Preclinical Neuroblastoma Models Research Articles

Abstract B34: Antitumor activity and sensitivity evaluation of novel BET inhibitors in neuroblastoma

Abstract Children with neuroblastoma exhibit marked variability in outcome based on age at diagnosis, disease stage and tumor biologic characteristics. Genomic amplification of MYCN is a potent oncogenic driver and negative prognostic marker in neuroblastoma patients. Therefore, there is a pressing need to identify therapeutic agents that target MYCN gene expression. The bromodomain and extra-terminal (BET) family of proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Previous reports demonstrate that BET inhibition (BETi) significantly attenuates cellular proliferation in numerous cancer models, perhaps via modulation of the MYC and/or MYCN oncogenes. Here, we show that potent BET inhibition induces anti-tumor effects in preclinical neuroblastoma models. Specifically, GSK726 and GSK762 (GlaxoSmithKline) were used for in vitro cytotoxicity and in vivo therapeutic studies, respectively. Neuroblastoma cell lines (n=22) were treated with GSK726 to calculate IC50 values in a luminescence-based cell viability assay and differential sensitivity was observed with an IC50 range of 27 nM to 9.5 µM. In sensitive cell lines, cell cycle distribution and induction of apoptosis were also measured. In these cell lines, GSK726 treatment resulted in MYCN depletion, G1 arrest within 24 hours, and apoptosis as measured by cleaved-PARP. To assess in vivo efficacy, GSK762 was subcutaneously administered in xenograft models and in genetically engineered neuroblastoma mouse models overexpressing MYCN and MYCN/ALK F1174L in the neural crest. In both models, GSK762 treatment resulted in tumor growth delay. Further assessment of results seen in vitro and in vivo indicated that MYCN amplification status did not fully predict sensitivity to GSK726 or GSK762. Thus, to determine additional biomarkers of sensitivity, we examined baseline gene expression data at the extremes of IC50 values by comparing sensitive (n=6; IC50<128 nM) and resistant (n=4; IC50>940 nM) neuroblastoma cell lines. Gene expression data from the neuroblastoma cell lines generated on HuGene1.0ST expression microarrays (Affymetrix) were utilized, and data were analyzed using the Limma package in R/Bioconductor. Univariate analysis, with a false discovery rate less than 0.25, revealed 6 genes (PTER, PCDHB14, RFTN1, JAK2, MYCBP, and DACH1) that were differentially expressed between sensitive and resistant cell lines in the MYCN amplified setting. While all 6 genes predicted BETi sensitivity in the MYCN-amplified subset of cell lines, only DACH1 expression predicted sensitivity to BET inhibition irrespective of MYCN amplification status (p=7.92x10-7). In addition, high DACH1 expression was correlated with poor patient outcome (p=1.74x10-5). As a result, high DACH1 expression serves as a candidate biomarker for future studies with respect to sensitivity to BETi. Ultimately, these studies will help to optimize the clinical utility of BETi in neuroblastoma and perhaps other MYC-driven malignancies. Citation Format: Jason R. Healy, Robert W. Schnepp, Lori S. Hart, Pichai Raman, Laura Danielson, Michael Russell, Priya Khurana, Maria Gagliardi, Ryan M. Kinsey, Anastasia Wyce, Olena Barbash, Peter J. Tummino, Louis Chesler, John M. Maris. Antitumor activity and sensitivity evaluation of novel BET inhibitors in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B34.

Abstract 4305: NMI as a biomarker of response to CDK4/6 inhibition in a preclinical model of neuroblastoma

Abstract Background Hyper-activation of the Cyclin D-CDK4/6 signaling pathway is a common feature of neuroblastoma. Previously, it has been shown that while a majority of preclinical models of neuroblastoma are highly sensitive to pharmacologic inhibition of CDK4/6 by LEE011 (Novartis Oncology), a subset are remarkably resistant. In an effort to aid in the selection of a patient population that will best benefit from this therapy, we sought to identify genomic biomarkers of response to CDK4/6 inhibition. Methods LEE011 IC50 values were determined using the RT-CES cell impedance assay, and gene expression was established at baseline using HuGene 1.0ST microarrays (Affymetrix). Expression data was then coupled with IC50 values to identify a set of genes significantly associated with LEE011 response via Pearson correlation. Using this gene set, a Lasso linear regression model was built in order to predict sensitivity to LEE011 based upon the expression of a subset of genes chosen to be most predictive of response. The model was tested in a separate cohort of neuroblastoma cell lines by comparing predicted responses to LEE011 with experimentally determined LEE011 IC50 values. Functional RNAi and over-expression studies are ongoing in order to validate genes identified to be correlated with or predictive of response. Results The Lasso model was able to predict the response of neuroblastoma cell lines to LEE011 based upon the expression pattern of 11 genes. A comparison of Lasso predictions to the LEE011 IC50 values of a separate panel of cell lines yielded a correlation coefficient of 0.63. The correlation of predicted to actual response across our entire cell line panel was 0.96, suggesting that response to CDK4/6 inhibition by LEE011 can be accurately predicted based on the expression of these genes. Of the 11 genes, NMI (N-myc and stat interactor) was prioritized for further validation and functional studies as it was identified by Pearson correlation to be the third most correlated gene with response (r = 0.86; p = 3.1×10-7) and because it interacts with MYCN, a known prognostic indicator in neuroblastoma. We next showed that NMI was expressed higher at both the mRNA and protein levels in neuroblastoma cell lines resistant (N = 6) to LEE011 compared to those demonstrating sensitivity to the compound (N = 16). Studies are ongoing to confirm the role of NMI as a predictor of resistance as well as to assess its contribution, if any, in desensitizing neuroblastoma to CDK4/6 inhibition. Conclusions We have identified NMI as a candidate biomarker of resistance to CDK4/6 inhibition in preclinical models of neuroblastoma. Further validation of this gene as a biomarker may ultimately help drive clinical decisions in selecting patients who would most benefit from CDK4/6 inhibition therapies, as well as in identifying novel drugs to combine with LEE011 for the treatment of neuroblastoma patients. Note: This abstract was not presented at the meeting. Citation Format: JulieAnn Rader, Pichai Raman, Lori Hart, Mike Russell, Kristina A. Cole, John M. Maris. NMI as a biomarker of response to CDK4/6 inhibition in a preclinical model of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4305. doi:10.1158/1538-7445.AM2015-4305

