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. 2018 Aug;17(8):1694-1704.
doi: 10.1158/1535-7163.MCT-18-0028. Epub 2018 May 10.

Novel Fluoroindenoisoquinoline Non-Camptothecin Topoisomerase I Inhibitors

Laetitia Marzi  1 , Keli Agama  1 , Junko Murai  1 , Simone Difilippantonio  2 , Amy James  2 , Cody J Peer  3 , William D Figg  3 , Daniel Beck  4 , Mohamed S A Elsayed  4 , Mark Cushman  4 , Yves Pommier  5
Affiliations

Novel Fluoroindenoisoquinoline Non-Camptothecin Topoisomerase I Inhibitors

Laetitia Marzi et al. Mol Cancer Ther. 2018 Aug.

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 To further improve metabolic stability, their methoxy groups have been replaced by fluorine, as in the fluoroindenoisoquinolines NSC 781517 (LMP517), NSC 779135 (LMP135), and NSC 779134 (LMP134).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 (γH2AX) compared with their parent compounds LMP744 and LMP776 in leukemia CCRF-CEM and colon carcinoma HCT116 cells. The fluoroindenoisoquinolines induced TOP1cc and γH2AX at nanomolar concentrations, and at higher levels than the parent indenoisoquinolines. The fluoroindenoisoquinoline LMP135 showed greater antitumor activity than topotecan in small-cell lung cancer cell H82 xenografts. It was also more potent than topotecan in the NCI-60 cancer cell line panel. Bioinformatics tools (http://discover.nci.nih.gov/cellminercdb) were used to investigate the following: (i) the correlations of fluoroindenoisoquinolines activity with other drugs, and (ii) genomic determinants of response in the NCI-60. The activity of the fluoroindenoisoquinolines was mostly correlated with camptothecin derivatives and the parent indenoisoquinolines, consistent with TOP1 targeting. Genomic analyses and activity assays in CCRF-CEM SLFN11-deleted cells showed that SLFN11 expression is a dominant determinant of response to LMP135. This study shows the potential value of the fluoroindenoisoquinolines for further development as novel anticancer agents targeting TOP1. Mol Cancer Ther; 17(8); 1694-704. ©2018 AACR.

