DNA replication stress (RS) causes genomic instability and vulnerability in cancer cells. To counteract RS, cells have evolved various mechanisms involving the ATR kinase signaling pathway, which regulates origin firing, cell cycle checkpoints, and fork stabilization to secure the fidelity of replication. However, ATR signaling also alleviates RS to support cell survival by driving RS tolerance, thereby contributing to therapeutic resistance. Cancer cells harboring genetic mutations and other changes that disrupt normal DNA replication increase the risk of DNA damage and the levels of RS, conferring addiction to ATR activity for sustainable replication and susceptibility to therapeutic approaches using ATR inhibitors (ATRis). Therefore, clinical trials are currently being conducted to evaluate the efficacy of ATRis as monotherapies or in combination with other drugs and biomarkers. In this review, we discuss recent advances in the elucidation of the mechanisms by which ATR functions in the RS response and its therapeutic relevance when utilizing ATRis.

The SWI/SNF chromatin remodeling complex regulates transcription through the control of chromatin structure and is increasingly thought to play an important role in human cancer. Lung adenocarcinoma (LADC) patients frequently harbor mutations in SMARCA4, a core component of this multisubunit complex. Most of these mutations are loss-of-function mutations, which disrupt critical functions in the regulation of chromatin architecture and can cause DNA replication stress. This study reports that LADC cells deficient in SMARCA4 showed increased DNA replication stress and greater sensitivity to the ATR inhibitor (ATRi) in vitro and in vivo. Mechanistically, loss of SMARCA4 increased heterochromatin formation, resulting in stalled forks, a typical DNA replication stress. In the absence of SMARCA4, severe ATRi-induced single-stranded DNA, which caused replication catastrophe, was generated on nascent DNA near the reversed forks around heterochromatin in an Mre11-dependent manner. Thus, loss of SMARCA4 confers susceptibility to ATRi, both by increasing heterochromatin-associated replication stress and by allowing Mre11 to destabilize reversed forks. These two mechanisms synergistically increase susceptibility of SMARCA4-deficient LADC cells to ATRi. These results provide a preclinical basis for assessing SMARCA4 defects as a biomarker of ATRi efficacy.

Activation of the KRAS oncogene is a source of replication stress, but how this stress is generated and how it is tolerated by cancer cells remain poorly understood. Here we show that induction of KRAS^G12V expression in untransformed cells triggers H3K27me3 and HP1-associated chromatin compaction in an RNA transcription dependent manner, resulting in replication fork slowing and cell death. Furthermore, elevated ATR expression is necessary and sufficient for tolerance of KRAS^G12V-induced replication stress to expand replication stress-tolerant cells (RSTCs). PrimPol is phosphorylated at Ser255, a potential Chk1 substrate site, under KRAS^G12V-induced replication stress and promotes repriming to maintain fork progression and cell survival in an ATR/Chk1-dependent manner. However, ssDNA gaps are generated at heterochromatin by PrimPol-dependent repriming, leading to genomic instability. These results reveal a role of ATR-PrimPol in enabling precancerous cells to survive KRAS-induced replication stress and expand clonally with accumulation of genomic instability.

Activation of oncogenes disturbs a wide variety of cellular processes and induces physiological dysregulation of DNA replication, widely referred to as replication stress (RS). Oncogene-induced RS can cause replication forks to stall or collapse, thereby leading to DNA damage. While the DNA damage response (DDR) can provoke an anti-tumor barrier to prevent the development of cancer, a small subset of cells triggers replication stress tolerance (RST), allowing precancerous cells to survive, thereby promoting clonal expansion and genomic instability (GIN). Genomic instability (GIN) is a hallmark of cancer, driving genetic alterations ranging from nucleotide changes to aneuploidy. These alterations increase the probability of oncogenic events and create a heterogeneous cell population with an enhanced ability to evolve. This review explores how major oncogenes such as RAS, cyclin E, and MYC induce RS through diverse mechanisms. Additionally, we delve into the strategies employed by normal and cancer cells to tolerate RS and promote GIN. Understanding the intricate relationship between oncogene activation, RS, and GIN is crucial to better understand how cancer cells emerge and to develop potential cancer therapies that target these vulnerabilities.

DNA replication stress (RS), a prevalent feature of various malignancies, arises from both genetic mutations and genotoxic exposure. Elevated RS levels increase the vulnerability of cancer cells to ataxia telangiectasia and Rad3-related kinase inhibitors (ATRis). Here, we screened for DNA damage response inhibitors that enhance ATRi-induced cytotoxicity using SWI/SNF complex-deficient cells and identified a potent synergy between ATRi and poly(ADP-ribose) polymerase inhibitor (PARPi), particularly in SMARCA4-deficient cells. PARP inhibition triggers chromatin changes, namely elevated histone H3 at lysine 9 di-methylation (H3K9me2), a hallmark of facultative heterochromatin, increasing dependence on ATR activity for replication fork progression and cell survival. Interestingly, SMARCA4 deficient cells, intrinsically vulnerable to replication stress, exhibited exacerbated DNA damage upon combined ATRi and PARPi treatment in a Mre11- and Mus81-mediated manner. In vivo, combined treatment with intermittent ATRi and continuous PARPi showed greater inhibition of tumor growth than ATRi alone in SMARCA4-deficient lung adenocarcinoma xenograft models. These findings demonstrate that PARPi-induced heterochromatin amplifies RS and ATRi susceptibility, providing a potential rationale for therapeutic strategies targeting SMARCA4-deficient tumors.

We previously demonstrated that 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induced apoptosis in primary cultured rat hepatocytes. In this study, we investigated apoptotic biomarkers in rat liver after perfusion with 30 microM Trp-P-1 as preliminary experiments for in vivo study. Induction of c-Myc and p53 protein and the activities of caspase-3, -6, and -8 were detected in Trp-P-1-perfused liver. In addition, Trp-P-1 modulated the DNA binding activity of the apoptosis-related transcription factors, NF-kappaB and AP-1. These results imply a possibility that Trp-P-1 would induce apoptosis in vivo.