Abstract 5333: TH-302 potentiates the antitumor activity of topotecan in neuroblastoma and rhabdomyosarcoma preclinical models

Abstract Background: There is strong evidence that tumor cells in the hypoxic regions cause drug resistance and tumor relapse. Hypoxia targeting agents may make cytotoxic therapy more effective. In this study, we investigated the efficacy of TH-302, a hypoxia-activated prodrug of bromo-isophosphoramide mustard, both alone and in combination with the cytotoxic agent topotecan, in preclinical models of neuroblastoma (NBL) and rhabdomyosarcoma (RMS). Materials and methods: A panel of four NBL and three RMS cell lines was tested in vitro under normoxic or hypoxic conditions to assess the effect of TH-302 on cell proliferation, both as a single agent and in combination with topotecan. In vivo activity was also evaluated in different xenograft models. TH-302 (50 mg/kg) and topotecan (1 mg/kg) were administered daily for 2 consecutive weeks, as single agents or in combination. Animal survival was studied with an NBL (SK-N-BE(2)) metastatic tumor model. Cleaved caspase-3 and the hypoxia staining agent pimonidazole were used as apoptotic and hypoxic markers in vivo. Results: Under normoxic conditions, all tested lines showed an anti-proliferative response to TH-302 with the IC50s ranging from 4.6 to 52μM. When exposed to hypoxia (1% O2) overnight, a dramatic decrease of IC50 was observed, ranging from 0.07 to 2.4μM. Adding topotecan to the TH-302 treatment significantly increased cytotoxicity in all tested tumor cell lines. In both NBL models (SK-N-BE(2) and CHLA-20), single-agent TH-302 treatment delayed tumor growth. Complete tumor regression was observed in the combined treatment group after a two-week treatment period, but all the regressed tumors relapsed after treatment ended. In the CHLA-20 xenograft model, single-agent topotecan also induced complete tumor regression. However, median time to relapse in the combination group was significantly longer than in the topotecan group (43 days vs. 16 days; p<0.01). In the combination group, all relapsed tumors remained responsive to the drug treatment when re-challenged with the original treatment regimen. In the neuroblastoma metastatic model, all three regimens prolonged animal survival, but the combined treatment group showed better survival than single-agent treatments (p<0.01). In a RMS xenograft model, combined treatment was also more effective than single-agent TH-302 or topotecan. Double immunostaining of apoptotic and hypoxic markers showed that TH-302 mostly targeted tumor cells residing at hypoxic regions while topotecan was more effective on tumor cells outside the hypoxic zones. The combined treatment induced tumor cell apoptosis in both normoxic and hypoxic regions. Conclusions: TH-302 showed antitumor effects in neuroblastoma and rhabdomyosarcoma xenograft models. Compared to single-agent TH-302 or topotecan, the combination treatment significantly improved tumor response, delayed tumor relapse, and enhanced animal survival. Citation Format: Libo Zhang, Bing Wu, Paula Marrano, Paul Thorner, Sylvain Baruchel. TH-302 potentiates the antitumor activity of topotecan in neuroblastoma and rhabdomyosarcoma preclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5333. doi:10.1158/1538-7445.AM2015-5333

Newimmunotherapeutic strategies for the treatment of neuroblastoma.

The prognosis of high-risk neuroblastoma (NB) is still poor, in spite of aggressive multimodal treatment. Recently, adjuvant immunotherapy with anti-GD2 antibodies combined with IL-2 or GM-CSF has been shown to improve survival. Several other immunotherapy strategies proved efficacy in preclinical models of NB, including different types of vaccines, adoptive cell therapies and combined approaches. The remarkable differences in the immunobiology of syngeneic models and human NB may, at least in part, limit the translation of preclinical therapies to a clinical setting. Nonetheless, several preliminary evidences suggest that new antibodies, cancer vaccines and adoptive transfer of lymphocytes, genetically engineered to acquire NB specificity, may result in clinical benefit, and clinical studies are currently ongoing.

