Induction Of DNA Damage Response Research Articles

Abstract PR13: Inhibition of RNA polymerase I transcription activates targeted DNA damage response and enhances the efficacy of PARP inhibitors in high-grade serous ovarian cancer

Abstract Introduction: PARP inhibitors (PARPi) have revolutionized disease management of patients with homologous recombination (HR) DNA repair-deficient high-grade serous ovarian cancer (HGSOC). However, acquired resistance to PARPi is a major challenge in the clinic. The specific inhibitor of RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rDNA) has demonstrated single-agent antitumor activity in p53 wild-type and p53-mutant hematologic malignancies (first-in-human trial, dose escalation study of CX-5461 at Peter MacCallum Cancer Centre) (Khot et al., Cancer Discov 2019). CX-5461 has also been reported to exhibit synthetic lethality with BRCA1/2 deficiency through stabilization of G-quadruplex DNA (GQ) structures. Here, we investigate the efficacy of CX-5461 in treating HGSOC. Experimental Design: The mechanisms by which CX-5461 induces DNA damage response (DDR) and displays synthetic lethality in HR-deficient HGSOC cells are explored. We present in vivo data of mice bearing two functionally and genomically profiled HGSOC-patient-derived xenograft (PDX)s treated with CX-5461 and olaparib, alone and in combination. We also investigate CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. Results: Utilizing ovarian cancer cell lines, we demonstrate that sensitivity to CX-5461 is associated with “BRCA1 mutation” and “MYC targets” gene expression signatures. In addition, sensitivity to CX-5461 is associated with high basal rates of Pol I transcription. Importantly, we demonstrate a novel mechanism for CX-5461 synthetic lethal interaction with HR deficiency mediated through the induction of replication stress at rDNA repeats. Our data reveal CX-5461-mediated DDR in HR-deficient cells does not involve stabilization of GQ structures as previously proposed. On the contrary, we show definitively that CX-5461 inhibits Pol I recruitment leading to rDNA chromatin defects including stabilization of R-loops, single-stranded DNA, and replication stress at the rDNA. Mechanistically, we demonstrate CX-5461 leads to replication-dependent DNA damage involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 has a different sensitivity spectrum to olaparib and cooperates with PARPi in exacerbating replication stress, leading to enhanced therapeutic efficacy in HR-deficient HGSOC-PDX in vivo compared to single-agent treatment of both drugs. Further, CX-5461 exhibits single-agent efficacy in olaparib-resistant HGSOC-PDX overcoming PARPi-resistance mechanisms involving fork protection. Importantly, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. Conclusions: CX-5461 is a promising therapy alone and in combination therapy with PARPi in HR-deficient HGSOC. CX-5461 also has exciting potential as a treatment option for patients with relapsed HGSOC tumors that have high MYC activity and poor clinical outcome; these patients currently have very limited effective treatment options. This abstract is also being presented as Poster A71. Citation Format: Elaine Sanij, Katherine Hannan, Jiachen Xuan, Shunfei Yan, Jessica A. Ahern, Anna S. Trigos, Natalie Brajanovski, Jinbae Son, Keefe T. Chan, Olga Kondrashova, Elizabeth Lieschke, Matthew J. Wakefield, Sarah Ellis, Carleen Cullinane, Gretchen Poortinga, Kum Kum Khanna, Linda Mileshkin, Grant A. McArthur, John Soong, Els M. Berns, Ross D. Hannan, Clare L. Scott, Karen E. Sheppard, Richard B. Pearson. Inhibition of RNA polymerase I transcription activates targeted DNA damage response and enhances the efficacy of PARP inhibitors in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR13.

Development and optimization of a cascade of screening assays for inhibitors of TRF2.

TRF2 is a telomere associated protein which plays an important role in telomere maintenance. Knockdown of TRF2 can cause chromosomal end to end fusions and induce DNA damage responses. TRF2 exerts its functions partially by recruiting a number of accessory proteins through its TRF homology domain (TRFH), therefore identification of small molecular compounds which can bind to the TRFH domain of TRF2 and block the interactions of TRF2 with its associated proteins is important to elucidate the molecular mechanism of these protein-protein interactions. Development of robust and sensitive screening and evaluation assays is critical to the identification of TRF2 inhibitors, in this paper we reported the development and optimization of a cascade of screening and binding affinity evaluation assays, including a competitive FP (Fluorescence Polarization) assay utilized in our previous research, and two novel label-free DSF (Differential Scanning Fluorescence) and BLI (Biolayer Interferometry) assays. A previously identified TRF2 inhibitor TRF2-27 was used as an internal reference compound and evaluated in all of these assays. According to the results, DSF assay is not suitable for TRF2 screening because of the low ΔTm, while the optimized labeled-free BLI assay was demonstrated to be an accurate and reproducible assay for TRF2 inhibitor screening and characterization.

TFEB Modulates p21/WAF1/CIP1 during the DNA Damage Response.

