The indenoisoquinoline LMP517: a novel antitumor agent targeting both TOP1 and TOP2

Abstract

The camptothecin derivatives topoisomerase I (TOP1) inhibitors, irinotecan and topotecan are US-FDA approved for the treatment of colorectal, ovarian, lung and breast cancers. Because of the chemical instability of camptothecins, short plasma half-life, drug efflux by the multidrug-resistance ABC transporters and the severe diarrhea produced by irinotecan, indenoisoquinoline TOP1 inhibitors (LMP400, LMP776 and LMP744), which overcome these limitations, have been developed and are in clinical development. Further modifications of the indenoisoquinolines led to the fluoroindenoisoquinolines; one of which, LMP517, is the focus of the present study. LMP517 showed better antitumor activity than its parent compound LMP744 against H82 (Small Cell Lung Cancer) xenografts. Genetic analyses in DT40 cells showed a dual TOP1 and TOP2 signature with selectivity of LMP517 for DNA repair-deficient tyrosyl DNA phosphodiesterase 2 (TDP2)- and Ku70-knockout cells. RADAR assays revealed that LMP517, and to a lesser extent LMP744, induce TOP2 cleavage complexes (TOP2ccs) in addition to TOP1ccs. Histone γH2AX detection showed that, unlike classical TOP1 inhibitors, LMP517 targets cells independently of their position in the cell cycle. Our study establishes LMP517 as a dual TOP1 and TOP2 inhibitor with therapeutic potential.

Introduction

Topoisomerases play critical roles in genome organization and stability. They remove supercoils and DNA intertwining induced by essential DNA processes including replication, transcription and chromatin remodeling (1–4). Topoisomerase I (TOP1) induces single-strand breaks, allowing the DNA to rotate on itself, while topoisomerases II (TOP2α and TOP2β) induce DNA double-strand breaks (DSBs), allowing a DNA helix to pass through another. Both classes of enzyme act by forming transient catalytic intermediates called topoisomerase cleavage complexes (TOPcc). Rejoining of the DNA strand(s) restores DNA integrity. Unresolved TOPccs lead to protein-linked DNA single- or double-strand breaks that are lethal as cells undergo replication or transcription (2). For this reason, TOP1 and TOP2 inhibitors are widely used for cancer treatment. TOP1ccs are targeted by the clinical camptothecin (CPT) derivatives topotecan and irinotecan, and camptothecin derivatives are the only chemical class of TOP1 inhibitor approved by the FDA (5). TOP2ccs are targeted by etoposide, doxorubicin (as well as other anthracycline derivatives) and mitoxantrone (4,6). Although TOP1 and TOP2 inhibitors are highly specific for their respective enzyme targets (TOP1 and TOP2, respectively), they share the same mechanism of action (7); they poison TOPccs by impeding DNA resealing as the drugs stack with the base pairs flanking the break made by the topoisomerases while making amino acid hydrogen bonds with the topoisomerases. This mode of inhibition, termed interfacial inhibition, was first discovered for the topoisomerase inhibitors and has been generalized to other natural products targeting macromolecular interfaces (7).

Following the trapping of TOPccs by topoisomerase inhibitors, cells must repair the irreversible topoisomerase-linked DNA breaks induced when replication and/or transcription machineries collide with the TOPccs. Tyrosyl DNA phosphodiesterases 1 and 2 (TDP1 and TDP2) remove the topoisomerase polypeptides covalently linked to the ends of the breaks (8–10); and the associated DSBs are repaired by two main pathways: homologous recombination (HR) and non-homologous-end-joining (NHEJ). HR primarily repairs TOP1ccs during S-phase when replicated DNA is available as repair template (11–14). By contrast, NHEJ relies on Ku70/80 and DNA-dependent protein kinase complexes (DNA-PKcs), which directly joins the broken ends in non-replicative cells. NHEJ is the primary repair pathway for TOP2ccs following their processing by tyrosyl-DNA phosphodiesterase 2 (TDP2) (8,14,15). Notably, inactivation of NHEJ by knocking-out Ku70 in chicken lymphoblastoid DT40 cells increases resistance to camptothecins (14). Indicating that toxic replication intermediates are generated by NHEJ in response to TOP1ccs while NHEJ effectively repairs TOP2ccs (14,15).

