DNA Repair Proteins Research Articles

Epigenetic silencing of HTATIP2 in glioblastoma contributes to treatment resistance by enhancing nuclear translocation of the DNA repair protein MPG.

Glioblastoma, the most malignant brain tumor in adults, exhibits characteristic patterns of epigenetic alterations that await elucidation. The DNA methylome of glioblastoma revealed recurrent epigenetic silencing of HTATIP2, which encodes a negative regulator of importin β-mediated cytoplasmic-nuclear protein translocation. Its deregulation may thus alter the functionality of cancer-relevant nuclear proteins, such as the base excision repair (BER) enzyme N-methylpurine DNA glycosylase (MPG), which has been associated with treatment resistance in GBM. We found that induction of HTATIP2 expression in GBM cells leads to a significant shift of predominantly nuclear to cytoplasmic MPG, whereas depletion of endogenous HTATIP2 results in enhanced nuclear MPG localization. Reduced nuclear MPG localization, prompted by HTATIP2 expression or by depletion of MPG, yielded less phosphorylated-H2AX-positive cells upon treatment with an alkylating agent. This suggested reduced MPG-mediated formation of apurinic/apyrimidinic sites, leaving behind unrepaired DNA lesions, reflecting a reduced capacity of BER in response to the alkylating agent. Epigenetic silencing of HTATIP2 may thus increase nuclear localization of MPG, thereby enhancing the capacity of the glioblastoma cells to repair treatment-related lesions and contributing to treatment resistance.

DNA repair and oxidative stress defense systems in radiation-resistant Deinococcus murrayi.

Deinococcus murrayi is a bacterium isolated from hot springs in Portugal, and named after Dr. Robert G.E. Murray in recognition of his research on the genus Deinococcus. Like other Deinococcus species, D. murrayi is extremely resistant to ionizing radiation. Repair of massive DNA damage and limitation of oxidative protein damage are two important factors contributing to the robustness of Deinococcus bacteria. Here, we identify, among others, the DNA repair and oxidative stress defense proteins in D. murrayi, and highlight special features of D. murrayi. For DNA repair, D. murrayi does not contain a standalone uracil-DNA glycosylase (Ung), but it encodes a protein in which Ung is fused to a DNA photolyase domain (PhrB). UvrB and UvrD contain large insertions corresponding to inteins. One of its endonuclease III enzymes lacks a [4Fe-4S] cluster. Deinococcus murrayi possesses a homolog of the error-prone DNA polymerase IV. Concerning oxidative stress defense, D. murrayi encodes a manganese catalase in addition to a heme catalase. Its organic hydroperoxide resistance protein Ohr is atypical because the redox active cysteines are present in a CXXC motif. These and other characteristics of D. murrayi show further diversity among Deinococcus bacteria with respect to resistance-associated mechanisms.

Recombinase-independent chromosomal rearrangements between dispersed inverted repeats in Saccharomyces cerevisiae meiosis.

DNA double-strand break (DSB) repair by homologous recombination (HR) uses a DNA template with similar sequence to restore genetic identity. Allelic DNA repair templates can be found on the sister chromatid or homologous chromosome. During meiotic recombination, DSBs preferentially repair from the homologous chromosome, with a proportion of HR events generating crossovers. Nevertheless, regions of similar DNA sequence exist throughout the genome, providing potential DNA repair templates. When DSB repair occurs at these non-allelic loci (termed ectopic recombination), chromosomal duplications, deletions and rearrangements can arise. Here, we characterize in detail ectopic recombination arising between a dispersed pair of inverted repeats in wild-type Saccharomyces cerevisiae at both a local and a chromosomal scale-the latter identified via gross chromosomal acentric and dicentric chromosome rearrangements. Mutation of the DNA damage checkpoint clamp loader Rad24 and the RecQ helicase Sgs1 causes an increase in ectopic recombination. Unexpectedly, additional mutation of the RecA orthologues Rad51 and Dmc1 alters-but does not abolish-the type of ectopic recombinants generated, revealing a novel class of inverted chromosomal rearrangement driven by the single-strand annealing pathway. These data provide important insights into the role of key DNA repair proteins in regulating DNA repair pathway and template choice during meiosis.

