Downregulation Of DNA Repair Pathways Research Articles

H3.3-G34 mutations impair DNA repair and promote cGAS/STING-mediated immune responses in pediatric high-grade glioma models.

Pediatric high-grade gliomas (pHGGs) are the leading cause of cancer-related deaths in children in the USA. Sixteen percent of hemispheric pediatric and young adult HGGs encode Gly34Arg/Val substitutions in the histone H3.3 (H3.3-G34R/V). The mechanisms by which H3.3-G34R/V drive malignancy and therapeutic resistance in pHGGs remain unknown. Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we demonstrate that this mutation led to the downregulation of DNA repair pathways. This resulted in enhanced susceptibility to DNA damage and inhibition of the DNA damage response (DDR). We demonstrate that genetic instability resulting from improper DNA repair in G34R-mutant pHGG led to the accumulation of extrachromosomal DNA, which activated the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway, inducing the release of immune-stimulatory cytokines. We treated H3.3-G34R pHGG-bearing mice with a combination of radiotherapy (RT) and DNA damage response inhibitors (DDRi) (i.e., the blood-brain barrier-permeable PARP inhibitor pamiparib and the cell-cycle checkpoint CHK1/2 inhibitor AZD7762), and these combinations resulted in long-term survival for approximately 50% of the mice. Moreover, the addition of a STING agonist (diABZl) enhanced the therapeutic efficacy of these treatments. Long-term survivors developed immunological memory, preventing pHGG growth upon rechallenge. These results demonstrate that DDRi and STING agonists in combination with RT induced immune-mediated therapeutic efficacy in G34-mutant pHGG.

DNA repair pathways are altered in neural cell models of frataxin deficiency

Friedreich's ataxia (FRDA) is a hereditary and predominantly neurodegenerative disease caused by a deficiency of the protein frataxin (FXN). As part of the overall efforts to understand the molecular basis of neurodegeneration in FRDA, a new human neural cell line with doxycycline-induced FXN knockdown was established. This cell line, hereafter referred to as iFKD-SY, is derived from the human neuroblastoma SH-SY5Y and retains the ability to differentiate into mature neuron-like cells. In both proliferating and differentiated iFKD-SY cells, the induction of FXN deficiency is accompanied by increases in oxidative stress and DNA damage, reduced aconitase enzyme activity, higher levels of p53 and p21, activation of caspase-3, and subsequent apoptosis. More interestingly, FXN-deficient iFKD-SY cells exhibit an important transcriptional deregulation in many of the genes implicated in DNA repair pathways. The levels of some crucial proteins involved in DNA repair appear notably diminished. Furthermore, similar changes are found in two additional neural cell models of FXN deficit: primary cultures of FXN-deficient mouse neurons and human olfactory mucosa stem cells obtained from biopsies of FRDA patients. These results suggest that the deficiency of FXN leads to a down-regulation of DNA repair pathways that synergizes with oxidative stress to provoke DNA damage, which may be involved in the pathogenesis of FRDA. Thus, a failure in DNA repair may be considered a shared common molecular mechanism contributing to neurodegeneration in a number of hereditary ataxias including FRDA.

Hypoxia increases mutational load of breast cancer cells through frameshift mutations

ABSTRACT Tumor hypoxia-induced downregulation of DNA repair pathways and enhanced replication stress are potential sources of genomic instability. A plethora of genetic changes such as point mutations, large deletions and duplications, microsatellite and chromosomal instability have been discovered in cells under hypoxic stress. However, the influence of hypoxia on the mutational burden of the genome is not fully understood. Here, we attempted to elucidate the DNA damage response and repair patterns under different types of hypoxic stress. In addition, we examined the pattern of mutations exclusively induced under chronic and intermittent hypoxic conditions in two breast cancer cell lines using exome sequencing. Our data indicated that hypoxic stress resulted in transcriptional downregulation of DNA repair genes which can impact the DNA repair induced during anoxic as well as reoxygenated conditions. In addition, our findings demonstrate that hypoxic conditions increased the mutational burden, characterized by an increase in frameshift insertions and deletions. The somatic mutations were random and non-recurring, as huge variations within the technical duplicates were recognized. Hypoxia also resulted in an increase in the formation of potential neoantigens in both cell lines. More importantly, these data indicate that hypoxic stress mitigates DNA damage repair pathways and causes an increase in the mutational burden of tumor cells, thereby interfering with hypoxic cancer cell immunogenicity.