The indenoisoquinolines are new TOP1 inhibitors, that have been developed to overcome the limitations of camptothecins (5,16). Three indenoisoquinolines derivatives are in Phase 1/Phase 2 clinical trials, LMP400 (indotecan), LMP776 (indimitecan) and LMP744 (5,17,18). Unlike the camptothecins, the indenoisoquinolines are chemically stable, induce persistent TOP1ccs, are not substrates of drug efflux by ABC transporters and have an extended plasma half-life (17,18). Moreover, unlike irinotecan they do not cause severe diarrhea (5,17,18). We recently reported the anticancer activities of second generation indenoisoquinolines, the fluoroindenoisoquinolines, in which the addition of a fluorine at position 3 replaces the methoxy groups at position 2, 3 of the clinical indenoisoquinolines (19–21) (Figure 1A). A recent study we showed that among these fluoroindenoisoquinolines, LMP517 generated the most DNA damage, which correlated with its potency for trapping TOP1cc (19). The aim of the present study was to identify the molecular mechanisms explaining the enhanced LMP517 potency and to compare it with its parent clinical derivative LMP744.

Figure 1: Antitumoral efficacy of LMP517 vs its parent compound LMP744.

A. Chemical structure of the fluoroindenoisoquinoline LMP517 and its parent compound LMP744. B. Antitumor efficacy of LMP517 vs LMP744 at 10 mg/kg with tumor volume in mm³ as y axis and time in days as the x axis. Mice were treated for one (left panel) or two cycles (right panel) of 5 days (with 2 days of rest; arrows under the plot). Bars: SEMs (n = 10 for all groups). P values: *, <0.05; **, <0.005. C. Kaplan Meyer representation of the experiment described in panel B.

Material and methods

Cells and reagents

The DT40 chicken lymphoma and the TK6 human lymphoma cell lines were obtained from Dr. Shunichi Takeda, Laboratory of Radiation Genetics, Graduate School of Medicine in Kyoto University (Kyoto, Japan). All the mutant cell lines were previously authenticated by Southern blotting and/or RT-PCR and/or Western blotting. DT40 cells were cultured at 37°C with 5% CO2 in RPMI-1640 medium (11875–093, Invitrogen, Carlsbad, CA) supplemented with 1% chicken serum (16110–082, Invitrogen, Carlsbad, CA), 10 nM β-mercaptoethanol (M-3148, Sigma-Aldrich, St. Louis, MO), penicillin-streptomycin (15140–122, Invitrogen), and 10% fetal bovine serum (100–106, Gemini Bio-Products, West Sacramento, CA). All cell lines were kept for 45 days maximum after thawing and tested for mycoplasma with MycoAlert™ Mycoplasma Detection Kit (Lonza). H82 cells were obtained from the NCI repository. For the HCT116 FUCCI cells, we obtained mKO2-hCdt1(30/120)/pCSII-EF-MCS and mAG-hGeminin(1/110)/pCSII-EF-MCS from Dr. Hiroyuki Miyoshi and Dr. Atsushi Miyawaki (RIKEN Institute) (22). HEK293T cells were transfected with either construct using Lenti-X packaging system (Takara). The harvested lentiviral particles from both constructs were used to coinfect HCT116 cells, which were then FACS sorted for dual-positive cells (FUCCI). The brightest single-clone was selected and used for this study.

Topotecan, camptothecin, etoposide and LMP744 were provided by the NCI Drug Developmental Therapeutics Program (DTP). LMP517 was synthesized in the Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (21).

Survival Assays

DT40 cells were seeded at 5,000 cells per well in 96-well white plates (#6005680 Perkin Elmer Life Sciences), and exposed to the indicated concentrations of LMP744, LMP517, etoposide or topotecan for 72 hours, performed at least 3 times with in-experiment triplicates. Cellular viability was determined using ATPlite 1-step kits (PerkinElmer). ATP levels of untreated cells were defined as 100%. Percent viability was defined as: [(ATP in treated cells) / (ATP in untreated cells)] × 100.

