Ebba U. Kurz

Doxorubicin Activates ATM-dependent Phosphorylation of Multiple Downstream Targets in Part through the Generation of Reactive Oxygen Species

The requirement for the serine/threonine protein kinase ATM in coordinating the cellular response to DNA damage induced by ionizing radiation has been studied extensively. Many of the anti-tumor chemotherapeutics in clinical use today cause DNA double strand breaks; however, few have been evaluated for their ability to modulate ATM-mediated pathways. We have investigated the requirement for ATM in the cellular response to doxorubicin, a topoisomerase II-stabilizing drug. Using several ATM-proficient and ATM-deficient cell lines, we have observed ATM-dependent nuclear accumulation of p53 and ATM-dependent phosphorylation of p53 on seven serine residues. This was accompanied by an increased binding of p53 to its cognate binding site, suggesting transcriptional competency of p53 to activate its downstream effectors. Treatment of cells with doxorubicin led to the phosphorylation of histone H2AX on serine 139 with dependence on ATM for the initial response. Doxorubicin treatment also stimulated ATM autophosphorylation on serine 1981 and the ATM-dependent phosphorylation of numerous effectors in the ATM-signaling pathway, including Nbs1 (Ser343), SMC1 (Ser957), Chk1 (Ser317 and Ser345), and Chk2 (Ser33/35 and Thr68). Although generally classified as a topoisomerase II-stabilizing drug that induces DNA double strand breaks, doxorubicin can intercalate DNA and generate reactive oxygen species. Pretreatment of cells with the superoxide scavenger ascorbic acid had no effect on the doxorubicin-induced phosphorylation and accumulation of p53. In contrast, preincubation of cells with the hydroxyl radical scavenger, N-acetylcysteine, significantly attenuated the doxorubicin-mediated phosphorylation and accumulation of p53, p53-DNA binding, and the phosphorylation of H2AX, Nbs1, SMC1, Chk1, and Chk2, suggesting that hydroxyl radicals contribute to the doxorubicin-induced activation of ATM-dependent pathways.

Altered chloride homeostasis removes synaptic inhibitory constraint of the stress axis.

In mammals, stress elicits a stereotyped endocrine response that requires an increase in the activity of hypothalamic parvocellular neuroendocrine neurons. The output of these cells is normally constrained by powerful GABA-mediated synaptic inhibition. We found that acute restraint stress in rats released the system from inhibitory synaptic drive in vivo by down-regulating the transmembrane anion transporter KCC2. This manifested as a depolarizing shift in the reversal potential of GABAA-mediated synaptic currents that rendered GABA inputs largely ineffective. Notably, repetitive activation of GABA synapses after stress resulted in a more rapid collapse of the anion gradient and was sufficient to increase the activity of neuroendocrine cells. Our data indicate that hypothalamic neurons integrate psychological cues to mount the endocrine response to stress by regulating anion gradients.

Sodium salicylate is a novel catalytic inhibitor of human DNA topoisomerase II alpha

We have previously reported that pretreatment of human lymphoblastoid cells with the hydroxyl radical scavenger, N-acetyl cysteine, attenuates doxorubicin-induced DNA damage signalling through the ATM protein kinase. We sought to extend these studies to examine the effects of other hydroxyl radical scavengers in human breast cancer cells. Using MCF-7 cells, we observed that doxorubicin treatment triggered autophosphorylation of ATM on serine 1981 and the ATM-dependent activation of its downstream effectors p53, Chk2, and SMC1. Furthermore, we demonstrate that this effect was attenuated by pretreatment of cells with the hydroxyl radical scavengers sodium benzoate, sodium salicylate and, to a lesser extent, N-acetyl cysteine, but not Trolox™. Intriguingly, these effects were independent of doxorubicins ability to redox cycle, were observed with multiple classes of topoisomerase II poisons, but did not represent a general damage-attenuating response. In addition, the observed effects were independent of the ability of sodium salicylate to inhibit cyclooxygenase-2 or NFκB. We demonstrate that sodium salicylate prevented doxorubicin-induced DNA double-strand break generation, which was attributable to inhibition of doxorubicin-stabilized topoisomerase IIα-DNA cleavable complex formation in vivo. Using topoisomerase IIα-DNA cleavage and decatenation assays, we determined that sodium salicylate is a catalytic inhibitor of topoisomerase IIα. Consistent with the observed inhibition of double-strand break formation, pretreatment of cells with sodium salicylate attenuated doxorubicin and etoposide cytotoxicity. These results demonstrate a novel mechanism of action for sodium salicylate and suggest that further study on the mechanism of topoisomerase II inhibition and the effects of related therapeutics on doxorubicin and etoposide cytotoxicity are warranted.

