Evaluation of clinically translatable MR imaging biomarkers of therapeutic response in the TH-MYCN transgenic mouse model of neuroblastoma.

PURPOSE: To evaluate noninvasive and clinically translatable magnetic resonance (MR) imaging biomarkers of therapeutic response in the TH-MYCN transgenic mouse model of aggressive, MYCN-amplified neuroblastoma. MATERIALS AND METHODS: All experiments were performed in accordance with the local ethical review panel and the UK Home Office Animals Scientific Procedures Act 1986 and with the UK National Cancer Research Institute guidelines for the welfare of animals in cancer research. Multiparametric MR imaging was performed of abdominal tumors found in the TH-MYCN model. T2-weighted MR imaging, quantitation of native relaxation times T1 and T2, the relaxation rate R2*, and dynamic contrast-enhanced MR imaging were used to monitor tumor response to cyclophosphamide (25 mg/kg), the vascular disrupting agent ZD6126 (200 mg/kg), or the antiangiogenic agent cediranib (6 mg/kg, daily). Any significant changes in the measured parameters, and in the magnitude of the changes after treatment between treated and control cohorts, were identified by using Student two-tailed paired and unpaired t test, respectively, with a 5% level of significance. RESULTS: Treatment with cyclophosphamide or cediranib induced a 54% or 20% reduction in tumor volume at 48 hours, respectively (P < .005 and P < .005, respectively; P < .005 and P < .005 versus control, respectively). Treatment with ZD6126 induced a 45% reduction in mean tumor volume 24 hours after treatment (P < .005; P < .005 versus control). The antitumor activity of cyclophosphamide, cediranib, and ZD6126 was consistently associated with a decrease in tumor T1 (P < .005, P < .005, and P < .005, respectively; P < .005, P < .005, and P < .005 versus control, respectively) and with a correlation between therapy-induced changes in native T1 and changes in tumor volume (r = 0.56; P < .005). Tumor response to cediranib was also associated with a decrease in the dynamic contrast-enhanced MR imaging-derived volume transfer constant (P = .07; P < .05 versus control) and enhancing fraction (P < .05; P < .01 versus control), and an increase in R2* (P < .005; P < .05 versus control). CONCLUSION: The T1 relaxation time is a robust noninvasive imaging biomarker of response to therapy in tumors in TH-MYCN mice, which emulate high-risk neuroblastoma in children. T1 measurements can be readily implemented on clinical MR systems and should be investigated in translational clinical trials of new targeted therapies for pediatric neuroblastoma. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12120128/-/DC1.

Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models.

PURPOSE: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.

The Promise of Patient-Derived Preclinical Models to Accelerate the Implementation of Personalised Medicine for Children with Neuroblastoma.

Patient-derived preclinical models are now a core component of cancer research and have the ability to drastically improve the predictive power of preclinical therapeutic studies. However, their development and maintenance can be challenging, time consuming, and expensive. For neuroblastoma, a developmental malignancy of the neural crest, it is possible to establish patient-derived models as xenografts in mice and zebrafish, and as spheroids and organoids in vitro. These varied approaches have contributed to comprehensive packages of preclinical evidence in support of new therapeutics for neuroblastoma. We discuss here the ethical and technical considerations for the creation of patient-derived models of neuroblastoma and how their use can be optimized for the study of tumour evolution and preclinical therapies. We also discuss how neuroblastoma patient-derived models might become avatars for personalised medicine for children with this devastating disease.

Targeting MYCN and ALK in resistant and relapsing neuroblastoma.

Neuroblastoma, a tumor of peripheral nerve, is the most common solid tumor of young children. In high-risk disease, which comprises approximately half of patients, death from chemotherapy-resistant, metastatic relapse is very frequent. Children who relapse exhibit clonal enrichment of two genomic alterations: high-level amplification of the MYCN oncogene, and kinase domain mutations of the anaplastic lymphoma kinase (ALK) gene. Overall survival in this patient cohort is less than 15% at 3 years, and there are few options for rationally targeted therapy. Neuroblastoma patients exhibit de novo resistance to many existing ALK inhibitors, and no clinical therapeutics to target MYCN have yet been developed. This review outlines the international efforts to uncover mechanisms of oncogenic action that are therapeutically targetable using small-molecule inhibitors. We describe a mechanistic interaction in which ALK upregulates MYCN transcription, and discuss clinical trials emerging to develop transcriptional inhibitors of MYCN, and to identify effective inhibitors of ALK in neuroblastoma patients.

Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus.

Neural crest migration is critical to its physiological function. Mechanisms controlling mammalian neural crest migration are comparatively unknown, due to difficulties accessing this cell population in vivo. Here we report requirements of glycogen synthase kinase 3 (GSK3) in regulating the neural crest in Xenopus and mouse models. We demonstrate that GSK3 is tyrosine phosphorylated (pY) in mouse neural crest cells and that loss of GSK3 leads to increased pFAK and misregulation of Rac1 and lamellipodin, key regulators of cell migration. Genetic reduction of GSK3 results in failure of migration. We find that pY-GSK3 phosphorylation depends on anaplastic lymphoma kinase (ALK), a protein associated with neuroblastoma. Consistent with this, neuroblastoma cells with increased ALK activity express high levels of pY-GSK3, and blockade of GSK3 or ALK can affect migration of these cells. Altogether, this work identifies a role for GSK3 in cell migration during neural crest development and cancer.

Immunoassays for the quantification of ALK and phosphorylated ALK support the evaluation of on-target ALK inhibitors in neuroblastoma.

Targeted inhibition of anaplastic lymphoma kinase (ALK) is a successful approach for the treatment of many ALK-aberrant malignancies; however, the presence of resistant mutations necessitates both the development of more potent compounds and pharmacodynamic methods with which to determine their efficacy. We describe immunoassays designed to quantitate phosphorylation of ALK, and their use in preclinical models of neuroblastoma, a pediatric malignancy in which gain-of-function ALK mutations predict a poor overall outcome to conventional treatment. Validation of the immunoassays is presented using a panel of neuroblastoma cell lines and evidence of on-target ALK inhibition provided by treatment of a genetically engineered murine model of neuroblastoma with two clinical ALK inhibitors, crizotinib and ceritinib, highlighting the superior efficacy of ceritinib.

Accelerating drug development for neuroblastoma - New Drug Development Strategy: an Innovative Therapies for Children with Cancer, European Network for Cancer Research in Children and Adolescents and International Society of Paediatric Oncology Europe Neuroblastoma project.

INTRODUCTION: Neuroblastoma, the commonest paediatric extra-cranial tumour, remains a leading cause of death from cancer in children. There is an urgent need to develop new drugs to improve cure rates and reduce long-term toxicity and to incorporate molecularly targeted therapies into treatment. Many potential drugs are becoming available, but have to be prioritised for clinical trials due to the relatively small numbers of patients. Areas covered: The current drug development model has been slow, associated with significant attrition, and few new drugs have been developed for neuroblastoma. The Neuroblastoma New Drug Development Strategy (NDDS) has: 1) established a group with expertise in drug development; 2) prioritised targets and drugs according to tumour biology (target expression, dependency, pre-clinical data; potential combinations; biomarkers), identifying as priority targets ALK, MEK, CDK4/6, MDM2, MYCN (druggable by BET bromodomain, aurora kinase, mTORC1/2) BIRC5 and checkpoint kinase 1; 3) promoted clinical trials with target-prioritised drugs. Drugs showing activity can be rapidly transitioned via parallel randomised trials into front-line studies. Expert opinion: The Neuroblastoma NDDS is based on the premise that optimal drug development is reliant on knowledge of tumour biology and prioritisation. This approach will accelerate neuroblastoma drug development and other poor prognosis childhood malignancies.

Tackling Crizotinib Resistance: The Pathway from Drug Discovery to the Pediatric Clinic.

Neuroblastoma is a childhood malignancy that has not yet benefitted from the rapid progress in the development of small-molecule therapeutics for cancer. An opportunity to take advantage of pharmaceutical innovation in this area arose when the identification of ALK fusion proteins in non-small cell lung cancer (NSCLC) occurred in parallel to the discovery of point mutations of ALK in neuroblastomas. ALK is now known to be a marker of poor outcome in neuroblastoma, and therefore, urgent development of specific ALK inhibitors to treat this devastating disease is a necessity. However, the translation of small molecules from adult directly into pediatric practice has thus far been challenging, due to mutation-specific structural variances in the ALK kinase domain. We discuss how the most recent structural and biological characterizations of ALK are directing preclinical and clinical studies of ALK inhibitors for both NSCLC and neuroblastoma.