Top1 Inhibitors Research Articles

Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) inhibitors

Inhibitors of topoisomerase I (Top1) that result in stalled Top1 cleavage complexes (Top1cc) are commonly employed against cancer. Combination chemotherapy with DNA repair inhibitors can potentially improve response to these widely used chemotherapeutics. One line of inquiry focuses on inhibitors of tyrosyl-DNA phosphodiesterase 1 (Tdp1), a repair enzyme for Top1cc. Tdp1 catalyzes the hydrolysis of DNA adducts covalently linked to the 3'-phosphate of DNA, including Top1-derived peptides and also 3'-phosphoglycolates. Tdp1 inhibitors should synergize not only with Top1-targeting drugs (camptothecins, indenoisoquinolines), but also with bleomycin, topoisomerase II (Top2) inhibitors (etoposide, doxorubicin) and DNA alkylating agents. Here, we summarize the structure–activity relationship obtained from the reported Tdp1 inhibitors. Better understanding of Top1cc repair in vivo coupled with detailed structural studies on Tdp1–inhibitor interaction will be crucial in guiding the rational design of Tdp1 inhibitors.

7-Azaindenoisoquinolines as Topoisomerase I Inhibitors and Potential Anticancer Agents

A series of 7-azaindenoisoquinoline topoisomerase I (Top1) inhibitors have been prepared to investigate the effect of increased electron affinity of the aromatic system on the ability to stabilize the Top1-DNA cleavage complex. Ab initio calculations suggest that introduction of nitrogen into the aromatic system of the indenoisoquinolines would facilitate charge transfer complex formation with DNA, thus improving the π-π stacking interactions. The present study shows that 7-azaindenoisoquinolines demonstrate improved water solubility without any decrease in Top1 inhibitory activity or cytotoxicity. Analysis of the biological results reveals that smaller lactam ring substituents enable intercalation into both free DNA and Top1-DNA cleavage complex, whereas larger substituents only allow binding to the cleavage complex but not free DNA. Free DNA binding suppresses Top1-catalyzed DNA cleavage at high drug concentrations, whereas DNA cleavage and inhibition of religation occurs at low drug concentration.

Molecular and cellular pharmacology of the novel noncamptothecin topoisomerase I inhibitor Genz-644282.

Camptothecin derivatives are powerful anticancer drugs because of their ability to trap topoisomerase I (Top1)-DNA cleavage complexes. However, they exhibit clinical limitations due to the instability of their α-hydroxylactone six-membered E-ring structure. In addition, they exhibit bone marrow and intestinal toxicity, especially in adults, and are drug efflux substrates. Here, we report a novel Top1 inhibitor, Genz-644282. We show that Genz-644282 and its metabolites induce Top1 cleavage at similar, as well as unique genomic positions, compared with camptothecin. The compound also induces protein-linked DNA breaks and Top1-DNA cleavage complexes that persist longer after compound removal than camptothecin. Concentration-dependent and persistent γH2AX formation was readily observed in cells treated with Genz-644282, and was present in greater than 50% of the cell population following 24 hours compound exposure. The compound shows partial cross-resistance in cell lines resistant to camptothecin. These cell lines include the human prostate DU145RC0.1 and the leukemic CEM/C2 cells. Limited cross-resistance to Genz-644282 was also found in the Top1 knockdown colon cancer (HCT116) and breast cancer (MCF7) cell lines and in human adenocarcinoma cells (KB31/KBV1) that overexpress (P-glycoprotein, ABCB1), a member of the ATP-binding cassette family of cell surface transport proteins known to confer MDR. Together, our results provide the first molecular and cellular characterization of Genz-644282 and its clinically relevant metabolites.

