Synthetic Lethal Screen Identifies NF-κB as a Target for Combination Therapy with Topotecan for patients with Neuroblastoma
© Tsang et al; licensee BioMed Central Ltd. 2012
Received: 19 August 2011
Accepted: 21 March 2012
Published: 21 March 2012
Despite aggressive multimodal treatments the overall survival of patients with high-risk neuroblastoma remains poor. The aim of this study was to identify novel combination chemotherapy to improve survival rate in patients with high-risk neuroblastoma.
We took a synthetic lethal approach using a siRNA library targeting 418 apoptosis-related genes and identified genes and pathways whose inhibition synergized with topotecan. Microarray analyses of cells treated with topotecan were performed to identify if the same genes or pathways were altered by the drug. An inhibitor of this pathway was used in combination with topotecan to confirm synergism by in vitro and in vivo studies.
We found that there were nine genes whose suppression synergized with topotecan to enhance cell death, and the NF-κB signaling pathway was significantly enriched. Microarray analysis of cells treated with topotecan revealed a significant enrichment of NF-κB target genes among the differentially altered genes, suggesting that NF-κB pathway was activated in the treated cells. Combination of topotecan and known NF-κB inhibitors (NSC 676914 or bortezomib) significantly reduced cell growth and induced caspase 3 activity in vitro. Furthermore, in a neuroblastoma xenograft mouse model, combined treatment of topotecan and bortezomib significantly delayed tumor formation compared to single-drug treatments.
Synthetic lethal screening provides a rational approach for selecting drugs for use in combination therapy and warrants clinical evaluation of the efficacy of the combination of topotecan and bortezomib or other NF-κB inhibitors in patients with high risk neuroblastoma.
Neuroblastoma is the most common extra-cranial solid tumor in childhood, accounting for 7-10% of childhood cancers . Based on age, staging, MYCN amplification status, histology, and DNA ploidy, neuroblastoma is classified into low, intermediate and high risk groups [2, 3]. At present, high risk neuroblastoma is treated with high dose chemotherapy, surgery, autologous stem cell transplantation, radiation, immune and differentiating therapy. Currently used chemotherapeutic agents in standard and salvage regimens include toposisomerase I and II inhibitors, topotecan, etoposide, irinotecan and doxorubicin; alkylating agents, cisplatin, carboplatin, melphalan and cyclophosphamide and the microtubule inhibitor vincristine [4, 5]. The differentiating agent 13-cis-retinoic acid is also administered during the maintenance period post chemotherapy. Recent clinical trials have shown that the combination of anti-GD2 antibodies and immunocytokines significantly increase the survival of patients with high risk neuroblastoma [6, 7]. Despite these aggressive combined multimodal treatments the survival rate for these high risk neuroblastoma patients remains less than 50%.
Topoisomerase inhibitors are currently a mainstay of many salvage regimens for neuroblastoma and are being evaluated as up-front therapy in an ongoing trial [8–11]. They function by perturbing the cellular machinery responsible for maintaining DNA structure during transcription and replication. Topotecan is an inhibitor for the enzyme topoisomerase-I which is involved in the replication and repair of nuclear DNA. As DNA is replicated in dividing cells, topoisomerase-I binds to super-coiled DNA causing single-stranded breaks. As a result, topoisomerase-I relieves the torsional stresses that are introduced into DNA ahead of the replication complex or moving replication fork. Topotecan inhibits topoisomerase-I by stabilizing the covalent complex of enzyme and strand-cleaved DNA, which is an intermediate of the catalytic mechanism, thereby inducing breaks in the protein-associated DNA single-strands, resulting in cell death . This agent is currently used for the treatment of many cancers including metastatic ovarian cancer and platinum-sensitive relapsed small-cell lung cancer , recurrent or persistent cervical cancer , and neuroblastoma . In addition, topotecan is being evaluated in pediatric cancer patients for treating leukemia, lymphoma, Ewing's sarcoma, rhabdomyosarcomas and gliomas (http://www.clinicaltrials.gov). However, the primary dose-limiting toxicity of topotecan is myelosuppression, restricting its use at high doses. Therefore, identification of other chemotherapeutic agents synergizing with topotecan may potentially maintain or increase efficacy while limiting toxicity.