422 CBL0137, a novel NFkB suppressor and p53 activator, is highly effective in pre-clinical models of neuroblastoma

422 CBL0137, a novel NFkB suppressor and p53 activator, is highly effective in pre-clinical models of neuroblastoma

Abstract B82: MEK 1/2 inhibition and biomarkers of response in preclinical models of neuroblastoma

Abstract Background: Despite an aggressive treatment regime, 40% of patients with high-risk neuroblastoma die of their disease. The mitogen-activated protein kinase (MAPK) signaling cascade is hyperactive in many cancers, including a subset of high-risk neuroblastomas. We, therefore, sought to determine the efficacy of MEK inhibition in neuroblastoma and develop a responder hypothesis of anti-tumor activity based on molecular genetic features. Methods: MEK162 (Novartis Pharmaceuticals), an orally available MEK1/2 inhibitor, is currently in adult clinical trials for advanced malignancies harboring RAS/RAF mutations. In determining MEK162 IC50 values, we employed the RT-CES cell impedance assay as it measures both cytostatic and cytotoxic responses in a temporal manner. Additional in vitro analysis of MEK162 activity included immunoblotting, cell cycle analysis with flow cytometric methods, and phosphorylation arrays across a panel of 23 neuroblastoma cell lines. Four of these cell lines (2 sensitive and 2 resistant) were also tested in vivo in subcutaneous xenograft models with tumor volume endpoints. The cell line IC50 ranking was cross-referenced with HuGene1.0ST expression microarrays (Affymetrix), phosphorylation arrays (Full Moon Biosystems), and exome sequencing in order to identify the genetic and proteomic underpinnings predictive of response to MEK inhibition. Microarray analysis was performed using the Limma package (Bioconductor/R), unsupervised clustering, and gene set enrichment (GSEA). Results: Neuroblastoma cell lines (N=23) showed a wide range of sensitivity to MEK162 across a 4-log dose range (median IC50 = 771 nM, range 5 nM-10 μM). Sensitive cell lines demonstrated G1 arrest within 24 hours of exposure to MEK162. In vivo subcutaneous xenograft experiments recapitulated the in vitro response of MEK162 sensitivity in each of the cell lines tested. In order to identify determinants of sensitivity, we profiled the genomic signature of 23 cell lines in the exponential growth phase prior to confluence. Of the ten most sensitive cell lines, six possessed mutations predicted to be damaging and indicative of RAS/MAPK pathway hyperactivation (NRAS-Q61K, NF1 copy number loss, KRAS-G12D). No resistant lines possessed genetic evidence of RAS hyperactivation. However, baseline phosphorylated-ERK status was not robustly predictive of drug activity. For the purpose of biomarker discovery, we considered a total of 4 cells lines as sensitive (IC50< 200 nM) and 4 resistant (IC50> 3,000 nM). Unsupervised clustering and GSEA of the most differentially expressed genes derived from comparing sensitive (N=4) and resistant (N=4) cell lines confirmed increased baseline MAPK pathway activity in the sensitive cell lines. In addition, resistance to MEK162 was found to be associated with MYCN expression (R=.70/p=0.00043), suggesting that MYCN may serve as a biomarker of MEK162 resistance. Independently, we have demonstrated a correlation between MYCN-amplification and sensitivity to CDK4/6 inhibition (p=0.0227), suggesting the combination of MEK and CDK4/6 inhibition may synergize in the inhibition of neuroblastoma growth. Conclusions: MEK inhibition is effective in a definable subset of human-derived neuroblastoma preclinical models. Canonical activation of the RAS/MAPK pathway via RAS mutation and/or NF1 inactivation partially account for sensitivity to MEK inhibition, however, biomarkers predictive of sensitivity to MEK inhibition remain to be identified. We demonstrate that both MYCN expression and CDK4/6 activity are associated with MEK resistance, providing a basis for defining MEK162 combination strategies in the treatment of neuroblastoma. Citation Format: Lori S. Hart, Vandana Batra, Pichai Raman, Maria Gagliardi, JulieAnn Rader, Lucy Chen, Christine Fritsch, Giordano Caponigro, Malte Peters, Markus Boehm, John M. Maris. MEK 1/2 inhibition and biomarkers of response in preclinical models of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B82.

Abstract B75: Defining the antitumor activity and sensitivity profiles of BET inhibitors in neuroblastoma

