pATM Levels Research Articles

Spatially resolved protein profiling in pancreatic adenosquamous carcinoma.

760 Background: Pancreatic adenosquamous carcinoma (PASC) is a rare histopathological type of pancreatic cancer that is more aggressive than pancreatic ductal adenocarcinoma (PDAC). Although PASC has a unique tumor biology and poorer prognosis, there are no guidelines for differentially treating PASC patients, and its rarity makes it significantly understudied. We used Nanostring GeoMX digital spatial profiling (DSP) to provide a targeted proteomic characterization of key tumor-promoting proteins in PASC and PDAC. Methods: FFPE tissue from PASC (n=10 primary, n= 4 met) and PDAC (n=21 primary, n=5 met) cases were analyzed using the DSP platform. Sections were stained with an 82-protein cocktail of antibodies conjugated to UV-photocleavable DNA barcodes. A pan-cytokeratin morphology marker was used to select tumor regions of interest (ROI) and inferred surrounding stromal (i.e., pan-CK negative) ROIs. UV excitation of the defined ROIs was used to release the DNA barcodes and were quantified on the NanoString nCounter system. For PASC, squamous enriched ROI were inferred from corresponding p40 IHC. Corresponding clinical and mutational status for all cases were abstracted from medical records. Results: TP53 (PDAC, 80%; PASC, 83%) and KRAS (PDAC, 76%; PASC, 100%) were the most prevalent mutations . In comparing the tumor ROIs of PASC and PDAC (all cases), DSP-assessed global expression and activation of EGFR-MAPK signaling is significantly increased in PASC, whereas PI3K-AKT signaling is significantly decreased. Compared to PDAC, EGFR expression and phosphorylation (p-EGFR), as well as p-MEK1, are significantly increased in PASC. PASC showed significantly lower levels of PLCG1 and INPP4B, while the majority of other AKT signaling effectors were unchanged. For DNA damage response signaling, PASC showed significantly higher levels of p-ATM, p-CHK2, and p21, but not p-ATR. Likewise, expression of oncoproteins c-MYC and BCL6 were significantly increased in PASC (vs. PDAC). Conclusions: Given that the normal pancreas lacks squamous cells, their histological presence is a uniquely aberrant feature that can be used to guide deep spatially-resolved interrogation of complex cellular and molecular programs. Our preliminary findings show that regions of squamous cell differentiation within PASC have intrinsically higher levels of active EGFR signaling. With emerging RAS-targeted agents poised to alter the treatment landscape, high EGFR signaling in PASC suggest a possible compensatory resistance mechanism that may require additional targeting. Likewise, reliance on ATM signaling highlights this as preferential target for DDR-directed therapies. Building on these results with additional multiomic assays will inform strategies for improving the management of patients with PASC.

S6K1 Controls DNA Damage Signaling Modulated by the MRN Complex to Induce Radioresistance in Lung Cancer

Radiation is a mainstay of lung cancer treatment; however, resistance frequently develops. Identifying novel therapeutic targets to increase radiation sensitivity is crucial. S6K1 is a serine/threonine kinase known to regulate protein translation which is associated with radioresistance, but the mechanisms involved are unknown. We proposed to determine whether S6K1 promotes radioresistance by regulating DNA repair in lung cancer. Colony formation, protein expression and proliferation were assessed. S6K1 was modulated pharmacologically by either PF-4708671 or genetically by Crispr-Cas9. Higher radioresistance levels in lung cancer cells were associated with lower phosphoactivation of MRN complex members, a key activator of radiation-induced DNA repair signaling. We also found lower levels of p-ATM, a target of the MRN complex, in more radioresistant cells, which was associated with a lower expression of γ-H2AX cafter radiation. Further, genetic and pharmacological S6K1 targeting sensitized lung cancer cells to low doses of radiation (p ≤ 0.01). Additionally, S6K1−/− deletion increased the phosphoactivation of MRN complex members, indicating that S6K1 itself can shut down DNA damage regulated by MRN signaling. This is the first report showing that S6K1 inhibition radiosensitizes lung cancer cells by decreasing MRN complex-regulated DNA repair signaling. Future studies should evaluate the role of S6K1 as a target to overcome radioresistance.

ATM Activation is Key in Vasculogenic Mimicry Formation by Glioma Stem-like Cells

Objective Vasculogenic mimicry (VM) is a novel vasculogenic process integral to glioma stem cells (GSCs) in glioblastoma (GBM). However, the relationship between VM and ataxia-telangiectasia mutated (ATM) serine/threonine kinase activation, which confers chemoradiotherapy resistance, remains unclear.Methods We investigated VM formation and phosphorylated ATM (pATM) levels by CD31/GFAPperiodic acid-Schiff dual staining and immunohistochemical staining in 145 GBM specimens. Glioma stem-like cells (GSLCs) derived from the formatted spheres of U87 and U251 cell lines and their pATM level and VM formation ability were examined using western blot and three-dimensional culture. For the examination of the function of pATM in VM formation by GSLCs, ATM knockdown by shRNAs and deactivated via ATM phosphorylation inhibitor KU55933 were studied.Results VM and high pATM expression occurred in 38.5% and 41.8% of tumors, respectively, and were significantly associated with reduced progression-free and overall survival. Patients with VM-positive GBMs exhibited higher pATM levels (rs = 0.425, P = 0.01). The multivariate analysis established VM as an independent negative prognostic factor (P = 0.002). Furthermore, GSLCs expressed high levels of pATM and formed vascular-like networks in vitro. ATM inactivation or knockdown hindered VM-like network formation concomitant with the downregulation of pVEGFR-2, VE-cadherin, and laminin B2.Conclusion VM may predict a poor GBM prognosis and is associated with pATM expression. We propose that pATM promotes VM through extracellular matrix modulation and VE-Cadherin / pVEGFR-2 activation, thereby highlighting ATM activation as a potential target for enhancing anti-angiogenesis therapies for GBM.

