Explore the vast range of titles available.

Replication stress is a major cause of genomic instability and a crucial vulnerability of cancer cells. This vulnerability can be therapeutically targeted by inhibiting kinases that coordinate the DNA damage response with cell cycle control, including ATR, CHK1, WEE1 and MYT1 checkpoint kinases. In addition, inhibiting the DNA damage response releases DNA fragments into the cytoplasm, eliciting an innate immune response. Therefore, several ATR, CHK1, WEE1 and MYT1 inhibitors are undergoing clinical evaluation as monotherapies or in combination with chemotherapy, poly[ADP-ribose]polymerase (PARP) inhibitors, or immune checkpoint inhibitors to capitalize on high replication stress, overcome therapeutic resistance and promote effective antitumour immunity. Here, we review current and emerging approaches for targeting replication stress in cancer, from preclinical and biomarker development to clinical trial evaluation.Replication stress is a cause of genome instability in cancer cells. This Review discusses strategies to increase replicative stress by inhibiting the checkpoint kinases that coordinate DNA damage response and cell cycle, as well as combination strategies with other targeted therapies.

Poly (ADP-ribose) polymerase inhibitor (PARPi, olaparib) impairs the repair of DNA single-strand breaks (SSBs), resulting in double-strand breaks (DSBs) that cannot be repaired efficiently in homologous recombination repair (HRR)-deficient cancers such as BRCA1/2-mutant cancers, leading to synthetic lethality. Despite the efficacy of olaparib in the treatment of BRCA1/2 deficient tumors, PARPi resistance is common. We hypothesized that the combination of olaparib with anticancer agents that disrupt HRR by targeting ataxia telangiectasia and Rad3-related protein (ATR) or checkpoint kinase 1 (CHK1) may be an effective strategy to reverse ovarian cancer resistance to olaparib. Here, we evaluated the effect of olaparib, the ATR inhibitor AZD6738, and the CHK1 inhibitor MK8776 alone and in combination on cell survival, colony formation, replication stress response (RSR) protein expression, DNA damage, and apoptotic changes in BRCA2 mutated (PEO-1) and HRR-proficient BRCA wild-type (SKOV-3 and OV-90) cells. Combined treatment caused the accumulation of DNA DSBs. PARP expression was associated with sensitivity to olaparib or inhibitors of RSR. Synergistic effects were weaker when olaparib was combined with CHK1i and occurred regardless of the BRCA2 status of tumor cells. Because PARPi increases the reliance on ATR/CHK1 for genome stability, the combination of PARPi with ATR inhibition suppressed ovarian cancer cell growth independently of the efficacy of HRR. The present results were obtained at sub-lethal doses, suggesting the potential of these inhibitors as monotherapy as well as in combination with olaparib.

Accumulating evidence suggests that exogenous cellular stress induces PD-L1 upregulation in cancer. A DNA double-strand break (DSB) is the most critical type of genotoxic stress, but the involvement of DSB repair in PD-L1 expression has not been investigated. Here we show that PD-L1 expression in cancer cells is upregulated in response to DSBs. This upregulation requires ATM/ATR/Chk1 kinases. Using an siRNA library targeting DSB repair genes, we discover that BRCA2 depletion enhances Chk1-dependent PD-L1 upregulation after X-rays or PARP inhibition. In addition, we show that Ku70/80 depletion substantially enhances PD-L1 upregulation after X-rays. The upregulation by Ku80 depletion requires Chk1 activation following DNA end-resection by Exonuclease 1. DSBs activate STAT1 and STAT3 signalling, and IRF1 is required for DSB-dependent PD-L1 upregulation. Thus, our findings reveal the involvement of DSB repair in PD-L1 expression and provide mechanistic insight into how PD-L1 expression is regulated after DSBs. PD-L1 is upregulated in many cancers due to exogenous cellular stress. Here the authors show that PD-L1 is upregulated in response to DNA double strand breaks via STAT and IRF1 signalling.

