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Keli Agama

National Institutes of Health, National Cancer Institute, Laboratory Manager

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Dynamic, results-driven scientific research advisor with an outstanding track record in leading and conducting health research. Over twelve years’ experience managing a premier pharmacology laboratory, partnering with the Branch Chief to set the strategic and policy framework of the National Cancer Institute (NCI) laboratory and achieve stated outcomes. Provides scientific leadership and strategic direction of the research portfolio and priorities of NCI. Supports and oversees the scientific aspects of the work performed by the research teams, and recommends strategies to build their research capacity. Develops and implements strategies to engage external researchers, including collaborative research proposals. Extensive experience working with cross-functional scientific and research teams. Excellent influencing and interpersonal skills. Unique ability to embrace different points of view and values the contributions of others. A global understanding of organizations and culture that goes beyond traditional boundaries of geography.
Supervisors: Dr. Yves Pommier
Address: Bethesda, Maryland, United States

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Middlesex University

Mary-Ann Bjornsti

Hongliang Zhang

Louisiana State University

Indian Association for the Cultivation of Science

SardarKrushinagar Dantiwada Agriculture University

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Papers by Keli Agama

Poisoning of Mitochondrial Topoisomerase I by Lamellarin D

Molecular Pharmacology, Jun 2, 2014

Lamellarin D (Lam-D) is a hexacyclic pyrole alkaloid isolated from marine invertebrates, whose bi... more Lamellarin D (Lam-D) is a hexacyclic pyrole alkaloid isolated from marine invertebrates, whose biologic properties have been attributed to mitochondrial targeting. Mitochondria contain their own DNA (mtDNA), and the only specific mitochondrial topoisomerase in vertebrates is mitochondrial topoisomerase I (Top1mt). Here, we show that Top1mt is a direct mitochondrial target of Lam-D. In vitro Lam-D traps Top1mt and induces Top1mt cleavage complexes (Top1mtcc). Using single-molecule analyses, we also show that Lam-D slows down supercoil relaxation of Top1mt and strongly inhibits Top1mt religation in contrast to the inefficacy of camptothecin on Top1mt. In living cells, we show that Lam-D accumulates rapidly inside mitochondria, induces cellular Top1mtcc, and leads to mtDNA damage. This study provides evidence that Top1mt is a direct mitochondrial target of Lam-D and suggests that developing Top1mt inhibitors represents a novel strategy for targeting mitochondrial DNA.

Development and Validation of an Immunoassay for Initial Target Engagement of Topoisomerase I in Mouse Xenografts for Use in Preclinical Modeling and Clinical Trials

Cellular Topoisomerase I Inhibition and Antiproliferative Activity by MJ-III-65 (NSC 706744), an Indenoisoquinoline Topoisomerase I Poison

Molecular Pharmacology, Nov 5, 2004

To overcome camptothecin's (CPT) lactone instability, reversibility of the drug-target interactio... more To overcome camptothecin's (CPT) lactone instability, reversibility of the drug-target interaction and drug resistance, attempts to synthesize compounds that are CPT-like in their specificity and potency yet display a unique profile have been underway. In this pursuit, we have identified one of the idenoisoquinoline derivatives (MJ-III-65; NSC 706744) with both similarities and differences from CPT. MJ-III-65 traps topoisomerase I (Top1) reversibly like CPT but with different DNA sequence preferences. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with MJ-III-65 at nanomolar concentrations. These MJ-III-65-induced protein-linked DNA breaks were resistant to reversal even after an hour of drug removal as compared to CPT, which completely reversed. Studies in human cells in culture found MJ-III-65 to be cytotoxic. Furthermore, limited cross-resistance was observed in camptothecin-resistant cell lines. MJ-III-65 also exhibits antitumor activity in mouse tumor xenografts.

Batracylin (NSC 320846), a Dual Inhibitor of DNA Topoisomerases I and II Induces Histone γ-H2AX as a Biomarker of DNA Damage

Cancer Research, Oct 15, 2007

Mus81-mediated DNA cleavage resolves replication forks stalled by topoisomerase I–DNA complexes

Journal of Cell Biology, Nov 28, 2011

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

Cellular and Molecular Biology, Apr 15, 2010

Poly(ADP-Ribose) polymerase (PARP) inhibitors represent a promising class of novel anticancer age... more 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.

