DNA Damage Research Articles

Genome streamlining of Pseudomonas putida B6-2 for bioremediation

ABSTRACT Microbial transformation is a favored approach for environmental remediation. However, the effectiveness of microbial remediation has been limited by the lack of chassis cells with satisfactory contaminant degradation performance. Pseudomonas putida B6-2, with a wide substrate spectrum and high solvent tolerance, is a chassis strain with great potential for application in environmental remediation. Here, guided by bioinformatic analyses and genome-scale metabolic model (GEM) predictions, we successfully optimized P. putida B6-2 by rationally reducing its nonessential genetic components and generating a more robust genome-streamlined strain, P. putida BGR4. Several improvements were observed compared with the original P. putida B6-2 strain, including a 1.4 × 10 5 -fold increase in electroporation efficiency, an 8.3-fold increase in conjugation efficiency, improved glycerol utilization capability, and increased phenol utilization after heterologous expression of the phenol monooxygenase encoded by dmpKLMNOP . Additionally, P. putida BGR4 exhibited enhanced tolerance to several stressors, including starvation, oxidative stress, and DNA damage. Transcriptomic analysis revealed that genome streamlining led to the upregulation of genes involved in the “carbon metabolism” and “tricarboxylic acid cycle” pathways in P. putida BGR4, which likely contributed to the superior phenotype of P. putida BGR4 in terms of carbon source utilization and contaminant degradation capabilities. Furthermore, the absence of four prophages was identified as a potential cause of the enhanced stress resistance observed in P. putida BGR4. Overall, we developed a combined genome-streamlining strategy involving bioinformatic analyses and GEM predictions and generated a more robust chassis strain, P. putida BGR4, which expands the repertoire of chassis cells for environmental remediation. IMPORTANCE Despite the development of many chassis cells, there is still a lack of robust chassis cells with satisfactory contaminant degradation performance. Targeted genome streamlining is an effective way to provide powerful chassis cells. However, genome streamlining does not always lead to the improved phenotypes of genome-streamlined chassis cells. In this research, a novel procedure that combined bioinformatic analyses and GEM predictions was proposed to guide genome streamlining and predict the effects of genome streamlining. This genome streamlining procedure was successfully applied to Pseudomonas putida B6-2, which was a chassis cell with great potential for application in environmental remediation and resulted in the generation of a more robust chassis cell, P. putida BGR4, thereby providing a superior chassis cell for efficient and sustainable environmental remediation and a valuable framework for guiding the genome streamlining of strains for other applications.

Isofraxidin alleviated radiation-induced testicular damage via the Nrf2/HO-1-NLRP3/ASC Axis

OBJECTIVE: Radiotherapy is used to treat patients with tumors; however, radiation (IR)-induced testicular injury, which has no effective treatment approved in clinical practice, significantly influences their prognosis and quality of life. The protective effects and underlying mechanisms of action of isofraxidin (IF) against IR-induced testicular injury were investigated. Methods: A mouse testis injury model was established using 5 Gy irradiation. H&E staining, immunofluorescence staining, and enzyme-linked immunosorbent assay were used to measure DNA damage, apoptosis, inflammatory reactions, and oxidative stress in the testes of mice after irradiation. The effectiveness of IF irradiation on testicular injury was evaluated, and the mechanisms of the related oxidative stress and inflammatory response pathways are discussed. Results: IF can improve IR-induced testicular injury by inhibiting the increased levels of DNA damage, apoptosis rate, oxidative stress, and inflammatory factors. The radioprotective effects of IF on testicular injury are mediated by the stimulation of Nrf2/HO-1 or suppression of NLRP3 inflammasome signaling pathways. In addition, crosstalk between the Nrf2/HO-1 and NLRP3 inflammasome signaling pathways was elucidated, in which the inhibition of the NLRP3 inflammasome was mediated by the activation of Nrf2 signaling with IF upon IR exposure. Conclusion: IF can be a potent radioprotective agent to mitigate testicular damage, and may provide a new therapeutic option to alleviate the side effects of radiotherapy in male patients with tumors.

