Non-homologous End Joining Activity Research Articles

BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment.MethodsGene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays.ResultsBUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t1/2, ~8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade.ConclusionsBUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

Abstract 711: HDAC3 sustains resistance to hypofractionated radiotherapy in fusion positve rhabdomyosarcoma cells

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. The fusion-positive (FP)-RMS variant expressing chimeric oncoproteins such as PAX3-FOXO1 and PAX7-FOXO1 shows a dismal prognosis with 5-year survival of less than 30% compared to non-metastatic fusion-negative (FN)-RMS variant. In the last years, a lot of interest has focused on new targets identification to improve the radiotherapy (RT) efficacy. HDAC inhibitors (HDACi) radio-sensitize different cancer cell types including RMS. Recently, we reported that MS-275, a Class I and IV HDACi, in combination with RT affected cell survival, reduced colony formation ability, increased DNA damage repair inhibition and reactive oxygen species formation in FP-RMS cells. However, despite promising preclinical studies, HDAC inhibitors (HDACi) achieved only modest success in human clinical trials for solid tumors, frequently showing several toxicities, probably because of the limited specificity of many HDACi. Thus, a major effort is being directed toward identification of HDACi which are selective for HDAC isoforms often uniquely implicated in the radioresistance of specific cancers. In order to identify the class I HDAC responsible of radioresistance in RMS we knocked-down HDAC1, HDAC2, HDAC3, and HDAC8 expression in combination with RT. Interestingly, we observed an increased radiosensitivity only in HDAC3-depleted FP-RMS cells. Thus, we focused on HDAC3 to understand the mechanisms by which it promotes radioresistance in RMS. We observed that HDAC3 is overexpressed in RMS patients and cell lines compared to the normal counterpart. Furthermore, RMS cells are strongly dependent by HDAC3 expression while slight dependency has been observed with other class I HDACs in DepMap portal. We demonstrated that HDAC3 depletion by CRISPR in combination with RT in FP-RMS cells increases apoptosis, reduces colony formation ability, cancer stem cells population and anchorage independent growth and reduces cell growth in vivo and in vitro. Accordingly, HDAC3 depleted cells in combination with RT reduces levels and activation of key player of FP-RMS biology such as MYCN, ERK and AKT. Moreover, HDAC3 KD increases radiotherapy-induced DNA double strand break and impairs DNA repair mechanisms reducing levels and activation of both homologous recombination (HR) and non-homologous end joining (NHEJ) factors such as ATM, RAD51 and DNA-PKcs. Thus, we developed a new potent and highly specific HDAC3 inhibitor (HDAC3i). The new HDAC3i is highly specific in targeting FP-RMS cell growth in vitro while no effects have been observed in normal cells such as myoblasts and lung fibroblast. Moreover, the drug treatment in combination with radiotherapy phenotypically and molecularly recapitulated what observed in HDAC3 depleted cells.The study has been founded by Italian Association for Cancer Research (AIRC) to FM. Citation Format: Matteo Cassandri, Antonella Porrazzo, Silvia Pomella, Simona Camero, Francesca A. Aiello, Lucrezia D'Archivio, Clemens Zwergel, Beatrice Noce, Miriam Tomaciello, Francesca Vulcano, Luisa Milazzo, Francesca Pedini, Michele Signore, Alessandro Fanzani, Cinzia Marchese, Giuseppe Minniti, Sergio Valente, Antonello Mai, Francesca Megiorni, Rossella Rota, Francesco Marampon. HDAC3 sustains resistance to hypofractionated radiotherapy in fusion positve rhabdomyosarcoma cells [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 711.

Repression of the SUMO-conjugating enzyme UBC9 is associated with lowered double minutes and reduced tumor progression

ABSTRACT Double minutes (DMs), extrachromosomal gene fragments found within certain tumors, have been noted to carry onco- and drug resistance genes contributing to tumor pathogenesis and progression. After screening for SUMO-related molecule expression within various tumor sample and cell line databases, we found that SUMO-conjugating enzyme UBC9 has been associated with genome instability and tumor cell DM counts, which was confirmed both in vitro and in vivo. Karyotyping determined DM counts post-UBC9 knockdown or SUMOylation inhibitor 2-D08, while RT-qPCR and Western blot were used to measure DM-carried gene expression in vitro. In vivo, fluorescence in situ hybridization (FISH) identified micronucleus (MN) expulsion. Western blot and immunofluorescence staining were then used to determine DNA damage extent, and a reporter plasmid system was constructed to detect changes in homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Our research has shown that UBC9 inhibition is able to attenuate DM formation and lower DM-carried gene expression, in turn reducing tumor growth and malignant phenotype, via MN efflux of DMs and lowering NHEJ activity to increase DNA damage. These findings thus reveal a relationship between heightened UBC9 activity, increased DM counts, and tumor progression, providing a potential approach for targeted therapies, via UBC9 inhibition.