Mouse antitumor experiments

Athymic nude mice (nu/nu, female, 20–25g, 8–12-week-old) from Charles River, were transplanted with 5 million H82 human small cell lung cancer cells. When the tumor volume reached between 100 and 125 mm³, the animals were randomized into treatment groups based on tumor volume and body weights using the StudyLog software. Ten mice for LMP744 and vehicle arm and ten mice for LMP517 were used. The animals were treated with either LMP744 (10 mg/kg) administered intravenous (i.v.) or with LMP517 (10 mg/kg) administered by intravenous (i.v.) push via tail vein once a day for 5 consecutive days once (1 cycle) or twice (2 cycles). LMP744 and LMP517 were dissolved in 10 mM citric acid, 5% dextrose. The three axes (millimeters) of tumors were measured with a caliper to calculate tumor volume. Measurement were made every 3 or 4 days. Maximum allowable weight loss tolerated of 20% was never reached. Mice were euthanized if tumors presented necrosis or exceeded 20 mm in diameter.

Frederick National Laboratory is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals” (National Research Council; 2011; National Academies Press; Washington, D.C.).

Recombinant proteins

Human TOP1 was purified from baculovirus as described (23). Human TOP2α was purified from yeast strains JEL1 top1Δ transformed with 12-URA-B 6×His-hTOP2α. Induction of TOP2 by galactose as described (24). Yeast cells were lysed in equilibration buffer (300 mM KCl, 10 mM imidazole, 20mM Tris HCl pH 7.7, 10% glycerol and protease inhibitor cocktail (Sigma Aldrich, Cat# P8215)) by glass bead homogenization. Lysates were incubated with Ni-NTA resin and washed using wash buffer #1 (300 mM KCL, 30 mM imidazole, 20M Tris HCl pH 7.7, 10% glycerol and protease inhibitors) then wash buffer #2 (150 mM KCl, 30 mM imidazole, 20 mM Tris HCl pH 7.7, 10% glycerol and protease inhibitor cocktail). hTOP2α and β were eluted on a Poly-Prep chromatography column (Bio-Rad, Cat# 7311550) with elution buffer (150 mM KCl, 300 mM imidazole, 20 mM Tris HCl pH 7.7, 10% glycerol and protease inhibitors). The peak protein fractions were dialyzed in dialysis buffer (750mM KCl, 50 mM Tris HCl pH 7.7, 20% glycerol, 0.1 mM EDTA and 0.5mM DTT) and His tag was removed using TEV protease

TOP1 and TOP2 cleavage assay

Topoisomerase plasmid cleavage assay was carried out as described (25). In brief, 250 ng of pBR322 supercoiled plasmid DNA and 1 μg of recombinant TOP1 or TOP2α were incubated in 20 μl TOP1 reaction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM DTT, 10 mM EDTA and 5 μg/ml acetylated bovine serum albumin (BSA) or in 20 μl TOP2 reaction buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 150 mM KCL, 1mM ATP, 1mM EDTA, 1 mM DTT and 30 μg/ml acetylated BSA (TOP2) in the presence of various concentrations of drugs at 37°C for 30 min. The reactions were terminated by adding 2 μl 10% SDS, 0.75 μl of 500 mM EDTA, pH 8.0, and 2 μl 0.8 mg/ml proteinase K and further incubated for 2 hours at 30°C. DNA samples were electrophoresed in 0.8% agarose gels containing 0.5 μg/ml ethidium bromide (EtBr).

Histone γH2AX detection

HCT116 and HCT116 FUCCI cells were plated at 50,000 cells per well in 4-well chamber slides, incubated for 72 hours and then treated with drugs. After treatment, slides were fixed in paraformaldehyde 4 % for 10 min at room temperature (RT) then permeabilized with 0.02 % Triton X-100 for 5 min at RT. After rinsing with PBS, the slides were blocked for 1 hour in Phosphate-Buffered Saline-BSA 8% at room temperature and incubated for 2 hours with primary antibody at RT (Abcam ab22551 mouse anti-γH2AX). After washing with PBS, cells were incubated with secondary antibody for 1 hour at room temperature, washed with PBS and mounted with Vectashield with DAPI (Vector Laboratories). The primary antibody for histone γH2AX was mouse monoclonal anti-γH2AX Ser140 antibody, clone JBW301 (Abcam: ab22551) and the secondary antibody was a chicken anti-mouse Alexa 647 (A21463 Invitrogen).