Bactericidal effect of far ultraviolet-C irradiation at 222 nm against bacterial peritonitis

Far ultraviolet-C irradiation at 222 nm has potent bactericidal effects against severe infections such as peritonitis, with minimal cytotoxicity. Bacterial peritonitis due to bowel perforation is a serious condition with high mortality despite current treatments. This study investigated the safety and efficacy of intraperitoneal far ultraviolet-C irradiation at 222 nm. In vitro experiments optimized the fluid conditions for bacterial or protein concentrations prior to in vivo evaluation. In vivo efficacy was assessed in a rat peritonitis model induced by Escherichia coli, measuring intra-abdominal bacterial concentration, blood cytokine levels, and mortality rates. Safety was evaluated by analyzing cyclobutane pyrimidine dimers as markers of DNA damage in five abdominal organs: stomach, small intestine, colon, liver, and spleen. Statistical analyses employed parametric methods for normally distributed data and non-parametric methods for data without normality. Optimal in vitro conditions included 106 CFU/mL bacteria, 0.5 mW/cm2 irradiation, and 10−3 mg/mL protein. In the rat model, far ultraviolet-C irradiation at 222 nm significantly decreased intra-abdominal bacteria, reduced blood cytokines (interleukin-1β and interleukin-6), and elevated survival rates from 20% to 60%, compared to lavage alone. The formation of cyclobutane pyrimidine dimers was significantly lower with 222 nm irradiation than with 254 nm, suggesting reduced DNA damage. These findings indicate that far ultraviolet-C irradiation at 222 nm, when combined with lavage, represents a promising therapeutic strategy for bacterial peritonitis, providing effective bacterial reduction and a favorable safety profile. Further research is needed to verify these findings and investigate long-term safety and potential clinical applications.

Effect of Mexel®432 on DNA Damage in Haliotis Discus Hannai and Mesocentrotus Nudus

Mexel®432 is a surfactant-like substance that is a biosuppressant against marine fouling organisms. Therefore, to study the toxic effect of Mexel®432 on marine organisms, the wrinkled abalone and echinococcus was exposed to concentrations of 0.175 mg·L-1, 0.350 mg·L-1, 0.700 mg·L-1, 0.275 mg·L-1, 0.550 mg∙L−1, 1.100 mg·L-1 Mexel®432 solution, and single-cell gel electrophoresis (comet experiment) was used to detect different exposure times (0, 10, 20 d) DNA damage effect of digestive gland cells and echinococcus pallidae intestinal cells in wrinkled disc abalone, and tail DNA content, tail length, tail distance, and Olive tail distance were used as evaluation indexes. The results showed that different concentrations of Mexel®432 could cause DNA disintegration and tailing in digestive gland cells and intestinal cells in a short period of time (10 d), and with the increase of Mexel®432 concentration and the extension of exposure time, DNA deformation was obvious, the fragmentation increased, and the degree of damage was aggravated. When the concentration of Mexel®432 reaches 0.350 mg·L-1, it produces certain genotoxicity to the digestive gland cells of wrinkled abalone, and when the concentration of Mexel®432 reaches 1.1000 mg·L-1, it also has certain genotoxicity to the intestinal cells of Mesocentrotus nudus. The experimental results showed that Mexel®432 had a significant dose effect relationship on DNA damage in the cells of two biological tissues.