Synthesis and biological evaluation of new fluorinated and chlorinated indenoisoquinoline topoisomerase I poisons

Bioorganic & Medicinal Chemistry, Apr 1, 2016

Fluorine and chlorine are metabolically stable, but generally less active replacements for a nitr... more Fluorine and chlorine are metabolically stable, but generally less active replacements for a nitro group at the 3-position of indenoisoquinoline topoisomerase IB (Top1) poisons. A number of strategies were employed in the present investigation to enhance the Top1 inhibitory potencies and cancer cell growth inhibitory activities of halogenated indenoisoquinolines. In several cases, the new compounds' activities were found to rival or surpass those of similarly substituted 3nitroindenoisoquinolines, and several unusually potent analogues were discovered through testing in human cancer cell cultures. A hydroxyethylaminopropyl side chain on the lactam nitrogen of two halogenated indenoisoquinoline Top1 inhibitors was found to also impart inhibitory activity against tyrosyl DNA phosphodiesterases 1 and 2 (TDP1 and TDP2), which are enzymes that participate in the repair of DNA damage induced by Top1 poisons.

Supplementary Figure Legend and Methods from The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Supplementary Figure 1 from The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Supplementary Table 1 from Batracylin (NSC 320846), a Dual Inhibitor of DNA Topoisomerases I and II Induces Histone γ-H2AX as a Biomarker of DNA Damage

Data from Batracylin (NSC 320846), a Dual Inhibitor of DNA Topoisomerases I and II Induces Histone γ-H2AX as a Biomarker of DNA Damage

Batracylin (8-aminoisoindolo [1,2-b]quinazolin-10(12H)-one; NSC320846) is an investigational clin... more Batracylin (8-aminoisoindolo [1,2-b]quinazolin-10(12H)-one; NSC320846) is an investigational clinical anticancer agent. Previous animal studies showed activity against solid tumors and Adriamycin-resistant leukemia. We initially sought to test the proposed Top2-mediated DNA cleavage activity of batracylin and identify potential biomarkers for activity. COMPARE analysis in the NCI-60 cell lines showed batracylin activity to be most closely related to the class of Top2 inhibitors. The 50% growth inhibition (GI50) value for batracylin in HT29 colon carcinoma cells was 10 μmol/L. DNA-protein cross-links, consistent with Top2 targeting, were measured by alkaline elution. DNA single-strand breaks were also detected and found to be protein associated. However, only a weak induction of DNA double-strand breaks was observed. Because batracylin induced almost exclusively DNA single-strand breaks, we tested batracylin as a Top1 inhibitor. Batracylin exhibited both Top1- and Top2α/β-mediated DNA cleavage in vitro and in cells. The phosphorylation of histone (γ-H2AX) was tested to measure the extent of DNA damage. Kinetics of γ-H2AX “foci” showed early activation with low μmol/L concentrations, thus presenting a useful early biomarker of DNA damage. The half-life of γ-H2AX signal reversal after drug removal was consistent with reversal of DNA-protein cross-links. The persistence of the DNA-protein complexes induced by batracylin was markedly longer than by etoposide or camptothecin. The phosphorylated DNA damage–responsive kinase, ataxia telangiectasia mutated, was also found activated at sites of γ-H2AX. The cell cycle checkpoint kinase, Chk2, was only weakly phosphorylated. Thus, batracylin is a dual Top1 and Top2 inhibitor and γ-H2AX could be considered a biomarker in the ongoing clinical trials. [Cancer Res 2007;67(20):9971–9]

Data from The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chela... more Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G1 cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (γ-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIα (top2α) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2α knockout cells (top2α+/−) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2α+/+ or top2β−/− cells. Specificity for top2α was confirmed using top2α and top2β small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2α. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2α activity. [Cancer Res 2009;69(3):948–57]

Correction to Synthesis and Biological Evaluation of the First Dual Tyrosyl-DNA Phosphodiesterase I (Tdp1)–Topoisomerase I (Top1) Inhibitors

Journal of Medicinal Chemistry, Aug 14, 2012

Abstract 4691: Rationale for combining PARP inhibitors with topotecan or irinotecan: taking advantage of the alternative DNA repair pathways involving XPF-ERCC1 and PARP-TDP1

