Repair Of Radiation-induced DNA Damage Research Articles

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.

242. BRD4 INHIBITION ENHANCES THE RADIOSENSITIVITY OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA THROUGH REGULATING ATF3-MEDIATED SERINE AND NUCLEOTIDE SYNTHESIS

Abstract Background Radioresistance is a major culprit for radiotherapy failure in esophageal squamous cell carcinoma (ESCC). This study aimed to investigate the underlying mechanism of Brd4 in radiosensitivity of ESCC. Methods Brd2/3/4 proteins were assessed in radiosensitive and radioresistant ESCC tissues using IHC. A serial of functional experiments was performed to verify the significance of Brd4 in ESCC. RNA-seq and bioinformatics analyses were used to determine the potential downstream targets. The dual-luciferase reporter and ChIP assay were further examined the underlying regulatory mechanism among targets. Besides, we further verified the importance of ATF3-mediated serine and nucleotide metabolism in radiated ESCC cells. Results Brd4 is highly expressed in radio-resistant ESCC tissue. Knockdown of Brd4 led to increased DNA damage and cell apoptosis in irradiated ESCC cells. RNA-seq analyses exhibited that ATF3 was a potential downstream target of Brd4. The dual-luciferase reporter and ChIP assay demonstrated that Brd4 upregulated ATF3 expression via activation its promoter region. Besides, we found that ATF3 could facilitate the enzyme activities involved in serine and nucleotide biosynthesis pathway to promote radiation-induced DNA damage repair. Conclusion Brd4 facilitates ATF3 expression via binding to and activating ATF3 promoter region. Enhanced ATF3 further increases crucial enzymes activity in serine and nucleotide biosynthesis pathway to promote radiation-induced DNA damage repair. Targeting Brd4 is a promising treatment strategy to improve radiosensitivity in ESCC.

Knockdown of the nucleoporin Nup50 protects cells against ionizing radiation through enhancing DNA-PKcs-mediated DNA damage repair

ObjectiveTo investigate the effect and mechanism of Nup50 on radiation-induced DNA damage repair to radiation and explore the potential role of Nup50 as radioprotective target. MethodsThe Nup50 gene was knocked down in HUVEC cells using lentiviruses. Colony formation, CCK-8, and flow cytometry were performed to determine the viability, proliferation and apoptosis of HUVEC cells treated with γ-rays，respectively. The extent of DNA damage was evaluated by using comet assay and immunofluorescence staining against γ-H2AX. In addition, we explored the role of Nup50 in DNA damage response (DDR) pathways through western blotting assay. Finally, nuclear and chromatin fractionation were performed to determine the potential molecular mechanism underlying the radiation protection function of Nup50 knockdown. ResultsNup50 knockdown increased the cellular resistance to ionizing radiation. The CCK-8 data showed that cell viability was significantly increased in the Nup50 knockdown group after radiation (t ​= ​4.23, P ​< ​0.01). The Nup50 knockdown group also showed more survived colonies (t ​= ​10.06, P ​< ​0.001), less apoptosis rate (t ​= ​3.78, P ​< ​0.05) and less unrepaired DNA damage. Furthermore, Nup50 knockdown increased radiation-activated phosphorylation levels of DNA-PKcs in HUVEC cells. Finally, the nuclear and chromatin fractionation data showed that inhibiting Nup50 increased the recruitment of DNA-PKcs to chromatin after DNA damage. ConclusionsOur findings revealed that Nup50 knockdown promoted radioresistance in normal HUVEC cells by regulating DNA-PKcs pathway, suggesting Nup50 as a potential target for radiation protection.

