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. Author manuscript; available in PMC: 2020 Apr 15.
Published in final edited form as: Clin Cancer Res. 2019 Aug 13;25(20):6206–6216. doi: 10.1158/1078-0432.CCR-19-0419

The indenoisoquinoline TOP1 inhibitors selectively target homologous recombination deficient- and Schlafen 11-positive cancer cells and synergize with olaparib

Laetitia Marzi 1, Ludmila Szabova 2, Melanie Gordon 2, Zoe Weaver Ohler 2, Shyam K Sharan 2,3, Michael L Beshiri 4, Moudjib Etemadi 1, Junko Murai 1, Kathleen Kelly 4, Yves Pommier 1,*
PMCID: PMC6801079  NIHMSID: NIHMS1535855  PMID: 31409613

Abstract

Purpose:

Irinotecan and topotecan are used to treat a variety of different cancers. However, they have limitations including chemical instability and severe side effects. To overcome these limitations, we developed the clinical indenoisoquinolines: LMP400 (indotecan), LMP776 (Indimitecan) and LMP744. The purpose of the study is to build the molecular rationale for phase 2 clinical trials.

Experimental design:

CellMinerCDB (http://discover.nci.nih.gov/cellminercdb) was used to mine the cancer cell lines genomic databases. The causality of Schlafen11 (SLFN11) was validated in isogenic cell lines. Because TOP1-mediated replication DNA damage is repaired by homologous recombination (HR), we tested the “synthetic lethality” of HR-deficient (HRD) cells. Survival and cell cycle alterations were performed after drug treatments in isogenic DT40, DLD1 and OVCAR cell lines with BRCA1, BRCA2 or PALB2 deficiencies and in organoids cultured from prostate cancer patient-derived xenografts with BRCA2 loss. We also used an ovarian orthotopic allograft model with BRCA1 loss to validate the efficacy of LMP400 and olaparib combination.

Results:

CellMinerCDB reveals that SLFN11, which kills cells undergoing replicative stress is a dominant drug determinant to the clinical indenoisoquinolines. In addition, BRCA1-, BRCA2- and PALB2-deficient cells were hypersensitive to the indenoisoquinolines. All three clinical indenoisoquinolines were also synergistic with olaparib, especially in the HRD cells. The synergy between LMP400 and olaparib was confirmed in the orthotopic allograft model harboring BRCA1 loss.

Conclusion:

Our results provide a rationale for molecularly designed clinical trials with the indenoisoquinolines as single agents and in combination with PARP inhibitors in HRD cancers expressing SLFN11.

Keywords: chemotherapy, DNA targeted drugs, DNA repair, topoisomerase inhibitors, biomarkers, PARP inhibitors

Introduction

Topoisomerases are essential enzymes in all organisms. They relax torsional strain and DNA supercoils, and they resolve DNA entanglements, thereby enabling life and genomic stability (1). Topoisomerase I (TOP1) is the target of camptothecin and its clinical derivatives irinotecan and topotecan. TOP1 relaxes DNA supercoiling by introducing transient DNA single-strand breaks, which are referred to as TOP1 cleavage complexes (TOP1ccs) (1). The selective trapping of TOP1ccs by camptothecins stabilizes TOP1ccs with TOP1 covalently attached to the 3’-end of the breaks. These stalled TOP1ccs lead to lethal DNA double-strand breaks when they produce collisions with DNA replication (1). Classical work in yeast revealed the critical importance for Rad52 and HR for the survival of cells with TOP1ccs (2,3). Thus, HRD synthetic lethality (4) was first demonstrated for camptothecins 30 years ago and confirmed more recently (5), well before the report of HRD synthetic lethality for poly(ADPribose)polymerase (PARP) inhibitors in 2005 (4,6,7).

BRCA1, BRCA2 and PALB2 (Partner And Localizer of BRCA2) are the most dominant tumor suppressor genes in familial breast and ovarian cancers. Antoniou et al. (8) calculated, in a combined analysis of 22 studies, that 39% of BRCA1 mutation carriers, are at risk of developing an ovarian cancer by the age of 70 and 11% for BRCA2 mutation carriers. Using the International BRCA½ Carrier Cohort Study (IBCCS), the Breast Cancer Family Registry (BCFR), and the Kathleen Cuningham Foundation Consortium for Research Into Familial Breast Cancer (kConFab), Kuchenbaecker et al. (9) found that the cumulative breast cancer risk to age 80 years was 72% (95% CI, 65%−79%) for BRCA1 and 69% (95% CI, 61%−77%) for BRCA2 carriers. BRCA1, BRCA2 and PALB2 act as key component of the homology directed repair pathways for the repair of DNA double-strand breaks (DSBs) and replication fork collapse with single-ended DSBs (10). Following its recruitment to the DNA breaks, BRCA1 induces 3’-end resection by recruiting the nucleases CtIP, Mre11, DNase2 and Exo1, and the scaffolding protein PALB2 bound to BRCA2. BRCA2 then mediates the displacement of the single-strand binding protein RPA from the 3’-DNA overhang by loading RAD51, which, in turn promotes strand invasion and replication fork stabilization (10). HRD is the focus of intense investigation with defects found across many cancer types including and not limited to prostate, pancreatic and kidney cancers.

