Purpose: MYCN-dependent neuroblastomas have low cure rates with current multimodal treatment regimens and novel therapeutic drugs are therefore urgently needed. In previous preclinical studies, we have shown that targeted inhibition of cyclin-dependent kinase 2 (CDK2) resulted in specific killing of MYCN-amplified neuroblastoma cells. This study describes the in vivo preclinical evaluation of the CDK inhibitor AT7519. Experimental Design: Preclinical drug testing was performed using a panel of MYCN-amplified and MYCN single copy neuroblastoma cell lines and different MYCN-dependent mouse models of neuroblastoma. Results: AT7519 killed MYCN-amplified neuroblastoma cell lines more potently than MYCN single copy cell lines with a median LC₅₀ value of 1.7 compared to 8.1 μmol/L (P = 0.0053) and a significantly stronger induction of apoptosis. Preclinical studies in female NMRI homozygous (nu/nu) mice with neuroblastoma patient-derived MYCN-amplified AMC711T xenografts revealed dose-dependent growth inhibition, which correlated with intratumoral AT7519 levels. CDK2 target inhibition by AT7519 was confirmed by significant reductions in levels of phosphorylated retinoblastoma (p-Rb) and nucleophosmin (p-NPM). AT7519 treatment of Th-MYCN transgenic mice resulted in improved survival and clinically significant tumor regression (average tumor size reduction of 86% at day 7 after treatment initiation). The improved efficacy of AT7519 observed in Th-MYCN mice correlated with higher tumor exposure to the drug. Conclusions: This study strongly suggests that AT7519 is a promising drug for the treatment of high-risk neuroblastoma patients with MYCN amplification. Clin Cancer Res; 21(22); 5100–9. ©2015 AACR.

For one-third of patients with pediatric cancer enrolled in precision medicine programs, molecular profiling does not result in a therapeutic recommendation. To identify potential strategies for treating these high-risk pediatric patients, we performed in vitro screening of 125 patient-derived samples against a library of 126 anticancer drugs. Tumor cell expansion did not influence drug responses, and 82% of the screens on expanded tumor cells were completed while the patients were still under clinical care. High-throughput drug screening (HTS) confirmed known associations between activating genomic alterations in NTRK, BRAF, and ALK and responses to matching targeted drugs. The in vitro results were further validated in patient-derived xenograft models in vivo and were consistent with clinical responses in treated patients. In addition, effective combinations could be predicted by correlating sensitivity profiles between drugs. Furthermore, molecular integration with HTS identified biomarkers of sensitivity to WEE1 and MEK inhibition. Incorporating HTS into precision medicine programs is a powerful tool to accelerate the improved identification of effective biomarker-driven therapeutic strategies for treating high-risk pediatric cancers. Significance: Integrating HTS with molecular profiling is a powerful tool for expanding precision medicine to support drug treatment recommendations and broaden the therapeutic options available to high-risk pediatric cancers.

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.

High efficacy of the BCL-2 inhibitor ABT199 (venetoclax) in BCL-2 high-expressing neuroblastoma cell lines and xenografts and rational for combination with MCL-1 inhibition

Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

Neuroblastoma tumors frequently overexpress the anti-apoptotic protein B-cell lymphoma/leukemia 2 (BCL-2). We previously showed that treating BCL-2–dependent neuroblastoma cells with the BCL-2 inhibitor venetoclax results in apoptosis, but unfortunately partial therapy resistance is observed. The current study describes the identification of drugs capable of resensitizing venetoclax-resistant neuroblastoma cells to venetoclax. To examine these effects, venetoclax resistance was induced in BCL-2–dependent neuroblastoma cell lines KCNR and SJNB12 by continuous exposure to high venetoclax concentrations. Non-resistant and venetoclax-resistant neuroblastoma cell lines were exposed to a 209-compound library in the absence and presence of venetoclax to identify compounds that were more effective in the venetoclax-resistant cell lines under venetoclax pressure. Top hits were further validated in combination with venetoclax using BCL-2–dependent neuroblastoma model systems. Overall, high-throughput drug screening identified the MDM2 inhibitor idasanutlin as a promising resensitizing agent for venetoclax-resistant neuroblastoma cell lines. Idasanutlin treatment induced BAX-mediated apoptosis in venetoclax-resistant neuroblastoma cells in the presence of venetoclax, whereas it caused p21-mediated growth arrest in control cells. In vivo combination treatment showed tumor regression and superior efficacy over single-agent therapies in a BCL-2–dependent neuroblastoma cell line xenograft and a patient-derived xenograft. However, xenografts less dependent on BCL-2 were not sensitive to venetoclax–idasanutlin combination therapy. This study demonstrates that idasanutlin can overcome resistance to the BCL-2 inhibitor venetoclax in preclinical neuroblastoma model systems, which supports clinical development of a treatment strategy combining the two therapies.