DPY30 is an epigenetic decoupler linking replication stress to immunoediting in pancreatic cancer.

Perturbation of DNA replication, for instance by hydroxyurea-dependent dNTP exhaustion, often leads to stalling or collapse of replication forks. This triggers a replication stress response that stabilizes these forks, activates cell cycle checkpoints, and induces expression of DNA damage response genes. While several factors are known to act in this response, the full repertoire of proteins involved remains largely elusive. Here, we develop Replication-IDentifier (Repli-ID), which allows for genome-wide identification of regulators of DNA replication in Saccharomyces cerevisiae. During Repli-ID, the replicative polymerase epsilon (Pol ε) is tracked at a barcoded origin of replication by chromatin immunoprecipitation (ChIP) coupled to next-generation sequencing of the barcode in thousands of hydroxyurea-treated yeast mutants. Using this approach, 423 genes that promote Pol ε binding at replication forks were uncovered, including LGE1 and ROX1. Mechanistically, we show that Lge1 affects replication initiation and/or fork stability by promoting Bre1-dependent H2B mono-ubiquitylation. Rox1 affects replication fork progression by regulating S-phase entry and checkpoint activation, hinging on cellular ceramide levels via transcriptional repression of SUR2. Thus, Repli-ID provides a unique resource for the identification and further characterization of factors and pathways involved in the cellular response to DNA replication perturbation.

Background Replication stress is a fundamental driver of genomic instability in cancer, reflecting chronic perturbations in DNA replication caused by oncogene activation, cell-cycle dysregulation, and impaired DNA damage response signaling. These processes shape tumor evolution and therapeutic vulnerability. In head and neck squamous cell carcinoma (HNSCC), the transcriptional consequences of replication stress states remain poorly defined. Methods Primary HNSCC tumors from TCGA-HNSC cohort with matched RNA sequencing, somatic mutation, copy number, and overall survival data were analyzed (N = 499). Replication stress was quantified using a multi-module transcriptomic signature spanning checkpoint signaling, fork stabilization, replication licensing, homologous recombination, Fanconi anemia pathways, and nucleotide metabolism. Innate immune activation was captured using a cGAS–STING axis signature (CGAS, TMEM173, TBK1, IRF3, IRF7, IFNB1, IFNA1, ISG15, MX1, OAS1). Tumors were stratified by median splits for replication stress and STING activity, yielding four groups. Immune activation was assessed using an interferon-γ response signature, cytolytic activity (GZMB, PRF1, GZMA), and PD-L1 expression (CD274). HPV status was obtained from TCGA PanCancer Atlas annotations. Associations were evaluated using Kruskal–Wallis tests, Mann–Whitney U tests, Fisher’s exact tests, and multivariable Cox regression adjusting for age, stage, and tumor mutational burden. Results RS-high/STING-high tumors comprised 25.9% of cases (129/499). Across RS/STING strata, there were highly significant differences in interferon-γ signaling (χ2 = 102.5, p < 2.2 × 10-16), cytolytic activity (χ2 = 73.4, p = 8.0 × 10-16), and PD-L1 expression (χ2 = 76.7, p < 2.2 × 10-16). RS-high/STING-high tumors were significantly enriched for HPV-positive disease (28/124 vs 44/363; OR 2.11, 95% CI 1.20–3.68; p = 0.0078). Importantly, within both HPV-positive and HPV-negative strata, RS-high/STING-high tumors retained significantly higher interferon-γ signaling, cytolytic activity, and PD-L1 expression compared with all other tumors (all p < 0.05), indicating that this immune-inflamed state is not simply a surrogate for viral etiology. In multivariable survival models (N = 474), RS-high/STING-high status was not independently prognostic (HR 1.34, 95% CI 0.96–1.87; p = 0.084),with no evidence of HPV-dependent effect modification (interaction HR 0.38, 95% CI 0.12–1.22; p = 0.11). HPV positivity showed a non-significant trend toward improved survival (HR 0.62, 95% CI 0.35–1.10; p = 0.10). Conclusions In HNSCC, high replication stress coupled with innate immune activation defines a distinct immune-inflamed transcriptional state that is enriched for HPV-positive tumors. These findings suggest that replication stress can demarcate tumors with coordinated interferon and cytolytic programs. Joint quantification of replication stress and innate immune activation may provide a rational biomarker framework for immune-priming strategies and rational combination therapies in HNSCC. Citation Format: Harold Nathan Tan, Michael Wotman, Luana Sousa, Khaled Sanber, Neal Akhave, George Blumenschein, Renata Ferrarotto, Maura Gillison, Faye Johnson, Timothy A. Yap. Replication stress coupled to cGAS–STING activation defines an innate immune–inflamed transcriptional state in head and neck squamous cell carcinoma that Is enriched for HPV-positive disease [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-C008.