PG545 sensitizes ovarian cancer cells to PARP inhibitors through modulation of RAD51-DEK interaction.

PG545 (Pixatimod) is a highly sulfated small molecule known for its ability to inhibit heparanase and disrupt signaling mediated by heparan-binding-growth factors (HB-GF). Previous studies indicated that PG545 inhibits growth factor-mediated signaling in ovarian cancer (OC) to enhance response to chemotherapy. Here we investigated the previously unidentified mechanisms by which PG545 induces DNA damage in OC cells and found that PG545 induces DNA single- and double-strand breaks, reduces RAD51 expression in an autophagy-dependent manner and inhibits homologous recombination repair (HRR). These changes accompanied the ability of PG545 to inhibit endocytosis of the heparan-sulfate proteoglycan interacting DNA repair protein, DEK, leading to DEK sequestration in the tumor microenvironment (TME) and loss of nuclear DEK needed for HRR. As a result, PG545 synergized with poly (ADP-ribose) polymerase inhibitors (PARPis) in OC cell lines in vitro and in 55% of primary cultures of patient-derived ascites samples ex vivo. Moreover, PG545/PARPi synergy was observed in OC cells exhibiting either de novo or acquired resistance to PARPi monotherapy. PG545 in combination with rucaparib also generated increased DNA damage, increased antitumor effects and increased survival of mice bearing HRR proficient OVCAR5 xenografts compared to monotherapy treatment in vivo. Synergistic antitumor activity of the PG545/rucaparib combination was likewise observed in an immunocompetent syngeneic ID8F3 OC model. Collectively, these results suggest that targeting DEK-HSPG interactions in the TME through the use of PG545 may be a novel method of inhibiting DNA repair and sensitizing cells to PARPis.

Epigenetic control of type III interferon expression by 8-oxoguanine and its reader 8-oxoguanine DNA glycosylase1.

Interferons (IFNs) are secreted cytokines with the ability to activate expression of IFN stimulated genes that increase resistance of cells to virus infections. Activated transcription factors in conjunction with chromatin remodelers induce epigenetic changes that reprogram IFN responses. Unexpectedly, 8-oxoguanine DNA glycosylase1 (Ogg1) knockout mice show enhanced stimuli-driven IFN expression that confers increased resistance to viral and bacterial infections and allergen challenges. Here, we tested the hypothesis that the DNA repair protein OGG1 recognizes 8-oxoguanine (8-oxoGua) in promoters modulating IFN expression. We found that functional inhibition, genetic ablation, and inactivation by post-translational modification of OGG1 significantly augment IFN-λ expression in epithelial cells infected by human respiratory syncytial virus (RSV). Mechanistically, OGG1 bound to 8-oxoGua in proximity to interferon response elements, which inhibits the IRF3/IRF7 and NF-κB/RelA DNA occupancy, while promoting the suppressor NF-κB1/p50-p50 homodimer binding to the IFN-λ2/3 promoter. In a mouse model of bronchiolitis induced by RSV infection, functional ablation of OGG1 by a small molecule inhibitor (TH5487) enhances IFN-λ production, decreases immunopathology, neutrophilia, and confers antiviral protection. These findings suggest that the ROS-generated epigenetic mark 8-oxoGua via its reader OGG1 serves as a homeostatic thresholding factor in IFN-λ expression. Pharmaceutical targeting of OGG1 activity may have clinical utility in modulating antiviral response.

Ent-kaurane-type Diterpenes Induce ROS-mediated Mitochondrial Dysfunction and Apoptosis by Suppress the Homologous Recombination DNA Repair in Triple-negative Breast Cancer Cells.

