High-risk Neuroblastoma Cell Lines Research Articles

Proliferative Effects of Mesenchymal Stromal Cells on Neuroblastoma Cell Lines: Are They Tumor Promoting or Tumor Inhibiting?

BackgroundNeuroblastoma is a common pediatric malignancy with poor survival for high-risk disease. Mesenchymal stromal cells (MSCs) have innate tumor-homing properties, enabling them to serve as a cellular delivery vehicle, but MSCs have demonstrated variable effects on tumor growth. We compared how placental MSCs (PMSCs) and bone marrow-derived MSCs (BM-MSCs) affect proliferation of neuroblastoma (NB) cells in vitro. MethodsIndirect co-culture assessed proliferative effects of 18 MSCs (early-gestation PMSCs (n = 9), term PMSCs (n = 5), BM-MSCs (n = 4) on three high-risk NB cell lines (NB1643, SH-SY5Y, and CHLA90). Controls were NB cells cultured in media alone. Proliferation was assessed using MTS assay and measured by fold change (fc) over controls. PMSCs were sub-grouped by neuroprotective effect: strong (n = 7), intermediate (n = 3), and weak (n = 4). The relationship between MSC type, PMSC neuroprotection, and PMSC gestational age on NB cell proliferation was assessed. ResultsNB cell proliferation varied between MSC groups. BM-MSCs demonstrated lower proliferative effects than PMSCs (fc 1.18 vs 1.44, p < 0.001). Neither gestational age nor neuroprotection significantly predicted degree of proliferation. Proliferative effects of MSCs varied among NB cell lines. BM-MSCs had less effect on CHLA90 (fc 1.01) compared to NB1643 (fc 1.33) and SH-SY5Y (fc 1.20). Only NB1643 showed a difference between early and term PMSCs (p = 0.04). ConclusionEffects of MSCs on NB cell proliferation vary by MSC source and NB cell line. BM-MSCs demonstrated lower proliferative effects than most PMSCs. MSC neuroprotection was not correlated with proliferation. Improved understanding of MSC proliferation-promoting mechanisms may provide valuable insight into selection of cells best suited as drug delivery vehicles. Level of EvidenceN/A. Type of StudyOriginal Research.

KDM5B expression in cisplatin resistant neuroblastoma cell lines.

Chemoresistance is a major problem in successful cancer therapy. Lysine-specific demethylase 5B (KDM5B), is a member of the KDM5 family of histone demethylases, whose dysregulation has been observed in numerous types of cancer and plays a role in drug tolerance. The present study examined KDM5B expression in high risk neuroblastoma cell lines. Its level was markedly reduced in cisplatin-resistant cells, UKF-NB-4CDDP, compared with parental sensitive cells UKF-NB-4. Moreover, KDM5B-silencing did not affect either viability nor the response to CDDP in resistant cells, and led to increase of proliferation and migration in CDDP resistant cells but not in sensitive ones. Compliant with these results, short interfering KDM5B transfection resulted in increased S phase in resistant cells. Overall, these findings suggested that KDM5B may be involved in the survival mechanisms of neuroblastoma cells, which makes KDM5B a promising factor for the prediction of sensitivity to CDDP that should therefore be considered for future research.

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

NAMPT Inhibition Induces Neuroblastoma Cell Death and Blocks Tumor Growth.

High-risk neuroblastoma (NB) portends very poor prognoses in children. Targeting tumor metabolism has emerged as a novel therapeutic strategy. High levels of nicotinamide-adenine-dinucleotide (NAD+) are required for rapid cell proliferation. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme for NAD+ salvage and is overexpressed in several cancers. Here, we determine the potential of NAMPT as a therapeutic target for NB treatment. NAMPT inhibition cytotoxicity was determined by trypan blue exclusion and LDH assays. Neuroblastoma stem cell self-renewal was evaluated by neurosphere assay. Protein expression was evaluated via Western blot. The effect of targeting NAMPT in vivo was determined using an NB1691-xenografted mouse model. Robust NAMPT expression was demonstrated in multiple N-MYC amplified, high-risk neuroblastoma cell lines. NAMPT inhibition with STF-118804 (STF) decreased ATP, induced apoptosis, and reduced NB stem cell neurosphere formation. STF treatment down-regulated N-MYC levels and abrogated AKT activation. AKT and glycolytic pathway inhibitors in combination with NAMPT inhibition induced robust, greater-than-additive neuroblastoma cell death. Lastly, STF treatment blocked neuroblastoma tumor growth in mouse xenograft models. NAMPT is a valid therapeutic target as inhibition promoted neuroblastoma cell death in vitro and prevented tumor growth in vivo. Further investigation is warranted to establish this therapy’s role as an adjunctive modality.

Heterogeneities in Cell Cycle Checkpoint Activation Following Doxorubicin Treatment Reveal Targetable Vulnerabilities in TP53 Mutated Ultra High-Risk Neuroblastoma Cell Lines.

