The research, conducted by a team at Mashhad University of Medical Sciences in Iran and published in the journal Molecular Biology Reports, examined bone marrow samples from 40 children who had been newly diagnosed with acute myeloid leukemia, one of the most aggressive cancers of the blood and bone marrow. The investigators compared the expression levels of two genes, BMAL1 and WEE1, against samples from 20 non-malignant control subjects. BMAL1, also known by its alternate name ARNTL, is widely regarded as the engine of the molecular circadian clock, forming a transcriptional complex that drives rhythmic gene expression throughout the body. WEE1, by contrast, is a kinase that acts as a gatekeeper at the G2/M checkpoint of the cell cycle, holding cells back from entering mitosis when their DNA is damaged or replication is incomplete.

The results were unambiguous. Using real-time quantitative PCR to measure messenger RNA levels, the team found that both genes were expressed at significantly lower levels in the leukemic samples than in the healthy controls, with statistical significance reaching a P value of less than 0.001. The magnitude of the difference was substantial: relative expression of BMAL1 in the patients’ bone marrow was just 0.18-fold that of the control group, while WEE1 expression stood at 0.31-fold. In other words, the core clock gene had been reduced to less than a fifth of its normal activity, and the cell cycle checkpoint kinase to less than a third.

Perhaps the most intriguing finding emerged when the researchers looked at how gene expression related to clinical measurements. BMAL1 expression showed a significant negative correlation with both white blood cell count and blast percentage, the proportion of immature, leukemic cells in the bone marrow. The correlation coefficients were -0.440 for white blood cell count, with a P value of 0.004, and -0.418 for blast percentage, with a P value of 0.007. This means that the more aggressive the disease appeared by standard hematological measures, the more profoundly the circadian clock gene was suppressed. WEE1 expression, in contrast, showed no significant correlation with any of the hematological parameters evaluated, including white blood cell count, hemoglobin, platelet count, and blast percentage.

To understand why these findings matter, it helps to appreciate how deeply the circadian clock and the cell cycle are intertwined. The clock operates through a transcription-translation feedback loop: BMAL1 pairs with CLOCK to activate expression of Period and Cryptochrome genes, whose protein products later feed back to inhibit BMAL1-CLOCK activity, creating an oscillation with a period of roughly 24 hours. Crucially, many of the genes controlled by this loop are involved in cell division. Landmark work published in Science in 2003 showed that the clock directly times cell division in vivo, and subsequent studies demonstrated that BMAL1 regulates the expression of p21, a key inhibitor of cell proliferation. When BMAL1 activity collapses, this timing coordination is lost, and cells may divide without the temporal safeguards that normally restrain them.

WEE1 sits at the intersection of this crosstalk. The kinase phosphorylates and inhibits CDK1, the master driver of mitotic entry, thereby enforcing the G2/M checkpoint that gives cells time to repair DNA damage before dividing. Loss of WEE1 function is known to produce genomic instability, and experiments in mice have shown that WEE1 deficiency leads to hyperactivation of the APC/C complex, chromosomal abnormalities, and ultimately carcinogenesis. At the same time, WEE1 has become a major target in cancer drug development, because many tumors become dependent on WEE1-mediated checkpoint control to survive the DNA damage inflicted by chemotherapy. WEE1 inhibitors are currently being explored in clinical trials across a range of malignancies, and prior work has suggested that WEE1 and PARP-1 play critical roles in the treatment of myelodysplastic syndrome and acute myeloid leukemia.

The downregulation of both genes in pediatric AML therefore paints a coherent, if preliminary, mechanistic picture. A silenced BMAL1 could mean that the leukemic cells have lost clock-driven control of proliferation and DNA damage responses, while reduced WEE1 might reflect a breakdown of checkpoint integrity that allows genetically damaged cells to progress through the cycle. It is worth noting, however, that the relationship between BMAL1 and cancer is not simple. Some studies have found that BMAL1 can actually accelerate certain tumors; one 2023 investigation reported that the clock protein promotes acute myeloid leukemia progression by inhibiting ferroptosis, a form of regulated cell death, through the EBF3/ALOX15 axis. In other cancers, including pancreatic, colorectal, and tongue squamous cell carcinoma, BMAL1 appears to act as a tumor suppressor, and its loss promotes growth and drug resistance. The new pediatric AML data, showing that lower BMAL1 tracks with higher tumor burden, leans toward the tumor-suppressive interpretation in this context, but the authors are careful to frame the finding as suggestive of a role rather than proof of one.

