The deSUMOylase SENP2 coordinates homologous recombination and nonhomologous end joining by independent mechanisms.

SUMOylation (small ubiquitin-like modifier) in the DNA double-strand break (DSB) response regulates recruitment, activity, and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and nonhomologous end joining (NHEJ) through the investigation of the deSUMOylase SENP2. We found that regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast, we show that HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 focus retention and increases NHEJ and radioresistance. Collectively, our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

Identification of plants' functional counterpart of the metazoan mediator of DNA Damage checkpoint 1.

Induction of DNA damage triggers rapid phosphorylation of the histone H2A.X (γH2A.X). In animals, mediator of DNA damage checkpoint 1 (MDC1) binds γH2A.X through a tandem BRCA1 carboxyl-terminal (tBRCT) domain and mediates recruitment of downstream effectors of DNA damage response (DDR). However, readers of this modification in plants have remained elusive. We show that from the Arabidopsis BRCT domain proteome, BCP1-4 proteins with tBRCT domains are involved in DDR. Through its tBRCT domain BCP4 binds γH2A.X in vitro and localizes to DNA damage-induced foci in an H2A.X-dependent manner. BCP4 also contains a domain that interacts directly with NBS1 and thus acts as a functional counterpart of MDC1. We also show that BCP1, that contains two tBRCT domains, co-localizes with γH2A.X but it does not bind γH2A.X suggesting functional similarity with human PAXIP1. A phylogenetic analysis supports that PAXIP1 and MDC1 in metazoa and their plant counterparts evolved independently from common ancestors with tBRCT domains. Collectively, our study reveals missing components and provides mechanistic and evolutionary insights into plant DDR.

ASF1a Promotes Non-homologous End Joining Repair by Facilitating Phosphorylation of MDC1 by ATM at Double-Strand Breaks.

Double-strand breaks (DSBs) of DNA in eukaryotic cells are predominantly repaired by non-homologous end joining (NHEJ). The histone chaperone anti-silencing factor 1a (ASF1a) interacts with MDC1 and is recruited to sites of DSBs to facilitate the interaction of phospho-ATM with MDC1 and phosphorylation of MDC1, which are required for the recruitment of RNF8/RNF168 histone ubiquitin ligases. Thus, ASF1a deficiency reduces histone ubiquitination at DSBs, decreasing the recruitment of 53BP1, and decreases NHEJ, rendering cells more sensitive to DSBs. This role of ASF1a in DSB repair cannot be provided by the closely related ASF1b and does not require its histone chaperone activity. Homozygous deletion of ASF1A is seen in 10%-15% of certain cancers, suggesting that loss of NHEJ may be selected in some malignancies and that the deletion can be used as a molecular biomarker for cancers susceptible to radiotherapy or to DSB-inducing chemotherapy.

A 5-year natural history study in LAMA2-related muscular dystrophy and SELENON-related myopathy: the Extended LAST STRONG study.

BACKGROUND: SELENON-related myopathy (SELENON-RM) is a rare congenital myopathy characterized by slowly progressive axial muscle weakness, rigidity of the spine, scoliosis, and respiratory insufficiency. Laminin-a2-related muscular dystrophy (LAMA2-MD) has a similar clinical phenotype, which ranges from severe, early-onset congenital muscular dystrophy type 1A (MDC1A) to milder forms presenting as childhood- or adult-onset limb-girdle type muscular dystrophy. The first 1.5-year natural history follow-up showed that 90% of the patients had low bone quality, respiratory impairments were found in all SELENON-RM and most of the LAMA2-MD patients, and many had cardiac risk factors. However, further extensive knowledge on long-term natural history data, and clinical and functional outcome measures is needed to reach trial readiness. Therefore, we extended the natural history study with 3- and 5-year follow-up visits (Extended LAST STRONG). METHODS: The Extended LAST STRONG is a long-term natural history study in Dutch-speaking patients of all ages diagnosed with genetically confirmed SELENON-RM or LAMA2-MD, starting in September 2023. Patients visit our hospital twice over a period of 2 years to complete a 5-year follow up from the initial LAST-STRONG study. At both visits, they undergo standardized neurological examination, hand-held dynamometry (age ≥ 5 years), functional measurements, muscle ultrasound, respiratory assessments (spirometry, maximal inspiratory and expiratory pressure, sniff nasal inspiratory pressure; age ≥ 5 years), Dual-energy X-ray absorptiometry (DEXA-)scan (age ≥ 2 years), X-ray of the left hand (age ≤ 17 years), lower extremity MRI (age ≥ 10 years), accelerometry for 8 days (age ≥ 2 years), and questionnaires (patient report and/or parent proxy; age ≥ 2 years). All examinations are adapted to the patient's age and functional abilities. Disease progression between all subsequent visits and relationships between outcome measures will be assessed. DISCUSSION: This study will provide valuable insights into the 5-year natural history of patients with SELENON-RM and LAMA2-MD and contribute to further selecting relevant and sensitive to change clinical and functional outcome measures. Furthermore, this data will help optimize natural history data collection in clinical care and help develop clinical care guidelines. TRIAL REGISTRATION: This study protocol including the patient information and consent forms has been approved by medical ethical reviewing committee ('METC Oost-Nederland'; https://www.ccmo.nl/metcs/erkende-metcs/metc-oost-nederland , file number: 2023-16401). It is registered at ClinicalTrials.gov (NCT06132750; study registration date: 2023-10-05; study first passed date: 2023-11-15).

