DNA Damage Checkpoint Pathway Research Articles

REV3 promotes cellular tolerance to 5-fluorodeoxyuridine by activating translesion DNA synthesis and intra-S checkpoint.

The drug floxuridine (5-fluorodeoxyuridine, FUdR) is an active metabolite of 5-Fluorouracil (5-FU). It converts to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP), which on incorporation into the genome inhibits DNA replication. Additionally, it inhibits thymidylate synthase, causing dTMP shortage while increasing dUMP availability, which induces uracil incorporation into the genome. However, the mechanisms underlying cellular tolerance to FUdR are yet to be fully elucidated. In this study, we explored the mechanisms underlying cellular resistance to FUdR by screening for FUdR hypersensitive mutants from a collection of DT40 mutants deficient in each genomic maintenance system. We identified REV3, which is involved in translesion DNA synthesis (TLS), to be a critical factor in FUdR tolerance. Replication using a FUdR-damaged template was attenuated in REV3-/- cells, indicating that the TLS function of REV3 is required to maintain replication on the FUdR-damaged template. Notably, FUdR-exposed REV3-/- cells exhibited defective cell cycle arrest in the early S phase, suggesting that REV3 is involved in intra-S checkpoint activation. Furthermore, REV3-/- cells showed defects in Chk1 phosphorylation, which is required for checkpoint activation, but the survival of FUdR-exposed REV3-/- cells was further reduced by the inhibition of Chk1 or ATR. These data indicate that REV3 mediates DNA checkpoint activation at least through Chk1 phosphorylation, but this signal acts in parallel with ATR-Chk1 DNA damage checkpoint pathway. Collectively, we reveal a previously unappreciated role of REV3 in FUdR tolerance.

γδ T cell-mediated cytotoxicity against patient-derived healthy and cancer cervical organoids.

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to αβ T cells and natural killer (NK) cells, γδ T cells represent an immune cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of γδ T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded γδ T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to γδ T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential γδ T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for γδ T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of γδ T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer.

Activation of the DNA damage checkpoint perturbs asymmetric localization of Kar9 to spindle pole bodies in Saccharomyces cerevisiae

During cell cycle progression in Saccharomyces cerevisiae, spindle pole bodies (SPBs) are duplicated during the G1/S-phase transition. SPBs are crucial for the organization of both the spindle and astral microtubules, and their orientation defines the direction of nuclear division. In this process, an old SPB, which serves as the template SPB during the duplication process, is oriented toward the bud side. The patterning microtubule plus-end tracking protein, Kar9, plays an important role in the orientation of SPBs by asymmetrically localizing to the old SPB. Here, methylglyoxal (MG), a metabolite derived from glycolysis, was found to perturb asymmetric Kar9 localization and influence proper positioning of the old SPB. MG activated the DNA damage checkpoint pathway, and MG-induced perturbation of asymmetric Kar9 localization was abolished by the deletion of MEC1, a sensor for the DNA damage checkpoint pathway. Methyl methanesulfonate, a DNA-alkylating agent, also perturbed asymmetric Kar9 localization. Our results suggest that activation of the DNA damage checkpoint pathway perturbs the asymmetric Kar9 localization required for proper positioning of SPBs.

The lipolysis inhibitor acipimox reverses the cardiac phenotype induced by electronic cigarettes

Electronic cigarettes (e-cigarettes) are a prevalent alternative to conventional nicotine cigarettes among smokers and people who have never smoked. Increased concentrations of serum free fatty acids (FFAs) are crucial in generating lipotoxicity. We studied the effects of acipimox, an antilipolytic drug, on e-cigarette-induced cardiac dysfunction. C57BL/6J wild-type mice on high fat diet were treated with saline, e-cigarette with 2.4% nicotine [e-cigarette (2.4%)], and e-cigarette (2.4%) plus acipimox for 12 weeks. Fractional shortening and ejection fraction were diminished in mice exposed to e-cigarettes (2.4%) compared with saline and acipimox-treated mice. Mice exposed to e-cigarette (2.4%) had increased circulating levels of inflammatory cytokines and FFAs, which were diminished by acipimox. Gene Set Enrichment Analysis revealed that e-cigarette (2.4%)-treated mice had gene expression changes in the G2/M DNA damage checkpoint pathway that was normalized by acipimox. Accordingly, we showed that acipimox suppressed the nuclear localization of phospho-p53 induced by e-cigarette (2.4%). Additionally, e-cigarette (2.4%) increased the apurinic/apyrimidinic sites, a marker of oxidative DNA damage which was normalized by acipimox. Mice exposed to e-cigarette (2.4%) had increased cardiac Heme oxygenase 1 protein levels and 4-hydroxynonenal (4-HNE). These markers of oxidative stress were decreased by acipimox. Therefore, inhibiting lipolysis with acipimox normalizes the physiological changes induced by e-cigarettes and the associated increase in inflammatory cytokines, oxidative stress, and DNA damage.