In this study, we performed a loss-of-function synthetic lethal siRNA screening of 418 apoptosis related genes with and without topotecan to identify genes or pathways whose inhibition synergized with topotecan to enhance growth suppression or apoptosis in neuroblastoma. The goal of the study was to identify drugs that would potentially be synergistic when used in combination with topotecan to inhibit the growth of neuroblastoma.
Cell lines and culture conditions
The neuroblastoma cell lines SK-N-AS and SH-SY5Y were maintained in RPMI-1640; and NB-1691 was maintained in DMEM, both supplemented with 10% FBS, 1% penicillin/streptomycin (P/S) and 1% L-glutamine (all from Quality Biological Inc., Gaithersburg, MD) at 37°C. To ensure consistency, a batch of cells was expanded, aliquoted and stored in liquid nitrogen prior to the screening. In each experiment, a vial of cells was defrosted and passaged 1:4 when 70% confluency was reached. Cells between passages 3 and 7 were used for all experiments.
Topotecan hydrocholoride (Hycamtin; GlaxoSmithKline, Philadelphia, PA) and Bortezomib (Velcade; Millenium Pharmaceuticals, Cambridge, MA) were reconstituted and stored according to the manufacturers' instructions. NSC 676914 was obtained from the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnostics, NCI/NIH.
High throughput siRNA screening
A set of synthetic siRNAs targeting 418 genes related to the apoptotic pathway (Qiagen Apoptosis Set V.1; Qiagen, Valencia, CA), with 2 siRNAs of different sequences per gene, was used for the first screen. For the second screen, 2 new siRNA pre-designed sequences were used (Qiagen). In the third confirmatory screen, one siRNA from each of the previous two screens was chosen. siRNAs were transfected at passage 4. Briefly, transfection reagent Dharmafect 1 (Dharmacon RNA Technologies, Lafayette, CO) was diluted in DCCR reagent medium at a ratio of 1:208 in volume. siRNA (20 nM) and 25 μL of the diluted transfection reagent were added to an individual well in a 96-well plate for complex formation with incubation for 20 min at RT. SK-N-AS cells were trypsinized, counted and resuspended in P/S free culture medium. 5000 cells were added to each individual well in 100 μL medium. The plate was incubated at RT for 30 min for cell attachment before being placed at 37°C for 24 h. Topotecan was then added to each well for additional 72 h incubation. Cell proliferation assay was performed at 96 h post siRNA transfection. The IC50 of topotecan for SK-N-AS cells at 24 h was 2 μM. In the 1st and 2nd screens, topotecan doses of 0, 1, 5 and 10 μM were used, whereas in the 3 rd screen, lower drug doses of topotecan at 0, 0.01, 0.1 and 1 μM were used to identify synergy. The criterion of hit selection for the enhancer genes was ≤0.8 fold cell growth compared to its own siRNA effect in the presence of topotecan. The final enhancer gene list was subjected to pathway analysis for identification of overrepresented genes within a target pathway. An inhibitor to the pathway was chosen and tested individually or in combination with topotecan in vitro and in vivo.
Cell proliferation assay
Cell proliferation was measured using Cell Titer Glo proliferation assay (Promega Corporation, Madison, WI) or a real-time cell sensing system (RT-CES; ACEA Biosciences, Inc. San Diego, CA) according to the manufacturer's instruction. We performed cell proliferation assays at a volume of 35 μL per well for 10 min and was measured at 562 nm on a Tecan plate reader (Tecan Inc., Durham, NC). Each treatment was performed in triplicates, averaged and normalized using untreated cells. For real-time cell electronic sensing assays, cells treated with drugs alone or in combinations or transfected with siRNA were added in triplicates to a 96-well plate device compatible with the real-time cell electronic sensing analyzer (RT-CES; ACEA Biosciences, Inc. San Diego, CA). Cell growth was monitored hourly for indicated durations via calculation of "cell index" (normalized impedance) for each well.
