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Novel Fluoroindenoisoquinoline Non-Camptothecin Topoisomerase I Inhibitors.

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 To further improve metabolic stability, their methoxy groups have been replaced by fluorine, as in the fluoroindenoisoquinolines NSC 781517 (LMP517), NSC 779135 (LMP135), and NSC 779134 (LMP134).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

SUBMITTER: Marzi L

PROVIDER: S-EPMC6072611 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Novel Fluoroindenoisoquinoline Non-Camptothecin Topoisomerase I Inhibitors.

Marzi Laetitia L Agama Keli K Murai Junko J Difilippantonio Simone S James Amy A Peer Cody J CJ Figg William D WD Beck Daniel D Elsayed Mohamed S A MSA Cushman Mark M Pommier Yves Y

Molecular cancer therapeutics 20180510 8

Contrary to other anticancer targets, topoisomerase I (TOP1) is targeted by only one chemical class of FDA-approved drugs: topotecan and irinotecan, the derivatives of the plant alkaloid, camptothecin. The indenoisoquinolines LMP400, LMP744, and LMP776 are novel noncamptothecin TOP1 inhibitors in clinical trial, which overcome the limitations of camptothecins. To further improve metabolic stability, their methoxy groups have been replaced by fluorine, as in the fluoroindenoisoquinolines NSC 7815 ...[more]

PMID: 29748210

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Project description:DNA topoisomerase I (Top1) and topoisomerase II (Top2) inhibitors are widely used to treat a variety of cancers. Their mechanism of action involves stabilization of otherwise transient ("cleavable") complexes between Top1 or Top2 and DNA; collisions of DNA replication forks with such stabilized complexes lead to formation of DNA double-strand breaks (DSBs). In this study, using 5-ethynyl-2'deoxyuridine (EdU) as a DNA precursor, we directly assessed the relationship between DNA replication and induction of DSBs revealed as ?H2AX foci in A549 cells treated with Top1 inhibitors topotecan (Tpt) or camptothecin (Cpt) and Top2 inhibitors mitoxantrone (Mxt) and etoposide (Etp). Analysis of cells by multiparameter laser scanning cytometry following treatment with Tpt or Cpt revealed that only DNA replicating cells showed induction of ?H2AX and a strong correlation between DNA replication and formation of DSBs (r = 0.86). In cells treated with Mxt or Etp, the correlation was weaker (r = 0.52 and 0.64). In addition, both Mtx and Etp caused induction of ?H2AX in cells not replicating DNA. Confocal imaging of nuclei of cells treated with Tpt revealed the presence of ?H2AX foci predominantly in DNA replicating cells and close association and co-localization of ?H2AX foci with DNA replication sites. In cells treated with Mxt or Etp, the ?H2AX foci were induced in DNA replicating as well as non-replicating cells but the close association between a large proportion of ?H2AX foci and DNA replication sites was also apparent. The data are consistent with the view that collision of DNA replication forks with cleavable Top1-DNA complexes stabilized by Tpt/Cpt is the sole cause of induction of DSBs. Additional mechanisms such as involvement of transcription and/or generation of oxidative stress may contribute to DSBs induction by Mxt and Etp. The confocal analysis of the association between DNA replication sites and the sites of DSBs (?H2AX foci) opens a new approach for mechanistic studies of the involvement of DNA replication in induction of DNA damage.

Project description:BACKGROUND: Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. METHODS: We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with ?H2AX staining and replication activity with molecular combing. RESULTS: In SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells. CONCLUSIONS: These results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.

Dataset Information

Novel Fluoroindenoisoquinoline Non-Camptothecin Topoisomerase I Inhibitors.

Publications

Novel Fluoroindenoisoquinoline Non-Camptothecin Topoisomerase I Inhibitors.

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