Expression Of DNA Repair Genes Research Articles

Chlorpromazine overcomes temozolomide resistance in glioblastoma by inhibiting Cx43 and essential DNA repair pathways

BackgroundIn the fight against GBM, drug repurposing emerges as a viable and time-saving approach to explore new treatment options. Chlorpromazine, an old antipsychotic medication, has recently arisen as a promising candidate for repositioning in GBM therapy in addition to temozolomide, the first-line standard of care. We previously demonstrated the antitumor efficacy of chlorpromazine and its synergistic effects with temozolomide in suppressing GBM cell malignant features in vitro. This prompted us to accomplish a Phase II clinical trial to evaluate the efficacy and safety of adding chlorpromazine to temozolomide in GBM patients with unmethylated MGMT gene promoter. In this in vitro study, we investigate the potential role of chlorpromazine in overcoming temozolomide resistance.MethodsIn our experimental set, we analyzed Connexin-43 expression at both the transcriptional and protein levels in control- and chlorpromazine-treated GBM cells. DNA damage and subsequent repair were assessed by immunofluorescence of γ-H2AX and Reverse-Phase Protein microArrays in chlorpromazine treated GBM cell lines. To elucidate the relationship between DNA repair systems and chemoresistance, we analyzed a signature of DNA repair genes in GBM cells after treatment with chlorpromazine, temozolomide and Connexin-43 downregulation.ResultsChlorpromazine treatment significantly downregulated connexin-43 expression in GBM cells, consequently compromising connexin-dependent cellular resilience, and ultimately contributing to cell death. In line with this, we observed concordant post-translational modifications of molecular determinants involved in DNA damage and repair pathways. Our evaluation of DNA repair genes revealed that temozolomide elicited an increase, while chlorpromazine, as well as connexin-43 silencing, a decrease in DNA repair gene expression in GBM cells.ConclusionsChlorpromazine potentiates the cytotoxic effects of the alkylating agent temozolomide through a mechanism involving downregulation of Cx43 expression and disruption of the cell cycle arrest essential for DNA repair processes. This finding suggests that chlorpromazine may be a potential therapeutic strategy to overcome TMZ resistance in GBM cells by inhibiting their DNA repair mechanisms.

Dulaglutide reduces oxidative DNA damage and hypermethylation in the somatic cells of mice fed a high-energy diet by restoring redox balance, inflammatory responses, and DNA repair gene expressions.

Obesity is an established risk factor for numerous malignancies, although it remains uncertain whether the disease itself or weight-loss drugs are responsible for a greater predisposition to cancer. The objective of the current study was to determine the impact of dulaglutide on genetic and epigenetic DNA damage caused by obesity, which is a crucial factor in the development of cancer. Mice were administered a low-fat or high-fat diet for 12 weeks, followed by a 5-week treatment with dulaglutide. Following that, modifications of the DNA bases were examined using the comet assay. To clarify the underlying molecular mechanisms, oxidized and methylated DNA bases, changes in the redox status, levels of inflammatory cytokines, and the expression levels of some DNA repair genes were evaluated. Animals fed a high-fat diet exhibited increased body weights, elevated DNA damage, oxidation of DNA bases, and DNA hypermethylation. In addition, obese mice showed altered inflammatory responses, redox imbalances, and repair gene expressions. The findings demonstrated that dulaglutide does not exhibit genotoxicity in the investigated conditions. Following dulaglutide administration, animals fed a high-fat diet demonstrated low DNA damage, less oxidation and methylation of DNA bases, restored redox balance, and improved inflammatory responses. In addition, dulaglutide treatment restored the upregulated DNMT1, Ogg1, and p53 gene expression. Overall, dulaglutide effectively maintains DNA integrity in obese animals. It reduces oxidative DNA damage and hypermethylation by restoring redox balance, modulating inflammatory responses, and recovering altered gene expressions. These findings demonstrate dulaglutide's expediency in treating obesity and its associated complications.

BET bromodomain inhibition potentiates radiosensitivity in models of H3K27-altered diffuse midline glioma.

Diffuse midline glioma (DMG) H3K27-altered is one of the devastating childhood cancers. Radiation therapy remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance radiation anti-tumor activity using radiosensitizing agents, although none have been successful. Given this, there is a critical need for identifying effective therapeutics to enhance radiation sensitivity for the treatment of DMG. Using high-throughput radiosensitivity screening, we identified bromo- and extra-terminal domain (BET) protein inhibitors as potent radiosensitizers in DMG cells. Genetic and pharmacologic inhibition of BET bromodomain activity reduced DMG cell proliferation and enhanced radiation-induced DNA damage by inhibiting DNA repair pathways. RNA-seq and CUT & RUN showed that BET bromodomain inhibitors regulate the expression of DNA repair genes mediated by H3K27 acetylation at enhancers. BET bromodomain inhibitors enhanced DMG radiation-response in patient-derived xenografts as well as genetically engineered mouse models. Together, our results highlight BET bromodomain inhibitors as radiosensitizer and provide a rationale for developing combination therapy with radiation for the treatment of DMG.