Another dominant determinant of response to camptothecins is SLFN11 (Schlafen 11), which was identified using cancer cell line pharmacogenomic databases by correlative analyses of drug response and genomic parameters (11-14). Lack of SLFN11 expression, which occurs in approximately 50% of cancer cell lines and many clinical cancers (15,16), also drives resistance to PARP inhibitors (17) and to the widely used chemotherapeutic agents that lead to replication damage, including cisplatin and carboplatin, gemcitabine, etoposide, doxorubicin and hydroxyurea (12,18-20). SLFN11 acts as a “restriction factor” (21,22). It irreversibly arrests replicating cells undergoing replicative stress by binding to single-stranded DNA filaments coated with replication protein A (RPA). Binding of SLFN11 at extended single-stranded-RPA segments irreversibly blocks the replication helicase complex (15,17), alters chromatin structure (15) and interferes with HR completion (23).

Non-camptothecin TOP1 inhibitors have been developed since the discovery of the first indenoisoquinoline NSC314622 as a TOP1 inhibitor phenocopying the cytototoxicity profile of camptothecins in the NCI-60 cancer cell line database (24). More potent indenoisoquinolines have been subsequently developed to overcome the limitations of camptothecin and its clinical derivatives topotecan and irinotecan (25). Those limitations include the chemical instability of the camptothecin α-hydroxy-lactone E-ring (26), drug resistance through active drug efflux by ABCG2-ABCB1 efflux pumps (27,28), short plasma half-life of topotecan and irinotecan (29) and severe diarrheas for irinotecan (29). Three indenoisoquinolines are in clinical development LMP400 (NSC724998; indotecan), LMP744 (NSC706744; MJ-III-65) and LMP776 (NSC725776; indimitecan) (25,30-33). By contrast with the camptothecins, the clinical indenoisoquinolines are chemically stable (25), induce stable TOP1ccs at nanomolar concentrations (32,34), have extended serum half-life and do not produce diarrheas (30,33). Hence, the indenoisoquinoline TOP1 inhibitors overcome the limitations of camptothecins.

In this study, we tested whether BRCA1, BRCA2 and PALB2 deficiencies and expression of SLFN11 determine the antiproliferative activity of the clinical indenoisoquinolines LMP400, LMP744 and LMP776 in preclinical models. In addition, we examined whether their combination with the FDA-approved PARP inhibitor olaparib (Lynparza®) is synergistic.

Material and methods

Cells and reagents

The DT40 chicken lymphoma cell lines used in this study were obtained from the Laboratory of Radiation Genetics, Graduate School of Medicine in Kyoto University (Kyoto, Japan) in 2011-2012. 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). DLD1 (human colorectal carcinoma cell line) parental and BRCA2 deleted were obtained from Horizon Discovery and were cultured in RPMI 1640 medium supplemented with 10% FBS. The murine serous epithelial ovarian cancer cell lines, OVCAR WT, p53 −/−, inactivation of Rb-TS and OVCAR Brca1 −/−, p53 −/−, inactivation of Rb-TS, were obtained from the Center for Advanced Preclinical Research (CAPR), Center for Cancer Research, National Institute of Health, Frederick, MD, USA. They were cultured in DMEM-F12 (Sigma- Cat. # D8437) supplemented with 4% FBS, penicillin-streptomycin, Insulin/Transferrin/Selenium (Invitrogen-Cat. # 51300), 0.5 μg/ml Hydrocortisone (Sigma-Cat. # H0135), anti-anti (Gibco-Cat.15240–062) and 10 ng/ml EGF (Sigma- Cat. # E4127). CCRF-CEM cells, were cultured in RPMI-1640 medium supplemented with 10 % FBS. All cell lines were kept for 45 days maximum after thawing and tested for mycoplasma with MycoAlert™ Mycoplasma Detection Kit (Lonza).

Topotecan, LMP400, LMP744 and LMP776 were provided by the NCI Drug Developmental Therapeutics Program (DTP). Olaparib was purchased from Selleckchem. LMP517, LMP135 and LMP134 were synthesized in the Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University.