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

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

γ-H2AX and phospho-ATM enzyme-linked immunosorbent assays as biodosimetry methods for radiation exposure assessment: a pilot study.

In the event of a radiological incident, a fast and accurate biological dosimetry (biodosimetry) method for evaluating people who have been potentially exposed to ionising radiation is crucial. Among the many biodosimetry methods available, the immunodetection of phosphorylated H2AX (γ-H2AX) stands as a promising method to be used in the triage of patients exposed to radiation. Currently, the most common way to measure γ-H2AX levels is through fluorescence microscopy. In this pilot study, we assessed the feasibility of using an enzyme-linked immunosorbent assay (ELISA) for quantifying γ-H2AX for biodosimetry purposes. Moreover, the usefulness of measuring phosphorylated ATM (pATM) levels through ELISA for biodosimetry was also evaluated. Blood samples were obtained from three male donors (38y) and were irradiated with 60Co (0, 1, 2 and 6Gy). Peripheral blood mononuclear cells (PBMCs) were isolated and lysed before measuring γ-H2AX, total H2AX protein and pATM using ELISA kits. The dicentric chromosome assay (DCA) using whole blood was also performed for comparison. Data from all donors at each dose were pooled before statistical analysis. The ratio of γ-H2AX/total H2AX and pATM levels increased in a radiation-dose-dependent manner. The average γ-H2AX/total H2AX ratios were 0.816±0.219, 0.830±0.685, 1.276±1.151 and 1.606±1.098, whereas the average levels of pATM were 59.359±3.740, 63.366±0.840, 66.273±2.603 and 69.936±4.439, in PBMCs exposed to 0, 1, 2 and 6Gy, respectively. The linear-quadratic dose-response calibration curve for DCA was Y=0.0017 (±0.0010)+0.0251 (±0.0142)×D+0.0342 (±0.0039)×D2 $\boldsymbol{Y}=\mathbf{0.0017}\left(\pm \mathbf{0.0010}\right)+\mathbf{0.0208}\left(\pm \mathbf{0.0218}\right)\times \boldsymbol{D}+\mathbf{0.0350}\left(\pm \mathbf{0.0050}\right)\times{\boldsymbol{D}}^{\mathbf{2}}$. Overall, despite a large variability in the ratio of γ-H2AX/total H2AX among donors, the present study revealed the suitability of using the ratio of γ-H2AX/total H2AX and pATM for biodosimetry. Still, more research with a larger group of subjects is necessary to construct a reliable calibration curve for the ratio of γ-H2AX/total H2AX and pATM levels for biodosimetry.

Enhanced efficacy of combined fluzoparib and chidamide targeting in natural killer/T-cell lymphoma.

The treatment of natural killer/T-cell lymphoma (NKTCL) presents an onerous challenge, and a search for new therapeutic targets is urgently needed. Poly ADP-ribose polymerase inhibitors (PARPi) were initially used to treat breast and ovarian cancers with BRCA1/2 mutations. Their excellent antitumor efficacy led to a series of clinical trials conducted in other malignancies. However, the exploration of PARPi and their potential use in combination treatments for NKTCL remains unexplored. We treated NKTCL cell lines with fluzoparib (a novel inhibitor of PARP) and chidamide (a classical inhibitor of HDACs) to explore their cytotoxic effects in vitro. Then, their antitumor efficacy in vivo was confirmed in YT-luciferin xenograft mouse models. Fluzoparib or chidamide alone inhibited NKTCL cell proliferation in a dose-dependent manner. Cotreatment with both drugs synergistically induced excessive accumulation of DNA double-strand breaks and massive apoptotic cell death by inhibiting the DNA damage repair pathway, as shown by the decreased protein levels of p-ATM, p-BRCA1, p-ATR, and Rad51. Moreover, the combination treatment apparently increased the level of intracellular reactive oxygen species (ROS) to enhance apoptosis, and pretreatment with an ROS scavenger reduced the proapoptotic effect by 30-60% in NKTCL cell lines. In vivo, this combined regimen also showed synergistic antitumor effects in xenograft mouse models. The combination of fluzoparib and chidamide showed synergistic effects against NKTCL both in vitro and in vivo and deserves further exploration in clinical trials.

