Mre11 Protein Research Articles

EZH1/2 plays critical roles in oocyte meiosis prophase I in mice

Backgroudabnormalities or defects in oocyte meiosis can result in decreased oocyte quality, reduced ovarian reserve, and female diseases. However, the mechanisms of oocyte meiosis remain largely unknown, especially epigenetic regulation. Here, we explored the role of EZH1/2 (histone methyltransferase of H3K27) in mouse oocyte meiosis by inhibiting its activity and deleting its gene.Resultswith embryonic ovary cultured in vitro, EZH1/2 was demonstrated to be essential for oocyte development during meiosis prophase I in mice. Activity inhibition or gene knockout of EZH1/2 resulted in cell apoptosis and a reduction in oocyte numbers within embryonic ovaries. By observing the expression of some meiotic marker protein (γ-H2AX, diplotene stage marker MSY2 and synapsis complex protein SCP1), we found that function deficiency of EZH1/2 resulted in failure of DNA double-strand breaks (DSBs) repair and break of meiotic progression in fetal mouse ovaries. Moreover, Ezh1/2 deficiency led to the suppression of ATM (Ataxia Telangiectasia Mutated kinase) phosphorylation and a decrease in the expression of key DNA repair proteins Hormad1, Mre11, Rad50, and Nbs1 in fetal mouse ovaries, underscoring the enzyme’s pivotal role in initiating DNA repair. RNA-seq analysis revealed that Ezh1/2-deletion induced abnormal expression of multiple genes involved into several function of oocyte development in embryonic ovaries. Knockout of Ezh1/2 in ovaries also affected the levels of H3K9me3 and H4K20me2, as well as the expression of their target genes L3mbtl4 and Fbxo44.Conclusionsour study demonstrated that EZH1/2 plays a role in the DSBs repair in oocyte meiosis prophase I via multiple mechanisms and offers new insights into the physiological regulatory role of histone modification in fetal oocyte guardianship and female fertility.

MRE11 as a plausible biomarker and prognostic bioindicator for head and neck squamous cell carcinoma

ObjectiveHead and Neck Squamous Cell Carcinoma (HNSCC) ranks as the sixth most prevalent form of cancer worldwide. MRE11 protein contains multiple domains that play a role in the initiation of DNA repair. This study aimed to elucidate the expression and prognostic significance of MRE11 in HNSCC. Material and methodsThe Cancer Genome Atlas (TCGA-HNSCC) dataset comprising 520 HNSCC tissues and 44 normal tissues was initially used to evaluate the association between MRE11 expression and clinicopathologic characteristics. Kaplan–Meier plot was utilized for survival analysis. MRE11-immune cell interaction was analyzed using Tumor Immune Estimation Resource (TIMER) database. Further, Insilco methods were used to explore the protein network and its association with other pathways. Quantitative reverse transcription PCR (RT-qPCR) was used to validate the MRE11 mRNA expression in oral squamous cell carcinoma (OSCC) tissues in patient samples. ResultsMRE11 expression was upregulated in HNSCC, and the expression significantly varied across different clinical stages, pathological grades, and initial treatment outcomes. Further, high MRE11 expression is associated with poorer survival outcomes. MRE11 overexpression is also linked to the activation of the HIPPO signaling pathway, the mTOR signaling pathway, and the MYC/MYCN signaling pathway. ConclusionMRE 11 can be considered a novel prognostic biomarker for HNSCC, which can be leveraged for promising treatment outcomes. This research highlights MRE11 as a novel molecular biomarker for HNSCC and offers a new direction for its treatment, explicitly targeting MRE11 and its network for therapeutic intervention.