The study also fits into a broader literature on clock gene disruption in blood cancers. Researchers have previously documented downregulation of circadian genes in chronic myeloid leukemia, where altered methylation patterns of the PER3 gene have been observed, and in chronic lymphocytic leukemia, where both clock genes and clock-controlled cell cycle genes show deregulated expression. More recently, the circadian clock circuitry has been shown to modulate the activity of leukemia-initiating cells in T-cell acute lymphoblastic leukemia, and BMAL1 and CLOCK expression patterns have been examined as potential diagnostic markers in multiple myeloma. Animal work adds further weight to the connection: disrupting the light-dark cycle in mice has been shown to impair the hematopoietic function of the bone marrow, the very tissue from which leukemia arises.

For clinicians and researchers, the immediate value of the study lies in its potential biomarker implications. Because BMAL1 expression correlated inversely with white blood cell count and blast percentage, it is conceivable that measuring clock gene activity could eventually help gauge disease burden or track response to therapy in pediatric AML, a disease in which risk stratification remains a major clinical challenge. The authors also point toward therapeutic possibilities, noting that the significant downregulation of BMAL1 and WEE1 suggests that disrupted circadian and cell cycle regulatory pathways may contribute to the pathogenesis of the disease. The emerging field of chronotherapy, which seeks to time drug administration to the patient’s internal rhythms to maximize efficacy and minimize toxicity, could also be informed by a better understanding of how the clock is rewired in leukemic cells.

Significant caveats remain. The study involved a relatively modest sample of 40 patients from a single center, and it measured messenger RNA rather than protein levels, leaving open the question of whether the observed transcriptional changes translate into altered protein function. Correlation does not establish causation, and it is not yet clear whether BMAL1 suppression drives leukemic progression or is merely a consequence of it. The authors note that the data supporting the findings are available from the corresponding author upon reasonable request, and the work was carried out under the ethical oversight of Mashhad University of Medical Sciences with written informed consent from the parents or legal guardians of all pediatric participants. Follow-up studies with larger cohorts, longitudinal sampling, and functional experiments in leukemic cell models will be needed to determine whether restoring clock gene activity could one day form part of a therapeutic strategy. For now, the message is clear and quietly profound: the machinery that tells our cells what time it is may also help decide whether they stay healthy, and in the bone marrow of children with acute myeloid leukemia, that machinery appears to have fallen silent.

Subject of Research: Dysregulation of the circadian clock gene BMAL1 and the cell cycle regulator WEE1 in pediatric acute myeloid leukemia

Article Title: Dysregulation of circadian clock gene BMAL1 and cell cycle regulator WEE1 in pediatric AML

Article References: Oraei Sajjadi, K., Ayatollahi, H., Sheikhi, M., & Ahmadi, M. H. (2026). Dysregulation of circadian clock gene BMAL1 and cell cycle regulator WEE1 in pediatric AML. Molecular Biology Reports, 53(1), Article 1624. https://doi.org/10.1007/s11033-026-12812-9

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12812-9

Keywords: acute myeloid leukemia, BMAL1, WEE1, circadian clock, cell cycle, pediatric cancer, gene expression, bone marrow, G2/M checkpoint, hematologic malignancy, chronotherapy, biomarker

The researchers, led by investigators at Nanjing Medical University, began by building a single-cell transcriptomic atlas spanning the disease continuum. They collected tissue from five patients undergoing gastrectomy for intestinal-type gastric cancer, sampling not only the tumors themselves but also matched intestinal metaplasia lesions near the tumor margin and non-atrophic gastritis tissue farther away. After rigorous quality control, the dataset comprised 55,785 individual cells from 13 specimens, which the team sorted into nine major cell types, including epithelial cells, malignant cells, T cells, B cells, plasma cells, myeloid cells, mast cells, fibroblasts, and endothelial cells. The composition shifted tellingly across disease stages: as lesions progressed, the fractions of myeloid cells, endothelial cells, and fibroblasts expanded, while malignant epithelial cells came to dominate the tumor samples.