Alcohol-, Diol-, and Carbohydrate-Substituted Indenoisoquinolines as Topoisomerase I Inhibitors: Investigating the Relationships Involving Stereochemistry, Hydrogen Bonding, and Biological Activity

The DNA-relaxing enzyme topoisomerase I (Top1) can be inhibited by heterocyclic compounds such as indolocarbazoles and indenoisoquinolines. Carbohydrate and hydroxyl-containing side chains are essential for the biological activity of indolocarbazoles. The current study investigated how similar functionalities could be "translated" to the indenoisoquinoline system and how stereochemistry and hydrogen bonding affect biological activity. Herein is described the preparation and assay of indenoisoquinolines substituted with short-chain alcohols, diols, and carbohydrates. Several compounds (including those derived from sugars) display potent Top1 poisoning and antiproliferative activities. The Top1 poisoning activity of diol-substituted indenoisoquinolines is dependent upon stereochemistry. Although the effect is striking, molecular modeling and docking studies do not indicate any reason for the difference in activity due to similar calculated interactions between the ligand and Top1-DNA complex and ambiguity about the binding mode. A stereochemical dependence was also observed for carbohydrate-derived indenoisoquinolines. Although similar trends were observed in other classes of Top1 inhibitors, the exact nature of this effect has yet to be elucidated.

Genz-644282, a Novel Non-Camptothecin Topoisomerase I Inhibitor for Cancer Treatment

Genz-644282 [8,9-dimethoxy-5-(2-N-methylaminoethyl)-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one] has emerged as a promising candidate for antitumor agents. This report describes the bone marrow colony-forming unit, granulocyte macrophage (CFU-GM) and tumor cell CFU activity of topoisomerase I (Top1) inhibitors, such as Genz-644282, topotecan, irinotecan/SN-38, and ARC-111, and examines their activity in several human tumor xenograft models. Colony-forming assays were conducted with mouse and human bone marrow and eight human tumor cell lines. In addition, 29 human tumor cell lines representing a range of histology and potential resistance mechanisms were assayed for sensitivity to Genz-644282 in a 72-hour exposure assay. The efficacy of Genz-644282 was compared with standard anticancer drugs (i.e., irinotecan, docetaxel, and dacarbazine) in human tumor xenografts of colon cancer, renal cell carcinoma, non-small cell lung cancer, and melanoma. Human bone marrow CFU-GM was more sensitive to the Top1 inhibitors than was mouse bone marrow CFU-GM. The ratio of mouse to human IC(90) values was more than 10 for the camptothecins and less than 10 for Genz-644282, which had more potency as a cytotoxic agent toward human tumor cells in culture than the camptothecins in the colony-forming and 72-hour proliferation assays. Genz-644282 has superior or equal antitumor activity in the human tumor xenografts than the standard drug comparators. On the basis of preclinical activity and safety, Genz-644282 was selected for development and is currently undergoing phase 1 clinical trial.

QM Scoring Function and its Application to TOP1 Inhibitors

between the amino acids and the DNA were replaced with hydrogen atoms to make the complex sufficiently small for the QM treatment. Three different sets of calculations were performed for each complex: TOP1_fragment-DNA_fragment–ligand (G Complex ), TOP1_fragment-DNA_fragment (G TOP1 ), and ligand (G Ligand ). Frequency calculations were introduced to calculate the entropic contribution to the total free energy. The calculations were carried out using GAUSSIAN03. 16 During the QM calculation, the coordinates of the heavy atoms of TOP1 and DNA parts were fixed whereas the other coordinates were relaxed. The total energy and interaction energy were evaluated at the HF level using the STO-3G and 4-21G basis sets. Relatively small basis sets were used because the systems are still fairly large for the application of QM. The binding energy was calculated as follows:

Abstract 4525: Discovery and characterization of a small chemical compound that shows exceptional antitumor activity and targets multiple antiapoptotic proteins in the inhibitor of apoptosis (IAP) and Bcl-2 families