Abstract A004: GTP signaling links metabolism, DNA repair, and responses to genotoxic stress

Abstract OBJECTIVE: We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS: Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION: The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome cancer resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues. Citation Format: Weihua Zhou, Zitong Zhao, Angelica Lin, John Yang, Jie Xu, Kari Wilder-Romans, Annabel Yang, Andrew J. Scott, Jing Li, Sumeet Solanki, Jennifer Speth, Natalie Walker, Ayesha U. Kothari, Yangyang Yao, Erik R. Peterson, Navyateja Korimerla, Christian K. Werner, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra M Obrien, Ameer L Elaimy, Sean P. Ferris, Shuang G. Zhao, Jann N. Sarkaria, Balázs Győrffy, Shuqun Zhang, Wajd N. Al-Holou, Yoshie Umemura, Meredith A Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Marc Peters-Golden, Yatrik M. Shah, Daniel R. Wahl. GTP signaling links metabolism, DNA repair, and responses to genotoxic stress [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A004.

The BAP1 nuclear deubiquitinase is involved in the nonhomologous end-joining pathway of double-strand DNA repair through interaction with DNA-PK.

BRCA1-associated protein 1 (BAP1) has emerged as a major tumor suppressor gene in diverse cancer types, notably in malignant pleural mesothelioma (DPM), and has also been identified as a germline cancer predisposition gene for DPM and other select cancers. However, its role in the response to DNA damage has remained unclear. Here, we show that BAP1 inactivation is associated with increased DNA damage both in Met-5A human mesothelial cells and human DPM cell lines. Through proteomic analyses, we identified PRKDC as an interaction partner of BAP1 protein complexes in DPM cells and 293 T human embryonic kidney cells. PRKDC encodes the catalytic subunit of DNA protein kinase (DNA-PKcs) which functions in the nonhomologous end-joining (NHEJ) pathway of DNA repair. Double-stranded DNA damage resulted in prominent nuclear expression of BAP1 in DPM cells and phosphorylation of BAP1 at serine 395. A plasmid-based NHEJ assay confirmed a significant effect of BAP1 knockdown on cellular NHEJ activity. Combination treatment with X-ray irradiation and gemcitabine (as a radiosensitizer) strongly suppressed the growth of BAP1-deficient cells. Our results suggest reciprocal positive interactions between BAP1 and DNA-PKcs, based on phosphorylation of BAP1 by the latter and deubiquitination of DNA-PKcs by BAP1. Thus, functional interaction of BAP1 with DNA-PKcs supports a role for BAP1 in NHEJ DNA repair and may provide the basis for new therapeutic strategies and new insights into its role as a tumor suppressor.

FSP1 inhibition enhances olaparib sensitivity in BRCA-proficient ovarian cancer patients via a nonferroptosis mechanism

Poly ADP-ribose polymerase inhibitors (PARPis) exhibit promising efficacy in patients with BRCA mutations or homologous repair deficiency (HRD) in ovarian cancer (OC). However, less than 40% of patients have HRD, it is vital to expand the indications for PARPis in BRCA-proficient patients. Ferroptosis suppressor protein 1 (FSP1) is a key protein in a newly identified ferroptosis-protective mechanism that occurs in parallel with the GPX4-mediated pathway and is associated with chemoresistance in several cancers. Herein, FSP1 is reported to be negatively correlated with the prognosis in OC patients. Combination therapy comprising olaparib and iFSP1 (a FSP1 inhibitor) strongly inhibited tumour proliferation in BRCA-proficient OC cell lines, patient-derived organoids (PDOs) and xenograft mouse models. Surprisingly, the synergistic killing effect could not be reversed by ferroptosis inhibitors, indicating that mechanisms other than ferroptosis were responsible for the synergistic lethality. In addition, cotreatment was shown to induce increased γH2A.X foci and to impair nonhomologous end joining (NHEJ) activity to a greater extent than did any single drug. Mass spectrometry and immunoprecipitation analyses revealed that FSP1 interacted with Ku70, a classical component recruited to and occupying the end of double-strand breaks (DSBs) in the NHEJ process. FSP1 inhibition decreased Ku70 PARylation, impaired subsequent DNA-PKcs recruitment to the Ku complex at DSB sites and was rescued by restoring PARylation. These findings unprecedentedly reveal a novel role of FSP1 in DNA damage repair and provide new insights into how to sensitize OC patients to PARPi treatment.

RBIO-08. GTP SIGNALING LINKS ALTERED METABOLISM, DNA REPAIR AND TREATMENT RESISTANCE IN GLIOBLASTOMA

Abstract OBJECTIVE We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome GBM resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues.

Abstract 3498: GTP-dependent signaling links metabolism, non-homologous end joining and treatment responses in cancer and normal tissues

