Persistent DNA Damage Research Articles

PARP Inhibitors for the Treatment of Ovarian Cancer.

The standard of treatment for advanced ovarian cancer is represented by optimal surgical debulking preceded or followed by chemotherapeutic regimens including taxanes and platinum agents, possibly associated with bevacizumab and/or intraperitoneal therapy. Despite this comprehensive treatment strategy, almost 75% of patients relapse or progress and are therefore candidates for a second-line treatment, showing, at this point, less chemo-sensitivity and worse prognosis. An interesting approach to improve outcomes of these patients has been developed in the last decade, in BRCA-related ovarian cancer. Mutations in one of the BRCA genes result in impaired homologousrecombination DNA repair, which causes genetic abnormalities that promote carcinogenesis. Interestingly, this defect has been exploited by the introduction of poly (ADP-ribose) polymerase (PARP) inhibitors to provide specific cancer cell cytotoxicity. Particularly, the inhibition of PARP in BRCAmutation carriers leads to the persistence of DNA damage usually repaired by the homologousrecombination system, resulting in cell cycle arrest and thus apoptosis. Despite the mechanism of action, an activity of PARP inhibitors was also observed in "BRCAness" ovarian tumors, and in BRCA-related tumors other than ovarian, suggesting that these agents may be active regardless of BRCA mutation status or site of origin. This review aims to describe the principal evidence that led to the development and the study of PARP inhibitors and to discuss their main implications in our daily clinical practice.

Clostridium difficile toxin B induces senescence in enteric glial cells: A potential new mechanism of Clostridium difficile pathogenesis

Clostridium difficile infection (CDI) causes nosocomial/antibiotic-associated diarrhea and pseudomembranous colitis, with dramatic incidence/mortality worldwide. C. difficile virulence factors are toxin A and toxin B (TcdB) which cause cytopathic/cytotoxic effects and inflammation. Until now studies were focused on molecular effects of C. difficile toxins (Tcds) on different cells while unexplored aspect is the status/fate of cells that survived their cytotoxicity. Recently we demonstrated that enteric glial cells (EGCs) are susceptible to TcdB cytotoxicity, but several EGCs survived and were irreversibly cell-cycle arrested and metabolically active, suggesting that EGCs could became senescent. This is important because allowed us to evaluate the not explored status/fate of cells surviving Tcds cytotoxicity, and particularly if TcdB induces senescence in EGCs.Rat-transformed EGCs were treated with 10 ng/ml TcdB for 6 h–48 h, or for 48 h, followed by incubation for additional 4 or 11 days in absence of TcdB (6 or 13 total days). Senescence markers/effectors were examined by specific assays.TcdB induces senescence in EGCs, as demonstrated by the senescence markers: irreversible cell-cycle arrest, senescence-associated-β‑galactosidase positivity, flat morphology, early and persistent DNA damage (ATM and H2AX phosphorylation), p27 overexpression, pRB hypophosphorylation, c‑Myc, cyclin B1, cdc2 and phosphorylated-cdc2 downregulation, Sirtuin‑2 and Sirtuin‑3 overexpression. TcdB-induced EGC senescence is dependent by JNK and AKT activation but independent by ROS, p16 and p53/p21 pathways.In conclusion, TcdB induces senescence in EGCs. The extrapolation of these results to CDI leads to hypothesize that EGCs that survived TcdB, once they have acquired a senescence state, could cause irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and tumors due to persistent inflammation, transfer of senescence status and stimulation of pre-neoplastic cells.

Recurrent DNA damage is associated with persistent injury in progressive radiation-induced pulmonary fibrosis

Purpose: Radiation-induced lung injuries (RILI), namely radiation pneumonitis and/or fibrosis, are dose-limiting outcomes following treatment for thoracic cancers. As part of a search for mitigation targets, we sought to determine if persistent DNA damage is a characteristic of this progressive injury.Methods: C57BL/6J female mice were sacrificed at 24 h, 1, 4, 12, 16, 24 and 32 weeks following a single dose of 12.5 Gy thorax only gamma radiation; their lungs were compared to age-matched unirradiated animals. Tissues were examined for DNA double-strand breaks (DSBs) (γ-H2A.X and p53bp1), cellular senescence (senescence-associated beta-galactosidase and p21) and oxidative stress (malondialdehyde).Results: Data revealed consistently higher numbers of DSBs compared to age-matched controls, with increases in γ-H2A.X positivity beyond 24 h post-exposure, particularly during the pathological phases, suggesting periods of recurrent DNA damage. Additional intermittent increases in both cellular senescence and oxidative stress also appeared to coincide with pneumonitis and fibrosis.Conclusions: These novel, long-term data indicate (a) increased and persistent levels of DSBs, oxidative stress and cellular senescence may serve as bioindicators of RILI, and (b) prevention of genotoxicity, via mitigation of free radical production, continues to be a potential strategy for the prevention of pulmonary radiation injury.