Abstract Anticancer efficacy and selectivity are the two critical factors for successful cancer treatment. The classical approach for anticancer drug discovery is the use of cytotoxicity as a drug selection marker. The drug candidates discovered via this approach usually show little selectivity to cancer versus normal tissues. The modern cancer drug discovery and research focus on cancer-associated gene or pathway-targeted approaches. One challenging issue for targeted cancer therapy is that while monogene or mono-pathway-targeted drug shows little toxicity, it possesses minimal antitumor efficacy. On the other hand, targeting multiple cancer-associated genes or pathways via multiple drug combination usually results in over toxicity to normal tissues. One strategy to solve this challenging issue is to apply one drug targeting multiple cancer-associated genes and/or survival pathways. In this report, we describe the discovery and characterization of a clinical favorable small chemical compound that targets multiple antiapoptotic proteins in the inhibitor of apoptosis (IAP) and Bcl-2 families. Using a high throughput screening (HTS) system (US Patent 7569221, 2009), we screened several small chemical compound libraries from various sources, followed by in vitro and in vivo analyses of HTS-derived hits-relevant analogs. The efforts finally resulted in an exceptional antitumor compound designated FL118. Although compound libraries were initially screened using the survivin gene as a target and biomarker, FL118 was found to selectively inhibit the expression of survivin, XIAP, c-IAP2 and Mcl-2, while it shows no inhibitory effects on promoter activity for the genes of p21WAF1/CIP1, DHFR, human thrombin receptor and TK. In addition, FL118 inhibited Akt activation, while showing no inhibitory effects on Erk1/2 activation. FL118 showed superior antitumor activity in human tumor animal models in comparison with other FDA-approved anticancer drugs including irinotecan, doxorubicin, 5-FU, gemcitabine, docetaxel, oxaliplatin, cytoxan, cisplatin and topotecan. Although the FL118 compound structurally has similarity to irinotecan, FL118 is a poor topoisomerase I (Top1) inhibitor in comparison with the active form of irinotecan (a known Top1 inhibitor), SN-38. Using the best schedule (weeklyx4) for irinotecan in a human head-&-neck tumor xenograft mouse model, we showed that while 40% mice treated with irinotecan show a temporary cure with rapid relapse, 100% mice treated with FL118 show permanent cure without relapse. Furthermore, FL118 effectively cures large and late stage tumors in animal models. These innovative properties for FL118 lay a foundation for its further development as an exceptional drug for effective cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4525. doi:10.1158/1538-7445.AM2011-4525

Abstract 4683: Inducible knockdown of DNA topoisomerase IIα affects drug sensitivity and cell growth

Abstract DNA topoisomerase IIα (Top2α) is an essential nuclear enzyme and target for important anti-cancer drugs, such as etoposide and doxorubicin (Dox). Our teniposide-resistant human leukemic lymphoblastic cells (CEM/VM-1-5) (Danks MK et al., Biochemistry 27(24):8861-8869, 1998) express reduced Top2α protein. To determine the role of Top2α in drug resistance, we knocked down Top2α in parental CEM cells by RNAi. Compared to control cells, CEMshTop2α cells were more resistant to the Top2 inhibitors, etoposide and Dox. The mechanism by which knockdown of Top2α caused resistance to Top2 inhibitors is probably caused by a reduction in Top2-DNA complexes leading to fewer broken DNA strands (Wang JC, Nat Rev Mol Cell Biol 3(6):430-440, 2002). The level of reduced Top2α expression in Top2α knockdown CEM cells was confirmed by Western blot. Importantly, we found no compensatory regulation by Top1 or Top2α overexpression under these conditions. It has been reported that knocking-out of Top2α will cause early embryonic death (Jenkins JR et al. Nucleic acids research 20(21):5587-5592, 1992), but in our cultured CEM cells, knocking Top2α down to 40% of control level had little or no impact on cell growth rate. We subsequently generated and characterized stable CEM-tTR-shTop2α cells where Top2α can be conditionally knocked down to 5% of control levels, as determined by Western blot, through a tetracycline-induced expression of short hairpin RNAs. CEM-tTR-shTop2α cells grow significantly more slowly than the control cells, which indicates that while cells can proliferate with ∼40% of Top2α expression, when Top2α is reduced by 95%, cells can apparently remain viable, but are severely impaired in their ability to grow. Present studies are focused on identifying genes that are targets of Top2α knockdown to provide insights into those genes involved in drug resistance. (Supported in part by CA40570 from NCI and in part by UIC) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4683. doi:10.1158/1538-7445.AM2011-4683