IGFBP3 regulates airway basal cell stemness and airway regeneration

Basal Cells (BCs) are multipotent progenitor cells of the upper airway and critical in airway epithelial regeneration. Impaired self-renewal and differentiation capacity of BCs is a significant cause of chronic respiratory diseases. However, mechanisms mediating these changes remain largely unexplored. We, and others, have observed attenuated growth and differentiation of BCs after repeated cell divisions in vitro. To investigate mechanisms regulating BC stemness, we performed bulk RNA-sequencing to analyze transcriptomes of early and late passage BCs maintained in culture. Our analyses reveal significant differences in the expression of genes involved in IGF- and IGFBP3-dependent pathways that are known to modulate cellular growth and ROS generation. The role of these pathways in regulating BC stemness have not been systematically investigated. We hypothesized that IGFBP3 regulates BC growth and differentiation by modulating cellular redox status and DNA damage repair. Herein, we demonstrate that passage of non-diseased human BCs over 5 passages increases IGFBP3 transcript in 3 independent growth conditions. Interestingly, continued passage decreased IGFBP3 expression and significantly altered its intracellular localization. In contrast to low passage cells where IGFBP3 is largely cytosolic, high passage cells almost exclusively express IGFBP3 in their nuclei where it is known to participate in DNA repair. Consistent with this, we observed increased DNA repair gene expression in high passage cells coupled with significant reduction in thioredoxin levels. Together, these results suggest that serial passage of BCs causes oxidative stress-mediated BC exhaustion and altered IGFBP3 expression and localization. To investigate the role of IGFBP3 in BC growth and differentiation, we knocked down IGFBP3 expression and, as hypothesized, IGFBP3 knock down of low passage cells attenuated growth, BC markers, and DNA damage repair gene expression. In addition, differentiation to secretory, ciliated and club cells at the air-liquid interphase was significantly attenuated. In conclusion, our data support an IGFBP3-dependent regulation of BC stemness by preserving growth and differentiation. This study was supported by the NIH R01HL139828 (ALR) and Cystic Fibrosis Foundation grants FIRTH17XX0 (ALR), RYAN21XX0, FIRTH21XX0 (ALR, CNM). BCs for this study were provided though the Center for Gene Therapy Cell Core funded by the NIH:NIDDK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibitory effect of malonic acid on Microcystis aeruginosa: Cell morphology, oxidative stress and gene expression

The utilization of allelochemicals, which are regarded as eco-friendly algaecides for mitigating harmful cyanobacterial blooms (HCB), has attracted increasing attention, owing to their negligible toxicity, high selectivity and excellent degradability. In the present study, the inhibitory effects of malonic acid on Microcystis aeruginosa were investigated in terms of growth indicators, cell morphology, antioxidant enzymes activities and gene expression. The results indicated that the exposure to 0.5 mmol L−1 malonic acid for 7 d caused a high microalgae inhibition rate of 86.88 %. Furthermore, the analysis of scanning electron microscopy (SEM) and transmission electron microscope (TEM) showed that the presence of malonic acid resulted in the disruption of cell integrity and the changes in cell morphology. Specifically, malonic acid was observed to cleave microalgae cells, while concurrently oxidizing the microalgae organic matter. In addition, under the exposure of 0.5 mmol L−1 malonic acid, the levels of malondialdehyde (MDA) were 3.28 times as the control group on d-5, while the activities of superoxide dismutase (SOD) and catalase (CAT) were first increased by 5.96 and 11.73 fold compare to the control group on d-5, and then decreased, due to oxidative stress induced by malonic acid. Transcriptome-based gene expression analysis revealed that the presence of malonic acid significantly inhibited the transcription of genes (rbcL, psbA1) associated with photosynthesis, and the expression of DNA repair gene (recA), stress response gene (ftsH) and microcystin synthesis genes (mcyD, mcyH) were up-regulated. These findings provide novel insights into the inhibitory effect of malonic acid on M. aeruginosa, suggesting that malonic acid has the potential to control M. aeruginosa blooms.

CAG repeat mosaicism is gene specific in spinocerebellar ataxias

Expanded CAG repeats in coding regions of different genes are the most common cause of dominantly inherited spinocerebellar ataxias (SCAs). These repeats are unstable through the germline, and larger repeats lead to earlier onset. We measured somatic expansion in blood samples collected from 30 SCA1, 50 SCA2, 74 SCA3, and 30 SCA7 individuals over a mean interval of 8.5 years, along with postmortem tissues and fetal tissues from SCA1, SCA3, and SCA7 individuals to examine somatic expansion at different stages of life. We showed that somatic mosaicism in the blood increases over time. Expansion levels are significantly different among SCAs and correlate with CAG repeat lengths. The level of expansion is greater in individuals with SCA7 who manifest disease compared to that of those who do not yet display symptoms. Brain tissues from SCA individuals have larger expansions compared to the blood. The cerebellum has the lowest mosaicism among the studied brain regions, along with a high expression of ATXNs and DNA repair genes. This was the opposite in cortices, with the highest mosaicism and lower expression of ATXNs and DNA repair genes. Fetal cortices did not show repeat instability. This study shows that CAG repeats are increasingly unstable during life in the blood and the brain of SCA individuals, with gene- and tissue-specific patterns.