A moderate exposure to perfluorooctanoic acid causes persistent DNA damage and senescence in human epidermal HaCaT keratinocytes

Perfluorooctanoic acid has been used widespread, during the last decades, in a number of consumer and industrial products. Although this compound has been subjected to extensive epidemiological and toxicological studies, limited data are available concerning its potential dermal toxicity in mammalian cells. In this study, we used a two-stage approach with relevant cytotoxicity endpoints including cell viability and proliferation, oxidative stress, DNA damage and cell senescence to assess the immediate and the long-lasting or delayed cytotoxicity caused by the compound in HaCaT keratinocytes.Our results suggest that a single exposure to perfluorooctanoic acid causes concentration-dependent changes in cell proliferation that were not restored during a 48 h recovery period. Furthermore, we demonstrate that a moderate treatment with this perfluorochemical causes persistent DNA damage, which ultimately leads to development of the senescence-associated secretory phenotype in HaCaT cells. This paper provides unprecedented data and insights regarding the cytotoxic effects of perfluorooctanoic acid in human cells that could be of special relevance for use in comparative in vitro-in vivo studies. Moreover, our findings highlight the importance of considering both the immediate and long-lasting or delayed cytotoxic responses caused by chemical exposure, to ensure the accurate identification of toxicity in cell-based systems.

Adaptation to chronic exposure to sepantronium bromide (YM155), a prototypical survivin suppressant is due to persistent DNA damage-response in breast cancer cells.

Sepantronium bromide (YM155), originally developed against the anti-apoptotic protein survivin, performed exceptionally well in pre-clinical and phase I clinical trials. However, in phase II trials of several cancer types including breast cancer it performed poorly. Additionally, no definitive correlation between survivin level and response to therapy was found. In an attempt to understand the true reason of the late-stage failure of this promising drug, we developed YM155-resistant MCF-7 breast cancer cell line and characterized side-by-side with the drug-naïve parental cell line. Chronic YM155 treatment resulted in downregulation of survivin expression yet triggered cellular responses typical of adaptation to persistent DNA damage. Lowering endogenous antioxidant glutathione level and activity of cell cycle check-point kinase restored YM155 activity. Thus, contrary to its development as a survivin suppressant, YM155 primarily acts as a chemotherapeutic drug causing oxidative stress-mediated DNA damage. Adaptation to long-term exposure to YM155 can be prevented and/or overcome by interfering with detoxification and DNA damage-response pathways. Finally, proteins associated with DNA damage-response pathway will be more appropriate as predictive biomarkers of YM155 in breast tumor cells.

Proton radiation-induced cancer progression

There are considerable health risks related to ionizing and proton radiation exposure. While there is a long history of health risks associated with ionizing (photon) radiation exposure, there is a limited understanding of the long-term health risks associated with proton radiation exposure. Since proton radiation is becoming more common in cancer therapy, the long-term biological effects of proton radiation remain less well characterized in terms of radiotherapy and well as for astronauts during deep space explorations. In this study, we compared the long-term side effects of proton radiation to equivalent doses of X-rays in the initiation and progression of premalignant lesions in a lung cancer susceptible mouse model (K-rasLA1). We show proton irradiation causes more complex DNA damage that is not completely repaired resulting in increased oxidative stress in the lungs both acutely and persistently. We further observed K-rasLA1 mice irradiated with protons had an increased number and size of initiated and premalignant lesions and adenomas that were often infiltrated with inflammatory cells. Proton irradiated mice had a lower median survival and increased carcinoma incidence as compared to unirradiated controls and X-rays exposed mice. Our conclusion is that exposure to proton irradiation enhances the progression of premalignant lesions to invasive carcinomas through persistent DNA damage, chronic oxidative stress, and immunosuppression.

AAV9-mediated telomerase activation does not accelerate tumorigenesis in the context of oncogenic K-Ras-induced lung cancer.