Novel Regulation of Nuclear Factor-YB by miR-485-3p Affects the Expression of DNA Topoisomerase IIα and Drug Responsiveness

Nuclear factor (NF)-YB, a subunit of the transcription factor nuclear factor Y (NF-Y) complex, binds and activates CCAAT-containing promoters. Our previous work suggested that NF-YB may be a mediator of topoisomerase IIα (Top2α), working through the Top2α promoter. DNA topoisomerase II (Top2) is an essential nuclear enzyme and the primary target for several clinically important anticancer drugs. Our teniposide-resistant human lymphoblastic leukemia CEM cells (CEM/VM-1-5) express reduced Top2α protein compared with parental CEM cells. To study the regulation of Top2α during the development of drug resistance, we found that NF-YB protein expression is increased in CEM/VM-1-5 cells compared with parental CEM cells. This further suggests that increased NF-YB may be a negative regulator of Top2α in CEM/VM-1-5 cells. We asked what causes the up-regulation of NF-YB in CEM/VM-1-5 cells. We found by microRNA profiling that hsa-miR-485-3p is lower in CEM/VM-1-5 cells compared with CEM cells. MicroRNA target prediction programs revealed that the 3'-untranslated region (3'-UTR) of NF-YB harbors a putative hsa-miR-485-3p binding site. We thus hypothesized that hsa-miR-485-3p mediates drug responsiveness by decreasing NF-YB expression, which in turn negatively regulates Top2α expression. To test this, we overexpressed miR-485-3p in CEM/VM-1-5 cells and found that this led to reduced expression of NF-YB, a corresponding up-regulation of Top2α, and increased sensitivity to the Top2 inhibitors. Results in CEM cells were replicated in drug-sensitive and -resistant human rhabdomyosarcoma Rh30 cells, suggesting that our findings represent a general phenomenon. Ours is the first study to show that miR-485-3p mediates Top2α down-regulation in part by altered regulation of NF-YB.

Design, Synthesis, and Evaluation of Dibenzo[c,h][1,6]naphthyridines as Topoisomerase I Inhibitors and Potential Anticancer Agents

Indenoisoquinoline topoisomerase I (Top1) inhibitors are a novel class of anticancer agents. Modifications of the indenoisoquinoline A, B, and D rings have been extensively studied in order to optimize Top1 inhibitory activity and cytotoxicity. To improve understanding of the forces that stabilize drug-Top1-DNA ternary complexes, the five-membered cyclopentadienone C-ring of the indenoisoquinoline system was replaced by six-membered nitrogen heterocyclic rings, resulting in dibenzo[c,h][1,6]naphthyridines that were synthesized by a novel route and tested for Top1 inhibition. This resulted in several compounds that have unique DNA cleavage site selectivities and potent antitumor activities in a number of cancer cell lines.

Development of a Validated Immunofluorescence Assay for γH2AX as a Pharmacodynamic Marker of Topoisomerase I Inhibitor Activity

Phosphorylated histone H2AX (γH2AX) serves as a biomarker for formation of DNA double-strand break repair complexes. A quantitative pharmacodynamic immunofluorescence assay for γH2AX was developed, validated, and tested in human tumor xenograft models with the use of clinically relevant procedures. The γH2AX immunofluorescence assay uses a novel data quantitation and image processing algorithm to determine the extent of nuclear-specific γH2AX staining in tumor needle biopsies and hair follicles collected from mice bearing topotecan-responsive A375 xenografts. After method validation with the topoisomerase I (Top1) inhibitor topotecan, the assay was used to compare pharmacodynamic properties of three structurally related indenoisoquinoline Top1 inhibitors. γH2AX response to topotecan was quantified over a 60-fold dose range (0.016-1.0 times the murine single-dose maximum tolerated dose), and significant pharmacodynamic response was measured at the mouse equivalent of the 1.5 mg/m(2) clinical dose as well as the lowest dose tested. Responses were within a time window amenable for biopsy collection in clinical trials. These studies enabled characterization of dose and time responses for three indenoisoquinolines, resulting in selection of two for clinical evaluation. γH2AX response to Top1 inhibitors in hair follicles was also observable above a minimal dose threshold. Our γH2AX assay is sufficiently accurate and sensitive to quantify γH2AX in tumor samples and will be used in correlative studies of two indenoisoquinolines in a phase I clinical trial at the National Cancer Institute. Data suggest that hair follicles may potentially serve as a surrogate tissue to evaluate tumor γH2AX response to Top1 inhibitors.