Exploring the combined anti-cancer effects of sodium butyrate and celastrol in glioblastoma cell lines: a novel therapeutic approach

Glioblastoma, a highly aggressive and lethal brain cancer, lacks effective treatment options and has a poor prognosis. In our study, we explored the potential anti-cancer effects of sodium butyrate (SB) and celastrol (CEL) in two glioblastoma cell lines. SB, a histone deacetylase inhibitor, and CEL, derived from the tripterygium wilfordii plant, act as mTOR and proteasome inhibitors. Both can cross the blood–brain barrier, and they exhibit chemo- and radiosensitive properties in various cancer models. GB cell lines LN-405 and T98G were treated with SB and CEL. Cell viability was assessed by MTT assay and IC50 values were obtained. Gene expression of DNA repair, apoptosis, and autophagy-related genes was analyzed by RT-PCR. Cell cycle distribution was determined using flow cytometry. Viability assays using MTT assay revealed IC50 values of 26 mM and 22.7 mM for SB and 6.77 μM, and 9.11 μM for CEL in LN-405 and T98G cells, respectively. Furthermore, we examined the expression levels of DNA repair genes (MGMT, MLH-1, MSH-2, MSH-6), apoptosis genes (caspase-3, caspase-8, caspase-9), and an autophagy gene (ATG-6) using real-time polymerase chain reaction. Additionally, flow cytometry analysis revealed alterations in cell cycle distribution following treatment with SB, CEL and their combination. These findings indicate that SB and CEL may act through multiple mechanisms, including DNA repair inhibition, apoptosis induction, and autophagy modulation, to exert their anti-cancer effects in glioblastoma cells. This is the first study providing novel insights into the potential therapeutic effects of SB and CEL in glioblastoma.

Chronic low-dose chromium VI exposure induces oxidative stress and apoptosis with altered expressions of DNA repair genes and promoter hypermethylation in the liver of Swiss albino mice.

The present investigation dealt with harmful effects of hexavalent chromium (Cr [VI]) on liver of Swiss albino mice. This variant exhibited cytotoxicity, mutagenicity, and carcinogenicity. Our study focused on elucidating the hepatotoxic effects of chronic low-dose exposure to Cr (VI) (2, 5, and 10ppm) administered via drinking water for 4 and 8months. The observed elevation in SGPT, ALP, and SGOT and increased oxidative stress markers unequivocally confirmed the severe disruption of liver homeostasis at these low treatment doses. Noteworthy alterations in histoarchitecture, body weight, and water intake provided further evidences of the harmful effects of Cr (VI). Production of reactive oxygen species (ROS) during metabolism led to DNA damages. Immunohistochemistry and qRT-PCR analyses revealed that chronic low-dose exposure of Cr (VI) induced apoptosis in liver tissue. Our study exhibited alterations in the expression pattern of DNA repair genes (Rad51, Mutyh, Mlh1, and Ogg1), coupled with promoter hypermethylation of Mutyh and Rad51, leading to transcriptional inhibition. Our findings underscored the potential of low-dose Cr (VI) exposure on hepatotoxicity by the intricate interplay between apoptosis induction and epigenetic alterations of DNA repair genes.

Abstract 5882: Targeting vulnerabilities of drug-tolerant persister cells in gastric cancer

Abstract Cytotoxic drugs often fail to eradicate cancers due to the presence of treatment-persistent tumor cells that represent a reservoir for relapse. These “residual” cancer cells escape from chemotherapy- induced death by entering a reversible slow proliferation state, known as drug tolerant persister (DTP) state. Although improvement of treatment options achieved a survival benefit, Gastric Cancer (GC) is still endowed with poor prognosis. Surgery and neo/adjuvant chemotherapy remain the keystone of treatment. However, most patients with advanced cancer relapse even after complete surgical resection, suggesting a critical role for DTP cells. We obtained GC DTP cells from primary cells upon first- and second-line chemotherapy regimens (FLOT and FOLFIRI). Cells were treated with doses corresponding to the drug maximal plasmatic concentration. At the end of the treatment, cells were left without drugs (washout). During this window of time, when cells had regrown, they were re-challenged with chemotherapy and resulted still sensitive to the treatments, thus fulfilling the criteria for bona fide DTP cells. GC DTPs showed increased expression of markers associated with gastric stem cells (i.e. LGR5), downregulation of proliferative markers (i.e. Ki67) and decreased levels of PS6, evaluated as a readout of mTOR activation. Interestingly, DTPs resulted positive to X-gal-based β-galactosidase staining, suggesting a chemotherapy-induced senescence-like status. RNA-seq analysis and gene expression arrays allowed the identification of genes specifically down/upregulated in DTP cells. Using the intuitive enrichment analysis web-based tool Enrichr, we discovered that 54/173 of the downregulated genes characterizing the persister state were targets of a single miRNA. By quantitative real-time PCR, we validated the expression of this miR in all the models of FOLFIRI- and FLOT-obtained persister cells and we found it strongly upregulated (up to 8-fold), highlighting its possible contribute in the establishment of the persister state. Since among the target genes of this miR there are genes involved in the Homolougus Recombination (HR) system, we performed TaqMan™ Array assay for Human DNA repair gene expression on FOLFIRI-obtained DTPs. We identified a significant downregulation of genes strictly correlated to the HR machinery, suggesting that the FOLFIRI-induced persister cells can temporarily enter in a “BRCAness” status that may be used for a synthetic lethality approach using PARP inhibitors. Targeting the drug tolerant persister state is an essential approach to diminish cancer cells when they are potentially in their most vulnerable state. Citation Format: Elisabetta Puliga, Lisa Negro, Martina Olivero, Claudia Orrù, Fabrizio Maina, Daniela Conticelli, Simona Corso, Silvia Giordano. Targeting vulnerabilities of drug-tolerant persister cells in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5882.