Most chemotherapeutics target DNA integrity and thereby trigger tumour cell death through activation of DNA damage responses that are tightly coupled to the cell cycle. Disturbances in cell cycle regulation can therefore lead to treatment resistance. Here, a comprehensive analysis of cell cycle checkpoint activation following doxorubicin (doxo) treatment was performed using flow cytometry, immunofluorescence and live-cell imaging in a panel of TP53 mutated ultra high-risk neuroblastoma (NB) cell lines, SK-N-DZ, Kelly, SK-N-AS, SK-N-FI, and BE(2)-C. Following treatment, a dose-dependent accumulation in either S- and/or G2/M-phase was observed. This coincided with a heterogeneous increase of cell cycle checkpoint proteins, i.e., phos-ATM, phos-CHK1, phos-CHK2, Wee1, p21Cip1/Waf1, and p27Kip among the cell lines. Combination treatment with doxo and a small-molecule inhibitor of ATM showed a delay in regrowth in SK-N-DZ, of CHK1 in BE(2)-C, of Wee1 in SK-N-FI and BE(2)-C, and of p21 in Kelly and BE(2)-C. Further investigation revealed, in all tested cell lines, a subset of cells arrested in mitosis, indicating independence on the intra-S- and/or G2/M-checkpoints. Taken together, we mapped distinct cell cycle checkpoints in ultra high-risk NB cell lines and identified checkpoint dependent and independent druggable targets.

Abstract 1374: Preclinical investigation of ATR inhibition alone and in combination with PARP inhibition in high risk neuroblastoma

Abstract Background:Neuroblastoma (NB) is the commonest extra-cranial malignant solid tumour of childhood and one of the most difficult to cure. DNA damage response (DDR) defects are frequently observed in high risk NB including allelic loss and loss of function mutations in key DDR genes, oncogene induced replication stress (RS) and cell cycle checkpoint dysfunction. Cancer cells with defective cell cycle checkpoint signalling and/or increased oncogene-driven RS are acutely dependent on the DNA damage sensor kinase ATR. This study aims to identify features of NB cell lines leading to ATR inhibitor sensitivity. As PARP inhibition causes RS through unrepaired single strand DNA breaks progressing to replication, we hypothesise that ATR inhibition will increase PARP inhibitor cytotoxicity. Aims: 1) To determine which molecular features lead to sensitivity to VE-821 (ATR inhibitor) in cell lines derived from high-risk NB tumours. 2) To assess synergism between PARP inhibition with olaparib and ATR inhibition in high risk NB cell lines and to measure RS. Materials and Methods: Cell proliferation in response to 72 hours treatment with VE-821 was assessed by XTT assay (Roche) in a panel of 11 NB cell lines and the effect of ATR inhibition on olaparib growth inhibition in 4 cell lines: SHSY5Y, SKNAS, NGP and N20_R1. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS respectively. RS was also measured by γH2AX foci formation using immunofluorescent microscopy. Results: VE-821 caused significantly more growth inhibition in MYCN amplified cell lines and cell lines with low ATM protein expression by XTT assay (p&amp;lt;0.05 Mann-Whitney U test). Olaparib (5 µM) treatment increased CHK1S345 and H2AXS129 phosphorylation after 24 hours treatment in all cell lines. H2AXS129 phosphorylation and foci number was further increased with the addition of VE-821 (1 µM). ATR inhibition prevented CHK1S345 phosphorylation. In cell proliferation assays, combination index analysis (Calcusyn) showed that ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (&amp;lt;1 µM) (CI value 0.04-0.89). Conclusion: MCYN amplification and low ATM protein expression are determinants of ATRi sensitivity in NB cell lines. ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (&amp;lt;1 µM) and further increases the replication stress caused by PARP inhibition. Citation Format: Harriet E. Southgate, Lindi Chen, Nicola J. Curtin, Deborah A. Tweddle. Preclinical investigation of ATR inhibition alone and in combination with PARP inhibition in high risk neuroblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1374.

Abstract A35: P-Glycoprotein is a resistance mechanism for conventional induction chemotherapy but not ALK inhibitors in high-risk neuroblastoma

