Abstract PR01: EMSY impairs DNA damage repair in a phosphorylation-dependent manner.

208 Background: Prostate cancer is the second leading cause of cancer-related deaths amongst men in North America. Data suggests that, following radiation therapy (XRT), androgen receptor (AR) enhances DNA damage repair and contributes to resistance of prostate cancer (PCa) cells to XRT. At present AR-pathway inhibition is the mainstay treatment of metastatic castration resistance prostate cancer (mCRPC). Enzalutamide (ENZA), a potent AR inhibitor is one of the approved drugs in this setting. The purpose of this study was to assess the potential radiosensitization of ENZA and its mechanism of action in hormone resistant PCa cells. Methods: The effect of ENZA alone or in combination with XRT was assessed on hormone-sensitive, (HS: LNCaP, PC3-T877A) and insensitive PCa cells (HI: PC3, PC3-AR V7, C4-2) using viability and clonogenic assays, cell cycle arrest and DNA damage analysis. Results: MTT assay demonstrates that ENZA significantly inhibits the proliferation of HS PCa cells in a dose dependent manner whereas CRPC required ENZA in combination with ADT (androgen deprivation therapy). Additionally, clonogenic assay proves that concurrent administration of ENZA or ADT+ENZA and XRT led to a supra-additive antitumor effect with the dose enhancement factor of 1.76±0.008 in LNCaP, 1.65±0.01 in PC3-T877A and 1.35±0.003 in C4-2 respectively at surviving fraction of 0.1. This effect was not observed in PC3 and PC3-AR V7 cells pre-treated with ENZA (in all cases DEF = 1 at SF = 0.1). Additionally, the level of γH2AX increased in HS cells and CRPC cells treated with ENZA/ADT+ENZA and XRT when compared to XRT alone. The enhanced H2AX activity remained unchanged up to 24 hours after combination treatment. Furthermore, there is an initial inhibition of DNA-PKcs in HS and CRPC cells treated with ENZA/ADT+ENZA administered before XRT. Conclusions: Our data suggest that the higher efficacy of ENZA/ENZA+ADT and XRT could be partially due to inhibition of DNA damage repair. Our results demonstrated a significant enhancement of XRT efficacy and confirms the rational for the ongoing combination clinical trials with XRT.

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Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma

Therapeutic Advances in Medical Oncology ◽

10.1177/1758835920956900 ◽

2020 ◽

Vol 12 ◽

pp. 175883592095690 ◽

Cited By ~ 2

Author(s):

Xiaoyang Li ◽

Dylan C. Dean ◽

Gregory M. Cote ◽

Lee Zou ◽

Francis J. Hornicek ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Damage ◽

Therapeutic Target ◽

Ex Vivo ◽

Dna Damage Repair ◽

Parp Cleavage ◽

Dependent Manner ◽

Cytoplasmic Vacuolization ◽

Damage Repair ◽

Metastatic Osteosarcoma

Background: Ataxia-telangiectasia and Rad3 related protein kinase (ATR) is an essential regulator of the DNA damage response in various cancers; however, its expression and roles in osteosarcoma are unclear. We therefore chose to evaluate the significance and mechanism of ATR in metastatic osteosarcoma, as well as its potential to be a therapeutic target. Methods: The osteosarcoma tissue microarrays constructed from 70 patient specimens underwent immunohistochemistry to quantify ATR and activated phospho-ATR (pATR) expression and their correlation with clinical outcomes. ATR sublocalization within the metastatic osteosarcoma cells was confirmed by immunofluorescence assay. Cell proliferation, apoptosis, and migration were evaluated following treatment with ATR siRNA or the selective inhibitor Berzosertib. Antitumor effects were determined with ex vivo three-dimensional (3D) culture models, and the impacts on the DNA damage repair pathways were measured with Western blotting. Results: Elevated ATR and activated pATR expression correlated with shorter patient survival and less necrosis following neoadjuvant chemotherapy. Intranuclear sublocalization of ATR and pATR suggested a mechanism related to DNA replication. ATR knockdown with siRNA or inhibition with Berzosertib suppressed cell proliferation in a time- and dose-dependent manner and induced apoptosis. In addition, ATR inhibition decreased Chk1 phosphorylation while increasing γH2AX expression and PARP cleavage, consistent with the interference of DNA damage repair. The ATR inhibitor Berzosertib also produced the characteristic cytoplasmic vacuolization preceding cell death, and suppressed ex vivo 3D spheroid formation and cell motility. Conclusion: The faithful dependence of cells on ATR signaling for survival and progression makes it an emerging therapeutic target in metastatic osteosarcoma.

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A DNA Damage-Repair Dynamic Model for HRS/IRR Effects of C.elegans Induced by Neutron Irradiation

Dose-Response ◽

10.1177/15593258211001254 ◽

2021 ◽

Vol 19 (1) ◽

pp. 155932582110012

Author(s):

Guangyan Feng ◽

Lianxin Zhang ◽

Zhanguo Yang ◽

Yong Zhang ◽

Siwei Zhang ◽

...

Keyword(s):

Dna Damage ◽

Neutron Irradiation ◽

Low Dose ◽

Biological Effects ◽

Dna Damage Repair ◽

Experimental Results ◽

Dependent Manner ◽

Survival Fraction ◽

C Elegans ◽

Damage Repair

Neutron irradiation which could trigger severe biological effects, is being applied in nuclear plants, radiotherapy, and aerospace gradually. Low dose hyper-radiosensitivity response of low Linear Energy Transfer (LET) irradiation on the cell survival has become a matter of great interest since its discovery, but a few research have been done on this response induced by neutron irradiation. To investigate this response induced by neutron irradiation, Caenorhabditis elegans ( C. elegans) was irradiated by neutron irradiation. The surviving fraction of C. elegans on the 12th day after irradiation was analyzed, which showed a hyper-radiosensitive response at low doses and followed by an increase in survival fraction at slightly higher doses. The finding of this work that neutron irradiation decreased the surviving fraction in a non-dose-dependent manner was different from previous low-LET irradiation studies. To understand the experimental results, a DNA damage-repair model was introduced. By comparing experimental results with theoretical analyses, we suggest that the low dose hyper-radiosensitivity response of neutron irradiation may possible related to different radiation types and DNA damage recognition proteins and immune system of C. elegans.

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Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia

Frontiers in Oncology ◽

10.3389/fonc.2021.752933 ◽

2021 ◽

Vol 11 ◽

Author(s):

Donna M. Edwards ◽

Dana K. Mitchell ◽

Zahi Abdul-Sater ◽

Ka-Kui Chan ◽

Zejin Sun ◽

...

Keyword(s):

Dna Damage ◽

Genomic Instability ◽

Fanconi Anemia ◽

Spindle Assembly Checkpoint ◽

Dna Damage Repair ◽

Spindle Assembly ◽

Dependent Manner ◽

Damage Repair

Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis in vivo, the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in Fancc-/- mice by introducing heterozygosity of the key spindle assembly checkpoint regulator Mad2. Fancc-/-;Mad2+/- mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than Fancc-/-mice. We utilized hematopoietic stem cell transplantation to propagate Fancc-/-; Mad2+/- AML in irradiated healthy mice to model FANCC-deficient AMLs arising in the non-FA population. Compared to cells from Fancc-/- mice, those from Fancc-/-;Mad2+/- mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of Fancc-/-;Mad2+/- mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in Fancc-/-; Mad2+/- mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies in vivo, show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.

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