DNA repair defects have been increasingly focused on as therapeutic targets. In hormone-positive breast cancer, XRCC1-deficient tumors have been identified and proposed as targets for combination therapies that damage DNA and inhibit DNA repair pathways. XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) polymerase 1, DNA polymerase β (POL β), and DNA ligases. Loss of XRCC1 confers BER defects and hypersensitivity to DNA damaging agents. BER defects have not been evaluated in triple negative breast cancers (TNBC), for which new therapeutic targets and therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects in TNBC, we examined XRCC1 expression in the TCGA database and its expression and localization in TNBC cell lines. The TCGA database revealed high XRCC1 expression in TNBC tumors and TNBC cell lines show variable, but mostly high expression of XRCC1. XRCC1 localized outside of the nucleus in some TNBC cell lines, altering their ability to repair base lesions and single-strand breaks. Subcellular localization of POL β also varied and did not correlate with XRCC1 localization. Basal levels of DNA damage correlated with observed changes in XRCC1 expression, localization, and measure repair capacity. The results confirmed that XRCC1 expression changes indicate DNA repair capacity changes but emphasize that basal DNA damage levels along with protein localization are better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in TNBC preclinical models and tumors, XRCC1 expression levels should be assessed when evaluating treatment responses of TNBC preclinical model cells.

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Background: The lack of molecular targets for triple negative breast cancer (TNBC) has limited treatment options and reduced survivorship. Identifying new molecular targets may help improve patient survival and decrease recurrence and metastasis. As DNA repair defects are prevalent in breast cancer, we evaluated the expression and repair capacities of DNA repair proteins in preclinical models. Methods: DNA repair capacity was analyzed in four TNBC cell lines, MDA-MB-157 (MDA-157), MDA-MB-231 (MDA-231), MDA-MB-468 (MDA-468), and HCC1806, using fluorescence multiplex host cell reactivation (FM-HCR) assays. Expression of DNA repair genes was analyzed with RNA-seq, and protein expression was evaluated with immunoblot. Responses to the combination of DNA damage response inhibitors and primary chemotherapy drugs doxorubicin or carboplatin were evaluated in the cell lines. Results: Defects in base excision and nucleotide excision repair were observed in preclinical TNBC models. Gene expression analysis showed a limited correlation between these defects. Loss in protein expression was a better indicator of these DNA repair defects. Over-expression of PARP1, XRCC1, RPA, DDB1, and ERCC1 was observed in TNBC preclinical models, and likely contributed to altered sensitivity to chemotherapy and DNA damage response (DDR) inhibitors. Improved cell killing was achieved when primary therapy was combined with DDR inhibitors for ATM, ATR, or CHK1. Conclusion: Base excision and nucleotide excision repair pathways may offer new molecular targets for TNBC. The functional status of DNA repair pathways should be considered when evaluating new therapies and may improve the targeting for primary and combination therapies with DDR inhibitors.

21 22 23DNA repair defects have been increasingly focused on as therapeutic targets. In hormone 24 positive breast cancer, XRCC1-deficient tumors have been identified and proposed as 25 targets for combination therapies that damage DNA and inhibit DNA repair pathways. 26XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating 27 essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) 28 polymerase 1, DNA polymerase β (POL β), and DNA ligases. Loss of XRCC1 confers 29 BER defects and hypersensitivity to DNA damaging agents. BER defects have not been 30 evaluated in triple negative breast cancer (TNBC), for which new therapeutic targets and 31 therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects 32 in TNBC, we examined XRCC1 expression and localization in the TCGA database and in 33 TNBC cell lines. High XRCC1 expression was observed for TNBC tumors in the TCGA 34 database and expression of XRCC1 varied between TNBC cell lines. We also observed 35 changes in XRCC1 subcellular localization in TNBCs that alter the ability to repair base 36 lesions and single-strand breaks. Subcellular localization changes were also observed for 37 POL β that did not correlate with XRCC1 localization. Basal levels of DNA damage were 38 also measured in the TNBC cell lines, and damage levels correlated with observed 39 changes in XRCC1 expression, localization, and repair functions. The results confirmed 40 that XRCC1 expression changes may indicate DNA repair capacity changes but 41 emphasize that basal DNA damage levels along with expression and localization are 42 better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in 43 TNBC preclinical models and the TCGA database, XRCC1 expression levels should be 44 considered when evaluating treatment responses of TNBC preclinical model cells. 45 3 Keywords: triple negative breast cancer, base excision repair, XRCC1, PARP1, DNA 46 repair, DNA damage, nuclear localization 47 48 Defects in DNA damage response and repair are driving factors in carcinogenesis 49and key determinants in the response to chemotherapy. Breast cancers may display 50 defects in DNA repair such as mutations in key DNA damage response and repair 51 proteins such as breast cancer-susceptibility (BRCA1/2) and tumor suppressor protein 52 p53 (TP53), and altered expression levels of DNA repair proteins thymine-DNA 53 glycosylase (TDG) and poly(ADP-ribose) polymerase 1 (PARP1) [1-3]. Therapeutic 54 outcomes may be improved by exploiting DNA repair defects present in cancer cells but 55 absent in normal cells, as in the use of PARP-inhibitors (PARPi) in cancers that have 56 BRCA1/2 deficiencies. However, characterization of DNA repair pathways often is lacking 57 in preclinical models and cell lines. Examining DNA repair defects in preclinical models 58 and patients is essential for evaluating the efficacy of therapeutic agents. 59 In breast cancer, DNA repair defects often extend beyond homologous 60 re...