The MiT/TFE family of transcription factors (MITF, TFE3, and TFEB), which control transcriptional programs for autophagy and lysosome biogenesis have emerged as regulators of energy metabolism in cancer. Thus, their activation increases lysosomal catabolic function to sustain cancer cell growth and survival in stress conditions. Here, we found that TFEB depletion dramatically reduces basal expression levels of the cyclin-dependent kinase (CDK) inhibitor p21/WAF1 in various cell types. Conversely, TFEB overexpression increases p21 in a p53-dependent manner. Furthermore, induction of DNA damage using doxorubicin induces TFEB-mediated activation of p21, delays G2/M phase arrest, and promotes cell survival. Pharmacological inhibition of p21, instead, abrogates TFEB-mediated protection during the DNA damage response. Together, our findings uncover a novel and direct role of TFEB in the regulation of p21 expression in both steady-state conditions and during the induction of DNA-damage response (DDR). Our observations might open novel therapeutic strategies to promote cancer cell death by targeting the TFEB-p21 pathway in the presence of genotoxic agents.

Hepatotoxic pyrrolizidine alkaloids induce DNA damage response in rat liver in a 28-day feeding study

Pyrrolizidine alkaloids (PA) are secondary plant metabolites that occur as food and feed contaminants. Acute and subacute PA poisoning can lead to severe liver damage in humans and animals, comprising liver pain, hepatomegaly and the development of ascites due to occlusion of the hepatic sinusoids (veno-occlusive disease). Chronic exposure to low levels of PA can induce liver cirrhosis and liver cancer. However, it is not well understood which transcriptional changes are induced by PA and whether all hepatotoxic PA, regardless of their structure, induce similar responses. Therefore, a 28-day subacute rat feeding study was performed with six structurally different PA heliotrine, echimidine, lasiocarpine, senecionine, senkirkine, and platyphylline, administered at not acutely toxic doses from 0.1 to 3.3 mg/kg body weight. This dose range is relevant for humans, since consumption of contaminated tea may result in doses of ~ 8 µg/kg in adults and cases of PA ingestion by contaminated food was reported for infants with doses up to 3 mg/kg body weight. ALT and AST were not increased in all treatment groups. Whole-genome microarray analyses revealed pronounced effects on gene expression in the high-dose treatment groups resulting in a set of 36 commonly regulated genes. However, platyphylline, the only 1,2-saturated and, therefore, presumably non-hepatotoxic PA, did not induce significant expression changes. Biological functions identified to be affected by high-dose treatments (3.3 mg/kg body weight) comprise cell-cycle regulation associated with DNA damage response. These functions were found to be affected by all analyzed 1,2-unsaturated PA.In conclusion, 1,2-unsaturated hepatotoxic PA induced cell cycle regulation processes associated with DNA damage response. Similar effects were observed for all hepatotoxic PA. Effects were observed in a dose range inducing no histopathological alterations and no increase in liver enzymes. Therefore, transcriptomics studies identified changes in expression of genes known to be involved in response to genotoxic compounds at PA doses relevant to humans under worst case exposure scenarios.

Integrative proteomic and functional analyses provide novel insights into the action of the repurposed drug candidate nitroxoline in AsPC-1 cells

We recently identified nitroxoline as a repurposed drug candidate in pancreatic cancer (PC) showing a dose-dependent antiproliferative activity in different PC cell lines. This antibiotic is effective in several in vitro and animal cancer models. To date, the mechanisms of nitroxoline anticancer action are largely unknown. Using shotgun proteomics we identified 363 proteins affected by nitroxoline treatment in AsPC-1 pancreatic cancer cells, including 81 consistently deregulated at both 24- and 48-hour treatment. These proteins previously unknown to be affected by nitroxoline were mostly downregulated and interconnected in a single highly-enriched network of protein-protein interactions. Integrative proteomic and functional analyses revealed nitroxoline-induced downregulation of Na/K-ATPase pump and β-catenin, which associated with drastic impairment in cell growth, migration, invasion, increased ROS production and induction of DNA damage response. Remarkably, nitroxoline induced a previously unknown deregulation of molecules with a critical role in cell bioenergetics, which resulted in mitochondrial depolarization. Our study also suggests that deregulation of cytosolic iron homeostasis and of co-translational targeting to membrane contribute to nitroxoline anticancer action. This study broadens our understanding of the mechanisms of nitroxoline action, showing that the drug modulates multiple proteins crucial in cancer biology and previously unknown to be affected by nitroxoline.

Eupalinolide J induces apoptosis, cell cycle arrest, mitochondrial membrane potential disruption and DNA damage in human prostate cancer cells.

Eupalinolide J (EJ) is a new sesquiterpene lactone isolated from Eupatorium lindleyanum DC. In the present study, we investigated the anti-cancer activity of EJ on cell proliferation in human prostate cancer cells. The MTT results indicated that EJ showed marked anti-proliferative activity in PC-3 and DU-145 cells in a dose- and time-dependent manner. DAPI staining analysis demonstrated that this effect was mediated by induction of cell apoptosis. Flow cytometric analysis indicated a significant increase in apoptotic cells, cell cycle arrest at G0/G1 phase and disruption of mitochondrial membrane potential (MMP) after EJ treatment. Meanwhile, the activation of caspase-3 and caspase-9 was visibly observed. Furthermore, our results demonstrated that the expression levels of γH2AX, p-Chk1 and p-Chk2 were significantly up-regulated, suggesting the induction of DNA damage responses in EJ-treated prostate cancer cells. The above results indicated that EJ exhibited effective anti-cancer activity in vitro. It could be a promising candidate agent for the clinical treatment of prostate cancer.