A novel Mre11 protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 possesses 5′–3′ exonuclease and endonuclease activities

Mre11 is one of important proteins that are involved in DNA repair and recombination by processing DNA ends to produce 3′-single stranded DNA, thus providing a platform for other DNA repair and recombination proteins. In this work, we characterized the Mre11 protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-Mre11) biochemically and dissected the roles of its four conserved residues, which is the first report on Mre11 proteins from Thermococcus. Tba-Mre11 possesses exonuclease activity for degrading ssDNA and dsDNA in the 5′–3′ direction, which contrasts with other reported Mre11 homologs. Maximum degradation efficiency was observed with Mn2+ at 80 °C and at pH 7.5–9.5. In addition to possessing 5′–3′ exonuclease activity, Tba-Mre11 has endonuclease activity that nicks plasmid DNA and circular ssDNA. Mutational data show that residues D10, D51 and N86 in Tba-Mre11 are essential for DNA degradation since almost no activity was observed for the D10A, D51A and N86A mutants. By comparison, residue D44 in Tba-Mre11 is not responsible for DNA degradation since the D44A mutant possessed the similar WT protein activity. Notably, the D44A mutant almost completely abolished the ability to bind DNA, suggesting that residue D44 is essential for binding DNA.

Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

Nano-Architecture of Persistent Focal DNA Damage Regions in the Minipig Epidermis Weeks after Acute γ-Irradiation.

Exposure to high acute doses of ionizing radiation (IR) can induce cutaneous radiation syndrome. Weeks after such radiation insults, keratinocyte nuclei of the epidermis exhibit persisting genomic lesions that present as focal accumulations of DNA double-strand break (DSB) damage marker proteins. Knowledge about the nanostructure of these genomic lesions is scarce. Here, we compared the chromatin nano-architecture with respect to DNA damage response (DDR) factors in persistent genomic DNA damage regions and healthy chromatin in epidermis sections of two minipigs 28 days after lumbar irradiation with ~50 Gy γ-rays, using single-molecule localization microscopy (SMLM) combined with geometric and topological mathematical analyses. SMLM analysis of fluorochrome-stained paraffin sections revealed, within keratinocyte nuclei with perisitent DNA damage, the nano-arrangements of pATM, 53BP1 and Mre11 DDR proteins in γ-H2AX-positive focal chromatin areas (termed macro-foci). It was found that persistent macro-foci contained on average ~70% of 53BP1, ~23% of MRE11 and ~25% of pATM single molecule signals of a nucleus. MRE11 and pATM fluorescent tags were organized in focal nanoclusters peaking at about 40 nm diameter, while 53BP1 tags formed nanoclusters that made up super-foci of about 300 nm in size. Relative to undamaged nuclear chromatin, the enrichment of DDR protein signal tags in γ-H2AX macro-foci was on average 8.7-fold (±3) for 53BP1, 3.4-fold (±1.3) for MRE11 and 3.6-fold (±1.8) for pATM. The persistent macro-foci of minipig epidermis displayed a ~2-fold enrichment of DDR proteins, relative to DSB foci of lymphoblastoid control cells 30 min after 0.5 Gy X-ray exposure. A lasting accumulation of damage signaling and sensing molecules such as pATM and 53BP1, as well as the DSB end-processing protein MRE11 in the persistent macro-foci suggests the presence of diverse DNA damages which pose an insurmountable problem for DSB repair.

MRE11:p.K464R mutation mediates olaparib resistance by enhancing DNA damage repair in HGSOC

BackgroundAlthough the clinical application of PARP inhibitors has brought hope to ovarian cancer, the problem of its resistance has become increasingly prominent. Therefore, clinical experts have been focused on finding specific indicators and therapeutic targets that can be used for resistance monitoring of PARP inhibitors.ResultsBy cfDNA detecting during Olaparib maintenance therapy in platinum-sensitive relapsed ovarian cancer, we found the presence of MRE11:p.K464R mutation was strongly associated with acquired Olaparib resistance. Structural analysis revealed that the MRE11:p.K464R mutation is situated at a critical site where the MRE11 protein interacts with other biomolecules, leading to potential structural and functional abnormalities of MRE11 protein. Functionally, MRE11:p.K464R mutation enhanced the tolerance of Olaparib by reducing the DNA damage. Mechanistically, MRE11:p.K464R mutation improved the efficiency of DNA damage repair and induce Olaparib resistance by enhancing its binding activity with the interacting proteins (including RAD50 and RPS3). Among them, the enhanced binding of MRE11:p.K464R mutation to RAD50/RPS3 facilitated non-homologous end joining (NHEJ) repair in tumor cells, thereby expanding the scope of research into acquired resistance to PARP inhibitors.ConclusionsOur findings provide a theoretical basis for MRE11:p.K464R mutation as a specific indicator of resistance monitoring in Olaparib treatment, and the exploration of its resistance mechanism provides a novel insights for the formulation of combination ther therapies after Olaparib resistance.