Abstract 689: BUB1 interferes with the repair of radiation-induced DNA damage to radiosensitize triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is associated with locoregional recurrence and low survival rates. Novel molecular targets are expected to improve the efficacy of radiation therapy (RT) in TNBC. BUB1, a mitotic checkpoint Serine/Threonine-protein kinase is known to impact chromosomal segregation during mitosis. However, its precise role in DNA damage repair in response to RT remains largely unknown. We demonstrated that BUB1 is overexpressed in BC (vs. normal breast) and considerably raised in TNBC with poor survival outcomes using differential gene expression analysis. In multiple TNBC cell lines, cell proliferation assays revealed that pharmacological (BAY1816032) or genomic (CRISPR) ablation of BUB1 was cytotoxic. Using clonogenic survival assays, we observed that BUB1 ablation radiosensitized TNBC cell lines (SUM159, MDA-MB-231, MDA-MB-468, BT-549) while its ablation did not radiosensitized breast epithelial cells (MCF10A). Furthermore, by depleting endogenous BUB1 (siRNAs and CRISPR) and reintroducing wild-type (WT) or kinase-dead (KD) BUB1, we demonstrated that inhibition of BUB1 kinase function is responsible for the radiosensitization phenotype. BUB1 ablation increased tumor doubling time in SUM159 xenografts when combined with radiation. Ki67 levels were also reduced in these tumors, confirming the efficacy of the combined treatment. BUB1 ablation led to persistent radiation-induced DNA DSB (gH2AX foci) indicating decreased DNA-DSB repair. BLRR assays confirmed that BUB1 impacts the non-homologous end joining (NHEJ) pathway. Biochemical analyses showed that BUB1 ablation stabilized total- and phospho- DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1i and DNAPKi prevented stabilization. To evaluate the recruitment of DDR proteins to the chromatin following RT, soluble nuclear and chromatin-enriched fractionations were performed which illustrated an increase in recruitment of phospho- and total-DNAPK, KAP1, and ATM to chromatin indicating that BUB1 is important in the activation and recruitment of critical NHEJ proteins to DSBs. In addition, BUB1 staining of TNBC TMAs demonstrated significant correlation between BUB1 protein expression with tumor grade and stage in TNBC patients. Our findings demonstrate that ablation of BUB1 sensitizes TNBC cell lines and xenograft to RT and BUB1 mediated radiosensitization may occur through NHEJ. Our results suggest that BUB1 may serve as a novel molecular target for radiosensitization in women with TNBC. Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Pin Li, Stephen L. Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. BUB1 interferes with the repair of radiation-induced DNA damage to radiosensitize triple-negative breast 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 689.

A spatial measure-valued model for radiation-induced DNA damage kinetics and repair under protracted irradiation condition

In the present work, we develop a general spatial stochastic model to describe the formation and repair of radiation-induced DNA damage. The model is described mathematically as a measure-valued particle-based stochastic system and extends in several directions the model developed in Cordoni et al. (Phys Rev E 103:012412, 2021; Int J Radiat Biol 1–16, 2022a; Radiat Res 197:218–232, 2022b). In this new spatial formulation, radiation-induced DNA damage in the cell nucleus can undergo different pathways to either repair or lead to cell inactivation. The main novelty of the work is to rigorously define a spatial model that considers the pairwise interaction of lesions and continuous protracted irradiation. The former is relevant from a biological point of view as clustered lesions are less likely to be repaired, leading to cell inactivation. The latter instead describes the effects of a continuous radiation field on biological tissue. We prove the existence and uniqueness of a solution to the above stochastic systems, characterizing its probabilistic properties. We further couple the model describing the biological system to a set of reaction–diffusion equations with random discontinuity that model the chemical environment. At last, we study the large system limit of the process. The developed model can be applied to different contexts, with radiotherapy and space radioprotection being the most relevant. Further, the biochemical system derived can play a crucial role in understanding an extremely promising novel radiotherapy treatment modality, named in the community FLASH radiotherapy, whose mechanism is today largely unknown.

Differential Distribution of the DNA-PKcs Inhibitor Peposertib Selectively Radiosensitizes Patient-derived Melanoma Brain Metastasis Xenografts.