Poly(ADP-Ribose) (PAR) polymerase (PARP) inhibitors represent an important class of novel antican... more Poly(ADP-Ribose) (PAR) polymerase (PARP) inhibitors represent an important class of novel anticancer agents. The present study explores the molecular rationale for combining veliparib (ABT-888) with camptothecin (CPT) and its clinical derivatives, topotecan and irinotecan. ABT-888 inhibited PAR induction by CPT and increased CPT-induced cell killing and DNA damage measured by histone gamma-H2AX and comet assays. Notably, increased DNA breaks by ABT-888 was not associated with a corresponding increase of topoisomerase I cleavage complexes (Top1cc). Also, ABT-888 failed to further enhance the gamma-H2AX response in tyrosyl-DNA-phosphodiesterase (TDP1) knockout cells, which indicates that PARP inhibition no longer enhances Top1-induced DNA damage once TDP1 is inactivated. To explore alternative endonuclease pathway for the repair of Top1cc upon PARP inhibition, we tested the role of the 3′-flap endonuclease XPF-ERCC1. XPF-ERCC1 inactivation reduced the ABT-888-induced gamma-H2AX response both in non-replicating and replicating cells, and further enhanced the antiproliferative effect of ABT-888 in CPT-treated cells. Together, our findings demonstrate that XPF-ERCC1 acts as an alternative endonuclease pathway for the repair of Top1-induced DNA damage. Its function becomes critical when the PARP-TDP1 excision repair pathway is inactivated. Our study suggests the rationale for combining Top1 and PARP inhibitors in tumors with XPF-ERCC1 deficiency. 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 4691. doi:1538-7445.AM2012-4691

4-Nitroquinoline-1-Oxide Induces the Formation of Cellular Topoisomerase I-DNA Cleavage Complexes

Cancer Research, Jul 1, 2006

RecQ helicase BLM-deficient cells are characteristically hypersensitive to 4-nitroquinoline-1-oxi... more RecQ helicase BLM-deficient cells are characteristically hypersensitive to 4-nitroquinoline-1-oxide (4NQO). We recently reported that isogenic BLM-deficient cells (PNSG13) are more sensitive than BLM-complemented cells (PNSF5) to camptothecin, which specifically traps topoisomerase I cleavage complexes (Top1cc). We now report that PNSG13 are also 3.5-fold more sensitive to 4NQO compared with PNSF5 and that 4NQO induces higher levels of Top1cc and reduced histone ;-H2AX in PSNG13 than in PNSF5. Similarly, 4NQO induces more Top1cc in primary fibroblasts from a patient with Bloom syndrome than in normal human fibroblasts. 4NQO also induces Top1cc in colon cancer HCT116 and HT29 cells in a time-and concentration-dependent fashion. Of note, distinct from camptothecin, the Top1cc produced by 4NQO accumulate progressively after 4NQO addition and persist following 4NQO removal. The Top1cc induced by 4NQO are detectable by alkaline elution. To examine the functional relevance of the Top1cc induced by 4NQO, we used two stable topoisomerase I small interfering RNA (siRNA) cell lines derived from HCT116 and MCF7 cells. Both topoisomerase I siRNA cell lines are resistant to 4NQO, indicating that Top1cc contribute to the cellular activity of 4NQO. Collectively, these data show that 4NQO is an effective inducer of cellular Top1cc. Because 4NQO does not directly trap Top1cc in biochemical assays, we propose that active metabolites of 4NQO trap Top1cc by forming DNA adducts. Induction of Top1cc and histone ;-H2AX by 4NQO may contribute to the cellular effects of 4NQO, including its selective activity toward RecQ helicase BLM-deficient cells.

The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Cancer Research, Jan 29, 2009

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chela... more Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G 1 cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (;-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIA (top2A) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2A knockout cells (top2A +/À) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2A +/+ or top2B À/À cells. Specificity for top2A was confirmed using top2A and top2B small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2A. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2A activity.

Supplemental Figure 1-3 from Single-Molecule Supercoil Relaxation Assay as a Screening Tool to Determine the Mechanism and Efficacy of Human Topoisomerase IB Inhibitors

Supplemental information contains three figures. Figure s1 displays plots of in vitro inhibition ... more Supplemental information contains three figures. Figure s1 displays plots of in vitro inhibition plotted versus IC50 values for inhibitors as measured for three individual cell lines. Figure S2 displays the low rate probability and religation inhibition measures of inhibition plotted versus the average normalized IC50 values for each of the four inhibitors. Figure S3 displays the fraction of relaxed DNA measured in the ensembled supercoil relaxation assay as a function of the inhibitor concentration for the four inhibitors.