Abstract Introduction/Objectives: Purines regulate DNA repair in brain tumors through uncertain mechanisms. We sought to define the mechanisms by which purines regulate DNA repair and therapy response. Methods: Crispr/Cas9, siRNA, and CDNA overexpression and immunoblot were used to modulate and confirm protein levels. γ-H2AX and Rad51 foci were enumerated using immunofluorescence. Fluorescent reporter assays were performed to detect DNA repair activity. Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after RT and antibodies generated against novel sites. Celltiter-glo or clonogenic assay were performed to evaluate drug/RT responses. IHC was used to detect protein expression. Animal models of glioblastoma and normal tissue (irradiation of GI system) were used to assess DNA repair and treatment responses in vivo. Results: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT, slowed the repair of RT-induced DSBs and inhibited the activity of non-homologous end joining (NHEJ), but not homologous recombination (HR). These effects could be rescued by GTP (but not ATP) supplementation. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced DSB repair and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to the small GTP-activated protein Rac1. Expression of constitutively active Rac1 promoted the dephosphorylation of Abi-1 (S323) and DSB repair while these effects were blocked by dominant negative Rac1. Pharmacogenomic inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. These findings have therapeutic relevance. In GBM PDX samples, levels of p-Abi-1 (S323) negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibition of Rac1 enhanced RT responses and suppressed Abi-1 S323 dephosphorylation. Abi-1 knockout enhanced efficacy of numerous genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in normal astrocytes and enteroids and protected mice from RT-mediated gastrointestinal injury. Conclusions: GTP promotes DNA repair by activating Rac1 and PP5 to dephosphorylate Abi-1 and stimulate NHEJ. Disrupting this regulation could improve brain tumor treatment responses while augmenting it could help protect normal tissues from genotoxic injury. Citation Format: Weihua Zhou, Angelica Lin, Zitong Zhao, John Yang, Kari Wilder-Romans, Jie Xu, Sumeet Solanki, Jing Li, Annabel Yang, Andrew Scott, Ayesha Kothari, Erik Peterson, Navyateja Korimerla, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra Obrien, Sean Ferris, Shuang Zhao, Jann N. Sarkaria, Meredith Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Yatrik Shah, Daniel R. Wahl. GTP-dependent signaling links metabolism, non-homologous end joining and treatment responses in cancer and normal tissues [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 3498.

Abstract 2836: H2S donors as radiosensitizers for glioblastoma multiforme (GBM)

Abstract Glioblastoma multiforme (GBM) is an extremely aggressive CNS cancer in adults. Radiation is a cornerstone therapy but poses high risk of healthy tissue necrosis. Despite maximal treatment, GBM results in death within 15-21 months of diagnosis. These poor statistics suggest the need for a novel therapy. The use of radiosensitizing agents is a novel approach to minimizing radiation mediated healthy tissue necrosis by allowing use of lower doses to achieve comparable cytotoxic effects. Our lab has shown that sodium sulfide (Na2S), a hydrogen sulfide (H2S) donor, selectively kills GBM (T98G) cells while sparing normal human brain endothelial (hCMEC/D3) cells. Na2S also selectively radiosensitizes GBM cells and enhances cell killing with photon or proton radiation. We are currently exploring Diallyl Trisulfide (DATS), another H2S donor, as a radiosensitizing agent and have demonstrated that DATS can kill GBM cells. At a given dose of 2 Gy, radiation alone decreases T98G survival by 10%. With DATS, survival decreases by 70%, suggesting an additive effect. To determine if DATS increases DNA damage mediated cell death, T98G cells were treated with DATS, radiation, or combined DATS + radiation treatment and then analyzed for γH2AX foci, an indicator of double stand breaks (DSB). Our results show that compared to no treatment, DATS increased the number of γH2AX foci per cell by 1.5-fold, by 3-fold with radiation, and by 4-fold with combined DATS + radiation, an additive response. To elucidate the mechanism of increased DNA damage, a novel fluorescence multiplex host cell reactivation (FM-HCR) reporter system was used to assess repair activity of DSBs in live cells. T98G cells pre-treated with DATS were transfected with reporter plasmids containing site-specific lesions, mimicking radiation-like DNA damage. Repair resulted in expression of a fluorescent reporter protein, which was quantitated using flow cytometry. Our results show that DATS did not alter non-homologous end joining (NHEJ) or homologous repair (HR) activity of DSB. However, DATS did alter base excision repair (BER) activity by decreasing repair of abasic lesions and increasing repair 8oxoG lesions. We hypothesize that this attempted BER via simultaneous strand cleavage by AP endonuclease (Ape1) or glycosylase (Ogg1) may lead to build up of repair intermediates, threatening genomic stability and inducing DSB. Pursuing specific DNA repair biochemical assays is required to further understand this effect of DATS on DNA damage and repair inhibition. Citation Format: Morni Modi, Camila Weber, Qinqin Xu, Kuanling Chen, Ana Cheong, Zachery Nagel, Lynn Harrison. H2S donors as radiosensitizers for glioblastoma multiforme (GBM) [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 2836.

APE1 promotes non-homologous end joining by initiating DNA double-strand break formation and decreasing ubiquitination of artemis following oxidative genotoxic stress

BackgroundApurinic/apyrimidinic endonuclease 1 (APE1) imparts radio-resistance by repairing isolated lesions via the base excision repair (BER) pathway, but whether and how it is involved in the formation and/or repair of DSBs remains mostly unknown.MethodsImmunoblotting, fluorescent immunostaining, and the Comet assay were used to investigate the effect of APE1 on temporal DSB formation. Chromatin extraction, 53BP1 foci and co-immunoprecipitation, and rescue assays were used to evaluate non-homologous end joining (NHEJ) repair and APE1 effects. Colony formation, micronuclei measurements, flow cytometry, and xenograft models were used to examine the effect of APE1 expression on survival and synergistic lethality. Immunohistochemistry was used to detect APE1 and Artemis expression in cervical tumor tissues.ResultsAPE1 is upregulated in cervical tumor tissue compared to paired peri-tumor, and elevated APE1 expression is associated with radio-resistance. APE1 mediates resistance to oxidative genotoxic stress by activating NHEJ repair. APE1, via its endonuclease activity, initiates clustered lesion conversion to DSBs (within 1 h), promoting the activation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key kinase in the DNA damage response (DDR) and NHEJ pathway. APE1 then participates in NHEJ repair directly by interacting with DNA- PKcs. Additionally, APE1 promotes NHEJ activity by decreasing the ubiquitination and degradation of Artemis, a nuclease with a critical role in the NHEJ pathway. Overall, APE1 deficiency leads to DSB accumulation at a late phase following oxidative stress (after 24 h), which also triggers activation of Ataxia-telangiectasia mutated (ATM), another key kinase of the DDR. Inhibition of ATM activity significantly promotes synergistic lethality with oxidative stress in APE1-deficient cells and tumors.ConclusionAPE1 promotes NHEJ repair by temporally regulating DBS formation and repair following oxidative stress. This knowledge provides new insights into the design of combinatorial therapies and indicates the timing of administration and maintenance of DDR inhibitors for overcoming radio-resistance.

