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.

Abstract Cytidine deaminase (CDA) functions in the pyrimidine salvage pathway for DNA and RNA syntheses and has been shown to protect cancer cells from deoxycytidine-based chemotherapies. In this study, we observed that CDA was overexpressed in pancreatic adenocarcinoma from patients at baseline and was essential for experimental tumor growth. Mechanistic investigations revealed that CDA localized to replication forks where it increased replication speed, improved replication fork restart efficiency, reduced endogenous replication stress, minimized DNA breaks, and regulated genetic stability during DNA replication. In cellular pancreatic cancer models, high CDA expression correlated with resistance to DNA-damaging agents. Silencing CDA in patient-derived primary cultures in vitro and in orthotopic xenografts in vivo increased replication stress and sensitized pancreatic adenocarcinoma cells to oxaliplatin. This study sheds light on the role of CDA in pancreatic adenocarcinoma, offering insights into how this tumor type modulates replication stress. These findings suggest that CDA expression could potentially predict therapeutic efficacy and that targeting CDA induces intolerable levels of replication stress in cancer cells, particularly when combined with DNA-targeted therapies. Significance: Cytidine deaminase reduces replication stress and regulates DNA replication to confer resistance to DNA-damaging drugs in pancreatic cancer, unveiling a molecular vulnerability that could enhance treatment response.

The exonuclease domain of DNA polymerases epsilon's catalytic subunit (POLE) removes misincorporated nucleotides, called proofreading. POLE-exonuclease mutations cause colorectal- and endometrial cancers with an extreme burden of single nucleotide substitutions. We recently reported that particularly the hereditary POLE exonuclease mutation N363K predisposes in addition to aggressive giant cell glioblastomas. We knocked-in this mutation homozygously into human cell lines and compared its properties to knock-ins of the likewise hereditary POLE L424V mutation and to a complete proofreading-inactivating mutation (exo-null). We found that N363K cells have higher mutation rates as both L424V- or exo-null mutant cells. In contrast to L424V cells, N363K cells expose a growth defect, replication stress and DNA damage. In non-transformed cells, these burdens lead to aneuploidy but macroscopically normal nuclei. In contrast, transformed N363K cells phenocopy the enlarged and disorganized nuclei of giant cell glioblastomas. Taken together, our data characterize a POLE exonuclease domain mutant that not only causes single nucleotide hypermutation, but in addition DNA damage and chromosome instability, leading to an extended tumor spectrum. Our results expand the understanding of the polymerase exonuclease domain and suggest that an assessment of both the mutational potential and the genetic instability might refine classification and treatment of POLE-mutated tumors.

Multiple myeloma (MM) is the second most frequent hematological malignancy, characterized by the accumulation of malignant plasma cells (PCs) within the bone marrow. To date, there is no definitive treatment for this pathology and a majority of patients will invariably relapse. Antibody secretion, the key biological function of PCs, is maintained in malignant PCs meaning that these cells display an elevated transcriptional stress. Besides, malignant PCs face oncogene-induced replication stress concomitantly with cell cycle deregulation. Consequently, transcription and replication in malignant PCs need to be tightly coordinated to avoid too much interferences that would increase replication stress and genomic instability. A failure to cope with these transcription/replication conflicts (TRCs) could have a significant impact on mutagenesis involved in MM development. Importantly, these effects might open the therapeutic possibility of TRCs enhancement to specifically kill malignant PCs.

Plasma cells (PCs) have an essential role in humoral immune response by secretion of antibodies, and represent the final stage of B lymphocytes differentiation. During this differentiation, the pre-plasmablastic stage is characterized by highly proliferative cells that start to secrete immunoglobulins (Igs). Thus, replication and transcription must be tightly regulated in these cells to avoid transcription/replication conflicts (TRCs), which could increase replication stress and lead to genomic instability. In this review, we analyzed expression of genes involved in TRCs resolution during B to PC differentiation and identified 41 genes significantly overexpressed in the pre-plasmablastic stage. This illustrates the importance of mechanisms required for adequate processing of TRCs during PCs differentiation. Furthermore, we identified that several of these factors were also found overexpressed in purified PCs from patients with multiple myeloma (MM) compared to normal PCs. Malignant PCs produce high levels of Igs concomitantly with cell cycle deregulation. Therefore, increasing the TRCs occurring in MM cells could represent a potent therapeutic strategy for MM patients. Here, we describe the potential roles of TRCs resolution factors in myelomagenesis and discuss the therapeutic interest of targeting the TRCs resolution machinery in MM.