RAD51 Inhibition Shows Antitumor Activity in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), the major type of liver cancer, causes a high annual mortality worldwide. RAD51 is the critical recombinase responsible for homologous recombination (HR) repair in DNA damage. In this study, we identified that RAD51 was upregulated in HCC and that RAD51 silencing or inhibition reduced the proliferation, migration, and invasion of HCC cells and enhanced cell apoptosis and DNA damage. HCC cells with the combinatorial treatments of RAD51 siRNA or inhibitor and sorafenib demonstrated a synergistic effect in inhibiting HCC cell proliferation, migration, and invasion, as well as inducing cell apoptosis and DNA damage. Single RAD51 silencing or sorafenib reduced RAD51 protein expression and weakened HR efficiency, and their combination almost eliminated RAD51 protein expression and inhibited HR efficiency further. An in vivo tumor model confirmed the RAD51 inhibitor's antitumor activity and synergistic antitumor activity with sorafenib in HCC. RNA-Seq and gene set enrichment analysis (GSEA) in RAD51-inactivated Huh7 cells indicated that RAD51 knockdown upregulated cell apoptosis and G1/S DNA damage checkpoint pathways while downregulating mitotic spindle and homologous recombination pathways. Our findings suggest that RAD51 inhibition exhibits antitumor activities in HCC and synergizes with sorafenib. Targeting RAD51 may provide a novel therapeutic approach in HCC.

Abstract 5496: Glutaminase inhibition induces replication stress in ovarian cancer cells and inhibition of replication checkpoint causes synthetic lethality

Abstract Ovarian cancer (OC) is a highly aggressive disease and the most lethal gynecologic malignancy in women. Although the majority of OC patients respond to chemotherapeutic drugs, more than 70% of the patients relapse and die as a result of chemoresistance. Therefore, novel therapeutics are warranted to prevent chemoresistance and treat the relapsed disease to improve prognosis. Many cancer cells depend on Glutamine as major carbon source and these cells have high Glutaminase (GLS) expression, an enzyme that converts Glutamine to Glutamate. Chemo-resistant OC cells have elevated levels of GLS, which confirms their increased dependency on glutamine metabolism. Based on these observations, we postulated that GLS inhibition may attenuate the aggressive growth of GLS high OC cells and may sensitize to the agents that can further potentiate these effects. Interestingly, GLS inhibition using a clinical-stage drug CB839 caused replication stress and activated DNA damage checkpoint protein 1 (CHK1) mediated cell cycle arrest. These novel findings suggested a role for CHK1 in protecting GLS inhibition-induced DNA damage by facilitating the timely repair of DNA breaks. Based on these observations, we hypothesized that GLS inhibition in combination with CHK1 inhibition may cause synergistic lethality in chemo-resistant and GLShigh OC cells. We evaluated the combination of GLS inhibitor CB839 and CHK1 inhibitor Prexasertib. Our results demonstrate that GLS inhibition-induced CHK1 phosphorylation is significantly attenuated by Prexasertib treatment. Similarly, combined treatment of CB839 and Prexasertib showed significantly elevated levels of DNA damage as measured by COMET assays, replication stress-mediated DNA damage responses, and synergistic OC cell lethality compared to individual drug treatments. Furthermore, CB839 and Prexasertib combination was more synergistic in cells that expressed high GLS compared to low GLS-expressing OC cells indicating the specificity of the combination of these drugs. Together, our studies identified a novel connection between metabolic and DNA damage checkpoint pathways in OC and propose a novel synergistic lethality-based combination therapy to treat chemo-resistant and aggressive OC. Citation Format: Pamela Luna, Ganesh Acharya, Damieanus Ochola, Swetha Peddibhotla, Chinnadurai Mani, Mark B. Reedy, Komaraiah Palle. Glutaminase inhibition induces replication stress in ovarian cancer cells and inhibition of replication checkpoint causes synthetic lethality. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5496.

