DNA Double-strand Break Repair Activity Research Articles

GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity.

DNA double-strand break (DSB) induction and repair are important events for determining cell survival and the outcome of cancer radiotherapy. The DNA-dependent protein kinase (DNA-PK) complex functions at the apex of DSBs repair, and its assembly and activity are strictly regulated by post-translation modifications (PTMs)-associated interactions. However, the PTMs of the catalytic subunit DNA-PKcs and how they affect DNA-PKcs's functions are not fully understood. Mass spectrometry analyses were performed to identify the crotonylation sites of DNA-PKcs in response to γ-ray irradiation. Co-immunoprecipitation (Co-IP), western blotting, in vitro crotonylation assays, laser microirradiation assays, in vitro DNA binding assays, in vitro DNA-PK assembly assays and IF assays were employed to confirm the crotonylation, identify the crotonylase and decrotonylase, and elucidate how crotonylation regulates the activity and function of DNA-PKcs. Subcutaneous xenografts of human HeLa GCN5 WT or HeLa GCN5 siRNA cells in BALB/c nude mice were generated and utilized to assess tumor proliferation in vivo after radiotherapy. Here, we reveal that K525 is an important site of DNA-PKcs for crotonylation, and whose level is sharply increased by irradiation. The histone acetyltransferase GCN5 functions as the crotonylase for K525-Kcr, while HDAC3 serves as its dedicated decrotonylase. K525 crotonylation enhances DNA binding activity of DNA-PKcs, and facilitates assembly of the DNA-PK complex. Furthermore, GCN5-mediated K525 crotonylation is indispensable for DNA-PKcs autophosphorylation and the repair of double-strand breaks in the NHEJ pathway. GCN5 suppression significantly sensitizes xenograft tumors of mice to radiotherapy. Our study defines K525 crotonylation of DNA-PKcs is important for the DNA-PK complex assembly and DSBs repair activity via NHEJ pathway. Targeting GCN5-mediated K525 Kcr of DNA-PKcs may be a promising therapeutic strategy for improving the outcome of cancer radiotherapy.

CDK2 phosphorylation of Werner protein (WRN) contributes to WRN's DNA double-strand break repair pathway choice.

Werner syndrome (WS) is an accelerated aging disorder characterized by genomic instability, which is caused by WRN protein deficiency. WRN participates in DNA metabolism including DNA repair. In a previous report, we showed that WRN protein is recruited to laser‐induced DNA double‐strand break (DSB) sites during various stages of the cell cycle with similar intensities, supporting that WRN participates in both non‐homologous end joining (NHEJ) and homologous recombination (HR). Here, we demonstrate that the phosphorylation of WRN by CDK2 on serine residue 426 is critical for WRN to make its DSB repair pathway choice between NHEJ and HR. Cells expressing WRN engineered to mimic the unphosphorylated or phosphorylation state at serine 426 showed abnormal DSB recruitment, altered RPA interaction, strand annealing, and DSB repair activities. The CDK2 phosphorylation on serine 426 stabilizes WRN’s affinity for RPA, likely increasing its long‐range resection at the end of DNA strands, which is a crucial step for HR. Collectively, the data shown here demonstrate that a CDK2‐dependent phosphorylation of WRN regulates DSB repair pathway choice and cell cycle participation.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F , and MPLW515L , or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.

The effect of eIF3a on anthracycline-based chemotherapy resistance by regulating DSB DNA repair