Functions of TopBP1 in preserving genome integrity during mitosis.

TopBP1/Rad4/Dpb11 is an essential eukaryotic protein with important roles in DNA replication, DNA repair, DNA damage checkpoint activation, and chromosome segregation. TopBP1 serves as a scaffold to assemble protein complexes in a phosphorylation-dependent manner via its multiple BRCT-repeats. Recently, it has become clear that TopBP1 is repurposed to scaffold different processes dependent on cell cycle regulated changes in phosphorylation of client proteins. Here we review the functions of human TopBP1 in maintaining genome integrity during mitosis.

MDC1 is essential for G2/M transition and spindle assembly in mouse oocytes.

Mammalian oocytes are particularly susceptible to accumulating DNA damage. However, unlike mitotic cells in which DNA damage induces G2 arrest by activating the ATM-Chk1/2-Cdc25 pathway, oocytes readily enter M-phase immediately following DNA damage. This implies a lack of a robust canonical G2/M DNA damage checkpoint in oocytes. Here we show that MDC1 plays a non-canonical role in controlling G2/M transition by regulating APC/C-Cdh1-mediated cyclin B1 degradation in response to DNA damage in mouse oocytes. Depletion of MDC1 impaired M-phase entry by decreasing cyclin B1 levels via the APC/C-Cdh1 pathway. Notably, the APC/C-Cdh1 regulation mediated by MDC1 was achieved by a direct interaction between MDC1 and APC/C-Cdh1. This interaction was transiently disrupted after DNA damage with a concomitant increase in Cdh1 levels, which, in turn, decreased cyclin B1 levels and delayed M-phase entry. Moreover, MDC1 depletion impaired spindle assembly by decreasing the integrity of microtubule organizing centers (MTOCs). Therefore, our results demonstrate that MDC1 is an essential molecule in regulating G2/M transition in response to DNA damage and in regulating spindle assembly in mouse oocytes. These results provide new insights into the regulation of the G2/M DNA damage checkpoint and cell cycle control in oocytes.

AP4 suppresses DNA damage, chromosomal instability and senescence via inducing MDC1/Mediator of DNA damage Checkpoint 1 and repressing MIR22HG/miR-22-3p.