The effect of mesenchymal stem cells administration on DNA repair gene expressions in critically ill COVID-19 patients: prospective controlled study

When the studies are evaluated, immunomodulatory effect of MSCs, administration in critically ill patients, obstacle situations in use and side effects, pulmonary fibrosis prevention, which stem cells and their products, regeneration effect, administration route, and dosage are listed under the main heading like. The effect of MSC administration on DNA repair genes in COVID-19 infection is unknown. Our aim is to determine the effect of mesenchymal stem cells (MSCs) therapy applied in critically ill patients with coronavirus infection on DNA repair pathways and genes associated with those pathways. Patients (n = 30) divided into two equal groups. Group-1: Patients in a critically ill condition, Group-2: Patients in critically ill condition and transplanted MSCs. The mechanism was investigated in eleven genes of five different pathways; Base excision repair: PARP1, Nucleotide excision repair (NER): RAD23B and ERCC1, Homologous recombinational repair (HR): ATM, RAD51, RAD52 and WRN, Mismatch repair (MMR): MLH1, MSH2, and MSH6, Direct reversal repair pathway: MGMT. It was found that MSCs application had a significant effect on 6 genes located in 3 different DNA damage response pathways. These are NER pathway genes; RAD23 and ERCC1, HR pathway genes; ATM and RAD51, MMR pathway genes; MSH2 and MSH6 (p < 0.05). Two main points were shown. First, as a result of cellular damage in critical patients with COVID-19, DNA damage occurs and then DNA repair pathways and genes are activated in reaction to this situation. Second, administration of MSC to patients with COVID-19 infection plays a positive role by increasing the expression of DNA repair genes located in DNA damage pathways.

Pharmacological modulation of RB1 activity mitigates resistance to neoadjuvant chemotherapy in locally advanced rectal cancer

Resistance to neoadjuvant chemotherapy leads to poor prognosis of locally advanced rectal cancer (LARC), representing an unmet clinical need that demands further exploration of therapeutic strategies to improve clinical outcomes. Here, we identified a noncanonical role of RB1 for modulating chromatin activity that contributes to oxaliplatin resistance in colorectal cancer (CRC). We demonstrate that oxaliplatin induces RB1 phosphorylation, which is associated with the resistance to neoadjuvant oxaliplatin-based chemotherapy in LARC. Inhibition of RB1 phosphorylation by CDK4/6 inhibitor results in vulnerability to oxaliplatin in both intrinsic and acquired chemoresistant CRC. Mechanistically, we show that RB1 modulates chromatin activity through the TEAD4/HDAC1 complex to epigenetically suppress the expression of DNA repair genes. Antagonizing RB1 phosphorylation through CDK4/6 inhibition enforces RB1/TEAD4/HDAC1 repressor activity, leading to DNA repair defects, thus sensitizing oxaliplatin treatment in LARC. Our study identifies a RB1 function in regulating chromatin activity through TEAD4/HDAC1. It also provides the combination of CDK4/6 inhibitor with oxaliplatin as a potential synthetic lethality strategy to mitigate oxaliplatin resistance in LARC, whereby phosphorylated RB1/TEAD4 can serve as potential biomarkers to guide the patient stratification.

Genomic and transcriptomic analysis of breast cancer identifies novel signatures associated with response to neoadjuvant chemotherapy

BackgroundNeoadjuvant chemotherapy (NAC) has become a standard treatment strategy for breast cancer (BC). However, owing to the high heterogeneity of these tumors, it is unclear which patient population most likely benefit from NAC. Multi-omics offer an improved approach to uncovering genomic and transcriptomic changes before and after NAC in BC and to identifying molecular features associated with NAC sensitivity.MethodsWe performed whole-exome and RNA sequencing on 233 samples (including matched pre- and post-treatment tumors) from 50 BC patients with rigorously defined responses to NAC and analyzed changes in the multi-omics landscape. Molecular features associated with NAC response were identified and validated in a larger internal, and two external validation cohorts, as well as in vitro experiments.ResultsThe most frequently altered genes were TP53, TTN, and MUC16 in both pre- and post-treatment tumors. In comparison with pre-treatment tumors, there was a significant decrease in C > A transversion mutations in post-treatment tumors (P = 0.020). NAC significantly decreased the mutation rate (P = 0.006) of the DNA repair pathway and gene expression levels (FDR = 0.007) in this pathway. NAC also significantly changed the expression level of immune checkpoint genes and the abundance of tumor-infiltrating immune and stroma cells, including B cells, activated dendritic cells, γδT cells, M2 macrophages and endothelial cells. Furthermore, there was a higher rate of C > T substitutions in NAC nonresponsive tumors than responsive ones, especially when the substitution site was flanked by C and G. Importantly, there was a unique amplified region at 8p11.23 (containing ADGRA2 and ADRB3) and a deleted region at 3p13 (harboring FOXP1) in NAC nonresponsive and responsive tumors, respectively. Particularly, the CDKAL1 missense variant P409L (p.Pro409Leu, c.1226C > T) decreased BC cell sensitivity to docetaxel, and ADGRA2 or ADRB3 gene amplifications were associated with worse NAC response and poor prognosis in BC patients.ConclusionsOur study has revealed genomic and transcriptomic landscape changes following NAC in BC, and identified novel biomarkers (CDKAL1P409L, ADGRA2 and ADRB3) underlying chemotherapy resistance and poor prognosis, which could guide the development of personalized treatments for BC.