NCI-60 and GDSC analyses

The NCI-60 and the GDSC (Genomics and Drug Sensitivity in Cancer; Massachusetts General Hospital-Wellcome Sanger Institute) databases were mined with the publicly available CellMinerCDB website (http://discover.nci.nih.gov/cellminercdb) (35). For the NCI-60, the drug activity z-score is calculated based on the -log10 of the GI50 (drug concentration producing 50% cell growth inhibition) across repeats for the drug response (35). GI50 were generated from at least 2 independent repeats for each of the LMP compounds and 18 independent repeats for topotecan.

Survival Assays

DT40, DLD1, OVCAR or CCR-CEM cells were seeded at 5,000 (suspension cells) or 1,000 (adherent cells) cells per well in 96-well white plates (#6005680 Perkin Elmer Life Sciences), and exposed to the indicated concentrations of LMP400, LMP744, LMP776, olaparib, talazoparib, topotecan, LMP517, LMP135 or LMP134 for 72 hours in 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)] x 100. Synergism was assessed by CompuSyn Software. All other statistical analyses were conducted in Microsoft Excel.

Cell cycle analysis

DT40 cells were seeded at 1 million cells per well in a 6 well plate then treated for indicated concentrations of drugs for 24h. After centrifugation and wash with phosphate buffer saline (PBS) they were resuspended in a 50μg/ml propidium iodide solution (P4170, Sigma-Aldrich) supplemented with RNase A (10109169001, Sigma-Aldrich). Labelled cells were then read with a FACS (fluorescence-activated cell sorting) instrument (BD LSRFortessa) and the result were analyze using FlowJo software.

Processing and plating of PDX tumor samples

LuCaP PDX lines were described by Holly M. Nguyen et al (36) and processed and cultured into organoids as described in (37). Generation-one organoids were plated in replicates of five on 384-well plates: 4,000 cells in 20 μL of growth factor reduced Matrigel per well, overlaid with 40 μL of media. Twenty-four hours after plating, the media were replaced with fresh media containing LMP400. Media containing drug was changed twice per week over the course of 2 weeks. Cell viability was quantified with CellTiter Glo 3D. GraphPad Prism 7 was used to plot dose-response curves.

Mouse antitumor experiments

Efficacy studies were performed on a murine orthotopic allograft model for Brca1-deficient ovarian cancer (38). Tumor fragments were implanted under the right ovarian bursa in FVB female recipient mice. Mice were enrolled into treatment groups when their tumor size reached 50–300mm3 (determined by ultrasound imaging). Compounds for in vivo dosing were dissolved in 1 part 20 mM HCl/10 mM citric acid and 9 parts dextrose water (for LMP400 and LMP744) or in 1 part 10 mM citric acid/9 parts dextrose water (for LMP776). Olaparib was dissolved in DMSO at 50mg/ml and further diluted to 2.5mg/ml in 10% DMSO, 10% (2-hydroxypropyl)-b-cyclodextrin (Santa Cruz) in water. For the efficacy study comparing the three LMP compounds LMP400 was administered at 10mg/kg, one dose per day for 5 days followed by 2 days of rest, repeated for 5 weeks. LMP744 was administered at 20mg/kg for 2 continuous weeks of 5 days on, 2 days off, followed by one week of rest after which dosing continued for another 2 weeks (5 days on, 2 days off). LMP776 was administered at 10mg/kg for 3 continuous weeks of 5 days on, 2 days off, followed by one week of rest after which dosing continued for 5 days. Vehicle (1 part of 20 mM HCl/10 mM citric acid and 9 parts dextrose water) was administered for 5 days followed by 2 days off, repeated for 5 weeks. All compounds were administered i.v. at the volume of 10 ml/kg.

For the LMP400 and olaparib combination efficacy study, LMP400 was administered i.v. at 5 mg/kg twice a week (day 1 and day 4) and olaparib p.o. at 25 mg/kg four times a week (days 1, 2, 4 and 5). Both compounds were administered to mice at a volume of 10 ml/kg. Vehicle treated mice received vehicle for olaparib p.o. at 10ml/kg followed by vehicle for LMP400 i.v. at 10ml/kg.

Tumor volume changes were followed throughout the duration of study by weekly ultrasound imaging until mice reached the pre-determined end points: presence of tumor ≥ 2000mm3, and/or presence of hemorrhagic ascites, labored breathing, 20% of weight loss or any other clinical signs that caused distress to the animal. 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.).

Statistical analysis: p value calculated between best treatment and other arms. p value calculated between the combination and the single treatment arms (with color code). P values: 0.05 > *, 0.005 > **, 0.0005 > ***, 0.00005 > ****

Tissues were fixed in 10% neutral buffered formalin and processed for paraffin embedding and sectioning. H&E stained sections were evaluated by veterinarian pathologist.