Within the epithelial compartment, the team re-clustered more than 34,000 cells into eight subtypes, ranging from chief and parietal cells, which dwindled as disease advanced, to tumor cells, which appeared only in cancerous tissue. The pivotal move came from pseudotime trajectory analysis, a computational technique that orders cells along an inferred developmental path based on their gene expression. The trajectory originated in neck cells and flowed toward enterocytes, the absorptive cells characteristic of intestinal metaplasia. One enterocyte population, which the researchers designated Enterocytes-S3, sat squarely between earlier epithelial states and the malignant cells along this trajectory, and tumor cells were heavily enriched in the same transcriptional state. To corroborate this intermediate identity, the team scored each cell against a published signature of incomplete intestinal metaplasia. The score climbed steadily from neck cells through successive enterocyte states, peaked in Enterocytes-S3, and then fell in cancer cells, exactly the pattern expected of a waystation on the road to malignancy.

What makes Enterocytes-S3 dangerous, the data suggest, is its cell-cycle program. Weighted gene co-expression network analysis identified 25 gene modules across the epithelial subtypes, and one of them, Module 4, was strongly upregulated in Enterocytes-S3 and enriched for genes governing mitotic nuclear division and mitotic cell-cycle phase transitions. The subpopulation also showed elevated activity of the G2/M checkpoint and E2F target gene sets, hallmarks of cells primed to proliferate. In short, this transitional population was not merely drifting toward a cancer-like identity; it was rewiring its division machinery, acquiring the proliferative momentum that tumors require.

To find the conductor of that rewiring, the researchers scored bulk transcriptomes from The Cancer Genome Atlas stomach adenocarcinoma cohort against the Enterocytes-S3 signature. PTTG1 emerged as the most significantly upregulated gene in tumors with high signature scores. The finding held up across multiple independent datasets, including the Asian Cancer Research Group cohort and the team’s own in-house transcriptomic data, with paired analyses confirming that PTTG1 expression was elevated in intestinal-type gastric cancer relative to matched normal mucosa. Pseudotime analysis showed the gene’s expression rising progressively from enterocytes toward malignant cells, and immunohistochemistry on a tissue microarray of 88 evaluable paired samples told the same story at the protein level: strong nuclear PTTG1 staining in tumors, weak or absent staining in adjacent normal tissue, and significantly higher levels in intestinal-type than in diffuse-type gastric cancer.

PTTG1 is no stranger to oncology. Originally discovered in rat pituitary tumor cells, the gene encodes securin, an inhibitor that prevents sister chromatids from separating until mitosis is properly timed, thereby safeguarding genomic integrity. It is barely expressed in most normal tissues apart from the testis, yet it is markedly overexpressed in hepatocellular, breast, lung, colon, testicular, and ovarian cancers, where it has been linked to proliferation, invasion, and metastasis through pathways ranging from PI3K/AKT to Wnt/beta-catenin and MAPK signaling. What the new study adds is a specific, mechanistic account of how PTTG1 reprograms the cell cycle in gastric epithelium, and evidence that its activity tracks the premalignant-to-malignant transition in a subtype-specific manner.

The functional experiments were decisive. When the team silenced PTTG1 in MKN1 and MKN7 gastric cancer cell lines using small interfering RNAs, proliferation measured by CCK-8 and EdU assays dropped significantly, and Transwell assays showed reduced migration and invasion. RNA sequencing of the knockdown cells revealed widespread downregulation of cell-cycle genes, with gene set enrichment analysis pinpointing the G2/M checkpoint pathway. Flow cytometry then delivered a subtle but important insight: PTTG1 depletion reduced the proportion of cells in G2 phase and increased the G1 fraction, indicating impaired progression through the G2/M transition rather than a classical arrest at the checkpoint. Western blotting confirmed the molecular signature of stalled mitotic entry, with Cyclin B1 and phosphorylated CDK1 both diminished, while levels of gamma-H2AX, a marker of DNA double-strand breaks, rose sharply, and immunofluorescence showed increased gamma-H2AX foci in the nuclei of PTTG1-deficient cells.