Abstract 273: KDM1A inhibition enhances chemotherapy response in glioblastoma via downregulation of DNA repair pathways

Abstract Background: Glioblastomas (GBM) have dismal survival rates (1 year-34.6% and 5 year-4.75%) and affect 13,000 patients yearly. Standard of care treatment consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide (TMZ), and tumor treating fields. Despite heavy investment in therapy, all patients will eventually succumb to their disease. Oncogenic and epigenetic signaling mechanisms may modulate DNA damage response (DDR) in tumors and contribute to chemo and radiation therapy resistance. Recently, we have shown that lysine-specific histone demethylase 1A (KDM1A), an epigenetic regulator, is overexpressed in GBM. In this study, we tested the hypothesis that KDM1A is essential for DDR, and that inhibition of KDM1A induces DNA repair deficiency and sensitizes GBM to TMZ therapy. Methods: To study the role of KDM1A in GBM cells, we have generated KDM1A knockout (KDM1A-KO) cells using the CRISPR/Cas9 system and KDM1A-shRNA transfected primary GBM cells (KDM1A-KD). Effect of KDM1A-KO, -KD or KDM1A inhibitor (NCD-38) on TMZ sensitization was studied using cell viability and survival assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR and western blot analysis. The blood-brain barrier (BBB) permeability of NCD-38 was studied by pharmacokinetic (PK) and brain bioavailability studies. Further, the in vivo efficacy of KDM1A inhibitor was studied using orthotopic models of GBM. Results: Cell viability and survival assays showed that knockout or pharmacological inhibition of KDM1A sensitized GBM cells to TMZ treatment. KDM1A expression is increased in GBM cells after treatment with TMZ. RNA-seq analysis revealed a decreased expression of several genes involved in DNA repair, including MGMT, RAD51, and MRE11A in NCD-38 treated cells compared to control. PK and brain bioavailability studies following a single intravenous dose (IV, 1 mg/kg) and per oral administration (PO, 10 mg/kg) of NCD-38 demonstrated that NCD-38 has favorable PK properties and exhibited a significant penetration of the BBB, with a total brain/plasma ratio of more than two at all tested time points. Further, KDM1A inhibition significantly reduced the in vivo tumor progression in established and primary GBM orthotopic models. Conclusions: Our results provide evidence that KDM1A contributes to chemotherapy resistance in GBM by modulating DNA repair pathways and the use of KDM1A inhibitor in conjunction with standard chemotherapy will serve as novel therapy for GBM patients. Citation Format: Bridgitte E. Palacios, Prabhakar Pitta-Venkata, Yihong Chen, Suryavathi Viswanadhapalli, Uday P. Pratap, Aleksandra Gruslova, Takayoshi Suzuki, Ratna K. Vadlamudi, Andrew Brenner, Gangadhara R. Sareddy. KDM1A inhibition enhances chemotherapy response in glioblastoma via downregulation of DNA repair pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 273.

Temozolomide Induces Senescence and Repression of DNA Repair Pathways in Glioblastoma Cells via Activation of ATR-CHK1, p21, and NF-κB.