Results

The fluoroindenoisoquinoline LMP517 has an improved antitumoral activity over LMP744

To test the antitumoral activity of LMP517, we xenografted nude mice with the small cell lung cancer cell line H82 (19) and compared LMP517 with its parent indenoisoquinoline LMP744 in one or two treatment cycles (5 days of treatment per cycle). LMP744 and LMP517 (chemical structures in Figure 1A) both had an MTD of 10 mg/kg. At this concentration, only LMP517 induced a reduction in tumor growth (Figure 1B) with marginal body weight lost (Supplementary Figure 1). LMP517 treatment resulted in an average survival of 30 days for 1 cycle and 36 days for the 2 cycles protocol vs 19 days for both one cycle and two cycle of LMP744 (Figure 1C). This result demonstrates the increased efficacy of the fluoroindenoisoquinoline LMP517 over LMP744 in this model system. The following experiments were performed to elucidate the molecular pharmacology of LMP517 and understand why it is more potent than its parent counterpart LMP744.

LMP517 displays a comparable phenotype to etoposide in isogenic TDP- and Ku70-knockout DT40 cells

Cells have redundant pathways to excise unresolved covalent complexes between topoisomerases and DNA (2). The most specific pathways involve TDP1 and TDP2, which hydrolyze the covalent bond between the tyrosine of the topoisomerase polypeptides (TOP1 and TOP2 respectively) and the DNA backbone. TDP1 inactivation inhibits the repair of TOP1cc and increases sensitivity to CPT, and to a lesser extent to etoposide (26,27). Conversely, TDP2 inactivation increases TOP2cc accumulation and sensitizes cells to etoposide (9,28). Here we used DT40 TDP1- or TDP2-deficient cells (referred as DT40 tdp1 and DT40 tdp2, respectively) (27,28) to determine the impact of TDP1 and TDP2 on the activity of LMP517.

As expected, DT40 tdp1 cells were hypersensitive to CPT with an IC50 of 2 nM vs 15 nM for DT40 WT (Figure 2A). DT40 tdp2 cells were also sensitive to CPT, consistent with the role of TDP2 as secondary repair pathway for TOP1ccs (9,28) (IC50: 5 nM for DT40 Tdp2) (Figure 2A). LMP744 showed a similar sensitivity pattern as CPT with an IC50 of 6 nM vs 25 nM for DT40 WT cells and 15 nM for DT40 tdp2 cells.

Figure 2: LMP517 is selective for cells with TDP1, TDP2 and Ku70 deficiencies.

A. DT40 WT, DT40 TDP1 knockout (tdp1) and DT40 TDP2 knockout (tdp2) cell viability curves after 72 hours treatment with increasing concentration of etoposide, CPT, LMP517 or LMP744. B. DT40 WT and DT40 Ku70 (XRCC6) knockout cell viability curves after 72 hours treatment with increasing concentration of etoposide, topotecan, LMP776, LMP517 or LMP744. Bars: standard deviation (SD) between three independent experiments. Statistically significant differences (p values < 0.05) between WT and KO cells are annotated with black stars.

Conversely, DT40 tdp2 cells were most sensitive to etoposide (IC50: 28 nM vs WT >125 nM) and tdp1 cells were also hypersensitive to etoposide, consistent with a role of TDP1 as alternative pathway for excising 5’-phosphotyrosyl adducts (27,29). Notably, among the three cells lines, tdp2 cells showed the highest sensitivity to LMP517 (IC50s: 11 nM, 18 nM and 32 nM in tdp2, tdp1 and WT cells, respectively) (Figure 2A). This unexpected result suggested that LMP517 might act like etoposide (by TOP2 poisoning) in addition to being a TOP1 poison.