BACKGROUND: AP4 (TFAP4) encodes a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor and is a direct target gene of the oncogenic transcription factor c-MYC. Here, we set out to determine the relevance of AP4 in human colorectal cancer (CRC) cells. METHODS: A CRISPR/Cas9 approach was employed to generate AP4-deficient CRC cell lines with inducible expression of c-MYC. Colony formation, ß-gal staining, immunofluorescence, comet and homologous recombination (HR) assays and RNA-Seq analysis were used to determine the effects of AP4 inactivation. qPCR and qChIP analyses was performed to validate differentially expressed AP4 targets. Expression data from CRC cohorts was subjected to bioinformatics analyses. Immunohistochemistry was used to evaluate AP4 targets in vivo. Ap4-deficient APCmin/+ mice were analyzed to determine conservation. Immunofluorescence, chromosome and micronuclei enumeration, MTT and colony formation assays were used to determine the effects of AP4 inactivation and target gene regulation on chromosomal instability (CIN) and drug sensitivity. RESULTS: Inactivation of AP4 in CRC cell lines resulted in increased spontaneous and c-MYC-induced DNA damage, chromosomal instability (CIN) and cellular senescence. AP4-deficient cells displayed increased expression of the long non-coding RNA MIR22HG, which encodes miR-22-3p and was directly repressed by AP4. Furthermore, Mediator of DNA damage Checkpoint 1 (MDC1), a central component of the DNA damage response and a known target of miR-22-3p, displayed decreased expression in AP4-deficient cells. Accordingly, MDC1 was directly induced by AP4 and indirectly by AP4-mediated repression of miR-22-3p. Adenomas and organoids from Ap4-deficient APCmin/+ mice displayed conservation of these regulations. Inhibition of miR-22-3p or ectopic MDC1 expression reversed the increased senescence, DNA damage, CIN and defective HR observed in AP4-deficient CRC cells. AP4-deficiency also sensitized CRC cells to 5-FU treatment, whereas ectopic AP4 conferred resistance to 5-FU in a miR-22-3p and MDC1-dependent manner. CONCLUSIONS: In summary, AP4, miR-22-3p and MDC1 form a conserved and coherent, regulatory feed-forward loop to promote DNA repair, which suppresses DNA damage, senescence and CIN, and contributes to 5-FU resistance. These findings explain how elevated AP4 expression contributes to development and chemo-resistance of colorectal cancer after c-MYC activation.

Oxidant stress-sensitive circRNA Mdc1 controls cardiomyocyte chromosome stability and cell cycle re-entry during heart regeneration.

Targeting cardiomyocyte plasticity has emerged as a new strategy for promoting heart repair after myocardial infarction. However, the precise mechanistic network underlying heart regeneration is not completely understood. As noncoding RNAs, circular RNAs (circRNAs) play essential roles in regulating cardiac physiology and pathology. The present study aimed to investigate the potential roles of circMdc1 in cardiac repair after injury and elucidate its underlying mechanisms. Here, we identified that circMdc1 levels were upregulated in postnatal mouse hearts but downregulated in the regenerative myocardium. The expression of circMdc1 in cardiomyocytes is sensitive to oxidative stress, which was attenuated by N-acetyl-cysteine. Enforced circMdc1 expression inhibited cardiomyocyte proliferation, while circMdc1 silencing led to cardiomyocyte cell cycle re-entry. In vivo, the cardiac-specific adeno-associated virus-mediated knockdown of circMdc1 promoted cardiac regeneration and heart repair accompanied by improved heart function. Conversely, circMdc1 overexpression blunted the regenerative capacity of neonatal hearts after apex resection. Moreover, circMdc1 was able to block the translation of its host gene Mdc1 specifically by binding to PABP, affecting DNA damage and the chromosome stability of cardiomyocytes. Furthermore, overexpression of Mdc1 caused damaged mouse hearts to regenerate and repair after myocardial infarction in vivo. Oxidative stress-sensitive circMdc1 plays an important role in cardiac regeneration and heart repair after injury by regulating DNA damage and chromosome stability in cardiomyocytes by blocking the translation of the host gene Mdc1.

The scaffold protein WRAP53ß orchestrates the ubiquitin response critical for DNA double-strand break repair.

The WD40 domain-containing protein WRAP53ß (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53ß as an essential regulator of DNA double-strand break (DSB) repair. WRAP53ß rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53ß targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53ß facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53ß controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53ß impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53ß as a novel regulator of DSB repair by providing a scaffold for DNA repair factors.

Natural history, outcome measures and trial readiness in LAMA2-related muscular dystrophy and SELENON-related myopathy in children and adults: protocol of the LAST STRONG study.