Abstract 6276: SKL27969, an oral selective PRMT5 inhibitor, sensitizes the effect of DNA-damaging agents in preclinical models of multiple solid tumors

Abstract Background: Protein arginine methyltransferase 5 (PRMT5) is overexpressed in various human malignancies and induces epigenetic and post-translational changes, which have a significant impact on cell growth, proliferation, apoptosis, and DNA damage repair. SKL27969 is a potent and selective brain-penetrating PRMT5 inhibitor demonstrating antitumor activity in the preclinical models of glioblastoma (GBM) and non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC) brain metastasis models. In this study, we evaluated the antitumor activity of SKL27969 alone or in combination with DNA-damaging agents in various cancer models. Methods: Cell cycle analysis, apoptosis, proliferation, and invasion assays were conducted to determine in vitro profiles of SKL27969. In vivo efficacy of SKL27969 was evaluated in human cancer cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) models. Immunohistochemistry and RNA-sequencing analysis in tumor samples from animal models were conducted to confirm the in vivo mechanism of action (MoA). In vitro synergistic effects of SKL27969 with combination partner drugs were analyzed using Combenefit software. Results: SKL27969 exhibits strong anti-proliferative effect by inducing cell cycle arrest and apoptosis and also inhibits invasion of cancer cells. An evaluation of SKL27969 in a panel of over 100 cancer cell lines revealed a broad spectrum of anti-proliferative activity in various cancer types, and an especially more sensitive response in cell lines harboring genetic alterations in the mitotic DNA damage checkpoint, DNA repair, or RNA processing pathways. Administration of SKL27969 demonstrated significant tumor growth inhibition, with a decrease in tumor symmetric dimethylarginine (SDMA) in NSCLC and TNBC subcutaneous xenograft models. Transcriptome and immunohistochemical analysis of SKL27969-treated tumors revealed that the cell cycle checkpoint pathways and DNA damage repair genes were downregulated, supporting the rationale for combination treatment with DNA-damaging agents such as chemotherapeutic agents. Robust antitumor effects were observed after treatment with SKL27969 in combination with paclitaxel or gemcitabine in NSCLC PDX or pancreatic cancer CDX models. Further ex vivo studies to confirm the in vivo MoA of this synergism, and in vivo combination studies of SKL27969 with standard chemotherapeutic agents in other solid cancer CDX models, are ongoing. Conclusion: This is the first preclinical study that suggests the therapeutic potential of SKL27969 in combination with currently approved DNA-damaging agents, and supports further clinical investigation of SKL27969’s role in improving the therapeutic benefit of standard therapies. Citation Format: Mijin Moon, Yongje Shin, Soyoung Ki, Jungtae Na, Ho Yeon Lee, Ilkyoo Koh, Beomjin Hong, Jiyeon Yun, Janice Laramy, Vijaykumar Vashi, Sook-Kyung Park. SKL27969, an oral selective PRMT5 inhibitor, sensitizes the effect of DNA-damaging agents in preclinical models of multiple solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6276.

LBSUN85 Acipimox Normalizes The Cardiac Phenotype Induced By Electronic Cigarettes