Radioresistance of melanoma brain metastases limits the clinical utility of conventionally fractionated brain radiation in this disease, and strategies to improve radiation response could have significant clinical impact. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is critical for repair of radiation-induced DNA damage, and inhibitors of this kinase can have potent effects on radiation sensitivity. In this study, the radiosensitizing effects of the DNA-PKcs inhibitor peposertib were evaluated in patient-derived xenografts of melanoma brain metastases (M12, M15, M27). In clonogenic survival assays, peposertib augmented radiation-induced killing of M12 cells at concentrations ≥100 nmol/L, and a minimum of 16 hours exposure allowed maximal sensitization. This information was integrated with pharmacokinetic modeling to define an optimal dosing regimen for peposertib of 125 mpk dosed just prior to and 7 hours after irradiation. Using this drug dosing regimen in combination with 2.5 Gy × 5 fractions of radiation, significant prolongation in median survival was observed in M12-eGFP (104%; P = 0.0015) and M15 (50%; P = 0.03), while more limited effects were seen in M27 (16%, P = 0.04). These data support the concept of developing peposertib as a radiosensitizer for brain metastases and provide a paradigm for integrating in vitro and pharmacokinetic data to define an optimal radiosensitizing regimen for potent DNA repair inhibitors.

Inhibition of demethylase by IOX1 modulates chromatin accessibility to enhance NSCLC radiation sensitivity through attenuated PIF1

Chromatin accessibility is a critical determinant of gene transcriptional expression and regulated by histones modification. However, the potential for manipulating chromatin accessibility to regulate radiation sensitivity remains unclear. Our findings demonstrated that the histone demethylase inhibitor, 5-carboxy-8-hydroxyquinoline (IOX1), could enhance the radiosensitivity of non-small cell lung cancer (NSCLC) in vitro and in vivo. Mechanistically, IOX1 treatment reduced chromatin accessibility in the promoter region of DNA damage repair genes, leading to decreased DNA repair efficiency and elevated DNA damage induced by γ irradiation. Notably, IOX1 treatment significantly reduced both chromatin accessibility and the transcription of phytochrome interacting factor 1 (PIF1), a key player in telomere maintenance. Inhibition of PIF1 delayed radiation-induced DNA and telomeric DNA damage repair, as well as increased radiosensitivity of NSCLC in vitro and in vivo. Further study indicated that the above process was regulated by a reduction of transcription factor myc-associated zinc finger protein (MAZ) binding to the distal intergenic region of the PIF1. Taken together, IOX1-mediated demethylase inactivation reduced chromatin accessibility, leading to elevated telomere damage which is partly due to PIF1 inhibition, thereby enhancing NSCLC radiosensitivity.

DNAR-02. MICROGLIA IN THE GLIOBLASTOMA MICROENVIRONMENT ASSIST REPAIR OF RADIATION INDUCED DNA DAMAGE IN A CONTACT INDEPENDENT MANNER

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 in the GBM TME 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 when 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 revealed that tumor regions with rapid DNA repair were rich in microglia and enriched for inflammatory response and epithelial to mesenchymal transition (EMT) pathway transcripts. Spatial transcriptomic analysis of patient tissue revealed that regions enriched in DNA damage repair pathways are also enriched for microglia and not other non-malignant cell types. Having nominated microglia, we then cocultured GBM cells and microglia and found that GBM cells cocultured with microglia repair DNA damage faster in a contact-independent manner. Hence, we show that microglia in the GBM microenvironment assist GBM cells to repair radiation induced DNA damage through inflammatory response and EMT and hence, mediate treatment resistance.

POT1 involved in telomeric DNA damage repair and genomic stability of cervical cancer cells in response to radiation.

Though telomeres play a crucial role in maintaining genomic stability in cancer cells and have emerged as attractive therapeutic targets in anticancer therapy, the relationship between telomere dysfunction and genomic instability induced by irradiation is still unclear. In this study, we identified that protection of telomeres 1 (POT1), a single-stranded DNA (ssDNA)-binding protein, was upregulated in γ-irradiated HeLa cells and in cancer patients who exhibit radiation tolerance. Knockdown of POT1 delayed the repair of radiation-induced telomeric DNA damage which was associated with enhanced H3K9 trimethylation and enhanced the radiosensitivity of HeLa cells. The depletion of POT1 also resulted in significant genomic instability, by showing a significant increase in end-to-end chromosomal fusions, and the formation of anaphase bridges and micronuclei. Furthermore, knockdown of POT1 disturbed telomerase recruitment to telomere, and POT1 could interact with phosphorylated ATM (p-ATM) and POT1 depletion decreased the levels of p-ATM induced by irradiation, suggesting that POT1 could regulate the telomerase recruitment to telomeres to repair irradiation-induced telomeric DNA damage of HeLa cells through interactions with p-ATM. The enhancement of radiosensitivity in cancer cells can be achieved through the combination of POT1 and telomerase inhibitors, presenting a potential approach for radiotherapy in cancer treatment.