BACKGROUND: SELENON (SEPN1)-related myopathy (SELENON-RM) is a rare congenital myopathy characterized by slowly progressive proximal muscle weakness, early onset spine rigidity and respiratory insufficiency. A muscular dystrophy caused by mutations in the LAMA2 gene (LAMA2-related muscular dystrophy, LAMA2-MD) has a similar clinical phenotype, with either a severe, early-onset due to complete Laminin subunit α2 deficiency (merosin-deficient congenital muscular dystrophy type 1A (MDC1A)), or a mild, childhood- or adult-onset due to partial Laminin subunit α2 deficiency. For both muscle diseases, no curative treatment options exist, yet promising preclinical studies are ongoing. Currently, there is a paucity on natural history data and appropriate clinical and functional outcome measures are needed to reach trial readiness. METHODS: LAST STRONG is a natural history study in Dutch-speaking patients of all ages diagnosed with SELENON-RM or LAMA2-MD, starting August 2020. Patients have four visits at our hospital over a period of 1.5 year. At all visits, they undergo standardized neurological examination, hand-held dynamometry (age ≥ 5 years), functional measurements, questionnaires (patient report and/or parent proxy; age ≥ 2 years), muscle ultrasound including diaphragm, pulmonary function tests (spirometry, maximal inspiratory and expiratory pressure, sniff nasal inspiratory pressure; age ≥ 5 years), and accelerometry for 8 days (age ≥ 2 years); at visit one and three, they undergo cardiac evaluation (electrocardiogram, echocardiography; age ≥ 2 years), spine X-ray (age ≥ 2 years), dual-energy X-ray absorptiometry (DEXA-)scan (age ≥ 2 years) and full body magnetic resonance imaging (MRI) (age ≥ 10 years). All examinations are adapted to the patient's age and functional abilities. Correlation between key parameters within and between subsequent visits will be assessed. DISCUSSION: Our study will describe the natural history of patients diagnosed with SELENON-RM or LAMA2-MD, enabling us to select relevant clinical and functional outcome measures for reaching clinical trial-readiness. Moreover, our detailed description (deep phenotyping) of the clinical features will optimize clinical management and will establish a well-characterized baseline cohort for prospective follow-up. CONCLUSION: Our natural history study is an essential step for reaching trial readiness in SELENON-RM and LAMA2-MD. TRIAL REGISTRATION: This study has been approved by medical ethical reviewing committee Region Arnhem-Nijmegen (NL64269.091.17, 2017-3911) and is registered at ClinicalTrial.gov ( NCT04478981 ).

New Synthetic Lethality Re-Sensitizing Platinum-Refractory Cancer Cells to Cisplatin In Vitro: The Rationale to Co-Use PARP and ATM Inhibitors.

The pro-apoptotic tumor suppressor BIN1 inhibits the activities of the neoplastic transcription factor MYC, poly (ADP-ribose) polymerase-1 (PARP1), and ATM Ser/Thr kinase (ATM) by separate mechanisms. Although BIN1 deficits increase cancer-cell resistance to DNA-damaging chemotherapeutics, such as cisplatin, it is not fully understood when BIN1 deficiency occurs and how it provokes cisplatin resistance. Here, we report that the coordinated actions of MYC, PARP1, and ATM assist cancer cells in acquiring cisplatin resistance by BIN1 deficits. Forced BIN1 depletion compromised cisplatin sensitivity irrespective of Ser15-phosphorylated, pro-apoptotic TP53 tumor suppressor. The BIN1 deficit facilitated ATM to phosphorylate the DNA-damage-response (DDR) effectors, including MDC1. Consequently, another DDR protein, RNF8, bound to ATM-phosphorylated MDC1 and protected MDC1 from caspase-3-dependent proteolytic cleavage to hinder cisplatin sensitivity. Of note, long-term and repeated exposure to cisplatin naturally recapitulated the BIN1 loss and accompanying RNF8-dependent cisplatin resistance. Simultaneously, endogenous MYC was remarkably activated by PARP1, thereby repressing the BIN1 promoter, whereas PARP inhibition abolished the hyperactivated MYC-dependent BIN1 suppression and restored cisplatin sensitivity. Since the BIN1 gene rarely mutates in human cancers, our results suggest that simultaneous inhibition of PARP1 and ATM provokes a new BRCAness-independent synthetic lethal effect and ultimately re-establishes cisplatin sensitivity even in platinum-refractory cancer cells.