Regulation of gene editing using T-DNA concatenation.

Transformation via Agrobacterium tumefaciens is the predominant method used to introduce exogenous DNA into plant genomes1,2. Transfer DNA (T-DNA) originating from Agrobacterium can be integrated as a single copy or in complex concatenated forms3,4, but the mechanisms affecting final T-DNA structure remain unknown. Here we demonstrate that inclusion of retrotransposon (RT)-derived sequences in T-DNA can increase T-DNA copy number by more than 50-fold in Arabidopsis thaliana. These additional T-DNA copies are organized into large concatemers, an effect primarily induced by the long terminal repeats (LTRs) of RTs that can be replicated using non-LTR DNA repeats. We found that T-DNA concatenation is dependent on the activity of the DNA repair proteins MRE11, RAD17 and ATR. Finally, we show that T-DNA concatenation can be used to increase the frequency of targeted mutagenesis and gene targeting. Overall, this work uncovers molecular determinants that modulate T-DNA copy number in Arabidopsis and demonstrates the utility of inducing T-DNA concatenation for plant gene editing.

The DNA Damage Sensor MRE11 Regulates Efficient Replication of the Autonomous Parvovirus Minute Virus of Mice.

Parvoviruses are single-stranded DNA viruses that utilize host proteins to vigorously replicate in the nuclei of host cells, leading to cell cycle arrest. The autonomous parvovirus, minute virus of mice (MVM), forms viral replication centers in the nucleus which are adjacent to cellular DNA damage response (DDR) sites, many of which are fragile genomic regions prone to undergoing DDR during the S phase. Since the cellular DDR machinery has evolved to transcriptionally suppress the host epigenome to maintain genomic fidelity, the successful expression and replication of MVM genomes at these cellular sites suggest that MVM interacts with DDR machinery distinctly. Here, we show that efficient replication of MVM requires binding of the host DNA repair protein MRE11 in a manner that is independent of the MRE11-RAD50-NBS1 (MRN) complex. MRE11 binds to the replicating MVM genome at the P4 promoter, remaining distinct from RAD50 and NBS1, which associate with cellular DNA break sites to generate DDR signals in the host genome. Ectopic expression of wild-type MRE11 in CRISPR knockout cells rescues virus replication, revealing a dependence on MRE11 for efficient MVM replication. Our findings suggest a new model utilized by autonomous parvoviruses to usurp local DDR proteins that are crucial for viral pathogenesis and distinct from those of dependoparvoviruses, like adeno-associated virus (AAV), which require a coinfected helper virus to inactivate the local host DDR. IMPORTANCE The cellular DNA damage response (DDR) machinery protects the host genome from the deleterious consequences of DNA breaks and recognizes invading viral pathogens. DNA viruses that replicate in the nucleus have evolved distinct strategies to evade or usurp these DDR proteins. We have discovered that the autonomous parvovirus, MVM, which is used to target cancer cells as an oncolytic agent, depends on the initial DDR sensor protein MRE11 to express and replicate efficiently in host cells. Our studies reveal that the host DDR interacts with replicating MVM molecules in ways that are distinct from viral genomes being recognized as simple broken DNA molecules. These findings suggest that autonomous parvoviruses have evolved distinct mechanisms to usurp DDR proteins, which can be used to design potent DDR-dependent oncolytic agents.

Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

Discovery of off-target CRISPR–Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR–Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate applicability to ex vivo knock-in of a cancer-directed transgenic T cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.

