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Background: Homologous recombination repair (HR) is the most accurate repair pathway for double-strand breaks and replication fork disruption that is capable of faithfully restoring the original nucleotide sequence of the broken DNA. The deficiency of this mechanism is a frequent event in tumorigenesis. Therapies that exploit defects in HR have been explored essentially in breast, ovarian, pancreatic, and prostate cancers, but poorly in colorectal cancers (CRC), although CRC ranks second in mortality worldwide. Methods: Tumor specimens and matched healthy tissues from 63 patients with CRC were assessed for gene expression of key HR components and mismatch repair (MMR) status, which correlated with clinicopathological features, progression-free survival, and overall survival (OS). Results: Enhanced expression of MRE11 homolog (MRE11A), the gene encoding a key molecular actor for resection, is significantly overexpressed in CRC, is associated with the occurrence of primary tumors, particularly T3-T4, and is found in more than 90% of the right-side of CRC, the location with the worst prognosis. Importantly, we also found that high MRE11A transcript abundance is associated with 16.7 months shorter OS and a 3.5 higher risk of death. Conclusion: Monitoring of MRE11 expression could be used both as a predictor of outcome and as a marker to select CRC patients for treatments thus far adapted for HR-deficient cancers.

Background: Homologous Recombination (HR) is the most accurate repair pathway for double-strand breaks and replication fork disruption that is capable of faithfully restoring the original nucleotide sequence of the broken DNA. The deficiency in this mechanism is a frequent event in tumorigenesis. Therapies that exploit defects in HR have been explored essentially in breast, ovarian, pancreas, and prostate cancers, but poorly in colorectal cancers (CRC) although CRC ranks second in mortality worldwide. Methods: Tumor specimens and matched healthy tissues from 63 patients with CRC were assessed for gene expression of HR key components and MMR status, which were correlated with clinicopathological features, progression-free survival, and overall survival (OS). Results: Enhanced expression of MRE11A, the gene encoding a key molecular actor for resection is significantly overexpressed in CRC, is associated with the occurrence of primary tumors particularly T3-T4 and is found in more than 90% of the right-side of CRC, the location with worst prognostic. Importantly, we also found that high MRE11A transcript abundance is associated with 16.7 months shorter OS and a 3.5 higher risk of death. Conclusion: Monitoring MRE11 expression could be used both as a predictor of outcome and as a marker to select CRC patients for treatments thus far adapted for HR deficient cancers.

Supplementary figures and figure legends

A characteristic of cancer development is the acquisition of genomic instability, which results from the inaccurate repair of DNA damage. Among double-strand break repair mechanisms induced by oncogenic stress, the highly mutagenic theta-mediated end-joining (TMEJ) pathway, which requires DNA polymerase theta (POLθ) encoded by the POLQ gene, has been shown to be overexpressed in several human cancers. However, little is known regarding the regulatory mechanisms of TMEJ and the consequence of its dysregulation. In this study, we combined a bioinformatics approach exploring both Molecular Taxonomy of Breast Cancer International Consortium and The Cancer Genome Atlas databases with CRISPR/Cas9-mediated depletion of the zinc finger E-box binding homeobox 1 (ZEB1) in claudin-low tumor cells or forced expression of ZEB1 in basal-like tumor cells, two triple-negative breast cancer (TNBC) subtypes, to demonstrate that ZEB1 represses POLQ expression. ZEB1, a master epithelial-to-mesenchymal transition–inducing transcription factor, interacted directly with the POLQ promoter. Moreover, downregulation of POLQ by ZEB1 fostered micronuclei formation in TNBC tumor cell lines. Consequently, ZEB1 expression prevented TMEJ activity, with a major impact on genome integrity. In conclusion, we showed that ZEB1 directly inhibits the expression of POLQ and, therefore, TMEJ activity, controlling both stability and integrity of breast cancer cell genomes.Significance:These findings uncover an original mechanism of TMEJ regulation, highlighting ZEB1 as a key player in genome stability during cancer progression via its repression of POLQ.See related commentary by Carvajal-Maldonado and Wood, p. 1441