27-hydroxycholesterol and DNA damage repair: implication in prostate cancer.

We previously reported that cholesterol homeostasis in prostate cancer (PC) is regulated by 27-hydroxycholesterol (27HC) and that CYP27A1, the enzyme that converts cholesterol to 27HC, is frequently lost in PCs. We observed that restoring the CYP27A1/27HC axis inhibited PC growth. In this study, we investigated the mechanism of 27HC-mediated anti-PC effects. We employed in vitro models and human transcriptomics data to investigate 27HC mechanism of action in PC. LNCaP (AR+) and DU145 (AR-) cells were treated with 27HC or vehicle. Transcriptome profiling was performed using the Affymetrix GeneChip™ microarray system. Differential expression was determined, and gene set enrichment analysis was done using the GSEA software with hallmark gene sets from MSigDB. Key changes were validated at mRNA and protein levels. Human PC transcriptomes from six datasets were analyzed to determine the correlation between CYP27A1 and DNA repair gene expression signatures. DNA damage was assessed via comet assays. Transcriptome analysis revealed 27HC treatment downregulated Hallmark pathways related to DNA damage repair, decreased expression of FEN1 and RAD51, and induced "BRCAness" by downregulating genes involved in homologous recombination regulation in LNCaP cells. Consistently, we found a correlation between higher CYP27A1 expression (i.e., higher intracellular 27HC) and decreased expression of DNA repair gene signatures in castration-sensitive PC (CSPC) in human PC datasets. However, such correlation was less clear in metastatic castration-resistant PC (mCRPC). 27HC increased expression of DNA damage repair markers in PC cells, notably in AR+ cells, but no consistent effects in AR- cells and decreased expression in non-neoplastic prostate epithelial cells. While testing the clinical implications of this, we noted that 27HC treatment increased DNA damage in LNCaP cells via comet assays. Effects were reversible by adding back cholesterol, but not androgens. Finally, in combination with olaparib, a PARP inhibitor, we showed additive DNA damage effects. These results suggest 27HC induces "BRCAness", a functional state thought to increase sensitivity to PARP inhibitors, and leads to increased DNA damage, especially in CSPC. Given the emerging appreciation that defective DNA damage repair can drive PC growth, future studies are needed to test whether 27HC creates a synthetic lethality to PARP inhibitors and DNA damaging agents in CSPC.

Prenatal exposure to polycyclic aromatic hydrocarbons (PAHs) augments neonatal hyperoxic lung injury: Role of cytochrome P450 (CYP)1A1, 1A2, and 1B1

Pregnant women exposed to polycyclic aromatic hydrocarbons (PAHs) are at increased risk for premature delivery. Premature infants often require supplemental oxygen, a known risk factor for bronchopulmonary dysplasia (BPD). Cytochrome P450 (CYP) enzymes have been implicated in hyperoxic lung injury. We hypothesize that prenatal PAH exposure exacerbates oxygen-mediated lung injury in neonatal mice, and that this effect is differentially altered in mice lacking the gene for (Cyp)1a1, 1a2, or 1b1. Timed pregnant wild type (WT) (C57BL/6J) mice were orally administered a PAH mixture of benzo[a]pyrene (BP) and benzo[b]fluoranthene (BbF) or the vehicle corn oil (CO) once daily on gestational days 16–19, and the dose response on postnatal lung injury was examined. In addition, timed pregnant mice with one of four genotypes, WT, Cyp1a1-null, Cyp1a2-null, and Cyp1b1-null, were treated orally with CO or PAH on gestational days 16–19 and exposed to hyperoxia or room air for 14 days. Lung injury was assessed on PND15 by radial alveolar count (RAC) and mean linear intercept (MLI) Gene expression of DNA repair genes in lung and liver were measured. Results showed that neonatal hyperoxic lung injury is augmented by prenatal PAH exposure in a dose-dependent manner. This effect was differentially altered in the Cyp-null mice, with Cyp1a2-null showing the greatest extent of lung injury. We concluded that newborn mice exposed to PAH in utero had more significant lung injury in response to hyperoxia than non-PAH exposed pups, and that CYP1A1 and CYP1A2 are protective against lung injury while CYP1B1 augments lung injury.

The DNA glycosylase NEIL2 is protective during SARS-CoV-2 infection

SARS-CoV-2 infection-induced aggravation of host innate immune response not only causes tissue damage and multiorgan failure in COVID-19 patients but also induces host genome damage and activates DNA damage response pathways. To test whether the compromised DNA repair capacity of individuals modulates the severity of COVID-19 infection, we analyze DNA repair gene expression in publicly available patient datasets and observe a lower level of the DNA glycosylase NEIL2 in the lungs of severely infected COVID-19 patients. This observation of lower NEIL2 levels is further validated in infected patients, hamsters and ACE2 receptor-expressing human A549 (A549-ACE2) cells. Furthermore, delivery of recombinant NEIL2 in A549-ACE2 cells shows decreased expression of proinflammatory genes and viral E-gene, as well as lowers the yield of viral progeny compared to mock-treated cells. Mechanistically, NEIL2 cooperatively binds to the 5’-UTR of SARS-CoV-2 genomic RNA to block viral protein synthesis. Collectively, these data strongly suggest that the maintenance of basal NEIL2 levels is critical for the protective response of hosts to viral infection and disease.