The DNA-methylating drug temozolomide, which induces cell death through apoptosis, is used for the treatment of malignant glioma. Here, we investigate the mechanisms underlying the ability of temozolomide to induce senescence in glioblastoma cells. Temozolomide-induced senescence was triggered by the specific DNA lesion O6-methylguanine (O6MeG) and characterized by arrest of cells in the G2-M phase. Inhibitor experiments revealed that temozolomide-induced senescence was initiated by damage recognition through the MRN complex, activation of the ATR/CHK1 axis of the DNA damage response pathway, and mediated by degradation of CDC25c. Temozolomide-induced senescence required functional p53 and was dependent on sustained p21 induction. p53-deficient cells, not expressing p21, failed to induce senescence, but were still able to induce a G2-M arrest. p14 and p16, targets of p53, were silenced in our cell system and did not seem to play a role in temozolomide-induced senescence. In addition to p21, the NF-κB pathway was required for senescence, which was accompanied by induction of the senescence-associated secretory phenotype. Upon temozolomide exposure, we found a strong repression of the mismatch repair proteins MSH2, MSH6, and EXO1 as well as the homologous recombination protein RAD51, which was downregulated by disruption of the E2F1/DP1 complex. Repression of these repair factors was not observed in G2-M arrested p53-deficient cells and, therefore, it seems to represent a specific trait of temozolomide-induced senescence. SIGNIFICANCE: These findings reveal a mechanism by which the anticancer drug temozolomide induces senescence and downregulation of DNA repair pathways in glioma cells.

Modified Citrus Pectin as a Potential Sensitizer for Radiotherapy in Prostate Cancer

Background: Radiotherapy is one of the primary therapies for localized prostatic carcinoma. Therefore, there is an emerging need to sensitize prostatic cancer cells to chemotherapy/radiotherapy. Modified citrus pectin (MCP) is an effective inhibitor of galectin-3 (Gal-3), which is correlated with tumor progression, proliferation, angiogenesis, and apoptosis. Purpose: This study was directed to evaluate the efficacy of combining ionizing radiation (IR) with MCP on PCa cells. Study Design: Effects of treatments on PCa cells survival were evaluated using XTT assay, flow cytometry, and clonogenic survival assay. Expression of selected proteins was estimated using western blotting. Cell motility, migration, and invasion were determined. Contribution of reactive oxygen species production to treatment effects on cell viability was tested. Results: Radiotherapy combined with MCP reduced viability and enhanced radiosensitivity associated with a decrease in Gal-3, cleavage of the precursor of caspase-3, increased expression of the pro-apoptotic protein Bax, and downregulation of DNA repair pathways, poly-ADP-ribose polymerase, and proliferating cell nuclear antigen. MCP significantly reduced the invasive and migratory potential of PCa cells. Combining sodium pyruvate with MCP and IR mitigated the effect on cell viability. Conclusion: Our findings demonstrated that MCP sensitized PCa cells to IR by downregulating anti-apoptotic Gal-3, modulating DNA repair pathways, and increasing ROS production. For the first time the correlation between MCP, radiotherapy, and Gal-3 for prostatic cancer treatment was found. In addition, MCP reduced the metastatic properties of PCa cells. These findings provide MCP as a radiosensitizing agent to enhance IR cytotoxicity, overcome radioresistance, and reduce clinical IR dose.

Nickel induces transcriptional down-regulation of DNA repair pathways in tumorigenic and non-tumorigenic lung cells.

The heavy metal nickel is a known carcinogen, and occupational exposure to nickel compounds has been implicated in human lung and nasal cancers. Unlike many other environmental carcinogens, however, nickel does not directly induce DNA mutagenesis, and the mechanism of nickel-related carcinogenesis remains incompletely understood. Cellular nickel exposure leads to signaling pathway activation, transcriptional changes and epigenetic remodeling, processes also impacted by hypoxia, which itself promotes tumor growth without causing direct DNA damage. One of the mechanisms by which hypoxia contributes to tumor growth is the generation of genomic instability via down-regulation of high-fidelity DNA repair pathways. Here, we find that nickel exposure similarly leads to down-regulation of DNA repair proteins involved in homology-dependent DNA double-strand break repair (HDR) and mismatch repair (MMR) in tumorigenic and non-tumorigenic human lung cells. Functionally, nickel induces a defect in HDR capacity, as determined by plasmid-based host cell reactivation assays, persistence of ionizing radiation-induced DNA double-strand breaks and cellular hypersensitivity to ionizing radiation. Mechanistically, we find that nickel, in contrast to the metalloid arsenic, acutely induces transcriptional repression of HDR and MMR genes as part of a global transcriptional pattern similar to that seen with hypoxia. Finally, we find that exposure to low-dose nickel reduces the activity of the MLH1 promoter, but only arsenic leads to long-term MLH1 promoter silencing. Together, our data elucidate novel mechanisms of heavy metal carcinogenesis and contribute to our understanding of the influence of the microenvironment on the regulation of DNA repair pathways.