International audience; Aim: The transcription factor RIP140 (receptor interacting protein of 140 kDa) is involved in intestinal tumorigenesis. It plays a role in the control of microsatellite instability (MSI), through the regulation of MSH2 and MSH6 gene expression. The aim of this study was to explore its effect on the expression of POLK, the gene encoding the specialized translesion synthesis (TLS) DNA polymerase κ known to perform accurate DNA synthesis at microsatellites.Methods: Different mouse models and engineered human colorectal cancer (CRC) cell lines were used to analyze by RT-qPCR, while Western blotting and luciferase assays were used to elucidate the role of RIP140 on POLK gene expression. Published DNA microarray datasets were reanalyzed. The in vitro sensitivity of CRC cells to methyl methane sulfonate and cisplatin was determined.Results: RIP140 positively regulates, at the transcriptional level, the expression of the POLK gene, and this effect involves, at least partly, the p53 tumor suppressor. In different cohorts of CRC biopsies (with or without MSI), a strong positive correlation was observed between RIP140 and POLK gene expression. In connection with its effect on POLK levels and the TLS function of this polymerase, the cellular response to methyl methane sulfonate was increased in cells lacking the Rip140 gene. Finally, the association of RIP140 expression with better overall survival of CRC patients was observed only when the corresponding tumors exhibited low levels of POLK, thus strengthening the functional link between the two genes in human CRC.Conclusion: The regulation of POLK gene expression by RIP140 could thus contribute to the maintenance of microsatellite stability, and more generally to the control of genome integrity.

Chronic DNA replication stress and genome instability are two hallmarks of cancer that fuel oncogenesis and tumor diversity. Therapeutic approaches aimed to leverage tumor-specific replication stress to intolerable levels or to expose vulnerabilities for synthetic lethality purposes have recently gained momentum, especially for pancreatic cancer, a disease with no cure. However, the current knowledge regarding the molecular mechanisms involved in the replication stress response in pancreatic tumors is limited. Cytidine deaminase (CDA) is involved in the pyrimidine salvage pathway for DNA and RNA synthesis. Loss of CDA induces genomic instability in Bloom Syndrome, and CDA protects tumor cells from chemotherapy with pyrimidine analogs. Here, we show that CDA is overexpressed in genetically unstable pancreatic tumors, associates with a DNA replication signature, and is instrumental for experimental tumor growth. In cancer cells, CDA promotes DNA replication, increases replication fork speed, and controls replication stress and genomic stability levels. CDA expression is predictive of DNA-damaging drug efficacy and targeting CDA relieves resistance to chemotherapy in patients models, both in vitro and in vivo. Our findings shed new light on the mechanisms by which pancreatic cancer cells control replication stress, and highlight targeting of CDA as a potential therapeutic strategy to defeat tumor resistance to treatment.

DNA polymerase kappa (Pol κ) has been well documented thus far for its specialized DNA synthesis activity during translesion replication, progression of replication forks through regions difficult to replicate, restart of stalled forks, and replication checkpoint efficiency. Pol κ is also required for the stabilization of stalled forks, although the mechanisms are poorly understood. In this study, we unveiled an unexpected role for Pol κ in controlling the stability and abundance of checkpoint kinase 1 (Chk1), an important actor for the replication checkpoint and fork stabilization. We found that loss of Pol κ decreased the Chk1 protein level in the nuclei of four human cell lines. Pol κ and not the other Y family polymerase members is required to maintain the Chk1 protein pool all along the cell cycle. We showed that Pol κ depletion affected the protein stability of Chk1 and protected it from proteasome degradation. Importantly, we also observed that the fork restart defects observed in Pol κ-depleted cells could be overcome by the reexpression of Chk1. Strikingly, this new function of Pol κ does not require its catalytic activity. We propose that Pol κ could contribute to the protection of stalled forks through Chk1 stability.