Effects of the PARP Inhibitor Niraparib on the Radiosensitivity of Human Lung and Cervical Cancer Cells

DNA damage repair (DDR) initiated after DNA damage may lead to a decreased radiosensitivity of tumor cells or even radiation resistance. Previous studies indicate that PARP inhibitors block the repair of DNA single-strand breaks (SSBs), and unrepaired SSBs may continuously accumulate and form double-strand breaks (DSBs) that promotes tumor cell death. Studies have also shown that PARP inhibitors act synergistically with RT to inhibit DDR. In this study, we aim to investigate the effect of Niraparib, a clinically approved PARP inhibitor on the radiosensitivity of human lung and cervical cancer cells, and preliminarily explore the underlying mechanism. The human lung cancer cell line A549 and human cervical cancer cell line Siha were both treated with niraparib (for 1h), or X-ray (4Gy), or Niraparib (for 1h) combined with X-ray (4Gy). The effect was examined via measurements of cell proliferation by the cell counting kit-8 assay, and cell viability was detected by clone formation assays. Cell apoptosis and cell cycle distribution were investigated by flow cytometry. In human lung and cervical cancer cell lines, Niraparib combined with radiation therapy significantly inhibited cell proliferation. The proportion of apoptotic cells in cell lines treated with Niraparib plus radiation (the combination group) was significantly higher compared with control, radiation-only, and Niraparib-only groups. Additionally, the proportion of A549 cells in the G2/M phase was significantly increased in the combination group compared with the radiation-only group. The results support that PARP inhibitor Niraparib increases the radiosensitivity of tumor cells, promotes their apoptosis, and induces cell cycle redistribution. The possible mechanism is associated with the inhibition of radiation-induced DNA damage repair.

On the Emergence of the Deviation from a Poisson Law in Stochastic Mathematical Models for Radiation-Induced DNA Damage: A System Size Expansion.

In this paper, we study the system size expansion of a stochastic model for radiation-induced DNA damage kinetics and repair. In particular, we characterize both the macroscopic deterministic limit and the fluctuation around it. We further show that such fluctuations are Gaussian-distributed. In deriving such results, we provide further insights into the relationship between stochastic and deterministic mathematical models for radiation-induced DNA damage repair. Specifically, we demonstrate how the governing deterministic equations commonly employed in the field arise naturally within the stochastic framework as a macroscopic limit. Additionally, by examining the fluctuations around this macroscopic limit, we uncover deviations from a Poissonian behavior driven by interactions and clustering among DNA damages. Although such behaviors have been empirically observed, our derived results represent the first rigorous derivation that incorporates these deviations from a Poissonian distribution within a mathematical model, eliminating the need for specific ad hoc corrections.

Effects of the PARP inhibitor Niraparib on the radiosensitivity of human lung and cervical cancer cells

ObjectiveTo investigate the effect of Niraparib, a clinically approved PARP inhibitor on the radiosensitivity of human lung and cervical cancer cells, and preliminarily explore the underlying mechanism. MethodsThe human lung cancer cell line A549 and human cervical cancer cell line Siha were both treated with Niraparib for 1 ​h, or X-ray irradiation with 4 ​Gy, or Niraparib for 1 ​h combined with X-ray irradiation with 4 ​Gy. The effect was examined via measurements of cell proliferation by the cell counting kit-8 assay, and cell viability was detected by clone formation assays. Cell apoptosis and cell cycle distribution were investigated by flow cytometry. ResultsIn human lung and cervical cancer cell lines, Niraparib combined with radiation therapy significantly inhibited cell proliferation. The proportion of apoptotic cells in cell lines treated with Niraparib plus radiation (the combination group) was significantly higher compared with control, radiation, and Niraparib groups (P ​< ​0.05). Additionally, the proportion of A549 ​cells in the G2/M phase was significantly increased in the combination group compared with the radiation group (P ​< ​0.05). ConclusionPARP inhibitor Niraparib increases the radiosensitivity of tumorcells, promotes their apoptosis, and induces cell cycle redistribution. The possible mechanism is associated with the inhibition of radiation-induced DNA damage repair.