C9orf72 expansion creates the unstable folate-sensitive fragile site FRA9A

Abstract The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and C9orf72, is linked to multiple diseases. C9orf72 (GGGGCC)n expansions (C9orf72Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. C9orf72Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immuno-stimulatory or damaged DNA is unknown. Here, we show C9orf72Exp in pre-symptomatic and ALS-FTD patient cells and brains cause the folate-sensitive chromosomal fragile site, FRA9A. FRA9A centers on > 33kb of C9orf72 as highly-compacted chromatin embedded in an 8.2Mb fragility zone spanning 9p21, encompassing 46 genes, making FRA9A one of the largest fragile sites. C9orf72Exp cells show chromosomal instability, heightened global- and Chr9p-enriched sister-chromatid exchanges, truncated-Chr9s, acentric-Chr9s and Chr9-containing micronuclei, providing endogenous sources of damaged and immunostimulatory DNA. Cells from one C9orf72Exp patient contained highly-rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic C9orf72Exp repeat instability and chromosomal fragility are sensitive to folate-deficiency. Age-dependent repeat instability, chromosomal fragility, and chromosomal instability can be transferred to CNS and peripheral tissues of transgenic C9orf72Exp mice, implicating C9orf72Exp as the source. Our results highlight unappreciated effects of C9orf72 expansions that trigger vitamin-sensitive chromosome fragility, adding structural variations to the disease-enriched 9p21 locus, and likely elsewhere.

DNA damage response inhibitors in cancer therapy: lessons from the past, current status and future implications

DNA damage response inhibitors in cancer therapy: lessons from the past, current status and future implications

Heterogeneous Formation of DNA Double-Strand Breaks and Cell-Free DNA in Leukemia T-Cell Line and Human Peripheral Blood Mononuclear Cells in Response to Topoisomerase II Inhibitors

Background: Improving precision medicine in chemotherapy requires highly sensitive and easily applicable diagnostic tools. In addition, non-invasive molecular real-time monitoring of cytotoxic response is highly desirable. Here, we employed the kinetics of DNA double-strand breaks (DSB) and cell-free DNA (cfDNA) in a cell model of topoisomerase II-inhibitors in T cell leukemia (Jurkat cells) compared to normal cells (peripheral blood mononuclear cells, PBMCs). Methods: We applied automated microscopy to quantify immuno-stained phosphorylated H2AX (γH2AX) as a marker for either DNA damage response (DDR) or cell death and quantitative PCR-based analysis of nuclear and mitochondrial cfDNA concentrations. Results: Jurkat cells displayed a DDR to cytotoxic drug treatment significantly earlier than PBMCs, and etoposide (ETP) induced DSB formation faster than doxorubicin (DOX) in both Jurkat and PBMCs. Jurkat cells exhibited an earlier cytotoxic response compared to PBMC, with a significantly increased mitochondrial cfDNA formation after 2 h of DOX application. In PBMCs, increased cell death was detected after 4 h of incubation with ETP, whereas DOX treatment was less effective. Conclusions: Both automated microscopy and mitochondrial cfDNA quantification analysis indicate that (malignant) Jurkat cells are more sensitive to DOX than (healthy) PBMC. Our real-time approach can improve DDR inducing drug selection and adaptation in cancer therapy and aids in decisions for optimal patient biosampling.

FoxO1 signaling in B cell malignancies and its therapeutic targeting

FoxO transcription factors (FoxO1, FoxO3a, FoxO4, FoxO6) are a highly evolutionary conserved subfamily of the ‘forkhead’ box proteins. They have traditionally been considered tumor suppressors, but FoxO1 also exhibits oncogenic properties. The complex nature of FoxO1 is illustrated by its various roles in B cell development and differentiation, immunoglobulin gene rearrangement and cell‐surface B cell receptor (BCR) structure, DNA damage control, cell cycle regulation, and germinal center reaction. FoxO1 is tightly regulated at a transcriptional (STAT3, HEB, EBF, FoxOs) and post‐transcriptional level (Akt, AMPK, CDK2, GSK3, IKKs, JNK, MAPK/Erk, SGK1, miRNA). In B cell malignancies, recurrent FoxO1 activating mutations (S22/T24) and aberrant nuclear export and activity have been described, underscoring the potential of its therapeutic inhibition. Here, we review FoxO1's roles across B cell and myeloid malignancies, namely acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and multiple myeloma (MM). We also discuss preclinical evidence for FoxO1 targeting by currently available inhibitors (AS1708727, AS1842856, cpd10).