Simple Summary Two to three percent of breast cancer patients harbor germline mutation of either BRCA1 or BRCA2 genes. Their tumor cells are deficient in homologous recombination, a BRCA-dependent DNA repair machinery. These deficient cells survive thanks to the PARP-mediated alternative pathway. Therefore, PARP inhibitors have already shown some level of efficiency in the treatment of metastatic breast cancer patients. Unfortunately, some tumor cells inevitably resist PARP inhibitors by different mechanisms. In this review, we (i) present the notion of homologous recombination deficiency and its evaluation methods, (ii) detail the PARP inhibitor clinical trials in breast cancer, (iii) briefly describe the mechanisms to PARP inhibitors resistance, and (iv) discuss some strategies currently under evaluation to enhance the therapeutic index of PARP inhibitors in breast cancer. Abstract As poly-(ADP)-ribose polymerase (PARP) inhibition is synthetic lethal with the deficiency of DNA double-strand (DSB) break repair by homologous recombination (HR), PARP inhibitors (PARPi) are currently used to treat breast cancers with mutated BRCA1/2 HR factors. Unfortunately, the increasingly high rate of PARPi resistance in clinical practice has dented initial hopes. Multiple resistance mechanisms and acquired vulnerabilities revealed in vitro might explain this setback. We describe the mechanisms and vulnerabilities involved, including newly identified modes of regulation of DSB repair that are now being tested in large cohorts of patients and discuss how they could lead to novel treatment strategies to improve the therapeutic index of PARPi.

Simple Summary DNA polymerase theta, encoded by the human POLQ gene, is upregulated in several cancers and is associated with poor clinical outcomes. The importance of POLQ, however, has yet to be elucidated in esophageal cancer. In this study, we explored the functional impacts of POLQ and looked into its underlying mechanisms. POLQ was overexpressed in esophageal squamous cell carcinoma (ESCC) tumors associated with unfavorable prognosis and contributed to malignant phenotypes by promoting genome stability, suggesting that targeting polymerase theta may provide a potential therapeutic approach for improving ESCC management. Abstract Overexpression of the specialized DNA polymerase theta (POLQ) is frequent in breast, colon and lung cancers and has been correlated with unfavorable clinical outcomes. Here, we aimed to determine the importance and functional role of POLQ in esophageal squamous cell carcinoma (ESCC). Integrated analysis of four RNA-seq datasets showed POLQ was predominantly upregulated in ESCC tumors. High expression of POLQ was also observed in a cohort of 25 Hong Kong ESCC patients and negatively correlated with ESCC patient survival. POLQ knockout (KO) ESCC cells were sensitized to multiple genotoxic agents. Both rH2AX foci staining and the comet assay indicated a higher level of genomic instability in POLQ-depleted cells. Double KO of POLQ and FANCD2, known to promote POLQ recruitment at sites of damage, significantly impaired cell proliferation both in vitro and in vivo, as compared to either single POLQ or FANCD2 KOs. A significantly increased number of micronuclei was observed in POLQ and/or FANCD2 KO ESCC cells. Loss of POLQ and/or FANCD2 also resulted in the activation of cGAS and upregulation of interferon-stimulated genes (ISGs). Our results suggest that high abundance of POLQ in ESCC contributes to the malignant phenotype through genome instability and activation of the cGAS pathway.

KDM5A and KDM5B histone-demethylases are overexpressed in many cancers and have been involved in drug tolerance. Here, we describe that KDM5A, together with KDM5B, contribute to replication stress (RS) response and tolerance. First, they positively regulate RRM2, the regulatory subunit of ribonucleotide reductase. Second, they are required for optimal levels of activated Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. We also found that KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1. Because RRM2 is a major determinant of replication stress tolerance, we developed cells resistant to HU, and show that KDM5A/B proteins are required for both RRM2 overexpression and tolerance to HU. Altogether, our results indicate that KDM5A/B are major players of RS management. They also show that drugs targeting the enzymatic activity of KDM5 proteins may not affect all cancer-related consequences of KDM5A/B overexpression., Summary: We describe that KDM5A and KDM5B histone-demethylases contribute to hydroxyurea-induced replication stress tolerance, in part by regulating expression of ribonuclease reductase subunit RRM2.

DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains This work was supported by “la Ligue nationale contre le cancer” (LNCC labellisation to JSH and VB and the grants RS16/75-108 and RS17/75-135 to JCC), the “Institut National du Cancer” (INCA, PLBIO2016-10493 to JSH, VB and JCC), the “Centre de Recherches en Cancérologie de Toulouse” to VB, the “Laboratoire d’excellence Toulouse Cancer—TOUCAN” to JSH, the “Groupement des Entreprises Françaises contre le Cancer” (GEFLUC) to JCC, the “IdEx Université de Paris” (ANR-18-IDEX-0001) to JCC, the generous legacy from Ms Suzanne Larzat to JCC, and “la Fondation pour la Recherche Médicale” to LC. MM and VP were funded by INSERM, the “Fondation Toulouse Cancer Santé” and “Pierre Fabre Research Institute” as part of the Chair of Bioinformatics in Oncology of the CRCT.

A characteristic of cancer development is the acquisition of genomic instability, which results from the inaccurate repair of DNA damage. Among double-strand break repair mechanisms induced by oncogenic stress, the highly mutagenic theta-mediated end-joining (TMEJ) pathway, which requires DNA polymerase theta (POLθ) encoded by the POLQ gene, has been shown to be overexpressed in several human cancers. However, little is known regarding the regulatory mechanisms of TMEJ and the consequence of its dysregulation. In this study, we combined a bioinformatics approach exploring both Molecular Taxonomy of Breast Cancer International Consortium and The Cancer Genome Atlas databases with CRISPR/Cas9-mediated depletion of the zinc finger E-box binding homeobox 1 (ZEB1) in claudin-low tumor cells or forced expression of ZEB1 in basal-like tumor cells, two triple-negative breast cancer (TNBC) subtypes, to demonstrate that ZEB1 represses POLQ expression. ZEB1, a master epithelial-to-mesenchymal transition–inducing transcription factor, interacted directly with the POLQ promoter. Moreover, downregulation of POLQ by ZEB1 fostered micronuclei formation in TNBC tumor cell lines. Consequently, ZEB1 expression prevented TMEJ activity, with a major impact on genome integrity. In conclusion, we showed that ZEB1 directly inhibits the expression of POLQ and, therefore, TMEJ activity, controlling both stability and integrity of breast cancer cell genomes. Significance: These findings uncover an original mechanism of TMEJ regulation, highlighting ZEB1 as a key player in genome stability during cancer progression via its repression of POLQ. See related commentary by Carvajal-Maldonado and Wood, p. 1441

DNA polymerase kappa (Pol κ) has been well documented thus far for its specialized DNA synthesis activity during translesion replication, progression of replication forks through regions difficult to replicate and replication checkpoint at stalled forks.Here we unveiled an unexpected role for Pol κ in controlling the stability and abundance of Chk1, the major mediator of the replication checkpoint. We found that loss of Pol κ decreased the Chk1 protein level in the nucleus of four human cell lines. Pol κ and not the other Y‐family polymerase members is required to maintain the Chk1 protein pool all along the cell cycle. We showed that Pol κ depletion affected the protein stability of Chk1 and protected it from proteasome degradation and the replication recovery defects observed in Pol κ-depleted cells could be overcome by the re-expression of Chk1. Importantly, this new function of Pol κ does not require its catalytic activity, revealing that in addition to its known roles in the replication process, Pol κ can contribute to the maintenance of genome stability independently of its DNA synthesis activity.

International audience; Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.

DNA replication is well orchestrated in mammalian cells through a tight regulation of the temporal order of replication origin activation, named the replication timing, a robust and conserved process in each cell type. Upon low replication stress, the slowing of replication forks induces delayed replication of fragile regions leading to genetic instability. The impact of low replication stress on the replication timing in different cellular backgrounds has not been explored yet. Here we analysed the whole genome replication timing in a panel of 6 human cell lines under low replication stress. We first demonstrated that cancer cells were more impacted than non-tumour cells. Strikingly, we unveiled an enrichment of specific replication domains undergoing a switch from late to early replication in some cancer cells. We found that advances in replication timing correlate with heterochromatin regions poorly sensitive to DNA damage signalling while being subject to an increase of chromatin accessibility. Finally, our data indicate that, following release from replication stress conditions, replication timing advances can be inherited by the next cellular generation, suggesting a new mechanism by which cancer cells would adapt to cellular or environmental stress.; DNA replication is well orchestrated in mammalian cells through a tight regulation of the temporal order of replication origin activation, named the replication timing, a robust and conserved process in each cell type. Upon low replication stress, the slowing of replication forks induces delayed replication of fragile regions leading to genetic instability. The impact of low replication stress on the replication timing in different cellular backgrounds has not been explored yet. Here we analysed the whole genome replication timing in a panel of 6 human cell lines under low replication stress. We first demonstrated that cancer cells were more impacted than non-tumour cells. Strikingly, we unveiled an enrichment of specific replication domains undergoing a switch from late to early replication in some cancer cells. We found that advances in replication timing correlate with heterochromatin regions poorly sensitive to DNA damage signalling while being subject to an increase of chromatin accessibility. Finally, our data indicate that, following release from replication stress conditions, replication timing advances can be inherited by the next cellular generation, suggesting a new mechanism by which cancer cells would adapt to cellular or environmental stress.