BackgroundAnthracycline are inhibitors of topoisomerase II leading to DNA double strand breaks, and it is widely used for treatment of breast cancer. eIF3a is the largest subunit of eukaryotic translation initiation factor 3 (eIF3) and highly expressed in breast cancer. In this study, we investigated the role of eIF3a in DSB DNA repair and the response of breast cancer patients to anthracycline-based chemotherapy. MethodsMTT assay was used to detect anthracycline sensitivity in cell lines. Real-time reverse transcriptase PCR, western blotting and immunofluorescence were performed to assess changes in gene expression levels. Cometassay and end-joining activity assay were conducted to explore the effect of eIF3a in NHEJ repair. Luciferase reporter assay was performed to detect LIG4 5′UTR activity. Immunohistochemistry was used to detect eIF3a, LIG4 and DNA-PKcs expression levels in breast cancer tissues. ResultsThe results showed that eIF3a increased cellular response to anthracyclines by regulating DSB repair activity via influencing the expression of LIG4 and DNA-PKcs at translational level. Breast cancer patients with high level of eIF3a or low level of LIG4 or low level of DNA-PKcs had better anthracycline-based chemotherapy prognosis compared. Moreover, Combined expressions of eIF3a, LIG4 and DNA-PKcs could be better to predict PFS in breast cancer patients with anthracycline-based chemotherapy. ConclusionOur findings suggest that eIF3a effects anthracycline-based chemotherapy response by regulating DSB DNA repair.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that both affect the epigenetic regulation of DNA methylation. Although the proteins antagonistically regulate the epigenetic mark of 5-methylcytosine (5-mC), where DNMT3A catalyzes addition of 5-mC while TET2 oxidizes 5-mC as a first step in DNA demethylation, mutations in both genes appear in a similar spectrum of human hematopoietic malignancies. Mutations in both genes often co-occur with activating mutations in oncogenic tyrosine kinases (OTKs) such as FLT3ITD, BCR-ABL1, JAK2V617F, MPLW515L or mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Moreover, while the mutations exert divergent effect on primitive hematopoietic progenitor cells, they lead to similar disease phenotypes, suggesting the roles of these mutations in hematopoietic malignancies may relate to mechanisms outside of DNA methylation. OTK-positive malignant cells accumulate high numbers of spontaneous and drug-induced DNA double-strand breaks (DSBs) in comparison to normal cells, but they manage to survive because of their enhanced/altered ability to repair these breaks. DSBs, the most lethal DNA lesions, are repaired by two major mechanisms, BRCA1/2-dependent homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (D-NHEJ). Both HR and D-NHEJ repair DSBs in proliferating cells, while D-NHEJ plays a major role in quiescent cells. PARP1 -dependent alternative NHEJ (Alt-NHEJ) serves as back-up in both proliferating and quiescent cells. The existence of these redundant pathways creates the opportunity to employ a phenomenon called "synthetic lethality", which was originally applied to eliminate cancer cells with mutations in BRCA1 and BRCA2 by PARP inhibitor (PARPi). Our previous report [M. Nieborowska-Skorska et al., Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells. J Clin Invest 127, 2392-2406 (2017)] suggested that certain leukemias are sensitive to PARPi-triggered synthetic lethality. Here we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing OTKs. Malignant TET2-deficient cells display downregulation of BRCA1 and LIG4 resulting in reduced activity of HR and D-NHEJ, respectively, and rely on Alt-NHEJ to protect them from the toxic effects of replication stress and drug-induced DSBs. Conversely, DNMT3A-deficient cells favor HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells are sensitive to PARPi treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity and/or TET2 - Wilms tumor 1 (WT1) binding ability were responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Our findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Tallman:Abbvie: Research Funding; Cellerant: Research Funding; Orsenix: Research Funding; ADC Therapeutics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Rafael: Research Funding; Amgen: Research Funding; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties. Martinelli:Roche: Consultancy; Pfizer: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Daichii Sankyo: Consultancy, Research Funding; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; AbbVie: Consultancy, Research Funding. Vassiliou:Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.

TGFβR-SMAD3 Signaling Induces Resistance to PARP Inhibitors in the Bone Marrow Microenvironment

Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately, tumor cells acquire PARPi resistance, which is usually associated with the restoration of homologous recombination, loss of PARP1 expression, and/or loss of DNA double-strand break (DSB) end resection regulation. Here, we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGFβR) kinase on malignant cells, which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-β1). Genetic and/or pharmacological targeting of the TGF-β1-TGFβR kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGFβR inhibitor in patients receiving PARPis.

Regulating BRCA1 protein stability by cathepsin S-mediated ubiquitin degradation

Cathepsin S (CTSS) is a cysteine protease that is thought to play a role in many physiological and pathological processes including tumor growth, angiogenesis, and metastasis; it has been identified as a radiation response gene. Here, we examined the role of CTSS in regulating the DNA damage response in breast cancer cells. Activating CTSS (producing the cleavage form of the protein) by radiation induced proteolytic degradation of BRCA1, which ultimately suppressed DNA double-strand break repair activity. Depletion of CTSS by RNAi or expression of a mutant type of CTSS enhanced the protein stability of BRCA1 by inhibiting its ubiquitination. CTSS interacted with the BRCT domain of BRCA1 and facilitated ubiquitin-mediated proteolytic degradation of BRCA1, which was tightly associated with decreased BRCA1-mediated DNA repair activity. Treatment with a pharmacological CTSS inhibitor inhibited proteolytic degradation of BRCA1 and restored BRCA1 function. Depletion of CTSS by shRNA delayed tumor growth in a xenograft mouse model, only in the presence of functional BRCA1. Spontaneously uced rat mammary tumors and human breast cancer tissues with high levels of CTSS expression showed low BRCA1 expression. From these data, we suggest that CTSS inhibition is a good strategy for functional restoration of BRCA1 in breast cancers with reduced BRCA1 protein stability.