Abstract Background: MYCN amplification is the most frequent somatically acquired genomic alteration in neuroblastoma and is a potent oncogenic driver. Targeting MYCN therapeutically has been complicated by the challenges inherent in targeting transcription factors. Recently, several groups have shown that inhibiting bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) and their ability to bind to acetylated lysine residues within histone tails resulted in silencing of MYC family protein expression and demonstrated therapeutic efficacy in preclinical cancer models. We hypothesized that potent inhibition of bromodomain-chromatin interactions would show anti-tumor activity in preclinical models of neuroblastoma and that reliable biomarkers of activity could be discovered. Methods: GlaxoSmithKline compounds GSK726 and GSK762 potently inhibit BET proteins (BETi) and were used for in vitro cytotoxicity and in vivo therapeutic studies, respectively. We exposed a panel of 15 cell lines to a 4-log dose range of GSK726 to determine IC50s in a luminescence-based cell viability assay. We also measured cell cycle changes by flow cytometry and apoptosis by immunoblotting for cleaved PARP. We tested the in vivo efficacy of the GSK762 in subcutaneous xenograft models (3 sensitive and 2 resistant cell lines) and in genetically engineered neuroblastoma mouse models (overexpressing MYCN and MYCN/ALK F1174L in the neural crest). For biomarker discovery, gene expression data from the neuroblastoma cell lines generated on HuGene1.0ST expression microarrays (Affymetrix) were utilized, and data were analyzed using the Limma package in R/Bioconductor. Results: Neuroblastoma cell lines (N=15) were differentially sensitive to GSK726, with a median IC50 of 128nM and a range of 17nM to >10uM. In sensitive cell lines, GSK726 treatment resulted in MYCN depletion, G1 arrest within 24 hours, and apoptosis. In subcutaneous xenograft models and genetically engineered neuroblastoma mouse models, GSK762 treatment resulted in tumor growth delay. Importantly, MYCN amplification status did not fully predict sensitivity to GSK726 or GSK762. Thus, to determine additional biomarkers of sensitivity, we examined baseline gene expression data at the extremes of IC50s by comparing sensitive (N=6; IC50<128 nM) and resistant (N=4; IC50>940 nM) neuroblastoma cell lines. Univariate analysis, with an FDR < 0.25, revealed 6 genes (PTER, PCDHB14, RFTN1, JAK2, MYCBP, and DACH1) differentially expressed between sensitive and resistant cell lines in the MYCN amplified setting. While all 6 genes predicted BETi sensitivity in the MYCN-amplified subset of cell lines, only DACH1 expression predicted sensitivity to BET inhibition irrespective of MYCN amplification status (7.92x10-7). Moreover, DACH1 levels were heterogeneously expressed in a panel of 88 primary neuroblastoma tumors (Versteeg-88) and high expression was correlated with poor patient outcome (1.74x10-5). Conclusions: BET inhibitors demonstrated significant anti-tumor activity in a subset of neuroblastoma preclinical models. Although MYCN protein levels decreased in neuroblastoma cell lines that were sensitive to BETi, MYCN amplification status alone did not fully explain BETi sensitivity. DACH1 expression serves as a candidate biomarker for sensitivity to BET inhibition. These studies will help to optimize the clinical utility of BETi in neuroblastoma and perhaps other MYC-driven malignancies. Citation Format: Robert Schnepp, Lori Hart, Pichai Raman, Laura Danielson, Maria Gagliardi, Ryan Kinsey, Anastasia Wyce, Olena Barbash, Peter Tummino, Louis Chesler, John Maris. Defining the antitumor activity and sensitivity profiles of BET inhibitors in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B75.

Aurora kinases as targets in drug-resistant neuroblastoma cells.

Aurora kinase inhibitors displayed activity in pre-clinical neuroblastoma models. Here, we studied the effects of the pan-aurora kinase inhibitor tozasertib (VX680, MK-0457) and the aurora kinase inhibitor alisertib (MLN8237) that shows some specificity for aurora kinase A over aurora kinase B in a panel of neuroblastoma cell lines with acquired drug resistance. Both compounds displayed anti-neuroblastoma activity in the nanomolar range. The anti-neuroblastoma mechanism included inhibition of aurora kinase signalling as indicated by decreased phosphorylation of the aurora kinase substrate histone H3, cell cycle inhibition in G2/M phase, and induction of apoptosis. The activity of alisertib but not of tozasertib was affected by ABCB1 expression. Aurora kinase inhibitors induced a p53 response and their activity was enhanced in combination with the MDM2 inhibitor and p53 activator nutlin-3 in p53 wild-type cells. In conclusion, aurora kinases are potential drug targets in therapy-refractory neuroblastoma, in particular for the vast majority of p53 wild-type cases.

455P - Phase I Study of Lee011 (Cdk4/6 Inhibitor) in Patients with Malignant Rhabdoid Tumors, Neuroblastoma, and Cyclin D–Cdk4/6 Pathway-Activated Tumors

ABSTRACT Aim: SMARCB1 mutations are observed in the majority of malignant rhabdoid tumors (MRTs) rendering them dependent on cyclin D1 (CCND1) for genesis and survival. Genetic aberrations in CCND1 and CDK4 are frequent in neuroblastoma cell lines. LEE011 is an orally bioavailable, selective inhibitor of CDK4/6 that has demonstrated tumor growth inhibition in MRT and neuroblastoma preclinical models. In this Phase I study, LEE011 is being investigated in patients (pts) with MRT, neuroblastoma, or other cancers with documented cyclin D–CDK4/6–INK4a–Rb pathway aberrations. Methods: Pts (aged 1–21 years) receive escalating once-daily doses of LEE011 on a 3-weeks-on, 1-week-off schedule. Dose escalation is guided by a Bayesian Logistic Regression Model with overdose control principle and real-time PK. Primary objective: MTD and/or recommended dose for expansion (RDE) determination. Secondary objectives: safety, PK, and efficacy. Results: As of April 8, 2014, 22 pts (11 MRT [9 primary central nervous system (CNS) and 2 extra-CNS], 10 neuroblastoma, and 1 CDK4-amplified alveolar rhabdomyosarcoma) have received LEE011: 5 at 280 mg/m2; 9 at 350 mg/m2; 8 at 470 mg/m2. The median age was 5 (1–20) years. Two DLTs were reported: 1 Grade (G)3 fatigue at 280 mg/m2; 1 G4 thrombocytopenia at 470 mg/m2. Study drug-related all-grade AEs (N = 20; ≥20%) included neutropenia (75%), leukopenia (65%), anemia (40%), thrombocytopenia (40%), lymphopenia (35%), vomiting (25%), decreased appetite (20%), electrocardiogram QT prolongation (20%), fatigue (20%), and nausea (20%). G3/4 AEs (>10%) included neutropenia (70%), leukopenia (35%), thrombocytopenia (25%), and lymphopenia (15%). Preliminary PK analysis showed that exposure following 280 and 350 mg/m2 is comparable to equivalent doses in adults and slightly higher at 470 mg/m2. LEE011 is rapidly absorbed with Tmax ranging from 1–4 hrs (median: 2–3 hrs) across dose levels. To date, best response obtained is stable disease. Conclusions: LEE011 demonstrated an acceptable safety profile and dose-dependent PK. Enrollment continues to identify the MTD/RDE and there will be expansion arms for pts with MRT or neuroblastoma at the RDE. Disclosure: S.G. Dubois: is the site PI for industry sponsored clinical trials (Merck and Novartis). He is also the study chair for a trial funded by Millennium; A. Marabelle: is a member of Advisory Boards for Novartis, BMS, Roche/Genentech, Celgene, Bayer, and Flexus Biotech; S. Modak: has served on an advisory board for Novartis for an unrelated drug; R. Kan: is an employee of and holds shares in Novartis Pharma AG A. Matano: is an employee of Novartis Pharma AG; S.G. Bhansali: is an employee of and holds shares in Novartis Pharmaceuticals Corporation; S. Parasuraman: is an employee of and holds shares in Novartis Pharma AG.All other authors have declared no conflicts of interest.

Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings

Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. Here we report the identification and characterization of novel peptide ligands specific for cells present in high-risk neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. To isolate such targeting moieties, we performed combined in vitro/ex-vivo phage display screenings on NB cell lines and on tumors derived from orthotopic mouse models of human NB.By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for their respective stromal components. Globally, we isolated 121 phage-displayed NB-binding peptides: 26 bound the primary tumor, 15 the metastatic mass, 57 and 23 their respective microenvironments. Of these, five phage clones were further validated for their specific binding ex-vivo to biopsies from stage IV NB patients and to NB tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into NB-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded liposomes led to a significant inhibition in tumor volume and enhanced survival in preclinical NB models, thereby paving the way to their clinical development.

Mechanisms of the Antitumor Activity of Human Vγ9Vδ2 T Cells in Combination With Zoledronic Acid in a Preclinical Model of Neuroblastoma

Low expression of surface major histocompatibility complex (MHC) class I molecules and defects in antigen processing machinery make human neuroblastoma (NB) cells appropriate targets for MHC unrestricted immunotherapeutic approaches. Human T-cell receptor (TCR) Vγ9Vδ2 lymphocytes exert MHC-unrestricted antitumor activity and are activated by phosphoantigens, whose expression in cancer cells is increased by aminobisphosphonates. With this background, we have investigated the in vivo anti-NB activity of human Vγ9Vδ2 lymphocytes and zoledronic acid (ZOL). SH-SY-5Y human NB cells were injected in the adrenal gland of immunodeficient mice. After 3 days, mice received ZOL or human Vγ9Vδ2 T cells or both agents by intravenous administration once a week for 4 weeks. A significantly improved overall survival was observed in mice receiving Vγ9Vδ2 T cells in combination with ZOL. Inhibition of tumor cell proliferation, angiogenesis and lymphangiogenesis, and increased tumor cell apoptosis were detected. Vγ9Vδ2 T lymphocytes were attracted to NB-tumor masses of mice receiving ZOL where they actively modified tumor microenvironment by producing interferon-γ (IFN-γ), that in turn induced CXCL10 expression in NB cells. This study shows that human Vγ9Vδ2 T cells and ZOL in combination inhibit NB growth in vivo and may provide the rationale for a phase I clinical trial in patients with high-risk NB.

Abstract 5620: Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings.

Abstract Purpose: Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. The identification of novel peptide ligands specific for cells present in high-risk neuroblastoma, a childhood tumor mostly refractory to current therapies, is needed. Experimental design: We performed combined in vitro/ex-vivo phage display screenings on human neuroblastoma cell lines and on tumors derived from orthotopic mouse models of human neuroblastoma. Binding validation and homing in vivo of selected phage clones were tested by immunohistochemistry/immunofluorescent analyses. Cell association experiments in vitro with the corresponding synthetic biotin-labeled peptides were performed. In vitro cytotoxicity and in vivo tumor accumulation and therapeutic experiments were performed using peptide-targeted, doxorubicin-loaded, nanocarriers. Results: By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for the stromal components. Globally, we isolated 121 phage-displayed neuroblastoma-binding peptides; of these, 26 bound the primary tumor, 15 the metastatic mass, 57 and 23 their respective microenvironments. Of these, five phage clones were further validated for their specific binding ex-vivo to biopsies from stage IV neuroblastoma patients and to neuroblastoma tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into neuroblastoma-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded nanocarriers led to a significant inhibition in tumor volume and enhanced survival in preclinical neuroblastoma models. Conclusions: Our findings demonstrate that novel ligands of neuroblastoma-associated markers are functional in the design of nanocarriers with therapeutic efficacy paving the way to their clinical development. Citation Format: Monica Loi, Daniela Di Paolo, Marco Soster, Chiara Brignole, Alice Bartolini, Laura Emionite, Jessica Sun, Pamela Becherini, Flavio Curnis, Andrea Petretto, Monica Sani, Alessandro Gori, Claudio Gambini, Renato Longhi, Michele Cilli, Theresa M. Allen, Federico Bussolino, Wadih Arap, Renata Pasqualini, Angelo Corti, Mirco Ponzoni, Serena Marchiò, Fabio Pastorino. Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5620. doi:10.1158/1538-7445.AM2013-5620