SUMMARYKDM5A and KDM5B histone-demethylases are overexpressed in many cancers and have been involved in drug tolerance. Here, we describe that KDM5A, together with KDM5B, contribute to replication stress (RS) response and tolerance. First, they positively regulate RRM2, the regulatory subunit of Ribonucleotide Reductase. Second, they are required for optimal activation of Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. This role in Chk1 activation is probably direct since KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1. Because RRM2 is a major determinant of replication stress tolerance, we developed cells resistant to HU, and show that KDM5A/B proteins are required for both RRM2 overexpression and tolerance to HU, in a manner that is independent of their demethylase activity. Altogether, our results indicate that KDM5A/B are major players of RS management. They also show that drugs targeting the enzymatic activity of KDM5 proteins may not affect all cancer-related consequences of KDM5A/B overexpression.

Oncogene-induced replication stress (RS) promotes cancer development but also impedes tumor growth by activating anti-cancer barriers. To determine how cancer cells adapt to RS, we have monitored the expression of different components of the ATR-CHK1 pathway in primary tumor samples. We show that unlike upstream components of the pathway, the checkpoint mediators Claspin and Timeless are overexpressed in a coordinated manner. Remarkably, reducing the levels of Claspin and Timeless in HCT116 cells to pretumoral levels impeded fork progression without affecting checkpoint signaling. These data indicate that high level of Claspin and Timeless increase RS tolerance by protecting replication forks in cancer cells. Moreover, we report that primary fibroblasts adapt to oncogene-induced RS by spontaneously overexpressing Claspin and Timeless, independently of ATR signaling. Altogether, these data indicate that enhanced levels of Claspin and Timeless represent a gain of function that protects cancer cells from of oncogene-induced RS in a checkpoint-independent manner., Oncogene-induced replication stress (RS) promotes cancer development. Here, the authors report that cancer cells adapt to oncogene-induced RS by overexpressing downstream components of ATR-CHK1 pathway, Claspin and Timeless, which have protective role at the replication forks independent of their checkpoint function.

Pancreatic ductal adenocarcinoma (PDAC) relies on hyperactivated protein synthesis. Consistently, human and mouse PDAC lose expression of the translational repressor and mTOR target 4E-BP1. Using genome-wide polysome profiling, we here explore mRNAs whose translational efficiencies depend on the mTOR/4E-BP1 axis in pancreatic cancer cells. We identified a functional enrichment for mRNAs encoding DNA replication and repair proteins, including RRM2 and CDC6. Consequently, 4E-BP1 depletion favors DNA repair and renders DNA replication insensitive to mTOR inhibitors, in correlation with a sustained protein expression of CDC6 and RRM2, which is inversely correlated with 4E-BP1 expression in PDAC patient samples. DNA damage and pancreatic lesions induced by an experimental pancreatitis model uncover that 4E-BP1/2-deleted mice display an increased acinar cell proliferation and a better recovery than WT animals. Targeting translation, independently of 4E-BP1 status, using eIF4A RNA helicase inhibitors (silvestrol derivatives) selectively modulates translation and limits CDC6 expression and DNA replication, leading to reduced PDAC tumor growth. In summary, 4E-BP1 expression loss during PDAC development induces selective changes in translation of mRNA encoding DNA replication and repair protein. Importantly, targeting protein synthesis by eIF4A inhibitors circumvents PDAC resistance to mTOR inhibition.