Abstract 4145: Concurrent treatment with Pim kinase inhibitor decreases alternative non-homologous end-joining repair of DNA damage induced by topoisomerase 2 inhibitors in cells with FLT3-ITD

Abstract Purpose: Internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) is a common (30%) molecular abnormality in acute myeloid leukemia (AML), associated with a high relapse rate and short disease-free survival. The oncogenic kinase Pim-1 is upregulated downstream of FLT3-ITD and contributes to its proliferative and anti-apoptotic effects. We recently showed that Pim kinase inhibition sensitizes AML cells with FLT3-ITD to apoptosis induction by topoisomerase 2 (TOP-2) inhibitors by increasing DNA double-strand breaks (DSBs) and oxidative stress. Here, we studied whether altered DSB repair activity contributes to the increase in DNA damage. Methods: Ba/F3 cells expressing FLT3-ITD (Ba/F3-ITD) or wild-type FLT3 (Ba/F3-WT) were studied. The pan-Pim kinase inhibitor AZD1208 was provided by AstraZeneca. TOP-2 inhibitors included daunorubicin (DNR), mitoxantrone (MXR) and etoposide (VP-16). Nuclear expression of repair proteins was measured by immunoblotting. Functional changes in DSB repair activities were measured in cell lines with stable chromosomal integration of the green fluorescent protein (GFP)-based DSB repair reporters DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical non-homologous end-joining (C-NHEJ) and alternative (Alt-) NHEJ, respectively. Results: Nuclear expression of the HR component Rad51 decreased progressively (to 40%) in Ba/F3-ITD cells after 12 hours of co-treatment with AZD1208 and DNR, compared to DNR alone. Progressive decrease (to 60%) in nuclear expression of the Alt-NHEJ component DNA ligase 3 and the C-NHEJ component Ku-70 was also seen with combination treatment. Functionally, co-treatment with AZD1208 and DNR led to a 50% decrease in Alt-NHEJ repair activity, compared to DNR alone, but no significant change in C-NHEJ or HR repair activity. Ba/F3-WT cells did not show a significant change in Alt-NHEJ, HR or C-NHEJ activity with combination treatment. Decreased Alt-NHEJ activity was also seen in Ba/F3-ITD, but not Ba/F3-WT, cells co-treated with AZD1208 and MXR or VP-16. Conclusions: While nuclear expression of multiple DNA DSB repair proteins decreased, only Alt-NHEJ repair activity decreased in FLT3-ITD cells co-treated with Pim kinase and TOP-2 inhibitors. Alt-NHEJ, a highly error-prone DNA DSB repair pathway, is upregulated in cells with FLT3-ITD and contributes to genomic instability in these cells. Decreasing Alt-NHEJ repair activity in cells treated with TOP-2 inhibitors has the potential to decrease the genomic instability that may contribute to disease progression in AML patients with FLT3-ITD. Citation Format: Kshama A. Doshi, Pratik K. Nagaria, Adriana E. Tron, Feyruz V. Rassool, Maria R. Baer. Concurrent treatment with Pim kinase inhibitor decreases alternative non-homologous end-joining repair of DNA damage induced by topoisomerase 2 inhibitors in cells with FLT3-ITD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4145. doi:10.1158/1538-7445.AM2017-4145

Aspirin inhibits the SHH/GLI1 signaling pathway and sensitizes malignant glioma cells to temozolomide therapy.