Ribosomal protein L6 (RPL6) is recruited to DNA damage sites in a poly(ADP-ribose) polymerase-dependent manner and regulates the DNA damage response.

Ribosomal proteins are the building blocks of ribosome biogenesis. Beyond their known participation in ribosome assembly, the ribosome-independent functions of ribosomal proteins are largely unknown. Here, using immunoprecipitation, subcellular fractionation, His-ubiquitin pulldown, and immunofluorescence microscopy assays, along with siRNA-based knockdown approaches, we demonstrate that ribosomal protein L6 (RPL6) directly interacts with histone H2A and is involved in the DNA damage response (DDR). We found that in response to DNA damage, RPL6 is recruited to DNA damage sites in a poly(ADP-ribose) polymerase (PARP)-dependent manner, promoting its interaction with H2A. We also observed that RPL6 depletion attenuates the interaction between mediator of DNA damage checkpoint 1 (MDC1) and H2A histone family member X, phosphorylated (γH2AX), impairs the accumulation of MDC1 at DNA damage sites, and reduces both the recruitment of ring finger protein 168 (RNF168) and H2A Lys-15 ubiquitination (H2AK15ub). These RPL6 depletion-induced events subsequently inhibited the recruitment of the following downstream repair proteins: tumor protein P53-binding protein 1 (TP53BP1) and BRCA1, DNA repair-associated (BRCA1). Moreover, the RPL6 knockdown resulted in defects in the DNA damage-induced G2-M checkpoint, DNA damage repair, and cell survival. In conclusion, our study identifies RPL6 as a critical regulatory factor involved in the DDR. These findings expand our knowledge of the extraribosomal functions of ribosomal proteins in cell physiology and deepen our understanding of the molecular mechanisms underlying DDR regulation.

H2AFX and MDC1 promote maintenance of genomic integrity in male germ cells.

In somatic cells, H2afx and Mdc1 are close functional partners in DNA repair and damage response. However, it is not known whether they are also involved in the maintenance of genome integrity in meiosis. By analyzing chromosome dynamics in H2afx-/- spermatocytes, we found that the synapsis of autosomes and X-Y chromosomes was impaired in a fraction of cells. Such defects correlated with an abnormal recombination profile. Conversely, Mdc1 was dispensable for the synapsis of the autosomes and played only a minor role in X-Y synapsis, compared with the action of H2afx This suggested that those genes have non-overlapping functions in chromosome synapsis. However, we observed that both genes play a similar role in the assembly of MLH3 onto chromosomes, a key step in crossover formation. Moreover, we show that H2afx and Mdc1 cooperate in promoting the activation of the recombination-dependent checkpoint, a mechanism that restrains the differentiation of cells with unrepaired DSBs. This occurs by a mechanism that involves P53. Overall, our data show that, in male germ cells, H2afx and Mdc1 promote the maintenance of genome integrity.This article has an associated First Person interview with the first author of the paper.

Epilepsy in LAMA2-related muscular dystrophy: An electro-clinico-radiological characterization.

OBJECTIVE: To delineate the epileptic phenotype of LAMA2-related muscular dystrophy (MD) and correlate it with the neuroradiological and muscle biopsy findings, as well as the functional motor phenotype. METHODS: Clinical, electrophysiological, neuroradiological, and histopathological data of 25 patients with diagnosis of LAMA2-related MD were analyzed. RESULTS: Epilepsy occurred in 36% of patients with LAMA2-related MD. Mean age at first seizure was 8 years. The most common presenting seizure type was focal-onset seizures with or without impaired awareness. Visual aura and autonomic signs, including vomiting, were frequently reported. Despite a certain degree of variability, bilateral occipital or temporo-occipital epileptiform abnormalities were by far the most commonly observed. Refractory epilepsy was found in 75% of these patients. Epilepsy in LAMA2-related MD was significantly more prevalent in those patients in whom the cortical malformations were more extensive. In contrast, the occurrence of epilepsy was not found to be associated with the patients' motor ability, the size of their white matter abnormalities, or the amount of residual merosin expressed on muscle. SIGNIFICANCE: The epileptic phenotype of LAMA2-related MD is characterized by focal seizures with prominent visual and autonomic features associated with EEG abnormalities that predominate in the posterior quadrants. A consistent correlation between epileptic phenotype and neuroimaging was identified, suggesting that the extension of the polymicrogyria may serve as a predictor of epilepsy occurrence.