The botanical drug PBI-05204, a supercritical CO2 extract of Nerium oleander, sensitizes alveolar and embryonal rhabdomyosarcoma to radiotherapy in vitro and in vivo.

Treatment of rhabdomyosarcoma (RMS), the most common a soft tissue sarcoma in childhood, provides intensive multimodal therapy, with radiotherapy (RT) playing a critical role for local tumor control. However, since RMS efficiently activates mechanisms of resistance to therapies, despite improvements, the prognosis remains still largely unsatisfactory, mainly in RMS expressing chimeric oncoproteins PAX3/PAX7-FOXO1, and fusion-positive (FP)-RMS. Cardiac glycosides (CGs), plant-derived steroid-like compounds with a selective inhibitory activity of the Na+/K+-ATPase pump (NKA), have shown antitumor and radio-sensitizing properties. Herein, the therapeutic properties of PBI-05204, an extract from Nerium oleander containing the CG oleandrin already studied in phase I and II clinical trials for cancer patients, were investigated, in vitro and in vivo, against FN- and FP-RMS cancer models. PBI-05204 induced growth arrest in a concentration dependent manner, with FP-RMS being more sensitive than FN-RMS, by differently regulating cell cycle regulators and commonly upregulating cell cycle inhibitors p21Waf1/Cip1 and p27Cip1/Kip1. Furthermore, PBI-05204 concomitantly induced cell death on both RMS types and senescence in FN-RMS. Notably, PBI-05204 counteracted in vitro migration and invasion abilities and suppressed the formation of spheroids enriched in CD133+ cancer stem cells (CSCs). PBI-05204 sensitized both cell types to RT by improving the ability of RT to induce G2 growth arrest and counteracting the RT-induced activation of both Non-Homologous End-Joining and homologous recombination DSBs repair pathways. Finally, the antitumor and radio-sensitizing proprieties of PBI-05204 were confirmed in vivo. Notably, both in vitro and in vivo evidence confirmed the higher sensitivity to PBI-05204 of FP-RMS. Thus, PBI-05204 represents a valid radio-sensitizing agent for the treatment of RMS, including the intrinsically radio-resistant FP-RMS.

The Frequency of Alternative Splicing in Multiple Myeloma Is Determined By the Non-Homologous End Joining Pathway

Introduction: Alternative Splicing (AS) plays a pivotal role in regulating cellular processes in both normal and malignant cells. Previous studies indicated mutations in the spliceosome complex, such as SF3B1, can cause aberrant AS in acute myeloid leukemia and multiple myeloma (MM) patients. However, there is also a known interaction between AS and the DNA damage response in other cancer types, which we investigate here. Materials and Methods: RNA-sequencing data of newly diagnosed MM patients (n=598) from the MMRF CoMMpass dataset were utilized to generate whole transcriptome AS profiles. For each patient, RNA-seq data was aligned to Hg38 reference genome using STAR while expression level was measured by Salmon. AS profiles were established from their alignment and expression profile using SUPPA2. Splicing level was defined by Percentage of Spliced-In (PSI). UMAP plots were generated for all patients using top 20,000 AS events ranked by variation in the sample set. High-quality differential AS events between groups of patients were obtained from the following thresholds: significant splicing level difference (|PSI|>10% and P <0.05 from T-test), sufficient expression level (TPM>1), not similarly spliced (|PSI|>10% and P-value<0.05 from T-test) in normal bone marrow plasma cells (N=5). Patient samples were annotated for cytogenetic, mutation, copy number and structural variation status. Pathway analysis was conducted for differentially expressed genes (DEGs). Patients' overall survival and progression-free survival was displayed with Kaplan-Meier curves. Survival difference was measured by log-rank test and cox-regression (when other well-known risk factors were incorporated). Results: We examined differential AS by defining and comparing high and low activity of key myeloma KEGG pathways including the spliceosome, proteasome, DNA repair and ubiquitination pathways. The DNA repair pathway generated significantly more (P<0.05) differential AS events (N=1,197) than the other pathways. To examine this further, we performed the analysis for the three main DNA repair mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) pathways. The number of differential AS events (N=2,999) between NHEJ-high (N=121) and -low (N=36) samples was significantly more than for the other DNA repair pathways (P<0.05). NHEJ-high samples were significantly enriched for TP53 mutations (15% vs. 6%, P=2x10-5, Fisher's exact test), biallelic-TP53 events (10% vs. 4%, P=3x10-5), and biallelic-TENT5C (21% vs. 10%, P=1x10-9) events (Figure 1). Additionally, expression of LIG4 and XRCC4, encoding DNA IV ligase complex, a key component of the NHEJ pathway, correlated highly (, Spearman correlation) with the number of differential AS events. The NHEJ-high group also harbored significantly more structural abnormalities than the NHEJ-low group (mean 87 vs. 19, P=6x10-4, Mann-Whitney U test), indicating their different status of DNA stability that was highly associated with this pathway. Survival analysis indicated that patients in the NHEJ-high group showed a significant worse progression-free survival (PFS) compared to others (Logrank statistic=7.3, P=0.007). In a multivariate analysis including other high-risk factors such as MGP-Double-Hit, t(4;14), t(14;16), TP53 mutation, 17p del, 1q gain/amp, and ISS=III, the NHEJ-high group retained significance (Hazard Ratio=1.8, P=1.6x10-5). To incorporate AS events we further applied a backward elimination approach to automatically select an optimal prognostic model from top 200 highly variant AS events ranked by variation and 7 well-known risk factors along with high NHEJ activity in PFS for the entire population. This model contained 16 significant risk factors (P<0.05, Multivariate cox regression) and 12 of them were AS events. NHEJ-high samples were significantly enriched (P<0.05, Fisher exact test) for eight of the AS events that were associated with poor prognosis. Conclusion: AS frequency is associated with the NHEJ pathway in MM. NHEJ activity is also associated with DNA instability through increased number of structural rearrangements and is a high-risk factor. Figure 1. High expression of the NHEJ pathways is associated with TP53 and TENT5C abnormalities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel KU70-mutant human leukemic cell line generated using CRISPR-Cas9 shows increased sensitivity to DSB inducing agents and reduced NHEJ activity