Digging deeper, the researchers traced a regulatory axis from PTTG1 to the machinery of mitosis. Knockdown reduced the expression of E2F1, a transcription factor known to bind directly the promoter of the CCNB1 gene, which encodes Cyclin B1. Using CUT&RUN-qPCR, a technique that maps where a protein sits on chromatin, the team showed that E2F1 occupied the CCNB1 promoter in control cells but that this occupancy fell markedly after PTTG1 silencing, accompanied by reduced CCNB1 messenger RNA. The model that emerges is one in which PTTG1 sustains E2F1-dependent transcription of Cyclin B1, thereby licensing the G2-to-M transition, while simultaneously helping to coordinate cell-cycle progression with DNA damage responses. The precise mechanism by which PTTG1 controls E2F1 remains an open question the authors acknowledge.

Two model systems extended the story beyond cell lines. In human paracancerous gastric organoids derived from intestinal-type gastric cancer patients, lentiviral overexpression of PTTG1 produced organoids that grew significantly larger than controls by days 6, 10, and 12 of culture, with more Ki67-positive proliferating cells and elevated Cyclin B1, much of it in the nucleus, a location associated with mitotic commitment. Strikingly, the overexpressing organoids also stained more intensely with Alcian blue and produced more MUC2, a goblet-cell mucin characteristic of intestinal metaplasia, suggesting that PTTG1 can push gastric epithelium toward an intestinal, mucin-producing identity. In the opposite direction, stable PTTG1 knockdown in MKN1 cells injected subcutaneously into nude mice markedly suppressed tumor growth, with smaller volumes and lower weights at the experimental endpoint, reduced Ki67 and Cyclin B1 staining, and altered AB-PAS mucin staining that, however, was not accompanied by a change in MUC2 expression, hinting that PTTG1’s effects on mucin phenotypes depend on the biological context.

The study is candid about its limits. Tissue dissociation erases the spatial geography of the gastric gland, the cross-sectional sampling of five patients cannot fully capture the temporal dynamics of a cascade that unfolds over years, and the cohort was too small to assess sex-associated differences. The tumor-initiating potential of Enterocytes-S3 remains to be proven, and more physiologically relevant culture systems will be needed to settle the mucin question. Notably, PTTG1 expression showed no significant association with overall survival in the TCGA or ACRG cohorts, suggesting the gene functions as an engine of tumor-intrinsic biology rather than an independent prognostic marker. Even so, by placing a chromosome-segregation guardian at the hinge between intestinal metaplasia and carcinoma, the work offers the clearest single-cell-resolved view yet of how the Correa cascade accelerates, and it flags PTTG1, and the G2/M checkpoint it commands, as a target worth pursuing in the fight against intestinal-type gastric cancer.

Subject of Research: The role of PTTG1 in G2/M checkpoint-mediated cell-cycle reprogramming during intestinal-type gastric cancer progression

Article Title: PTTG1 drives intestinal-type gastric cancer progression via G2/M checkpoint-mediated cell-cycle reprogramming

Article References: Hu, C., Bian, L., Luo, L., Zheng, Z., Chen, Y., Gao, X., Li, F., Yang, D., Liu, Y., Li, G., Xu, H., Yan, C., & Jin, G. (2026). PTTG1 drives intestinal-type gastric cancer progression via G2/M checkpoint-mediated cell-cycle reprogramming. iScience, 29(11), Article 117742. https://doi.org/10.1016/j.isci.2026.117742

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117742

Keywords: gastric cancer, PTTG1, securin, G2/M checkpoint, single-cell RNA sequencing, intestinal metaplasia, Correa cascade, Cyclin B1, E2F1, organoids, xenograft, cell cycle