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. Fluoroindenoisoquinolines are potent inhibitors of human TOP1
A, Structures of the clinical first generation indenoisoquinolines (LMP744, LMP776 and LMP400) and of the three fluoroindenoisoquinolines (LMP517, LMP135 and LMP134). The methoxy groups of LMP744 and LMP776 are replaced by a fluorine substituent in LMP517 and LMP135, respectively. B, Representative gel showing TOP1cc-associated DNA breaks induced by the indicated compounds. The substrate DNA (3′ end-labeled PvuII/HindIII fragment of pBluescript SK (–) phagemid DNA (pSK), lane 1) was reacted with recombinant TOP1 in the absence of drug (lane 2) or in the presence of the indicated concentrations (micromolar) of CPT, LMP400 (lanes 20 to 23), LMP744 (lanes 4 to 7), LMP776 (lanes 12 to 15), LMP517 (lanes 8 to 11), LMP134 (lanes 24 to 27) and LMP135 (lanes 16 to 19). Reactions were performed at 30°C for 20 min and stopped by adding 0.5% SDS. DNA fragments were separated in 16% denaturing polyacrylamide gels. Cleavage sites are indicated on the right. The sequence of the DNA substrate and TOP1cc sites are shown at the bottom. The asterisk indicates the position of the 32P-end-labelling.
Figure 2
Figure 2. Cellular TOP1cc induced by the fluoroindenoisoquinolines in human cancer cells
A, TOP1cc detected by ICE bioassay. Human leukemia CCRF-CEM and colon carcinoma HCT116 cells were treated with the indicated drugs (1 μM) for 1 h at 37°C. DNA-containing fractions were blotted and TOP1 was detected using TOP1 C21 monoclonal antibody. B, Quantitative analysis of TOP1cc detected as DNA-protein complexes (DPC) by alkaline elution. Human leukemia CCRF-CEM cells were treated for 1 h at 37°C as indicated.
Figure 3
Figure 3. Cellular DNA damage induced by the fluoroindenoisoquinolines
A, Representative immunofluorescence confocal microscopy images. HCT116 cells were treated with the indicated drugs (1 μM for 1 h at 37°C). Following fixation, cells were stained for histone γH2AX and DAPI. B, Quantitative analysis of γH2AX as a function of time. Cells were treated with 1 μM of topotecan, LMP744, LMP517, LMP134 and LMP135 for the indicated times (h). γH2AX signal intensities of 50 cells (each individual cell represented as a dot) were measured by image-J. Same sized area signal was quantified in each nucleus and plotted for each condition. C and D, Quantitative analysis of γH2AX induction as a function of drug concentration. Cells were treated as indicated for 1 h. γH2AX signal intensities of 70 cells were measured by image-J as in panel B. Signals below the horizontal dotted lines are within background signal for untreated cells.
Figure 4
Figure 4. Persistent DNA damage and TOP1cc in cells treated with the fluoroindenoisoquinolines
A, Quantitative analysis of TOP1cc after drug removal in CCRF-CEM cells. Cells were treated with 1 μM CPT, LMP744, LMP776, LMP517, LMP134 or LMP135 for 1 h at 37°C. Drugs were removed and cells were grown for another hour in drug-free medium (R1). TOP1cc were determined as DPC by alkaline elution. Percent reversal for each drug is indicated in the text. B, Quantitative analysis of γH2AX persistence 6 h after drug removal. HCT116 were treated for 1 h with 1 μM drug concentration, and either fixed at that point (1 h) or grown for an additional 6 h in drug-free medium (R). γH2AX signal intensities of 50 cells (each individual cell represented as a dot/circle) were measured by image-J. Same sized area signal was quantified in each nucleus. Percent reversal for each drug is indicated in the text. Points below the horizontal dotted lines are γH2AX signals within background signal for untreated cells.
Figure 5
Figure 5. LMP135 is the most potent fluoroindenoisoquinoline in the NCI-60 and shows antitumor activity in small cell lung cancer H82 xenografts model
A, Comparison of the NCI-60 responses to the indicated TOP1 inhibitors. Each dot represents the growth inhibitory 50% concentration (GI50) of a given NCI-60 cell line for the indicated drug. Data were obtained from the NCI Developmental Therapeutics Program (DTP). Average GI50 for all 60 cell lines is indicated below the graph. LMP744 and LMP776 were not tested under 10 nM and LMP135 under 5 nM, and in those cell lines in which the GI50 was less than the minimum concentration tested, the GI50 values were recorded as 10 and 5 nM, respectively. The “averages” in these cases therefore represent mean-graph midpoint (MGM) values rather than true GI50 averages. B, Comparison of the sensitivity of the NCI-60 cells to LMP135 vs. topotecan. GI50 of each drug for each cell line is indicated as well as equipotency curve. LMP135 is approximately 10-fold more potent than topotecan. C-D, Antitumor activity of LMP135 (20 mg/kg) compared to topotecan (1.5 mg/kg) represented as tumor volume (C) or as Kaplan Meyer curves (D). Bars represent standard deviations (n = 10 for vehicle and topotecan; n = 6 for LMP135).
Figure 6
Figure 6. SLFN11 is a dominant determinant of response to the fluoroindenoisoquinolines
A, Relationship between SLFN11 gene expression and the antiproliferative activity of LMP135 across the NCI-60 cancer cell lines. Gene expression of the drug efflux transporters ABCB1 and ABCG2 is shown for comparison. Cell lines (individual columns) are ranked by drug sensitivity. Color scale: red represents high drug sensitivity and high gene expression. Green is the opposite. B, Correlation between SLFN11 expression and the antiproliferative activity of LMP135 across the NCI-60. C, Causal relationship between SLFN11 expression and sensitivity to LMP135. Growth inhibition of CCRF-CEM parental and SLFN11-deleted cells (24) was measured by ATPlite® assay after treatment with the indicated drugs for 72 h.
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