DNA replication initiates with pre-replication complex (pre-RC) formation at replication origins in G1 (replication origin licensing), followed by activation of a pre-RC subset in the S phase. It has been suggested that a checkpoint prevents S phase entry when too few origins are licensed. Yet, we found that in normal cells, complete DNA synthesis inhibition by overexpression of a non-degradable geminin variant, or by CDT1 silencing prevents DNA replication without inducing any checkpoint. Cells continue cycling and enter mitosis, despite the absence of replicated DNA. Most of these unlicensed cells exit mitosis without dividing and enter senescence; however, about 25% of them successfully divide without previous DNA replication, producing daughter cells with half the normal diploid complement of chromosomes (1C). This suggests a potentially attractive strategy to derive haploid cells from any somatic cell type and unveil undescribed aspects of the coordination between DNA replication and cell division in mammals. ### Competing Interest Statement The authors have declared no competing interest.

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors’ combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression.

We generated knockout mice for MCM8 and MCM9 and show that deficiency for these genes impairs homologous recombination (HR)-mediated DNA repair during gametogenesis and somatic cells cycles. MCM8(-/-) mice are sterile because spermatocytes are blocked in meiotic prophase I, and females have only arrested primary follicles and frequently develop ovarian tumors. MCM9(-/-) females also are sterile as ovaries are completely devoid of oocytes. In contrast, MCM9(-/-) testes produce spermatozoa, albeit in much reduced quantity. Mcm8(-/-) and Mcm9(-/-) embryonic fibroblasts show growth defects and chromosomal damage and cannot overcome a transient inhibition of replication fork progression. In these cells, chromatin recruitment of HR factors like Rad51 and RPA is impaired and HR strongly reduced. We further demonstrate that MCM8 and MCM9 form a complex and that they coregulate their stability. Our work uncovers essential functions of MCM8 and MCM9 in HR-mediated DSB repair during gametogenesis, replication fork maintenance, and DNA repair.

Nuclear pore complexes (NPCs) embedded in the double nuclear membrane mediate the entire nucleocytoplasmic transport between the nucleus and cytoplasm. Each NPC is composed of about 30 different proteins (nucleoporins or Nups), which exist in multiple (8, 16 or 32) copies within the NPC scaffold. Recently, we have identified and characterized the large structural Nups, Nup188 and Nup192, from the thermophilic eukaryote Chaetomium thermophilum, which exhibited superior properties for biochemical and structural studies, when compared to their mesophilic orthologs. Here, we study the large structural Nups from the model organism yeast Saccharomyces cerevisiae. Our data show that yeast Nup188 like its thermophilic orthologue ctNup188 exhibits a twisted S-like structure, which flexibly binds the linker nucleoporin Nic96 via a short conserved α-helix motif. Using bioinformatic methods, we have generated a pseudo-atomic structural model of Nup188 and its related Nup192, which further strengthens the view that the large α-solenoid structural Nups are related to karyopherins.