BRCT-domain protein BRIT1 influences class switch recombination.

DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs.

Karyopherin α-2 Mediates MDC1 Nuclear Import through a Functional Nuclear Localization Signal in the tBRCT Domain of MDC1.

Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in DNA damage response (DDR) by coordinating the repair of double strand breaks (DSBs). Here, we identified a novel interaction between MDC1 and karyopherin α-2 (KPNA2), a nucleocytoplasmic transport adaptor, and showed that KPNA2 is necessary for MDC1 nuclear import. Thereafter, we identified a functional nuclear localization signal (NLS) between amino acid residues 1989-1994 of the two Breast Cancer 1 (BRCA1) carboxyl-terminal (tBRCT) domain of MDC1 and demonstrated disruption of this NLS impaired interaction between MDC1 and KPNA2 and reduced nuclear localization of MDC1. In KPNA2-depleted cells, the recruitment of MDC1, along with the downstream signaling p roteins Ring Finger Protein 8 (RNF8), 53BP1-binding protein 1 (53BP1), BRCA1, and Ring Finger Protein 168 (RNF168), to DNA damage sites was abolished. Additionally, KPNA2-depleted cells had a decreased rate of homologous recombination (HR) repair. Our data suggest that KPNA2-mediated MDC1 nuclear import is important for DDR signaling and DSB repair.

Loss of NFBD1/MDC1 disrupts homologous recombination repair and sensitizes nasopharyngeal carcinoma cells to PARP inhibitors.

BACKGROUND: Nasopharyngeal carcinoma (NPC), a highly invasive tumor, exhibits a distinctive racial and geographic distribution. As options of agents for effective combination chemoradiotherapy for advanced NPC are limited, novel therapeutic approaches are desperately needed. Here the potential of silencing NFBD1 in combination with PARP inhibition as a novel therapeutic strategy for NPC was investigated. METHODS: To investigate the function of NFBD1, we created NFBD1-depleted NPC cell lines via lentivirus mediated shRNA, and the colony formation, MTS assay, comet assay and apoptosis analysis were used to evaluate the sensitivity of NFBD1 knockdown on PARP inhibition. The signaling change was assessed by western blot, Immunofluorescence and flow cytometry. Furthermore, Xenografts model was used to evaluate the role of silencing NFBD1 in combination with PARP inhibition. RESULTS: We find that silencing NFBD1 in combination with PARP inhibition significantly inhibits the cell proliferation and cell cycle checkpoint activity, and increases the apoptosis and DNA damage. Mechanistic studies reveal that NFBD1 loss blocks olaparib-induced homologous recombination repair by decreasing the formation of BRCA1, BRCA2 and RAD51 foci. Furthermore, the xenograft tumor model demonstrated significantly increases sensitivity towards PARP inhibition under NFBD1 deficiency. CONCLUSIONS: We show that NFBD1 depletion may possess sensitizing effects of PARP inhibitor, and consequently offers novel therapeutic options for a significant subset of patients.

Mutations in the TP53 gene affected recruitment of 53BP1 protein to DNA lesions, but level of 53BP1 was stable after γ-irradiation that depleted MDC1 protein in specific TP53 mutants.