Short and dysfunctional telomeres are sufficient to induce a persistent DNA damage response at chromosome ends, which leads to the induction of senescence and/or apoptosis and to various age-related conditions, including a group of diseases known as “telomere syndromes”, which are provoked by extremely short telomeres owing to germline mutations in telomere genes. This opens the possibility of using telomerase activation as a potential therapeutic strategy to rescue short telomeres both in telomere syndromes and in age-related diseases, in this manner maintaining tissue homeostasis and ameliorating these diseases. In the past, we generated adeno-associated viral vectors carrying the telomerase gene (AAV9-Tert) and shown their therapeutic efficacy in mouse models of cardiac infarct, aplastic anemia, and pulmonary fibrosis. Although we did not observe increased cancer incidence as a consequence of Tert overexpression in any of those models, here we set to test the safety of AAV9-mediated Tert overexpression in the context of a cancer prone mouse model, owing to expression of oncogenic K-ras. As control, we also treated mice with AAV9 vectors carrying a catalytically inactive form of Tert, known to inhibit endogenous telomerase activity. We found that overexpression of Tert does not accelerate the onset or progression of lung carcinomas, even when in the setting of a p53-null background. These findings indicate that telomerase activation by using AAV9-mediated Tert gene therapy has no detectable cancer-prone effects in the context of oncogene-induced mouse tumors.

Abstract B049: DNA damage echoes are key to prostate cancer susceptibility to supraphysiologic androgen levels

Abstract Purpose: Prostate cancer cells are dependent on AR for growth and survival, but recent reports have shown a paradoxical role of supraphysiologic androgen in growth inhibition, known as hormone interference. Androgen stimulation in AR-overexpressing prostate cancer cells leads to formation of double-strand breaks (DSBs). Here, we investigate the potential mechanism of high androgen induced DSBs and growth inhibition and its effect on survival following androgen stimulation in combination with DNA damage repair inhibitors or defects. Experimental Design: AR-dependent LNCaP, AR overexpressing LNCaP and PC3, AR-independent PC3, and APIPC were used for this study. High levels of androgen (R1881)/ DHT and olaparib-induced DNA damage were evaluated by gH2AX and 53BP1 foci formation after 6 or 24h. For cell death assays the drug was administered continuously for 3 days following the plating day. DNA PKcs activity was determined by Ser2056foci formation and dissociation from chromatin was measured by Thr2609 foci. AR activity or DNA binding was evaluated by Ser81 AR phosphorylation. An ex vivo PDX model (LuCaP 96CR) was utilized to mimic the BRCA2 defect condition. Results: We demonstrated that supraphysiologic androgen treatment on androgen-dependent and AR-overexpressing prostate cancer cell lines resulted in a dose-dependent induction of widespread DSBs, which was augmented after PARP inhibition (olaparib). AR expression correlated with persistent DNA damage as AR-independent cells lacked DSBs. High androgen treatment significantly induced caspase activity and reduced survival in AR-overexpressing cells. Hyperactivated DNA PKcs was retained on chromatin following high androgen exposure in these cells, leading to impaired dissociation of DNA PKcs from chromatin and inducing DNA damage. AR phosphorylation at Ser81 was also in concordance with hyperphosphorylated DNA PKcs, particularly in AR-overexpressing cells following high androgen and olaparib treatment. HR-deficient, BRCA2-null cells induced more DSBs after high androgen treatment with or without olaparib and were susceptible to DNA PKcs inhibition following androgen treatment. In contrast to this, AR-overexpressing cells did not exhibit any DNA damage following DNA PKcs inhibition after high androgen exposure. Ex vivo PDX models demonstrated a synergistic interaction between olaparib and high levels of androgen-induced DSB formation as well as hyperactive DNA PKcs. Conclusions: These results suggest a potential mechanism of supraphysiologic androgen stimulated AR-induced DNA damage and proliferation inhibition. AR binds to DNA PKcs following high androgen and until AR levels become saturated DNA PKcs and AR stay on the chromatin, which leads to inefficient DNA damage repair. HR-deficient cells were particularly susceptible to high androgen. Our data provide a mechanistic rationale for the response of AR upregulated cells as well as DNA repair deficient cells to supraphysiologic androgen therapy in combination with PARP inhibition. Citation Format: Payel Chatterjee, Jared M. Lucas, Michael D. Nyquist, Eva Corey, Peter S. Nelson. DNA damage echoes are key to prostate cancer susceptibility to supraphysiologic androgen levels [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B049.

Novel Fluoroindenoisoquinoline Non-Camptothecin Topoisomerase I Inhibitors.