Alternative Lengthening of Telomeres (ALT) in Tumors and Pluripotent Stem Cells.

A telomere consists of repeated DNA sequences (TTAGGG)n as part of a nucleoprotein structure at the end of the linear chromosome, and their progressive shortening induces DNA damage response (DDR) that triggers cellular senescence. The telomere can be maintained by telomerase activity (TA) in the majority of cancer cells (particularly cancer stem cells) and pluripotent stem cells (PSCs), which exhibit unlimited self-proliferation. However, some cells, such as telomerase-deficient cancer cells, can add telomeric repeats by an alternative lengthening of the telomeres (ALT) pathway, showing telomere length heterogeneity. In this review, we focus on the mechanisms of the ALT pathway and potential clinical implications. We also discuss the characteristics of telomeres in PSCs, thereby shedding light on the therapeutic significance of telomere length regulation in age-related diseases and regenerative medicine.

MEDI2228, a Novel Bcma Antibody-PBD Conjugate, Sensitizes Human Multiple Myeloma Cells to NK Cell-Mediated Cytotoxicity and Upregulates CD38 Expression in MM Cells

MEDI2228, an antibody-drug conjugate (ADC) comprised of an anti-BCMA antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine dimer, is currently under clinical development for the treatment of human multiple myeloma (MM) (NCT03489525). MEDI2228 induces DNA damage responses (DDR) prior to apoptosis, as demonstrated by phosphorylation of ATM/ATR, CHK1/2, and gH2AX in MM cells regardless of p53 status and responsiveness to current MM therapies including bortezomib and IMiDs. Since activation of DDR alters expression of ligands for NKG2D receptors critical for NK-mediated immune surveillance, we here examined whether the ATM/ATR-CHK1/2 signaling cascades activated by MEDI2228 treatment would increase NKG2D ligands in MM cells. Using real-time quantitative RT-PCR and flow cytometry analysis, we found that treatment with MEDI2228 increased the expression of major histocompatibility complex (MHC) class I chain-related proteins A and B (MICA/B) in MM cell lines (n>5) and CD138+ MM cells from patients with relapsed and refractory disease (n=4). In addition, expression of the MHC class I molecules/NKG2D ligands ULBP-1, -3, -2/5/6 increased following MEDI2228 treatment. Next, we evaluated NK cell-mediated lysis of MM target cells (n>3) with or without pretreatment with MEDI2228 and found increased NK cell-mediated lysis of MEDI2228-pretreated vs control MM cells in an effector-target ratio-dependent manner. In parallel, we examined whether MEDI2228 stimulates STAT1- and IFN-related signaling pathways since they are activated by DDR and play a crucial role in innate and adaptive immunity. We found that MEDI2228 treatment significantly increases phosphorylation of STAT1 in H929 and its derived IMiD-resistant cells, and further augments expression of IFN-induced genes (IFITs), IFIT1, 2, 3, and 5, which have been shown to inhibit proliferation and promote apoptosis in cancer cells. Significantly, CD38 is upregulated by MEDI2228 treatment, with increased mRNA expression as well as membrane expression detected by flow cytometry in MM cell lines and MM cells from newly diagnosed and refractory patients (n=5). Consequently, MEDI2228-pretreated MM cells (n>3) are more susceptible to NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) by daratumumab, which targets CD38. Taken together, our data show that MEDI2228-induced DDR primes MM cells to NK cell-mediated cytotoxicity by increasing expression of MICA/B in MM cells to enhance binding and activating NK cytolytic activity. Simultaneously, MEDI2228 induces IFN-stimulated genes, including CD38, resulting in enhanced MM cell lysis by daratumumab. These results indicate additional mechanisms of anti-MM activity for MEDI2228 and suggest that a combination of MEDI2228 and anti-CD38 mAbs may further improve outcome for MM patients. Disclosures Kinneer: AstraZeneca: Employment. Munshi:Janssen: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy. Anderson:Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau.

Glutaminase Inhibition Overcomes Acquired Resistance to Mitochondrial Complex I in NOTCH1-Driven T-Cell Acute Lymphoblastic Leukemias (T-ALL) Via Block of Glutamine Driven Reductive Metabolism