Six ent-kaurane-type diterpenes were isolated from the roots of Isodon ternifolia. Previous studies have shown that compounds 1 and 2 exhibited cytotoxicity against three human cancer cell lines (MCF-7, A549, and HCT116), but its molecular mechanism has not been studied yet. In the present study, the inhibited proliferation of compounds 1 and 2 of two triple-negative breast cancer (TNBC) cell lines (4T1 and MDA-MB-231) have been demonstrated by MTT and colony formation assay. Flow cytometry, western blotting, and qPCR were used to further demonstrate the apoptosis process in TNBCs. Importantly, the following mitochondrial membrane potential (MMP) decrease during apoptosis was demonstrated to correlate to reactive oxygen species (ROS) production. In addition, DNA damage induced by compounds 1 and 2 was illustrated by detect of homologous recombination (HR) DNA repair genes and proteins expression, such as RAD51. These results indicated that compounps 1 and 2 could trigger the TNBCs apoptosis mediated by ROS-induced mitochondrial dysfunction and induce DNA double-strand breaks (DSBs) by down regulating HR DNA repair. Furthermore, this research reveals that the mechanism between mitochondria dysfunction and DNA damage is deserved to be investigated for elucidating the dynamic signal transduction between the nucleus and the cellular matrix during apoptosis.

MAGI1 Prevents Senescence and Promotes the DNA Damage Response in ER+ Breast Cancer.

MAGI1 acts as a tumor suppressor in estrogen receptor-positive (ER+) breast cancer (BC), and its loss correlates with a more aggressive phenotype. To identify the pathways and events affected by MAGI1 loss, we deleted the MAGI1 gene in the ER+ MCF7 BC cell line and performed RNA sequencing and functional experiments in vitro. Transcriptome analyses revealed gene sets and biological processes related to estrogen signaling, the cell cycle, and DNA damage responses affected by MAGI1 loss. Upon exposure to TNF-α/IFN-γ, MCF7 MAGI1 KO cells entered a deeper level of quiescence/senescence compared with MCF7 control cells and activated the AKT and MAPK signaling pathways. MCF7 MAGI1 KO cells exposed to ionizing radiations or cisplatin had reduced expression of DNA repair proteins and showed increased sensitivity towards PARP1 inhibition using olaparib. Treatment with PI3K and AKT inhibitors (alpelisib and MK-2206) restored the expression of DNA repair proteins and sensitized cells to fulvestrant. An analysis of human BC patients' transcriptomic data revealed that patients with low MAGI1 levels had a higher tumor mutational burden and homologous recombination deficiency. Moreover, MAGI1 expression levels negatively correlated with PI3K/AKT and MAPK signaling, which confirmed our in vitro observations. Pharmacological and genomic evidence indicate HDACs as regulators of MAGI1 expression. Our findings provide a new view on MAGI1 function in cancer and identify potential treatment options to improve the management of ER+ BC patients with low MAGI1 levels.

Transmission disequilibrium analysis of whole genome data in childhood-onset systemic lupus erythematosus.

Childhood-onset systemic lupus erythematosus (cSLE) patients are unique, with hallmarks of Mendelian disorders (early-onset and severe disease) and thus are an ideal population for genetic investigation of SLE. In this study, we use the transmission disequilibrium test (TDT), a family-based genetic association analysis that employs robust methodology, to analyze whole genome sequencing data. We aim to identify novel genetic associations in an ancestrally diverse, international cSLE cohort. Forty-two cSLE patients and 84 unaffected parents from 3 countries underwent whole genome sequencing. First, we performed TDT with single nucleotide variant (SNV)-based (common variants) using PLINK 1.9, and gene-based (rare variants) analyses using Efficient and Parallelizable Association Container Toolbox (EPACTS) and rare variant TDT (rvTDT), which applies multiple gene-based burden tests adapted for TDT, including the burden of rare variants test. Applying the GWAS standard threshold (5.0 × 10-8) to common variants, our SNV-based analysis did not return any genome-wide significant SNVs. The rare variant gene-based TDT analysis identified many novel genes significantly enriched in cSLE patients, including HNRNPUL2, a DNA repair protein, and DNAH11, a ciliary movement protein, among others. Our approach identifies several novel SLE susceptibility genes in an ancestrally diverse childhood-onset lupus cohort.

Proliferating cell nuclear antigen inhibitors block distinct stages of herpes simplex virus infection.