Cytidine deaminase (CDA) converts cytidine and deoxycytidine into uridine and deoxyuridine within the pyrimidine salvage pathway for DNA and RNA synthesis. In this regard, loss of CDA provokes genomic instability in Bloom Syndrome, and CDA overexpression is associated with tumor resistance to chemotherapy with pyrimidine analogs. However, the precise role of CDA per se in cancer has been totally underexplored so far. Patient cohort analysis demonstrate that CDA is overexpressed in PDAC, a disease with no cure with increasing incidence, and associated with a worse prognosis in patient. Functional studies demonstrate that CDA is essential to PDAC cell proliferation and tumor growth. We found that CDA expression is associated with gene set enrichment in DNA replication signature, in both PDAC tumors and experimental models. Using enforced expression, genetic or pharmacologic targeting, we demonstrate that CDA promotes DNA replication, localizes to the replication fork and increases replication fork fitness. These effects are strictly dependent on CDA deaminase activity. Hence, we found that CDA controls replication stress as CDA expression is inversely correlated with the level of DNA breaks during S-phase, and that CDA targeting is associated with gene set enrichment in replication stress signature and CHK1 protein activation. Next, we demonstrate that CDA preserves genomic stability of PDAC cells, as CDA expression is inversely correlated with micronuclei formation and DNA damage transfer to daughter cells. We next explored PDAC cell lines from the Cancer Cell Lines Encyclopedia and found that CDA expression is associated with resistance to drug targeting DNA synthesis. Functional studies demonstrate that overexpressing CDA, and not CDA catalytically inactive mutant, protects PDAC cell lines from camptothecin, a topoisomerase inhibitor, while targeting CDA sensitizes PDAC cells to treatment by camptothecin and oxaliplatin, that forms platinum-DNA adducts. To gain further insights into the clinical potential of such finding, we targeted CDA expression in primary cells from patient with PDAC and found that CDA is essential to primary cell growth, and that CDA targeting sensitizes primary cells to treatment by oxaliplatin. Taken together, our results reveal for the first time that CDA controls DNA replication, replication stress level and genomic stability of PDAC cells, and that this new role of CDA is involved in tumor resistance to drugs inducing DNA damages. Thus, our work stems for new strategies based on CDA targeting to defeat PDAC resistance to treatment. Citation Format: Audrey Lumeau, Nicolas Bery, Cyril Ribeyre, Samad Elkaoutari, Guillaume Labrousse, Miguel Madrid-Mencia, Vera Pancaldi, Marie-Jeanne Pillaire, Valérie Bergoglio, Nelson Dusseti, Jean-Sébastien Hoffmann, Louis Buscail, Malik Lutzmann, Pierre Cordelier. Cytidine deaminase protects pancreatic cancer cells from replicative stress and drive response to DNA-targeting drugs [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-043.

Summary: Hematopoiesis is particularly sensitive to DNA damage. Myeloid tumor incidence increases in patients with DNA repair defects and after chemotherapy. It is not known why hematopoietic cells are highly vulnerable to DNA damage. Addressing this question is complicated by the paucity of mouse models of hematopoietic malignancies due to defective DNA repair. We show that DNA repair-deficient Mcm8- and Mcm9-knockout mice develop myeloid tumors, phenocopying prevalent myelodysplastic syndromes. We demonstrate that these tumors are preceded by a lifelong DNA damage burden in bone marrow and that they acquire proliferative capacity by suppressing signaling of the tumor suppressor and cell cycle controller RB, as often seen in patients. Finally, we found that absence of MCM9 and the tumor suppressor Tp53 switches tumorigenesis to lymphoid tumors without precedent myeloid malignancy. Our results demonstrate that MCM8/9 deficiency drives myeloid tumor development and establishes a DNA damage burdened mouse model for hematopoietic malignancies. : Lutzmann et al. show that MCM8- or MCM9-deficient mice suffer chronic DNA damage, causing myeloid tumors, resembling human myelodysplastic syndromes, during aging. These tumors lose RB-mediated cell cycle control, cause splenomegaly, and preclude progressively normal hematopoiesis. Additional loss of the tumor suppressor Tp53 switches tumor development to T cell lymphoma. Keywords: MCM8, MCM9, DNA damage, DNA repair, myelodysplastic syndrome, cancer, homologous recombination, hematopoiesis