Abstract 6212: Inhibition of ATM-dependent checkpoint control and DNA double-strand break repair enhances the efficacy of ATR inhibitors

Abstract Ataxia telangiectasia and Rad3 related (ATR) and ataxia telangiectasia mutated (ATM) protein kinases play key roles in the DNA damage response (DDR) by responding to replication stress and double-strand DNA breaks respectively to pause the cell cycle and promote DNA repair. ATR kinase inhibition in cancer cells disrupts cell cycle control and causes unrepaired DNA lesions and cytotoxicity. Accordingly, several ATR inhibitors (ATRi) are in clinical development as monotherapies and in combination with DNA damaging chemotherapies and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that while treatment of cancer cells with an ATRi inhibits the ATR pathway, it simultaneously activates the ATM signaling pathway as shown by increased levels of p-ATM, p-CHK2, p-KAP1 and p-p53. In p53 wild-type cancer cells, ATR inhibition causes an ATM-mediated G1 cell cycle arrest which diminishes the DNA lesions and cytotoxic effects of ATR inhibition. Combination of an ATRi with a selective ATM inhibitor (ATMi) synergistically potentiated efficacy in cancer cells in vitro and increased efficacy in vivo at doses that did not show overt toxicities. In a panel of patient-derived xenograft (PDX) models of triple-negative breast cancer treated with an ATRi/ATMi combination, substantial improvement in efficacy was observed. Thus, activation of the ATM pathway by an ATRi acts as a compensatory resistance pathway for ATR inhibition. These results suggest a novel and efficacious combination approach for cancer therapy by dual inhibition of these two key DDR kinases. Citation Format: Audrey Turchick, Astrid Zimmermann, Li-Ya Chiu, Heike Dahmen, Brian Elenbaas, Frank T. Zenke, Andree Blaukat, Lyubomir T. Vassilev. Inhibition of ATM-dependent checkpoint control and DNA double-strand break repair enhances the efficacy of ATR inhibitors. [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 6212.

Efficacy of Vcp/p97 Inhibitor, CB-5339, Alone and in Combinations Against High-Risk AML, Including Those with Genetic Lesion in TP53

Valosin-containing protein (VCP)/p97 or p97 is an AAA+ type ATPase involved in quality control of cellular proteins in normal and transformed cells, especially under cellular stress. VCP/p97 alleviates proteotoxic stress by facilitating bulk protein degradation through the ubiquitin-proteasome system (UPS), harnessing the energy of ATP hydrolysis to unfold and extract its ubiquitylated protein substrates from protein complexes or organelles and targeting them for proteasomal degradation, e.g., ER-associated degradation (ERAD). Targeted inhibition of p97 induces lethal ER stress. The vital role of p97 in protein homeostasis has been recognized as one of the key non-oncogenic addictions in transformed cells. Targeting p97 has been previously shown to yield preferential lethality in cancer versus normal cells and exhibit in vivo efficacy against xenograft models of cancers. By probing the CRISPR-screen dependency map (DepMap), we found that p97 is a dependency across AML cell lines, including those harboring alterations in genes that indicate an increased risk or poor prognosis. CB-5339 is a selective and potent, ATP-competitive inhibitor of p97, with only low inhibitory activity against other ATPases, AAA+ ATPases, and kinases. CB-5339 was previously shown to induce accumulation of polyubiquitylated proteins, retention of ERAD substrates, and lethal ER stress in cancer cells mediated by CHOP, DR5 and NOXA. In present studies, we determined that treatment with CB-5339 dose-dependently (200 to 1000 nM for 48 hours) induces in vitro loss of viability in AML cell lines (OCI-AML3, MOLM13, SET2 and HEL92.1.7 cells), as well as in fresh, patient-derived (PD) AML cells, including those with TP53 mutations and/or TP53 allelic loss, or with 3q26 (MECOM locus) lesions and EVI1 overexpression. Utilizing MOLM13 cells edited to express the missense mutation R175H or R248Q, or the null allele of TP53, introduced via CRISPR/Cas9 into the endogenous TP53 locus, we determined that CB-5339 was active against MOLM13 cells (with MLL-AF9, FLT3-ITD and wild-type TP53) and retained significant activity against MOLM13 cells with TP53-R175H, TP53-R248Q and MOLM13-TP53-/-. Comparing ATAC-Seq and RNA-Seq findings in CB-5339-treated versus -untreated MOLM13 cells we found concordant, significantly altered chromatin accessibility and mRNA expressions of gene-loci, e.g., increase in ATF4, ATF3, HSPA5 (GRP78), NOXA, BBC3 (PUMA), FBXO32, ITGAM, and CD86, but decrease in FOXA1, TLR4, CLEC12A, CXCR4 and IL7R. Following CB-5339 treatment, gene-set enrichment analyses (GSEA) showed significantly increased normalized enrichment scores of the gene-set involved in ER stress response, not only in MOLM13 with WT-TP53 but also in those with the TP53 mutations or allelic loss of TP53. However, treatment with CB-5339 increased levels of p-ATM, p-TP53 and TP53 only in MOLM13 and OCI-AML3 (each expressing only WT-TP53), without altering the protein levels of MDM2/4. In contrast, CB-5339-induced loss of viability correlated with augmented levels of ATF4, CHOP, as well as of pro-death proteins NOXA, DR5 and active BAX in AML cells regardless of TP53 status. Importantly, similar effects were also noted in PD AML cells with or without TP53 mutation. Co-treatment with relatively lower concentrations of CB-5339 (100 to 225 nM) and venetoclax (5 to 20 nM) exerted synergistic in vitro lethality in not only MOLM13 but also PD AML cells with WT-TP53, as well as in SET2 and PD AML cells harboring mutant TP53. Co-treatment with CB-5339 and pan-BET inhibitor OTX015 was also synergistically lethal against MOLM13 and PD AML cells with or without mutant TP53. Finally, in a tail-vein infused, luciferase transduced, aggressive xenograft model of MOLM13 cells, after AML engraftment, co-treatment for 3 weeks with CB-5339 (50 mg/kg/day, PO) and either venetoclax (30 mg/kg/day, PO) or OTX015 (30 mg/kg/day, PO), as compared to treatment with vehicle or each drug alone, significantly reduced the AML burden and improved median and overall survival of the NSG mice, without inducing significant toxicity. Taken together, these findings highlight that CB-5339 induces lethal ER stress in AML cells regardless of the TP53 status, and underscore the promise of CB-5339 treatment alone and in rational combinations in exerting efficacy against AML, including those with high-risk genetic alterations in TP53 or chromosome 3q26 lesions and EVI1 overexpression.