Prediction of CHO cell line stability using expression of DNA repair genes.

Chinese hamster ovary (CHO) cells are essential to biopharmaceutical manufacturing and production instability, the loss of productivity over time, is a long-standing challenge in the industry. Accurate prediction of cell line stability could enable efficient screening to identify clones suitable for manufacturing saving significant time and costs. DNA repair genes may offer biomarkers to address this need. In this study, over 40 cell lines representing various host lineages from three companies/organizations were evaluated for expression of five DNA repair genes (Fam35a, Lig4, Palb2, Pari, and Xrcc6). Expression measured in cells with less than 30 population doubling levels (PDLs) was correlated to stability profiles at 60+ PDL. Principal component analysis identified markers which separate stable and unstable CHO-DG44 cell lines. Notably, two genes, Lig4 and Xrcc6, showed higher expression in unstable CHO-DG44 cell lines with copy number loss identified as the mechanism of production instability. Expression levels across all cell ages showed lower DNA repair gene expression was associated with increased cell age. Collectively, DNA repair genes provide critical insight into long-term behavior of CHO cells and their expression levels have potential to predict cell line stability in certain cases.

Computational analysis of regulation and expression of plant DNA repair genes under various abiotic stresses reveals a stochastic correlation

Biotic/abiotic stresses are known to induce genome instability in all organisms. Plants being sessile, are more vulnerable to stresses. Thus, they have evolved mechanisms to protect their genome from the unpredictably changing environment. DNA repair genes play a critical role in preserving genome integrity. Plants have about 15 different DNA repair pathways comprising more than 100 genes. To gain a better understanding of how plant DNA repair genes perceive stress and regulate their expression, we performed a computational analysis of the putative binding sites of different abiotic stress-responsive transcription factors (ASRTFs) on their promoters, in the model plant Arabidopsis thaliana. Of the 12 different ASRTFs that we analysed, MYC2 had the highest number of 1876 putative binding sites. Diversity study in 135 DNA repair gene promoters revealed that these had about 6 to 12 types of putative binding sites for the ASRTFs. While RAD51 promoter had the highest number of 207 putative binding sites, RECQ4B had only 11. Comparison of the Arabidopsis eFP browser-based temporal/spatial expression profiles of these genes under nine different abiotic stresses revealed that all of these genes had altered expression under various stresses. Some genes exhibited similar expression profiles. To identify the reasons for their similar expression pattern, we compared the promoter sequences and putative cis-regulatory elements such as binding sites for ASRTFs and methylation-prone nucleotide repeats. Our analysis indicated that the gene expression and gene regulation mediated by ASRTFs, and DNA methylation have a stochastic correlation

Genomic Stability, DNA Repair and the SKP1-CUL1-F-Box Complex in HIV Associated Diffuse Large B-Cell Lymphoma

Introduction: Previously we demonstrated that HIV associated diffuse large B-cell lymphomas (DLBCL) of Germinal Center B-cell subtype (GCB) are more proliferative, with enhanced genomic stability and increased expression of DNA repair genes, particularly Fanconi anemia (FA) and homologous recombination (HR) genes compared to their HIV negative counterparts (Maguire et al, 2019). It is unknown if this signature is unique to the GCB subtype or if it is also observed in Activated B-cell (ABC) and Unclassified (UNC) DLBCL. Herein we validate our HIV associated GCB DLBCL findings in a larger independent cohort and report for the first time the aberrational profile of molecularly profiled HIV associated ABC DLBCL compared to their HIV negative counterparts respectively. Methods: An independent cohort of 353 DLBCL FFPE cases was assembled, including 125 HIV-DLBCL samples. All samples underwent hematopathological review to confirm diagnosis and determine tumor content. Samples with &amp;lt;60% tumor content were macro-dissected before they were RNA/DNA extracted. Cell of origin (COO) profiles were assessed using the Lymph3Cx molecular assay which can distinguish primary mediastinal B-cell lymphoma (PMBL) like from GCB, ABC and UNC DLBCL (Mottok et al 2018). Copy number (CN) analyses were performed on samples with sufficient DNA (41 HIV neg, 32 HIV pos) using the OncoScan FFPE assay kit and the resulting data analyzed using Chromosome Analysis Suite (ChAS) v4.1 and the multisample viewer module of Reproductive Health Research Analysis Software (RHAS) v1.1. Additional digital gene expression profiling was performed using the Pan-Cancer panel (NanoString, Seattle, WA) customized to include 9 BCL2 family genes for a total of 739 genes and analyzed using the NanoString nSolver Advanced Analysis module. Results: GCB, ABC, UNC and PMBL-like proportions were 50%, 30%, 14% and 6% in HIV unassociated (HIV neg) and 50%, 23%, 25% and 2% in HIV pos tumors respectively, confirming both cohorts were GCB-enriched by molecular profiling. PMBL-like cases were excluded from further analyses. OncoScan CN analyses showed that HIV pos DLBCL were significantly less aberrant than HIV neg DLBCL (median CN per sample 0.56 vs 1.01, p&amp;lt;0.001). HIV pos GCB tumors were significantly less aberrant than both HIV neg GCB tumors (median CN per sample 0.50 vs 1.11, p&amp;lt;0.001), and HIV pos ABC tumors (median CN per sample 0.50 vs 0.81, p=0.009). HIV pos ABC tumors were also less aberrant than HIV neg ABC tumors, however, the difference was not significant overall (median CN per sample 0.81 vs 1.03, p=0.162) or for gain/amplifications (median CN per sample 0.38 vs 0.42, p=0.814), but was borderline significant for loss/deletions (median CN per sample 0.31 vs 0.47, p=0.06). GEP analyses identified Cell Cycle and DNA repair as the top two upregulated pathways in HIV pos GCB tumors compared to HIV neg GCB tumors. In contrast, Cell Cycle and DNA repair were downregulated in HIV pos ABC tumors compared to HIV neg ABC tumors. However, one gene ( RBX1) was identified as significantly upregulated in both pathways in HIV pos GCB (adjusted p-value 0.0215) and HIV pos ABC (adjusted p-value 0.0046) tumors compared to their HIV neg counterparts. A second gene ( SKP1) was also significantly upregulated in HIV pos GCB (adjusted p-value 3.94E-5) and HIV pos ABC (adjusted p-value 0.0031) tumors. Together RBX1 and SKP1 encode the two principal functional subunits of the SKP1, CUL1, F-box protein (SCF) complex which is part of the ubiquitin proteasome system (UPS) and has been reported as essential for maintaining genome and chromosome stability (Thompson et al 2021). Conclusions: These results demonstrate that HIV pos DLBCL tumors are less aberrant than their HIV neg counterparts irrespective of COO subtype. However, the degree of genomic stability differs by COO, with HIV pos GCB tumors being more genomically stable than HIV pos ABC tumors. Although both have enhanced expression of UPS related SCF genes ( RBX1, SKP1), only HIV pos GCB tumors have enhanced expression of key DNA repair genes as well including Fanconi Anemia ( BRCA2/FANCD1, FANCL) and mismatch repair ( MSH2, MSH6) genes (adjusted p-values of ≤ 0.0263). Given the reported role of SCF in genomic stability maintenance, the HIV pos COO CN differences could be due to the contributions of both DNA repair and SCF mechanisms in HIV pos GCB and mainly SCF mechanisms in HIV pos ABC tumors.