Proliferating cell nuclear antigen (PCNA) forms a homotrimer that encircles replicating DNA and is bound by DNA polymerases to add processivity to cellular DNA synthesis. In addition, PCNA acts as a scaffold to recruit DNA repair and chromatin remodeling proteins to replicating DNA via its interdomain connecting loop (IDCL). Despite encoding a DNA polymerase processivity factor UL42, it was previously found that PCNA associates with herpes simplex virus type 1 (HSV-1) replication forks and is necessary for productive HSV-1 infection. To define the role that PCNA plays during viral DNA replication or a replication-coupled process, we investigated the effects that two mechanistically distinct PCNA inhibitors, PCNA-I1 and T2AA, have on the HSV-1 infectious cycle. PCNA-I1 binds at the interface between PCNA monomers, stabilizes the homotrimer, and may interfere with protein-protein interactions. T2AA inhibits select protein-protein interactions within the PCNA IDCL. Here we demonstrate that PCNA-I1 treatment results in reduced HSV-1 DNA replication, late gene expression, and virus production, while T2AA treatment results in reduced late viral gene expression and infectious virus production. To pinpoint the mechanisms by which PCNA inhibitors affect viral processes and protein recruitment to replicated viral DNA, we performed accelerated native isolation of proteins on nascent DNA (aniPOND). Results indicate that T2AA inhibits recruitment of the viral uracil glycosylase UL2 and transcription regulatory factors to viral DNA, likely leading to a defect in viral base excision repair and the observed defect in late viral gene expression and infectious virus production. In addition, PCNA-I1 treatment results in decreased association of the viral DNA polymerase UL30 and known PCNA-interacting proteins with viral DNA, consistent with the observed block in viral DNA replication and subsequent processes. Together, we conclude that inhibitors of cellular PCNA block recruitment of key viral and cellular factors to viral DNA to inhibit viral DNA synthesis and coupled processes.

Phase-separated ribosome-nascent chain complexes in genotoxic stress response.

Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered amino-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of Saccharomyces cerevisiae protein complexes according to their amino-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, SGS1 mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together, these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human.

UBA80 and UBA52 fine-tune RNF168-dependent histone ubiquitination and DNA repair

The ubiquitin signaling pathway is crucial for the DNA damage response pathway. More specifically, RNF168 is integral in regulating DNA repair proteins at damaged chromatin. However, the detailed mechanism by which RNF168 is regulated in cells is not fully understood. Here, we identify the ubiquitin-ribosomal fusion proteins UBA80 (also known as RPS27A) and UBA52 (also known as RPL40) as interacting proteins for H2A/H2AX histones and RNF168. Both UBA80 and UBA52 are recruited to laser-induced micro-irradiation DNA damage sites and are required for DNA repair. Ectopic expression of UBA80 and UBA52 inhibits RNF168-mediated H2A/H2AX ubiquitination at K13/15 and impairs 53BP1 recruitment to DNA lesions. Mechanistically, the C-terminal ribosomal fragments of UBA80 and UBA52, S27A and L40, respectively, limit RNF168-nucleosome engagement by masking the regulatory acidic residues at E143/E144 and the nucleosome acidic patch. Together, our results reveal that UBA80 and UBA52 antagonize the ubiquitination signaling pathway and fine-tune the spatiotemporal regulation of DNA repair proteins at DNA damage sites.

The roles of non-productive complexes of DNA repair proteins with DNA lesions

A multitude of different types of lesions is continuously introduced into the DNA inside our cells, and their rapid and efficient repair is fundamentally important for the maintenance of genomic stability and cellular viability. This is achieved by a number of DNA repair systems that each involve different protein factors and employ versatile strategies to target different types of DNA lesions. Intriguingly, specialized DNA repair proteins have also evolved to form non-functional complexes with their target lesions. These proteins allow the marking of innocuous lesions to render them visible for DNA repair systems and can serve to directly recruit DNA repair cascades. Moreover, they also provide links between different DNA repair mechanisms or even between DNA lesions and transcription regulation. I will focus here in particular on recent findings from single molecule analyses on the alkyltransferase-like protein ATL, which is believed to initiate nucleotide excision repair (NER) of non-native NER target lesions, and the base excision repair (BER) enzyme hOGG1, which recruits the oncogene transcription factor Myc to gene promoters under oxidative stress.

Methadone Potentiates the Cytotoxicity of Temozolomide by Impairing Calcium Homeostasis and Dysregulation of PARP in Glioblastoma Cells.