Contrary to other anticancer targets, topoisomerase I (TOP1) is targeted by only one chemical class of FDA-approved drugs: topotecan and irinotecan, the derivatives of the plant alkaloid, camptothecin. The indenoisoquinolines LMP400, LMP744, and LMP776 are novel noncamptothecin TOP1 inhibitors in clinical trial, which overcome the limitations of camptothecins. To further improve metabolic stability, their methoxy groups have been replaced by fluorine, as in the fluoroindenoisoquinolines NSC 781517 (LMP517), NSC 779135 (LMP135), and NSC 779134 (LMP134). We tested the induction and stability of TOP1 cleavage complexes (TOP1cc), and the induction and persistence of DNA damage measured by histone H2AX phosphorylation (γH2AX) compared with their parent compounds LMP744 and LMP776 in leukemia CCRF-CEM and colon carcinoma HCT116 cells. The fluoroindenoisoquinolines induced TOP1cc and γH2AX at nanomolar concentrations, and at higher levels than the parent indenoisoquinolines. The fluoroindenoisoquinoline LMP135 showed greater antitumor activity than topotecan in small-cell lung cancer cell H82 xenografts. It was also more potent than topotecan in the NCI-60 cancer cell line panel. Bioinformatics tools (http://discover.nci.nih.gov/cellminercdb) were used to investigate the following: (i) the correlations of fluoroindenoisoquinolines activity with other drugs, and (ii) genomic determinants of response in the NCI-60. The activity of the fluoroindenoisoquinolines was mostly correlated with camptothecin derivatives and the parent indenoisoquinolines, consistent with TOP1 targeting. Genomic analyses and activity assays in CCRF-CEM SLFN11-deleted cells showed that SLFN11 expression is a dominant determinant of response to LMP135. This study shows the potential value of the fluoroindenoisoquinolines for further development as novel anticancer agents targeting TOP1. Mol Cancer Ther; 17(8); 1694-704. ©2018 AACR.

Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA

Neurons are highly vulnerable to DNA damage induced by genotoxic agents such as topoisomerase activity, oxidative stress, ionizing radiation (IR) and chemotherapeutic drugs. To avert the detrimental effects of DNA lesions in genome stability, transcription and apoptosis, neurons activate robust DNA repair mechanisms. However, defective DNA repair with accumulation of unrepaired DNA are at the basis of brain ageing and several neurodegenerative diseases. Understanding the mechanisms by which neurons tolerate DNA damage accumulation as well as defining the genomic regions that are more vulnerable to DNA damage or refractory to DNA repair and therefore constitute potential targets in neurodegenerative diseases are essential issues in the field. In this work we investigated the nuclear topography and organization together with the genome-wide distribution of unrepaired DNA in rat cortical neurons 15 days upon IR. About 5% of non-irradiated and 55% of irradiated cells accumulate unrepaired DNA within persistent DNA damage foci (PDDF) of chromatin. These PDDF are featured by persistent activation of DNA damage/repair signaling, lack of transcription and localization in repressive nuclear microenvironments. Interestingly, the chromatin insulator CTCF is concentrated at the PDDF boundaries, likely contributing to isolate unrepaired DNA from intact transcriptionally active chromatin. By confining damaged DNA, PDDF would help preserving genomic integrity and preventing the production of aberrant proteins encoded by damaged genes.ChIP-seq analysis of genome-wide γH2AX distribution revealed a number of genomic regions enriched in γH2AX signal in IR-treated cortical neurons. Some of these regions are in close proximity to genes encoding essential proteins for neuronal functions and human neurodegenerative disorders such as epm2a (Lafora disease), serpini1 (familial encephalopathy with neuroserpin inclusion bodies) and il1rpl1 (mental retardation, X-linked 21). Persistent γH2AX signal close to those regions suggests that nearby genes could be either more vulnerable to DNA damage or more refractory to DNA repair.

Defenses against Pro-oxidant Forces - Maintenance of Cellular and Genomic Integrity and Longevity.

There has been enormous recent progress in understanding how human cells respond to oxidative stress, such as that caused by exposure to ionizing radiation. We have witnessed a significant deciphering of the events that underlie how antioxidant responses counter pro-oxidant damage to key biological targets in all cellular compartments, including the genome and mitochondria. These cytoprotective responses include: 1. The basal cellular repertoire of antioxidant capabilities and its supporting cast of facilitator enzymes; and 2. The inducible phase of the antioxidant response, notably that mediated by the Nrf2 transcription factor. There has also been frenetic progress in defining how reactive electrophilic species swamp existing protective mechanisms to augment DNA damage, events that are embodied in the cellular "DNA-damage response", including cell cycle checkpoint activation and DNA repair, which occur on a time scale of hours to days, as well as the implementation of cellular responses such as apoptosis, autophagy, senescence and reprograming that extend the time period of damage sensing and response into weeks, months and years. It has become apparent that, in addition to the initial oxidative insult, cells typically undergo further waves of secondary reactive oxygen/nitrogen species generation, DNA damage and signaling and that these may reemerge long after the initial events have subsided, probably being driven, at least in part, by persisting DNA damage. These reactive oxygen/nitrogen species are an integral part of the pathological consequences of radiation exposure and may persist across multiple cell divisions. Because of the pervasive nature of oxidative stress, a cell will manifest different responses in different subcellular compartments and to different levels of stress injury. Aspects of these compartmentalized responses can involve the same proteins (such as ATM, p53 and p21) but in different functional guises, e.g., in cytoplasmic versus nuclear responses or in early- versus late-phase events. Many of these responses involve gene activation and new protein synthesis as well as a plethora of post-translational modifications of both basal and induced response proteins. It is these responses that we focus on in this review.