Aberrant activation of sonic hedgehog (SHH)/glioma-associated oncogene homolog 1 (GLI1) pathway plays an important role in the tumorigenicity of malignant glioma cells and resistance to temozolomide (TMZ). Here we investigated the aspirin's antineoplastic molecular route by targeting SHH/GLI1 pathway and examined the feasibility of aspirin combined with TMZ therapy. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the activity of the SHH/GLI1 pathway was strongly inhibited by aspirin. Aspirin acted as the glioma growth-inhibitory and pro-apoptosis roles by inhibiting the SHH/GLI1 pathway and reprogramming the epithelial to mesenchymal transition (EMT). The immunofluorescence assay showed aspirin could prevent the nuclear translocation of GLI1 to inhibit its transcriptional regulation. The stable lentiviral overexpression of GLI1 reversed the DNA double strand breaks (DSBs) caused by the GANT61 and TMZ. Furthermore, aspirin combined with TMZ enhanced chemosensitivity and GLI1-induced chemoprotection was partly blocked by aspirin in vitro and in vivo. Collectively, aspirin has a therapeutic potential for SHH/GLI1 targeted therapy against glioma cells. Acquired activation of GLI1 protects glioma cells against TMZ therapy. Impairment of DNA DSBs repair activity might be involved in the route of aspirin-induced chemosensitivity. Combined aspirin with TMZ may be a promising strategy against malignant glioma.

Abstract PR04: Mechanism for PARPi resistance: Homologous recombination without BRCA1

Abstract Approximately 15-20% of all epithelial ovarian cancers harbor germline or somatic mutations in BRCA1/2. Despite PARP inhibitor induce synthetic lethality in non-cancerous mouse embryo fibroblast cells that harbor BRCA1/2 mutations, a substantial fraction of BRCA1/2-mutated tumors patients do not respond to PARPi treatment, and a large fraction of BRCA1/2-mutated tumors patients eventually develop resistance to PARPi suggest significant frequency of acquired mutation lead to PARPi resistance preventing complete cure. We use CRISPR based library screen identified ATMIN/DYNLL1 loss as strong candidate for PARPi and cisplatin resistance mechanism. Targeted mutation on ATMIN or its transcription target DYNLL1 lead to increase of Cisplatin IC50 up to 200% and Olaparib IC50 up to 400%. More interestingly, ATMIN/DYNLL1 loss lead to restoration of Homologous recombination in BRCA1 mutant cells. ATMIN is under-expressed in about 30% ovarian cancer patient and low ATMIN expression correlate with shorter survival. Since ATMIN is important for DNA base damage repair but not Double Strand Break repair, we hypothesis ATMIN play important role in DNA damage repair pathway choice to suppress DSB repair while promote DNA base damage repair. Cells with mutant BRCA1 and loss ATMIN/DYNLL1 may result in abnormal DSB repair activity and lead to resistance to DNA damage agent such as Olaparib and Cisplatin. This abstract is also being presented as Poster A18. Citation Format: Yizhou Joseph He, Khyati Meghani, Dipanjan Chowdhury. Mechanism for PARPi resistance: Homologous recombination without BRCA1 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR04.

Abstract PR09: c-MYC preserves genomic integrity during DNA replication: a paradigm shift of c-MYC

Abstract During DNA synthesis, single-stranded DNA breaks (SSBs) are easily produced by mitogenic and oxidative stresses. Although SSBs are naturally converted to double-stranded DNA breaks (DSBs) at the collapse of replication forks, the integrity of chromosomal DNA is regularly maintained during DNA synthesis. Because DNA replication is a fundamental biological process involved in homeostasis, we hypothesized that an S-phase-associated guardian of the genome constantly stimulates cellular DSB repair machinery during DNA replication. The p53 tumor suppressor is known to act as the guardian of the genome. However, in response to DSBs, p53 not only activates G1 arrest, but also represses RAD51, a key component of homologous recombination (HR), an error-free DSB repair mechanism. Moreover, p53 is regularly dormant in actively growing cells. Thus, p53 cannot be the S-phase-associated DNA-repair enhancer. In this study, we show that the c-MYC transcription factor, which promotes DNA replication, concomitantly stimulates ataxia telangiectasia mutated kinase (ATM), γH2AX formation, and DSB repair by repressing BIN1. As an E2F1 corepressor, BIN1 suppressed the human ATM promoter, whereas via protein-protein interaction, BIN1 negatively regulated ATM auto-phosphorylation at serine 1981, a critical step for ATM activation. Accordingly, even before DSBs were formed, impaired BIN1 was sufficient to enhance ATM-dependent phosphorylation of histone H2AX at serine 139 (forming γH2AX), which has been widely used as a biomarker of DSBs. Reduced BIN1-dependent γH2AX foci formation was accompanied by the phosphorylation of MDC1 (mediator of DNA damage checkpoint protein 1) and 53BP1 foci. Furthermore, loss of BIN1 substantially accelerated cellular DSB-repair activity, which was reversed by the depletion of BRCA2, a critical player of HR. Intriguingly, activated c-MYC recruited the chromatin-remodeling factor BRG1 to and transcriptionally repressed the BIN1 promoter, thus indirectly liberating ATM expression. Because c-MYC robustly increases γH2AX foci, the oncoprotein has been thought to induce real DSBs. However, c-MYC silencing did not diminish γH2AX foci in the absence of BIN1, suggesting that c-MYC-induced γH2AX foci formation is chiefly attributable to c-MYC-induced decrease in BIN1 and following ATM reactivation in a manner independent of DSBs. Our study identifies c-MYC-associated γH2AX as a novel warning beacon of imminent DSBs and establishes a new paradigm for c-MYC in facilitating DSB-repair signaling during DNA replication. This abstract is also being presented as Poster B20. Citation Format: Alpana Kumari, Tetsushi Iwasaki, Walson P. Folk, Amy L. Abdulovic-Cui, Slovénie Pyndiah, George C. Prendergast, John M. Sedivy, Daitoku Sakamuro. c-MYC preserves genomic integrity during DNA replication: a paradigm shift of c-MYC. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR09.