Repair of TOP2cc by TDP2 generates direct substrate for NHEJ (2,8,9,15) whereas NHEJ exerts the opposite effect in response to TOP1cc by initiating toxic NHEJ (14). We used this difference to further determine whether LMP517 could be acting as a TOP2 poison (Figure 2B). DT40 Ku70-deficient cells showed the expected resistance to topotecan while being hypersensitive to etoposide, LMP517 and LMP744 (Figure 2B). The phenotypes observed in TDP1-, TDP2- and Ku-deficient DT40 cells suggest that LMP517 could act as a TOP2 poison.

LMP517 is a dual TOP1 and TOP2 inhibitor in biochemical assays and in HCT116 and TK6 cells

To directly determine the effects of LMP517 on TOP1 and TOP2, we performed biochemical assays with negatively supercoiled plasmid pBR322 in the presence of recombinant TOP1 or TOP2, using CPT and etoposide as positive controls. As expected, both LMP517 and LMP744 trapped TOP1ccs (Figure 3A). Indeed, when TOP1 was applied to supercoiled DNA (Sc) in the presence of LMP744 or LMP517, we observed extensive accumulation of nicked DNA, as in the case of CPT (Figure 3A, lane C) (30). Notably, in the presence of TOP2, we observed both linearized and nicked DNA with LMP517 or LMP744, which are characteristic results obtained with trapping of TOP2 by etoposide (Figure 3A, lane E at right) (31). The induction of nicked and linear DNA species is consistent with TOP2 trapping by LMP517 and LMP744.

Figure 3: LMP517 induces TOP1cc and TOP2cc both with purified enzymes and in human cancer cells.

A. Recombinant TOP1 and TOP2 applied to the plasmid pBR322 in the presence of CPT (C), etoposide (E), LMP744 or LMP517. B. TOP1, TOP2α and TOP2β cleavage complexes detected in lymphoblast TK6 cells after treatment with CPT, etoposide (ETP), LMP517 or LMP744. C. TOP1, TOP2α and TOP2β cleavage complexes detected in colon cancer HCT116 cells after treatment with CPT, ETP, LMP517 or LMP744.

To test whether LMP517 also acts as a dual TOP1 and TOP2 inhibitor in cells, we treated human lymphoblast TK6 and colon carcinoma HCT116 cells for 1 hour with LMP517, CPT, etoposide or LMP744 and detected TOP1cc and TOP2cc by Radar assay (32). Figure 3B shows that LMP517 induces both TOP2α and TOP2βccs in addition to TOP1ccs in both cell line tested (Figure 3B–C). In HCT116, we also tested the indenoisoquinoline LMP744 and found that it also induced TOP2ccs, although to a lesser extent than LMP517 (Figure 3C).

Together these results demonstrate that LMP517 is a dual inhibitor of TOP1 and TOP2 (both TOP2α and TOP2β) in biochemical and cellular assays.

LMP517 induces histone γH2AX in both G1- and S/G2-cell cycle phase cells, consistent with its dual activity against TOP1 and TOP2

It is well established that TOP1ccs trapped by camptothecins induce DNA damage primarily in replicative cells (S-phase) (2,33) while etoposide targets cells in all phase of the cell cycle by trapping both TOP2α and TOP2β (2,34). To test the induction of DNA damage by LMP517 and whether such damage is dependent on replication, we used HCT116 FUCCI cells, which express tagged cell-cycle reporter peptides, mAzami-Green-tagged N-terminus of geminin (green channel) or mKusabira-Orange2-tagged N-terminus of CDT1 (red channel). This dual labeling allows the detection of cells in the S/G2 or G1 phase, respectively (22).

We treated HCT116 FUCCI cells with CPT (1 μM), etoposide (50 μM), LMP517 (1 μM) or LMP744 (1 μM) for 1 hour and detected DNA damage with γH2AX antibodies (Figure 4A). LMP517 induced DNA damage starting from 50 nM with extensive γH2AX at 1 μM (Figure 4A and Supplementary Figure 2). When treated with CPT, HCT116 cells were divided in two groups: γH2AX-positive and γH2AX-negative (Figure 4A). γH2AX-negative cells were mainly mKO2-positive cells (G1 phase cells) (35) (Supplementary Figure 3). By contrast, etoposide treatment induced γH2AX signal in nearly all cells (Figure 4 and Supplementary Figure 3), demonstrating that etoposide targets cells regardless of their cell cycle phase. LMP744 induced γH2AX-positive cells, similar to CPT, mainly in S/G2-phase cells (GFP positive; Figure 4B and Supplementary Figure 3). This result is consistent with the conclusion that LMP744 acts primarily as a TOP1 inhibitor in HCT116 cells (36).