SK-N-AS cells were collected at 60 h post siRNA transfection. Total RNA was reverse transcribed using SuperScript II reverse transcriptase system (Life Technologies, Foster City, CA). The target regions were then pre-amplified using a standard PCR for 10 cycles. The pre-amplified cDNA was quantified by Taqman gene expression assay (Life Technologies) using Fluidigm digital array (South San Francisco, CA) according to the manufacturer's protocol. Fold expression was calculated using a comparative threshold cycle method (2-Δ ΔCT) .
The pathway analysis was performed using MetaCore (http://www.genego.com/metacore.php, GeneGo Inc., St Joseph, MI). MetaCore is an integrated software suite for functional analysis of experimental data and it contains curated protein interaction networks on the basis of manually curated database of human protein-protein, protein-DNA, protein-RNA and protein-compound interactions. Metacore uses a hypergeometric model to determine the significance of enrichment. The enhancer genes from our experiment and the genes in the GeneGo maps from the MetaCore database were used to identify the enriched GeneGo pathway maps.
Gene Set Enrichment Analysis
To investigate gene set enrichment, GSEA (http://www.broad.mit.edu/gsea/) was performed for genes, ranked by log2 ratio of gene expressions between topotecan-treated (1 μM and 10 μM) and untreated control SK-N-AS cells, using a weighted Kolmogorov-Smirnov-like statistics . The gene set of NF-κB target genes used in GSEA were downloaded from http://bioinfo.lifl.fr/NF-KB.
Cells were seeded on a 100 mm2 culture dish for overnight and were treated with topotecan alone or with bortezomib. For topotecan alone effect, SK-N-AS were treated with 5 μM of topotecan for 0, 3, 6, or 24 h at 37°C. For the combined effect of bortezomib and topotecan, cells were treated with bortezomib at 0, 1 and 10 nM for 24 h, followed by the addition of topotecan at 5 μM for 6 h. Total cell lysate was collected with RIPA buffer containing 1% phosphatase inhibitor and 1% protease inhibitor (all from Thermo Fisher Scientific, Rockford, IL). Protein concentration was measured by BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA) and protein lysates (20 ug per lane) were resolved on 4-12% TRIS-gradient gel (Invitrogen Life Technologies, Carlsbad, CA) and were transferred to nitrocellulose membranes by iBlot blotting system (Invitrogen Life Technologies). The membranes were blocked with 5% non-fat dry milk in PBS with 0.1% Tween 20 (PBST) for 1 h at RT, followed by incubation with mouse monoclonal antibodies against total or phosphorylated IκB-α (Ser32/36), rabbit monoclonal antibodies against total or phosphorylated p65/RelA or rabbit monoclonal antibody against NFKB1 (all from Cell Signaling Technology, Danvers, MA) at 4°C overnight. Peroxidase-conjugated goat anti-mouse or anti-rabbit antibody was used as secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h incubation at RT. Immunoreactive bands were visualized by ECL analysis system (GE Healthcare, Piscataway, NJ) and enhanced chemiluminescence. For loading control, the membranes were washed with Restore Western blot stripping buffer (Thermo Fisher Scientific) and probed with goat anti-human actin HRP conjugated antibody (Santa Cruz Biotechnology).
In vitro drug combination
Neuroblastoma cells were trypsinized, counted and resuspended in P/S free culture medium. 5000 cells per well in 100 μL medium were seeded in 96-well white plates for overnight. Topotecan and NSC 676914 or bortezomib were added individually or in combination at various doses and the plates were incubated at 37°C for 24 h and 48 h respectively. Cell proliferation assay was performed as described above. Combination index was calculated using CompuSyn software (ComboSyn Inc., Paramus, NJ). Briefly, the combination index theorem was used to quantify synergy or antagonism for two drugs by the formula C.I. = (D)1/(Dx)1 + (D)2/(Dx)2, where D1 and D2 are drug 1 and drug 2, and × is growth inhibition by X% .
The caspase-3 activity was measured using the PE Active Caspase-3 Apoptosis kit (BD Pharmingen, San Diego, CA). Briefly, SK-N-AS cells (untreated or treated with topotecan and bortezomib alone or in combination for 24 h) were trypsinized, fixed, and stained with PE rabbit anti-active caspase-3 antibody. Fluorescence intensity was measured by FACS Calibur and data were analyzed using CellQuest software (BD Biosciences, Franklin Lakes, NJ).