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

Bioorganic & Medicinal Chemistry, Aug 1, 2010

Aromathecins are inhibitors of human topoisomerase I (Top1). These compounds are composites of se... more 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 2,3ethylenedioxy) 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.

Optimization of the Lactam Side Chain of 7-Azaindenoisoquinoline Topoisomerase I Inhibitors and Mechanism of Action Studies in Cancer Cells

Journal of Medicinal Chemistry, Feb 6, 2014

Optimization of the lactam ω-aminoalkyl substituents in a series of 7azaindenoisoquinolines resul... more Optimization of the lactam ω-aminoalkyl substituents in a series of 7azaindenoisoquinolines resulted in new anticancer agents with improved Top1 inhibitory potencies and cancer cell cytotoxicities. The new compounds 14−17 and 19 exhibited mean graph midpoint cytotoxicity (GI 50) values of 21−71 nM in the NCI panel of 60 human cancer cell cultures. Ternary 7-azaindenoisoquinoline−DNA−Top1 cleavage complexes that persist for up to 6 h were detected in HCT116 colon cancer cells. Ternary complexes containing 7-azaindenoisoquinolines were significantly more stable than those in which camptothecin was incorporated. DNA content distribution histograms showed S-phase block 3 h after drug removal. Drug-induced DNA damage in HCT116 cells was revealed by induction of the histone γ-H2AX marker. The 7-azaindenoisoquinolines were able to partially overcome resistance in several drug-resistant cell lines, and they were not substrates for the ABCB1 drug efflux transporter. Molecular modeling studies indicate that the 7-azaindenoisoquinolines intercalate at the DNA cleavage site in DNA−Top1 covalent complexes with the lactam side chain projecting into the major groove. Overall, the results indicate that the 7azaindenoisoquinolines are promising anticancer agents that merit further development.

Phenylpyrazolo[1,5-<i>a</i>]quinazolin-5(4<i>H</i>)-one: A Suitable Scaffold for the Development of Noncamptothecin Topoisomerase I (Top1) Inhibitors

Journal of Medicinal Chemistry, Sep 16, 2013

In search for a novel chemotype to develop Topoisomerase I (Top1) inhibitors, the pyrazolo[1,5a]q... more In search for a novel chemotype to develop Topoisomerase I (Top1) inhibitors, the pyrazolo[1,5a]quinazoline nucleus, structurally related to the indenoisoquinoline system precursor of wellknown Top1 poisons, was variously decorated (i.e. a substituted phenyl ring at 2-or 3-position, a protonable side chain at 4-or 5-position) affording a number of Top1 inhibitors with cleavage patterns common to CPT and MJ-III-65. SARs data were rationalized by means of an advanced docking protocol.

Poisoning of Mitochondrial Topoisomerase I by Lamellarin D

Molecular Pharmacology, Jun 2, 2014

Lamellarin D (Lam-D) is a hexacyclic pyrole alkaloid isolated from marine invertebrates, whose bi... more Lamellarin D (Lam-D) is a hexacyclic pyrole alkaloid isolated from marine invertebrates, whose biologic properties have been attributed to mitochondrial targeting. Mitochondria contain their own DNA (mtDNA), and the only specific mitochondrial topoisomerase in vertebrates is mitochondrial topoisomerase I (Top1mt). Here, we show that Top1mt is a direct mitochondrial target of Lam-D. In vitro Lam-D traps Top1mt and induces Top1mt cleavage complexes (Top1mtcc). Using single-molecule analyses, we also show that Lam-D slows down supercoil relaxation of Top1mt and strongly inhibits Top1mt religation in contrast to the inefficacy of camptothecin on Top1mt. In living cells, we show that Lam-D accumulates rapidly inside mitochondria, induces cellular Top1mtcc, and leads to mtDNA damage. This study provides evidence that Top1mt is a direct mitochondrial target of Lam-D and suggests that developing Top1mt inhibitors represents a novel strategy for targeting mitochondrial DNA.