Importance of Germline and Somatic Alterations in Human MRE11, RAD50, and NBN Genes Coding for MRN Complex

The MRE11, RAD50, and NBN genes encode for the nuclear MRN protein complex, which senses the DNA double strand breaks and initiates the DNA repair. The MRN complex also participates in the activation of ATM kinase, which coordinates DNA repair with the p53-dependent cell cycle checkpoint arrest. Carriers of homozygous germline pathogenic variants in the MRN complex genes or compound heterozygotes develop phenotypically distinct rare autosomal recessive syndromes characterized by chromosomal instability and neurological symptoms. Heterozygous germline alterations in the MRN complex genes have been associated with a poorly-specified predisposition to various cancer types. Somatic alterations in the MRN complex genes may represent valuable predictive and prognostic biomarkers in cancer patients. MRN complex genes have been targeted in several next-generation sequencing panels for cancer and neurological disorders, but interpretation of the identified alterations is challenging due to the complexity of MRN complex function in the DNA damage response. In this review, we outline the structural characteristics of the MRE11, RAD50 and NBN proteins, the assembly and functions of the MRN complex from the perspective of clinical interpretation of germline and somatic alterations in the MRE11, RAD50 and NBN genes.

Drug repurposing of propafenone to discover novel anti-tumor agents by impairing homologous recombination to delay DNA damage recovery of rare disease conjunctival melanoma

Conjunctival melanoma (CM), a rare and fatal malignant ocular tumor, lacks proper diagnostic biomarkers and therapy. Herein, we revealed the novel application of propafenone, an FDA-approved antiarrhythmic medication, which was identified effective in inhibiting CM cells viability and homologous recombination pathway. Detailed structure-activity relationships generated D34 as one of the most promising derivatives, which strongly suppressed the proliferation, viability, and migration of CM cells at submicromolar concentrations. Mechanically, D34 had the potential to increase γ-H2AX nuclear foci and aggravated DNA damage by suppressing homologous recombination pathway and its factors, particularly the complex of MRE11-RAD50-NBS1. D34 bound to human recombinant MRE11 protein and inhibited its endonuclease activity. Moreover, D34 dihydrochloride significantly suppressed tumor growth in the CRMM1 NCG xenograft model without obvious toxicity. Our finding shows that propafenone derivatives modulating the MRE11-RAD50-NBS1 complex will most likely provide an approach for CM targeted therapy, especially for improving chemo- and radio-sensitivity for CM patients.

Mild photothermal therapy boosts nanomedicine antitumor efficacy by disrupting DNA damage repair pathways and modulating tumor mechanics

A variety of Poly (ADP-ribose) polymerase inhibitors (PARPi) have been approved for the clinical treatment of breast cancers. However, pre-clinical and clinical evidences indicate that PARPi only benefits patients with homologous recombination (HR)-deficient breast cancers. Besides, the abnormal mechanical microenvironment of breast cancers severely restricts drug transport to tumor cells. Leveraging efforts from nanomedicine, mesoporous polydopamine (mP) with excellent biocompatibility and large specific surface area was employed to deliver both Olaparib (Ola), an FDA approved PARPi, and Doxorubicin (Dox), a typical DNA-damaging drug. In this nano drug delivery system (NDDS), mP operates not only as a drug carrier but also as a photothermal generator. Mechanistically, we revealed that locally mild photothermal therapy (M-PTT, around 43 ℃) on tumors was capable of inhibiting the HR repair pathway via dramatically downregulating the expressions of key HR-related proteins MRE11, RAD51 and BRCA2. Concomitantly, in vivo results indicated that M-PTT could effectively repress cancer associated fibroblasts (CAFs) by relieving hypoxia, resulting in diminution of dense extracellular matrix (ECM) of breast cancer (collagen Ⅰ and fibronectin decreased by 77.1% and 36.7%, respectively). Furthermore, the reduction of ECM normalized both tumor mechanics and tumor vasculature, facilitating drug delivery and penetration. Therefore, with the aid of M-PTT, this NDDS induces potent DNA damage, thereby enhancing antitumor efficacy (tumor inhibition rate of 86.1%) while minimizing systemic side effects. This work not only highlights the great potential of M-PTT-induced on-demand HR deficiency in clinical cancer therapy, but also reveals the potential mechanisms of M-PTT in inhibiting DNA damage repair and regulating tumor mechanics.