Figure 4: LMP517 induces DNA damage in all phases of the cell cycle like etoposide.

A. Total γH2AX signal after 1 hour treatments with CPT (1 μM), etoposide (ETP) (50 μM), LMP517 (1 μM) or LMP744 (1 μM). Dots correspond to individual cells (n = 888 for each of the 5 sets). The shaded area corresponds to γH2AX-positive cells defined by a γH2AX signal above a threshold set at 24,000 AU (arbitrary unit) (5% of the non-treated (NT) cells). Dose-response for LMP517 at lower concentrations is included in Supplementary Figure 2. B. Left panel: Quantification of the ratio of γH2AX-positive G1-cells over the total G1-cells after 1 hour treatments with CPT, etoposide, LMP744 or LMP517. Right panel: Quantification of the ratio of γH2AX-positive S/G2-cells over the total S/G2-cells after 1 hour treatments with CPT, etoposide, LMP744 or LMP517. Bars: SD between three independent experiments. Significance between CPT and other treatment is displayed. P values: ***, <0.0005; ****<0.00005.

Notably, cells treated with LMP517 did not show a bimodal γH2AX signal distribution (Figure 4A). Analyses of the GFP and RFP signals and cell cycle distribution showed that, like etoposide, LMP517 induced γH2AX signal in G1-phase cells (88% and 89% of total G1-phase cells, respectively) (Figure 4B, left panel). Only a small percentage of G1-phase cells showed γH2AX signal when treated with CPT or LMP744 (29% and 23% of total G1-phase cells, respectively) (Figure 4B, left panel). These results demonstrate that LMP517, like etoposide and contrary to CPT and LMP744, induces DNA damage in cells in G1-phase of the cell cycle, consistent with cellular damage induced by TOP2 trapping.

Schlafen 11 (SLFN11), NHEJ and BRCAness are determinants of response to LMP517

SLFN11 is an established dominant determinant of response to both TOP1 and TOP2 inhibitors (37–39). We recently reported, in isogenic human leukemia CCRF-CEM cells, an increased resistance to LMP517 in the absence of SLFN11 expression (19). To determine the predictive value of SLFN11 expression in non-isogenic cells, we analyzed the activity of LMP517 across the NCI-60 cell line panel using CellMinerCDB (40,41) (Figure 5A). A significant correlation was observed between the activity of LMP517 and SLNF11 expression. However, some cells seem to respond in the absence of SLFN11 and other, SLFN11-positive, were not hypersensitive to LMP517, indicating other cellular determinants of response. Using the CellMinerCDB tools (40,41) (http://discover.nci.nih.gov/cellminercdb), we identified two other determinants of response: XRCC6 (KU70) copy number and Aprataxin and PNKP Like Factor (APLF) expression, which were both negatively correlated to LMP517 response (Supplementary Figure 4A and B). These results are consistent with those shown in Figure 2B, demonstrating that NHEJ plays a major role in LMP517 cytotoxicity. The predicted response with all three determinants of response (SLFN11, Ku70 and APLF) is represented in Figure 5B. Figure 5C shows the correlation between SLFN11 expression, XRCC6 DNA copy number and APLF expression with LMP517 using the CellMinerCDB “Multivariate analyses” tool (http://discover.nci.nih.gov/cellminercdb) (41).

Figure 5: SLFN11 and homologous recombination deficiencies (HRD) are determinants of response to LMP517.