Molecular Imaging of Neuroblastoma Progression in TH-MYCN Transgenic Mice

TH-MYCN transgenic mice represent a valuable preclinical model of neuroblastoma. Current methods to study tumor progression in these mice are inaccurate or invasive, limiting the potential of this murine model. The aim of our study was to assess the potential of small animal positron emission tomography (SA-PET) to study neuroblastoma progression in TH-MYCN mice. Serial SA-PET scans using the tracer 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F-FDG) have been performed in TH-MYCN mice. Image analysis of tumor progression has been compared with ex vivo evaluation of tumor volumes and histological features. [(18)F]FDG-SA-PET allowed to detect early staged tumors in almost 100% of TH-MYCN mice positive for disease. Image analysis of tumor evolution reflected the modifications of the tumor volume, histological features, and malignancy during disease progression. Image analysis of TH-MYCN mice undergoing chemotherapy treatment against neuroblastoma provided information on drug-induced alterations in tumor metabolic activity. These data show for the first time that [(18)F]FDG-SA-PET is a useful tool to study neuroblastoma presence and progression in TH-MYCN transgenic mice.

Abstract 1436: Therapeutic targeting of the DNA damage mediators CHK1 and Wee1 in neuroblastoma

Abstract Background: High-risk neuroblastoma accounts for only 4% of all pediatric cancer diagnoses, yet is responsible for 12% of pediatric cancer deaths, despite significant intensification of therapy. Through an unbiased RNAi screen, the checkpoint kinase CHK1 was identified as a neuroblastoma therapeutic target (PNAS 2010), and sensitivity to single agent CHK1 inhibition was likely due to myc-induced replication stress - an effect that was synergistically enhanced in combination with conventional chemotherapy. To identify potential mediators of this effect, other members of the pathway were analyzed and Wee1 emerged as an additional target (AACR abstract 4758, 2011). Like CHK1, Wee1 was constitutively phosphorylated in neuroblastoma primary tumors and RNAi depletion in cell lines caused apoptosis. To translate these findings, we have evaluated small molecule inhibitors of these kinases in pre-clinical models of neuroblastoma. Methods: A panel of 10 human and 2 murine neuroblastoma cell lines were selected to screen for Wee1 (MK-1775) & CHK1 (SCH 900776) growth inhibition using the CellTiter-Glo viability assay. The cell lines represented the genomic variation of clinically relevant neuroblastoma. The compounds were tested at ten dose levels to determine the IC50 across a 5-log range. By the method of Chau-Talalay, the compounds were then tested with each other, and in combination with chemotherapy agents to calculate a combination index (CI). At the average IC50, four cell lines were tested for inhibition of downstream CHK1 and Wee1 signaling. Results: Neuroblastoma cell lines (n=10) were sensitive to single agent SCH 900776 (CHK1) and MK-1775 (Wee1) inhibition, with median IC50s of 888 nM and 243 nM, respectively. The murine cell lines were also significantly inhibited by single agent nanomolar CHK1 and Wee1 inhibition and sensitivity correlated with MYCN dosage. SCH 900776 and MK-1775 combined with each other, SN-38 or gemcitabine, demonstrate broad synergistic cytotoxicity, with the combination of the Wee1 and CHK1 inhibitors being the greatest (average CI = 0.3). In four representative neuroblastoma cell lines treated with SCH 900776 or MK-1775, there was decreased phosphorylation of the respective downstream targets p-CHK1 and p-CDC2. However, the induction of DNA damage, as determined by p-γ H2Ax, was apparent only when the two small molecule inhibitors were combined. Conclusion: Neuroblastomas show significant cytotoxicity to the kinase inhibitors SCH 900776 (CHK1) and MK-1775 (Wee1) in vitro as single agents, and this is enhanced in combination with each other or chemotherapy, at doses that are likely to be physiologically feasible. Current efforts are focused on developing the optimal combinatorial strategy in murine models including determining the optimal biomarker for anti-tumor activity prior to translation to an early phase clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1436. doi:1538-7445.AM2012-1436

Enhanced cell cycle perturbation and apoptosis mediate the synergistic effects of ST1926 and ATRA in neuroblastoma preclinical models