Nuclear IMPDH2 controls the DNA damage response by modulating PARP1 activity

Nuclear IMPDH2 controls the DNA damage response by modulating PARP1 activity

Abstract 4137913: Role of serial evaluation of myocardial inflammatory activity and oxidative stress in determining the therapeutic efficacy of immunosuppression and clinical outcome in patients with cardiac sarcoidosis

Background: Cardiac sarcoidosis (CS) was characterized by formation of granulomas in the heart. Enhancement in myocardial inflammatory activity and oxidative stress is a crucial cause of major cardiovascular adverse events (MACE). Although immunosuppressive therapy is recommended for active CS, there is no established markers for treatment and prognosis. Hypothesis: We hypothesized that the inflammation and oxidative stress in heart were associated with MACE after steroid therapy. Aims: We identified prognostic markers for MACE in patients with CS after steroid therapy. Methods: This study was a prospective observational cohort study. Patients with CS diagnosed according to Japanese criteria were enrolled in this study. Patients with abnormal accumulation of 18 F-FDG in heart were treated with steroids with standard guideline-recommended protocol. 18 F-FDG PET were performed after more than 6 months of induction. Urinary 8-hydroxy-2'-deoxyguanosine (U-8-OHdG), a marker of oxidative DNA damage, and indices of cardiac and renal function were measured. The major outcome was a composite of the first sustained ventricular tachycardia (sVT) /sudden cardiac death (SCD), hospitalization for heart failure, and radiological relapse with exacerbation of clinical manifestation. Results: Fifty consecutive patients with CS underwent steroid therapy and followed up (median follow-up period of 58 months). 39 of 50 patients underwent 18F-FDG PET/CT more than 6 months after steroid therapy. During the follow-up period, 17 of 39 patients showed MACE, consisting of sVT/SCD (N= 11), hospitalization (N= 2) and radiological relapse (N= 4). A Cox proportional-hazard model showed that the U-8-OHdG and SUV max value of FDG-PET were independent predictors of MACE. ROC analysis revealed that the cut-off values of U-8-OHdG and SUV max for predicting the MACE were 11.6 ng/mg Cr (AUC 0.913, sensitivity 86.7%, specificity 90.0%) and 4.64 (AUC 0.878, sensitivity 76.5%, specificity 91.0%), respectively (Fig.1). Patients with a U-8-OHdG ≧11.6 ng/mg Cr or SUV max ≧4.64 had a significantly higher MACE risk (P values for U-8-OHdG and SUV max were both 0.001 by Log Rank analysis) (Fig.2). It is noted that U-8-OHdG <11.6 ng/mg Cr and SUV max <4.64 after steroid therapy indicate the good responses to the immunosuppressive therapy and better prognosis (Fig.3). Conclusion: U-8-OHdG and SUV max may be useful markers in determining the therapeutic efficacy and clinical outcome in patients with CS.

TMIC-25. MICROGLIA IN THE GLIOBLASTOMA MICROENVIRONMENT SECRETE ACETYLATED AMINO ACIDS WHICH ASSIST IN THE REPAIR OF RADIATION INDUCED DNA DAMAGE

Abstract Rapid repair of DNA damage mediates genotoxic therapy resistance and poor prognosis in glioblastoma (GBM). The GBM tumor microenvironment (TME) is heterogenous, and we hypothesized that non-malignant cells support the repair of DNA damage in glioblastoma cells. Consistent with this hypothesis, we found that GBM tumors grown intracranially in mice had rapid DNA repair and were resistant to radiation compared to tumors of identical genotype grown extracranially in mouse flanks. Further interrogation of irradiated intracranial tumors using gamma-H2AX immunofluorescence revealed spatial heterogeneity in DNA damage repair, with spatially separate regions of rapid and slow gamma-H2AX resolution. Paired spatial transcriptomic analysis and gamma-H2AX immunofluorescence showed that tumor regions with rapid DNA repair were rich in microglia. Having nominated microglia from these results, we cocultured GBM cells and microglia in direct contact as well as in transwell coculture. We found that GBM cells cocultured with microglia repair DNA damage faster in a contact-independent manner. We then assessed the supernatant metabolome of microglia and GBM monocultures and cocultures and found that acetylated amino acids are secreted by microglia and consumed by GBM cells. Supplementation with acetylated amino acids promotes radiation resistance by speeding the repair of radiation-induced DNA damage. Mechanistically, acetylated amino acid supplementation allows glioma cells to re-route glucose utilization away from the TCA cycle and towards nucleotide synthesis. Hence, we show that microglia in the GBM microenvironment assist GBM cells to repair radiation induced DNA damage by secreting acetylated amino acids which are consumed by GBM cells and used to fuel the TCA cycle allowing utilization of glucose for nucleotide synthesis, thereby making the GBM cells resistant to radiation therapy.