High MRE11 Expression Level Predicts Poor Survival in Upper Tract Urothelial Carcinomas.

Upper tract urothelial carcinoma (UTUC) is an aggressive malignancy with characteristics of high metastasis and poor prognosis. There are some particularly different features of UTUC between the Asian and Western countries. Double-strand break repair protein MRE11 is a component of the MRN complex that is involved in the DNA repair pathway. Emerging studies have focused on the role of MRE11 in human malignancies with conflicting results. We aimed to establish the relationship between MRE11 expression and the oncological outcome of UTUC. This study retrospectively reviewed 150 patients who underwent radical nephroureterectomy with pathologically confirmed UTUC. Pathologic slides were reviewed, and clinical parameters were collected. An immunohistochemical study was performed, and the cytoplasmic and nuclear-staining results of UTUC were recorded. The expression of MRE11 was analyzed to identify correlations with various clinicopathological parameters, metastasis-free survival, and cancer-specific survival (CSS). MRE11 expression was significantly correlated with patients with a high pathologic stage ( P =0.001), perineural invasion ( P =0.015), and tumor necrosis ( P =0.034). Upon univariate analysis, a high MRE11 expression was associated with poor metastasis-free survival ( P =0.014, 95% CI 1.18, 4.38) and poor CSS ( P =0.001, 95% CI 2.45, 27.75). Upon multivariable analysis, a high MRE11 expression was associated with poor CSS ( P =0.019, 95% CI 1.28, 15.65). In summary, MRE11 expression could serve as a potential predictor of prognosis in patients with UTUC.

Analysis of MRE11 and Mortality Among Adults With Muscle-Invasive Bladder Cancer Managed With Trimodality Therapy

Bladder-preserving trimodality therapy can be an effective alternative to radical cystectomy for treatment of muscle-invasive bladder cancer (MIBC), but biomarkers are needed to guide optimal patient selection. The DNA repair protein MRE11 is a candidate response biomarker that has not been validated in prospective cohorts using standardized measurement approaches. To evaluate MRE11 expression as a prognostic biomarker in MIBC patients receiving trimodality therapy using automated quantitative image analysis. This prognostic study analyzed patients with MIBC pooled from 6 prospective phase I/II, II, or III trials of trimodality therapy (Radiation Therapy Oncology Group [RTOG] 8802, 8903, 9506, 9706, 9906, and 0233) across 37 participating institutions in North America from 1988 to 2007. Eligible patients had nonmetastatic MIBC and were enrolled in 1 of the 6 trimodality therapy clinical trials. Analyses were completed August 2020. Trimodality therapy with transurethral bladder tumor resection and cisplatin-based chemoradiation therapy. MRE11 expression and association with disease-specific (bladder cancer) mortality (DSM), defined as death from bladder cancer. Pretreatment tumor tissues were processed for immunofluorescence with anti-MRE11 antibody and analyzed using automated quantitative image analysis to calculate a normalized score for MRE11 based on nuclear-to-cytoplasmic (NC) signal ratio. Of 465 patients from 6 trials, 168 patients had available tissue, of which 135 were analyzable for MRE11 expression (median age of 65 years [minimum-maximum, 34-90 years]; 111 [82.2%] men). Median (minimum-maximum) follow-up for alive patients was 5.0 (0.6-11.7) years. Median (Q1-Q3) MRE11 NC signal ratio was 2.41 (1.49-3.34). Patients with an MRE11 NC ratio above 1.49 (ie, above first quartile) had a significantly lower DSM (HR, 0.50; 95% CI, 0.26-0.93; P = .03). The 4-year DSM was 41.0% (95% CI, 23.2%-58.0%) for patients with an MRE11 NC signal ratio of 1.49 or lower vs 21.0% (95% CI, 13.4%-29.8%) for a ratio above 1.49. MRE11 NC signal ratio was not significantly associated with overall survival (HR, 0.84; 95% CI, 0.49-1.44). Higher MRE11 NC signal ratios were associated with better DSM after trimodality therapy. Lower MRE11 NC signal ratios identified a poor prognosis subgroup that may benefit from intensification of therapy.