Ultrasound imaging

A high frequency (40 MHz) Ultrasound imaging system (Vevo 2100, VisualSonics, Toronto, Canada), was used in these studies. 3D B-mode images were acquired using the MS-550S or for larger volumes the MS250 transducer: axial resolution of 40μm and 75 μm, respectively, and a lateral spatial resolution of 80μm and 165 μm, respectively. The acoustic focus was placed at the center of the ovary. Throughout the imaging session, mice were kept anesthetized with 1–2% isoflurane on a heated stage according to the manufacturer’s protocol. Ovarian volumes were analyzed using the vendor analysis software (VisualSonics, Toronto, Canada).

Results

The clinical indenoisoquinolines selectively kill SLFN11-expressing cell lines

Schlafen 11 (SLFN11) has recently been identified as a dominant response determinant to DNA-targeted drugs both in non-isogenic (11,12,35) and isogenic cancer cell lines (15,17). SLFN11 has a broad range of expression across cancers (11,12,35) [see Supplementary Figure 1 and Figure S1 in reference (15)] making it a potential translational biomarker. Moreover, lack of SLFN11 expression has been associated with tumor resistance to cisplatin, etoposide, camptothecins and PARP inhibitors (17,19,39).

Figure 1 shows the activity of LMP400 across the NCI-60 cancer cell lines using the “Regression Models” tool of CellMinerCDB (http://discover.nci.nih.gov/cellminercdb) (35). It illustrates the correlation between the cytotoxicity of LMP400 and the expression of SLFN11 and to a lesser extent of TOP1 across each of the NCI-60 cell lines (Figure 1A). The corresponding growth inhibitory 50% (GI50) values for LMP400 are included in Supplementary Table 1 and GI50 plots across the NCI60 for LMP400, LMP744 and LMP776 in comparison to topotecan are shown in Supplementary Figure 2. High correlations (r) can also be found using the “Univariate Analysis” tools of CellMinerCDB for all three LMP compounds across the NCI-60 cells (Supplementary Figure 1). For LMP400, the Pearson correlation coefficient is r = 0.65, p-value = 2.8e-08; for LMP776: r = 0.51, p-value = 3.7e-051; and for LMP744 [MJ-III-65]: r = 0.68, p-value = 1.7e-09). LMP744 (MJ-III-65) was also tested across 712 MGH-Sanger cancer cell lines (GDSC database) (35) and showed a highly significant correlation with SLFN11 expression (r = 0.48; p-value = 1.7e-41; Supplementary Figure 1. These results demonstrate the predictive value of SLFN11 expression for the indenoisoquinolines across non-isogenic cell lines.

Figure 1: Schlafen 11 (SLFN11)-dependent antiproliferative activity of the clinical indenoisoquinoline TOP1 inhibitors.

Figure 1:

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A. Snapshot image of CellMiner analysis (http://discover.nci.nih.gov/cellminercdb) for LMP400 in the NCI-60 cell lines (“Regression Model” tab with LMP400 [NSC724998] chosen for drug activity) showing that high expression of SLFN11 correlates with the cell line sensitivity to LMP400. The least sensitive cell lines are colored in dark blue and the most sensitive in red. SK-MEL-2 has the lowest z-score (−1.43 corresponding to a GI50 of 100 μM) and the most sensitive cell line leukemia SR has a z-score or 2.28 corresponding to a GI50 of 10 nM) (see Supplementary Table 1). SLFN11 exhibits a wide range of expression with the highest expression for MOLT4 (8.355 Affymetrix units) and lowest expression for SK-MEL2 (3.609). The range of expression for TOP1 is more consistent across cell lines than for SLFN11, as expected for an essential gene. HCT116 have the highest Affymetrix value for TOP1 (9.381) while the lowest TOP1 expression value is 7.033 for SF-539. B. Resistance of human leukemia CCRF-CEM wild-type (WT) and SLFN11-knockout (SLFN11-KO) cells to topotecan, LMP400, LMP744 and LMP776. Cell viability was determined by ATPlite after 3-day drug treatments. C. Western blotting showing SLFN11 expression in CCRF-CEM parent (WT) and SLFN11 knockout (SLFN11-KO) cells.

To establish the causality between SLFN11 expression and sensitivity to the indenoisoquinolines, we next tested the three clinical LMP compounds in SLFN11-expressing and knockout isogenic human leukemia CCRF-CEM cells (15,17) (Fig. 1B). Knocking-out SLFN11 rendered the cells highly resistant to all three LMPs. Topotecan was also included as control (11,12). Together, these results demonstrate the importance of SLFN11 expression as a determinant of response to the three clinical indenoisoquinolines, LMP400, LMP776 and LMP744 in a broad range of cancer cells.