Programmed death ligand-1 (PD-L1) inhibitors are currently under investigation as a potential treatment option for ovarian cancer. Although this therapy has shown promise, its efficacy is highly variable among patients. Evidence suggests that genomic instability influences the expression of PD-L1, but little is known about this relationship in ovarian cancer. To examine the relationship between PD-L1 expression and genomic instability, we measured DNA damage using Repair Assisted Damage Detection (RADD). We then correlated the presence of persistent DNA damage in the ovarian tumor with protein expression of PD-L1 using immunohistochemistry. Ovarian tumors showed a high prevalence of oxidative DNA damage. As the level of oxidative DNA damage increased, we saw a significant correlation with PD-L1 expression. The highest correlation between DNA damage and PD-L1 expression was observed for mucinous ovarian tumors (r = 0.82), but a strong correlation was also observed for high grade serous and endometrioid tumors (r = 0.67 and 0.69, respectively). These findings link genomic instability to PD-L1 protein expression in ovarian cancer and suggest that persistent DNA damage can be used as a potential biomarker for patient selection for immunotherapy treatment.

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The purpose of this study was to investigate changes in neuropeptide Y (NPY) protein and dipeptidyl peptidase IV (DPP‐IV) activity in the plasma and saliva in normally cycling women and women after menopause. We recruited seven premenopausal women and seven post‐menopausal women for the study. Blood via venipuncture and saliva samples were taken at each point in the menstrual cycle (pre‐menopausal) or once per week (post‐menopausal) for 2 months. Blood and saliva were analyzed for estrogen, neuropeptide Y using ELISA's and DPP‐IV activity using a fluorometric assay. Plasma β‐estradiol was an average of 96.45±57.04 pg/ml over 2 cycles in the pre‐menopausal group and 1.72±0.35 pg/ml over 2 months in the post‐menopausal group (p<0.05). In the pre‐menopausal group, there were no significant differences in saliva or plasma NPY or DPP‐IV over the cycle. For the post‐menopausal group, salivary NPY and DPP‐IV did not change over 2 months. Plasma NPY was lowest in the middle 2 weeks (average: 0.52±0.10 ng/ml) compared to the first and 4th weeks (average of week 1 and 4: 0.60±0.14 ng/ml; p<0.05). Plasma NPY in postmenopausal women was higher overall (0.56±0.13 ng/ml) compared to pre‐menopausal women (0.30±0.11 ng/ml; p<0.05). Plasma DPP‐IV activity was unchanged by time in the post‐menopausal group. Saliva DPP‐IV and saliva NPY in the pre‐menopausal group had a significant negative correlation (R=−0.95; p<0.05). We found that saliva measures of NPY and DPP‐IV activity are poor estimates of plasma concentrations and activities. Differences in plasma NPY concentrations between the groups and the relationship between salivary NPY and DPP‐IV suggests that there are some unique differences between these groups.Support or Funding InformationThe work presented in this poster session was supported by Award Number 1R03DC013664‐01A1 from the National Institute on Deafness and Other Communication Disorders (NIDCD). The content of this presentation is solely the responsibility of the authors and does not necessarily represent the official views of the NIDCD or the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Despite the majority of ovarian cancer patients achieving a complete clinical response with first‐line therapies, nearly 80% will recur within 24 months. There is an urgent need for novel therapeutic strategies to treat ovarian cancer effectively. Renewed interest in immunotherapy in ovarian cancer has shown that tumor‐infiltrating lymphocytes are associated with high‐grade serous, clear cell, and endometrioid histology. As a result, immune checkpoint inhibitors have emerged as promising treatments for ovarian cancer. Programmed death ligand‐1 (PD‐L1) is expressed by cancer cells to evade host surveillance. Its interactions with Programmed death‐1 (PD‐1) negatively regulate CD8+ T‐cells to control inflammatory responses and maintain self‐tolerance. Blockade of PD‐1/PDL‐1 restores anti‐tumor cell immunity and has been successfully used to treat some cancers, including non‐small cell lung cancer and melanoma. The use of PD‐1/PDL‐1 blockade in ovarian cancer has been limited by our lack of understanding of the mechanisms that control PD‐L1 expression. Emerging evidence has suggested that DNA repair defects and genomic instability influence the expression of PDL‐1. However, little is known about the relationship between genomic instability and PDL‐1 expression in ovarian cancer, despite the high prevalence of DNA repair defects reported. We hypothesized that genomic instability increases the expression of PD‐L1; therefore, PD‐L1 expression levels will correlate with high levels of DNA damage in the tumor genome. To test this hypothesis, we have measured DNA repair defects and genomic instability in ovarian tumors using Repair Assisted Damage Detection (RADD). RADD measures persistent DNA lesion and strand breaks within tumor cells using a cocktail of DNA repair enzymes. Lesion sites are then tagged with nucleotide analog and fluorescently labeled for detection. DNA repair defects are revealed by the presence of DNA damage within the tumor genome. In parallel, we also measured PDL‐1 expression within these tumor cells using immunohistochemistry. Tumors were categorized and stratified based on the basal levels of DNA damage, and PDL‐1 expression was correlated to the type and quantity of DNA damage present in each tumor sample. Using this method, we have established that differences in DNA damage and PD‐L1 expression exist among ovarian tumor grade, stage, and pathology. Using different enzyme cocktails in our RADD protocol, we were also able to establish that oxidative lesions are primarily retained within the genome of ovarian tumors. Correlations in DNA damage and PD‐L1 expression suggest that oxidative lesions influence the expression of PD‐L1 in ovarian cancers. This study establishes a potential biomarker for assessing the utility of PD‐L1 inhibitors in ovarian tumors.