KU70 (XRCC6 gene in humans) is one of the proteins in the KU70-KU80 heterodimer which is the first component recruited to broken DNA ends during DNA double-strand break repair through nonhomologous end joining (NHEJ). Previous studies have shown that Ku70 deficient mouse cells are defective in NHEJ and V(D)J recombination. In contrast, heterozygous KU70 mutant human cell lines did not show any significant change in cell viability and sensitivity towards ionizing radiation. In this study, we used CRISPR-Cas9 technique to generate a KU70 mutant (heterozygous) human pre-B leukemic cell line (N6-KU70–2-DG). We observed that the N6-KU70–2-DG cells showed a prominent reduction in the expression of both KU70 mRNA and protein. The mutant cells showed reduced cell viability, increased sensitivity to DSB inducing agents such as ionizing radiation (IR) and etoposide, and increased number of unrepaired DSBs after exposure to IR. In addition, the mutant cells showed a reduction in the NHEJ activity and increased rate of microhomology mediated joining (MMEJ) activity. KU70 mutant cells also revealed enhanced level of senescence markers following irradiation. Thus, we report a novel KU70-mutant leukemic cell line (heterozygous) with reduced NHEJ, which is sensitive to DNA damaging agents, unlike the previously reported other KU heterozygous mutant cell lines.

Loss of claudin-4 reduces DNA damage repair and increases sensitivity to PARP inhibitors (228)