Retinoic acid therapy is nowadays an important component of treatment for residual disease of stage IV neuroblastoma after multimodal therapy. Nevertheless, arising resistance and treatment toxicity could represent relevant limiting factors. In the present study, we show that retinoic acid enhances the cytostatic and apoptogenic properties of the novel adamantyl retinoid ST1926 in a panel of neuroblastoma cells with different p53 status and caspase 8 expression, resulting in synergistic effects as assessed by Combination Index and Isobologram analysis. Under conditions where the two drugs alone produced no toxic effects, their combination resulted in enhanced G2-M arrest and sub-G1 population as shown by BrdU pulse-chase and labeling experiments. PARP cleavage, caspase 3, 8 and 9 activation and modulation of DR4 and FAS were indicative of enhanced apoptosis triggered by the co-incubation of the two drugs whereas neither ST1926-mediated genotoxic damage nor ATRA-differentiating effects were affected by the combined treatment. Caspase-3 and 8-mediated apoptosis appeared to play an important role in the drugs synergism. In fact, the addition of a pan-caspase inhibitor ZVAD-FMK reverted this effect in SK-N-DZ cells, and synergism was confined to limited drugs doses in HTLA cells not expressing caspase-8. Although not modulated, p53 appeared to enhance cells responsiveness to retinoid/ATRA combination. In vivo studies in the most sensitive neuroblastoma model SK-N-DZ, confirmed enhanced activity of the drugs combination vs single treatments. The study provides important lines of evidence that such a drugs combination could represent a less toxic and more effective approach for maintenance treatment in children with neuroblastoma.

Abstract 4342: The allosteric Akt inhibitor MK-2206 inhibits neuroblastoma tumor cell growth in vitro and in vivo

Abstract Background: Activated Akt in tumor tissue of NB patients is a marker of decreased event-free as well as overall survival. Our studies indicate activated Akt induces chemoresistance in NB cells and chemoresistance is a major problem in the treatment of Neuroblastoma (NB). The allosteric Akt inhibitor, MK-2206 inhibits the activation of the 3 Akt isoforms with IC50-Akt1 = 5.3nM, -Akt2 = 12nM and -Akt3 = 65nM. In this study we tested the effects of MK-2206 in vitro and in vivo pre-clinical models of NB. Methods: NB cell lines (NGP, SY5Y, KCNR, BE2, SKNFI, and AS) were treated with increasing concentrations of MK-2206 for 48 hours. MK-2206-induced cell death was evaluated by MTS assay. Phosphorylated Akt and its downstream target S6 were determined by Western Blot analysis. The effect of MK-2206 (100 or 200mg/kg/3 days/wk) on NB tumor growth was evaluated in a xenograft nude mouse model. Results: NB cell lines express all 3 Akt isoforms to varying levels. In in vitro studies, MK-2206 inhibited cell growth in all NB cell lines although with different sensitivities. The growth of NGP, SY5Y, KCNR and BE2 cells were more sensitive to MK-2206, with IC50<4μM; while the growth of AS and SKNFI were less sensitive, with IC50>11μM. MK-2206 treatment decreased phosphorylated-Akt and its target phosphorylated-S6 in NB cells to a comparable level in all tested cell lines regardless of biologic sensitivity to MK-2206. In in vivo studies, mice bearing the sensitive BE2 NB tumors were treated with MK-2206 (100 or 200mg/kg) while mice bearing the less sensitive AS tumors received MK-2206 (200mg/kg). There was an approx. 30% inhibition of tumor growth in each tumor model only in mice treated with 200mg/kg MK-2206 (P<0.01). MK-2206 (200mg/kg) treatment led to a comparable increase in survival in both models (P<0.01). Conclusion: This study indicates that despite differences in in vitro growth sensitivities, the Akt inhibitor, MK-2206 has a comparable anti-tumor cell growth inhibiting activity in vivo. This study supports the future evaluation of the combination of MK-2206 with chemotherapeutic drugs to improve the treatment efficacy in NB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4342. doi:10.1158/1538-7445.AM2011-4342

Phase I trial of lestaurtinib for children with refractory neuroblastoma: a new approaches to neuroblastoma therapy consortium study

TrkB acts as an oncogenic kinase in a subset of human neuroblastomas. Lestaurtinib, a multi-kinase inhibitor with potent activity against Trk kinases, has demonstrated activity in preclinical models of neuroblastoma. Patients with refractory high-risk neuroblastoma received lestaurtinib twice daily for 5days out of seven in 28-day cycles, starting at 70% of the adult recommended Phase 2 dose. Lestaurtinib dose was escalated using a 3+3 design. Pharmacokinetics and plasma phospho-TrkB inhibitory activity were evaluated in the first cycle. Forty-seven subjects were enrolled, and 10 dose levels explored starting at 25mg/M(2)/dose BID. Forty-six subjects were evaluable for response, and 42 subjects were fully evaluable for determination of dose escalation. Asymptomatic and reversible grade 3-4 transaminase elevation was dose limiting in 4 subjects. Reversible pancreatitis (grade 2) was observed in 3 subjects after prolonged treatment at higher dose levels. Other toxicities were mild and reversible. Pharmacokinetic analyses revealed rapid drug absorption, however inter-patient variability was large. Plasma inhibition of phospho-TrkB activity was observed 1h post-dosing at 85mg/M(2) with uniform inhibition at 120mg/M(2). There were two partial responses and nine subjects had prolonged stable disease at dose levels≥5, (median: 6cycles). A biologically effective and recommended phase 2 dose of 120mg/M(2)/dose BID was established. Lestaurtinib was well tolerated in patients with refractory neuroblastoma, and a dose level sufficient to inhibit TrkB activity was established. Safety and signs of activity at the higher dose levels warrant further evaluation in neuroblastoma.