The clinical indenoisoquinolines selectively kill BRCA1-, BRCA2- and PALB2-deficient DT40 lymphoma chicken cells and murine Brca1-deficient ovarian cancer cells

To test whether HRD is another predictor of response to the clinical indenoisoquinolines, we tested the selectivity of LMP400, LMP744 and LMP776 in isogenic avian B-cell lymphoma DT40 cells (40,41). Indeed, the NCI-60 does not contain homozygously deficient BRCA½ or PAPB2 cells (41). Viability assays and cell cycle analyses were performed in parallel with olaparib, known to selectively kill BRCA-deficient cells (6,7,41). The viability results shown in Figure 2A demonstrate the selectivity of all three indenoisoquinolines in BRCA1-, BRCA2- and PALB2-deficient cells. The IC50 of LMP400 was around ≈ 15 nM in the HRD vs. 45 nM in the WT cells. For LMP744, it was ≈ 7 nM in HRD vs. 40 in WT cells; and for LMP776, ≈ 5 nM in HRD vs. 18 nM in WT cells. As expected, olaparib exhibited high selectivity for the HRD DT40 cells (41).

Figure 2: Selective activity of the indenoisoquinolines TOP1 inhibitors for isogenic BRCA1-, BRCA2- and PALB2-deficient DT40 cells and Brca1-deficient murine serous ovarian cancer (OVCAR) cells.

Figure 2:

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A. Cell viability curves of LMP400, LMP744 and LMP776 in WT DT40 cells and in the isogenic BRCA1−/−, BRCA2−/− and PALB2−/− cells. Experiments were performed in triplicate and error bars represent standard deviation of the mean. B. Representative cell cycle analysis of DT40 WT, BRCA1-, BRCA2- or PALB2-deficient treated with LMP400 (40 nM), LMP744 (40 nM), LMP776 (25 nM) and olaparib (200 nM) for 24 hours. C. Quantitation of sub-G1 population following treatment with LMP400 (40 nM), LMP744 (40 nM) and LMP776 (25 nM) in DT40 WT, BRCA1-, BRCA2- or PALB2-deficient cells. Colored bars and brackets represent the mean and errors bars for 3 independent experiments. D. Cell viability of WT OVCAR cells and the isogenic Brca1−/− cells treated with LMP400, LMP744, LMP776 and olaparib. Cell viability was determined by the ATPlite assay after 3-day treatments. Experiments were performed in triplicate and error bars represent standard deviation of the mean.

To establish that the viability changes were due to cell death, we performed cell cycle analyses after 24-hour treatments with LMP400 (40 nM), LMP744 (40 nM), LMP776 (25 nM) or olaparib (200 nM). All three indenoisoquinolines induced an accumulation of the BRCA1-, BRCA2- and PALB2-deficient DT40 cells in sub-G1 phase (Fig. 1B-C). Together the cell survival and cell cycle analyses demonstrate that LMP400, LMP744 and LMP776 selectively kill HRD DT40 cells.

To consolidate these results, we tested murine serous epithelial ovarian cancer (SEOC) cells (OVCAR) derived from genetically engineered mice (GEM) based on inactivation of Tp53 and Rb with and without Brca1 (38) (Fig. 2D). All three indenoisoquinolines, like olaparib, showed selective activity in the Brca1-deficient OVCAR cells at nanomolar concentrations.

The human colon cancer cell line DLD1 and PDX-derived prostate cancer organoids presenting BRCA2 loss are also hypersensitive to LMP400, LMP776 and LMP744

Next we tested the human colorectal adenocarcinoma cell line DLD1 WT and isogenic BRCA2 −/− to confirm the results obtained in the isogenic chicken and murine cell lines. Figure 3A shows the selective activity of all three indenoisoquinolines and olaparib at nanomolar concentrations for the BRCA2-deficient DLD1 cells. LMP400 IC50 around 12.5 nM in BRCA2-deficient DLD1 vs 35 nM in WT, LMP744 about 15 nM vs 45 nM and for LMP776 10 nM vs 40 nM.

Figure 3: The indenoisoquinolines TOP1 inhibitors show selective cytotoxicity for BRCA2-deficient human colon cancer DLD1 and BRCA2-deficient prostate PDX LuCaP organoids.

Figure 3:

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A. Cell viability curves of LMP400, LMP744, LMP776 and olaparib in WT DLD1 cells and in their isogenic BRCA2−/− counterpart. B. Cell viability curves of LMP400 in WT BRCA2 LuCaP 147 organoids compared to LuCaP 145.1 BRCA2+/− organoids (left), and LuCaP 86.2 (BRCA2 +/−) compared to LuCaP 86.2CR (BRCA2 −/−) organoids (right). Cell viability was determined by the CellTiter Glo 3D assay after 2-week treatments. Experiments were performed in triplicate and error bars represent standard deviation of the mean.