Topoisomerase I Inhibitors in the Treatment of Primary CNS Malignancies: An Update on Recent Trends

High grade primary CNS gliomas hold some of the worst prognoses of any malignancy, with the vast majority of patients dying within two years of diagnosis, even with aggressive modern treatments. Surgical resection and radiotherapy are cornerstones of treatment when possible. In spite of many years of research, only recently has management with chemotherapy been able to prolong survival in patients with high grade gliomas, albeit only modestly at best. Topoisomerase I (TOP1) inhibitors target an enzyme critical for DNA replication and cell-cycle progression; they cross the blood-brain barrier and have antitumor activity against glioblastoma cells in vitro. The most frequently associated toxicities are neutropenia and diarrhea, but are often manageable. The two most used agents are irinotecan and topotecan. Due to enhanced cytochrome CY3A4/5 enzyme activity, irinotecan dose must be adjusted with concomitant enzyme-inducing antiepileptic drug usage; the data is less clear regarding the effects on topotecan. Clinical trials in patients with recurrent malignant glioma have evaluated TOP1 inhibitors as monotherapy and in combination with other agents. There is evidence for using topotecan with radiotherapy. Irinotecan has limited efficacy as monotherapy, but shows promise in combination with other agents, particularly temozolomide and bevacizumab. Newer generation TOP1 inhibitors are currently being evaluated in phase I trials. TOP1 inhibitors show promising activity in patients with primary CNS malignancies and warrant further study.

Identification of the MMS22L-TONSL Complex that Promotes Homologous Recombination

Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NFκBIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S phase-associated DSBs, such as topoisomerase (TOP) inhibitors. In this light, MMS22L and TONSL are required for the HR-mediated repair of replication fork-associated DSBs. In cells depleted of either protein, DSBs induced by the TOP1 inhibitor camptothecin are resected normally, but the loading of the RAD51 recombinase is defective. Therefore, MMS22L and TONSL are required for the maintenance of genome stability when unscheduled DSBs occur in the vicinity of DNA replication forks.

Genome-wide Analysis of Novel Splice Variants Induced by Topoisomerase I Poisoning Shows Preferential Occurrence in Genes Encoding Splicing Factors

RNA splicing is required to remove introns from pre-mRNA, and alternative splicing generates protein diversity. Topoisomerase I (Top1) has been shown to be coupled with splicing by regulating serine/arginine-rich splicing proteins. Prior studies on isolated genes also showed that Top1 poisoning by camptothecin (CPT), which traps Top1 cleavage complexes (Top1cc), can alter RNA splicing. Here, we tested the effect of Top1 inhibition on splicing at the genome-wide level in human colon carcinoma HCT116 and breast carcinoma MCF7 cells. The RNA of HCT116 cells treated with CPT for various times was analyzed with ExonHit Human Splice Array. Unlike other exon array platforms, the ExonHit arrays include junction probes that allow the detection of splice variants with high sensitivity and specificity. We report that CPT treatment preferentially affects the splicing of splicing-related factors, such as RBM8A, and generates transcripts coding for inactive proteins lacking key functional domains. The splicing alterations induced by CPT are not observed with cisplatin or vinblastine and are not simply due to reduced Top1 activity, as Top1 downregulation by short interfering RNA did not alter splicing like CPT treatment. Inhibition of RNA polymerase II (Pol II) hyperphosphorylation by 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) blocked the splicing alteration induced by CPT, which suggests that the rapid Pol II hyperphosphorylation induced by CPT interferes with normal splicing. The preferential effect of CPT on genes encoding splicing factors may explain the abnormal splicing of a large number of genes in response to Top1cc.