DNAR-12. STEAROYL-COA DESATURASE REGULATES DNA DAMAGE REPAIR IN GBM BY MODULATING PARP1 ACTIVITY

Abstract Glioblastoma (GBM) is the most prevalent malignant brain cancer in adults, notorious for its aggressiveness and resistance to therapy. GBM patients commonly receive radiation therapy combined with temozolomide (TMZ), which engages tumor cells to promote lethal DNA damage and subsequent cytotoxicity. Active DNA repair mechanisms, particularly in a subset population of GBM stem-like cells (GSCs), drive therapeutic resistance, thus largely contributing to the bleak prognosis of GBM. Poly (ADP-ribose) polymerases (PARPs) are essential in various cellular processes, including DNA repair. We have previously reported that fatty acid desaturation mediated by Stearoyl-CoA desaturase 1 (SCD1) plays a cytoprotective role in GSCs. Inhibiting SCD prevents tumor initiation and inhibits DNA damage repair, hence sensitizing to radiation and TMZ. Yet, the mechanistic role of SCD in DNA damage regulation remained unknown. Here, we found that the expression of both human isoforms of SCD, SCD1, and SCD5, directly correlates with PARP protein levels and subcellular localization. Downregulating SCD1/5 in GSCs resulted in significantly lower levels of PARP1, coupled with increased DNA damage, as well as reduced DNA damage repair and chromosome assembly. Conversely, cells overexpressing SCD1/5 exhibited higher PARP1 expression and lower levels of basal DNA damage compared to control GSCs. Notably, SCD1/5 overexpression also led to increased resistance to TMZ. We show that SCD1/5 regulates PARP activity by controlling PARP1 subcellular localization. In addition to decreased total levels of PARP following SCD1/5 downregulation, we also found increased PARP1 self-PARylation. Using confocal microscopy, we show that when SCD1/5 is repressed, PARylated PARP translocates from the nucleus to the cytosol, rendering it unable to bind DNA and recruit DNA-repairing machinery. In conclusion, our study provides a direct link between fatty acid metabolism and PARP1-mediated DNA damage repair.

L-Tryptophan improves the sperm quality of threatened Abant trout (Salmo abanticus) against glyphosate toxicity: impact on oxidative stress and DNA damage

The current study focused on the ameliorating impact of TRP (5 mM) on Salmo abanticus spermatozoa exposed to sub‑lethal concentrations of GLY (2.5, 5, and 10 mg/L). Sperm quality parameters, oxidative stress indices, and DNA damage were analyzed in the post‑exposure sperm. Our data reveal that GLY has an adverse effect on motility rate and duration, sperm kinetics parameters, antioxidant status and DNA damage, and triggers antioxidant defense mechanisms at the highest dose (10 mg/L) and it has insufficient therapeutic effect. Overall, GLY poses potential hazards to male reproductive cells and may adversely affect subsequent generations of natural populations.