A. Correlation between SLFN11 expression and the antiproliferative activity of LMP517 across the NCI-60. Each dot corresponds to a cell line (see key to the right and http://discover.nci.nih.gov/cellminercdb). B. Predicted response of LMP517 across the NCI-60 by including SLFN11 expression, XRCC6 (KU70) copy number and APLF expression [CellMinerCDB “Multivariate Analyses” (41). C. Relationship between SLFN11 expression, Ku70 (XRCC6) copy number and APLF expression and the antiproliferative activity of LMP517 across the NCI-60 cell lines. Cell lines (individual columns) are ranked by drug sensitivity. Color scale: red represents high drug sensitivity and high gene expression. Blue is the opposite. D. Cell viability of DT40 WT, BRCA1-, BRCA2- and PALB2-knockout cells after 72 hours treatments with increasing concentration of LMP517. Bars: standard deviation (SD) between three independent experiments. Statistically significant differences (p values < 0.05) between WT and KO cells are annotated with black stars. E. Cell viability of DT40 WT and BRCA1-knockout cells after 72 hours treatments with increasing concentration of olaparib without or with 3 nM of LMP517. Bars: SD between three independent experiments.

We also recently demonstrated a selectivity of the first generation indenoisoquinolines for homologous recombination deficient (HRD; BRCA1, BRCA2 and PALB2 deficient) cells and their synergistic combination with the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib (11). To determine whether LMP517 was also selective for BRCA1-, BRCA2- and PALB2-deficient cells, we tested isogenic DT40 cells inactivated for those genes. Hypersensitivity of DT40 cells lacking BRCA1, BRCA2 and PALB2 was observed (Figure 5D). Next, we combined LMP517 with olaparib (Figure 5E), and found additive effects but no synergism between the two drugs. This result is consistent with previous studies showing additive activities but no synergism between TOP2 inhibitors and PARP inhibitors (42), consistent with the conclusion that TOP2 is a prominent cellular target of LMP517.

Discussion

Our results demonstrate that the fluoroindenoisoquinoline LMP517 (19,20) displays improved antitumoral efficacy in murine H82 (SCLC) xenograft compared to its parent compound LMP744 (see Figure 1), which was recently introduced in Phase 1 clinical trials based on its antitumor activity in dog lymphomas (18). Hence, LMP517 has the potential to be developed as second generation indenoisoquinoline inhibitor. Further studies are warranted to determine LMP517 potency against other models and determine its clinical potential.

We show that LMP517 acts by dual targeting of TOP1 and TOP2 at nanomolar concentrations. This conclusion is based on our current results. First, LMP517 exhibits selective antiproliferative activity toward TDP2-defective cells compared to TDP1-deficient cells and also toward Ku70-defective cells (see Figure 2) like the TOP2 inhibitor etoposide and unlike the TOP1 inhibitors: CPT, topotecan, LMP400 (indotecan) and LMP776 (indimitecan) (5,14). Secondly, LMP517 traps both TOP1 and TOP2 cleavage complexes in biochemical assays and in human colon carcinoma HCT116 and lymphoma TK6 cells (see Figure 3). Thirdly, LMP517 induces DNA damage at nanomolar concentrations (19) in non-replicating G1-phase cells, similar to etoposide and distinct from CPT (34,43,44) (see Figure 4). And finally, similar to the other established TOP1 and TOP2 inhibitors, SLFN11 (37,39) and BRCAness (HRD) (11,14) are dominant determinants of response to LMP517 (see Figure 5).

Comparing LMP517 to its parent indenoisoquinoline LMP744 reveals that the addition of a fluorine and removal of the methoxy groups in the A-ring (see Figure 1) increased the ability of LMP517 to target TOP2. In our recent study of LMP517, we also noticed the ability of LMP517 to intercalate into DNA at high concentration as it has been previously established for LMP744 (36). This intercalating effect of LMP517 and LMP744 may explain the inhibitory effect on TOP2 as several chemical classes of DNA intercalating agents effectively trap TOP2ccs (45–47) by stacking with the base pairs flanking the TOP2cc, including the clinically used anthracyclines (doxorubicin, daunorubicin, epirubicin, idarubicin), mitoxantrone and amsacrine, as well as ellipticine derivatives (6,43). Indeed, DNA intercalation of drugs and ligands such as benzo[a]pyrene carcinogens at a TOP2 cleavage site can directly block the DNA religation by TOP2 (48),(7,49).

Our results provide groundwork for the potential clinical development of LMP517 as a dual TOP1 and TOP2 inhibitor and potentially as a tumor-targeted delivery payload (5,50).