Mrc1 (mediator of replication checkpoint), Tof1 (topoisomerase I interacting factor), and Csm3 (chromosome segregation in meiosis) are checkpoint-mediator proteins that function during DNA replication and activate the effector kinase Rad53. We reported previously that Mrc1 and Tof1 are constituents of the replication machinery and that both proteins are required for the proper arrest and stabilization of replication forks in the presence of hydroxyurea. In our current study, we show that Csm3 is a component of moving replication forks and that both Tof1 and Csm3 are specifically required for the association of Mrc1 with these structures. In contrast, the deletion of mrc1 did not affect the association of Tof1 and Csm3 with the replication fork complex. In agreement with previous observations in yeast cells, the results of a baculovirus coexpression system showed that these three proteins interact directly with each other to form a mediator complex in the absence of replication forks.

Background: Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal cancer, both for lack of effective screening method and for resistance to chemotherapy (CTX) and radiotherapy. However, some chemotherapeutic agents, such as gemcitabine, have immune modulatory effects. We started from the hypothesis that more immunogenic antigens can be induced by CTX and targeted by passive or active immunotherapy. To discover TAAs that might be selected for immunotherapy, antibody response in PDA patients' sera was analyzed before and after CTX. TAAs mostly recognized after CTX were selected and used to evaluate whether PDA patients' T cells have an increased TAA-specific response after CTX and if the immune checkpoint blockade (ICB) could enhance T cells activation. Material and methods: Antibody response in sera of PDA patients, before and after CTX treatments, was analyzed by Serological Proteome Analysis (SERPA) and the antigens recognized were identified by mass spectrometry. T cell proliferation and IFNγ and IL10 production were evaluated on patients' PBMCs (peripheral blood mononuclear cells) stimulated with TAAs in presence or not of ICB. Results: After CTX the level of antibody recognition increased, both in term of intensity and number of recognized TAAs. For some of these TAAs the increased antibody recognition showed a positive correlation with patients' survival. Of note, after CTX an increased complement dependent cytotoxicity against PDA cell lines was demonstrated in 48% of PDA patients' sera. In most cases after CTX PDA patients' T cells stimulated in vitro with recombinant selected TAAs proliferated more. Moreover, about 50% of TAA-specific T cell responses switched from a protumor regulatory to an antitumor effector phenotype after CTX. Interestingly, this shift was better elicited by ICB after CTX than before. Conclusions: These data indicate that in PDA patients CTX induces an increase of TAAs-specific antibodies. Furthermore, CTX can switch several TAA-specific T cell responses from regulatory to effector phenotype and ICB could potentiate this polarization. Based on these results the combination of DNA vaccination against TAA and CTX is currently investigated in a PDA mouse model. Citation Format: Sara Bulfamante, Giorgia Mandili, Moitza Principe, Daniele Giordano, Emanuela Mazza, Claudia Curcio, Laura Follia, Giulio Ferrero, Andrea Evangelista, Maria Antonietta Satolli, Paola Cappello, Francesco Novelli. Antibody and T cell response profiling in pancreatic cancer patients before and after chemotherapy identify tumor associated antigens suitable for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2761.

FBH1 Catalyzes Regression of Stalled Replication Forks.

Precise replication of genetic material is essential to maintain genome stability. DNA replication is a tightly regulated process that ensues faithful copies of DNA molecules to daughter cells during each cell cycle. Perturbation of DNA replication may compromise the transmission of genetic information, leading to DNA damage, mutations, and chromosomal rearrangements. DNA replication stress, also referred to as DNA replicative stress, is defined as the slowing or stalling of replication fork progression during DNA synthesis as a result of different insults. Oncogene activation, one hallmark of cancer, is able to disturb numerous cellular processes, including DNA replication. In fact, extensive work has indicated that oncogene-induced replication stress is an important source of genomic instability in human carcinogenesis. In this review, we focus on main oncogenes that induce DNA replication stress, such as RAS, MYC, Cyclin E, MDM2, and BCL-2 among others, and the molecular mechanisms by which these oncogenes interfere with normal DNA replication and promote genomic instability.

Precise replication of genetic material is essential to maintain genome stability. DNA replication is a tightly regulated process that ensues faithful copies of DNA molecules to daughter cells during each cell cycle. Perturbation of DNA replication may compromise the transmission of genetic information, leading to DNA damage, mutations, and chromosomal rearrangements. DNA replication stress, also referred to as DNA replicative stress, is defined as the slowing or stalling of replication fork progression during DNA synthesis as a result of different insults. Oncogene activation, one hallmark of cancer, is able to disturb numerous cellular processes, including DNA replication. In fact, extensive work has indicated that oncogene-induced replication stress is an important source of genomic instability in human carcinogenesis. In this review, we focus on main oncogenes that induce DNA replication stress, such as RAS, MYC, Cyclin E, MDM2, and BCL-2 among others, and the molecular mechanisms by which these oncogenes interfere with normal DNA replication and promote genomic instability.