DNAR-05. VALIDATING THE ERK5 PROTAC OS11 AS A POTENTIAL NOVEL THERAPEUTIC INEX VIVO GLIOMA STEM CELL MODELS OF INTRATUMOURAL HETEROGENEITY AND RESIDUAL DISEASE

Abstract Glioblastoma is a grade IV brain tumour with poor survival rates of 15 months post-diagnosis, and 5-year survival rates of 10%. Limited progress over the past 40 years to improve the current treatment regimen of surgery and chemo-/radio-therapy are mainly due to tumour heterogeneity that drive resistance mechanisms. The persistence of glioma stem-like cells (GSCs) sub-populations, particularly within post-surgical residual tumour tissue. ERK5 is an emerging oncology drug target that we previously highlighted as a potential target for combined therapy with temozolomide (TMZ) in glioblastoma due to heightened ERK5 expression, which is associated with poor survival. This current project aims to expand on our previous findings by establishing whether targeting ERK5 can sensitise glioblastoma cells to TMZ using primary derived ex vivo GSC models of intratumor heterogeneity and residual disease. Primary GSC models were derived from patients at the Royal Hallamshire Hospital. The PROTAC OS11 (CRUK & University of Manchester) was used to degrade ERK5. DNA damage and cell survival were evaluated following treatment of TMZ alone and in combination with OS11. Consistent with our findings in established glioma cell lines, OS11 was able to robustly degrade ERK5 in a 3D GSC model for at least 72hrs. Combining OS11 and TMZ in GSCs led to a significant increase in DNA damage, with MGMT negative cells exhibiting a greater increase in DNA damage compared to MGMT positive cells. However, unlike our previous findings in established cell lines, this did not lead to reduced cell survival in combination with TMZ. These findings further support the preclinical use of ex vivo models that better represent intratumoural heterogeneity and post-surgical GCS niches. Overall, these data highlight the value of preclinical validation in more clinically relevant models to identify the best possible novel therapeutic approaches to pursue further towards clinical delivery.

DNAR-11. INHIBITION OF RAD52 ACTIVITY INDUCES DNA DAMAGE BY ACCUMULATING R-LOOPS IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma (DMG) is one of the most devastating childhood cancers with a median survival of < 1year from diagnosis. There is a critical need for new therapeutics for improving treatment outcomes for DMG patients. We performed genome-wide CRISPR/Cas9 screening and identified RAD52 as a potential therapeutic target in DMG cells. RAD52 inhibition, using shRNA-mediated RAD52 depletion and treatment with RAD52 inhibitor, D-I03, suppressed DMG cell proliferation. Western blotting revealed that RAD52 inhibition increased DNA double-starand breaks (DSBs) marker γH2AX while it decreased repair markers BRCA1 and RAD51. Also, RAD52 inhibition causes a sustained level of phosphorylated RAD50 and γH2AX in irradiated DMG cells over 24 hours. Immunocytochemistry (ICC) of γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. DNA repair assay showed that RAD52 inhibition suppressed DNA DSB repair through a homologous recombination pathway. RNA sequencing showed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Importantly, RAD52 inhibition increased the radiosensitivity of DMG cells. To understand the mechanism of RAD52 inhibition on DNA damage, we performed dot plot assay and ICC of S9.6 which recognizes DNA-RNA hybrids (also known as R-loops) and found accumulation of R-loop formation in DMG cells treated with RAD52 inhibitor. In addition, RAD52 inhibition decreased the expression of RNA polymerase II. Finally, the combination therapy of RAD52 inhibitor and radiation inhibited tumor growth and increased survival of mice bearing DMG patient-derived xenografts, outperforming either monotherapy. Together, our results highlight that RAD52 inhibition induces DNA damage by accumulating R-loops, results in reducing DMG cell proliferation, while also increase DMG radiosensitivty, and providing a rationale for developing combination therapy with radiation for the treatment of DMG.