In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals elevated histone H2AX phosphorylated on Ser139 damage removal in epithelial cells, not EMTs, and poly(ADP-ribose)polymerase inhibitor sensitivities, which suggested a DSB repair pathway shift with increasing patient age. Of interest, we found elevated microhomology-mediated end joining in EMTs, not epithelial cells, from patients who received a treatment recommendation of adjuvant chemotherapy, that is, those with high-risk tumors. Our discoveries of altered DSB repair activities in cells may serve as a method to further classify breast cancer to predict responsiveness to adjuvant chemotherapy and/or therapeutics that target DSB repair-dysfunctional tumors.-Deniz, M., Kaufmann, J., Stahl, A., Gundelach, T., Janni, W., Hoffmann, I., Keimling, M., Hampp, S., Ihle, M., Wiesmüller, L. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

CELLULAR RESPONSES TO TRITIUM EXPOSURE IN RAINBOW TROUT: HTO- AND OBT-SPIKED FEED EXPOSURE EXPERIMENTS

Biological effects were evaluated in rainbow trout (Oncorhynchus mykiss) exposed to tritiated water (HTO) or food spiked with organically bound tritium (OBT). An HTO exposure study was conducted using a tritium activity concentration of 7000 Bq/L, and an OBT exposure study was conducted using a tritium activity concentration of 30 000 Bq/L. Following 140 days of in vivo HTO exposure, liver, heart, spleen, kidney, and brain cells did not show statistically significant differences in viability; kidney, liver, and spleen cells did not show significant differences in DNA double-strand break repair activity compared with control cells. Membrane fatty acid composition analysis was conducted on liver cells and no effects of HTO exposure could be detected. Following 140 days of in vivo OBT exposure, viability and DNA double-strand break repair activity were not statistically different from controls in liver, heart, spleen, kidney, and brain cells. Changes, however, were noted in the fatty acid composition of liver and muscle tissues.For both studies, all measurements were performed on each tissue and on a fraction of the same tissue that was exposed to a gamma 4 Gy dose in vitro to test for adaptive responses, and no effects were observed except for fatty acid composition. The findings demonstrated that membrane fatty acid composition is a sensitive marker and that microscopic evaluation of gamma-H2AX foci is more sensitive than the flow cytometric approach. These studies are the first to correlate uptake and depuration with biological health indicators in edible fish for tritium exposures within worldwide drinking water guidelines.

Calcium influx-mediated translocation of m-calpain induces Ku80 cleavage and enhances the Ku80-related DNA repair pathway.

Proteomic analysis of ionomycin-treated and untreated mammary epithelial MCF10A cells elucidated differences in Ku80 cleavage. Ku80, a subunit of the Ku protein complex, is an initiator of the non-homologous, end-joining (NHEJ), double-strand breaks (DSBs) repair pathway. The nuclear Ku80 was cleaved in a calcium concentration-dependent manner by m-calpain but not by m-calpain. The cleavage of nuclear Ku80 at its α/β domain was validated by Western blotting analysis using flag-tagged expression vectors of truncated versions of Ku80 and a flag antibody and was confirmed in m-calpain knock-down cells and in vitro cell-free evaluation with recombinant proteins of calpains, Ku70, and Ku80. In addition, the cleaved Ku80 still formed a Ku heterodimer and promoted DNA DSB repair activity. Taken together, these findings indicate that translocated m-calpain enhances the NHEJ pathway through the cleavage of Ku80. Based on the present study, m-calpain in DNA repair pathways might be a novel anticancer drug target, or its mechanism might be a possible route for resistance acquisition of DNA damage-inducing chemotherapeutics.