Development and Validation of an Immunoassay for Initial Target Engagement of Topoisomerase I in Mouse Xenografts for Use in Preclinical Modeling and Clinical Trials

Cellular Topoisomerase I Inhibition and Antiproliferative Activity by MJ-III-65 (NSC 706744), an Indenoisoquinoline Topoisomerase I Poison

Molecular Pharmacology, Nov 5, 2004

To overcome camptothecin's (CPT) lactone instability, reversibility of the drug-target interactio... more To overcome camptothecin's (CPT) lactone instability, reversibility of the drug-target interaction and drug resistance, attempts to synthesize compounds that are CPT-like in their specificity and potency yet display a unique profile have been underway. In this pursuit, we have identified one of the idenoisoquinoline derivatives (MJ-III-65; NSC 706744) with both similarities and differences from CPT. MJ-III-65 traps topoisomerase I (Top1) reversibly like CPT but with different DNA sequence preferences. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with MJ-III-65 at nanomolar concentrations. These MJ-III-65-induced protein-linked DNA breaks were resistant to reversal even after an hour of drug removal as compared to CPT, which completely reversed. Studies in human cells in culture found MJ-III-65 to be cytotoxic. Furthermore, limited cross-resistance was observed in camptothecin-resistant cell lines. MJ-III-65 also exhibits antitumor activity in mouse tumor xenografts.

Batracylin (NSC 320846), a Dual Inhibitor of DNA Topoisomerases I and II Induces Histone γ-H2AX as a Biomarker of DNA Damage

Cancer Research, Oct 15, 2007

Mus81-mediated DNA cleavage resolves replication forks stalled by topoisomerase I–DNA complexes

Journal of Cell Biology, Nov 28, 2011

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

Cellular and Molecular Biology, Apr 15, 2010

Poly(ADP-Ribose) polymerase (PARP) inhibitors represent a promising class of novel anticancer age... more 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.

Synthesis and biological evaluation of new fluorinated and chlorinated indenoisoquinoline topoisomerase I poisons

Bioorganic & Medicinal Chemistry, Apr 1, 2016

Fluorine and chlorine are metabolically stable, but generally less active replacements for a nitr... more Fluorine and chlorine are metabolically stable, but generally less active replacements for a nitro group at the 3-position of indenoisoquinoline topoisomerase IB (Top1) poisons. A number of strategies were employed in the present investigation to enhance the Top1 inhibitory potencies and cancer cell growth inhibitory activities of halogenated indenoisoquinolines. In several cases, the new compounds' activities were found to rival or surpass those of similarly substituted 3nitroindenoisoquinolines, and several unusually potent analogues were discovered through testing in human cancer cell cultures. A hydroxyethylaminopropyl side chain on the lactam nitrogen of two halogenated indenoisoquinoline Top1 inhibitors was found to also impart inhibitory activity against tyrosyl DNA phosphodiesterases 1 and 2 (TDP1 and TDP2), which are enzymes that participate in the repair of DNA damage induced by Top1 poisons.

Supplementary Figure Legend and Methods from The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Supplementary Figure 1 from The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Supplementary Table 1 from Batracylin (NSC 320846), a Dual Inhibitor of DNA Topoisomerases I and II Induces Histone γ-H2AX as a Biomarker of DNA Damage

Data from Batracylin (NSC 320846), a Dual Inhibitor of DNA Topoisomerases I and II Induces Histone γ-H2AX as a Biomarker of DNA Damage

Batracylin (8-aminoisoindolo [1,2-b]quinazolin-10(12H)-one; NSC320846) is an investigational clin... more Batracylin (8-aminoisoindolo [1,2-b]quinazolin-10(12H)-one; NSC320846) is an investigational clinical anticancer agent. Previous animal studies showed activity against solid tumors and Adriamycin-resistant leukemia. We initially sought to test the proposed Top2-mediated DNA cleavage activity of batracylin and identify potential biomarkers for activity. COMPARE analysis in the NCI-60 cell lines showed batracylin activity to be most closely related to the class of Top2 inhibitors. The 50% growth inhibition (GI50) value for batracylin in HT29 colon carcinoma cells was 10 μmol/L. DNA-protein cross-links, consistent with Top2 targeting, were measured by alkaline elution. DNA single-strand breaks were also detected and found to be protein associated. However, only a weak induction of DNA double-strand breaks was observed. Because batracylin induced almost exclusively DNA single-strand breaks, we tested batracylin as a Top1 inhibitor. Batracylin exhibited both Top1- and Top2α/β-mediated DNA cleavage in vitro and in cells. The phosphorylation of histone (γ-H2AX) was tested to measure the extent of DNA damage. Kinetics of γ-H2AX “foci” showed early activation with low μmol/L concentrations, thus presenting a useful early biomarker of DNA damage. The half-life of γ-H2AX signal reversal after drug removal was consistent with reversal of DNA-protein cross-links. The persistence of the DNA-protein complexes induced by batracylin was markedly longer than by etoposide or camptothecin. The phosphorylated DNA damage–responsive kinase, ataxia telangiectasia mutated, was also found activated at sites of γ-H2AX. The cell cycle checkpoint kinase, Chk2, was only weakly phosphorylated. Thus, batracylin is a dual Top1 and Top2 inhibitor and γ-H2AX could be considered a biomarker in the ongoing clinical trials. [Cancer Res 2007;67(20):9971–9]