Increased Levels of Oxidative Damage in Liver Metastases Compared with Corresponding Primary Colorectal Tumors: Association with Molecular Subtype and Prior Treatment

High levels of oxidative stress in disseminated colorectal cancer tumor cells may form a therapeutically exploitable vulnerability. However, it is unclear whether oxidative stress and damage persist in metastases. Therefore, we analyzed markers of oxidative damage in primary colorectal tumors and their corresponding liver metastases. Markers of generic and oxidative DNA damage [phosphorylated histone H2AX (γH2AX) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] were significantly higher in liver metastases compared with their corresponding primary tumors. Chemotherapy and/or radiotherapy before tumor resection was associated with increased persistent oxidative DNA damage, and this effect was more pronounced in metastases. Immunohistochemistry-based molecular classification into epithelial- and mesenchymal-like molecular subtypes revealed that untreated mesenchymal-like tumors contained lower levels of oxidative DNA damage compared with epithelial-like tumors. Treated mesenchymal-like tumors, but not epithelial-like tumors, showed significantly higher levels of γH2AX and 8-OHdG. Mesenchymal-like tumors expressed significantly lower levels of phosphorylated nuclear factor erythroid 2-related factor2, a master regulator of the antioxidant response, and nuclear factor erythroid 2-related factor 2-controlled genes. Of interest, a positive 8-OHdG status identified a subgroup of mesenchymal-like metastases with a poor overall survival. An increased capacity to tolerate therapy-induced oxidative damage in mesenchymal-like colorectal cancer may explain, at least in part, the poor responsiveness of these tumors to chemotherapy, which could contribute to the poor survival of this patient subgroup.

Silica - A trace geogenic element with emerging nephrotoxic potential

Silica is a trace-geogenic compound with limited-bioavailability. It inflicts health-perils like pulmonary-silicosis and chronic kidney disease (CKD), when available via anthropogenic-disturbances. Amidst silica-imposed pathologies, pulmonary toxicological-mechanisms are well-described, ignoring the renal-pathophysiological mechanisms. Hence, the present-study aimed to elucidate cellular-cum-molecular toxicological-mechanisms underlying silica-induced renal-pathology in-vitro.Various toxicity-assessments were used to study effects of silica on the physiological-functions of HK-cells (human-kidney proximal-tubular cells - the toxin's prime target) on chronic (1–7 days) sub-toxic (80 mg/L) and toxic (100–120 mg/L) dosing.Results depicted that silica triggered dose-cum-time dependent cytotoxicity/cell-death (MTT-assay) that significantly increased on long-term dosing with ≥100 mg/L silica; establishing the nephrotoxic-potential of this dose. Contrarily, insignificant cell-death on sub-toxic (80 mg/L) dosing was attributed to rapid intracellular toxin-clearance at lower-doses preventing toxic-effects. The proximal-tubular (HK-cells) cytotoxicity was found to be primarily mediated by silica-triggered incessant oxidative-stress (elevated ROS).·This enhanced ROS inflicted severe inflammation and subsequent fibrosis, evident from increased pro-inflammatory-cum-fibrogenic cytokines generation (IL-1β, IL-2, IL-6, TNF-α and TGF-β). Simultaneously, ROS induced persistent DNA-damage (Comet-assay) that stimulated G2/M arrest for p53-mediated damage-repair, aided by checkpoint-promoter (Chk1) activation and mitotic-inducers (i.e. Cdc-25, Cdk1, cyclinB1) inhibition. However, DNA-injuries surpassed the cellular-repair, which provoked the p53-gene to induce mitochondrial-mediated apoptotic cell-death via activation of Bax, cytochrome-c and caspase-cascade (9/3). This persistent apoptotic cell-death and simultaneous incessant inflammation culminated in the development of tubular-atrophy and fibrosis, the major pathological-manifestations of CKD.These findings provided novel-insights into the pathological-mechanisms (cellular and molecular) of silica-induced CKD, inflicted on chronic toxic-dosing (≥100 mg/L).Thereby, encouraging the development of therapeutic-strategies (e.g. anti-oxidant treatment) for specific molecular-targets (e.g. ROS) to retard silica-induced CKD-progression, for reduction in the global-CKD burden.