Methadone is commonly used as an alternative to morphine in patients with pain associated with glioblastoma and other cancers. Although concomitant administration of methadone and cytostatics is relatively common, the effect of methadone on the efficacy of cytostatic drugs has not been well studied until recently. Moreover, the mechanism behind the effect of methadone on temozolomide efficacy has not been investigated in previous studies, or this effect has been automatically attributed to opioid receptors. Our findings indicate that methadone potentiates the effect of temozolomide on rat C6 glioblastoma cells and on human U251 and T98G glioblastoma cells and increases cell mortality by approximately 50% via a mechanism of action independent of opioid receptors. Our data suggest that methadone acts by affecting mitochondrial potential, the level of oxidative stress, intracellular Ca2+ concentration and possibly intracellular ATP levels. Significant effects were also observed on DNA integrity and on cleavage and expression of the DNA repair protein PARP-1. None of these effects were attributed to the activation of opioid receptors and Toll-like receptor 4. Our results provide an alternative perspective on the mechanism of action of methadone in combination with temozolomide and a potential strategy for the treatment of glioblastoma cell resistance to temozolomide.

Nei-like DNA glycosylase 2 selectively antagonizes interferon-β expression upon respiratory syncytial virus infection

As part of the antiviral response, cells activate the expressions of type I interferons (IFNs) and proinflammatory mediators to control viral spreading. Viral infections can impact DNA integrity; however, how DNA damage repair coordinates antiviral response remains elusive. Here we report Nei-like DNA glycosylase 2 (NEIL2), a transcription-coupled DNA repair protein, actively recognizes the oxidative DNA substrates induced by respiratory syncytial virus (RSV) infection to set the threshold of IFN-β expression. Our results show that NEIL2 antagonizes nuclear factor κB (NF-κB) acting on the IFN-β promoter early after infection, thus limiting gene expression amplified by type I IFNs. Mice lacking Neil2 are far more susceptible to RSV-induced illness with an exuberant expression of proinflammatory genes and tissue damage, and the administration of NEIL2 protein into the airway corrected these defects. These results suggest a safeguarding function of NEIL2 in controlling IFN-β levels against RSV infection. Due to the short- and long-term side effects of type I IFNs applied in antiviral therapy, NEIL2 may provide an alternative not only for ensuring genome fidelity but also for controlling immune responses.

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Impact of Manganese and Chromate on Specific DNA Double-Strand Break Repair Pathways.

Manganese is an essential trace element; nevertheless, on conditions of overload, it becomes toxic, with neurotoxicity being the main concern. Chromate is a well-known human carcinogen. The underlying mechanisms seem to be oxidative stress as well as direct DNA damage in the case of chromate, but also interactions with DNA repair systems in both cases. However, the impact of manganese and chromate on DNA double-strand break (DSB) repair pathways is largely unknown. In the present study, we examined the induction of DSB as well as the effect on specific DNA DSB repair mechanisms, namely homologous recombination (HR), non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ). We applied DSB repair pathway-specific reporter cell lines, pulsed field gel electrophoresis as well as gene expression analysis, and investigated the binding of specific DNA repair proteins via immunoflourescence. While manganese did not seem to induce DNA DSB and had no impact on NHEJ and MMEJ, HR and SSA were inhibited. In the case of chromate, the induction of DSB was further supported. Regarding DSB repair, no inhibition was seen in the case of NHEJ and SSA, but HR was diminished and MMEJ was activated in a pronounced manner. The results indicate a specific inhibition of error-free HR by manganese and chromate, with a shift towards error-prone DSB repair mechanisms in both cases. These observations suggest the induction of genomic instability and may explain the microsatellite instability involved in chromate-induced carcinogenicity.

The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment.