DNAR-08. THY1 MEDIATES TREATMENT RESISTANCE IN GLIOBLASTOMA THROUGH RAPID REPAIR OF DNA DAMAGE

Abstract Glioblastoma (GBM) is a uniformly lethal primary intracranial neoplasm that inevitably recurs despite treatment by DNA damaging agents radiation (RT) and temozolomide (TMZ). Recurrence may be driven by inherently resistant glioma stem-like cells (GSCs). GSCs have been shown to have constitutive upregulation of DNA damage response (DDR) pathways, resulting from elevated DNA replication stress. This baseline elevated DDR has consequently been shown to confer inherent resistance to DNA damaging agents, TMZ/RT. However, factors mediating elevated replication stress and upregulated DDR in GSCs are poorly defined. Previously, we identified the cell-surface protein, THY1, as a mediator of GSC phenotype and treatment resistance in GBM. To determine mechanisms mediating treatment resistance in THY1+ cells, we engineered patient-derived neurosphere and organoid models, to either overexpress or silence THY1 expression. These models were treated with RT and collected at various time points to measure DNA repair efficiency by assessing the formation and resolution of yH2AX foci and ATM/ATR activation. In parallel, patient-derived GBM tissue from over 20 patients was analyzed using single cell RNA sequencing and spatial transcriptomics to assess pathways associated with THY1 expression. Interestingly, spatial transcriptomics and single cell RNA sequencing data demonstrates significant upregulation of GSC pathways, as well as constitutive upregulation of DDR pathways (p<2e-16) in THY1-high cells. Exploiting our patient-derived lines we determined that DNA repair activation was upregulated at baseline, measured using yH2AX foci and immunoblotting. We then measured DDR in response to radiation and found that high THY1 expression mediated rapid repair of DNA. Overall, we demonstrate that THY1 mediates therapy resistance by constitutively upregulating DDR, likely by upregulating DNA replication stress. We are now working to determine the mechanisms mediating the GSC phenotype and DNA replication stress in THY1 cells in hopes of future translation.

NCOG-37. SINGLE CENTER EXPERIENCE OF IDH INHIBITORS IN THE TREATMENT OF RECURRENT HIGH GRADE GLIOMAS

Abstract BACKGROUND The antitumor effect of IDH inhibitors in high grade gliomas (HGG) is not well known, with conflicting views regarding the role of IDH status and the potential for resistance to DNA damage treatments. We present the outcomes of patients with HGG that were treated with IDH inhibitors at our institution. METHODS We performed a retrospective review of patients with IDH mutant gliomas treated with FDA approved IDH inhibitors (ivosidenib and enasidenib) in the past 5 years at our institution. One cycle of IDH inhibitor was defined as treatment for 28 days. RESULTS We identified 10 HGG patients (both oligodendrogliomas and astrocytomas). Median age at diagnosis was 31 years and 7 were male. Seven patients were diagnosed as WHO grade 3. Six patients were WHO grade 3 oligodendrogliomas, three WHO grade 4 astrocytomas, and one patient WHO grade 3 astrocytoma. Only one patient received enasidenib and the rest were treated with ivosidenib. Eight patients were started on IDH inhibitors after 3 recurrences. Median number of cycles was 5. One patient received 23 cycles, and one patient received 11 cycles. All patients underwent resection, 4 received chemotherapy (including temozolomide, bevacizumab, and CCNU), and 4 radiation prior to starting IDH inhibitors. One patient was treated with Tumor Treating Fields. Five patients progressed during the study follow up, and 3 patients died. Four patients are still on treatment. No serious adverse events. Median PFS after starting an IDH inhibitor was 6 months. DISCUSSION IDH inhibitors were well tolerated in IDH mutant HGG patients previously treated with DNA-damaging agents. It may have a role in the treatment of HGG with multiple recurrences with 3 patients showing stable disease for more than 18 months. More studies are needed to clarify the role of IDH inhibitors in patients with IDH mutant HGG.