The effectiveness of checkpoint kinase 1 (Chk1) inhibitors at killing cancer cells is considered to be fully dependent on their effect on DNA replication initiation. Chk1 inhibition boosts origin firing, presumably limiting the availability of nucleotides and in turn provoking the slowdown and subsequent collapse of forks, thus decreasing cell viability. Here we show that slow fork progression in Chk1-inhibited cells is not an indirect effect of excess new origin firing. Instead, fork slowdown results from the accumulation of replication barriers, whose bypass is impeded by CDK-dependent phosphorylation of the specialized DNA polymerase eta (Polη). Also in contrast to the linear model, the accumulation of DNA damage in Chk1-deficient cells depends on origin density but is largely independent of fork speed. Notwithstanding this, origin dysregulation contributes only mildly to the poor proliferation rates of Chk1-depleted cells. Moreover, elimination of replication barriers by downregulation of helicase components, but not their bypass by Polη, improves cell survival. Our results thus shed light on the molecular basis of the sensitivity of tumors to Chk1 inhibition. Fil: González, Marina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Calzetta, Nicolás Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Loureiro, Sofía M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Habif, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Bétous, Rémy. University Paul Sabatier; Francia Fil: Pillaire, Marie Jeanne. University Paul Sabatier; Francia Fil: Maffia, Antonio. Istituto di Genetica Molecolare “Luigi Luca Cavalli-Sforza"; Italia Fil: Sabbioneda, Simone. Istituto di Genetica Molecolare “Luigi Luca Cavalli-Sforza"; Italia Fil: Hoffmann, Jéan Sebastién. University Paul Sabatier; Francia Fil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20⁻30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or “dormant„ origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress.

DNA polymerase theta (Polθ) is a specialized A-family DNA polymerase that functions in processes such as translesion synthesis (TLS), DNA double-strand break repair and DNA replication timing. Overexpression of POLQ, the gene encoding Polθ, is a prognostic marker for an adverse outcome in a wide range of human cancers. While increased Polθ dosage was recently suggested to promote survival of homologous recombination (HR)-deficient cancer cells, it remains unclear whether POLQ overexpression could be also beneficial to HR-proficient cancer cells. By performing a short interfering (si)RNA screen in which genes encoding druggable proteins were knocked down in Polθ-overexpressing cells as a means to uncover genetic vulnerabilities associated with POLQ overexpression, we could not identify genes that were essential for viability in Polθ-overexpressing cells in normal growth conditions. We also showed that, upon external DNA replication stress, Polθ expression promotes cell survival and limits genetic instability. Finally, we report that POLQ expression correlates with the expression of a set of HR genes in breast, lung and colorectal cancers. Collectively, our data suggest that Polθ upregulation, besides its importance for survival of HR-deficient cancer cells, may be crucial also for HR-proficient cells to better tolerate DNA replication stress, as part of a global gene deregulation response, including HR genes., Summary: Our work suggests that Polθ upregulation may be crucial for homologous recombination (HR)-proficient cells to better tolerate DNA replication stress, as part of a global gene deregulation response, including HR genes.

Nature Communications 8: Article number: 15983 (2017); Published: 17 July 2017; Updated: 26 October 2017 In this Article, there are errors in the labelling of the y axis in Fig. 1a and Supplementary Fig. 1b. The labels ‘20’, ‘40’, ‘60’, ‘80’ and ‘100’ should have been ‘0.5’, ‘1.0’ and ‘1.5’ in Fig. 1a and the labels ‘50’, ‘100’, ‘150’ and ‘200’ should have been ‘1’, ‘2’ and ‘3’ in Supplementary Fig.