Abstract 4810: HIF1α is involved in radiation-induced DNA damage repair and anti-apoptotic pathways in pancreatic ductal adenocarcinoma

Abstract Purpose/Objective(s): Pancreatic ductal adenocarcinoma (PDAC) is characterized by an unusually high degree of hypoxia in the tumor microenvironment, which activates hypoxic signaling pathways orchestrated by hypoxia-inducible factor 1α (HIF1α). It has been shown previously that HIF1α significantly increases PDAC radioresistance and growth, though its mechanism of action is unknown. Here, we test the hypothesis that HIF1α confers these traits in PDAC through involvement in DNA damage repair and KRAS signaling in vitro. Materials/Methods: We performed studies with the KPC cell line (KrasLSL-G12D/+;TP53LSL-R172H/+). We used CRISPR-Cas9, a genome editing tool, to generate a HIF1α knockout (KO) cell line. Hypoxic microenvironments were induced using 100uM of the oxygen scavenger CoCl2. Radioresistance and double-strand DNA damage was evaluated and compared between wild-type (WT) and KO cells after treatment with increasing doses of ionizing radiation using neutral comet assays. Tumor signaling pathways were mapped and analyzed using western blot and co-immunoprecipitation. The KRASG12D inhibitor, BI-2852, was obtained from Boehringer-Ingelheim. Cancer cells were evaluated for growth and migration using cell proliferation assays and scratch wound assays following treatment with 50 uM BI-2852. Bioinformatic analysis was conducted using TCGA (The Cancer Genome Atlas) data; high and low expression were defined as samples with mRNA expression z-scores ≥ 1 and ≤ -1, respectively, Results: HIF1α KO cells demonstrated significantly increased radioresistance compared to WT cells following 8 and 10 Gy of radiation in hypoxic conditions (t-test, P&amp;lt;0.05). The olive moment of KO cells treated with the IC50 radiation dose (4 Gy) was significantly higher than that of WT cells (t-test, P&amp;lt;0.0001), suggesting that the DNA damage repair was upregulated by HIF1α. In addition, WT cells demonstrated higher tumor proliferation (t-test, P&amp;lt;0.01) and migration (t-test, P&amp;lt;0.05) compared to KO cells under hypoxia, suggesting HIF1α promoted tumor growth. WT cells treated with BI-2852 demonstrated significantly lower levels of TP53; using coimmunoprecipitation, we show that TP53 was likely degraded through Mdm2. HIF1α KO was necessary to induce high levels of apoptosis in cells treated with BI-2852. Our results suggest that HIF1α may act synergistically with KRAS to promote tumor survival through anti-apoptotic pathways that target TP53 for degradation. TCGA data also suggest that tumors with high expression of HIF1α is are associated with significantly poorer prognosis coimpared to tumors with low expression of HIF1α (Logrank test, P=0.003). Conclusion: Overall, this study demonstrates that HIF1α promotes DNA damage repair in PDAC and reveals a HIF1α/KRAS/p53 signaling axis. Future studies will test anti-HIF1α and anti-KRAS combination therapies in PDAC mouse models. Citation Format: Kevin J. Tu, Sanjit K. Roy, Amit Sawant, Hem D. Shukla. HIF1α is involved in radiation-induced DNA damage repair and anti-apoptotic pathways in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4810.