We also tested BRCA2-deficient human prostate cancer organoids derived from the LuCaP-series of PDX tumors (37): LuCaP 147 (BRCA2-WT), LuCaP 145.1 (BRCA2 1 copy loss), LuCaP 86.2 (BRCA2 1 copy loss) and LuCaP 86.2CR (BRCA2 2 copy loss). LuCaP 86.2CR is a castration-resistant subline derived from LuCaP 86.2(36). Figure 3B shows that heterozygous BRCA2 LuCaP 145.1 exhibited greater sensitivity than BRCA2-WT LuCaP 147 with an IC50 of 12.5 nM vs. 85 nM. In addition, BRCA2-null LuCaP 86.2CR cells were greatly more sensitive to LMP400 than the LuCaP 86.2 BRCA2 heterozygous cells, with IC50s of ≈12.5 nM and >200 nM respectively. LuCaP 145.1 and LuCaP 86.2 are both heterozygous for BRCA2, but originated from different patients. The high SLFN11 expression status of LuCaP 145.1 BRCA2 −/+ cells may account for its greater sensitivity to LMP400 than the LuCaP 86.2 BRCA2 −/+ cells that do not express SLFN11.

Together, the results in the panel of chicken B-cell lymphoma DT40, murine ovarian carcinoma (OVCAR), and human colon carcinoma DLD1 and prostate PDX-derived organoids demonstrate the selective activity of the three clinical LMPs: LMP400, LMP776 and LMP744 in BRCA-deficient cancer cells.

Indenoisoquinolines are synergistic with olaparib in DT40 BRCA1 −/− cells

As BRCA1-deficient cells are sensitive to the indenoisoquinolines as well as olaparib, we tested whether these treatments can act synergetically. To do so, we measured cell survival in DT40 BRCA1−/− and WT cells treated with combinations of each of the three clinical indenoisoquinolines LMP400, LMP744 or LMP776 and olaparib (Fig. 4).

Figure 4: The indenoisoquinolines TOP1 inhibitors are synergistic with olaparib in DT40 BRCA1−/− cells.

Figure 4:

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A. Cell viability curves of olaparib in DT40 WT (blue symbols and curves) and BRCA1−/− cell lines (red) with the indicated concentrations of LMP400, LMP744 and LMP776. Open symbols and dashed lines correspond to olaparib alone. B. Combination index (CI) of the combination of indenoisoquinolines with olaparib.

Based on the data shown in Figure 2, we chose low nanomolar concentrations of the indenoisoquinolines with minimal (if any) effect on the WT cells (3.1, 6.2 and 12.5 nM for each of the LMP compounds) and concentrations below 50 nM for olaparib, which are clinically relevant. Figure 4A shows that the addition of each of the LMP compounds to olaparib induced significantly greater cell death than olaparib alone. This synergy was especially notable at the 12.5 nM concentrations of LMP400, LMP744 and LMP776 (right plots in Fig. 4A). We also tested synergism between talazoparib, another PARP inhibitor, and LMP776, and also found a synergistic effect (supplementary Figure 3).

Combination index calculations with the CompuSyn Software showed that both in WT and in BRCA1 deficient cells the combination was synergistic (42). In WT cells, the synergy of LMP400, LMP744 and LMP776 with olaparib was moderate to strong (Fig. 4B, left). In the BRCA1-deficient cells all indenoisoquinoline treatments exhibited even greater synergy (Fig. 4B, right). Notably, comparison of the synergy in WT and BRCA1-deficient cells showed that at same concentrations of LMP400, LMP744, LMP776 and olaparib, the fraction affected (Fa) was overall greater in the BRCA1-deficient compared to the WT DT40 cells. Seventy three percent of the Fa values were above 0.5 in BRCA1-deficient vs. 13% in the WT cells. Together, these results show that the combinations of LMP400, LMP744 or LMP776 with olaparib target BRCA1-deficient cancer cells and has a markedly less effect on normal cells.

LMP400 synergize with olaparib in murine orthotopic allograft model for Brca1-deficient serous epithelial ovarian cancer (SEOC)

To extend these results to the in vivo setting, we evaluated a murine orthotopic allograft model derived from genetically engineered mice (GEM) based on inactivation of Rb and loss of Tp53 and Brca1 (38). Mice were implanted with the BRCA1-deficient ovarian tumor fragments and treatments were initiated when tumors reached 50–300 mm3. The maximum tolerated doses (MTD) in this model were 10 mg/kg for LMP400, 20 mg/kg for LMP744 and 10 mg/kg for LMP776.