Abstract Background: Despite intensive treatment, patients with high-risk neuroblastoma (HR-NB) have a five-year survival rate of only 50%, largely due to intrinsic or acquired drug resistance. The multidrug transporter P-glycoprotein (P-gp; ABCB1) effluxes conventional agents used in HR-NB induction therapy, including vincristine, etoposide, and doxorubicin, and has been reported as a resistance mechanism to the ALK inhibitors crizotinib and ceritinib. We assessed the prevalence of P-gp expression and its role in resistance to standard-of-care chemotherapies and molecularly targeted therapies. Methods and Results: Analysis of ABCB1 levels in large patient tumor datasets demonstrates that high expression is more common in neuroblastoma than in other cancers and may be attributable to the sympathoadrenal lineage of the disease. Moreover, high relative expression of ABCB1 in HR-NB tumors is associated with poorer outcome, consistent with its multidrug transporter function. Frequent high ABCB1/P-gp expression in HR-NB was confirmed using RNA-sequencing, immunohistochemistry, and Western blot on panels of neuroblastoma tumor samples, patient-derived xenograft models, and cell lines. We demonstrated that the P-gp inhibitor tariquidar and P-gp knockdown (shRNA) both strongly sensitize high P-gp expressing neuroblastoma cells to vincristine, doxorubicin, and etoposide but not to ALK inhibitors or to relapse therapy regimens. Further, P-gp knockdown sensitized human neuroblastoma xenografts to vincristine, substantially extending animal survival. We have generated ABCB1-null HR-NB cell lines by CRISPR/Cas9 gene editing to further assess the role of P-gp in HR-NB drug resistance, have established flow-based assays to measure P-gp activity in tumor samples, and are investigating the benefits of tariquidar as adjuncts to conventional chemotherapy in PDX models. Conclusions: Elevated P-gp expression is common in HR-NB. In laboratory models, P-gp can confer resistance to standard-of-care chemotherapies but not to ALK inhibitors. Our findings suggest that tumor P-gp activity might be used to guide treatment options for individual patients in order to avoid ineffective treatments. The potential of P-gp inhibitors as adjuncts to conventional chemotherapy for HR-NB should be further investigated. Citation Format: Caroline Atkinson, Carole Tactacan, Alvin Kamili, Federica Saletta, Christine Gana, Georgina Eden, Chelsea Mayoh, Richard Lock, Murray Norris, Michelle Haber, Andrew Gifford, Toby Trahair, Jamie Fletcher. P-Glycoprotein is a resistance mechanism for conventional induction chemotherapy but not ALK inhibitors in high-risk neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A35.

Telomere Maintenance Mechanisms Define Clinical Outcome in High-Risk Neuroblastoma.

Neuroblastoma is a childhood cancer with heterogeneous clinical outcomes. To comprehensively assess the impact of telomere maintenance mechanism (TMM) on clinical outcomes in high-risk neuroblastoma, we integrated the C-circle assay [a marker for alternative lengthening of telomeres (ALT)], TERT mRNA expression by RNA-sequencing, whole-genome/exome sequencing, and clinical covariates in 134 neuroblastoma patient samples at diagnosis. In addition, we assessed TMM in neuroblastoma cell lines (n = 104) and patient-derived xenografts (n = 28). ALT was identified in 23.4% of high-risk neuroblastoma tumors and genomic alterations in ATRX were detected in 60% of ALT tumors; 40% of ALT tumors lacked genomic alterations in known ALT-associated genes. Patients with high-risk neuroblastoma were classified into three subgroups (TERT-high, ALT+, and TERT-low/non-ALT) based on presence of C-circles and TERT mRNA expression (above or below median TERT expression). Event-free survival was similar among TERT-high, ALT+, or TERT-low/non-ALT patients. However, overall survival (OS) for TERT-low/non-ALT patients was significantly higher relative to TERT-high or ALT patients (log-rank test; P < 0.01) independent of current clinical and molecular prognostic markers. Consistent with the observed higher OS in patients with TERT-low/non-ALT tumors, continuous shortening of telomeres and decreasing viability occurred in low TERT-expressing, non-ALT patient-derived high-risk neuroblastoma cell lines. These findings demonstrate that assaying TMM with TERT mRNA expression and C-circles provides precise stratification of high-risk neuroblastoma into three subgroups with substantially different OS: a previously undescribed TERT-low/non-ALT cohort with superior OS (even after relapse) and two cohorts of patients with poor survival that have distinct molecular therapeutic targets. SIGNIFICANCE: These findings assess telomere maintenance mechanisms with TERT mRNA and the ALT DNA biomarker C-circles to stratify neuroblastoma into three groups, with distinct overall survival independent of currently used clinical risk classifiers.

ATR Inhibition Potentiates PARP Inhibitor Cytotoxicity in High Risk Neuroblastoma Cell Lines by Multiple Mechanisms.