Enhanced Genomic Stability in Monomorphic Post-Transplant Lymphoproliferative Disorders Is Driven By Distinct Mechanisms Stratified By EBV Status

Introduction: We previously showed that HIV associated EBV negative (-) diffuse large B-cell lymphomas (DLBCL) of germinal center origins (GCB) have enhanced genomic stability and increased expression of DNA repair genes compared to their HIV negative counterparts (Maguire et al 2019). Given that immune deficiency can be induced virally or therapeutically, including the administration of immunosuppressive drugs post solid organ or hematopoietic stem cell transplant, we questioned whether these observations are specific to HIV or if they represent a more global immune impaired signature. Here we assessed the genomic stability of monomorphic DLBCL post-transplant lymphoproliferative disorders (PTLD) and the potential role of DNA repair. Methods: A total of 49 monomorphic DLBCL PTLDs (26 EBV(-), 23 EBV positive (+)) and 170 immune competent (IC) DLBCL (EBV status unknown) were studied. All were pathologically reviewed and samples with &amp;lt;60% tumor content were macro-dissected before RNA/DNA extraction. Cell of origin (COO) profiles were assessed using the DLBCL90 molecular assay which can distinguish primary mediastinal B-cell lymphoma (PMBL) from GCB, Activated B-cell (ABC) and unclassified (UNC) DLBCL (Ennishi et al 2019). NanoString gene expression profiling (GEP) was performed using the PanCancer Pathways panel and analyzed using NanoString nSolver Advanced Analysis. Copy number (CN) analysis was performed on a subset of samples (45 PTLD, 41 DLBCL) using the OncoScan CN assay and analyzed using Chromosome Analysis Suite (ChAS) v4.1. All p-values listed were Benjamini-Hochberg adjusted and Mann-Whitney tested for the GEP and CN data respectively. Results: In contrast to IC-DLBCL which are GCB enriched (50%), COO profiling revealed PTLD to be ABC enriched (43%). CN analyses revealed that the median CN/sample for PTLD (0.57, p&amp;lt;0.001), EBV(+)PTLD (0.25, p&amp;lt;0.001) and EBV(-)PTLD (0.80, p = 0.050) tumors were significantly less aberrant than IC-DLBCL (1.01). The data also showed that EBV(+)PTLD are less aberrant than EBV(-)PTLD tumors (0.25 vs 0.80, p&amp;lt;0.001). GEP pathway analysis identified DNA repair as downregulated in PTLD and EBV(+)PTLD but upregulated in EBV(-)PTLD compared IC-DLBCL. Nine DNA repair genes ( RAD51, CCNA2a, CHEK1b, POLR2Da, PCNAa, WHSC1a, GTF2H3a, ATM c, EP300 c) were differentially expressed (DE) in EBV(-)PTLD compared to IC-DLBCL (7 increased, 2 decreased, p ≤ 0.0425): where the most overexpressed gene RAD51, a critical mediator of Homologous Recombination (HR) and the Fanconi Anemia (FA) DNA repair pathways, was not DE in PTLD or EBV(+)PTLD compared to IC-DLBCL. Five a genes were increased in PTLD (p ≤ 0.0204) but not EBV(+)PTLD, 1 b was decreased in EBV(+)PTLD (p = 0.01) but not PTLD and 2 c were reduced in PTLD regardless of EBV status compared to IC-DLBCL (p ≤ 0.0024). Twenty-8 genes were DE in EBV(+)PTLD compared to IC-DLBCL. Of these, 23 were downregulated (p ≤ 0.0418) and included HR, FA and mismatch repair genes ( CHEK1, CHEK2, BRAC1/FANCS, FANCA, FANCB, FANCE, MSH2, MSH6). All but 2/23 were similarly expressed in PTLD compared to IC-DLBCL of which 11 were significant (p ≤ 0.0356). The five upregulated genes ( ZBTB32, DDB2, NBN, POLB, RBX1, p ≤ 0.0092) were also upregulated in PTLD (p ≤ 0.0042) but not EBV(-)PTLD compared to IC-DLBCL. Two additional genes ( SKP1, FBXW7) were also identified as increased in EBV(+)PTLD and PTLD (p ≤ 0.0204) compared to IC-DLBCL. In contrast, SKP1 was increased (p = 2.7E-4) and FBXW7 was decreased (p = 0.3610) in EBV(-)PTLD compared to IC-DLBCL. Together the protein products of RBX1, SKP1 and FBXW7 comprise the functional cores of a complete UPS complex, the SCF FBXW7 E3 ubiquitin ligase which have been reported to regulate the DNA damage response and promote genomic stability. Conclusions: Like HIV associated DLBCL, these results show that PTLD is more genomically stable than IC-DLBCL, and that genomic stability is further enhanced in EBV(+)PTLD which have increased expression of UPS related genes. In contrast, EBV(-)PTLD have increased expression of DNA damage repair genes, including RAD51. In summary, monomorphic DLBCL PTLD tumors have enhanced genomic stability that appears to be mediated by two distinct mechanisms stratified by EBV status. Further work is required to assess if this stratification of mechanisms is unique to PTLD or reflects a more global immune impaired EBV related signature.