Activation of ATM/Chk2 by Zanthoxylum armatum DC extract induces DNA damage and G1/S phase arrest in BRL 3A cells.

Zanthoxylum armatum DC is a traditional medicinal plant. It is widely used in clinical treatment and disease prevention in China, India and other regions. Modern studies have reported the phytotoxicity, cytotoxicity and the animal toxicity of Zanthoxylum armatum DC, and the damage of genetic material has been observed in plants, but the detailed mechanism has not been explored. Besides, the toxicity of normal mammalian cells has not been evaluated. To evaluate the effects and underlying mechanism of genetic material damage in BRL 3A cells induced by Zanthoxylum armatum DC. Ultra-High Performance Liquid Chromatography and Orbitrap High-Resolution Mass Spectrometry was used for identification of compounds in methanol extract of Zanthoxylum armatum DC. BRL 3A cells were incubated with different concentrations of methanol extract of Zanthoxylum armatum DC (24h). The cytotoxicity of extract was assessed with cell viability, LDH release rate, and ROS production. The damage of genetic material was assessed with OTM value of comet cells, cell cycle and the expression levels of p-ATM, p- Chk2, Cdc25A, and CDK2. Ultra-High Performance Liquid Chromatography and Orbitrap High-Resolution Mass Spectrometry investigation revealed the presence of compounds belonging to flavonoid, fatty acid and alkaloid groups. The viability of BRL 3A cells was reduced in a time-dose dependent manner treated by methanol extract of Zanthoxylum armatum DC. It increased LDH release rate and ROS production, activated the DNA double strand damage marker of γH2AX and produced comet cells. In addition, methanol extract of Zanthoxylum armatum DC caused ATM-mediated DNA damage, further phosphorylated Chk2, inhibited cell cycle related proteins, and arrested the G1/S cycle. Methanol extract of Zanthoxylum armatum DC induces DNA damage and further leads G1/S cell cycle arrest by triggering oxidative stress in the BRL 3A cells. This study provides some useful evidences for its development as an antitumor drug via activation of ATM/Chk2.

Abnormal expression of p-ATM/CHK2 in nasal extranodal NK/T cell lymphoma, nasal type, is correlated with poor prognosis

AimsThe aim of this study is to investigate the expression profiles of cell cycle related proteins in nasal extranodal NK/T cell lymphoma, nasal type (ENKTCL).MethodsThe expression profiles of cell cycle...

CBX8 Promotes Cell Proliferation and Metastasis and Leads to Radiotherapy Tolerance of Glioma Cells.

To illustrate the role of CBX8 -a protein involved in protein metabolism and chromatin regulation/acetylation- in glioma cells, especially in DNA damage repair pathways. Detect CBX8 expression in glioma cells and clinical samples by qPCR and Western blot. Overexpression and knockdown CBX8 cell lines were constructed by lentivirus infection. CCK8, wound healing, and transwell assays were used to verify the effects of CBX8 on proliferation, migration, and invasion of glioma cells. After radiation treatment, CCK8 and colony formation assays were used to detect cell sensitivity of CBX8 expression levels to radiotherapy. Western blot detected expression levels of p-ATM, p-ATR, BRCA-1, RAD51, and P53 in various cells after radiation treatment, demonstrating CBX8?s effect on DNA damage and repair proteins. Finally, the sensitivity of tumors with different CBX8 expression levels to radiotherapy was verified in vivo. CBX8 expression is significantly increased in glioma. High CBX8 expression promotes proliferation, invasion, and migration of glioma cells. It also causes glioma cells to resist radiotherapy. CBX8 affects protein expression related to DNA damage repair. In vivo, tumors with low CBX8 expression are more sensitive to radiotherapy. CBX8 promotes proliferation and metastasis of glioma cells and reduces cell sensitivity to radiotherapy by affecting DNA damage repair pathways.