Background: High risk neuroblastoma (HR-NB) is one the most difficult childhood cancers to cure. These tumours frequently present with DNA damage response (DDR) defects including loss or mutation of key DDR genes, oncogene-induced replication stress (RS) and cell cycle checkpoint dysfunction. Aim: To identify biomarkers of sensitivity to inhibition of Ataxia telangiectasia and Rad3 related (ATR), a DNA damage sensor, and poly (ADP-ribose) polymerase (PARP), which is required for single strand break repair. We also hypothesise that combining ATR and PARP inhibition is synergistic. Methods: Single agent sensitivity to VE-821 (ATR inhibitor) and olaparib (PARP inhibitor), and the combination, was determined using cell proliferation and clonogenic assays, in HR-NB cell lines. Basal expression of DDR proteins, including ataxia telangiectasia mutated (ATM) and ATR, was assessed using Western blotting. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS, respectively. RS and homologous recombination repair (HRR) activity was also measured by γH2AX and Rad51 foci formation using immunofluorescence. Results: MYCN amplification and/or low ATM protein expression were associated with sensitivity to VE-821 (p < 0.05). VE-821 was synergistic with olaparib (CI value 0.04–0.89) independent of MYCN or ATM status. Olaparib increased H2AXS129 phosphorylation which was further increased by VE-821. Olaparib-induced Rad51 foci formation was reduced by VE-821 suggesting inhibition of HRR. Conclusion: RS associated with MYCN amplification, ATR loss or PARP inhibition increases sensitivity to the ATR inhibitor VE-821. These findings suggest a potential therapeutic strategy for the treatment of HR-NB.

CCI52 sensitizes tumors to 6-mercaptopurine and inhibits MYCN-amplified tumor growth

The antimetabolite 6-mercaptopurine (6-MP) is an important component in the treatment of specific cancer subtypes, however, the development of drug resistance and dose-limiting toxicities can limit its effectiveness. The therapeutic activity of 6-MP requires cellular uptake, enzymatic conversion to thio-GMP and incorporation of thio-GTP into RNA and DNA, as well as inhibition of de novo purine synthesis by methyl-thio-IMP. Mechanisms that prevent 6-MP entry into the cell, prevent 6-MP metabolism or deplete thiopurine intermediates, can all lead to 6-MP resistance. We previously conducted a high-throughput screen for inhibitors of the multidrug transporter MRP4 using 6-MP sensitivity as the readout. In addition to MRP4-specific inhibitors, we identified a compound, CCI52, that sensitized cell lines to 6-MP independent of this transporter. CCI52 and its more stable analogue CCI52-14 also function as effective chemosensitizers in vivo, substantially extending survival in a transgenic mouse cancer model treated with 6-MP. Chemosensitization was associated with an increase in thio-IMP, suggesting that CCI52 functions directly on 6-MP uptake or metabolism. In addition to its chemosensitizing effects, CCI52 and CCI52-14 inhibited the growth of MYCN-amplified high-risk neuroblastoma cell lines and delayed tumor progression in a MYCN-driven, transgenic mouse model of neuroblastoma. These multifunctional inhibitors may be useful for the further development of anticancer agents and as tools to better understand 6-MP metabolism.

Abstract 4741: Re-evaluating the role of P-glycoprotein in the resistance of high-risk neuroblastoma to standard-of-care chemotherapies

Abstract Background: Neuroblastoma is the most common extracranial solid malignancy in children, comprising 15% of cancer related deaths. Despite intensive treatment, patients with high-risk neuroblastoma (HR-NB) have a survival rate of ~50%, largely due to intrinsic or acquired drug resistance. The multidrug transporter P-glycoprotein (P-gp; ABCB1) effluxes several conventional agents used in HR-NB induction therapy, including doxorubicin, vincristine and etoposide, as well as the ALK inhibitor crizotinib. We observed high P-gp expression in HR-NB cell lines and patient-derived xenograft (PDX) models, so sought to assess the prevalence of P-gp expression and its role in resistance to standard-of-care chemotherapies and relevant targeted agents. Methods and Results: Using RNA-sequencing, immunohistochemistry and western blot on panels of neuroblastoma tumour samples, PDX models and cell lines, we demonstrated that high ABCB1/P-gp expression is frequent in HR-NB. Analysis of ABCB1 levels in large patient tumor datasets suggests that high expression is attributable to the sympathoadrenal lineage of the disease, that high expression is more common in HR-NB than in most other cancers, and that high relative expression of ABCB1 in HR-NB tumours is associated with poorer outcome, consistent with its multidrug transporter function. We demonstrated that the P-gp inhibitor tariquidar and P-gp knockdown (shRNA) both strongly sensitize cultured high P-gp expressing neuroblastoma cells to vincristine, doxorubicin and etoposide but not to ALK inhibitors. Further, P-gp knockdown sensitized human neuroblastoma xenografts to vincristine, substantially extending survival. Conclusions: Elevated P-gp expression is common in HR-NB and can be sufficient to confer resistance to standard-of-care chemotherapies in model systems. Our findings suggest that tumour P-gp levels might be used to guide treatment options for individual patients, and to avoid ineffective treatments. The potential of P-gp inhibitors as adjuncts to conventional chemotherapy for HR-NB should be further investigated. Citation Format: Caroline Atkinson, Carole M. Tactacan, Alvin Kamili, Federica Saletta, Christine Gana, Georgina L. Eden, Chelsea Mayoh, Richard B. Lock, Murray D. Norris, Michelle Haber, Andrew J. Gifford, Toby N. Trahair, Jamie I. Fletcher. Re-evaluating the role of P-glycoprotein in the resistance of high-risk neuroblastoma to standard-of-care chemotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4741.