53BP1 is a very well-known protein that is recruited to DNA lesions. The focal accumulation of p53 binding protein, 53BP1, is a main feature indicating the repair of spontaneous or irradiation-induced foci (IRIF). Thus, here, we addressed the question of whether mutations in the TP53 gene, which often affect the level of p53 protein, can change the recruitment of 53BP1 to γ- or UVA-irradiated chromatin. In various TP53 mutants, we observed a distinct accumulation of 53BP1 protein to UV-induced DNA lesions: in R273C mutants, 53BP1 appeared transiently at DNA lesions, during 10-30 min after irradiation; the mutation R282W was responsible for accumulation of 53BP1 immediately after UVA-damage; and in L194F mutants, the first appearance of 53BP1 protein at the lesions occurred during 60-70 min. These results showed that specific mutations in the TP53 gene stand behind not only different levels of p53 protein, but also affect the localized kinetics of 53BP1 protein in UVA-damaged chromatin. However, after γ-irradiation, only G245S mutation in TP53 gene was associated with surprisingly decreased level of 53BP1 protein. In other mutant cell lines, levels of 53BP1 were not affected by γ-rays. To these effects, we conversely found a distinct number of 53BP1-positive irradiation-induced foci in various TP53 mutants. The R280K, G245S, L194F mutations, or TP53 deletion were also characterized by radiation-induced depletion in MDC1 protein. Moreover, in mutant cells, an interaction between MDC1 and 53BP1 proteins was abrogated when compared with wild-type counterpart. Together, the kinetics of 53BP1 accumulation at UV-induced DNA lesions is different in various TP53 mutant cells. After γ-irradiation, despite changes in a number and a volume of 53BP1-positive foci, levels of 53BP1 protein were relatively stable. Here, we showed a link between the status of MDC1 protein and TP53 gene, which specific mutations caused radiation-induced MDC1 down-regulation. This observation is significant, especially with regard to radiotherapy of tumors with abrogated function of TP53 gene.

Phosphorylation of the Cajal body protein WRAP53ß by ATM promotes its involvement in the DNA damage response.

The cellular response to DNA double-strand breaks is orchestrated by the protein kinase ATM, which phosphorylates key actors in the DNA repair network. WRAP53ß is a multifunctional protein that controls trafficking of factors to Cajal bodies, telomeres and DNA double-strand breaks but what regulates the involvement of WRAP53ß in these separate processes remains unclear. Here, we show that in response to various types of DNA damage, including IR and UV, WRAP53ß is phosphorylated on serine residue 64 by ATM with a time-course that parallels its accumulation at DNA lesions. Interestingly, recruitment of phosphorylated WRAP53ß (pWRAP53ßS64) to sites of such DNA damage promotes its interaction with γH2AX at these locations. Moreover, pWRAP53ßS64 stimulates the accumulation of the repair factor 53BP1 at DNA double-strand breaks and enhances repair of this type of damage via homologous recombination and non-homologous end joining. At the same time, phosphorylation of WRAP53ß is dispensable for its localization to Cajal bodies, where it accumulates even in unstressed cells. These findings not only reveal ATM to be an upstream regulator of WRAP53ß, but also indicates that phosphorylation of WRAP53ß at serine 64 controls its involvement in the DNA damage response and may also restrict its other functions.

Mediator of DNA damage checkpoint protein 1 (MDC1) as a prognostic marker for patients with oral squamous cell carcinoma.

BACKGROUND: The mediator of DNA damage checkpoint protein 1 (MDC1) is involved in the regulation of cell cycle checkpoints and recruitment of several repair proteins to the site of DNA double-stranded breaks (DSBs). This study aimed to correlate the expression of MDC1 protein with clinicopathological parameters and to evaluate its prognostic significance in patients with oral squamous cell carcinoma (OSCC). METHODS: MDC1 protein expression was evaluated immunohistochemically from untreated 100 patients with OSCC using modified H-score method. The association of MDC1 immunostaining was evaluated with clinicopathological parameters and disease outcome using univariate and multivariate survival analysis for relapse-free survival (RFS) and overall survival (OS). RESULTS: Incidence of nuclear and cytoplasmic expression of MDC1 protein was 85% & 92%, respectively. Strong nuclear MDC1 protein expression was found to be significantly correlated with lymph node metastasis (P = 0.032). For RFS, Kaplan-Meier survival analysis demonstrated that presence of metastatic lymph node (P = 0.001), lymphatic permeation (P = 0.020), and nuclear MDC1 (P = 0.005) remained significant risk predictors. In multivariate survival analysis, nuclear MDC1 (P = 0.027) entered at step 2 after presence of metastatic lymph node (P = 0.002) at step 1 for predicting reduced RFS. In relation to treatment, OSCC patients exhibiting weak expression of nuclear MDC1 protein were benefited significantly when treated with surgery followed by radiation therapy (P = 0.001). CONCLUSION: Thus, this study showed that MDC1 protein expression could be used as a prognostic marker in predicting relapse-free survival in patients with OSCC. OSCC patients expressing weak MDC1 protein could be benefited by adjuvant radiotherapy instead chemo-radiotherapy.