Notch1-mutated T-ALL is an aggressive hematologic malignancy lacking targeted therapeutic options. Genomic alterations in Notch1-gene and its activated downstream pathways are associated with metabolic stress response and heightened glutamine (Gln) utilization to fuel oxidative phosphorylation (OxPhos) (Kishton at al., Cell Metabolism 2016, 23:649, Herranz at al., Nat Med, 2015, 21(10): 1182-1189). Hence, targeting NOTCH1-associated OxPhos and/or Gln dependency could constitute a plausible therapeutic strategy for T-ALL. In this study we examined metabolic vulnerabilities of NOTCH1-driven T-ALL and tested pre-clinical efficacy of novel mitochondrial complex I (OxPhosi) IACS-010759 and of glutaminase inhibitor CB-839 (GLSi) in T-ALL models including Notch1-mutated T-ALL cell lines, patient-derived xenograft (PDX) and primary T-ALL cells. We have previously reported and confirmed in this expanded study the anti-leukemia efficacy of IACS-010759 (EC50s 0.1-15 nM) (Molina at al., Nat Med, 2018, 24: 1036; Baran at al., Blood, 2018, 132:4020). Metabolic characterization demonstrated that OxPhosi caused striking dose-dependent decrease in basal and maximal oxygen consumption rate (OCR), ATP and NADH generation in T-ALL cell lines and primary T-ALL samples (p<0.001). OxPhosi, similar to knockout of complex I subunit NDUFS4 using CRISPR-CAS9, induced profound changes in T-ALL mitochondria, with induction of mitochondrial reactive oxygen species (ROS), DNA damage, activation of AMPK and inhibition of mTOR pathway. OxPhosi altered cellular energy homeostasis by reduction of TCA cycle intermediates, glutathione and reduction of intracellular nucleotides ATP, CTP, GTP, and UTP, translating into inhibition of DNA and RNA synthesis (p<0.0001) (by UPLC-MS/MS). IACS-010759 significantly reduced the glucose flux through the TCA cycle, redirected it towards lactate production and triggered increased utilization of Gln for fueling of the TCA cycle and reductive metabolism (Fig.1, Flux metabolic analysis SIRM). In concert with these findings, supplementation with Gln partially rescued growth-inhibitory effects of OxPhos inhibition. These results uncover metabolic gap used by T-ALL to escape OxPhos block, and identifying reliance on glutaminolysis as a critical therapeutic target. To confirm that blockade of Gln entry into TCA cycle with GLSi synergistically reduced viable ALL cell numbers, we studied potential synergy of OxPhosi and GLSi. The key role of Gln in maintaining energy production and cell proliferation via OxPhos in Notch1-mutated T-ALL cells was confirmed by the findings that Gln starvation or pharmacological GLS inhibition by CB-839 reduced ATP production and OCR and decreased cell proliferation by more than 50% in vitro (Fig.2, Fig.3). Dual blockade of OxPhos together with GLS induced DNA damage response via accumulation of ROS upon glutathione deprivation, induced AMPK signaling through profound reduction of all adenosine related intermediates and inhibited mTOR signaling. This translated into significant reduction of leukemia burden and extension of overall survival in vivo (p<0.0001) in Notch1-mutated T-ALL PDX models (n=2) with IACS-010759/CB-839 co-treatment and in the Notch1-mutated GLS fl/fl murine model upon tamoxifen-induced GLS knockout (p<0.0001) (Fig.4, Fig.5). In summary, our findings indicate that dual blockade of metabolic processes by inhibiting complex I of mitochondria and restricting Gln utilization results in metabolic catastrophe in Notch1-mutated T-ALL associated with energy depletion and oxidative stress, which combined severely inhibit T-ALL growth and survival. We postulate that targeting this unique metabolic vulnerability of Notch1-mutated T-ALL cells constitutes a novel therapeutic modality in this aggressive malignancy. Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. Jabbour:AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. Konopleva:Agios: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Astra Zeneca: Research Funding; Kisoji: Consultancy, Honoraria; Ascentage: Research Funding; Genentech: Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Eli Lilly: Research Funding; Cellectis: Research Funding; Forty-Seven: Consultancy, Honoraria; Ablynx: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; AbbVie: Consultancy, Honoraria, Research Funding.

Preliminary Lasso Regression Analysis Identifies DNA-Damage Gene Expression Signature Predictive of Clinical Outcomes in Patients Using Gemtuzumab Ozogamicin