Abstract 1371: Tumor treating fields exposure causes an imbalance of reactive oxygen homeostasis likely through the cytosolic function of the fanconi′s anemia genes

Abstract The use of Tumor Treating Fields (TTFields) has been shown to significantly benefit patients with non-resectable locally advanced or metastatic glioblastoma and malignant pleural mesothelioma. Although the disruption of mitosis that was initially attributed as the mechanism of tumor cell killing, in subsequent studies, it has been shown that TTFields inhibits DNA damage repair by downregulating DNA repair genes and proteins, including those associated with Fanconi's Anemia (FA). As a result, radiation-induced DNA damage repair is reduced and DNA replication fork protection is impaired, leading to replication stress and replication fork collapse. As the FA genes play a critical role in multiple repair pathways, TTFields is likely to increase susceptibility to other chemo agents besides radiation that cause DNA damage which is repaired by FA-dependent DNA repair and replication fork maintenance pathways in nuclei. In addition to their nuclear role, the FA proteins also regulate mitophagy in the cytosol. Using a panel of non-small cell lung cancer cell lines, it was found that TTFields exposure disrupted the clearance of damaged mitochondria despite the presence of a well-defined membrane markers for autophagosomal destruction of damaged mitochondria, that being PINK1 and parkin. Analysis of electron micrographs revealed disrupted mitochondrial cristae, while radical oxygen metabolism studies identified an imbalance leading to the production of reactive oxygen species (ROS) and ultimately, cell death. The downregulation of key mitochondrial genes including DAPIT, OSCP1, ATP5S, ATP5B and COXIV that are related to electron transport and mitochondrial integrity was confirmed by gene expression analysis. The small molecule dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, changes the metabolic state of cancer cells from cytoplasmic glycolysis to mitochondrial glucose oxidation, resulting in a decrease in mitochondrial membrane potential and an increase in ROS production in cancer cells. A combination of DCA with TTFields is therefore being investigated as a means of exploiting mitochondrial dysfunction caused by TTFields. The inhibition of mitophagy suggests a new mechanism of action for TTFields exposure tied to the downregulation of the FA pathway genes, the result being both nuclear and cytosolic perturbations in cell homeostasis. This study offers an evidence-based rationale for combining TTFields with various chemotherapy agents that may cause DNA damage, induce replication stress, and expose cells to radical oxygen byproducts of disrupting electron transport chains and the disruption of mitochondrial integrity. Citation Format: Narasimha Kumar Karanam, Michael Story. Tumor treating fields exposure causes an imbalance of reactive oxygen homeostasis likely through the cytosolic function of the fanconi′s anemia genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1371.

JNJ-64619178 radiosensitizes and suppresses fractionated ionizing radiation-induced neuroendocrine differentiation (NED) in prostate cancer.

Radiation therapy (RT) is a standard treatment regimen for locally advanced prostate cancer; however, its failure results in tumor recurrence, metastasis, and cancer-related death. The recurrence of cancer after radiotherapy is one of the major challenges in prostate cancer treatment. Despite overall cure rate of 93.3% initially, prostate cancer relapse in 20-30% patients after radiation therapy. Cancer cells acquire radioresistance upon fractionated ionizing radiation (FIR) treatment, eventually undergo neuroendocrine differentiation (NED) and transform into neuroendocrine-like cells, a mechanism involved in acquiring resistance to radiation therapy. Radiosensitizers are agents that inhibit the repair of radiation-induced DNA damage. Protein arginine methyltransferase 5 (PRMT5) gets upregulated upon ionizing radiation treatment and epigenetically activates DNA damage repair genes in prostate cancer cells. In this study, we targeted PRMT5 with JNJ-64619178 and assessed its effect on DNA damage repair gene activation, radiosensitization, and FIR-induced NED in prostate cancer. γH2AX foci analysis was performed to evaluate the DNA damage repair after radiation therapy. RT-qPCR and western blot were carried out to analyze the expression of DNA damage repair genes. Clonogenic assay was conducted to find out the surviving fraction after radiation therapy. NED was targeted with JNJ-64619178 in androgen receptor (AR) positive and negative prostate cancer cells undergoing FIR treatment. JNJ-64619178 inhibits DNA damage repair in prostate cancer cells independent of their AR status. JNJ-64619178 impairs the repair of ionizing radiation-induced damaged DNA by transcriptionally inhibiting the DNA damage repair gene expression and radiosensitizes prostate, glioblastoma and lung cancer cell line. It targets NED induced by FIR in prostate cancer cells. JNJ-64619178 can radiosensitize and suppress NED induced by FIR in prostate cancer cells and can be a potential radiosensitizer for prostate cancer treatment.