Abstract 2881: Antiproliferative effect of copper complex of clotam in neuroblastoma cell lines

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor in infants and young children. NB often metastasizes to lymph nodes and bone marrow. When compared to low or intermediate risk tumors, high risk neuroblastoma (HRNB) is more aggressive with low (40-50%) 5 year survival rates. Furthermore, current intensive treatment methods induce higher toxicity and side effects. In search of identifying less-toxic agents to induce anti-cancer activity in NB cells, our group reported the anti-cancer activity of a non-steroidal anti-inflammatory drug, Clotam (TA) using HRNB cell lines potentially inhibiting the transcription factor, Specificity protein 1 (Sp1) and an inhibitor of apoptosis protein, survivin. Recently, our laboratories tested a copper complex of TA (Cu-TA) using 12 cell lines and found that Cu-TA’s IC50 values were 30-80% lower when compared to TA. In this study, we investigated the cytotoxicity of SH-SY 5Y, LA1-55n and SMS-KCNR cells in the presence of vehicle (Dimethyl sulfoxide) or TA or Cu-TA using CellTiter-Glo kit and determined the IC50 values using SigmaPlot software. The effect of TA or Cu-TA on apoptosis and cell cycle phase distribution was evaluated by flow cytometry using Annexin-V and Propidium Iodide kits respectively. The characterization and stability of the Cu-TA was also determined. SH SY5Y and SMS-KCNR cells were treated with IC50 values of TA or Cu-TA and the expression of Sp1 and survivin was measured by qPCR and Western blot analysis. The results showed that while both TA and Cu-TA inhibited NB cell growth, Cu-TA’s IC50 values were 60-70% less than TA. The inhibition of NB cells with Cu-TA was accompanied by induced apoptotic cell population and cell cycle arrest at G2M phase. Western blot and qPCR results confirmed that Cu-TA inhibited the expression of both Sp1 and survivin with predominant effect on SMS-KCNR cells. These results demonstrate that Cu-TA is more effective than TA for inhibiting Sp1 and survivin in NB cells, which was consistent with the anti-proliferative activity against NB cells. Since TA and Cu-TA are less toxic than standard chemotherapeutic agents, employing these agents for the treatment of pediatric malignancies could be useful. Eventually, combination of Cu-TA and chemotherapeutic agents will be tested. If Cu-TA sensitizes NB cells to chemotherapy, it will help to address the issues associated with the side effects of chemotherapy. Citation Format: Yazmin Hernandez, Sarah Sarah Grebennikov, Emily Zakhary, Ashni Dudhia, Rajasekhar Maram, Diego Casanova, Umesh T. Sankpal, W. Paul Bowman, Deondra T. Brown, Jaya Chhabra, Alvin A. Holder, Riyaz M. Basha. Antiproliferative effect of copper complex of clotam in neuroblastoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2881.

Telomere Trimming and DNA Damage as Signatures of High Risk Neuroblastoma

Telomeres play important roles in genome stability and cell proliferation. High risk neuroblastoma (HRNB), an aggressive childhood cancer, is especially reliant on telomere maintenance. Three recurrent genetic aberrations in HRNB (MYCN amplification, TERT re-arrangements, and ATRX mutations) are mutually exclusive and each capable of promoting telomere maintenance mechanisms (i.e., through telomerase or ALT). We analyzed a panel of 5 representative HRNB cell lines and 30 HRNB surgical samples using assays that assess average telomere lengths, length distribution patterns, single-stranded DNA on the G- and C-strand, as well as extra-chromosomal circular telomeres. Our analysis pointed to substantial and variable degrees of telomere DNA damage in HRNB, including pervasive oxidative lesions. Moreover, unlike other cancers, neuroblastoma consistently harbored high levels of C-strand ssDNA overhangs and t-circles, which are consistent with active “telomere trimming”. This feature is observed in both telomerase- and ALT-positive tumors and irrespective of telomere length distribution. Moreover, evidence for telomere trimming was detected in normal neural tissues, raising the possibility that TMMs in HRNB evolved in the face of a canonical developmental program of telomere shortening. Telomere trimming by itself appears to distinguish neuroectodermal derived tumors from other human cancers, a distinguishing characteristic with both biologic and therapeutic implications.

Clinically Relevant Biomarker Discovery in High-Risk Recurrent Neuroblastoma.

Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High-risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. The need for biomarkers that can select patients not responding well to therapy in an early phase is therefore needed. In this study, we used next generation sequencing technology to determine the expression profiles in high-risk neuroblastoma cell lines established before and after therapy. Using partial least squares-discriminant analysis (PLS-DA) with least absolute shrinkage and selection operator (LASSO) and leave-one-out cross-validation, we identified a panel of 55 messenger RNAs and 17 long non-coding RNAs (lncRNAs) which were significantly altered in the expression between cell lines isolated from primary and recurrent tumors. From a neuroblastoma patient cohort, we found 20 of the 55 protein-coding genes to be differentially expressed in patients with unfavorable compared with favorable outcome. We further found a twofold increase or decrease in hazard ratios in these genes when comparing patients with unfavorable and favorable outcome. Gene set enrichment analysis (GSEA) revealed that these genes were involved in proliferation, differentiation and regulated by Polycomb group (PcG) proteins. Of the 17 lncRNAs, 3 upregulated (NEAT1, SH3BP5-AS1, NORAD) and 3 downregulated lncRNAs (DUBR, MEG3, DHRS4-AS1) were also found to be differentially expressed in favorable compared with unfavorable outcome. Moreover, using expression profiles on both miRNAs and mRNAs in the same cohort of cell lines, we found 13 downregulated and 18 upregulated experimentally observed miRNA target genes targeted by miR-21, -424 and -30e, -29b, -138, -494, -181a, -34a, -29b, respectively. The advantage of analyzing biomarkers in a clinically relevant neuroblastoma model system enables further studies on the effect of individual genes upon gene perturbation. In summary, this study identified several genes, which may aid in the prediction of response to therapy and tumor recurrence.

Abstract 3190: DFMO preferentially drives cancer stem cells in neuroblastoma towards senescence

Abstract Background: High-risk neuroblastoma (HRNB) is an aggressive form of the most common extracranial solid tumor cancer in children, often characterized by amplified levels of MYCN. HRNB patients in remission have a high relapse rate, resulting in poor survival. Recent studies have shown that cancer stem cells (CSCs) play a role in the progression, chemoresistance, and relapse of many cancers. Difluoromethylornithine (DFMO) is being evaluated to prevent relapse in clinical trials. Our current study examines the in vitro and in vivo effects of DFMO on tumor-initiating cells through evaluating CSC viability, CSC markers and senescence. Methods: BE(2)-C and SMS-KCNR established HRNB cell lines were injected subcutaneously at limiting cell dilutions into nude mice. DFMO was administered in the drinking water (2%, by volume) for 50 and 75 days for BE(2)-C and SMS-KCNR, respectively. Tumor formation was monitored, and LDA was performed. In a second xenograft model BE(2)-C cells were injected into nude mice, once tumors were palpable, the mice were given either normal drinking water (vehicle) or DFMO (2%)-containing water. Following 7 days of treatment, the tumors were resected and measured for volumes/weights as well as the harvested for Western blot analysis of CSC markers (OCT4, SOX2, NANOG, KLF4). BE(2)-C cells were cultured for 48 and 72h with or without 5mM DFMO and stained for SA-βGal activity as a marker of senescence. NB cell lines treated for 48 and 72h with a range of DFMO concentrations (0, 10μM, 50μM, 100μM, 1mM, 2.5mM, and 5mM) were harvested and labeled with cell surface antibodies for CSCs (CD133, CD114), senescence (CD148), differentiation (CD24), and NB (CD56) for 30min in the dark, and cell viability was determined by 7AAD staining immediately before flow cytometric analysis. Results: Limiting dilution analysis (LDA) on xenograft mice receiving DFMO showed both prevention of tumor formation (p value&amp;lt;0.001) and a decrease in frequency of tumor-initiating cells (p value p&amp;lt;0.04). Tumors harvested from mice treated with DFMO for 7 days showed decrease in volume and weight relative to control (p value &amp;lt;0.001) as well as a decrease in the presence of CSC markers shown by Western blot. Galactosidase (SA-βGal) staining of cultured HRNB cells showed the induction of senescence in HRNB cells when treated with DFMO by 72h (p value &amp;lt;0.001). Flow cytometric analysis determined that DFMO specifically targeted the CSC subpopulation at as low as 50uM concentrations (100uM p value&amp;lt;0.05); CSCs had decreased viability and higher levels of senescence-associated protein expression than the overall NB population (p value&amp;lt;0.05). Conclusions: Our results show the preferential induction of senescence and overall decrease in the CSC population at very low doses of DFMO. This may expose a novel treatment mechanism to inhibit CSC function and prevent relapse in HRNB patients. Citation Format: Tracey Avequin, Austin Goodyke, Joseph Zagorski, Tyler Maser, Giselle L. Saulnier Sholler. DFMO preferentially drives cancer stem cells in neuroblastoma towards senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3190.