Background: In September 2017, the FDA announced re-approval of gemtuzumab ozogamicin (GO), a CD33-directed antibody-drug conjugate, for treatment of newly diagnosed and relapsed/refractory AML setting the pace for a new era of personalized therapeutic options for AML. While the future of GO as a therapy in AML is bright, studies have shown that clinical response to GO is subject to interpatient variability (Gbadamosi 2018). Hitherto, efforts to understand the interpatient variation have focused on profiling CD33 expression levels (Pollard et al. 2016) and more recently on genetic variation in CD33 and ABCB1 predicative of GO response (Chauhan et al. 2019, Rafiee et al. 2019). However, efforts to understand interpatient variability in regards to calicheamicin, the DNA-damaging cytoxin linked to the antibody portion of GO, have been limited. Thus, we hypothesized that interpatient differences in expression levels of genes involved in pharmacodynamic effects of calicheamicin may impact GO response/resistance. Methods: Herein, our group has used the least absolute shrinkage and selection operator (LASSO) regression analysis method to identify critical genes with expression levels predictive of clinical outcomes in response to GO. Using data from the TARGET database, the expression levels of 18 genes, selected for their role in calicheamicin induced DNA-damage response, were extracted for AML patients treated with GO in the AAML03P1 and AAML0531 clinical trials (N=128; Table 1). Using a penalized LASSO regression algorithm (glmnet R-package), a Cox regression model was fit on to the expression levels of the selected gene. A thousand bootstraps of LASSO regression were performed and genes included in greater than 95% of the models were further selected. Average coefficients from the LASSO model were used to generate a gene signature equation designated as the DNA-damage response score (DDRS) given the nature of the genes included. Patients were classified into high (DDRS High) or low (DDRS Low) values for DDRS according to the median value and evaluated using Cox-proportional hazard model for survival data analysis, while the Chi-square test was used to examine the differences between categories, and the Wilcoxon rank-sum test was used to assess the differences in averages where appropriate. Results: The DDRS equation was defined as DDRS = (AKT1*-0.070) + (CASP9*-0.091) + (H2AFX*-0.160) + (XRCC4*0.373) where gene expression levels are multiplied by the coefficients obtained from the LASSO regression (Figure 1). Patients in the DDRS High group had significantly worse event free survival (EFS; HR = 2.22, P < 0.001; Figure 2A), lower complete remission rates (67.7% vs 94.6%, P < 0.001; Figure 3A), and a trend towards worse overall survival (OS; HR = 1.70, P = 0.058) as compared to patients within DDRS Low group. DDRS score was also significantly different amongst patients that were MRD+ve vs. MRD-ve after induction 1 therapy (21% difference, P = 0.038; Figure 3B). Consistent results were seen within standard risk group patients where patients in the DDRS High group had significantly worse EFS (HR = 2.29, P = 0.01; Figure 2B) as compared to those in the DDRS Low group. In preliminary multivariate Cox regression analysis, the DDRS remained a significant predictor of EFS amongst age, risk group, FLT3 status, and WBC (HR 2.42, P < 0.001; Table 2). Conclusion: Our preliminary investigation using LASSO regression model defined DDRS, a gene signature predictive of clinical response in patients treated with GO. The model included four genes well known for their involvement in DNA-damage response: AKT1, a kinase that regulates cell growth and division; CASP9, The initiator caspase involved in apoptosis; H2AFX, a DNA-damage marker that recruits other DNA-damage response proteins to damaged loci; and XRCC4, a ligase for DNA damage repair. Our future work focuses on expanding this investigation in bigger cohorts of patients representing different risk groups of AML as well as in vitro mechanistic studies. Once validated for its sensitivity and specificity to calicheamicin response, our results hold promise towards developing strategies for understanding interpatient variability in GO response and personalizing GO therapy based on diagnostic gene expression signatures. Disclosures No relevant conflicts of interest to declare.

346O - Association of BAP1 with ATR protein and their clinical significance with patient outcome in uveal melanoma

BackgroundUveal melanoma (UM) is an intraocular malignancy commonly arising from the choroid. There is a lack of effective therapy to detect and treat early metastatic spread in these patients. Therefore, it is important to find new biomarkers that help in early detection of metastasis. ATR is localized at position 23 on the same chromosome 3 where BAP1 is located at position 21.1. Loss of BAP1 is a known poor prognostic marker of uveal melanoma. There is no current study available on UM with respect to ATR protein that induces DNA damage response. Therefore, the aim of the study is to detect the expression of ATR protein in the UM patients and correlate its expression with BAP1 loss. MethodsExpression of nuclear ATR was investigated on sixty-nine UM patients which were divided into two groups on the basis of BAP1 expression. Formalin-fixed paraffin embedded choroidal melanoma samples were taken to evaluate the expression of ATM and BAP1 by immunohistochemistry and validated by semi-quantitative PCR. Results were then correlated with clinicopathological parameters. To determine the prognostic significance, Kaplan–Meier and multivariate analysis were performed. ResultsPatients with BAP1 loss also showed ATR loss in 62% of the cases which was statistically significant with high tumor pigmentation, LTD >10mm and cell type. At transcription level, down-regulation of ATR gene was found in 45% of cases and these results were in line with the IHC results. On multivariate analysis, advanced tumor staging and loss of ATR protein found to be independent prognostic factors. ConclusionsOur study emphasized the fact that loss of ATR could be a potential biomarker to detect the early metastasis in uveal melanoma. Hence, it could have a key role for therapeutic purpose. However, further studies are required in a larger cohort of patients with longer follow up and translational validation needs to be performed. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Targeting the Chromosomal Passenger Complex Subunit INCENP Induces Polyploidization, Apoptosis, and Senescence in Neuroblastoma.