FoxM1 knockdown enhanced radiosensitivity of esophageal cancer by inducing apoptosis.

Radioresistance is a main reason for local recurrence of esophageal squamous cell carcinoma (ESCC). Forkhead box M1 (FoxM1) is implicated in cancer progression and chemoresistance. This study aims to determine the role of FoxM1 in ESCC radioresistance. We found that FoxM1 protein was upregulated in ESCC tissues compared with adjacent normal tissues. In vitro assays revealed that following irradiation, Eca-109, TE-13, and KYSE-150 cells had increased levels of FoxM1 protein. FoxM1 knockdown resulted in significantly reduced colony formation and increased cell apoptosis following irradiation. Moreover, FoxM1 knockdown induced ESCC cells to accumulate in the radiosensitive G2 /M phase and impeded the repair of radiation-induced DNA damage. Mechanistic studies indicated that radiosensitization of ESCC enhanced by FoxM1 knockdown was associated with increased BAX/BCL2 ratio as well as downregulated Survivin and XIAP, followed by the activation of both extrinsic and intrinsic apoptosis pathways. In xenograft mouse model, the combination of radiation and FoxM1-shRNA led to a synergistic anti-tumor effect. In conclusion, FoxM1 is a promising target to enhance radiosensitivity of ESCC.

Glioblastoma cells have increased capacity to repair radiation-induced DNA damage after migration to the olfactory bulb.

The invasive nature of GBM combined with the diversity of brain microenvironments creates the potential for a topographic heterogeneity in GBM radioresponse. Investigating the mechanisms responsible for a microenvironment-induced differential GBM response to radiation may provide insights into the molecules and processes mediating GBM radioresistance. Using a model system in which human GBM stem-like cells implanted into the right striatum of nude mice migrate throughout the right hemisphere (RH) to the olfactory bulb (OB), the radiation-induced DNA damage response was evaluated in each location according to γH2AX and 53BP1 foci and cell cycle phase distribution as determined by flow cytometry and immunohistochemistry. RNAseq was used to compare transcriptomes of tumor cells growing in the OB and the RH. Protein expression and neuron-tumor interaction were defined by immunohistochemistry and confocal microscopy. After irradiation, there was a more rapid dispersal of γH2AX and 53BP1 foci in the OB versus in the RH, indicative of increased double strand break repair capacity in the OB and consistent with the OB providing a radioprotective niche. With respect to the cell cycle, by 6h after irradiation there was a significant loss of mitotic tumor cells in both locations suggesting a similar activation of the G2/M checkpoint. However, by 24h post-irradiation there was an accumulation of G2 phase cells in the OB, which continued out to at least 96h. Transcriptome analysis showed that tumor cells in the OB had higher expression levels of DNA repair genes involved in non-homologous end joining and genes related to the spindle assembly checkpoint. Tumor cells in the OB were also found to have an increased frequency of soma-soma contact with neurons. GBM cells that have migrated to the OB have an increased capacity to repair radiation-induced double strand breaks and altered cell cycle regulation. These results correspond to an upregulation of genes involved in DNA damage repair and cell cycle control. Because the murine OB provides a source of radioresistant tumor cells not evident in other experimental systems, it may serve as a model for investigating the mechanisms mediating GBM radioresistance.

Effect of Autophagy Inhibitors on Radiosensitivity in DNA Repair-Proficient and -Deficient Glioma Cells.