Abstract A04: DNA double-strand break repair assay in primary cultures of patient-derived ovarian carcinoma xenografts.

Abstract Introduction: Epithelial ovarian cancer (EOC) represents the most fatal gynecological malignancy. The DNA repair capacity is a key determinant for the cellular response to DNA damaging agents and is inversely correlated with their cytotoxicity. It has been reported that half of high grade EOC display defects in DNA repair, accounting for the sensitivity of this tumor to the first line chemotherapy. The aim of the present work was to evaluate the functional DNA repair activity, focusing on DNA double strand-break (DSB) repair, in EOC cell lines with different sensitivity to cisplatin (DDP) and in EOC primary cultures obtained from patient-derived ovarian carcinoma xenografts (PDX), recently established in our laboratory. Material and Method: Primary cultures derived from intraperitoneal ascites or from subcutaneous EOC xenograft solid masses were mechanically disaggregated and cultured in RPMI. The experiments were performed within the third in vitro passage. To functionally evaluate DSB repair pathways, cells were transfected with various reporter substrates for EGFP-based quantification of specific DSB repair activities: homologous recombination (HR), total non-homologous end-joining (tNHEJ), and micro-homology mediated NHEJ (mmEJ) pathway. Briefly, 10^6 cells were transfected with different plasmid mixtures, containing the meganuclease expression plasmid (pCMV-I-SceI) together with one of the recombination substrates, and wtEGFP plasmid in split samples for determination of transfection efficiencies. Cellular fluorescence was quantified by flow cytometry and recombination frequencies were calculated from the fraction of wtEGFP positive cells normalized for transfection efficiency. Results: Among cell lines, OVCAR5 showed very low DSB repair frequencies in all the three sub-pathways investigated. Interestingly, OVCAR433 showed the highest DSB repair frequencies and also the lowest sensitivity to DDP. MmEJ repair frequencies were lower than HR and tNHEJ both in cell lines and in primary EOC cultures derived from PDX. In EOC primary cultures tNHEJ frequencies were 2 to 10 times higher than HR. Conclusion: We were able to analyze distinct DSB repair activities in EOC primary cultures obtained from PDX. The application of such DNA repair assays to a panel of PDX xenografts with known sensitivity to DDP treatment will allow to assess the power of these tests to predict the response to chemotherapy. Citation Format: Federica Guffanti, Julia Kaufmann, Francesca Bizzaro, Francesca Ricci, Monica Ganzinelli, Monica Lupi, Raffaella Giavazzi, Lisa Wiesmüller, Giovanna Damia. DNA double-strand break repair assay in primary cultures of patient-derived ovarian carcinoma xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A04.

XRCC4 deficiency in human subjects causes a marked neurological phenotype but no overt immunodeficiency

Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair mechanism in human cells. The final rejoining step requires DNA ligase IV (LIG4) together with the partner proteins X-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor. Patients with mutations in genes encoding LIG4, XRCC4-like factor, or the other NHEJ proteins DNA-dependent protein kinase catalytic subunit and Artemis are DSB repair defective and immunodeficient because of the requirement for NHEJ during V(D)J recombination. We found a patient displaying microcephaly and progressive ataxia but a normal immune response. We sought to determine pathogenic mutations and to describe the molecular pathogenesis of the patient. We performed next-generation exome sequencing. We evaluated the DSB repair activities and V(D)J recombination capacity of the patient's cells, as well as performing a standard blood immunologic characterization. We identified causal mutations in the XRCC4 gene. The patient's cells are radiosensitive and display the most severe DSB repair defect we have encountered using patient-derived cell lines. In marked contrast, a V(D)J recombination plasmid assay revealed that the patient's cells did not display the junction abnormalities that are characteristic of other NHEJ-defective cell lines. The mutant protein can interact efficiently with LIG4 and functions normally in in vitro assays and when transiently expressed in vivo. However, the mutation makes the protein unstable, and it undergoes proteasome-mediated degradation. Our findings reveal a novel separation of impact phenotype: there is a pronounced DSB repair defect and marked clinical neurological manifestation but no clinical immunodeficiency.