Chromosomal passenger complex (CPC) has been demonstrated to be a potential target of cancer therapy by inhibiting Aurora B or survivin in different types of cancer including neuroblastoma. However, chemical inhibition of either Aurora B or survivin does not target CPC specifically due to off-target effects or CPC-independent activities of these two components. In a previous chromatin-focused siRNA screen, we found that neuroblastoma cells were particularly vulnerable to loss of INCENP, a gene encoding a key scaffolding component of the CPC. In this study, INCENP was highly expressed by neuroblastoma cells, and its expression decreased following retinoic acid-induced neuroblastoma differentiation. Elevated levels of INCENP were significantly associated with poor prognosis in primary tumors of neuroblastoma patients with high-risk disease. Genetic silencing of INCENP reduced the growth of both MYCN-wild-type and MYCN-amplified neuroblastoma cell lines in vitro and decreased the growth of neuroblastoma xenografts in vivo, with significant increases in murine survival. Mechanistically, INCENP depletion suppressed neuroblastoma cell growth by inducing polyploidization, apoptosis, and senescence. In most neuroblastoma cell lines tested in vitro, apoptosis was the primary cell fate after INCENP silencing due to induction of DNA damage response and activation of the p53-p21 axis. These results confirm that CPC is a therapeutic target in neuroblastoma, and targeting INCENP is a novel way to disrupt the activity of CPC and inhibit tumor progression in neuroblastoma. SIGNIFICANCE: Dysregulation of INCENP contributes to neuroblastoma tumorigenesis and targeting INCENP presents a novel strategy to disrupt the activity of chromosomal passenger complex and inhibit neuroblastoma progression.

Zika Virus Infection Induces DNA Damage Response in Human Neural Progenitors That Enhances Viral Replication.

Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication.IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.

WEE1 inhibition enhances sensitivity to hypoxia/reoxygenation in HeLa cells.

Hypoxia/reoxygenation (H/R) treatment reportedly induces DNA damage response (DDR), including DNA double-strand break (DSB) repair and G2 arrest, resulting in reduction of clonogenic survival. Because WEE1 plays a key role in the G2/M checkpoint along with CHK1/2, we investigated the effect of WEE1 inhibition on H/R-induced DDR using HeLa cells. The H/R treatment combined with WEE1 inhibitor abrogated G2 arrest, subsequently leading to the cells entering the M phase, and finally resulting in mitotic catastrophe after prolonged mitosis. Colony-forming assay showed an enhanced decrease in the surviving fraction and the focus formation of BRCA1 was significantly reduced. We demonstrate for the first time that WEE1 inhibition enhances H/R-induced cell death accompanied by mitotic catastrophe and that the process may be mediated by homologous recombination.

Bacterial quorum-sensing signal IQS induces host cell apoptosis by targeting POT1-p53 signalling pathway.

Pseudomonas aeruginosa, an opportunistic life-threatening human bacterial pathogen, employs quorum-sensing (QS) signal molecules to modulate virulence gene expression. 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS) is a recently identified QS signal that integrates the canonical lasR-type QS of P. aeruginosa and host phosphate stress response to fine-tune its virulence production for a successful infection. To address the role of IQS in pathogen-host interaction, we here present that IQS inhibits host cell growth and stimulates apoptosis in a dosage-dependent manner. By downregulating the telomere-protecting protein POT1 in host cells, IQS activates CHK1, CHK2, and p53 in an Ataxia telangiectasia mutated (ATM)/ATM and RAD3-related (ATR)-dependent manner and induces DNA damage response. Overexpression of POT1 in host cells presents a resistance to IQS treatment. These results suggest a pivotal role of IQS in host apoptosis, highlighting the complexity of pathogenesis mechanisms developed by P. aeruginosa during infection.

Abstract 2965: Induction of DNA damage responses by T-oligo and 6-thio-dG via modulating telomere associated proteins and telomerase

Abstract T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3′ telomeric overhang, has been shown to elicit potent DNA-damage responses (DDRs) in numerous cancer cell lines. However, the detailed molecular mechanism by which T-oligo induces these responses in cancer cells remains elusive. Additionally, it has recently been reported that the novel drug 6-Thio-2’-Deoxyguanosine (6-thio-dG), a nucleoside analogue of the approved drug 6-thioguanine, inhibits growth of cancer cells by its incorporation into the telomere via telomerase followed by subsequent shelterin disruption. This study aims to investigate the mechanism of action of T-oligo and 6-thio-dG at the telomere, as well as to assess the therapeutic potential of T-oligo in combination with 6-thio-dG. Finally, we also investigated the therapeutic efficacy of T-oligo in combination with Vemurafenib, an FDA-approved BRAF inhibitor, in BRAF-mutant melanoma cells as a potential combinatorial therapy for aggressive BRAF-mutant melanoma. Previous studies have shown that T-oligo is able to reduce expression of hTERT, the catalytic subunit of telomerase, by 50% in MM-AN melanoma cells. Results from this study showed that 2.5µM of 6-thio-dG increased expression of hTERT by 50% in this cell line, as seen by qPCR. This suggests that the mechanism of action of 6-thio-dG and T-oligo may involve modulation of telomerase in different ways. Treatment of MM-AN melanoma cells with 2.5μM 6-thio-dG induced a 1.54- and 1.75-fold upregulation of TRF2 at 48 and 72 hours, respectively, and an upregulation of POT1 by 1.63- and 2.15-fold at 48 and 72 hours, respectively, as seen by immunoblotting. These results suggest that 6-thio-dG may induce shelterin dissociation events similar to T-oligo, which has been shown to upregulate TRF2 and POT1 in this cell line. Our studies showed that 1.25μM and 2.5μM 6-thio-dG significantly inhibited growth of melanoma cells by 1.6- and 3.8-fold, respectively (p<0.01), by MTT assay. However, treatment of melanoma cells with 10μM T-oligo in combination with 6-thio-dG (1.25μM or 2.5μM) did not significantly inhibit cell growth in comparison to T-oligo alone, suggesting that these drugs may induce DDRs by similar mechanisms. Preliminary results indicate that, in MU melanoma cells, which are positive for the V600E BRAF mutation, treatment with T-oligo and Vemurafenib induced an additive effect in comparison to Vemurafenib or T-oligo alone. These results suggest that T-oligo and Vemurafenib may be potential candidates for combinatorial therapy for aggressive BRAF-mutant melanomas. Citation Format: Zachary Schrank, Nabiha Khan, Joseph Kellen, Sanjana Singh, Chike Osude, Neelu Puri. Induction of DNA damage responses by T-oligo and 6-thio-dG via modulating telomere associated proteins and telomerase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2965.