In vivo xenograft model
All animal experiments have been reviewed and approved by the NIH Animal Care and User Committees. A minimal residual disease xenograft model in mice bearing neuroblastoma was established in 8-10 week-old female SCID Beige mice (Charles River Laboratories, Fredrick, MD). Briefly, five million SK-N-AS cells expressing luciferase (gift from Dr. Bryan Clary, Duke University Medical Center) were injected intravenously via the lateral tail vein into the mice. Tumors were allowed to grow for 7 d, and then mice were randomly assigned to cohorts treated with topotecan and bortezomib administered individually or in combination, or with saline solution (control mice). Bortezomib (0.6 mg/kg) was injected intraperitoneally three times a week for two weeks, rested for two weeks and repeated with another course of treatment. Topotecan (0.5 mg/kg) was injected intraperitoneally five times a week for two weeks, rested for two weeks, followed by another course of treatment. Body weight and general wellness of the mice were monitored, and tumor size was monitored by Xenogen IVIS 100 imaging system (Caliper Life Sciences Inc., Hopkinton, MA). The in vivo xenograft experiment was repeated, and results from two independent experiments were combined (n = 43).
Non-parametric Mann-Whitney test was used to compare among various groups in cell growth assay. For relative luciferase intensity results from two independent in vivo experiments, we normalized the log2 transformed intensities from each experiment using median-centered method and then combined the results. T-test was used to compare the difference of two groups.
Identification and validation of enhancer genes
The common enhancer genes that potentiated the effect of topotecan from three screens
Genes that enhance the effect of topotecan-induced cell death
BIRC4 *, **
CTSD *, **
NFKB1 *, **
NOS2A *, **
RIPK1 *, **
TGFB1 *, **
TNFRSF10A *, **
TNFRSF25 *, **
TNFRSF8 *, **
Inhibition of NF-κB pathway enhanced topotecan-mediated growth inhibition in neuroblastoma cells
Pathway analysis of the 9 common enhancer genes (P < 0.01)
Number of genes in the map
Number of overlapped enhancer genes
Anti-apoptotic TNFs/NF-κB/IAP pathway
Apoptotic TNF-family pathways
HTR1A signaling pathway
APRIL and BAFF signaling pathway
Synergistic growth inhibition in three neuroblastoma cell lines treated with topotecan and NF-κB inhibitors
Delayed tumor progression in human neuroblastoma xenograft treated with topotecan and bortezomib
More than half of the neuroblastoma patients over 1 year old have advanced metastatic disease at the time of diagnosis . For these patients, the overall survival rate remains less than 50%. Therefore, a new therapeutic strategy is critically needed. Current treatment regimens used in high risk neuroblastoma include topotecan, a topoisomerase I inhibitor and cyclophosphamide, a nitrogen mustard alkylating agent. Cyclophosphamide induces DNA cross-linking and DNA single-strand breaks; while topotecan inhibits religation of the topoisomerase I-mediated DNA single-strand breaks. Both result in increased numbers of strand breaks and stabilization of these unrepaired breaks, leading to enhanced cytotoxicity. The combination was first proven effective in a phase II trial in neuroblastoma, in which there were six partial responses in 13 patients with neuroblastoma with the combination of cyclophosphamide and topotecan compared with two responses (one complete and one partial) in 37 patients treated with topotecan alone.. Subsequently, topotecan together with other topoisomerase inhibitors have become the basis of many salvage regimens and is being evaluated as up-front therapy in ongoing trials in neuroblastoma and other cancers.