The S-phase checkpoint kinases Mec1 and Rad53 in the budding yeast, Saccharomyces cerevisiae, are activated in response to replication stress that induces replication fork arrest. In the absence of a functional S-phase checkpoint, stalled replication forks collapse and give rise to chromosome breakage. In an attempt to better understand replication dynamics in S-phase checkpoint mutants, we developed a replication origin array for budding yeast that contains 424 of 432 previously identified potential origin regions. As expected, mec1-1 and rad53-1 mutants failed to inhibit late origin activation. Surprisingly however, 17 early-firing regions were not replicated efficiently in these mutants. This was not due to a lack of initiation, but rather to problems during elongation, as replication forks arrested in close proximity to these origins, resulting in the accumulation of small replication intermediates and eventual replication fork collapse. Importantly, these regions were not only prone to chromosome breakage in the presence of exogenous stress but also in its absence, similar to fragile sites in the human genome.

Ubiquitination of the C-terminus of histone H2B (H2BK120ub) is a key histone modification with functions in a wide array of DNA-related processes, best characterized in gene transcription and repair. A role for H2B ubiquitination in DNA replication has been postulated and investigated in yeast but is still elusive in human cells. Here, we uncovered a critical function of H2BK120ub in replication fork dynamics. H2BK120ub is present at replication forks and accumulates upon replication stress in a manner dependent on ATR and RAD51. Loss of RNF20, the main ubiquitin ligase promoting H2BK120ub, leads to RECQ1-mediated unrestrained replication fork progression and defective fork reversal upon mild replication stress, restoring fork stability in BRCA2-deficient cells. Furthermore, we identified RNF169, a factor involved in the DNA damage response and repair, as a reader of the H2BK120ub mark at stalled replication forks, where it is required to protect the nascent DNA from excessive nucleolytic degradation. Hence, RNF20, H2BK120ub and RNF169 are key novel players orchestrating replication stress response and fork plasticity in human cells.

ABSTRACTA balance in the deoxyribonucleotide (dNTPs) intracellular concentration is critical for the DNA replication and repair processes. In the model yeast Saccharomyces cerevisiae, the Mec1-Rad53-Dun1 kinase cascade mainly regulates the ribonucleotide reductase (RNR) gene expression during DNA replication and DNA damage stress. However, the RNR regulatory mechanisms in basidiomycete fungi during DNA replication and damage stress remain elusive. Here, we observed that in C. neoformans, RNR1 (large RNR subunit) and RNR21 (one small RNR subunit) were required for cell viability, but not RNR22 (another small RNR subunit). RNR22 overexpression compensated for the lethality of RNR21 suppression. In contrast to the regulatory mechanisms of RNRs in S. cerevisiae, Rad53 and Chk1 kinases cooperatively or divergently controlled RNR1 and RNR21 expression under DNA damage and DNA replication stress. In particular, this study revealed that Chk1 mainly regulated RNR1 expression during DNA replication stress, whereas Rad53, rather than Chk1, played a significant role in controlling the expression of RNR21 during DNA damage stress. Furthermore, the expression of RNR22, not but RNR1 and RNR21, was suppressed by the Ssn6-Tup1 complex during DNA replication stress. Notably, we observed that RNR1 expression was mainly regulated by Mbs1, whereas RNR21 expression was cooperatively controlled by Mbs1 and Bdr1 as downstream factors of Rad53 and Chk1 during DNA replication and damage stress. Collectively, the regulation of RNRs in C. neoformans has both evolutionarily conserved and divergent features in DNA replication and DNA damage stress, compared with other yeasts.IMPORTANCE Upon DNA replication or damage stresses, it is critical to provide proper levels of deoxynucleotide triphosphates (dNTPs) and activate DNA repair machinery. Ribonucleotide reductases (RNRs), which are composed of large and small subunits, are required for synthesizing dNTP. An imbalance in the intracellular concentration of dNTPs caused by the perturbation of RNR results in a reduction in DNA repair fidelity. Despite the importance of their roles, functions and regulations of RNR have not been elucidated in the basidiomycete fungi. In this study, we found that the roles of RNR1, RNR21, and RNR22 genes encoding RNR subunits in the viability of C. neoformans. Furthermore, their expression levels are divergently regulated by the Rad53-Chk1 pathway and the Ssn6-Tup1 complex in response to DNA replication and damage stresses. Therefore, this study provides insight into the regulatory mechanisms of RNR genes to DNA replication and damage stresses in basidiomycete fungi.