Abstract 1526: WEE1 inhibition suppresses esophageal adenocarcinoma tumor growth both in vitro and in vivo

Abstract Esophageal adenocarcinoma (EAC) has seen a 400% increase in incidence over the past 30 years. The 5-year survival rate is under 20% due to ineffective therapeutics and a lack of actionable oncogenic drivers, necessitating novel therapeutic avenues in this disease. Genomic analysis indicates that a subset of EAC tumors have alterations in the G2/M cell cycle check point and DNA damage response governed by mutations in TP53 or other hits in this pathway, suggesting targeting the G2/M pathway might be a viable option in EAC. We and others have demonstrated that the WEE1 inhibitor AZD1775 is effective both in vitro and in vivo against tumors with DNA damage response alterations, and this drug is currently in clinical trials across a number of tumor types. We hypothesized that inhibition of WEE1 would induce DNA damage and cell death in EAC tumors, and provide a rational therapeutic avenue against this deadly disease. Across multiple EAC cell lines, AZD1775 suppressed tumor cell growth either as a monotherapy or in combination with DNA-damaging therapies. The growth suppression was accompanied by persistent DNA damage as determined by γH2AX and the induction of apoptosis. FLO-1 cells, responsive to monotherapy AZD1775, showed a dramatic reduction in G2 cells 24 hours post AZD1775 exposure with concomitant increases in cells in G0 and S phases of the cycle; while SK-GT-4, resistant to monotherapy AZD1775, showed an initial reduction in cells in G2 but recovered to normal levels 24 hours post-exposure. To assess AZD1775 in vivo, we generated a patient-derived xenograft (PDX) model of an EAC tumor excised at our institution. Combinations of AZD1775 with radiation or cisplatin significantly reduced in vivo tumor growth compared to vehicle, and the combination of AZD1775 with cisplatin was as effective as standard-of-care treatment (cisplatin + docetaxel + radiation), a therapeutic avenue plagued by toxicity in humans. Exposure to AZD1775 in combination with cisplatin showed suppression of phospho-CDC2 (target-hit) and induction of γH2AX in vivo compared to vehicle. Towards understanding a mechanism for lack of response of certain EAC cell lines to AZD1775 monotherapy, we explored the PI3K pathway that has been previously implicated in AZD1775 resistance. SK-GT-4 cells, which are unresponsive to AZD1775 monotherapy, demonstrated elevated phospho-AKT and phospho-p70S6K protein levels compared to other cell lines and were found more sensitive to PI3K or MTOR inhibition, suggesting a dependence on this pathway for tumor cell survival. Collectively, our data suggest that AZD1775, both alone and in combination with standard-of-care DNA damage, may be an effective therapeutic strategy for those EAC tumors dependent on the G2/M checkpoint. Citation Format: Mylan Blomquist, Vashti M. Carson, Ross M. Bremner, Timothy G. Whitsett, Landon J. Inge. WEE1 inhibition suppresses esophageal adenocarcinoma tumor growth both in vitro and in vivo [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 1526.

Abstract 4811: Inhibition of PLK1 abrogates side population and increases radiation-induced DNA damage in human glioblastoma