Perhaps the two most fundamental rules in DNA replication say firstly to replicate the entire genome without leaving even small parts unreplicated and secondly, not to replicate any part, even a small one, more than just once during each cell cycle. In light of the size of an average eukaryotic genome and the time of S-phase, this is an enormous challenge. If in a gedankenexperiment a mammalian cell were scaled up to the size of a football and its DNA proportionally expanded in length, the DNA would span more than the distance from the earth to the sun more than 1700 millions of kilometers in a football that has to be replicated in approximately eight hours. In contrast to a (small) bacterial genome on a single chromosome, the replication of an eukaryotic genome (portioned in several chromosomes) has to start from multiple starting points, called origins of replication, in order to be accomplished in a reasonable time. Around 30.000 such origins are activated in an average mamalian cell and give rise to replication forks in each S-phase. In order to hold the fundamental rules of once per cell cycle replication, this complexity demands an extremely sophisticated regulation of replication initiation events. The formation of the pre-replication complex (pre-RC) on potential orgins of replication at a time different from active replication is a key step in this regulation. Through the formation of the pre-RC, the replicative helicase, the MCM2-7 complex, is loaded onto chromatin. The activity of this helicase separates the DNA double strand at the beginning and during S-phase and permits access of the polymerase machinery to the template strands. In addition, only the presence of the pre-RC proteins on chromatin permits the recruitment of other proteins that are necessary to load the polymerases. Therefore, in order to avoid that replication can start more than once per cell cycle at the same site, which would lead to rereplication, the formation of the pre-RC is restricted to a period with low S-phase kinase activity, that is, at the exit from mitosis and in G1. This system normally assures the once-per cell cycle replication: The license to start replication can only be given before replication itself has started. The cell only has to safeguard in addition that a pre-RC that once gave rise to a replication fork is by this action deactivated for the remaining cell cycle. However, this very efficient system to avoid rereplication comprises on the other hand the danger that not sufficient replication forks will be created to replicate the entire genome if the cell encounters stress situations that block replication forks or lead to DNA breaks.

In all eukaryotes, the heterohexameric MCM2-7 complex functions as the main replicative helicase during S phase. During early G(1) phase, it is recruited onto chromatin in a sequence of reactions called pre-replication complex (pre-RC) formation or DNA licensing. This process is ATP-dependent and at least two different chromatin-bound ATPase activities are required besides several others essential, but not enzymatically active, proteins. Although functionally conserved during evolution, pre-RC formation and the way the MCM2-7 helicase is loaded onto DNA are more complex in metazoans than in single-cell eukaryotes. Recently, we characterized a new essential factor for pre-RC assembly and DNA licensing, the vertebrate-specific MCM9 protein that contains not only an ATPase but also a helicase domain. MCM9 adds another layer of complexity to how vertebrates achieve and regulate the loading of the MCM2-7 helicase and DNA replication.

When observing living cells, only mitosis is easily distinguishable from other phases of the cell cycle. In this issue, Sakaue-Sawano et al. (2008) present a method to visually distinguish cells at different phases of the cell cycle by the expression of colored fusion proteins that are under the control of the ubiquitin ligases SCF and APC.

Prereplication complexes (pre-RCs) define potential origins of DNA replication and allow the recruitment of the replicative DNA helicase MCM2-7. Here, we characterize MCM9, a member of the MCM2-8 family. We demonstrate that MCM9 binds to chromatin in an ORC-dependent manner and is required for the recruitment of the MCM2-7 helicase onto chromatin. Its depletion leads to a block in pre-RC assembly, as well as DNA replication inhibition. We show that MCM9 forms a stable complex with the licensing factor Cdt1, preventing an excess of geminin on chromatin during the licensing reaction. Our data suggest that MCM9 is an essential activating linker between Cdt1 and the MCM2-7 complex, required for loading the MCM2-7 helicase onto DNA replication origins. Thus, Cdt1, with its two opposing regulatory binding factors MCM9 and geminin, appears to be a major platform on the pre-RC to integrate cell-cycle signals.

The transport receptor Mex67-Mtr2 functions in mRNA export, and also by a loop-confined surface on the heterodimer binds to and exports pre-60S particles. We show that Mex67-Mtr2 through the same surface that recruits pre-60S particles interacts with the Nup84 complex, a structural module of the nuclear pore complex devoid of Phe-Gly domains. In vitro, pre-60S particles and the Nup84 complex compete for an overlapping binding site on the loop-extended Mex67-Mtr2 surface. Chemical crosslinking identified Nup85 as the subunit in the Nup84 complex that directly binds to the Mex67 loop. Genetic studies revealed that this interaction is crucial for mRNA export. Notably, pre-60S subunit export impaired by mutating Mtr2 or the 60S adaptor Nmd3 could be partially restored by second-site mutation in Nup85 that caused dissociation of Mex67-Mtr2 from the Nup84 complex. Thus, the Mex67-Mtr2 export receptor employs a versatile binding platform on its surface that could create a crosstalk between mRNA and ribosome export pathways.