The structure–activity relationships of A-ring-substituted aromathecin topoisomerase I inhibitors strongly support a camptothecin-like binding mode

Aromathecins are inhibitors of human topoisomerase I (Top1). These compounds are composites of several heteroaromatic systems, namely the camptothecins and indenoisoquinolines, and they possess notable Top1 inhibition and cytotoxicity when substituted at position 14. The SAR of these compounds overlaps with indenoisoquinolines, suggesting that they may intercalate into the Top1-DNA complex similarly. Nonetheless, the proposed binding mode for aromathecins is purely hypothetical, as an X-ray structure is unavailable. In the present communication, we have synthesized eight novel series of A-ring-substituted (positions 1–3) aromathecins, through a simple, modular route, as part of a comprehensive SAR study. Certain groups (such as 2,3-ethylenedioxy) moderately improve Top1 inhibition, and, often, antiproliferative activity, whereas other groups (2,3-dimethoxy and 3-substituents) attenuate bioactivity. Strikingly, these trends are very similar to those previously observed for the A-ring of camptothecins, and this considerable SAR overlap lends further support (in the absence of crystallographic data) to the hypothesis that aromathecins bind in the Top1 cleavage complex as interfacial inhibitors in a ‘camptothecin-like’ pose.

Proteomic analysis of nuclei isolated from cancer cell lines treated with indenoisoquinoline NSC 724998, a novel topoisomerase I inhibitor.

The indenoisoquinoline NSC724998 is a novel topoisomerase I (Top1) inhibitor entering Phase I clinical trials at the National Cancer Institute, USA. In this study, 2-D PAGE analysis was performed on nuclear lysates prepared from HCT-116 and A375 cells treated with 1 microM NSC724998 for 24 h and the differentially regulated spots identified by LC-MS/MS. One-hundred fourteen protein spot differentials were identified, 66 from A375 cells and 48 from HCT-116 cells. Proteins related to apoptosis changed specifically in A375 cells, whereas proteins involved in the ubiquitin-proteasome system were highly enriched in treated HCT-116 cells. Importantly, 12 differentially expressed proteins (ETFA, HCC1, HNRCL, KAP1, NPM, NUCL, PRDX1, PRP19, PSB6, RAE1L, RU2A, and SFRS9) were common to both cell lines. Western blotting and immunocytochemistry confirmed significant nuclear upregulation of both the proteasome subunit PSB6 and the transcriptional repressor KAP1. Interestingly, increased KAP1 polypeptide was accompanied by enhanced phosphorylation at Ser824. Similar to gammaH2AX, KAP1 phosphorylation was consistently enhanced in a panel of 12 cell lines and in A375 xenografts following NSC 724998 treatment. In summary, these data enhance our understanding of protein dynamics in the nucleus following DNA damage and provide an alternate marker (pKAP1) with potential for monitoring clinical responses to Top1 poisons.

Abstract 3525: Molecular determinants of the anticancer efficacy of indenoisoquinolines