Abstract Electronic nicotine delivery systems or electronic cigarettes (e-cigarettes) are becoming exceptionally popular in the world as an alternative to conventional nicotine cigarettes, both among smokers and people who have never smoked. Nicotine can induce lipolysis in adipose tissue, leading to increased serum free fatty acids (FFAs). Increased levels of FFAs are one of the key elements in generating a pro-inflammatory response and lead to ectopic lipid accumulation, lipotoxicity, mitochondrial dysfunction, and DNA damage. Our laboratory has shown that e-cigarettes induce cardiac dysfunction associated with oxidative stress and inflammatory phenotype. We investigated the effects of acipimox, an antihyperlipidemic drug that blocks lipolysis, on e-cigarettes-induced cardiac dysfunction and its associated inflammatory signals and oxidative stress. C57BL/6J wild type mice on high fat diet were exposed to saline, e-cigarette with nicotine (2.4%), e-cigarette (2.4%) plus acipimox for 12 weeks. Fractional shortening and ejection fraction was decreased in mice exposed to e-cigarette (2.4%) compared with saline and acipimox. Therefore, acipimox normalized the e-cigarette-induced cardiac dysfunction. Transcriptomic evaluation with Gene Set Enrichment Analysis revealed that e-cigarette treated mice had genes enriched in the G2/M DNA damage checkpoint pathways. These transcriptomic changes were normalized by acipimox. Mice exposed to e-cigarettes had increased circulating levels of M-CSF, IL-6, and FFAs, which were decreased by acipimox. Mice exposed to e-cigarette (2.4%) had increased cardiac Heme oxygenase 1, a marker of oxidative stress. Additionally, treatment with e-cigarette (2.4%) increased the apurinic/apyrimidinic sites. This marker of oxidative DNA damage was normalized by acipimox. Understanding the consequences of e-cigarette use on the cardiovascular system is directly relevant to developing policies related to e-cigarette use. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Label-Free Direct Mass Spectrometry Analysis of the Bystander Effects Induced in Chondrocytes by Chondrosarcoma Cells Irradiated with X-rays and Carbon Ions.

Radiation-induced bystander effects are induced changes in cells that were not themselves directly irradiated but were in the vicinity of a radiation path. Such effects, which occur in the microenvironment of an irradiated tumor, remain poorly understood and depend on the cell type and irradiation quality. This study aimed to evaluate bystander effects in non-irradiated chondrocytes that received conditioned medium from irradiated chondrosarcoma cells. SW1353 chondrosarcoma cells were irradiated with X-rays and carbon ions, each at 0.1 Gy and 2 Gy, and the conditioned media of the irradiated cells were transferred to T/C-28A2 chondrocytes and Human Umbilical Venous Endothelial Cells (HUVECs). The whole proteome of bystander chondrocytes was analyzed by label-free mass spectrometry, and a comparative study was performed by dose and irradiation quality. HUVECs were evaluated for inflammatory cytokine secretion. The bystander response of chondrocytes to X-ray irradiation primarily affected the protein translation pathway (DHX36, EIF3B, EIF3D, EIF3M, EIF5, RPL6, RPLP0, RPS24, SYNCRIP), IL-12 (AIP, BOLA2, MIF, GAS6, MIF, PDGFRB) and the oxidative stress pathway (MGST3, PRDX2, PXDN, SOD2, TXN, TXNL1). Following carbon-ion irradiation, the G1/S pathway (PCBP4, PSMD12, PSME, XIAP) and mitotic G2 DNA damage checkpoint pathway (MRE11, TAOK1, UIMC1) were engaged. Changes in the regulation of chromosome separation (BCL7C, BUB3, CENPF, DYNC1LI1, SMARCA4, SMC4) were associated with only low-dose X-ray and carbon-ion irradiation. Modification of the protein translation pathway represented at least 30% of bystander effects and could play a role, possibly along with stress granules, in reduction in cellular metabolism to protect proteins. Stress granules were significantly enriched according to an interaction map. All these accessions corresponded to a window of the proteins modulated in response to the bystander effect. Our chondrosarcoma model clarified the nature of the bystander response of chondrocytes and may suggest several interesting new mechanisms that are specific to particular irradiation doses and qualities.

Caenorhabditis elegans NHR-14/HNF4α regulates DNA damage-induced apoptosis through cooperating with cep-1/p53

BackgroundNuclear hormone receptors are involved in transcriptional regulation and many important cellular processes including development and metabolism. However, its role in DNA damage-induced apoptosis remains elusive.MethodsSynchronized young adult animals were irradiated with different doses of gamma-Ray, and then put back to culture at 20 °C. Germline cell apoptosis was scored at different time point.ResultsDeletion of nhr-14 led to decreased DNA damage-induced germline apoptosis, but not the physiological programmed cell death. We also demonstrate that nhr-14 functions downstream of the DNA damage checkpoint pathway. Moreover, we show that nhr-14 regulates egl-1 and ced-13 transcription upon DNA damage. Mechanistically, NHR-14 forms a complex with CEP-1/p53 and binds directly to the egl-1 promoter to promote egl-1 transcription..ConclusionsOur results indicate that NHR-14/HNF4α cooperates with CEP-1/p53 to regulate DNA damage-induced apoptosis.Graphic abstract3SXEF2qkT2QHPu1N_hpJxUVideo abstract