Abstract 1712: Estrogen receptor beta signaling sensitizes glioblastoma cells to chemo and radiation therapy

Abstract Current treatment options for glioblastoma (GBM) are poor, the mortality rates are very high and therapy resistance is a major clinical problem. Standard treatment consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide (TMZ), and tumor treating fields. Nonetheless, despite a heavy investment in therapy, all patients eventually succumb to their disease. Recent studies suggest that estrogen receptor beta (ERβ) may function as a tumor suppressor in GBM. However, the mechanism(s) by which ERβ contributes to chemo- and radiation therapy response remains unknown. The objective of this study is to examine whether ERβ sensitizes GBM to chemo- and radiation therapy and to understand the mechanistic insights of ERβ mediated tumor suppression in GBM. Methods: To study the functions of endogenous ERβ in GBM cells, we have utilized multiple ERβ overexpressing GBM model (GBM- ERβ) cells using lentiviral transduction. As a second model we have also generated ERβ knockout (ERβKO) cells using CRISPR/Cas9 system, and as a third model we used lentiviral-ERβshRNA transfected primary GBM cells (ERβKD). We then examined the effect of TMZ and radiation on the expression of ERβ using qRT-PCR. The effect of TMZ and radiation on ERβ overexpression and knockout models was examined using MTT cell viability assays. Mechanistic studies were conducted using RNA-seq, HR reporter gene assays, confocal microscopy, western blot, and qRT-PCR analysis. The in vivo role of ERβ on chemo sensitivity of TMZ was studied using orthotopic models of GBM and mouse survival was determined using Kaplan-Meier survival curves. Results: Cell viability and survival assays using multiple established and primary GBM cells demonstrated that ERβ sensitizes GBM cells to DNA damaging agents including TMZ and radiation therapy. qRT-PCR analysis demonstrated that ERβ expression was decreased following chemo- and radiation treatment. Combination analysis of RNA-seq studies using ERβ overexpression, and ERKO models, revealed an alteration in the number of genes involved in DNA recombination and repair, ATM signaling, and cell cycle check point control. Mechanistic studies showed that ERβ plays a significant role in homologous recombination (HR) mediated repair; and ERβ reduced expression and activation of ATM upon DNA damage. Generation of optimal γH2AX foci following TMZ treatment is dependent on the status of ERβ. More importantly, GBM cells expressing ERβ had increased survival when compared to control GBM cells in orthotopic GBM models. ERβ overexpression further enhanced the survival of mice to TMZ therapy in both TMZ sensitive and TMZ resistant GBM models. Conclusion: Our results provided evidence that ERβ is required for optimal chemo- and radiation- induced DNA damage response in GBM cells. Citation Format: Uday P. Pratap, Gangadhara Reddy Sareddy, Mengxing Li, Yiliao Luo, Mei Zhou, Suryavathi Viswanadhapalli, Rajeshwar Rao Tekmal, Andrew Brenner, Ratna K. Vadlamudi. Estrogen receptor beta signaling sensitizes glioblastoma cells to chemo and radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1712.

Abstract 2537: Targeting chromosomal passenger complex by disruption of INCENP function inhibits tumor progression in neuroblastoma

Abstract Chromosomal passenger complex (CPC), which is composed of Aurora B and Survivin, INCENP and Borealin, regulates crucial mitotic events including chromosome alignment, segregation and cytokinesis during the mitosis. Disruption of CPC function by Aurora B and Survivin inhibition has been demonstrated to be effective at killing tumor cells and show promising results in many different cancer types including neuroblastoma (NB). However, chemical inhibition of either Aurora B or Survivin is unable to target CPC specifically due to off-target effects of Aurora B inhibitors on the other kinases and antiapoptotic activities of Survivin independent of this complex. In a chromatin-focused siRNA screen, we found that NB cells were particularly vulnerable to the silencing of INCENP, a gene encoding a key scaffolding and regulatory component of the CPC. In this study, we found that INCENP was highly expressed in NB cells and its expression levels were decreased upon Retinoic Acid (RA)-induced NB cell differentiation. Genetic silencing of INCENP reduced the growth of both MYCN single copy and MYCN amplified NB cell lines in vitro and led to significant decreases in NB xenograft growth and increases in murine survival in vivo. Elevated levels of INCENP were significantly associated with poor prognosis in primary NB tumors whereas low INCENP expression levels were predictive of better outcomes. Mechanistically, we found that INCENP depletion suppressed NB cell growth by inducing massive polyploidization, mitotic arrest, senescence and cell death (apoptosis). We also observed that in the majority of NB cell lines tested in vitro, cell death represented the primary cell fate after INCENP silencing due to a strong induction of DNA damage response and activation of the p53-p21 axis. Therefore, targeting INCENP phenocopies treatment with Aurora B inhibitor, providing a novel strategy to disrupt the activity of CPC and inhibit tumor progression in NB. Citation Format: Ming Sun, Veronica Veschi, Norris Lam, Sukriti Bagchi, Man Xu, Arnulfo Mendoza, Zhihui Liu, Carol J. Thiele. Targeting chromosomal passenger complex by disruption of INCENP function inhibits tumor progression in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2537.