Multi-Level Control of Myeloma Cell Proliferation and Genomic Instability By the PDZ Binding Kinase (PBK)

Genomic instability and cell proliferation play important roles in the development and progression of cancer. We identified a six-gene kinase signature that correlated with overall genomic instability in multiple human cancers including multiple myeloma (MM). PDZ Binding Kinase (PBK) was one of the top kinases in this signature. Inhibiting PBK downregulated E2F and FOXM1-related pathways and impacted several members of the DREAM complex, which represses cell cycle genes during quiescence. We further evaluated whether PBK affects entry into mitosis by controlling FOXM1 phosphorylation and affects genome stability. Using co-immunoprecipitation and chromatin immunoprecipitation assays, we observed that PBK binds to and phosphorylates FOXM1, leading to upregulation of FOXM1 target genes. Overexpression of PBK increased the association of LIN54 (a major component of the MuvB complex) with phosphorylated-FOXM1 and inhibited LIN54 interaction with E2F4 in the DREAM complex. Consistently, PBK knockdown or inhibitor increased the LIN54-E2F4 interaction and inhibited the LIN54 and phosphorylated-FOXM1 interaction. The inhibitor also impaired tumor growth in vitro and significantly increased the efficacy of a chemotherapeutic agent ( P = 0.0011) in a subcutaneous mouse model of MM. Overall, these data indicate that PBK phosphorylates FOXM1, then FOXM1 interacts with MuvB which is associated with the start of mitosis, dissociating it from E2F4 in the DREAM complex, thus allowing entry into mitosis and subsequent tumor growth. Given that phosphorylation of FOXM1 also activates the expression of major DNA repair genes (including RAD51, EXO1), we investigated whether PBK affects genome stability. Surprisingly, we observed that PBK also directly phosphorylates DNA repair/HR genes, such as flap structure-specific nuclease 1 (FEN1), and that PBK inhibition reduces spontaneous and chemotherapy (melphalan)-induced genomic instability ( P &amp;lt; 0.05). A significant upregulation of PBK has also been observed in MM patient samples at relapse (after melphalan treatment) relative to paired samples collected at diagnosis (GSE19554). To further investigate the consequences of elevated PBK in vivo, we transformed the normalized PBK expression into a Z-score and used this to divide patients (in MMRF-COMPASS) into three groups based on PBK expression (i.e., high, medium and low PBK expression groups). Patients with high PBK expression had poor overall survival as compared to those with medium and low PBK expression ( P=1.127e−13). DNA replication, mismatch repair, homologous recombination, cell cycle and Fanconi anemia were the top upregulated pathways in patients with high PBK expression. Known drivers of DNA replication, including those involved in the initiation of once-per-cell cycle replication and several DNA repair and HR genes, which are part of different genomic instability signatures (such as RAD51, RAD51C, RAD54B, RAD54L, EXO1, BRCA1, FEN1, ATAD2, CENPA, MAD2L1, TTK), were significantly overexpressed in patients with high PBK expression. Interestingly, FOXM1, which can impact both replication and genome stability was also overexpressed (Log2 Fold change of 1.8; P adj = 1.27E-86). In summary, we demonstrate that PBK drives DNA replication and genomic instability in MM cells by acting at multiple levels. Therefore, PBK inhibitors investigated here have the potential to impair growth and increase the efficacy of chemotherapeutic agents while reducing spontaneous as well as chemotherapeutic agent-induced genomic instability in MM.