OBJECTIVES A proportion of resectable pancreatic ductal adenocarcinoma (PDAC) patients display poorer survival due to profound local immune suppression. However, a pathological/morphological parameter that could functionally read out immune evasion and predict patient survival has not been defined. This study investigated the feasibility of heterotypic cell-in-cell (CIC) structures for immune cell cannibalism by tumor cells to serve as a parameter for survival prediction in resectable PDAC patients. METHODS A total of 410 samples from PDAC patients were examined using the methods of “EML” multiplex staining or immunohistochemistry (IHC). Prognostic CIC candidates were initially identified in samples plotted in tissue microarray (n=300), then independently validated in specimens from the First Affiliated Hospital of Sun Yat-Sen University (n=110). The Kaplan–Meier estimator and/or the Cox regression model were used for univariate and multivariate analysis. A nomogram was made using the Regression Modeling Strategies. RESULTS CICs were prevalent in cancerous (203/235) but not non-malignant tissues (15/147). Among the 4 CIC subtypes identified, 2 heterotypic subtypes with tumor cells internalizing CD45+ lymphocytes (LiT, mOS = 8 vs. 14.5 months, p = 0.008) or CD68+ monocytes (MiT, mOS = 7.5 vs. 15 months, p = 0.001), and overall CICs (oCIC, mOS = 10 vs. 27 months, p = 0.021), but not homotypic CICs (TiT, p = 0.089), were identified in univariate analysis as adverse prognostic factors of overall survival (OS) of PDAC. Notably, through cannibalism of immune cells by tumor cells, heterotypic CICs (L/MiT: LiT plus MiT) could independently predict shorter OS (HR = 1.85, p = 0.008) in multivariate analysis, with a performance comparable or even superior to traditional clinicopathological parameters such as histological grade (HR = 1.78, p = 0.012) and TNM stage (HR=1.64, p = 0.108). This was confirmed in the validation cohort, where L/MiT (HR = 1.71, p = 0.02) and tumor–node–metastasis (TNM) stage (HR = 1.66, p = 0.04) were shown to be independent adverse prognostic factors. Moreover, L/MiT stood out as the most prominent contributor in nomogram models constructed for survival prediction (area under the curve = 0.696 at 14 months), the dropout of which compromised prediction performance (area under the curve = 0.661 at 14 months). Furthermore, stratification analysis indicated that L/MiT tended preferentially to impact young and female patients (HR = 11.61, p CONCLUSION This was the first CIC profiling to be performed in PDAC, and is currently largest for human tumors. Subtyped CICs, as a valuable input to the traditional variables such as TNM stage, represent a novel type of prognostic factor. The formation of heterotypic L/MiT may be a surrogate for local immune evasion and predict poor survival, particularly in young female patients of resectable PDAC. Study Highlights Prior knowledge The post-operation survival periods of resectable pancreatic ductal adenocarcinoma (PDAC) patients range widely, and the search for reliable prognostic biomarkers is warranted. Although profound local immune suppression is implicated in PDAC progression and poor patient survival, a prognostic marker to read immune evasion in situ is not yet available. The impact of subtyped cell-in-cell (CIC) structures, which target either tumor or immune cells for internalization and death, on PDAC patient survival is not clear. Novelty of study This study presents the first CIC subtype profiling in PDAC, which is currently the largest of its type for human cancers. Subtyped CIC structures were identified and confirmed independently as a valuable prognostic factor for PDAC patients, with a performance comparable or superior to traditional variables such as tumor–node–metastasis (TNM) stage. The L/MiT heterotypic CIC subtype, surrogating a type of cellular immune evasion, could independently predict poor survival, particularly for young female patients of resectable PDAC.

STAG2 Loss Gives Rise to Therapeutically Targetable DNA Damage Repair Defects and Altered Replication Fork Dynamics in Acute Myeloid Leukaemia

BackgroundAlthough platinum-based chemotherapy is standard treatment for pancreatic ductal adenocarcinoma (PDAC) patients with either germline/somatic deficiencies in homologous recombination (mutHRD), a subset become platinum-resistant. We have shown that patients with...

Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.

HUWE1 is a multi‐faceted E3 ubiquitin ligase of the HECT family with many confirmed substrates, but mechanistic understanding of its functional roles in signaling pathways remains limited. In this issue of EMBO Reports, Choe et al demonstrate a novel function for HUWE1 in promoting DNA damage tolerance mechanisms to bypass DNA lesions during replication stress, thereby preserving genome stability. The authors connect this role for HUWE1 with its function in maintaining H2AX monoubiquitination levels for efficient signaling at stalled replication forks . Thus, this work highlights HUWE1 as a novel player in the replication stress response and prompts further investigation of its regulation during replication and other cellular processes.