Extrachromosomal DNA (ecDNA) presents a major challenge for cancer patients. ecDNA renders tumours treatment resistant by facilitating massive oncogene transcription and rapid genome evolution, contributing to poor patient survival 1 – 7 . At present, there are no ecDNA-specific treatments. Here we show that enhancing transcription–replication conflict enables targeted elimination of ecDNA-containing cancers. Stepwise analyses of ecDNA transcription reveal pervasive RNA transcription and associated single-stranded DNA, leading to excessive transcription–replication conflicts and replication stress compared with chromosomal loci. Nucleotide incorporation on ecDNA is markedly slower, and replication stress is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. pRPA2-S33, a mediator of DNA damage repair that binds single-stranded DNA, shows elevated localization on ecDNA in a transcription-dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition causes extensive and preferential tumour cell death in ecDNA-containing tumours. We advance a highly selective, potent and bioavailable oral CHK1 inhibitor, BBI-2779, that preferentially kills ecDNA-containing tumour cells. In a gastric cancer model containing FGFR2 amplified on ecDNA, BBI-2779 suppresses tumour growth and prevents ecDNA-mediated acquired resistance to the pan-FGFR inhibitor infigratinib, resulting in potent and sustained tumour regression in mice. Transcription–replication conflict emerges as a target for ecDNA-directed therapy, exploiting a synthetic lethality of excess to treat cancer. Extrachromosomal DNA makes cancerous tumours resistant to treatment, but this research demonstrates that increasing transcription–replication conflict allows for targeted elimination of cancer cells containing extrachromosomal DNA, and thus sustained tumour regression in mice.

Checkpoint kinases 1 and 2 (CHK1 and CHK2) are enzymes that are involved in the control of DNA damage. At the present time, these enzymes are some of the most important targets in the fight against cancer since their inhibition produces cytotoxic effects in carcinogenic cells. This paper proposes the use of spirostans (Sp), natural compounds, as possible inhibitors of the enzymes CHK1 and CHK2 from an in silico analysis of a database of 155 molecules (S5). Bioinformatics studies of molecular docking were able to discriminate between 13 possible CHK1 inhibitors, 13 CHK2 inhibitors and 1 dual inhibitor for both enzymes. The administration, distribution, metabolism, excretion and toxicity (ADMETx) studies allowed a prediction of the distribution and metabolism of the potential inhibitors in the body, as well as determining the excretion routes and the appropriate administration route. The best inhibition candidates were discriminated by comparing the enzyme-substrate interactions from 2D diagrams and molecular docking. Specific inhibition candidates were obtained, in addition to studying the dual inhibitor candidate and observing their stability in dynamic molecular studies. In addition, Highest Occupied Molecular Orbital—Lowest Unoccupied Molecular Orbital (HOMO-LUMO) interactions were analyzed to study the stability of interactions between the selected enzymes and spirostans resulting in the predominant gaps from HOMOCHKs to LUMOSp (Highest Occupied Molecular Orbital of CHKs—Lowest Unoccupied Molecular Orbital of spirostan). In brief, this study presents the selection inhibitors of CHK1 and CHK2 as a potential treatment for cancer using a combination of molecular docking and dynamics, ADMETx predictons, and HOMO-LUMO calculation for selection.

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in Drosophila sensory neurons removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhances regeneration. Our findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma. The Atr-Check1 pathway is involved in cell cycle and the DNA damage response. Here, the authors show that the Atr-Check1 pathway can inhibit axon regeneration in response to Piezo-mediated mechanosensation, affecting functional recovery.

Colorectal cancer (CRC) ranks third among the most frequent malignancies and represents the second most common cause of cancer-related deaths worldwide. By interfering with the DNA replication process of cancer cells, several chemotherapeutic molecules used in CRC therapy induce replication stress (RS). At the cellular level, this stress is managed by the ATR-CHK1 pathway, which activates the replication checkpoint. In recent years, the therapeutic value of targeting this pathway has been demonstrated. Moreover, MSI + (microsatellite instability) tumors frequently harbor a nonsense, heterozygous mutation in the ATR gene. Using isogenic HCT116 clones, we showed that this mutation of ATR sensitizes the cells to several drugs, including SN-38 (topoisomerase I inhibitor) and VE-822 (ATR inhibitor) and exacerbates their synergistic effects. We showed that this mutation bottlenecks the replication checkpoint leading to extensive DNA damage. The combination of VE-822 and SN-38 induces an exhaustion of RPA and a subsequent replication catastrophe. Surviving cells complete replication and accumulate in G2 in a DNA-PK-dependent manner, protecting them from cell death. Together, our results suggest that RPA and DNA-PK represent promising therapeutic targets to optimize the inhibition of the ATR-CHK1 pathway in oncology. Ultimately, ATR frameshift mutations found in patients may also represent important prognostic factors.