Hypoxia-induced alterations of G2 checkpoint regulators

Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, genome stability is also surveilled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can influence the DNA damage-induced cell cycle checkpoints. Here, we show that hypoxia (24 h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, the levels of several G2 checkpoint regulators, in particular Cyclin B, were reduced in G2 phase cells after hypoxic exposure, as shown by flow cytometric barcoding analysis of individual cells. These effects were accompanied by decreased phosphorylation of a Cyclin dependent kinase (CDK) target in G2 phase cells after hypoxia, suggesting decreased CDK activity. Furthermore, cells pre-exposed to hypoxia showed increased G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (∼0.03% O2 20 h and 0.2% O2 72 h). These results demonstrate that the DNA damage-induced G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors.

Functional genomics of acclimation and adaptation in response to thermal stress in Daphnia.

BackgroundGene expression regulation is one of the fundamental mechanisms of phenotypic plasticity and is expected to respond to selection in conditions favoring phenotypic response. The observation that many organisms increase their stress tolerance after acclimation to moderate levels of stress is an example of plasticity which has been long hypothesized to be based on adaptive changes in gene expression. We report genome-wide patterns of gene expression in two heat-tolerant and two heat-sensitive parthenogenetic clones of the zooplankton crustacean Daphnia pulex exposed for three generations to either optimal (18°C) or substressful (28°C) temperature.ResultsA large number of genes responded to temperature and many demonstrated a significant genotype-by-environment (GxE) interaction. Among genes with a significant GxE there were approximately equally frequent instances of canalization, i.e. stronger plasticity in heat-sensitive than in heat-tolerant clones, and of enhancement of plasticity along the evolutionary vector toward heat tolerance. The strongest response observed is the across-the-board down-regulation of a variety of genes occurring in heat-tolerant, but not in heat-sensitive clones. This response is particularly obvious among genes involved in core metabolic pathways and those responsible for transcription, translation and DNA repair.ConclusionsThe observed down-regulation of metabolism, consistent with previous findings in yeast and Drosophila, may reflect a general compensatory stress response. The associated down-regulation of DNA repair pathways potentially creates a trade-off between short-term benefits of survival at high temperature and long-term costs of accelerated mutation accumulation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-859) contains supplementary material, which is available to authorized users.

Excision of an oxidative DNA lesion (8-oxo-deoxyguanosine) in Mytilus edulis populations from two sites in the eastern Irish Sea

Mussels (Mytilus edulis) from the urban/industrialised site of New Brighton, Merseyside, UK and the reference site of Llandudno, North Wales, UK were collected at various time-points in 2005–2006. The excision capacity of mussel gill towards a radio-labelled probe containing the oxidative lesion 8-oxo-dG (8-oxo-deoxyguanosine) was quantified for both populations within 24 h of collection. Fpg (formamidopyrimidine glycosylase)-sensitive sites (which are proportional to 8-oxo-dG levels) were also quantified via the Fpg-modified Comet assay. Generally, excision capacity in New Brighton mussels was lower compared to Llandudno mussels, with the exception of May when activities from both groups were at their lowest. No substantial differences between Fpg-sensitive sites were noted but the ratio “mean Fpg-sensitive sites/mean 8-oxo-dG excision capacity” was elevated for New Brighton mussels. It is possible that the evident oxidative stress these mussels undergo is linked to pollutants found in their natural environment. These pollutants may act not only by direct oxidative DNA damage but also by down-regulation of DNA repair pathways.