DNA double-strand breaks are the most toxic lesions repaired through the non-homologous and joining (NHEJ) or the homologous recombination (HR), which is dependent on the generation of single-strand tails, by the DNA end resection mechanism. The resolution of the HR intermediates leads to error-free repair (Gene Conversion) or the mutagenic pathways (Single Strand Annealing and Alternative End-Joining); the regulation of processes leading to the resolution of the HR intermediates is not fully understood. Here, we used a hydrophilic extract of a new tomato genotype (named DHO) in order to modulate the Camptothecin (CPT) DNA damage response. We demonstrated increased phosphorylation of Replication Protein A 32 Serine 4/8 (RPA32 S4/8) protein in HeLa cells treated with the CPT in combination with DHO extract with respect to CPT alone. Moreover, we pointed out a change in HR intermediates resolution from Gene Conversion to Single Strand Annealing through the modified DNA repair protein RAD52 homolog (RAD52), DNA excision repair protein ERCC-1 (ERCC1) chromatin loading in response to DHO extract, and CPT co-treatment, with respect to the vehicle. Finally, we showed an increased sensitivity of HeLa cell lines to DHO extract and CPT co-treatment suggesting a possible mechanism for increasing the efficiency of cancer therapy. We described the potential role of DHO extract in the modulation of DNA repair, in response to Camptothecin treatment (CPT), favoring an increased sensitivity of HeLa cell lines to topoisomerase inhibitor therapy.

C-Jun N-terminal kinase signaling in cellular senescence.

Cellular senescence leads to decreased tissue regeneration and inflammation and is associated with diabetes, neurodegenerative diseases, and tumorigenesis. However, the mechanisms of cellular senescence are not fully understood. Emerging evidence has indicated that c-Jun N-terminal kinase (JNK) signaling is involved in the regulation of cellular senescence. JNK can downregulate hypoxia inducible factor-1α to accelerate hypoxia-induced neuronal cell senescence. The activation of JNK inhibits mTOR activity and triggers autophagy, which promotes cellular senescence. JNK can upregulate the expression of p53 and Bcl-2 and accelerates cancer cell senescence; however, this signaling also mediates the expression of amphiregulin and PD-LI to achieve cancer cell immune evasion and prevents their senescence. The activation of JNK further triggers forkhead box O expression and its target gene Jafrac1 to extend the lifespan of Drosophila. JNK can also upregulate the expression of DNA repair protein poly ADP-ribose polymerase 1 and heat shock protein to delay cellular senescence. This review discusses recent advances in understanding the function of JNK signaling in cellular senescence and includes a comprehensive analysis of the molecular mechanisms underlying JNK-mediated senescence evasion and oncogene-induced cellular senescence. We also summarize the research progress in anti-aging agents that target JNK signaling. This study will contribute to a better understanding of the molecular targets of cellular senescence and provides insights into anti-aging, which may be used to develop drugs for the treatment of aging-related diseases.

DIPG-24. BRD4 INHIBITION AS A RADIOSENSITIZER THROUGH BLOCKING DNA REPAIR FOR THE TREATMENT OF DIFFUSE MIDLINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) is one of the devastating childhood cancers. Radiation therapy (RT) remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance RT efficacy by combining it with radiosensitizing agents, though none have been successful in doing so. Given this, there is a critical need to identify effective therapeutics to enhance the RT anti-tumor activity in DIPG. Here, we identified BRD4 inhibitors as candidate radiosensitizers from a high throughput drug screening. DIPG cells show increased H3 K27 acetylation (H3K27ac) levels, which bind to BET bromodomain protein 4 (BRD4) and are strongly associated with active transcription. We tested two BRD4 inhibitors (BRD4i: AZD5153 and JQ-1) as well as genetic BRD4 depletion, and observed enhancement of radiation-induced DNA damage, confirming BRD4i as potential radiosensitizers in the treatment of DIPG. We evaluated the effects of BRD4i on gene expression using RNAseq, and observed significant inhibition of DNA-repair proteins such as BRCA1 and RAD51. CUT-RUN qPCR showed decreased H3K27ac at BRCA1 and RAD51 promoters. Combination of BRD4i and RT inhibited cell viability significantly using clonogenic survival assay, apoptosis assay, and also showed cell cycle arrest. Radiation-induced DNA double-strand break (DSB) repair was prolonged with BRD4i with high levels of gH2AX and 53BP1 likely due to inhibition of DNA-repair. In vivo studies revealed increased survival of animals treated with combination therapy of RT and BRD4i in comparison to either monotherapy. Together, these results highlight BRD4i as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.