Activity of irinotecan and temozolomide in the presence of O6-methylguanine-DNA methyltransferase inhibition in neuroblastoma pre-clinical models

Background:The combination of temozolomide (TMZ) and irinotecan is a regimen used in neuroblastoma patients with recurrent disease. O6-methylguanine-DNA methyltransferase (MGMT) may have a function in resistance to TMZ. Using neuroblastoma pre-clinical models, we determined whether the inhibition of MGMT by O6-benzylguanine (O6-BG) could enhance the anti-tumour activity of TMZ and irinotecan.Methods:The cytotoxicity of TMZ and irinotecan, either alone or in combination, was measured in five neuroblastoma cell lines in the presence or absence of O6-BG with a fluorescence-based cell viability assay (DIMSCAN). Anti-tumour activity was measured in three neuroblastoma xenograft models.Results:MGMT mRNA and protein were expressed in 9 out of 10 examined cell lines. Pretreatment of cells with 25 μM O6-BG decreased MGMT protein expression and enhanced The TMZ cytotoxicity by up to 0.3–1.4 logs in four out of five tested cell lines. TMZ (25 mg kg−1 per day for 5 days every 3 weeks for four cycles) did not significantly improve mice survival, whereas the same schedule of irinotecan (7.5 mg kg−1 per day) significantly improved survival (P<0.0001) in all three xenograft models. Combining O6-BG and/or TMZ with irinotecan further enhanced survival.Conclusion:Our in vitro and in vivo findings suggest that irinotecan drives the activity of irinotecan and TMZ in recurrent neuroblastoma. Inhibitors of MGMT warrant further investigation for enhancing the activity of regimens that include TMZ.

Tumor Regression and Curability of Preclinical Neuroblastoma Models by PEGylated SN38 (EZN-2208), a Novel Topoisomerase I Inhibitor

Treatment of neuroblastoma is successful in less than half of patients with high-risk disease. The antitumor activity of a water soluble pegylated SN38 drug conjugate, EZN-2208, was compared with CPT-11 (a prodrug for SN38) in preclinical models of human neuroblastoma. The in vitro cytotoxicity of EZN-2208 was tested by counting trypan blue dye- and Annexin V-positive cells, whereas its therapeutic efficacy was evaluated, in terms of survival, and antitumor and antiangiogenic activities, in s.c. luciferase-transfected, pseudometastatic, and orthotopic neuroblastoma animal models. EZN-2208 was about 100-fold more potent than CPT-11 in vitro, by inducing apoptosis/necrosis and p53 expression and by reducing hypoxia-inducible factor (HIF)-1α/HIF-2α expression. EZN-2208 gave superior antitumor effects compared with CPT-11 in neuroblastoma xenografts. EZN-2208 treatment always resulted in lack of tumor detection at the end of trials whereas only small therapeutic effects were observed with CPT-11, as assessed by luciferase assay or tumor size, or even by staining histologic sections of tumors with antibodies recognizing neuroblastoma cells and cell proliferation. In a neuroblastoma model resistant to doxorubicin, cisplatin, vincristine, fenretinide, and topotecan, EZN-2208 induced 100% curability. It also blocked tumor relapse after topotecan-vincristine-doxorubicin combined treatment. Mechanistic experiments showed statistically significantly enhanced terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Histone H2ax staining as well as decreased vascular endothelial growth factor, CD31, matrix metalloproteinase (MMP)-2, and MMP-9 expression in tumors removed from EZN-2208-treated mice and radiating vessels invading the tumor implanted onto the chorioallantoic membranes. EZN-2208 should be considered a most promising novel antineuroblastoma agent. An ongoing phase I study in pediatric patients should identify the optimal dose for a phase II study.

Slug (SNAI2) Down-Regulation by RNA Interference Facilitates Apoptosis and Inhibits Invasive Growth in Neuroblastoma Preclinical Models

We assessed the relevance of Slug (SNAI2) for apoptosis resistance and invasion potential of neuroblastoma cells in vitro and in vivo. We evaluated the effect of imatinib mesylate on invasion and analyzed the genes modulated by imatinib mesylate treatment in neuroblastoma cells. Slug expression, inhibited by imatinib mesylate treatment, was knocked down in neuroblastoma cells by RNA interference, and the effects on invasion and apoptosis were evaluated in vitro. A pseudometastatic model of neuroblastoma in severe combined immunodeficient mice was used to assess the effects of Slug silencing alone or in combination with imatinib mesylate treatment on metastasis development. Microarray analysis revealed that several genes, including Slug, were down-regulated by imatinib mesylate. Slug expression was detectable in 8 of 10 human neuroblastoma cell lines. Two Slug-expressing cell lines were infected with a vector encoding a microRNA to Slug mRNA. Infected cells with reduced levels of Slug were tested for the expression of apoptosis-related genes (p53, Bax, and Bcl-2) identified previously as Slug targets. Bcl-2 was down-regulated in Slug-interfered cells. Slug down-regulation increased sensitivity to apoptosis induced by imatinib mesylate, etoposide, or doxorubicin. Invasion of Slug-silenced cells was reduced in vitro. Animals injected with Slug-silenced cells had fewer tumors than controls and the inhibition of tumor growth was even higher in animals treated with imatinib mesylate. Slug down-regulation facilitates apoptosis induced by proapoptotic drugs in neuroblastoma cells and decreases their invasion capability in vitro and in vivo. Slug inhibition, possibly combined with imatinib mesylate, may represent a novel strategy for treatment of metastatic neuroblastoma.