Development of a novel method to create double-strand break repair fingerprints using next-generation sequencing.

Efficient DNA double-strand break (DSB) repair is a critical determinant of cell survival in response to DNA damaging agents, and it plays a key role in the maintenance of genomic integrity. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. We now understand that HR and NHEJ repair are composed of multiple sub-pathways, some of which still remain poorly understood. As such, there is great interest in the development of novel assays to interrogate these key pathways, which could lead to the development of novel therapeutics, and a better understanding of how DSBs are repaired. Furthermore, assays which can measure repair specifically at endogenous chromosomal loci are of particular interest, because of an emerging understanding that chromatin interactions heavily influence DSB repair pathway choice. Here, we present the design and validation of a novel, next-generation sequencing-based approach to study DSB repair at chromosomal loci in cells. We demonstrate that NHEJ repair "fingerprints" can be identified using our assay, which are dependent on the status of key DSB repair proteins. In addition, we have validated that our system can be used to detect dynamic shifts in DSB repair activity in response to specific perturbations. This approach represents a unique alternative to many currently available DSB repair assays, which typical rely on the expression of reporter genes as an indirect read-out for repair. As such, we believe this tool will be useful for DNA repair researchers to study NHEJ repair in a high-throughput and sensitive manner, with the capacity to detect subtle changes in DSB repair patterns that was not possible previously.

The DNA repair component Metnase regulates Chk1 stability.

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. Metnase (also termed SETMAR) is a SET histone methylase and transposase nuclease protein that promotes both DNA double strand break (DSB) repair and re-start of stalled replication forks. We previously found that Chk1 phosphorylation of Metnase on S495 enhanced its DNA DSB repair activity but decreased its ability to re-start stalled replication forks. Here we show that phosphorylated Metnase feeds back to increase the half-life of Chk1. Chk1 half-life is regulated by DDB1 targeting it to Cul4A for ubiquitination and destruction. Metnase decreases Chk1 interaction with DDB1, and decreases Chk1 ubiquitination. These data define a novel pathway for Chk1 regulation, whereby a target of Chk1, Metnase, feeds back to amplify Chk1 stability, and therefore enhance replication fork arrest.

Oncogene-Induced DNA Repair Defects Promote PARP1-Mediated “Dual Synthetic Lethality” To Eradicate Quiescent and Proliferating Leukemia Stem and Progenitor Cells