Identifying Deubiquitinase Regulating the DNA Damage Response Pathway

In response to DNA damage, cells often activate Mec1‐dependent DNA damage checkpoint, resulting in the activation of the effector kinase Rad53 that promotes cell cycle arrest and DNA repair. The checkpoint proteins and the DNA damage response pathway are often regulated by ubiquitination, a reversible post‐translational modification, raising the possibility that checkpoint activation may be regulated by a deubiquitinating enzyme. To test this, we screened a collection of yeast deubiquitinating enzyme deletion mutants and monitored their responses to DNA damage agents. Interestingly, we find that one deubiquitinating enzyme deletion mutant displayed consistently decreased levels of checkpoint activation. Further analysis indicated that this mutant does not affect either the abundance or the stability of checkpoint activators, but it rather has a profound effect on Mec1 itself. This analysis suggests deubiquitinating enzyme as a novel regulator for Mecl1 and DNA damage checkpoint pathway.

Fission yeast Ase1PRC1 is required for the G2-microtubule damage response.

Schizosaccharomyces pombe delays entry into mitosis following G2 microtubule damage. This pathway is dependent on Rad26ATRIP, the regulatory subunit of the Rad26ATRIP/Rad3ATR DNA damage response (DDR) complex. However, this G2 microtubule damage response pathway acts independently of the G2 DNA damage checkpoint pathway. To identify other proteins in this G2 microtubule damage pathway, we previously screened a cDNA overexpression library for genes that rescued the sensitivity of rad26Δ cells to the microtubule poison thiabendazole. A partial cDNA fragment encoding only the C-terminal regulatory region of the microtubule bundling protein Ase1PRC1 was isolated. This fragment lacks the Ase1PRC1 dimerization and microtubule binding domains and retains the conserved C-terminal unstructured regulatory region. Here, we report that ase1Δ cells fail to delay entry into mitosis following G2 microtubule damage. Microscopy revealed that Rad26ATRIP foci localized alongside Ase1PRC1 filaments, although we suggest that this is related to microtubule-dependent double strand break mobility that facilitates homologous recombination events. Indeed, we report that the DNA repair protein Rad52 co-localizes with Rad26ATRIP at these foci, and that localization of Rad26ATRIP to these foci depends on a Rad26ATRIP N-terminal region containing a checkpoint recruitment domain. To our knowledge, this is the first report implicating Ase1PRC1 in regulation of the G2/M transition.

Efficacy of platinum-based chemotherapy in advanced triple-negative breast cancer in association with homologous recombination deficiency.

e13051 Background: Previous studies have identified that at least 50% of triple negative breast cancer (TNBC) harbor mutation characteristics of homologous recombination deficiency (HRD). Thus, more sophisticated research into comprehensive genomic profiling of HRD is urgently needed. Whereas BRCA1/2-deficient advanced TNBC patients are sensitive to treatment with platinum, it is not yet clear whether HRD status could predict platinum response. Methods: 3DMed-HRD algorithm was developed based on loss of heterozygosity score (LOH), telomeric allelic imbalance score (TAI) and large-scale state transition score (LST) as previously described. HRD status was defined as HRD positive (deleterious mutation in BRCA1/2 or HRD score ≥30) or HRD negative (no deleterious mutation in BRCA1/2 and HRD score < 30). Tumor samples from 207 TNBC patients were analyzed by next-generation sequencing. Deleterious or suspected deleterious mutations were included for analysis. Among the overall cohort, 34 patients with advanced TNBC treated with chemotherapy were analyzed. Cox regression model was applied to evaluate the relationship between HRD status and clinical outcomes. Results: Deleterious BRCA1/2 mutations were detected in 22.2% (46/207) of TNBC patients as well as 54.6% (113/207) were defined as HRD positive. The most frequent mutations in HRD-positive patients were TP53 (93.5%), MYC (29.0%), PIK3R1 (22.6%), PTEN (22.6%) and MCL1 (19.4%), while TP53 (77.8%), MYC (29.6%), PIK3CA (18.5%), KMT2C (14.8%) and RIT1 (14.8%) enriched in HRD-negative patients. Mutations in DNA Damage Response (DDR), P53, Checkpoint and Receptor Tyrosine Kinase (RTK) pathways were most involved in HRD positive patients. In advanced TNBC cohort, 19 patients received platinum-based and 15 received platinum-free chemotherapy in the first-line treatment. The progression-free survival (PFS) of the platinum-based group was longer than that of the platinum-free group (media PFS 9.1 vs 2.2 months, HR 0.44, 95%CI, 0.20-0.94, P = 0.009). In HRD-positive patients, median PFS was significantly longer in platinum-based group (N = 6) than platinum-free group (N = 8) (media PFS 13.6 vs 1.9 months, HR 0.30, 95%CI, 0.09-0.95, P = 0.008). No significant difference in PFS between platinum-based and platinum-free group (p = 0.332) in patients with HRD-negative tumors. Patients with mutations in homologous recombination (HR) pathway had a worse PFS compared to wild-type in platinum-free group (1.4 vs 3.0 months, p = 0.050). Conclusions: Our findings illustrate the potential of HRD status as a marker to guide chemotherapy in advanced TNBC. In HRD positive patients, platinum-based chemotherapy might be a preferable regimen. Patients with mutations in HR pathway had a shorter PFS in platinum-free group. Prospective study with a larger sample-size is needed for further validation of our findings.