The logic of the new study rests on a vulnerability embedded in the biology of cancer itself: the DNA damage checkpoint. When anticancer drugs inflict lesions on DNA, healthy cells respond by halting the cell cycle through checkpoint pathways, buying time for repair before replication resumes. Two major checkpoints govern this process. The G1 checkpoint depends on the ATM–p53–p21 axis, while the G2/M checkpoint is controlled by the ataxia telangiectasia and Rad3-related kinase, or ATR, acting through Chk1 and Cdc25. Because malignant cells frequently carry p53 mutations and cannot arrest in G1, they become unusually dependent on the ATR-driven G2/M checkpoint to survive genotoxic stress. Normal cells, by contrast, retain a functional ATM–p53–p21 pathway. This asymmetry creates a therapeutic window: blocking ATR should selectively cripple cancer cells while sparing healthy tissue.

AZD6738, developed as an oral ATR inhibitor, does exactly that. By suppressing ATR-dependent phosphorylation—most notably Chk1 phosphorylation at serine 345—it disables the G2/M checkpoint that resistant tumors rely upon. The drug has already attracted attention across oncology, with Phase II trials suggesting clinical activity in combination with the immunotherapy durvalumab in previously treated advanced gastric cancer, particularly in patients lacking ATM expression, and in non-small cell lung cancer. Emerging data also support combinations with the PARP inhibitor olaparib in triple-negative breast cancer, with durvalumab in melanoma, and with radiotherapy. But in colorectal cancer, preclinical work on ATR inhibition had remained sparse. The Nagoya team had previously shown that AZD6738 potentiates both 5-FU and trifluridine in treatment-naïve colorectal cancer cells. The critical open question was whether that synergy survives the acquisition of 5-FU resistance—the setting in which trifluridine is actually given to patients.

To answer it, the researchers built a rigorous experimental system using two genetically defined human colorectal cancer cell lines, HCT116 and DLD-1, alongside 5-FU–resistant derivatives established through more than 100 passages of stepwise drug escalation. Both resistant sublines, HCT116/5FUR and DLD-1/5FUR, exhibited roughly 100-fold higher IC50 values for 5-FU than their parental counterparts, confirming profound acquired resistance. All cell lines were authenticated by short tandem repeat profiling and confirmed free of mycoplasma contamination. The team then designed experiments around a deliberately conservative dosing strategy: AZD6738 was used at 0.5 micromolar, a concentration shown to have no effect on proliferation after 72 hours in any of the four cell lines, so that any enhancement of cell killing could be attributed to genuine potentiation rather than simple additive toxicity.

The in vitro results were striking. As expected, 5-FU and its combination with AZD6738 lost all effectiveness in the resistant sublines—the combination that worked in parental cells was completely abrogated by acquired resistance, echoing earlier findings with Chk1 inhibitors. Trifluridine, however, told a different story. Although trifluridine alone was modestly weakened in the resistant cells, adding noncytotoxic AZD6738 significantly reduced cell viability across a wide dose range, from 1 to 1000 micromolar. Combination index analysis confirmed true synergy, with values of 0.735 in HCT116/5FUR and 0.418 in DLD-1/5FUR, where values below 1 indicate synergistic interaction. In time-course assays, the combination suppressed proliferation significantly at 72 hours in both resistant lines—p = 6.0 × 10⁻⁵ for HCT116/5FUR and p = 4.0 × 10⁻⁷ for DLD-1/5FUR—and restored a level of growth inhibition comparable to what trifluridine achieved in the parental, drug-sensitive cells. DLD-1/5FUR, the most resistant line, saw its trifluridine IC50 collapse from 155 micromolar to just 3.5 micromolar in the presence of AZD6738.

The molecular fingerprints of the combination revealed its mechanism. Western blotting showed that trifluridine alone produced little induction of γH2AX, a marker of DNA double-strand break signaling, but cotreatment with AZD6738 triggered a marked surge. Cleaved caspase-3, the executioner enzyme of apoptosis, rose far more strongly with the combination than with trifluridine alone, indicating that stalled checkpoint signaling had collapsed into programmed cell death. Meanwhile, the trifluridine-induced phosphorylation of Chk1 at Ser345—the very signal AZD6738 is designed to block—was suppressed by the drug, confirming on-target activity. Flow cytometry added a cell-cycle dimension: trifluridine alone drove a pronounced G2/M arrest, effectively locking damaged cells in a state of suspended repair, while the combination relieved that arrest, pushing cells past the checkpoint and into catastrophic division with unrepaired DNA damage.