The histone deacetylase SIRT1 mediates the proper DNA repair response by targeting histone H2AX to protect against doxorubicin-induced cardiotoxicity

Abstract Background Failure of DNA repair and accumulation of damaged DNA have been implicated in the pathogenesis of doxorubicin-induced cardiotoxicity. SIRT1, an NAD+-dependent histone deacetylase, is known to positively regulate DNA repair. One of the earliest events in DNA damage response (DDR) is phosphorylation of histone H2AX on Ser139 catalyzed by kinases including ATM (mutated in ataxia-telangiectasia). However, it remains unknown whether SIRT1 protects the heart from doxorubicin-induced cardiotoxicity by regulating histone H2AX. Purpose In this study, we investigated whether SIRT1 plays a role against doxorubicin-induced cardiotoxicity by regulating histone H2AX and therefore DDR. Methods and results We used tamoxifen-inducible cardiomyocyte-specific SIRT1 knockout (SIRT1-cKO) mice. Knockout was induced at 2 month of age, and mice without Cre recombinase served as wild type (WT). Mice were treated with vehicle (Veh) or doxorubicin (4 IP injections of 5 mg/kg/week) starting at 3 month of age. Echocardiography showed that fractional shortening (FS) before doxorubicin was similar in WT (34%) and SIRT1-cKO (34%). However, FS at 1 week after final doxorubicin was lower in SIRT1-cKO than WT (26% vs. 30%, P<0.05). Myocardial ANP mRNA level was 2.4-fold higher in SIRT1-cKO than WT after doxorubicin. Apoptotic cells analyzed by TUNEL-positive nuclei were similar in Veh-treated SIRT1-cKO and WT (0.125 vs. 0.073%) but were more increased by doxorubicin in SIRT1-cKO than WT (0.384 vs 0.194%, P<0.05). Immunoblotting showed that doxorubicin significantly increased myocardial levels of phospho-Ser139-histone H2AX (p-H2AX) and phospho-Ser1981-ATM (p-ATM) in WT. However, the doxorubicin-induced increase in p-H2AX level was significantly attenuated in SIRT1-cKO despite similar p-ATM levels after doxorubicin between WT and SIRT1-cKO. In H9c2 cardiomyocytes, doxorubicin treatment (10 μM) increased both p-ATM and p-H2AX levels, but siRNA-mediated knockdown (KD) of SIRT1 attenuated doxorubicin-induced phosphorylation of H2AX without changing p-ATM level. Cell death after doxorubicin was enhanced in SIRT1-KD cells compared to control cells (13.2% vs 8.6%, P<0.05). Immunostaining showed that SIRT1 colocalized with histone H2AX in the nucleus. Treatment with a SIRT1 inhibitor Ex527 increased level of acetylated H2AX at 5th lysine residue, suggesting that SIRT1 regulates histone H2AX via deacetylation. Conclusion These data suggest that SIRT1 plays a protective role against doxorubicin-induced cardiotoxicity via regulation of H2AX phosphorylation to mediates proper DDR. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Japan Society for the Promotion of Science

Abstract 5914: URI participates in cisplatin-induced DNA damage repair of gastric cancer cells via the ATM/CHK2 pathway

Abstract Gastric cancer is the most common gastrointestinal malignant tumor and also the second leading cause of cancer-related mortality worldwide. Given its high rate of non- symptoms at early stage, gastric cancer is usually diagnosed at middle or advanced stage with metastasis. Therefore, chemotherapy, including Cisplatin, becomes a main treatment option, in addition to surgery. However, it is not uncommon for patients to develop cisplatin resistance which limits its clinical applications. URI, a prefoldin family member and also an oncoprotein, has been shown to promote multiple cancer development, including gastric cancer. The aim of this study was to investigate how URI may participate in the DNA damage repair of gastric cancer cells induced by Cisplatin and its possible mechanism via the ATM/CHK2 pathway. URI knockdown cell lines from gastric cancer cells MGC-803 and SGC-7901 were established and treated with different concentrations of Cisplatin. Cell viability, proliferation and apoptosis were analyzed by CCK-8, EDU assay, and flow cytometry respectively. DNA injury marker H2AX were examined by qRT-PCR and Western blot analysis. Comet assay was used to detect the DNA damage. The results showed that URI knockdown reduces cell viability and proliferation, as well as the Cisplatin-induced apoptosis of gastric cancer cells. Cisplatin dose responsively induced DNA damage in gastric cancer cells and URI knockdown enhanced this damage as indicated by comet assay and increased γH2AX expression. We have also detected increased levels of P-ATM and P-CHK2 in gastric cancer cells treated with Cisplatin, while URI knockdown decreased the P-ATM and P-CHK2 expression after the cells were treated with cisplatin for 12 hours. Taken together, our results support that URI reduces DNA damage and promotes DNA repair of gastric cancer cells induced by cisplatin and therefore, enhances its resistance possibly via the ATM/CHK2 pathway Citation Format: Huiqin Bian, Yaojuan Lu, Yu Gu, Xiaojing Zhang, Lichun Sun, Junxia Gu, Qiping Zheng. URI participates in cisplatin-induced DNA damage repair of gastric cancer cells via the ATM/CHK2 pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5914.