Abstract Topoisomerase I (top1) is a validated target for the anticancer alkaloid camptothecin and its analogs (CPTs). CPTs are the only class of topoisomerase I (top1) inhibitors approved for cancer treatment in clinic. However, CPTs have pharmacologic and clinical limitations. This leads to the development of noncamptothecins, such as indenoisoquinolines (INDs), as novel top1 inhibitors. Currently three INDs have been selected as leads for clinical development by the National Cancer Institute [1]. INDs are endowed with favorable characteristics. Persistence of top1 cleavage complexes induced by INDs is higher than that of induced by CPTs. The top1 cleavage sites trapped by the INDs have different genomic locations, implying differential targeting of cancer cell genomes. Presently, we demonstrate that molecular dynamics and bioinformatics are valuable tools for the study of molecular determinants of anticancer drugs. Our previous molecular dynamics studies revealed that the most persistent CPTs have higher calculated free-energy barriers for drug dissociation from the flanking base pairs [2]. We have recently extended our studies to INDs. We found the INDs have higher calculated free-energy barriers when compared with that of CPTs. This may explain why INDs experimentally produce more persistent top1 cleavage complexes when compared with that of CPTs [3]. The National Cancer Institute (NCI)-60 panel consists of 60 human tumor cell lines established for screening anticancer efficacy of a larger number of molecules, including INDs. To deciphering the relationships between anticancer efficacy of INDs and the characteristics of the cancer cells, the molecular profile data on the NCI-60 were downloaded from the CellMiner database [4]. Integromic and bioinformatics analyses were carried out on the markers that correlated with the responses of INDs-treatments. Same and unique markers, when compared to that of CPTs, were identified for INDs. Further studies are required for the validation of these predictive markers.

Abstract 2973: Poly(ADP)ribose inhibition forces the repair of topoisomerase I cleavage complexes through homologous recombination and non-homologous end joining

Abstract Poly(ADP-Ribose) polymerase (PARP) inhibitors represent a promising class of novel anticancer agents. In the present study, we explored the strategy of combining the classical topoisomerase I (Top1) inhibitor camptothecin (CPT) and the novel PARP inhibitor ABT-888 in human cancer cells. ABT-888 potentiated cell killing by CPT. By colony forming assay, the survival ratio of CPT-treated colon cancer HT-29 cells was 52%, while the survival fraction were decreased to 35% when HT-29 cells were co-treated with CPT and ABT-888. By measuring γH2AX, a DNA double-strand break marker, we found that ABT-888 enhanced the γH2AX level induced by CPT. The enhanced γH2AX response in the presence of ABT-888 affected both replication-related γH2AX but also transcription-related γH2AX. In osteosarcoma U2OS cells, ABT-888 also enhanced γH2AX in both EdU (5-ethynyl-2′- deoxyuridine) positive and negative cells. Even in non-replicating human primary lymphocytes, ABT-888 enhanced CPT-induced γH2AX foci (1.3 ± 1.2 vs. 5.1 ± 3.1 γH2AX foci/per cell in the absence and presence of co-treatment with ABT-888, respectively). Proteasome inhibition with MG-132 blocked the γH2AX induction by CPT, and PARP inhibition could not overcome the block, which demonstrates that PARP acts downstream from the proteasome. Neutral Comet assays showed that the enhanced γH2AX response was related to DNA breaks. Alkaline elution assays revealed that ABT-888 increased CPT-induced DNA single-strand breaks (SSBs) but without a corresponding increase in DNA protein crosslinks indicating that ABT-888 was inducing DNA breaks in addition to the Top1-DNA cleavage complexes trapped by CPT. These additional DNA breaks were dependent on XPF/ERCC1 endonuclease. Furthermore, after removing CPT, reversal of γH2AX in the absence or presence of ABT-888 showed the same reversal rate, although there was more γH2AX in the presence of ABT-888, suggesting that PARP inhibition did not affect the repair of double-strand breaks. Moreover, PARP inhibition enhanced DNA-PK and ATM activation with increased formation of Rad51 foci. Therefore, we propose that PARP inhibition forces the cells to activate alternative endonucleolytic and recombination pathways in response to Top1-mediated DNA damage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2973.

Abstract 5370: Potentiation of the novel topoisomerase I inhibitor indenoisoquinoline NSC743400 by Chk1/2 inhibitors in p53-deficient cells