A gene dosage-dependent effect unveils NBS1 as both a haploinsufficient tumour suppressor and an essential gene for SHH-medulloblastoma.

AimsInherited or somatic mutations in the MRE11, RAD50 and NBN genes increase the incidence of tumours, including medulloblastoma (MB). On the other hand, MRE11, RAD50 and NBS1 protein components of the MRN complex are often overexpressed and sometimes essential in cancer. In order to solve the apparent conundrum about the oncosuppressive or oncopromoting role of the MRN complex, we explored the functions of NBS1 in an MB‐prone animal model.Materials and methodsWe generated and analysed the monoallelic or biallelic deletion of the Nbn gene in the context of the SmoA1 transgenic mouse, a Sonic Hedgehog (SHH)‐dependent MB‐prone animal model. We used normal and tumour tissues from these animal models, primary granule cell progenitors (GCPs) from genetically modified animals and NBS1‐depleted primary MB cells, to uncover the effects of NBS1 depletion by RNA‐Seq, by biochemical characterisation of the SHH pathway and the DNA damage response (DDR) as well as on the growth and clonogenic properties of GCPs.ResultsWe found that monoallelic Nbn deletion increases SmoA1‐dependent MB incidence. In addition to a defective DDR, Nbn +/− GCPs show increased clonogenicity compared to Nbn +/+ GCPs, dependent on an enhanced Notch signalling. In contrast, full Nbn KO impairs MB development both in SmoA1 mice and in an SHH‐driven tumour allograft.ConclusionsOur study indicates that Nbn is haploinsufficient for SHH‐MB development whereas full Nbn KO is epistatic on SHH‐driven MB development, thus revealing a gene dosage‐dependent effect of Nbn inactivation on SHH‐MB development.

Targeting Mre11 overcomes platinum resistance and induces synthetic lethality in XRCC1 deficient epithelial ovarian cancers

Platinum resistance is a clinical challenge in ovarian cancer. Platinating agents induce DNA damage which activate Mre11 nuclease directed DNA damage signalling and response (DDR). Upregulation of DDR may promote chemotherapy resistance. Here we have comprehensively evaluated Mre11 in epithelial ovarian cancers. In clinical cohort that received platinum- based chemotherapy (n = 331), Mre11 protein overexpression was associated with aggressive phenotype and poor progression free survival (PFS) (p = 0.002). In the ovarian cancer genome atlas (TCGA) cohort (n = 498), Mre11 gene amplification was observed in a subset of serous tumours (5%) which correlated highly with Mre11 mRNA levels (p < 0.0001). Altered Mre11 levels was linked with genome wide alterations that can influence platinum sensitivity. At the transcriptomic level (n = 1259), Mre11 overexpression was associated with poor PFS (p = 0.003). ROC analysis showed an area under the curve (AUC) of 0.642 for response to platinum-based chemotherapy. Pre-clinically, Mre11 depletion by gene knock down or blockade by small molecule inhibitor (Mirin) reversed platinum resistance in ovarian cancer cells and in 3D spheroid models. Importantly, Mre11 inhibition was synthetically lethal in platinum sensitive XRCC1 deficient ovarian cancer cells and 3D-spheroids. Selective cytotoxicity was associated with DNA double strand break (DSB) accumulation, S-phase cell cycle arrest and increased apoptosis. We conclude that pharmaceutical development of Mre11 inhibitors is a viable clinical strategy for platinum sensitization and synthetic lethality in ovarian cancer.