High pATM is Associated With Poor Local Control in Supraglottic Cancer Treated With Radiotherapy.

ObjectivesMost early stage laryngeal squamous cell carcinomas (LSCC) are treated with radiotherapy. Discovery of new biomarkers are needed to improve prediction of outcome after radiotherapy and to identify potential targets for systemic targeted therapy. The ataxia telangiectasia mutated (ATM) gene plays a critical role in DNA damage response induced by ionizing radiation.MethodsThe prognostic value of immunohistochemical expression of pATM, pChk2, and p53 were investigated in 141 patients with T1‐T2 LSCC curatively treated with external beam radiotherapy. Uni‐ and multivariable Cox regression analyses were performed to examine the relation between expression levels of markers and local control.ResultsLocal control was significantly worse in cases with high levels of pATM (HR 2.14; 95% CI, 1.08–4.24; P = .03). No significant associations with local control were found for pChk2 and p53 expression. The association of high pATM expression with poor local control was only found for supraglottic LSCC (HR 10.9; 95% CI, 1.40–84.4; P = .02).ConclusionOur findings suggest a potential role for ATM in response to radiotherapy in early stage supraglottic LSCC and imply ATM inhibition as a possibility to improve response to radiotherapy.Level of Evidence NA Laryngoscope, 130: 1954–1960, 2020

Evidence that Tip60 Induces the DDR & Cardiomyocyte Replicative Senescence in the Neonatal Heart

Tip60 (Tat interactive protein, 60 kD) is a tumor suppressor protein that acetylates histone and non‐histone proteins. Among the latter, ATM (Ataxia Telangiectasia Mutated), a kinase that induces the DNA damage response (DDR), is activated in cancer cells by Tip60‐mediated acetylation. We are investigating the function of Tip60 in the heart, using global and conditional knockout mouse models to disrupt the Kat5 gene encoding Tip60. Because global knockout (i.e. Tip60−/−) causes early embryonic lethality, we used Tip60+/− heterozygotes to show that, when under cardiac stress, modest Tip60 depletion correlates with re‐activation of the cardiomyocyte (CM) cell‐cycle in the adult heart. More recently, using a knockout model wherein Tip60 is constitutively and specifically depleted in CMs, we observed CM dropout and lethality in three‐month‐old mice, wherein hearts exhibited features of mitotic catastrophe. Because the latter phenotype was preceded by increased CM density, it was suggested that Tip60‐depletion may extend the duration of CM proliferation during neonatal stages of heart development. Moreover, considered in the context of recent reports that ATM is activated at birth to induce the DDR, which in turn causes replicative senescence of CMs by mid‐neonatal stages, these findings suggest that Tip60 induces these events. To examine these possibilities we are conditionally depleting Tip60 in CMs of Tip60F/F;Cre neonatal mice by injecting tamoxifen at postnatal day 0 (P0). To date, immunostaining of sections from neonatal hearts with antibodies that recognize 5′‐BrdU, phospho‐histone‐H3, and phospho‐ATM (pATM) has revealed that Tip60 depletion significantly increases CM proliferation as early as P7, by which time CM proliferation is normally diminished, and that this is accompanied by significantly decreased pATM levels, indicative of a diminished DDR. These findings provide further support for the notion that DDR activation at the time of birth induces replicative senescence of neonatal CMs, implicating Tip60 as the inducer of this process.Support or Funding InformationNIH HL131788 (JL & JA), and a grant from the MCW Cardiovascular CenterThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P1-06-04: Small-molecule screening nominates diverse combination therapies that sensitize BRCA mutant and wild-type triple negative breast cancer to PARP inhibition

