How replication stress shapes cancer evolution and opens new doors for therapy

Every time a cell divides, it must copy roughly three billion letters of DNA with near-perfect fidelity. That copying process, DNA replication, is among the most demanding operations a cell performs, and it is under constant assault in cancer cells. A new review published in Nature Reviews Cancer by Junjie Chen of The University of Texas MD Anderson Cancer Center and Lee Zou of Duke University School of Medicine synthesizes three decades of research into replication stress, the condition in which DNA synthesis is slowed, stalled or derailed. The authors argue that replication stress is not merely collateral damage in tumours but a central engine of cancer genome evolution and, paradoxically, one of the most promising frameworks for precision oncology. Their central insight is a biological double bind: the very pressure that destabilizes cancer genomes also creates dependencies on stress-response pathways that drugs can now target.

Replication stress arises from many sources, and the review methodically maps them. Oncogene activation is a principal culprit. When growth-promoting signals such as MYC or cyclin E are unleashed, they push cells through the cell cycle faster than replication machinery can be properly licensed, forcing replication to begin in inappropriate places. Shortened G1 phases leave origin licensing incomplete, and transcription programs fire at full blast while forks attempt to copy the same sequences. The resulting collisions between replication and transcription complexes are among the most potent sources of chromosome breakage, particularly at long, highly transcribed genes known as common fragile sites, which have been recognized as breakage hotspots since experiments with the polymerase inhibitor aphidicolin in the 1980s.

Transcription-replication conflicts come in mechanistically distinct flavors. Head-on conflicts, where the replication fork meets an oncoming RNA polymerase, and co-directional conflicts produce different R-loop levels and activate different DNA damage responses. R-loops, three-stranded structures containing an RNA-DNA hybrid and a displaced single strand, have emerged as pivotal intermediates that can either protect or poison genomes depending on how they are resolved. Studies of BRCA1 and BRCA2 have shown that these breast and ovarian cancer tumor suppressors do more than repair double-strand breaks; they actively suppress R-loop accumulation by coordinating transcription elongation and RNA processing. Similarly, chromatin remodelers such as the SWI/SNF complex and spliceosome factors help clear conflicts, explaining why mutations in these complexes, common in myelodysplastic syndromes and Ewing sarcoma, produce cells exquisitely sensitive to ATR inhibition.

Beyond collisions, the review highlights metabolic and structural pressures. Nucleotide pools are a striking example: early work demonstrated that deoxyribonucleotide deficiency distorts origin choice and spacing, while later studies showed that oncogene-induced senescence is partly a consequence of nucleotide starvation. Cancer cells also accumulate unprocessed uracil and ribonucleotides in DNA, both of which impede fork progression. DNA secondary structures add another layer. Unstable inverted repeats fold into hairpins and cruciforms, and expanded TA-dinucleotide repeats in microsatellite-unstable colorectal cancers form structures that grind forks to a halt. These repetitive obstacles, rather than mismatch repair failure alone, are now understood to be why such tumours become fatally dependent on the WRN helicase, which unwinds these structures and prevents catastrophic chromosome shattering.

The consequences of unresolved replication stress reverberate through the cancer genome. Stalled forks are vulnerable to nuclease degradation unless BRCA2 protects the nascent DNA, and collapse generates breaks that drive chromosomal instability, linking structural and numerical chromosome chaos. Under-replicated regions persist into mitosis, producing anaphase bridges, micronuclei and copy-number changes that mirror the polymorphic and pathogenic variants seen in human populations. Several avenues of rescue have been characterized, including fork reversal, repriming by the PRIMPOL primase-polymerase and specialized translesion polymerases that replicate past damaged bases, but these repair routes are intrinsically error-prone. Translesion synthesis underlies acquired chemotherapy resistance, and repriming leaves single-stranded DNA gaps that, recent work shows, are a key determinant of PARP inhibitor sensitivity in BRCA-deficient cells.

The ATR-CHK1 checkpoint pathway is the master guardian that keeps replication stress survivable. ATR is recruited to RPA-coated single-stranded DNA through its partner ATRIP and activated by TopBP1 or ETAA1, with the MRN complex and the 9-1-1 clamp sharpening its activation. Once active, ATR suppresses excess origin firing, stabilizes stalled forks, enforces an intrinsic S/G2 checkpoint that prevents cells from entering mitosis with unfinished replication, and prevents global exhaustion of RPA, a failure mode that culminates in replication catastrophe and genome fragmentation. Without ATR, forks collapse and single-stranded DNA accumulates beyond rescue, a finding that transformed the enzyme from a basic research curiosity into a top-priority drug target.

That targeting has now reached the clinic. ATR inhibitors such as ceralasertib and elimusertib have produced durable responses in tumours with high replication stress, including small cell lung cancer, and synthetic lethal combinations with ATM loss, ERCC1 deficiency, CCNE1 amplification and APOBEC3 activity are being pursued. WEE1 inhibitors override the G2 checkpoint and force stressed cells into lethal mitosis, with combination strategies including sequential PARP and WEE1 dosing designed to minimize toxicity. The clearest success story remains PARP inhibition, introduced in landmark 2005 studies demonstrating synthetic lethality in BRCA-mutant cells. Mechanistic work has since refined the model: PARP inhibitors trap PARP1 and PARP2 on DNA and act through persistent single-stranded gaps that BRCA-deficient cells cannot fill, and next-generation PARP1-selective agents such as AZD5305 promise better tolerability. WRN inhibition in microsatellite-unstable cancers represents the newest synthetic lethal frontier, with resistance mechanisms beginning to be mapped by CRISPR screens.

The review also reframes replication stress as a chronic, sublethal condition that sculpts tumour evolution over years rather than an acute lethal event. This slow burn generates mutational signatures, including those of APOBEC3 cytidine deaminases, that fuel heterogeneity, therapy resistance and metastasis, and therapy itself can amplify APOBEC3A-driven evolution in persistent cancer cells. Importantly, replication stress intersects with the immune system. Under-replicated DNA in micronuclei activates the cGAS-STING pathway, and chromosomal instability promotes metastasis through cytosolic DNA sensing, while SAMHD1 and MYC normally suppress inflammatory signaling from replication-derived nucleic acids. ATR inhibition can potentiate the inflammatory tumour microenvironment, opening rational combinations with immune checkpoint blockade, an approach already validated in microsatellite-unstable and BRCA-mutated tumour settings.

The authors position replication stress not as a single weakness but as a dynamic, context-dependent vulnerability landscape shaped by each tumour’s oncogenic, metabolic and chromatin state. Biomarkers such as replication stress signatures, gap formation and fork protection status may soon guide which patients receive ATR, WEE1, PARP or WRN inhibitors, and in which combinations. The therapeutic opportunity, they argue, lies in recognizing that tumours pay a permanent price for their genomic chaos, and that the rescue pathways they depend upon are now precisely druggable targets.

Subject of Research: Replication stress in cancer: origins, consequences and therapeutic opportunities

Article Title: Replication stress in cancer: origins, consequences and therapeutic opportunities

Article References: Chen, J., & Zou, L. (2026). Replication stress in cancer: origins, consequences and therapeutic opportunities. Nature Reviews Cancer. https://doi.org/10.1038/s41568-026-00979-z

Image Credits: AI Generated

DOI: 10.1038/s41568-026-00979-z

Keywords: replication stress, cancer, ATR, CHK1, PARP inhibitors, synthetic lethality, R-loops, genomic instability, WRN helicase, WEE1 inhibitors, precision oncology, DNA replication