T-LAK-originated protein kinase (TOPK) overexpression is a feature of multiple cancers, yet is absent from most phenotypically normal tissues. As such, TOPK expression profiling and the development of TOPK-targeting pharmaceutical agents have raised hopes for its future potential in the development of targeted therapeutics. Results presented in this paper confirm the value of TOPK as a potential target for the treatment of solid tumours, and demonstrate the efficacy of a TOPK inhibitor (OTS964) when used in combination with radiation treatment. Using H460 and Calu-6 lung cancer xenograft models, we show that pharmaceutical inhibition of TOPK potentiates the efficacy of fractionated irradiation. Furthermore, we provide in vitro evidence that TOPK plays a hitherto unknown role during S phase, showing that TOPK depletion increases fork stalling and collapse under conditions of replication stress and exogenous DNA damage. Transient knockdown of TOPK was shown to impair recovery from fork stalling and to increase the formation of replication-associated single-stranded DNA foci in H460 lung cancer cells. We also show that TOPK interacts directly with CHK1 and Cdc25c, two key players in the checkpoint signalling pathway activated after replication fork collapse. This study thus provides novel insights into the mechanism by which TOPK activity supports the survival of cancer cells, facilitating checkpoint signalling in response to replication stress and DNA damage.

Checkpoint kinase 1 (CHK1) is a central mediator of the DNA damage response (DDR) at the S and G2/M cell cycle checkpoints, and plays a crucial role in preserving genomic integrity. CHK1 overexpression is thought to contribute to cancer aggressiveness, and several selective inhibitors of this kinase are in clinical development for various cancers, including neuroblastoma (NB). Here, we examined the sensitivity of MYCN-amplified NB cell lines to the CHK1 inhibitor PF-477736 and explored mechanisms to increase its efficacy. PF-477736 treatment of two sensitive NB cell lines, SMS-SAN and CHP134, increased the expression of two pro-apoptotic proteins, BAX and PUMA, providing a mechanism for the effect of the CHK1 inhibitor. In contrast, in NB-39-nu and SK-N-BE cell lines, PF-477736 induced DNA double-strand breaks and activated the ataxia telangiectasia mutated serine/threonine kinase (ATM)-p53-p21 axis of the DDR pathway, which rendered the cells relatively insensitive to the antiproliferative effects of the CHK1 inhibitor. Interestingly, combined treatment with PF-477736 and the ATM inhibitor Ku55933 overcame the insensitivity of NB-39-nu and SK-N-BE cells to CHK1 inhibition and induced mitotic cell death. Similarly, co-treatment with PF-477736 and NU7441, a pharmacological inhibitor of DNA-PK, which is also essential for the DDR pathway, rendered the cells sensitive to CHK1 inhibition. Taken together, our results suggest that synthetic lethality between inhibitors of CHK1 and the DDR drives G2/M checkpoint abrogation and could be a novel potential therapeutic strategy for NB.

Checkpoint kinase 1 (CHK1) is critical for intrinsic cell cycle control and coordination of cell cycle progression in response to DNA damage. Despite its essential function, CHK1 has been identified as a target to kill cancer cells and studies using Chk1 haploinsufficient mice initially suggested a role as tumor suppressor. Here, we report on the key role of CHK1 in normal B-cell development, lymphomagenesis and cell survival. Chemical CHK1 inhibition induces BCL2-regulated apoptosis in primary as well as malignant B-cells and CHK1 expression levels control the timing of lymphomagenesis in mice. Moreover, total ablation of Chk1 in B-cells arrests their development at the pro-B cell stage, a block that, surprisingly, cannot be overcome by inhibition of mitochondrial apoptosis, as cell cycle arrest is initiated as an alternative fate to limit the spread of damaged DNA. Our findings define CHK1 as essential in B-cell development and potent target to treat blood cancer. Checkpoint kinase 1 (CHK1) is critical for intrinsic cell cycle control and coordination of cell cycle progression. Here the authors show that CHK1 loss or chemical inhibition impacts on normal B cell development, lymphomagenesis and cancer cell survival.