Context-dependent pro- and anti-resection roles of ZKSCAN3 in the regulation of fork processing during replication stress

Uncontrolled resection of replication forks under stress can cause genomic instability and influence cancer formation. Extensive fork resection has also been implicated in the chemosensitivity of “BReast CAncer gene” BRCA-deficient cancers. However, how fork resection is controlled in different genetic contexts and how it affects chromosomal stability and cell survival remains incompletely understood. Here, we report a novel function of the transcription repressor ZKSCAN3 in fork protection and chromosomal stability maintenance under replication stress. We show disruption of ZKSCAN3 function causes excessive resection of replication forks by the exonuclease Exo1 and homologous DNA recombination/repair protein Mre11 following fork reversal. Interestingly, in BRCA1-deficient cells, we found ZKSCAN3 actually promotes fork resection upon replication stress. We demonstrate these anti- and pro-resection roles of ZKSCAN3, consisting of a SCAN box, Kruppel-associated box, and zinc finger domain, are mediated by its SCAN box domain and do not require the Kruppel-associated box or zinc finger domains, suggesting that the transcriptional function of ZKSCAN3 is not involved. Furthermore, despite the severe impact on fork structure and chromosomal stability, depletion of ZKSCAN3 did not affect the short-term survival of BRCA1-proficient or BRCA1-deficient cells after treatment with cancer drugs hydroxyurea, PARPi, or cisplatin. Our findings reveal a unique relationship between ZKSCAN3 and BRCA1 in fork protection and add to our understanding of the relationships between replication fork protection, chromosomal instability, and chemosensitivity.

Anti-inflammatory and anti-apoptotic effects of Shaoyao decoction on X-ray radiation-induced enteritis of C57BL/6 mice

Ethnopharmacological relevanceAs a typical heat-clearing prescription, Shaoyao decoction (SYD) has a robust function of clearing viscera heat for the treatment of several intestinal discomfort symptoms. Clinical evidence indicated that it had the potential to cure radiation enteritis. However, its underlying mechanisms remain unclear. Aim of the studyThe present study was designed to probe the protective effects and the involved mechanisms of SYD on X-ray radiation-induced enteritis of C57BL/6 mice. Materials and methodsX-ray irradiation were used to establish the radiation enteritis model. Forty-eight male C57BL/6 mice (20 ± 2 g) were randomly divided into six groups: the control group, model group, dexamethasone group (DEX, 0.12 mg/kg) and SYD groups (0.12, 0.24 and 0.36 g/mL), respectively. All mice (except the control group) were intragastrically administrated for a continuous 7 days. H&E and Masson staining were employed to evaluate the morphological and collagen fibers changes of the colon. ELISA was performed to assess the levels of MDA, SOD, COX, LPS, IL-6, IL-1β and TNF-α in serum. Moreover, TUNEL fluorescence, western blot and qRT-PCR were used to detect the levels of apoptosis-related proteins and genes of Dclk-1, ATM, MRE-11, Bcl-2, Bax, Caspase-3, and Cyto-c. Furthermore, immunofluorescent staining was applied to detect the protein levels of p53 and Claudin-1 in colon. ResultsTreatment with SYD decreased the exfoliated and necrotic epithelial cells and prevent the proliferate from damaged fibrous tissue in the crypt layer of mucos. The levels of serum peroxidation and pro-inflammatory cytokines (MDA, COX, LPS, IL-6, IL-1β and TNF-α) were obviously inhibited, while SOD sharply increased in serum after administration. Moreover, SYD can significantly ameliorate the apoptosis of colon cells, evidenced by the reduced positive expression of TUNEL staining. Meanwhile, the results of qRT-PCR and western blot demonstrated that SYD can dramatically stimulate the expression of genes and proteins Dclk-1, ATM and MRE-11, thus promoting the expression of mitochondrial pro-apoptotic proteins Bax, Caspase-3 and Cyto-c, while increasing the level of anti-apoptotic protein Bcl-2. Furthermore, immunofluorescence revealed that SYD can notably decreased the protein level of p53 while reverse the reduction of Claudin-1. ConclusionsThese results indicated that radiation enteritis in C57BL/6 mice can be ameliorated by treatment with SYD. The potential protection mechanisms may be involved in ameliorating tissue fibrosis by decreasing inflammatory and apoptotic events.