VE-822 mediated inhibition of ATR signaling sensitizes chondrosarcoma to cisplatin via reversion of the DNA damage response.

IntroductionCisplatin has been reported to elicit the DNA damage response (DDR) via activation of the ATR-Chk1 pathway, which in turn contributes to the induction of cisplatin resistance. Inhibition of ATR-Chk1 signaling reverses cisplatin resistance in some cancers. However, the influence of inhibiting ATR-Chk1 signaling on cisplatin resistance in chondrosarcoma cancer has not been reported.Materials and methodsWe compared the expression levels of ATR kinases in human nasopharyngeal carcinoma, choriocarcinoma and chondrosarcoma cell lines. We inhibited ATR kinase function with VE-822, a selective ATR inhibitor, and suppressed ATR kinase expression with shRNA. Western blotting, the CCK-8 assay, cell cycle distribution assay and apoptosis analysis were used to study the influence of inhibiting ATR-Chk1 signaling on reversing cisplatin resistance in chondrosarcoma cell lines.ResultsWe found that chondrosarcoma cells expressed very low basal levels of phosphorylated ATR, but cisplatin treatment induced the activation of ATR-Chk1 signaling in a dose- and time-dependent manner, suggesting the induction of DDR. As expected, ATR inhibition with VE-822 reversed cisplatin-induced DDR and enhanced cisplatin-induced activation of H2AX, which is an important marker of DNA damage. Meanwhile, ATR inhibition by RNA interference also reversed DDR and promoted DNA damage. Furthermore, both pharmacological and molecular inhibition of ATR accelerated cisplatin-induced inhibition of cell proliferation and cell death.ConclusionOur results suggested that inhibiting ATR activation promoted cisplatin-induced cell death via reversion of DDR, and VE-822 may be a valuable strategy for the prevention of cisplatin resistance in chondrosarcoma.

Ribosomal DNA as DAMPs Signal for MCF7 Cancer Cells

Introduction: The cell free ribosomal DNA (cf-rDNA) is accrued in the total pool of cell free DNA (cfDNA) in some non-cancer diseases and demonstrates DAMPs characteristics. The major research questions: (1) How does cell free rDNA content change in breast cancer; (2) What type of response in the MCF7 breast cancer cells is caused by cf-rDNA; and (3) What type of DNA sensors (TLR9 or AIM2) is stimulated in MCF7 in response to the action of cf-rDNA?Materials and Methods: CfDNA and gDNA were isolated from the blood plasma and the cells derived from 38 breast cancer patients and 20 healthy female controls. The rDNA content in DNA was determined using non-radioactive quantitative hybridization. In order to explore the rDNA influence on MCF7 breast cancer cells, the model constructs (GC-DNAs) were applied: pBR322-rDNA plasmid (rDNA inset 5836 bp long) and pBR322 vector. ROS generation, DNA damage, cell cycle, expression of TLR9, AIM2, NF-kB, STAT3, and RNA for 44 genes affecting the cancer cell viability were evaluated. The methods used: RT-qPCR, fluorescent microscopy, immunoassay, flow cytometry, and siRNA technology.Results: The ratio R = cf-rDNA/g-rDNA for the cases was higher than for the controls (median 3.4 vs. 0.8, p < 10−8). In MCF7, GC-DNAs induce a ROS burst, DNA damage response, and augmentation of NF-kB and STAT3 activity. The number of the apoptotic cells decreases, while the number of cells with an instable genome (G2/M– arrest, micronuclei) increase. Expression of anti-apoptotic genes (BCL2, BCL2A1, BCL2L1, BIRC3, MDM2) is elevated, while expression of pro-apoptotic genes (BAX, BID, BAD, PMAIP1, BBC3) is lowered. The cells response for pBR322-rDNA is much more intense and develops much faster, than response for pBR322, and is realized through activation of TLR9- MyD88 - NF-kB- signaling. This difference in response speed is owing to the heightened oxidability of pBR322-rDNA and better ability to penetrate the cell. Induction of TLR9 expression in MCF7 is followed by blocking AIM2 expression.Conclusion: (1) Ribosomal DNA accumulates in cfDNA of breast cancer patients; (2) Cell free rDNA induce DNA damage response and stimulates cells survival, including cells with an instable genome; (3) Cell free rDNA triggers TLR9- MyD88- NF-kB- signaling, with significantly repressing the expression of AIM2.