Abstract Glioblastoma multiforme (GBM), a highly malignant form of brain tumor has limited treatment modalities with poor patient survival. Expression of CD133, a marker of cancer stem cells (CSCs), has been linked to GBM recurrence, metastasis and radiation/drug resistance. The GBM CSCs exhibit high level of ABC transporters, DNA repair enzymes and anti-apoptotic proteins. The side population (SP) phenotype of CSCs with high levels of ABC transporters is associated with chemo-resistance. Overexpression of Polo-like kinase-1 (PLK1), a key cell cycle regulator, has been linked to poor prognosis of various cancers. However, the role of PLK1 in radio/chemo-resistance of GBM is poorly understood. In this study, we explored the use of PLK1 inhibitor Volasertib in combination with temozolomide (TMZ) followed by radiation to overcome resistance in GBM, both in vitro and in vivo. Our results revealed that Volasertib and TMZ act synergistically at a sub-lethal dose to inhibit cell proliferation and induce apoptosis. Both Volasertib and TMZ strongly induce reactive oxygen species (ROS) which may cause cell death by reducing repair capacity. Combined treatment with TMZ and Volasertib followed by radiation showed persistent DNA damage after removal of drugs for 24 hrs. In contrast to earlier reports, significant decrease in the side population was also observed, even after radiation treatment. We also observed that Volasertib alone or in combination with TMZ activated STAT1 pathways and inhibited MAPK/AKT activation. As the latter pathway promotes apoptosis, our results suggest higher tumor growth inhibitory effect of combinatorial treatment compared to single agents in xenograft mouse model. In summary, while Volasertib can be used alone or in the combination with radiation, Volasertib combined with TMZ may be useful to inhibit recurrence of GBM after radiation therapy. Furthermore, increased STAT1 expression may modulate the tumor-microenvironment and improve immunotherapy by MHC class 1 upregulation. Citation Format: Arvind Pandey, Junhua Mai, Satyendra C. Tripathi, Samir M. Hanash, Haifa Shen, Sankar Mitra. Inhibition of PLK1 abrogates side population and increases radiation-induced DNA damage in human glioblastoma [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 4811.

Abstract 4163: Space radiation-induced persistent DNA damage and alterations in cytoskeletal remodeling pathways affect intestinal epithelial cell migration in mice

Abstract Lombardi Comprehensive Cancer Center, and Department of Biochemistry and Molecular & Cellular Biology Georgetown University, Washington, DC 20057, USA. Heavy ion space radiation is considered as a risk factor of gastrointestinal (GI) cancer and perturbed intestinal epithelial cell (IEC) migration has been implicated in GI carcinogenesis. The purpose of the current study was to assess rate of IEC migration as well as dissect key IEC migration signaling events after exposure to heavy ion 56Fe irradiation and compare the results to sham- and γ-irradiation. Male C57BL/6J mice were exposed to 0.5 Gy of either γ or 56Fe radiation and control mice were sham irradiated. IEC migration distance was measured 7 and 60 d after exposure using BrdU pulse labeling. We also analyzed cell polarity, microtubule dynamics, and adhesion dynamics using immunoblots and immunofluorescence. Measure of IEC migration rate showed a greater decrease after 56Fe relative to γ radiation at both the time points. At the molecular level, compared to sham and γ radiation, 56Fe radiation exhibited significantly greater impairment of cell adhesion dynamics evidenced by altered integrins and Rock1 as well as cytoskeletal dynamics evidenced by altered MLCK expression. Similarly, perturbed cell polarity, tight junction, and microtubule dynamics was revealed by the deregulation of Par3, Claudin1, Scribble, Occludin, ZO-1, phospho-Tau and MAP1B. Also, the Wnt/β-catenin and EphB/EphrinB pathways critical for IEC migration consistently showed greater deregulation at both the time points tested after 56Fe radiation relative to γ -rays. Taken together, our molecular findings correlate with the BrdU pulse labeling data, and demonstrate greater effects of heavy ion 56Fe on IEC migration relative to γ -rays. In summary, our results have demonstrated for the first time that radiation can disrupt cell migration dynamics through alteration of signaling molecules involved in crucial cellular processes and these results have implications for heavy ion space radiation-induced gastrointestinal homeostatic deregulation and carcinogenesis. Citation Format: Santosh Kumar, Shubhankar Suman, Albert Fornace, Kamal Datta. Space radiation-induced persistent DNA damage and alterations in cytoskeletal remodeling pathways affect intestinal epithelial cell migration in mice [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 4163.

Abstract LB-398: DNA-PKcs protects irradiated cells from persistent DNA damage signaling and accelerated senescence