Here we utilized a high throughput loss of function approach using siRNAs to identify genes whose inhibition would synergize with topotecan with the ultimate goal of discovering potent synergistic drug combinations for treating patients with neuroblastoma. SiRNA screening can identify genes, and pathways critical for cancer cell growth and survival. This approach provides a rational method of choosing inhibitors to target the identified genes and pathways. The objective of combination chemotherapy is to simultaneously target multiple pathways that are important for cancer cell growth and survival, in the hope to synergistically inhibit tumor cell growth. In our study, there was an enrichment of NF-κB pathway genes in the positive hits and an induction of NF-κB gene signature upon treatment with topotecan. This pathway plays an important role in inflammation, autoimmune response, cell proliferation, and apoptosis depending on the cell type and context . In cancers including neuroblastoma, the NF-κB pathway is found to activate transcription of genes encoding tumor-promoting cytokines, playing critical roles in neoplastic transformation and cancer cell survival . Hence, targeting this signaling pathway should be effective for growth inhibition and increasing apoptosis for cancers. However, NF-κB inhibitors are generally used as adjuvants because inhibition of NF-κB alone may be insufficient for a pronounced apoptotic response unless it is combined with apoptosis-inducing drugs or radiation . Our results indicated that the NF-κB pathway was activated as a possible protective mechanism against topoisomerase-I inhibition resulting in single stranded DNA breaks. This together with our siRNA screening results suggested that the combination of topotecan and an NF-κB inhibitor might be a good combination to kill neuroblastoma cells.
In this study, we found that a specific NF-κB inhibitor, NSC 676914, synergized growth inhibition mediated by topotecan in both MYCN and non-MYCN amplified neuroblastoma cell lines. NSC 676914 has been identified as a novel, specific and stable small-molecular NF-κB inhibitor through the inhibition of IKK-β to induce growth inhibition of multiple myeloma cells in vitro and in vivo [19, 26]. Our findings were consistent with a recent publication which suggested that inhibition of NF-κB pathway could lead to increased chemosensitivity . In addition, we used bortezomib, a FDA-approved drug known to have NF-κB inhibition effects through preventing IκB-α protein degradation in the proteasome , for the subsequent in vitro and in vivo studies. Bortezomib, the first FDA-approved proteasome inhibitor, is a boronic-acid derivative that reversibly inhibits the active sites in the 20S proteasome [29, 30]. Down-regulation of NF-κB pathway is its prevailing mechanism of action in multiple myeloma and relapsed mantle cell lymphoma . In addition to NF-κB inhibition, bortezomib has been shown to inhibit other pathways which may partially contribute to the effect observed in this study. For example, the topoisomerase I cleavable complex also serves as a substrate for the proteasome , an inhibitory target of bortezomib. Stabilization of these complexes has been shown to enhance the cytotoxic effect of another topoisomerase I inhibitor camptothecin, an analog of topotecan . Furthermore, bortezomib inhibits tumor growth, causing cell cycle arrest in colon, ovarian, breast, renal cell and pancreatic carcinomas , as well as in lymphomas and leukemias and pediatric tumors including neuroblastoma in vitro and in vivo .
Our in vivo xenograft experiments demonstrated a delay in tumor progression when topotecan was combined with bortezomib. Therefore, bortezomib could be a logical choice of NF-κB inhibitors to be used with topotecan as a rational combination therapy. With regards to drug toxicity, myelosuppression, primarily reversible, noncumulative neutropenia, is the predominant toxicity observed with topotecan treatment [11, 36]. For bortezomib, while peripheral neuropathy is the most often seen toxicity, recent study suggests that it is manageable and reversible . Bortezomib-induced thrombocytopenia and neutropenia are cyclic, reversible, typically do not lead to treatment discontinuation and recover prior to initiation of the subsequent cycle [38, 39]. The combination of topotecan and bortezomib is currently under investigation in clinical trials for other advanced solid tumors in adults . Thus this combination should be well tolerated in patients with neuroblastoma.
In conclusion, our synthetic lethal siRNA screening led to the discovery that NF-κB inhibition synergized cell death when used with topotecan in neuroblastoma. Furthermore we showed that the NF-κB pathway was induced in neuroblastoma cells treated with topotecan. Finally we demonstrated evidence of the synergistic effects of topotecan and bortezomib in in-vitro and in a pre-clinical mouse model. Our study therefore provides the rationale for future clinical trials evaluating this combination therapy for patients with high risk neuroblastoma.
We would like to thank Dr Paul Meltzer, Dr Natasha Caplen and Dr Scott Martin for providing the siRNA library and for useful discussions; and Dr David Azorsa for helpful technical advice. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. 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.
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