MYBL2 and ATM suppress replication stress in pluripotent stem cells

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR‐dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand‐fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2‐MRN‐ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.

Correction: Mck1 kinase is a new player in the DNA damage checkpoint pathway

[This corrects the article DOI: 10.1371/journal.pgen.1008372.].

Mck1 kinase is a new player in the DNA damage checkpoint pathway.

Mck1 kinase is a new player in the DNA damage checkpoint pathway.

Kaiso is required for MTG16-dependent effects on colitis-associated carcinoma.

The myeloid translocation gene family member MTG16 is a transcriptional corepressor that relies on the DNA-binding ability of other proteins to determine specificity. One such protein is the ZBTB family member Kaiso, and the MTG16:Kaiso interaction is necessary for repression of Kaiso target genes such as matrix metalloproteinase-7. Using the azoxymethane and dextran sodium sulfate (AOM/DSS) murine model of colitis-associated carcinoma, we previously determined that MTG16 loss accelerates tumorigenesis and inflammation. However, it was unknown whether this effect was modified by Kaiso-dependent transcriptional repression. To test for a genetic interaction between MTG16 and Kaiso in inflammatory carcinogenesis, we subjected single and double knockout (DKO) mice to the AOM/DSS protocol. Mtg16−/− mice demonstrated increased colitis and tumor burden; in contrast, disease severity in Kaiso−/− mice was equivalent to wild type controls. Surprisingly, Kaiso deficiency in the context of MTG16 loss reversed injury and pro-tumorigenic responses in the intestinal epithelium following AOM/DSS treatment, and tumor numbers were returned to near to wild type levels. Transcriptomic analysis of non-tumor colon tissue demonstrated that changes induced by MTG16 loss were widely mitigated by concurrent Kaiso loss, and DKO mice demonstrated downregulation of metabolism and cytokine-associated gene sets with concurrent activation of DNA damage checkpoint pathways as compared with Mtg16−/−. Further, Kaiso knockdown in intestinal enteroids reduced stem- and WNT-associated phenotypes, thus abrogating the induction of these pathways observed in Mtg16−/− samples. Together, these data suggest that Kaiso modifies MTG16-driven inflammation and tumorigenesis and suggests that Kaiso deregulation contributes to MTG16-dependent colitis and CAC phenotypes.

ATM activation is impaired in human cells defective in RecQL4 helicase activity

RecQL4 has been shown to be involved in DNA replication and repair, but its role in DNA damage checkpoint pathway has not been reported. Here, we show that RecQL4 plays an important role in the activation of ataxia telangiectasia mutated (ATM)-dependent checkpoint pathway in human cells. Cells depleted with RecQL4 or Rothmund-Thomson syndrome cells showed significant impairment in the activation of ATM and the downstream effector proteins such as checkpoint kinase 2 and p53 after DNA damage. This defect was recovered with the expression of wild type RecQL4 but not any mutant RecQL4 proteins with defective helicase activities. While RecQL4 failed to show any direct interaction with ATM, it stably interacted with the Mre11-Rad50-Nbs1 complex that is essential for the activation of ATM and was localized on the DNA damage foci. Thus, our results suggest that the helicase activity of RecQL4 plays an important role in the activation of ATM-dependent checkpoint pathway against DNA double strand breaks in human cells.