Abstract The DNA-dependent protein kinase catalytic subunit DNA-PKcs has long been considered as a promising target in cancer, based on its roles in DNA damage response and the potential for inhibitors to enhance sensitivity to genotoxic therapy. Multiple investigational DNA-PK inhibitors remain under evaluation, though several have failed in development and none have reached the clinic. DNA-PKcs has been assigned diverse roles in DNA damage response that overlap with ATM including direct phosphorylation of H2AX to form γH2AX and promoting double-strand break (DSB) repair by conventional non-homologous end-joining (NHEJ). Nonetheless, a recent report implicated DNA-PKcs as a negative regulator of ATM. This may resolve the paradox that while ATM inhibitors block H2AX phosphorylation, prevent ionizing radiation induced foci (IRIF) formation and suppress terminal arrest and onset of cellular senescence, DNA-PK inhibitors induce persistent γH2AX, delay IRIF resolution and promote senescence. Toward better defining DNA-PKcs roles in radiation response, we examined MCF7 mammary carcinoma cells for effects of DNA-PKcs shRNA knockdown or treatment with specific inhibitors on DNA damage response and senescence after irradiation. We confirmed a requirement for DNA-PK in resolution of γH2AX and IRIF by 24 h after irradiation. Rather than return to proliferation, surviving cells with DNA-PK defects developed characteristic features of senescence within five days in culture. To examine if this effect of DNA-PKcs inhibition was mediated by blocking DSB repair, we irradiated MCF7 cell lines expressing shRNAs targeting NHEJ factors Ku70, Ku80, XLF, XRCC4, PAXX, and LIG4 as well as scrambled control in the presence or absence of DNA-PKcs inhibitor NU7441. Strikingly, IRIF resolution at 24 h was similar to control in cell lines lacking NHEJ factors while NU7441 increased IRIF persistence and enhanced senescence in all cell lines. These results suggest that a critical role for DNA-PKcs in radiation response is to attenuate DNA damage signaling and prevent cellular senescence. Treating irradiated DNA-PKcs knockdown cells with the ATM inhibitor KU55933 at 24 h resolved persistent γH2AX and IRIF and blocked senescence. Taken together, our data demonstrate that DNA-PKcs regulates IRIF resolution and cell senescence by targeting ATM functions rather than directly affecting DSB repair. Citation Format: Yue Liu, Elena V. Efimova, Aishwarya Ramamurthy, Stephen J. Kron. DNA-PKcs protects irradiated cells from persistent DNA damage signaling and accelerated senescence [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 LB-398.

INK4a/ARF Expression Impairs Neurogenesis in the Brain of Irradiated Mice.

SummaryBrain neurogenesis is severely impaired following exposure to ionizing radiation (IR). We and others have shown that the expression of the tumor suppressor gene p16INK4a is increased in tissues exposed to IR and thus hypothesized that its expression could limit neurogenesis in the irradiated brain. Here, we found that exposure to IR leads to persistent DNA damage and the expression of p16INK4a in the hippocampus and subventricular zone regions. This was accompanied by a decline in neurogenesis, as determined by doublecortin expression and bromodeoxyuridine incorporation, an effect partially restored in Ink4a/arf-null mice. Increased neurogenesis in the absence of INK4a/ARF expression was independent of apoptosis and activation of the microglia. Moreover, treatment of irradiated mice with a superoxide dismutase mimetic or clearance of p16INK4a-expressing cells using mouse genetics failed to increase neurogenesis. In conclusion, our results suggest that IR-induced p16INK4a expression is a mechanism that limits neurogenesis.

1215 RAGE mediates UVB-induced persistent DNA Damage Response (DDR) and resistance to apoptosis in human melanocytes

1215 RAGE mediates UVB-induced persistent DNA Damage Response (DDR) and resistance to apoptosis in human melanocytes

Genetic ablation of histone deacetylase 2 leads to lung cellular senescence and lymphoid follicle formation in COPD/emphysema.

Histone deacetylase 2 (HDAC2), a critical determinant of chromatin remodeling, is reduced as a consequence of oxidative stress-mediated DNA damage and impaired repair. Cigarette smoke (CS) exposure causes DNA damage and cellular senescence. However, no information is available on the role of HDAC2 in CS-induced DNA damage, stress-induced premature senescence (SIPS), and senescence-associated secretory phenotype (SASP) during the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. We hypothesized that CS causes persistent DNA damage and cellular senescence via HDAC2-dependent mechanisms. We used HDAC2 global knockout (KO) and HDAC2 lung epithelial cell-specific KO [Clara cell-specific HDAC2 deletion (HDAC2 CreCC10)] mice to determine whether HDAC2 is a major player in CS-induced oxidative stress, SIPS, and SASP. HDAC2 KO mice exposed to CS show exaggerated DNA damage, inflammatory response, and decline in lung function leading to airspace enlargement. Chronic CS exposure augments lung senescence-associated β-galactosidase activity in HDAC2 KO, but not in HDAC2 CreCC10 mice. HDAC2 lung epithelial cell-specific KO did not further augment CS-induced inflammatory response and airspace enlargement but instead caused an increase in lymphoid aggregate formation. Our study reveals that HDAC2 is a key player regulating CS-induced DNA damage, inflammatory response, and cellular senescence leading to COPD/emphysema.-Sundar, I. K., Rashid, K., Gerloff, J., Rangel-Moreno, J., Li, D., Rahman, I. Genetic ablation of histone deacetylase 2 leads to lung cellular senescence and lymphoid follicle formation in COPD/emphysema.