All three indenoisoquinolines inhibited tumor growth in the GEM model, and treatment with LMP400 resulted in the longest overall survival (Fig. 5A and 5B and Supplementary Figure 4A for individual curves) without notable toxicity (body weight change in Supplementary Figure 4B and D). LMP400 was therefore chosen to determine the antitumor activity of the combination with olaparib. Tumor growth was delayed by the olaparib/LMP400 combination, which resulted in a greater increase in survival than with either drug alone (Fig. 5B-D and Supplementary Figure 4C for individual curves). In the combination study, the median survival was 50 days for each of the single treatments and 73 days for the combination (Fig. 5B-D).

Figure 5: Antitumor activity of the indenoisoquinolines LMP400, LMP744 and LMP776 in Brca1-deficient murine orthotopic allograft model for ovarian cancer and synergy of LMP400 with olaparib.

Figure 5:

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A-B. Antitumor activity of LMP400, LMP744 and LMP776 represented as average tumor volume (A) and as Kaplan-Meier survival plots (B). p value calculated between best treatment and other arms. LMP400 was administered at 10 mg/kg i.v., one dose per day for 5 days followed by 2-day rest, repeated for 5 weeks. LMP744 was administered at 20 mg/kg i.v. for 2 continuous weeks of 5 days on, 2 days off, followed by one week of rest after which dosing continued for another 2 weeks (5 days on, 2 days off). LMP776 was administered at 10 mg/kg i.v. for 3 continuous weeks of 5 days on, 2 days off, followed by one week of rest after which dosing continued for 5 days. C-D. Anti-tumor activity of the combination of LMP400 with olaparib represented as average tumor volume (C) and Kaplan-Meier survival plots (D). LMP400 was administered i.v. at 5 mg/kg twice a week (day 1 and day 4) and olaparib at 25mg/kg p.o. four times a week (days 1, 2, 4 and 5) for 5 consecutive weeks. p value calculated between the combination and the single treatment arms (with color code). P values: 0.05 > *, 0.005 > **, 0.0005 > ***, 0.00005 > ****

Discussion

It has been 20 years since the FDA approved the camptothecin derivatives topotecan (hycamtin) for ovarian and small cell lung cancer and irinotecan (camptosar) for colorectal cancers. In spite of the well-established limitations of camptothecins (chemical instability, resistance by drug efflux transporters, transient trapping of TOP1ccs, short plasma half-lives, severe diarrheas for irinotecan) (25,27-29), until now non-camptothecin TOP1 inhibitors have not been pursued by pharmaceutical companies. The successful development and FDA approval of 5 PARP inhibitors targeting DNA repair-deficient (HRD) tumors has revived the interest in DNA-targeted agents (4,43).

The indenoisoquinolines TOP1 inhibitors were developed to overcome the above listed limitations of camptothecins and to provide an alternative to the camptothecin chemical class (24,25). The three indenoisoquinolines examined in the present report (LMP400, LMP744 and LMP776) are in Phase 1 clinical trials (30,33) with LMP400 (indotecan) being the most clinically advanced (30). The purpose of the present study was to inform the design of the Phase 2 trials by defining dominant molecular determinants of response to the clinical indenoisoquinolines, as well as explore combination with PARP inhibitors.

Here we demonstrates that the three clinical indenoisoquinolines are active at nanomolar concentrations across a variety of cancer cell lines, and that both HRD (BRCA1, BRCA2 and PALB2 genomic defects) and SLFN11 are dominant response determinants to the indenoisoquinolines. Although TOP1 is the target of the indenoisoquinolines (25), its expression correlates poorly with cell killing (Fig. 1A). LMP776 gives the highest correlation with TOP1 expression in the NCI-60 (r = 0.253; p = 0.0551). In the MGH-Sanger database, because of the large number of cell lines tested, LMP744 (MJ-III-65) give a slightly more significant correlation with TOP1 expression (r = 0.184; p = 7.69e-7). These results are in agreement the conclusion that TOP1 trapping and cleavage complex formation is necessary but not sufficient to predict response of cancer cells to TOP1 inhibitors (44). That HRD and SLFN11 determine response to the indenoisoquinolines is consistent with the selective targeting of TOP1 by the indenoisoquinolines (25) and the biologically established fact that camptothecins are selectively active in SLFN11-expressing cancer cells (11,12,15,23) and HRD cells both in yeast (2,3) and human cancer models (5). SLFN11 and homologous recombination mechanisms of action involved in indenoisoquinolines response are likely similar to the previous findings for camptothecin response (15,17,23). Further clinical studies are warranted to confirm the value of SLFN11 and HR-deficiency in patients, ongoing clinical studies are underway to test the potential predictive value of SLFN11 expression and HRD.