Data from The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chela... more Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G1 cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (γ-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIα (top2α) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2α knockout cells (top2α+/−) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2α+/+ or top2β−/− cells. Specificity for top2α was confirmed using top2α and top2β small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2α. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2α activity. [Cancer Res 2009;69(3):948–57]

Correction to Synthesis and Biological Evaluation of the First Dual Tyrosyl-DNA Phosphodiesterase I (Tdp1)–Topoisomerase I (Top1) Inhibitors

Journal of Medicinal Chemistry, Aug 14, 2012

Abstract 4691: Rationale for combining PARP inhibitors with topotecan or irinotecan: taking advantage of the alternative DNA repair pathways involving XPF-ERCC1 and PARP-TDP1

Poly(ADP-Ribose) (PAR) polymerase (PARP) inhibitors represent an important class of novel antican... more Poly(ADP-Ribose) (PAR) polymerase (PARP) inhibitors represent an important class of novel anticancer agents. The present study explores the molecular rationale for combining veliparib (ABT-888) with camptothecin (CPT) and its clinical derivatives, topotecan and irinotecan. ABT-888 inhibited PAR induction by CPT and increased CPT-induced cell killing and DNA damage measured by histone gamma-H2AX and comet assays. Notably, increased DNA breaks by ABT-888 was not associated with a corresponding increase of topoisomerase I cleavage complexes (Top1cc). Also, ABT-888 failed to further enhance the gamma-H2AX response in tyrosyl-DNA-phosphodiesterase (TDP1) knockout cells, which indicates that PARP inhibition no longer enhances Top1-induced DNA damage once TDP1 is inactivated. To explore alternative endonuclease pathway for the repair of Top1cc upon PARP inhibition, we tested the role of the 3′-flap endonuclease XPF-ERCC1. XPF-ERCC1 inactivation reduced the ABT-888-induced gamma-H2AX response both in non-replicating and replicating cells, and further enhanced the antiproliferative effect of ABT-888 in CPT-treated cells. Together, our findings demonstrate that XPF-ERCC1 acts as an alternative endonuclease pathway for the repair of Top1-induced DNA damage. Its function becomes critical when the PARP-TDP1 excision repair pathway is inactivated. Our study suggests the rationale for combining Top1 and PARP inhibitors in tumors with XPF-ERCC1 deficiency. 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 4691. doi:1538-7445.AM2012-4691

4-Nitroquinoline-1-Oxide Induces the Formation of Cellular Topoisomerase I-DNA Cleavage Complexes

Cancer Research, Jul 1, 2006

RecQ helicase BLM-deficient cells are characteristically hypersensitive to 4-nitroquinoline-1-oxi... more RecQ helicase BLM-deficient cells are characteristically hypersensitive to 4-nitroquinoline-1-oxide (4NQO). We recently reported that isogenic BLM-deficient cells (PNSG13) are more sensitive than BLM-complemented cells (PNSF5) to camptothecin, which specifically traps topoisomerase I cleavage complexes (Top1cc). We now report that PNSG13 are also 3.5-fold more sensitive to 4NQO compared with PNSF5 and that 4NQO induces higher levels of Top1cc and reduced histone ;-H2AX in PSNG13 than in PNSF5. Similarly, 4NQO induces more Top1cc in primary fibroblasts from a patient with Bloom syndrome than in normal human fibroblasts. 4NQO also induces Top1cc in colon cancer HCT116 and HT29 cells in a time-and concentration-dependent fashion. Of note, distinct from camptothecin, the Top1cc produced by 4NQO accumulate progressively after 4NQO addition and persist following 4NQO removal. The Top1cc induced by 4NQO are detectable by alkaline elution. To examine the functional relevance of the Top1cc induced by 4NQO, we used two stable topoisomerase I small interfering RNA (siRNA) cell lines derived from HCT116 and MCF7 cells. Both topoisomerase I siRNA cell lines are resistant to 4NQO, indicating that Top1cc contribute to the cellular activity of 4NQO. Collectively, these data show that 4NQO is an effective inducer of cellular Top1cc. Because 4NQO does not directly trap Top1cc in biochemical assays, we propose that active metabolites of 4NQO trap Top1cc by forming DNA adducts. Induction of Top1cc and histone ;-H2AX by 4NQO may contribute to the cellular effects of 4NQO, including its selective activity toward RecQ helicase BLM-deficient cells.