Salicylate, a Catalytic Inhibitor of Topoisomerase II, Inhibits DNA Cleavage and Is Selective for the α Isoform

Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (α and β), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme–DNA interaction, nor does it promote stabilization of topo IIα in closed clamps on DNA. Although salicylate decreased topo IIα ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate’s effects using purified human topo IIα and topo IIβ revealed that salicylate selectively inhibits the α isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIα and provide support for further studies determining the basis for its isoform selectivity.

In vitro screen of a small molecule inhibitor drug library identifies multiple compounds that synergize with oncolytic myxoma virus against human brain tumor-initiating cells

BACKGROUND Brain tumor-initiating cells (BTICs) are stem-like cells hypothesized to form a disease reservoir that mediates tumor recurrence in high-grade gliomas. Oncolytic virotherapy uses replication-competent viruses to target and kill malignant cells and has been evaluated in clinic for glioma therapy with limited results. Myxoma virus (MyxV) is a safe and highly effective oncolytic virus (OV) in conventional glioma models but, as seen with other OVs, is only modestly effective for patient-derived BTICs. The objective of this study was to determine whether MyxV treatment against human BTICs could be improved by combining chemotherapeutics and virotherapy. METHODS A 73-compound library of drug candidates in clinical use or preclinical development was screened to identify compounds that sensitize human BTICs to MyxV treatment in vitro, and synergy was evaluated mathematically in lead compounds using Chou-Talalay analyses. The effects of combination therapy on viral gene expression and viral replication were also assessed. RESULTS Eleven compounds that enhance MyxV efficacy were identified, and 6 were shown to synergize with the virus using Chou-Talalay analyses. Four of the synergistic compounds were shown to significantly increase viral gene expression, indicating a potential mechanism for synergy. Three highly synergistic compounds (axitinib, a VEGFR inhibitor; rofecoxib, a cyclooxygenase-2 inhibitor; and pemetrexed, a folate anti-metabolite) belong to classes of compounds that have not been previously shown to synergize with oncolytic viruses in vitro. CONCLUSIONS This study has identified multiple novel drug candidates that synergistically improve MyxV efficacy in a preclinical BTIC glioma model.

Abstract LB-140: Targeting brain tumour initiating cells using a dual-pronged approach of oncolytic virotherapy and chemotherapeutics