EXTH-65. EXPLORING ANTIHISTAMINES TO TREAT GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive and lethal primary malignant brain tumor with a 5-year survival of 6.8%. Interestingly, people with allergies, atopy, or asthma are less likely to develop GBM. A recent study recognized that high histamine receptor 1 (HRH1) expression is inversely related to overall and progression-free survival. METHODS We used a bioinformatics tool, iLINCS, which, after analyzing 99 GBM and 38 healthy samples, identified 36 drugs that can reverse GBM signatures. Based on the concordance score and blood-brain barrier permeability, we selected brompheniramine (Brom), a first-generation HRH1 antagonist, as a potential candidate. HRH1 expression analysis was done on human and mouse GBM cell lines by immunoblotting. The effect of Brom alone or in combination with temozolomide (TMZ) was investigated on proliferation, DNA damage and apoptosis in human (U118 and U251) and three syngeneic (EGFRvIII, p16-/- & GFAP Cre, PTEN+/-, p53R172H+/-, EGFRvIII & GFAP Cre, PTEN-/-, p53R172H-/- & GFAP Cre) cell lines. GBM orthograft mouse models were used to test the efficacy of combination therapy. RESULTS HRH1 is highly expressed in human and syngeneic GBM cells. This expression is further increased in TMZ-resistant cells. Interestingly, the addition of histamine, induced TMZ resistance in GBM cells. TMZ with Brom decreases proliferation in mouse and human GBM cells, and Brom is non-toxic to non-transformed cells. Combination treatment increases DNA damage and decreases the DNA repair mechanism, possibly by downregulating the expression of O6methylguanine-DNA methyltransferase (MGMT). In the GBM orthograft mouse model, the combination therapy reduced tumor volume and significantly increased survival compared to single-agent treatment. CONCLUSIONS Brom is non-toxic; Brom, in combination with TMZ, enhanced the anti-tumor efficacy of TMZ both in in vitro and in vivo GBM models. Further studies in spontaneous mouse models are planned.

Targeting G-quadruplex by TMPyP4 for inhibition of colorectal cancer through cell cycle arrest and boosting anti-tumor immunity

AbstractG-quadruplex (G4) is a noncanonical DNA secondary structure known to induce DNA damage and regulate the expression of immune-related genes. We aim to exploit the G4 folding as a treatment strategy to trigger anti-tumor immune response. In this study, we observe that the abundant genomic G4 in epithelial cells coexists with increased infiltration of CD8+ T cells in colorectal cancer tissue. Furthermore, our data substantiate the inhibitory effect of the G4 ligand TMPyP4 on cancer progression while concurrently stimulating anti-tumor immunity. Mechanistically, TMPyP4 impedes cancer cell proliferation and induces G2/M cell cycle arrest. Additionally, in vivo experiments demonstrate that TMPyP4 enhances the anti-tumor immune response by triggering DNA damage and activating the cGAS-STING pathway, which fosters CD8+ T cell activation and dendritic cell maturation. Importantly, the combined treatment of TMPyP4 and anti-PD1 exhibits a synergistic therapeutic effect on colorectal cancer. In summary, our findings underscore the potential of the G4 ligand TMPyP4 as a dual strategy to target colorectal cancer: inhibiting cancer progression and augmenting anti-tumor immunity through the activation of cGAS-STING pathway.

Loss of Brcc3 in Zebrafish Embryos Increases Their Susceptibility to DNA Damage Stress

DNA double-strand breaks (DSBs) represent one of the most severe forms of genetic damage in organisms, yet vertebrate models capable of monitoring DSBs in real-time remain scarce. BRCA1/BRCA2-containing complex subunit 3 (BRCC3), also known as BRCC36, functions within various multiprotein complexes to mediate diverse biological processes. However, the physiological role of BRCC3 in vertebrates, as well as the underlying mechanisms that govern its activity, are not well understood. To explore these questions, we generated brcc3-knockout zebrafish using CRISPR/Cas9 gene-editing technology. While brcc3 mutant zebrafish appear phenotypically normal and remain fertile, they exhibit significantly increased rates of mortality and deformity following exposure to DNA damage. Furthermore, embryos lacking Brcc3 display heightened p53 signaling, elevated γ-H2AX levels, and increased apoptosis in response to DNA-damaging agents such as ultraviolet (UV) light and Etoposide (ETO). Notably, genetic inactivation of p53 or pharmacological inhibition of Ataxia-telangiectasia mutated (ATM) activity rescues the hypersensitivity to UV and ETO observed in Brcc3-deficient embryos. These findings suggest that Brcc3 plays a critical role in DNA damage response (DDR), promoting cell survival during embryogenesis. Additionally, brcc3-null mutant zebrafish offer a promising vertebrate model for real-time monitoring of DSBs.