Dendrogenin A (DDA), a mammalian cholesterol metabolite with tumor suppressor properties, has recently been shown to exhibit strong anti-leukemic activity in acute myeloid leukemia (AML) cells by triggering lethal autophagy. Here, we demonstrated that DDA synergistically enhanced the toxicity of anthracyclines in AML cells but not in normal hematopoietic cells. Combination index of DDA treatment with either daunorubicin or idarubicin indicated a strong synergism in KG1a, KG1 and MV4-11 cell lines. This was confirmed in vivo using immunodeficient mice engrafted with MOLM-14 cells as well as in a panel of 20 genetically diverse AML patient samples. This effect was dependent on Liver X Receptor &beta, a major target of DDA. Furthermore, DDA plus idarubicin strongly increased p53BP1 expression and the number of DNA strand breaks in alkaline comet assays as compared to idarubicin alone, whereas DDA alone was non-genotoxic. Mechanistically, DDA induced JNK phosphorylation and the inhibition of AKT phosphorylation, thereby maximizing DNA damage induced by idarubicin and decreasing DNA repair. This activated autophagic cell death machinery in AML cells. Overall, this study shows that the combination of DDA and idarubicin is highly promising and supports clinical trials of dendrogenin A in AML patients.

Simple Summary Higher eukaryotic cells frequently enter mitosis with a certain load of under-replicated DNA, also referred to as unreplicated DNA, due to incomplete genomic DNA replication during the previous S phase. Double replication fork stalling events, when two converging forks irreversibly stall with no replication origin in between them, seem to be one of the major drivers of incomplete genomic replication in S phase. Genome stability is yet maintained in the vast majority of cells implying that cells must possess dedicated post-replicative mechanisms that allow for faithful repair of these seemingly unavoidable errors. Here, we provide a comprehensive overview of the mechanisms or events that cause, regulate and repair under-replicated DNA in eukaryotic cells. Abstract In this review, we provide an overview of how proliferating eukaryotic cells overcome one of the main threats to genome stability: incomplete genomic DNA replication during S phase. We discuss why it is currently accepted that double fork stalling (DFS) events are unavoidable events in higher eukaryotes with large genomes and which responses have evolved to cope with its main consequence: the presence of under-replicated DNA (UR-DNA) outside S phase. Particular emphasis is placed on the processes that constrain the detrimental effects of UR-DNA. We discuss how mitotic DNA synthesis (MiDAS), mitotic end joining events and 53BP1 nuclear bodies (53BP1-NBs) deal with such specific S phase DNA replication remnants during the subsequent phases of the cell cycle.

International audience; Alteration in the DNA replication, repair or recombination processes is a highly relevant mechanism of genomic instability. Despite genomic aberrations manifested in hematologic malignancies, such a defect as a source of biomarkers has been underexplored. Here, we investigated the prognostic value of expression of 82 genes involved in DNA replication-repair-recombination in a series of 99 patients with chronic lymphocytic leukemia without detectable 17p deletion or TP53 mutation. We found that expression of the POLN gene, encoding the specialized DNA polymerase ν (Pol ν) correlates with time to relapse after first-line therapy with fludarabine. Moreover, we found that POLN was the only gene up-regulated in primary patients’ lymphocytes when exposed in vitro to proliferative and pro-survival stimuli. By using two cell lines that were sequentially established from the same patient during the course of the disease and Pol ν knockout mouse embryonic fibroblasts, we reveal that high relative POLN expression is important for DNA synthesis and cell survival upon fludarabine treatment. These findings suggest that Pol ν could influence therapeutic resistance in chronic lymphocytic leukemia. (Patients’ samples were obtained from the CLL 2007 FMP clinical trial registered at: clinicaltrials.gov identifer: 00564512).

Failure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity., BRCA2-deficient cells fail to complete replication in S-phase, which results in DNA bridges and chromosome mis-segregation at mitosis. Here the authors show that MUS81 helps BRCA2-deficient cells survive replication stress by activating mitotic DNA synthesis and resolving mitotic DNA bridges.

Replication stress is a strong and early driving force for genomic instability and tumor development. Beside replicative DNA polymerases, an emerging group of specialized DNA polymerases is involved in the technical assistance of the replication machinery in order to prevent replicative stress and its deleterious consequences. During S-phase, altered progression of the replication fork by endogenous or exogenous impediments induces replicative stress, causing cells to reach mitosis with genomic regions not fully duplicated. Recently, specific mechanisms to resolve replication intermediates during mitosis with the aim of limiting DNA damage transmission to daughter cells have been identified. In this review, we detail the two major actions of specialized DNA polymerases that limit DNA damage transmission: the prevention of replicative stress by non-B DNA replication and the recovery of stalled replication forks.