Objectives: Claudin-4 is aberrantly expressed in nearly 70% of all ovarian cancer tumors, and increased claudin-4 expression conveys a worse overall survival. Poly (ADP-ribose) polymerase (PARP) inhibitors are a therapeutic option for patients with ovarian cancer. PARP inhibitors exploit deficiencies in DNA damage repair (e.g., BRCA-mutation) by inducing replication stress and DNA damage. This study aimed to define the relationship between claudin-4 expression and response to PARP inhibitors. Results: Elevated claudin-4 mRNA expression is associated with ovarian cancer, DNA damage repair pathways, and resistance to platinum-based chemotherapies and PARP inhibitors. Claudin-4 knockdown significantly sensitized ovarian cancer cells to PARP inhibitors. Specifically, loss of claudin-4 reduced the 50% inhibitory concentration (IC50) of olaparib by 70% (IC50 - 378 nM to 88 nM) and rucaparib by 85% (IC50 - 189 nM to 27.5 nM). Furthermore, loss of claudin-4 expression sensitized ovarian cancer cells to several FDA-approved chemotherapies that induce DNA damage. Claudin-4 downregulation resulted in the decrease of several DNA damage repair effectors, including 53BP1 and XRCC1. Claudin-4/53BP1/XRCC1 expression was mainly confined to the tumor cells. Claudin-4 knockdown did not change homology-directed repair but reduced both non-homologous end-joining activity by 41% (p<0.001) and 53BP1 foci formation by an average of 50% (p<0.05). In the TCGA, lower claudin-4 expression correlated to increased mutational burden (Claudin-4 “High”, 45.2 mutations vs Claudin-4 “Low” 134.6 mutations; p<0.01). In 15 primary ovarian cancer tumors (12 high-grade serous, one low-grade serous, one mucinous, and one granulosa cell), “low”/null claudin-4 expressing tumors had a significant reduction in Ki67 after PARP inhibitor treatment alone (p<0.01). In contrast, “High” claudin-4 tumors only had significantly reduced Ki67 in the claudin-4 inhibitor alone (p<0.0001) and in the CMP/olaparib combination (p<0.0001). Conclusions: Claudin-4 is related to non-homologous end-joining DNA damage repair and 53BP1 foci formation. Claudin-4 inhibition in “high” claudin-4 tumors sensitized tumors to PARP inhibition. Claudin-4 expression in high-grade ovarian cancer tumors could potentially serve as both a marker of PARP inhibitor response and a target to improve PARP inhibitor response. Objectives: Claudin-4 is aberrantly expressed in nearly 70% of all ovarian cancer tumors, and increased claudin-4 expression conveys a worse overall survival. Poly (ADP-ribose) polymerase (PARP) inhibitors are a therapeutic option for patients with ovarian cancer. PARP inhibitors exploit deficiencies in DNA damage repair (e.g., BRCA-mutation) by inducing replication stress and DNA damage. This study aimed to define the relationship between claudin-4 expression and response to PARP inhibitors. Results: Elevated claudin-4 mRNA expression is associated with ovarian cancer, DNA damage repair pathways, and resistance to platinum-based chemotherapies and PARP inhibitors. Claudin-4 knockdown significantly sensitized ovarian cancer cells to PARP inhibitors. Specifically, loss of claudin-4 reduced the 50% inhibitory concentration (IC50) of olaparib by 70% (IC50 - 378 nM to 88 nM) and rucaparib by 85% (IC50 - 189 nM to 27.5 nM). Furthermore, loss of claudin-4 expression sensitized ovarian cancer cells to several FDA-approved chemotherapies that induce DNA damage. Claudin-4 downregulation resulted in the decrease of several DNA damage repair effectors, including 53BP1 and XRCC1. Claudin-4/53BP1/XRCC1 expression was mainly confined to the tumor cells. Claudin-4 knockdown did not change homology-directed repair but reduced both non-homologous end-joining activity by 41% (p<0.001) and 53BP1 foci formation by an average of 50% (p<0.05). In the TCGA, lower claudin-4 expression correlated to increased mutational burden (Claudin-4 “High”, 45.2 mutations vs Claudin-4 “Low” 134.6 mutations; p<0.01). In 15 primary ovarian cancer tumors (12 high-grade serous, one low-grade serous, one mucinous, and one granulosa cell), “low”/null claudin-4 expressing tumors had a significant reduction in Ki67 after PARP inhibitor treatment alone (p<0.01). In contrast, “High” claudin-4 tumors only had significantly reduced Ki67 in the claudin-4 inhibitor alone (p<0.0001) and in the CMP/olaparib combination (p<0.0001). Conclusions: Claudin-4 is related to non-homologous end-joining DNA damage repair and 53BP1 foci formation. Claudin-4 inhibition in “high” claudin-4 tumors sensitized tumors to PARP inhibition. Claudin-4 expression in high-grade ovarian cancer tumors could potentially serve as both a marker of PARP inhibitor response and a target to improve PARP inhibitor response.

Analysis of DNA Repair-Related Prognostic Function and Mechanism in Gastric Cancer.

DNA repair mechanisms have been proven to be essential for cells, and abnormalities in DNA repair could cause various diseases, such as cancer. However, the diversity and complexity of DNA repair mechanisms obscure the functions of DNA repair in cancers. In addition, the relationships between DNA repair, the tumor mutational burden (TMB), and immune infiltration are still ambiguous. In the present study, we evaluated the prognostic values of various types of DNA repair mechanisms and found that double-strand break repair through single-strand annealing (SSA) and nonhomologous end-joining (NHEJ) was the most prognostic DNA repair processes in gastric cancer (GC) patients. Based on the activity of these two approaches and expression profiles, we constructed a HR-LR model, which could accurately divide patients into high-risk and low-risk groups with different probabilities of survival and recurrence. Similarly, we also constructed a cancer-normal model to estimate whether an individual had GC or normal health status. The prognostic value of the HR-LR model and the accuracy of the cancer-normal model were validated in several independent datasets. Notably, low-risk samples, which had higher SSA and NHEJ activities, had more somatic mutations and less immune infiltration. Furthermore, the analysis found that low-risk samples had higher and lower methylation levels in CpG islands (CGIs) and open sea regions respectively, and had higher expression levels of programmed death-ligand 1 (PD-L1) and lower methylation levels in the promoter of the gene encoding PD-L1. Moreover, low-risk samples were characterized primarily by higher levels of CD4+ memory T cells, CD8+ naive T cells, and CD8+ TEM cells than those in high-risk samples. Finally, we proposed a decision tree and nomogram to help predict the clinical outcome of an individual. These results provide an improved understanding of the complexity of DNA repair, the TMB, and immune infiltration in GC, and present an accurate prognostic model for use in GC patients.

Inhibiting nonhomologous end-joining repair would promote the antitumor activity of gemcitabine in nonsmall cell lung cancer cell lines.