The synergy between camptothecins and PARP inhibitors is also well established in preclinical models (43,45,46). Three molecular mechanisms can explain this synergy: 1/ PARP1 binding to TOP1 and poly(ADPribosylation) of TOP1 reverse TOP1ccs (47), 2/ PARP1 stimulates the excision of TOP1ccs by recruiting the repair enzyme tyrosyl-DNA-phosphodiesterase 1 (TDP1) to TOP1ccs (48), and 3/ PARP1 stabilizes stressed replicons, enabling replication fork regression and stabilization (49,50). However, the combination of camptothecins with PARP inhibitors has been difficult to translate to the clinic as PARP inhibitors also enhance the toxicity of camptothecins toward the normal bone marrow precursor cells. Our finding that the synergism of the clinical indenoisoquinoline TOP1 inhibitors is greater in HRD cells suggests that combination of TOP1 inhibitors (indenoisoquinolines but also camptothecins) with PARP inhibitors is worth testing in clinical trials for HRD tumors. Results in the NCI60 and GDSC databases (35) indicate that cells resistant to olaparib or platinum tend to be also less sensitive to the indenoisoquinolines, which is consistent with the importance of homologous recombination and SLFN11 in the response to TOP1 inhibitors. Yet, a large number of cancer cells not responding to PARP inhibitors are sensitive to the indenoisoquinolines and topotecan (see Supplementary Figure 5) indicating the broad spectrum of activity of the indenoisoquinolines.

In conclusion, the present study demonstrates the potential value of developing additional non-camptothecin TOP1 inhibitors as anticancer agents for patients with SLFN11-expressing and BRCA1-, BRCA2- and PALB2-deficient (HRD) tumors. It also paves the way for developing phase 2 clinical trials of LMP400 (indotecan), the most advanced indenoisoquinoline TOP1 inhibitor, in combination with PARP inhibitors.

Supplementary Material

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Statement of significance: Contrary to other anticancer targets, topoisomerase I (TOP1) is targeted by only one chemical class of FDA approved drugs: topotecan and irinotecan, which are derivatives of camptothecin. Our study establishes a rationale for clinical trials and presents the preclinical results of the indenoisoquinoline non-camptothecin TOP1 inhibitors currently in clinical development.

Statement of translational relevance.

Topoisomerase I (TOP1) inhibitors act as anticancer drugs by selectively trapping TOP1 cleavage complexes and generating DNA damage. However, camptothecin derivatives, which are the only FDA-approved TOP1 inhibitors, have well-established limitations (chemical instability, drug efflux by ABC transporters, short half-life, diarrhea). The indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan) and LMP744 are non-camptothecin TOP1 inhibitors that overcome these limitations and are currently in clinical trials. Here we explore the rationale for their selectivity against cancer cells and for their clinical development. We demonstrate: 1/ the selectivity of the clinical indenoisoquinolines for homologous recombination-deficient (BRCA1-, BRCA2- and PALB2-defective) and SLFN11-positive cancer cells; 2/ the synergistic combination of the clinical indenoisoquinolines with the PARP inhibitor olaparib both in BRCA1-deficient cancer cells and in an in vivo orthotopic model.

Acknowledgements:

Our studies are supported by the Center for Cancer Research, the Intramural Program of the National Cancer Institute (Z01-BC006161). We wish to thank the Developmental Therapeutics Branch (DTP) of the NCI for the cellular testing of the indenoisoquinolines in the NCI-60, and the CellMiner Genomics and Pharmacology Facility (CGPF) of the Developmental Therapeutics Branch, CCR, NCI (William Reinhold) for the development of pharmacogenomic tools (http://discover.nci.nih.gov/cellminer) for our analyses of the NCI-60. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. In vivo studies have been conducted by the Center for Advanced Preclinical Research (CAPR) (https://ccr.cancer.gov/capr) of the Center for Cancer Research, National Cancer Institute. We would like to thank Caitlin Tice of the NIH/NCI/CCR/LGCP for technical support.

Footnotes

The authors declare no potential conflicts of interest.

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Supplementary Materials

1
NIHMS1535855-supplement-1.pdf (1.6MB, pdf)
2
NIHMS1535855-supplement-2.pdf (878.3KB, pdf)
3
NIHMS1535855-supplement-3.pdf (852KB, pdf)
4
NIHMS1535855-supplement-4.pdf (1MB, pdf)
5
NIHMS1535855-supplement-5.pdf (2.4MB, pdf)
6
NIHMS1535855-supplement-6.xlsx (19.2KB, xlsx)

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