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL BACKGROUND: Brain tumour initiating cells (BTICs) are stem-like cells hypothesized to mediate tumorigenesis and recurrence in glioblastoma multiforme (GBM). Patient-derived BTICs represent highly relevant preclinical GBM models, forming aggressive, infiltrative tumours in nude mice that closely recapitulate the phenotypic heterogeneity of patient GBMs. BTICs have also been shown to possess mechanisms mediating both chemo- and radio-resistance. Recently, success has been demonstrated in treating BTICs with oncolytic virotherapy (OV), which uses replication-competent viruses to specifically target and kill malignant cells. Myxoma virus (MyxV) is a promising oncolytic candidate, which our lab has shown to be highly efficacious in preclinical GBM models, effectively curing GBM xenografts with a single intra-cranial injection. By contrast, long-term survival is not obtained in MyxV-treated, BTIC-tumour-bearing mice, though survival is prolonged. HYPOTHESIS: We hypothesize that MyxV treatment of BTICs can be improved by utilizing clinically relevant chemotherapeutics identified via high-throughput pharmacoviral screens. RESULTS: We utilized a diverse, comprehensive panel of 80 small molecule inhibitors with preclinical and clinical anti-cancer efficacy to screen for synergy with MyxV treatment in vitro. Multiple candidates have been identified and are currently being validated for synergy (Chou-Talalay method) using a panel of genetically distinct, patient-derived BTICs. Our results implicate multiple potential targets for OV combination therapy, including topoisomerase I and the PI-3K/Akt/mTOR pathway. Target validation is currently underway using shRNA knockdowns and additional targeted inhibitors of these pathways. Further, the mechanism of synergistic cell death during these combination treatments is being explored. In vivo experiments utilizing the dual-pronged pharmacoviral approach are also underway. SIGNIFICANCE: We describe the nature and identity of compounds that carry the potential to sensitize BTICs to MyxV infection. Our findings offer an effective avenue to elucidate resistance mechanisms and develop efficacious combination therapies for targeting disease reservoirs within highly refractory GBMs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-140. doi:1538-7445.AM2012-LB-140

Abstract C39: Catalytic inhibition of human DNA topoisomerase II α by salicylate and related nonsteroidal anti-inflammatory drugs

Salicylate-based drugs, including aspirin (acetylsalicylic acid), have long been used in the treatment of mild to moderate cancer pain and have been shown in population-based studies to have cancer chemopreventive properties. Several mechanisms have been hypothesized to underlie these effects, including the inhibition of cyclooxygenases and the inhibition of the transcription factor NF-\#954;B. We have recently demonstrated, however, that salicylate, the primary metabolite of aspirin, is a novel catalytic inhibitor of human DNA topoisomerase IIα (topo II), a nuclear enzyme essential for cell proliferation and division and the target of several widely used anti-cancer chemotherapeutics. As a consequence of this inhibition, and independent of its capacity to inhibit cyclooxygenases and NF-\#954;B, we have demonstrated that a brief pretreatment of human breast cancer cells with salicylate attenuates the cytotoxicity of the topo II poisons, doxorubicin and etoposide. We have observed that salicylate prevents doxorubicin-induced DNA double-strand break generation, which is attributable to salicylate-mediated inhibition of doxorubicin-stabilized topo II-DNA cleavable complex formation in vivo. Together, these data suggest that co-administration of salicylates could negatively impact the efficacy of cancer treatment regimens incorporating topo II-targeting therapeutics. We have now extended our investigation of salicylate, using a biochemical approach to determine the mechanism whereby salicylate inhibits the catalytic activity of topo II. As the inhibition of topo II can occur at one of several stages in its catalytic cycle, we have undertaken multiple independent approaches, including an examination of DNA binding, DNA intercalation, measurement of ATPase activity and evaluation of salicylate9s capacity to stabilize topo II in a closed clamp formation without causing DNA double-strand breaks. In addition to delineating the mechanism of salicylate-mediated inhibition of topo II, we have investigated whether common salicylate- and non-salicylate-based non-steroidal anti-inflammatory drugs (NSAIDs) possess similar topo II inhibitory properties, initially by examining the effects of short-term exposure on doxorubicin-induced DNA damage signaling followed by a direct examination of their effects on topo II catalytic activity. Our experiments demonstrate that inhibition of topo II is readily observed with multiple salicylate-based therapies at clinically achieved concentrations. These investigations identify a novel cellular target of salicylate and will inform future studies that may reveal evidence warranting the discouragement of NSAID co-administration in patients undergoing treatment for any of the broad-reaching malignancies using topo II poisons in their therapeutics regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C39.