Abstract Background: Triple negative breast cancer (TNBC) remains a heterogeneous clinical phenotype with few, known therapeutic targets. PARP inhibitors (PARPi) are the first approved, targeted therapy in TNBC, limited to germline BRCA mutant (BRCAm) cancers that lack homologous recombination repair capacity. Even in this context, resistance quickly emerges via secondary mutations that restore DNA repair ability. While DNA damage repair is an intriguing target in BRCA wild type (BRCAwt) TNBC due to inherent, genomic instability, PARPi alone have been ineffective in unselected populations. Systematic approaches to define novel drugs that sensitize BRCAwt and BRCAm TNBC to PARPi would greatly improve therapeutic efficacy and durability. Methods: BRCAwt (HCC1806) and BRCAm (SUM149PT) cell lines were screened in duplicate using a 2,100-compound small molecule library. Cell lines were plated in media containing DMSO or sub-lethal doses of the PARPi, olaparib, onto Selleck Bioactive drug plates. Cell viability was assessed after 72 hours, then normalized to vehicle control. Hit cut-offs were predefined as log2 drug/DMSO of ≤ -0.7 with a viability difference greater than 20% -where stringent scoring thresholds were chosen to exceed the full range of scores observed in 816 empty control wells. Hits were sorted by target and pathway to provide mechanistic insight into the synergy of combinations. Drug combinations with the highest potential for near term translation were validated using GI50 viability assays in 9 BRCAwt and BRCAm TNBC cell lines. The most promising combination was further validated via immunoblotting, colony formation, and apoptosis assays. Results: Several drug classes affecting well-known oncogenic signaling pathways conferred sensitivity to PARPi, with more hits in the BRCAm cell line. Relevant druggable targets sensitizing cells to olaparib in BRCAm TNBC that met the predefined cut-point were inhibitors of PI3K (pan-PI3K, PI3Kα and PI3Kβ specific), VEGFR, MEK, EGFR, NF-kB, aurora kinase and several DNA damaging agents. Aurora kinase, EGFR, and NF-kB inhibition sensitized cells to olaparib, yet upon further validation, synergy was mild. The screen identified ATM inhibitors, KU-55933 and KU-60019, as sensitizers of BRCAm cells to olaparib. The potent ATM inhibitor, AZD0156, and olaparib were a highly synergistic combination validated in all 9 BRCAm and BRCAwt TNBC cell lines via cell viability, annexin V, and colony formation assays. Immunoblotting of relevant DNA damage repair proteins showed that olaparib caused upregulation of p-ATM in BRCAm and BRCAwt cells. p-ATM expression decreased in response to combination ATM and PARP inhibition. Attenuated levels of p-ATM resulted in increased levels of p- and T-γH2AX, indicating an accumulation of double stranded DNA breaks. Conclusion: In vitro, inhibition of several relevant, oncogenic pathways yielded sensitivity to PARPi in TNBC. We identified the ATM inhibitor, AZD0156, and olaparib as a potent combination regardless of BRCA status, a finding currently being evaluated in patient-derived in vivo models. Combination ATM plus PARP inhibitor therapy is a promising and feasible approach for near term translation in metastatic TNBC. Citation Format: Sammons S, Yip C, Anderson G, Force J, Marcom K, Westbrook K, Anders CK, Blackwell K, Wood K. Small-molecule screening nominates diverse combination therapies that sensitize BRCA mutant and wild-type triple negative breast cancer to PARP inhibition [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-06-04.

Protective effects of ginsenoside Rg2 and astaxanthin mixture against UVB-induced DNA damage

ABSTRACTUltraviolet B (UVB) radiation induces skin damage, skin matrix degradation, and wrinkle formation through photochemical reaction and oxidative stress. Therefore, protecting the skin from UVB can prevent skin aging. In this study, we investigated the effects of a mixture (RA) of Rg2, a ginsenoside, and astaxanthin, an antioxidant, on the responses of HaCaT cells exposed to UVB (700 J/m2). The cells were incubated for 24 h after UVB exposure and cell viability was determined by MTT assay. UVB decreased cell viability by 60% compared to that of untreated control cells, whereas RA increased cell viability in a concentration-dependent manner, and this increase was significantly higher than that in the single treatment groups. Further, UVB increased the levels of DNA lesions such as cyclobutane pyrimidine dimer (CPD) and 8-hydroxyguanine (8-OHdG). Conversely, RA decreased both CPD and 8-OHdG levels in a concentration-dependent manner. UVB exposure also increased phosphorylation of ataxia-telangiectasia mutated (ATM) protein kinase and p53 and subsequently increased the levels of GADD45α, p21, and matrix metalloproteinases (MMPs)-3, -9, and -13. Additionally, UVB exposure decreased the level of COL1A1. However, RA treatment decreased the levels of p-ATM, p-p53, GADD45α, p21, MMP-3, -9, and -13 and increased the level of COL1A1 in a concentration-dependent manner. These results suggest that RA reduces UVB-induced cytotoxicity and genotoxicity through up-regulation of DNA repair via the combined effects of Rg2 and astaxanthin.

Abstract 323: DNA damage response (DDR)-targeting strategy by targeting WEE1 and or ATM/ATR works in biliary tract cancer

Abstract Background: Currently, targeting DNA damage response (DDR) is one strategy for new cancer drug development. DDR pathway involves DNA damage repair, cell cycle progression, apoptosis, and regulate the immune system. Biliary tract cancer (BTC) has a poor prognosis with a huge unmet medical need. In BTC, DNA repair pathway, which includes BAP1, MSH6, BRCA1, ATM, MLH1, and MSH2, is altered in about 20 % of cases. TP53 module is observed in 33.9% of cases (Nat Genet 2015). The purpose of this study is to evaluate DNA damage response (DDR)-targeting strategy in BTC. Methods: Using 10 kinds of BTC cell lines (SNU245, SNU308, SNU478, SNU869, SNU1079, SNU1196, HuCCT-1, TFK-1, SNU2670, and SNU2773). AZD1775 (Wee1 inhibitor), AZD6738 (ATR inhibitor) and AZD0156 (ATM inhibitor) were tested. Results: Among 10 cell lines, SNU308 and HuCCT1 were very sensitive to AZD1775, and SNU2670 and SNU2773 were relatively resistant to AZD1775. AZD1775 blocked phosphorylation of CDK1 (Y15) and CDK2 (Y15) in sensitive cells and but increased rH2AX in all cells. AZD1775 significantly increased apoptosis (cleavage of PARP and caspase-7) and G2/M arrest. Interestingly, Wee1 inhibitor increased pATR and pATM levels in resistant cells. AZD1775 in combination with AZD6738 or AZD0156 showed more potent antitumor effect than monotherapy of each drug. Conclusion: Inhibition of Wee1 has an antitumor effect in some BTC cells. In combination with ATM/ATR inhibitors, the resistance to Wee1 inhibition could be overcome. Taken together, this study supports the further clinical development of DDR-targeting strategy in BTC as monotherapy or in combination. Citation Format: Ah Rong Nam, Ji Eun Park, Ju-Hee Bang, Mei Hua Jin, Yung-Jue Bang, Do-Youn Oh. DNA damage response (DDR)-targeting strategy by targeting WEE1 and or ATM/ATR works in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 323.