Overexpression of Nitrogen Permease Regulator Like-2 (NPRL2) Enhances Sensitivity to Irinotecan (CPT-11) in Colon Cancer Cells by Activating the DNA Damage Checkpoint Pathway.

BackgroundColorectal cancer (CRC) is the third most common cancer worldwide, making it is a serious threat to human health. It is imperative to develop new therapeutics to improve the CRC treatment efficiency. The aim of this study was to investigate the role of NPRL2 in improving sensitivity to CPT-11 in colon cancer cells.Material/MethodsNPRL2 overexpression was established by transfecting the recombinant lentivirus-encoding NPRL2 gene into HCT116 colon cancer cells. Cell proliferation was identified using Cell Counting Kit-8 (CCK8) assay. Cell cycle and apoptosis were examined by flow cytometry. An immunofluorescence staining assay was conducted to examine the expression of γ-H2AX. Wound-healing and Transwell assays were utilized to show cell migration and invasion capability. The expression of apoptosis-related proteins (cleaved caspase-3, caspase-9, cleaved PARP, BAX, and Bcl-2), invasion-related proteins (MMP2, MMP9, p-PI3K, and p-AKT), and DNA damage checkpoint pathway proteins (p-ATM, p-Chk2, Cdc25C, Cdc2, and Cyclin B1) were quantified by Western blotting.ResultsA CCK8 assay revealed that the overexpression of NPRL2 improved the sensitivity of CPT-11 in HCT116 cells (P<0.05). Functionally, NPRL2 overexpression elevated the sensitivity of CPT-11 by preventing colon cancer cell proliferation, cell movement, and invasion, and promoting cell apoptosis and G2/M cell cycle arrest. Mechanistically, NPRL2 overexpression enhanced CPT-11 sensitivity by activating the DNA damage checkpoint pathway.ConclusionsNPRL2 overexpression enhances sensitivity to CPT-11 treatment in colon cancer cells, and it may serve as a molecular therapeutic agent to treat patients with CRC.

DNA damage checkpoint pathway modulates the regulation of skeletal growth and osteoblastic bone formation by parathyroid hormone-related peptide.

We previously demonstrated that parathyroid hormone-related peptide (PTHrP) 1-84 knockin (Pthrp KI) mice, which lacked a PTHrP nuclear localization sequence (NLS) and C-terminus, displayed early senescence, defective osteoblastic bone formation, and skeletal growth retardation. However, the mechanism of action of the PTHrP NLS and C-terminus in regulating development of skeleton is still unclear. In this study, we examined alterations of oxidative stress and DNA damage response-related molecules in Pthrp KI skeletal tissue. We found that ROS levels, protein expression levels of γ-H2AX, a DNA damage marker, and the DNA damage response markers p-Chk2 and p53 were up-regulated, whereas gene expression levels of anti-oxidative enzymes were down-regulated significantly. We therefore further disrupted the DNA damage response pathway by deleting the Chk2 in Pthrp KI (Chk2-/-KI) mice and did comparison with WT, Chk2-/- and Pthrp KI littermates. The Pthrp KI mice with Chk2 deletion exhibited a longer lifespan, improvement in osteoblastic bone formation and skeletal growth including width of growth plates and length of long bones, trabecular and epiphyseal bone volume, BMD, osteoblast numbers, type I collagen and ALP positive bone areas, the numbers of total colony-forming unit fibroblasts (CFU-f), ALP+ CFU-f and the expression levels of osteogenic genes. In addition, the genes associated with anti-oxidative enzymes were up-regulated significantly, whereas the tumor suppressor genes related to senescence were down-regulated in Chk2-/- KI mice compared to Pthrp KI mice. Our results suggest that Chk2 deletion in Pthrp KI mice can somewhat rescue defects in osteoblastic bone formation and skeletal growth by enhancing endochondral bone formation and osteogenesis. These studies therefore indicate that the DNA damage checkpoint pathway may be a target for the nuclear action of PTHrP to regulate skeletal development and growth.