Nonsmall cell lung cancer (NSCLC) is a major type of lung cancer. In current study, we aim to evaluate whether the combination of Ku70/80 heterodimer protein inhibitor STL127705 and gemcitabine would be more favorable approach for the treatment of NSCLC compared with monotreatment with gemcitabine. Clongenic survival assay was used to determine the survival and sensitivity to irradiation. H1299 was stained with fluorescein isothiocyanate-Annexin V, and cell apoptosis was measured by flow cytometry. H1299 cells were transfected with nonhomologous end-joining (NHEJ) repair reporter, and stable cell line was selected by puromycin. NHEJ activity was determined based on the intensity of green fluorescent protein. DNA double-strand breaks (DSBs) were determined by the fluorescence intensity of γH2AX using flow cytometry. The mRNA expressions of Ku70 and Ku80 were determined using quantitative real-time PCR. Combination of STL127705 enhanced sensitivity of NSCLC cell lines to irradiation when compared with treatment with gemcitabine alone. However, small cell lung cancer cell line was not affected. H1299 cells treated with STL127705 in combination with gemcitabine showed a significantly increased apoptosis compared with H1299 cells treated with gemcitabine alone. Moreover, STL127705 treatment dramatically reduced NHEJ activity in H1299 cells when compared with gemcitabine single treatment. Increased DSBs were consistently observed in H1299 when treated with the combination of STL127705 and gemcitabine. However, the mRNA levels of Ku70 and Ku80 were upregulated by the combination treatment. It demonstrated that STL127705 enhanced antitumor activity of gemcitabine. Mechanistically, treatment with STL127705 enhanced DNA damage via inhibiting NHEJ pathway, blocking DNA-PK, and forming Ku70/80 heterodimer, eventually leading to tumor cells apoptosis.

DNA Repair Capacity for Personalizing Risk and Treatment Response − Assay Development and Optimization in Human Peripheral Blood Mononuclear Cells (PBMCs)

DNA repair capacity (DRC) is the ability of a cell to repair DNA damage. Differential DRC plays an important role in human disease, including lung and other cancers. Measuring DRC could aid in translational disease research and in personalizing treatment. We developed and optimized a flow cytometry-based assay to measure individual DRC using GFP-expressing plasmids modified by ultraviolet (UV) light for nucleotide excision repair (NER) and restriction enzyme digestion to induce a blunt double-strand cut between promoter and GFP expression regions for nonhomologous end joining (NHEJ). Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy volunteers were used to measure DRC and optimize the assay. Pathway specificity of the NHEJ DRC assay was confirmed using Ku80-/- MEF cells, which showed a 6-fold reduction in NHEJ compared to Ku80+/+. Using a cell mixing assay, we show a linear correlation between NHEJ DRC and the expected concentration of Ku80. NHEJ DRC measurements in cryopreserved PBMCs are quantifiable with low interindividual and inter-assay variability, and a titratable decrease in NHEJ activity was observed in PBMCs treated with the DNA-PK inhibitor NU7441. Pathway specificity of the NER DRC assay was confirmed by a decrease in measured NER activity in human XPC deficient compared to XPC proficient fibroblasts, with a linear correlation measured between NER DRC and expected XPC concentration by cell mixing assay. NER DRC is quantifiable, reproducible, and titratable in PBMCs from healthy volunteers. We measured both NER and NHEJ DRC in PBMCs obtained from newly diagnosed, untreated lung cancer patients; measured DRC differed in these PBMCs compared to healthy volunteers. With further investigation, measurement of NER and NHEJ DNA repair capacity may be useful in personalizing disease risk and response to DNA damaging therapies and small molecular inhibitors of DNA repair pathways using readily available human PBMCs.

Targeted Gene Insertion and Replacement in the Basidiomycete Ganoderma lucidum by Inactivation of Nonhomologous End Joining Using CRISPR/Cas9.

Targeted gene insertion or replacement is a promising genome-editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum, targeted gene insertion and replacement remain a serious challenge due to the low efficiency of homologous recombination (HR) in this species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the nonhomologous end joining (NHEJ) pathway, at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma, we first inactivated the NHEJ pathway via disruption of the Ku70 gene (ku70) using a dual single guide RNA (sgRNA)-directed gene deletion method. Disruption of the ku70 gene significantly decreased NHEJ activity in G. lucidum. Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of targeted gene insertion and replacement, respectively, when target DNA with the orotidine 5'-monophosphate decarboxylase (ura3) gene and 1.5-kb homologous 5'- and 3'-flanking sequences was used as a donor template, compared to 3.3% and 0%, respectively, at these targeted sites for a control strain (Cas9 strain). Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum. IMPORTANCE Functional genomic studies in Ganoderma have been hindered by the absence of adequate genome-engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum, targeted gene insertion and replacement have remained a serious challenge due to the low efficiency of HR in these species, although such precise genome modifications, including site mutations, site-specific integrations, and allele or promoter replacements, would be incredibly valuable. In this work, we inactivated the NHEJ repair mechanism in G. lucidum by disrupting the ku70 gene using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70-disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

Tyrosine Kinase Inhibitor-Induced Defects in DNA Repair Sensitize FLT3(ITD)-Positive Acute Myeloid Leukemia Quiescent and Proliferative Cells to PARP Inhibitors