The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Cancer Research, Jan 29, 2009

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chela... more Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G 1 cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (;-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIA (top2A) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2A knockout cells (top2A +/À) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2A +/+ or top2B À/À cells. Specificity for top2A was confirmed using top2A and top2B small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2A. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2A activity.

Supplemental Figure 1-3 from Single-Molecule Supercoil Relaxation Assay as a Screening Tool to Determine the Mechanism and Efficacy of Human Topoisomerase IB Inhibitors

Supplemental information contains three figures. Figure s1 displays plots of in vitro inhibition ... more Supplemental information contains three figures. Figure s1 displays plots of in vitro inhibition plotted versus IC50 values for inhibitors as measured for three individual cell lines. Figure S2 displays the low rate probability and religation inhibition measures of inhibition plotted versus the average normalized IC50 values for each of the four inhibitors. Figure S3 displays the fraction of relaxed DNA measured in the ensembled supercoil relaxation assay as a function of the inhibitor concentration for the four inhibitors.

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

Bioorganic & Medicinal Chemistry, Aug 1, 2010

Aromathecins are inhibitors of human topoisomerase I (Top1). These compounds are composites of se... more 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 2,3ethylenedioxy) 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.

Optimization of the Lactam Side Chain of 7-Azaindenoisoquinoline Topoisomerase I Inhibitors and Mechanism of Action Studies in Cancer Cells

Journal of Medicinal Chemistry, Feb 6, 2014

Optimization of the lactam ω-aminoalkyl substituents in a series of 7azaindenoisoquinolines resul... more Optimization of the lactam ω-aminoalkyl substituents in a series of 7azaindenoisoquinolines resulted in new anticancer agents with improved Top1 inhibitory potencies and cancer cell cytotoxicities. The new compounds 14−17 and 19 exhibited mean graph midpoint cytotoxicity (GI 50) values of 21−71 nM in the NCI panel of 60 human cancer cell cultures. Ternary 7-azaindenoisoquinoline−DNA−Top1 cleavage complexes that persist for up to 6 h were detected in HCT116 colon cancer cells. Ternary complexes containing 7-azaindenoisoquinolines were significantly more stable than those in which camptothecin was incorporated. DNA content distribution histograms showed S-phase block 3 h after drug removal. Drug-induced DNA damage in HCT116 cells was revealed by induction of the histone γ-H2AX marker. The 7-azaindenoisoquinolines were able to partially overcome resistance in several drug-resistant cell lines, and they were not substrates for the ABCB1 drug efflux transporter. Molecular modeling studies indicate that the 7-azaindenoisoquinolines intercalate at the DNA cleavage site in DNA−Top1 covalent complexes with the lactam side chain projecting into the major groove. Overall, the results indicate that the 7azaindenoisoquinolines are promising anticancer agents that merit further development.

Phenylpyrazolo[1,5-<i>a</i>]quinazolin-5(4<i>H</i>)-one: A Suitable Scaffold for the Development of Noncamptothecin Topoisomerase I (Top1) Inhibitors

Journal of Medicinal Chemistry, Sep 16, 2013

In search for a novel chemotype to develop Topoisomerase I (Top1) inhibitors, the pyrazolo[1,5a]q... more In search for a novel chemotype to develop Topoisomerase I (Top1) inhibitors, the pyrazolo[1,5a]quinazoline nucleus, structurally related to the indenoisoquinoline system precursor of wellknown Top1 poisons, was variously decorated (i.e. a substituted phenyl ring at 2-or 3-position, a protonable side chain at 4-or 5-position) affording a number of Top1 inhibitors with cleavage patterns common to CPT and MJ-III-65. SARs data were rationalized by means of an advanced docking protocol.