Background and Objectives: The development of radioresistance is a fundamental barrier to successful glioblastoma therapy. Autophagy is thought to play a role in facilitating the DNA repair of DNA damage foci in radiation-exposed tumor cells, thus, potentially contributing to their restoration of proliferative capacity and development of resistance in vitro. However, the effect of autophagy inhibitors on DNA damage repair is not fully clear and requires further investigation. Materials and Methods: In this work, we utilized M059K (DNA-PKcs proficient) and M059J (DNA-PKcs deficient) glioma cell lines to investigate the role of autophagy inhibitors in the DNA repair of radiation-induced DNA damage. Cell viability following radiation was determined by trypan blue exclusion in both cell lines. Cell death and autophagy assays were performed to evaluate radiation-induced cell stress responses. DNA damage was measured as based on the intensity of phosphorylated γ-H2AX, a DNA double-stranded breaks (DSBs) marker, in the presence or absence of autophagy inhibitors. Results: The cell viability assay showed that M059J cells were more sensitive to the same dose of radiation (4 Gy) than M059K cells. This observation was accompanied by an elevation in γ-H2AX formation in M059J but not in M059K cells. In addition, the DAPI/TUNEL and Senescence-associated β-galactosidase (SA-β-gal) staining assays did not reveal significant differences in apoptosis and/or senescence induction in response to radiation, respectively, in either cell line. However, acridine orange staining demonstrated clear promotion of acidic vesicular organelles (AVOs) in both cell lines in response to 4 Gy radiation. Moreover, DNA damage marker levels were found to be elevated 72 h post-radiation when autophagy was inhibited by the lysosomotropic agent bafilomycin A1 (BafA1) or the PI3K inhibitor 3-methyl adenine (3-MA) in M059K cells. Conclusions: The extent of the DNA damage response remained high in the DNA-PKcs deficient cells following exposure to radiation, indicating their inability to repair the newly formed DNA-DSBs. On the other hand, radioresistant M059K cells showed more DNA damage response only when autophagy inhibitors were used with radiation, suggesting that the combination of autophagy inhibitors with radiation may interfere with DNA repair efficiency.

Accumulation of oncometabolite D-2-Hydroxyglutarate by SLC25A1 inhibition: A metabolic strategy for induction of HR-ness and radiosensitivity

Oncogenic mutations in metabolic genes and associated oncometabolite accumulation support cancer progression but can also restrict cellular functions needed to cope with DNA damage. For example, gain-of-function mutations in isocitrate dehydrogenase (IDH) and the resulting accumulation of the oncometabolite D-2-hydroxyglutarate (D-2-HG) enhanced the sensitivity of cancer cells to inhibition of poly(ADP-ribose)-polymerase (PARP)1 and radiotherapy (RT). In our hand, inhibition of the mitochondrial citrate transport protein (SLC25A1) enhanced radiosensitivity of cancer cells and this was associated with increased levels of D-2-HG and a delayed repair of radiation-induced DNA damage. Here we aimed to explore the suggested contribution of D-2-HG-accumulation to disturbance of DNA repair, presumably homologous recombination (HR) repair, and enhanced radiosensitivity of cancer cells with impaired SLC25A1 function. Genetic and pharmacologic inhibition of SLC25A1 (SLC25A1i) increased D-2-HG-levels and sensitized lung cancer and glioblastoma cells to the cytotoxic action of ionizing radiation (IR). SLC25A1i-mediated radiosensitization was abrogated in MEFs with a HR-defect. D-2-HG-accumulation was associated with increased DNA damage and delayed resolution of IR-induced γH2AX and Rad51 foci. Combining SLC25A1i with PARP- or the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs)-inhibitors further potentiated IR-induced DNA damage, delayed DNA repair kinetics resulting in radiosensitization of cancer cells. Importantly, proof of concept experiments revealed that combining SLC25A1i with IR without and with PARPi also reduced tumor growth in the chorioallantoic membrane (CAM) model in vivo. Thereby SLC25A1i offers an innovative strategy for metabolic induction of context-dependent lethality approaches in combination with RT and clinically relevant inhibitors of complementary DNA repair pathways.

PTEN mutant non-small cell lung cancer require ATM to suppress pro-apoptotic signalling and evade radiotherapy

BackgroundDespite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy.ResultsWe demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA-PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model.ConclusionPTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.