Abstract 5197: Targeting PARP-1 to deliver alpha-particles to cancer chromatin

Abstract Introduction: Neuroblastoma (NB) is a radiosensitive pediatric cancer that develops in the sympathetic nervous system and typically affects children under the age of 10. High-risk NB is associated with a 40% 5-year survival rate. Nuclear enzyme poly (ADP-ribose) polymerase 1 (PARP-1) is overexpressed in high-risk NB, making it an attractive target for alpha-particle therapy. Alpha-particles have a short path length and high linear energy transfer, causing dense ionizations across DNA, inducing double stranded breaks that result in cell death. The purpose of this study was to explore a newly developed radiotherapeutic ([211At]MM4) that combines the targeting potential of a small molecule PARP inhibitor (PARPi) with the cytotoxic effects of 211At in high-risk NB. Methods: In vitro cytotoxicity was performed in a panel of high-risk NB cell lines to evaluate the relative potency of [211At]MM4. Next, DNA damage was assessed by measuring gH2AX foci formation at 1, 4, and 24 hrs after [211At]MM4 treatment. In parallel, PARP-1 expression was measured in response to therapy and cleaved PARP-1 was quantified to assess apoptosis. Cell cycle analysis was performed after treatment to identify therapy related effects. The in-vivo biodistribution of [211At]MM4 was performed alongside ex-vivo autoradiography. Tumor cytology for PARP-1, gH2AX, and Ki-67 was performed in response to therapy. Finally, in-vivo therapy experiments were performed. Results: Cytotoxicity data indicated a significant reduction in cell viability following treatment for all six NB cell lines, in-vitro co-incubation studies confirmed the specificity of [211At]-MM4 to its drug target. Immunofluorescence analysis showed a dose-dependent increase in both gH2AX and PARP-1 expression. The in-vivo biodistribution of [211At]MM4 revealed rapid tumor targeting at 1 hr and clearance from all tissues at 4 hrs. Ex-vivo autoradiography showed a tumor-muscle ratio greater than 6. Tumor cytology revealed DNA damage measured by gH2AX and PARP-1 expression increased following treatment. Small colonies of proliferating tumor cells were detected after treatment using Ki-67 staining. In-vivo therapy efficacy studies revealed low fractionated doses were tolerable and resulted in significant delay in tumor regrowth. Conclusion: [211At]MM4 is a novel alpha-emitting radiotherapeutic that specifically targets nuclear PARP-1 overexpression in neuroblastoma and incites double-stranded breaks in cancer DNA. The cytotoxic effects of [211At]MM4 have been experimentally validated both in-vitro and in-vivo; the results of these experiments confirm the therapeutic potential of [211At]MM4 as a viable treatment option for high-risk neuroblastoma. Citation Format: Laura Puentes, Kuiying Xu, Catherine Hou, Robert H. Mach, John M. Maris, Daniel A. Pryma, Mehran Makvandi. Targeting PARP-1 to deliver alpha-particles to cancer chromatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5197. doi:10.1158/1538-7445.AM2017-5197

Translational compensation of genomic instability in neuroblastoma.

Cancer-associated gene expression imbalances are conventionally studied at the genomic, epigenomic and transcriptomic levels. Given the relevance of translational control in determining cell phenotypes, we evaluated the translatome, i.e., the transcriptome engaged in translation, as a descriptor of the effects of genetic instability in cancer. We performed this evaluation in high-risk neuroblastomas, which are characterized by a low frequency of point mutations or known cancer-driving genes and by the presence of several segmental chromosomal aberrations that produce gene-copy imbalances that guide aggressiveness. We thus integrated genome, transcriptome, translatome and miRome profiles in a representative panel of high-risk neuroblastoma cell lines. We identified a number of genes whose genomic imbalance was corrected by compensatory adaptations in translational efficiency. The transcriptomic level of these genes was predictive of poor prognosis in more than half of cases, and the genomic imbalances found in their loci were shared by 27 other tumor types. This homeostatic process is also not limited to copy number-altered genes, as we showed the translational stoichiometric rebalance of histone genes. We suggest that the translational buffering of fluctuations in these dose-sensitive transcripts is a potential driving process of neuroblastoma evolution.

Combination of 13 cis-retinoic acid and tolfenamic acid induces apoptosis and effectively inhibits high-risk neuroblastoma cell proliferation

