

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WEE1 kinase phosphorylates CDK1 and CDK2 to regulate origin firing and mitotic entry. Inhibition of WEE1 has become an attractive target for cancer therapy due to the simultaneous induction of replication stress and inhibition of the G2/M checkpoint. WEE1 inhibition in cancer cells with high levels of replication stress results in induction of replication catastrophe and mitotic catastrophe. To increase potential as a single agent chemotherapeutic, a better understanding of genetic alterations that impact cellular responses to WEE1 inhibition is warranted. Here, we investigate the impact of loss of the helicase, FBH1, on the cellular response to WEE1 inhibition. FBH1-deficient cells have a reduction in ssDNA and double strand break signaling indicating FBH1 is required for induction of replication stress response in cells treated with WEE1 inhibitors. Despite the defect in the replication stress response, FBH1-deficiency sensitizes cells to WEE1 inhibition by increasing mitotic catastrophe. We propose loss of FBH1 is resulting in replication-associated damage that requires the WEE1-dependent G2 checkpoint for repair.

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DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.

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Replication stress is a common feature of cancer cells. Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signalling, a DNA damage repair (DDR) pathway, is activated by regions of single-stranded DNA (ssDNA) that can arise during replication stress. ATR delays cell cycle progression and prevents DNA replication fork collapse, which prohibits cell death and promotes proliferation. Several ATR inhibitors have been developed in order to restrain this protective mechanism in tumours. It is known, however, that despite other effective anticancer chemotherapy treatments targeting DDR pathways, resistance occurs. This begets the need to identify combination treatments to overcome resistance and prevent tumour cell growth. We conducted a drug screen to identify potential synergistic combination treatments by screening an ATR inhibitor (VE822) together with compounds from a bioactive small molecule library. The screen identified adefovir dipivoxil, a reverse transcriptase inhibitor and nucleoside analogue, as a compound that has increased cytotoxicity in the presence of ATR, but not ATM or DNA-dependant protein kinase (DNA-PK) inhibition. Here we demonstrate that adefovir dipivoxil induces DNA replication stress, activates ATR signalling and stalls cells in S phase. This simultaneous induction of replication stress and inhibition of ATR signalling lead to a marked increase in pan-nuclear γH2AX-positive cells, ssDNA accumulation and cell death, indicative of replication catastrophe.

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It has been long assumed that normally leading strand synthesis must proceed coordinated with the lagging strand to prevent strand uncoupling and the pathological accumulation of single-stranded DNA (ssDNA) in the cell, a dogma recently challenged by in vitro studies in prokaryotes. Here, we report that human DNA polymerases can function independently at each strand in vivo and that the resulting strand uncoupling is supported physiologically by a cellular tolerance to ssDNA. Active forks rapidly accumulate ssDNA at the lagging strand when POLA1 is inhibited without triggering a stress response, despite ssDNA formation being considered a hallmark of replication stress. Acute POLA1 inhibition causes a lethal RPA exhaustion, but cells can duplicate their DNA with limited POLA1 activity and exacerbated strand uncoupling as long as RPA molecules suffice to protect the elevated ssDNA. Although robust, this uncoupled mode of DNA replication is also an in-built weakness that can be targeted for cancer treatment.

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Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.

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Inhibitors of poly(ADP-ribose) polymerase (PARP) have demonstrated efficacy in women with BRCA-mutant ovarian cancer. However, only 15%-20% of ovarian cancers harbor BRCA mutations, therefore additional therapies are required. Here, we show that a subset of ovarian cancer cell lines and ex vivo models derived from patient biopsies are sensitive to a poly(ADP-ribose) glycohydrolase (PARG) inhibitor. Sensitivity is due to underlying DNA replication vulnerabilities that cause persistent fork stalling and replication catastrophe. PARG inhibition is synthetic lethal with inhibition of DNA replication factors, allowing additional models to be sensitized by CHK1 inhibitors. Because PARG and PARP inhibitor sensitivity are mutually exclusive, our observations demonstrate that PARG inhibitors have therapeutic potential to complement PARP inhibitor strategies in the treatment of ovarian cancer.

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Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.

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Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)- and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive single-strand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells.

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Cancer stem cells (CSCs) are subpopulations of multipotent stem cells (SCs) responsible for the initiation, long-term clonal maintenance, growth and spreading of most human neoplasms. Reportedly, CSCs share a very robust DNA damage response (DDR) with embryonic and adult SCs, which allows them to survive endogenous and exogenous genotoxins. A range of experimental evidence indicates that CSCs have high but heterogeneous levels of replication stress (RS), arising from, and being boosted by, endogenous causes, such as specific genetic backgrounds (e.g., p53 deficiency) and/or aberrant karyotypes (e.g., supernumerary chromosomes). A multipronged RS response (RSR) is put in place by CSCs to limit and ensure tolerability to RS. The characteristics of such dedicated cascade have two opposite consequences, both relevant for cancer therapy. On the one hand, RSR efficiency often increases the reliance of CSCs on specific DDR components. On the other hand, the functional redundancy of pathways of the RSR can paradoxically promote the acquisition of resistance to RS- and/or DNA damage-inducing agents. Here, we provide an overview of the molecular mechanisms of the RSR in cancer cells and CSCs, focusing on the role of CHK1 and some emerging players, such as PARP1 and components of the homologous recombination repair, whose targeting can represent a long-term effective anti-CSC strategy.

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Cdc45 is an essential protein that together with Mcm2-7 and GINS forms the eukaryotic replicative helicase CMG. Cdc45 seems to be rate limiting for the initial unwinding or firing of replication origins. In line with this view, Cdc45-overexpressing cells fired at least twice as many origins as control cells. However, these cells displayed an about 2-fold diminished fork elongation rate, a pronounced asymmetry of replication fork extension, and an early S phase arrest. This was accompanied by H2AX-phosphorylation and subsequent apoptosis. Unexpectedly, we did not observe increased ATR/Chk1 signaling but rather a mild ATM/Chk2 response. In addition, we detected accumulation of long stretches of single-stranded DNA, a hallmark of replication catastrophe. We conclude that increased origin firing by upregulated Cdc45 caused exhaustion of the single-strand binding protein RPA, which in consequence diminished the ATR/Chk1 response; the subsequently occurring fork breaks led to an ATM/Chk2 mediated phosphorylation of H2AX and eventually to apoptosis.

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