O-GlcNAc transferase couples MRE11 to transcriptionally active chromatin to suppress DNA damage

BackgroundTranscription, metabolism and DNA damage response are tightly regulated to preserve the genomic integrity, and O-GlcNAc transferase (OGT) is positioned to connect the three. Prostate cancer is the most common cancer in men, and androgen-ablation therapy halts disease progression. However, a significant number of prostate cancer patients develop resistance against anti-androgens, and this incurable disease is termed castration-resistant prostate cancer (CRPC). We have shown that combined inhibition of OGT and the transcription elongation kinase CDK9 induce CRPC-selective anti-proliferative effects. Here, we explain the functional basis for these combinatorial effects.MethodsWe used comprehensive mass spectrometry profiling of short-term CDK9 inhibitor effects on O-GlcNAcylated proteins in an isogenic cell line system that models transition from PC to CRPC. In addition, we used both ChIP-seq and RNA-seq profiling, and pulldown experiments in multiple CRPC models. Finally, we validated our findings in prostate cancer patient samples.ResultsInhibition of CDK9 results in an OGT-dependent remodeling of the proteome in prostate cancer cells. More specifically, the activity of the DNA damage repair protein MRE11 is regulated in response to CDK9 inhibition in an OGT-dependent manner. MRE11 is enriched at the O-GlcNAc-marked loci. CDK9 inhibition does not decrease the expression of mRNAs whose genes are bound by both O-GlcNAc and MRE11. Combined inhibition of CDK9 and OGT or MRE11 further decreases RNA polymerase II activity, induces DNA damage signaling, and blocks the survival of prostate cancer cells. These effects are seen in CRPC cells but not in normal prostate cells. Mechanistically, OGT activity is required for MRE11 chromatin-loading in cells treated with CDK9 inhibitor. Finally, we show that MRE11 and O-GlcNAc are enriched at the prostate cancer-specific small nucleotide polymorphic sites, and the loss of MRE11 activity results in a hyper-mutator phenotype in patient tumors.ConclusionsBoth OGT and MRE11 are essential for the repair of CDK9 inhibitor-induced DNA damage. Our study raises the possibility of targeting CDK9 to elicit DNA damage in CRPC setting as an adjuvant to other treatments.Graphical

RAG enhances BCR-ABL1-positive leukemic cell growth through its endonuclease activity in vitro and in vivo.

BCR‐ABL1 gene fusion associated with additional DNA lesions involves the pathogenesis of chronic myelogenous leukemia (CML) from a chronic phase (CP) to a blast crisis of B lymphoid (CML‐LBC) lineage and BCR‐ABL1 + acute lymphoblastic leukemia (BCR‐ABL1 + ALL). The recombination‐activating gene RAG1 and RAG2 (collectively, RAG) proteins that assemble a diverse set of antigen receptor genes during lymphocyte development are abnormally expressed in CML‐LBC and BCR‐ABL1 + ALL. However, the direct involvement of dysregulated RAG in disease progression remains unclear. Here, we generate human wild‐type (WT) RAG and catalytically inactive RAG‐expressing BCR‐ABL1 + and BCR‐ABL1 − cell lines, respectively, and demonstrate that BCR‐ABL1 specifically collaborates with RAG recombinase to promote cell survival in vitro and in xenograft mice models. WT RAG‐expressing BCR‐ABL1 + cell lines and primary CD34+ bone marrow cells from CML‐LBC samples maintain more double‐strand breaks (DSB) compared to catalytically inactive RAG‐expressing BCR‐ABL1 + cell lines and RAG‐deficient CML‐CP samples, which are measured by γ‐H2AX. WT RAG‐expressing BCR‐ABL1 + cells are biased to repair RAG‐mediated DSB by the alternative non–homologous end joining pathway (a‐NHEJ), which could contribute genomic instability through increasing the expression of a‐NHEJ‐related MRE11 and RAD50 proteins. As a result, RAG‐expressing BCR‐ABL1 + cells decrease sensitivity to tyrosine kinase inhibitors (TKI) by activating BCR‐ABL1 signaling but independent of the levels of BCR‐ABL1 expression and mutations in the BCR‐ABL1 tyrosine kinase domain. These findings identify a surprising and novel role of RAG in the functional specialization of disease progression in BCR‐ABL1 + leukemia through its endonuclease activity.