▪Leukemia stem cells (LSCs), including quiescent cells, are disease initiating and therapy-refractory cells. Therefore, even if a treatment clears a disease burden consisting mostly of leukemia progenitor cells (LPCs), it usually fails to eradicate LSCs and residual LPCs which developed therapy-resistance. Leukemia cells expressing BCR-ABL1 (CML, Ph+ALL), FLT3(ITD) (AML), and AML1-ETO (AML) accumulate high numbers of spontaneous DNA double strand breaks (DSBs). Leukemia cells can survive numerous DSB, the most lethal DNA lesions, due to enhanced DSB repair activity by homologous recombination (HR; active in S and G2 cell cycle phase) and/or non-homologous end-joining (NHEJ; active in G0/G1 and S cell cycle phase). Altogether, leukemia cells may be “addicted” to DSB repair pathways.Normal cells usually employ BRCA1/2-RAD51 -mediated HRR and Ku70/86-DNA-PKcs -dependent NHEJ. BCR-ABL1 causes BRCA1 and DNA-PKcs deficiency, FLT3(ITD) inhibits Ku86, and AML1-ETO downregulates DNA-PKcs and RAD51. Accordingly, leukemia cells expressing these oncogenes are forced to employ alternative DSB repair pathways, such as PARP-LigIII –mediated NHEJ. Since NHEJ plays a predominant role in quiescent cells and also supports HR in proliferating cells, we postulated that targeting PARP should exert “dual synthetic lethality” to eradicate quiescent LSCs and proliferating LSCs/LPCs, with negligible effect on normal cells.We showed that PARP inhibitor olaparib, which is in clinical trials for the treatment of solid tumors displaying BRCA1/2 mutations, abrogated NHEJ activity in BCR-ABL1 cells. Olaparib reduced the number of imatinib-naïve and imatinib-treated Lin-CD34+CD38-CTVmax quiescent CML-CP LSCs in hypoxia and normoxia mimicking bone marrow niche and arterial peripheral blood, respectively. In addition, olaparib enhanced the anti-proliferative effect of imatinib in Lin-CD34+CML-CP and CML-AP LPCs. These effects are probably due to imatinib-mediated inhibition of BCR-ABL1 kinase-dependent anti-apoptotic activity and olaparib-induced increase of the number of lethal DSBs, resulting in accumulation of annexin V-positive apoptotic quiescent LSCs and hyper-activation of caspase-3 in imatinib+olaparib treated LPCs. Inhibition of PARP almost completely abrogates DSB repair in DNA-PKcs-deficient quiescent LSCs and diminishes resolution of ROS-induced stalled replication forks in BRCA1-deficient proliferating LSCs/LPCs. The combination of imatinib+olaparib did not affect normal quiescent hematopoietic stem cells, but exerted modest inhibitory effect on normal proliferating progenitors.Since olaparib does not discriminate neither between PARP family members nor other enzymatic pathways involving NAD+, its long-term application may generate side-effects. Our genetic studies involving Parp1-/- mice and PARP1(E988K) catalytic-deficient mutant identified PARP1 as major player in NHEJ in BCR-ABL1 cells. Using high throughput screening we identified 5F2, a small molecule which abrogated histone 4-dependent PARP1 activation and exerted synthetic lethality in BRCA1-deficient, but not BRCA1-proficient carcinoma cells. 5F2, similarly to olaparib, reduced the number of imatinib-naïve and imatinib-treated Lin-CD34+CD38-CTVmax quiescent LSCs and inhibited colony formation by Lin-CD34+LPCs. However 5F2, in contrast to olaparib, did not affect normal cells.In addition, olaparib and/or 5F2 reduced the number of imatinib-naïve and imatinib-treated Ph+ALL cells harvested from patients at diagnosis. Moreover, PARP1 inhibitors exerted anti-leukemia effect against ponatinib-naïve and ponatinib-treated Ph+ALL cells carrying BCR-ABL1 T315I mutation, and against lestaurtinib/quizartinib-naïve and lestaurtinib/quizartinib-treated FLT3(ITD)-positive AML cells. PARP1 inhibitors also abrogated the growth of leukemia cells expressing AML1-ETO (AML), but not of these expressing PML-RAR (APL) or overexpressing HOXA9+MEIS1 (AML).In conclusion, targeting PARP1 resulted in the induction of “dual synthetic lethality” and eradication of quiescent and proliferating CML cells displaying specific defects in DSB repair pathways. Similar effect is induced in other leukemias carrying specific, oncogene-induced DSB repair deficiencies. PARP1 inhibitors are currently tested in vivo using primary leukemia xenografts. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

Functional analysis—Make or break for cancer predictability

Copy number variations (CNVs) encompass a variety of genetic alterations including deletions and amplifications and cluster in regions of the human genome with intrinsic instability. Small-sized CNVs can act as initial genetic changes giving rise to larger CNVs such as acquired somatic copy number aberrations (CNAs) promoting cancer formation. Previous studies provided evidence for CNVs as an underlying cause of elevated breast cancer risk when targeting breast cancer susceptibility genes and of accelerated breast cancer progression when targeting oncogenes. With the development of novel techniques for genome-wide detection of CNVs at increasingly higher resolution, it became possible to qualitatively and quantitatively analyse manifestation of DNA damage resulting from defects in any of the large variety of DNA double-strand break (DSB) repair mechanisms. Breast carcinogenesis, particularly in familial cases, has been linked with a defect in the homologous recombination (HR) pathway, which in turn switches damage removal towards alternative, more error-prone DSB repair pathways such as microhomology-mediated non-homologous end joining (mmNHEJ). Indeed, increased error-prone DSB repair activities were detected in peripheral blood lymphocytes from individuals with familial breast cancer risk independently of specific gene mutations. Intriguingly, sequence analysis of breakpoint regions revealed that the majority of genome aberrations found in breast cancer specimens are formed by mmNHEJ. Detection of pathway-specific error-prone DSB repair activities by functional testing was proposed to serve as biomarker for hereditary breast cancer risk and responsiveness to therapies targeting HR dysfunction. Identification of specific error-prone DSB repair mechanisms underlying CNAs and ultimately mammary tumour formation highlights potential targets for future breast cancer prevention regimens.