Chemotherapeutic regimens used for the treatment of Neuroblastoma (NB) cause long-term side effects in pediatric patients. NB arises in immature sympathetic nerve cells and primarily affects infants and children. A high rate of relapse in high-risk neuroblastoma (HRNB) necessitates the development of alternative strategies for effective treatment. This study investigated the efficacy of a small molecule, tolfenamic acid (TA), for enhancing the anti-proliferative effect of 13 cis-retinoic acid (RA) in HRNB cell lines. LA1-55n and SH-SY5Y cells were treated with TA (30μM) or RA (20μM) or both (optimized doses, derived from dose curves) for 48h and tested the effect on cell viability, apoptosis and selected molecular markers (Sp1, survivin, AKT and ERK1/2). Cell viability and caspase activity were measured using the CellTiter-Glo and Caspase-Glo kits. The apoptotic cell population was determined by flow cytometry with Annexin-V staining. The expression of Sp1, survivin, AKT, ERK1/2 and c-PARP was evaluated by Western blots. The combination therapy of TA and RA resulted in significant inhibition of cell viability (p<0.0001) when compared to individual agents. The anti-proliferative effect is accompanied by a decrease in Sp1 and survivin expression and an increase in apoptotic markers, Annexin-V positive cells, caspase 3/7 activity and c-PARP levels. Notably, TA+RA combination also caused down regulation of AKT and ERK1/2 suggesting a distinct impact on survival and proliferation pathways via signaling cascades. This study demonstrates that the TA mediated inhibition of Sp1 in combination with RA provides a novel therapeutic strategy for the effective treatment of HRNB in children.

Targeting histone deacetylases (HDACs) and Wee1 for treating high-risk neuroblastoma.

Despite advances in treatment regimens, patients with high-risk neuroblastoma have long-term survival rates of < 40%. Wee1 inhibition in combination with CHK1 inhibition has shown promising results in neuroblastoma cells. In addition, it has been demonstrated that panobinostat can downregulate CHK1. Therefore, combination of panobinostat and MK-1775 may result in synergistic cytotoxicity against neuroblastoma cell lines. In vitro cytotoxicities of panobinostat and MK-1775 at clinically achievable concentrations, either alone or in combination, were evaluated in SK-N-AS, SK-N-DZ, and SK-N-BE(2) high-risk neuroblastoma cell lines using MTT assays. The mechanism of antitumor interaction was investigated using propidium iodide (PI) staining and flow cytometry analysis to determine apoptosis, as well as Western blotting to assess expression of phosphorylated CDK1/2, CHK1, and H2AX. Treatment of neuroblastoma cell lines with 500 nM MK-1775 caused growth arrest and apoptosis in SK-N-DZ and SK-N-AS, while it had minimal effect on the SK-N-BE(2) cell line. The combination of panobinostat and MK-1775 resulted in synergistic antitumor interactions in all three of the cell lines tested. MK-1775 treatment in SK-N-BE(2) cells induced increased levels of p-CHK1(S345) , which could be decreased by the addition of panobinostat. This was accompanied by increased DNA damage and apoptosis. The combination of panobinostat and MK-1775 has synergistic antitumor activity against neuroblastoma cell lines and holds promise as a potential treatment strategy for the management of high-risk neuroblastoma patients.

Panobinostat Synergistically Enhances the Cytotoxic Effects of Cisplatin, Doxorubicin or Etoposide on High-Risk Neuroblastoma Cells

High-risk neuroblastoma remains a therapeutic challenge with a long-term survival rate of less than 40%. Therefore, new agents are urgently needed to overcome chemotherapy resistance so as to improve the treatment outcome of this deadly disease. Histone deacetylase (HDAC) inhibitors (HDACIs) represent a novel class of anticancer drugs. Recent studies demonstrated that HDACIs can down-regulate the CHK1 pathway by which cancer cells can develop resistance to conventional chemotherapy drugs. This prompted our hypothesis that combining HDACIs with DNA damaging chemotherapeutic drugs for treating neuroblastoma would result in enhanced anti-tumor activities of these drugs. Treatment of high-risk neuroblastoma cell lines with a novel pan-HDACI, panobinostat (LBH589), resulted in dose-dependent growth arrest and apoptosis in 4 high-risk neuroblastoma cell lines. Further, the combination of panobinostat with cisplatin, doxorubicin, or etoposide resulted in highly synergistic antitumor interactions in the high-risk neuroblastoma cell lines, independent of the sequence of drug administration. This was accompanied by cooperative induction of apoptosis. Furthermore, panobinostat treatment resulted in substantial down-regulation of CHK1 and its downstream pathway and abrogation of the G2 cell cycle checkpoint. Synergistic antitumor interactions were also observed when the DNA damaging agents were combined with a CHK1-specific inhibitor, LY2603618. Contrary to panobinostat treatment, LY2603618 treatments neither resulted in abrogation of the G2 cell cycle checkpoint nor enhanced cisplatin, doxorubicin, or etoposide-induced apoptosis in the high-risk neuroblastoma cells. Surprisingly, LY2603618 treatments caused substantial down-regulation of total CDK1. Despite this discrepancy between panobinostat and LY2603618, our results indicate that suppression of the CHK1 pathway by panobinostat is at least partially responsible for the synergistic antitumor interactions between panobinostat and the DNA damaging agents in high-risk neuroblastoma cells. The results of this study provide a rationale for clinical evaluation of the combination of panobinostat and cisplatin, doxorubicin, or etoposide for treating children with high-risk neuroblastoma.