Crucially, the synergy translated into living tumors. In a mouse xenograft model bearing DLD-1/5FUR tumors, neither TAS-102 alone nor AZD6738 alone significantly shrank tumors compared with controls. But the combination significantly suppressed tumor growth over the two-week treatment period, and at necropsy only the combination group showed a significant reduction in tumor weight relative to all other groups, including TAS-102 monotherapy (p = 0.039). Immunohistochemistry on excised tumors corroborated the mechanism in vivo: 25.8 percent of tumor cell nuclei stained positive for γH2AX in the combination group, compared with only 4.1 percent in the TAS-102 group. Equally important, safety signals were reassuring. Body weight did not differ among groups, and organ weights and blood counts showed no exacerbation of the hematologic toxicity that TAS-102 alone can induce, suggesting the addition of AZD6738 did not compound harm in this preclinical model.

The pharmacological reasoning behind the finding illuminates why trifluridine, unlike 5-FU, retains its potency in resistant tumors. Both drugs inhibit thymidylate synthase, an enzyme essential for DNA synthesis, and both can be incorporated into DNA and RNA. However, overexpression of thymidylate synthase is a major driver of 5-FU resistance, neutralizing the TS-inhibitory arm of both drugs. Trifluridine’s distinguishing feature is its exceptionally high rate of direct incorporation into DNA during S phase in place of thymidine, and earlier work by Emura and colleagues demonstrated that trifluridine retains substantial cytotoxicity in 5-FU–resistant colorectal cancer cells precisely because its activity depends largely on DNA incorporation rather than TS inhibition. That massive misincorporation generates replication stress, which the ATR–Chk1 axis manages at the G2/M checkpoint—making trifluridine and ATR inhibition a mechanistically rational pairing even after 5-FU resistance has taken hold.

The authors are candid about the study’s limitations. Mechanistic conclusions rest solely on pharmacological ATR inhibition without genetic validation; normal colon epithelial cells were not evaluated, leaving the therapeutic window uncertain; and only a single concentration of AZD6738 was tested, so broader dose–response studies will be needed. The roles of p53 and ATM also remain unresolved, as synergy appeared in both p53 wild-type and p53-mutant models, and no direct experiments interrogated these determinants. Nevertheless, the clinical relevance of the setting is hard to overstate: trifluridine is administered as TAS-102, known commercially as Lonsurf, almost exclusively after failure of fluoropyrimidine-based first- and second-line regimens, meaning most patients receiving it already harbor 5-FU resistance. By providing proof of concept that pharmacological ATR inhibition can restore—and even rescue—the antitumor activity of trifluridine in that refractory context, the study lays the groundwork for mechanistic follow-up and, ultimately, translational trials of a ceralasertib–TAS-102 combination in patients whose options are running out.

Subject of Research: ATR inhibition with AZD6738 to enhance trifluridine efficacy in 5-FU–resistant colorectal cancer

Article Title: AZD6738 (Ceralasertib) Enhances Trifluridine's Antitumor Effect in 5‐FU–Resistant Colorectal Cancer Cells

Article References: Uehara, S., Suzuki, T., Kato, J., Asai, H., Kato, A., Harata, S., Ushigome, H., Yamakawa, Y., Hirokawa, T., Takahashi, H., Matsuo, Y., & Takiguchi, S. (2026). AZD6738 (Ceralasertib) Enhances Trifluridine's Antitumor Effect in 5‐ FU –Resistant Colorectal Cancer Cells. Cancer Reports, 9(9), Article e70668. https://doi.org/10.1002/cnr2.70668

Image Credits: AI Generated

DOI: 10.1002/cnr2.70668

Keywords: AZD6738, ceralasertib, trifluridine, TAS-102, colorectal cancer, 5-FU resistance, ATR inhibitor, DNA damage checkpoint, Chk1, G2/M checkpoint, drug resistance, preclinical study