Abstract Clincally available camptothecin derivatives, though highly effective against a broad range tumors, are limited by their chemical instability, solubility, ABCG2-mediated efflux and reversibility of Top1-DNA complexes. Indenoisoquinolines were identified as topoisomerase I (Top1) inhibitors by the COMPARE analysis of the NCI drug screen database. Although structurally similar to camptothecin, idenoisoquinolines are chemically stable, able to overcome ABCG2 efflux while forming more persistent Top1-DNA complexes and maintaining potent antitumor activity. The potential for indenoisoquinoline derivatives as possible clinical agents is further aided by the synergistic effect observed between camptothecin and Chk1/2 inhibitors. Chk1/2 kinases regulate cell cycle, DNA damage response via ATM, ATR and DNA-PK, which are activated by genomic instability. Previous studies have shown Chk1/2 inhibitors, such as UCN-01, can potentiate camptothecin-mediated cytotoxicity in p53 mutant cells by eliminating the DNA replication checkpoint. Further synthesis has led to the water-soluble indenoisoquinoline derivative, NSC743400 for clinical development. Current checkpoint kinase inhibitors, like PV1019 or AZD7762, offer a higher level of specificity than their predecessors that can be exploited by combination with indenoisoquinolines. Checkpoint inhibition showed synergistic antiproliferative activity with NSC743400 in human colon carcinoma HT-29 cells. Furthermore, AZD7762 was able to abrogate the late S-phase arrest induced by NSC743400 as early as 3h post treatment. Inhibition of checkpoint kinases also abrogated the DNA replication fork progression response to NSC743400. The effect of these inhibitors on the autophosphorylation of both Chk1 and Chk2 post NSC743400 was examined by Western blot. These results further substantiate the idea that checkpoint inhibition is a viable strategy in enhancing the antiproliferative effect of novel non-camptothecinn inhibitors such as the indenoisoquinolines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5370.

Abstract 3519: The polyphenolic ellagitanin vescalagin is a specific inhibitor of the alpha isoform of human DNA topoisomerase II

Abstract Polyphenolic ellagitanins are natural structurally complex molecules that are often associated with the therapeutic activity of plant extracts used in traditional medicine. Studies have reported their cancer-preventing activity in animal models at concentrations found in diets. In vitro, some derivatives display cytotoxic activities at micromolar concentrations and induce apoptosis by a mechanism which remains unclear. Potential targets have been proposed, including topoisomerases (top) I and II, which are involved in the regulation of DNA topology during replication, transcription and chromosome segregation. The two human isoforms of top2: top2 alpha (top2a) and top2 beta (top2b) are the target of anticancer agents such as doxorubicin or etoposide. It is known that antitumoral activity of etoposide is primarily due the inhibition of the alpha isoform whereas inhibition of the beta isoform is primarily responsible for the development of secondary malignancies, pointing to the need for more selective top2a inhibitors. Here, we report the differential activity on top2a and top2b of new polyphenolic ellagitanins: vescalagin, methylvescalagin, acutissimin B (acuB) and epiacutissimin B (epiacuB). Using kinetoplast DNA as a substrate, we showed that top2a-mediated decatenation was strongly inhibited by all 4 derivatives at 1 µM concentration, whereas no significant inhibition of top2b could be seen. When used at 10 µM, acuB and epiacuB inhibited both isoforms and were even more active on top2b (>80% inhibition), whereas vescalagin and methylvescalagin specifically targeted top2a with a reduced effect on top2b (<20% inhibition). Vescalagin could also specifically poison top2a in treated CEM cells, since it could induce an increase in covalent top2a-DNA but not top2b-DNA cleavage complexes as detected by the immuno complex of enzyme (ICE) assay. We showed that top2a mutated CEM-VM1 etoposide-resistant cells were also resistant to vescalagin, but were still sensitive to ICRF193 suggesting that vecsalagin is a top2a poison. Using alkaline elution technique we also showed that treatment of CEM cells with 50 µM vescalagin (2h) did not induce detectable double-strand breaks conversely to etoposide. This was confirmed by the absence of gH2AX detection. Surprisingly, vescalagin could induce a significant amount of single-strand breaks. Whether generation of these single-strand breaks is mediated by top2a poisoning and is the cause of cell growth inhibition remains to be investigated. Together our preliminary data identified vescalagin as a new potent and specific top2a poison that could be used as a lead to generate alternative top2 inhibitors with reduced chances to develop secondary malignancies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3519.