Mutations in the FMS-like tyrosine-kinase 3 (FLT3) such as internal tandem duplications (ITD) can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors, which are not curative. Poly (ADP-ribose) polymerase (PARP) inhibitors have been recently successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying deficiency in a major DNA double strand break (DSB) repair pathway - homologous recombination (HR). Using personalized medicine approach we recently reported that a cohort of AMLs displayed deficiencies in HR and also in another major DSB repair pathway - non-homologous end-joining (NHEJ) (Nieborowska-Skorska et al., J.Clin.Invest., 2017). HR and NHEJ play a key roles in DSB repair in proliferating (HR and NHEJ) and quiescent (NHEJ) cells, and HR/NHEJ deficiency sensitized quiescent and proliferating AML stem cells to synthetic lethality triggered by PARP inhibitors olaparib and BMN673 (talazoparib). Although AML cells expressing FLT3(ITD) displayed activation of HR and NHEJ activities, we show here that inhibition of FLT3(ITD) kinase by tyrosine kinase inhibitor AC220 caused downregulation of selected DNA repair proteins such as BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in simultaneous inhibition of HR and NHEJ. PARP inhibitors olaparib and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells thus mimicking synthetic lethality. In addition, combination of FLT3 inhibitor and PARP inhibitor in vitro eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells and leukemic progenitors from human and mouse leukemia samples. Moreover, AC220 combined with BMN673 were highly effective in vivo against FLT3(ITD)-positive primary AML xenograft cells, including leukemia initiating cells. In conclusion, we postulate that FLT3 inhibitor-induced deficiencies in HR and NHEJ sensitized FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP inhibitor. Therefore FLT3(ITD) could be used as a precision medicine marker identifying patients with AMLs, which may benefit from therapeutic regimen combining FLT3 and PARP inhibitors. DisclosuresKoschmieder:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Roche: Other: Clinical Trial participation. Valent:BMS: Honoraria; Deciphera: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Blueprint: Research Funding; Celgene: Honoraria, Research Funding; Pfizer: Honoraria; Teva: Honoraria; Ariad: Honoraria, Research Funding; Incyte: Honoraria.

Impact of Bioflavonoid Exposure on DNA Damage Response and of Non-Homologous End Joining (NHEJ) Pathway Repair Proteins in Stem Cells

DNA double-strand breaks (DSBs) activate DNA damage response signaling pathways and multiple repair pathways depending on cell cycle stage and differentiation state. An array of recurrent, non-random chromosomal translocations are associated with hematologic malignancies, and we are interested in determining the role of specific DNA repair proteins or pathway choice in the etiology of these rearrangements. Classic non-homologous end joining (NHEJ) utilizes LIG4, XRCC4, and XLF during the final steps; LIG4 and XRCC4 deficiency is implicated in both leukemais and lymphomas but the role of XLF deficiency in leukemia etiology is not clear. Activation of alternative NHEJ (Alt-NHEJ) is largely dependent on CtIP that promotes the resection of DNA ends to uncover micro-homologies promoting the formation of large deletions and chromosomal translocations. Studies suggest that loss of efficient activity in one NHEJ repair pathway may promote use of the other more error-prone pathway. The topoisomerase II poison etoposide is a cytotoxic chemotherapeutic agent associated with therapy-related leukemias characterized by translocations involving the MLL gene at 11q23. Leukemias with MLL gene fusions have been detected in newborns indicating that these translocations can occur in utero, and additional evidence from our lab and others suggests maternal exposure to environmental topoisomerase II inhibitors such as bioflavonoids have the potential to promote these events. We are interested in understanding the crosstalk involved in bioflavonoid exposure, DNA damage response signaling, and repair pathway choice as a model system to understand the underlying mechanisms of oncogenic transformation in infant leukemias.Using established in vitro differentiation protocols, we expose embryonic stem (ES) cells and hematopoietic stem cells (HSC) derived from them to a panel of bioflavonoids alone or in combination for one hour at increasing doses ranging from 0μM-200μM based on determined LD50 values. As a control, cells are exposed to etoposide at the same doses. Following exposure, cells recovered for a period of 0-24hrs, followed by protein extraction and Western blotting. Quantitation of increases in total gH2AX confirm DNA damage, presence of Ku70 and XLF to support classical NHEJ, and CtIP to support Alt-NHEJ. Cells exposed to bioflavonoids genistein and quercetin as well as etoposide show elevated γH2AX levels by 4 hrs post-treatment compared to untreated cells. Parallel studies show dose-dependent increase in the presence of γH2AX foci in these cells. These results confirm that although bioflavonoids have pleiotropic effects, they directly promote DSBs in a short period of time post-exposure. Protein levels of Ku70 and XLF in cells exposed to genistein, quercetin, and etoposide at doses of 25, 50, 75 and 100μM are not significantly different from untreated cells. However, CtIP levels are elevated in cells exposed to quercetin compared to all other groups suggesting a specific potential for this bioflavonoid to promote Alt-NHEJ repair and genome instability.To correlate the activity of these DNA repair protein activities with downstream repair products, we use a model cell system in which parental ES cells were engineered to contain two constructs: GFP exons inserted into MLL and AF9 bcr transgene fragments. Aberrant repair resulting in chromosomal translocations are scored by GFP+ fluorescence. In both ES and derived HSC populations from them translocations can be scored following exposure to bioflavonoids and etoposide. Ongoing experiments include downregulation of XLF or CtIP by siRNA in cells prior to exposure to these compounds. Results will be presented on impact of downregulation of these two NHEJ proteins on 1) amount and persistence of γH2AX; 2) protein levels of other DNA repair pathway proteins, and 3) frequency of generating chromosomal translocations.Understanding the integrated activity and role of these proteins in preventing or promoting translocations, particularly in the context of exposure to a wide range of environmental and dietary compounds, will be critical to understanding the potential for specific isoforms or mutants to increase leukemia risk. DisclosuresNo relevant conflicts of interest to declare.