Abstract 4900: Reactive oxygen species scavenger extends the efficacy of BRAF inhibitors in BRAF-mutant melanoma

Abstract We aim to identify whether reactive oxygen species (ROS) targeted therapy using a ROS scavenger, A100, will inhibit tumor growth and evade BRAF inhibitor induced resistance in melanoma cells harboring the endogenous BRAFV600E mutation. We also propose to investigate the mechanism by which A100 sensitizes the dabrafenib-mediated resistance. Dabrafenib-resistant (DR) melanoma cells (WM-115DR, SK-MEL-24DR and A375DR) were generated using increasing doses of dabrafenib. We conducted flow cytometry experiments to measure ROS levels before and after acquisition of resistance. DCF-DA assay indicated upregulation of hydrogen peroxide levels in dabrafenib resistant cell lines versus parental cell lines. Furthermore, dabrafenib resistant cell lines demonstrated elevated superoxide levels as analyzed by MitoSOX. Crystal violet and three-dimensional matrigel assays were performed to analyze the effect of A100 and dabrafenib on cell growth of dabrafenib resistant and parental melanoma cell lines. We observed a statistically significant decrease in cell proliferation and colony formation of dabrafenib resistant cells when subjected to the combination of A100 and dabrafenib compared to single agent dabrafenib or A100. We also noted a trend of inhibition of cell proliferation in response to combined treatment of A100 and dabrafenib in parental cell lines versus the single agent. There was significant reduction in colony formation under similar condition in parental cells. Dabrafenib as a single agent had similar effect in reducing phosphorylated ERK1/2 as compared to co-treatment with A100 in A375, SK-MEL-24 and WM-115 parental cells. As expected, there was no alteration of MAPK signaling in resistant WM-115DR, SK-MEL-24DR and A375DR cells upon treatment with dabrafenib. A100 has been reported to cause DNA damage in leukemia cells. We analyzed the effect of A100 in the presence or absence of dabrafenib on markers of DNA damage. The data suggested that DNA damage is induced by A100 as assessed by increased levels of p-ATM, p-ATR and γ-H2AX. Further, A100 in combination with dabrafenib increased levels of p-ATM, p-ATR and γ-H2AX in parental and dabrafenib resistant cell lines. Proteomics analysis on WM-115 versus WM-115DR cell line was performed to determine alterations in mitochondrial protein expression upon resistance to BRAF inhibitors. The data indicated upregulation of proteins with antioxidant functions including SOD2 (superoxide dismutase [Mn]) and Peroxiredoxin-1 (PRDX1) in WM-115DR cells compared to WM-115 parental which was confirmed by immunoblotting analysis. Overall, this study suggested that the combination of ROS quenching agent (A100) with BRAF inhibitor (dabrafenib) could be a potential strategy to treat BRAF-mutant melanoma patients. Citation Format: Long Yuan, Rosalin Mishra, Hima Patel, Samar Alanazi, Joan Garrett. Reactive oxygen species scavenger extends the efficacy of BRAF inhibitors in BRAF-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4900.

Radiation-Induced DNA Damage in Operators Performing Endovascular Aortic Repair.

Radiation exposure during fluoroscopically guided interventions such as endovascular aortic repair (EVAR) is a growing concern for operators. This study aimed to measure DNA damage/repair markers in operators perfoming EVAR. Expression of the DNA damage/repair marker, γ-H2AX and DNA damage response marker, phosphorylated ataxia telangiectasia mutated (pATM), were quantified in circulating lymphocytes in operators during the peri-operative period of endovascular (infrarenal, branched, and fenestrated) and open aortic repair using flow cytometry. These markers were separately measured in the same operators but this time wearing leg lead shielding in addition to upper body protection and compared with those operating with unprotected legs. Susceptibility to radiation damage was determined by irradiating operators' blood in vitro. γ-H2AX and pATM levels increased significantly in operators immediately after branched endovascular aortic repair/fenestrated endovascular aortic repair (P<0.0003 for both). Only pATM levels increased after infrarenal endovascular aortic repair (P<0.04). Expression of both markers fell to baseline in operators after 24 hours (P<0.003 for both). There was no change in γ-H2AX or pATM expression after open repair. Leg protection abrogated γ-H2AX and pATM response after branched endovascular aortic repair/fenestrated endovascular aortic repair. The expression of γ-H2AX varied significantly when operators' blood was exposed to the same radiation dose in vitro (P<0.0001). This is the first study to detect an acute DNA damage response in operators performing fluoroscopically guided aortic procedures and highlights the protective effect of leg shielding. Defining the relationship between this response and cancer risk may better inform safe levels of chronic low-dose radiation exposure.