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1.  Distinct Neuroblastoma-associated Alterations of PHOX2B Impair Sympathetic Neuronal Differentiation in Zebrafish Models 
PLoS Genetics  2013;9(6):e1003533.
Heterozygous germline mutations and deletions in PHOX2B, a key regulator of autonomic neuron development, predispose to neuroblastoma, a tumor of the peripheral sympathetic nervous system. To gain insight into the oncogenic mechanisms engaged by these changes, we used zebrafish models to study the functional consequences of aberrant PHOX2B expression in the cells of the developing sympathetic nervous system. Allelic deficiency, modeled by phox2b morpholino knockdown, led to a decrease in the terminal differentiation markers th and dbh in sympathetic ganglion cells. The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects. We demonstrate that Phox2b is capable of regulating itself as well as ascl1, and that phox2b deficiency uncouples this autoregulatory mechanism, leading to inhibition of sympathetic neuron differentiation. This effect on terminal differentiation is associated with an increased number of phox2b+, ascl1+, elavl3− cells that respond poorly to retinoic acid. These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.
Author Summary
Neuroblastoma, a tumor of the peripheral sympathetic nervous system, is the most common cancer diagnosed in infancy. Although most cases arise sporadically, familial predisposition also occurs in association with mutations in a single copy of the PHOX2B gene, a “master regulator” of sympathetic neuronal development. The exact mechanisms by which these mutations increase susceptibility to neuroblastoma are unclear, primarily because of the paucity of optimal models in which to study very early development of the sympathetic nervous system. We took advantage of the ex vivo development and transparent nature of zebrafish embryos to study the roles of both normal and mutated PHOX2B in development of the sympathetic nervous system. We present data indicating that aberrant PHOX2B expression causes an arrest in the normal maturation of sympathetic neurons, leading to immature cells that are resistant to drug-induced differentiation. Indeed, we demonstrate that phox2b gene “dosage” is important for normal differentiation of sympathetic neurons in the zebrafish and suggest that the population of immature cells resulting from a decreased dosage of this pivotal factor may be susceptible to secondary mutations that could ultimately lead to neuroblastoma.
doi:10.1371/journal.pgen.1003533
PMCID: PMC3675015  PMID: 23754957
2.  Promising therapeutic targets in neuroblastoma 
Clinical Cancer Research  2012;18(10):2740-2753.
Neuroblastoma, the most common extra- cranial solid tumor in children, is derived from neural crest cells. Nearly half of patients present with metastatic disease, and have 5-year EFS of less than 50%. New approaches with targeted therapy may improve efficacy without increased toxicity. The current review will evaluate three promising targeted therapies, including 131I-metaiodobenzylguanidine (MIBG), a radiopharmaceutical taken up by the human norepinephrine transporter expressed in 90% of neuroblastomas, immunotherapy with monoclonal antibodies targeting the GD2 ganglioside, expressed on 98% of neuroblastoma cells, and inhibitors of ALK, a tyrosine kinase which is mutated or amplified in approximately 10% of neuroblastoma and expressed on the surface of most neuroblastoma cells. Early phase trials have confirmed the activity of 131I-MIBG in relapsed neuroblastoma, with response rates of about 30%, but the technical aspects of administration of large amounts of radioactivity in young children and the limited access have hindered incorporation into treatment of newly diagnosed patients. Anti-GD2 antibodies have also demonstrated activity in relapsed disease, and a recent phase III randomized trial showed a significant improvement in event-free survival for patients receiving chimeric anti-GD2 (ch14.18) combined with cytokines and isotretinoin after myeloablative consolidation therapy. A recently approved small molecule inhibitor of ALK has promising pre-clinical activity for neuroblastoma, and is currently in phase I and II trials. This is the first agent directed to a specific mutation in neuroblastoma, and marks a new step toward personalized therapy for neuroblastoma. Further clinical development of targeted treatments offers new hope for children with neuroblastoma.
doi:10.1158/1078-0432.CCR-11-1939
PMCID: PMC3382042  PMID: 22589483
3.  Activated ALK Collaborates with MYCN in Neuroblastoma Pathogenesis 
Cancer Cell  2012;21(3):362-373.
SUMMARY
Amplification of the MYCN oncogene in childhood neuroblastoma is often accompanied by mutational activation of ALK (anaplastic lymphoma kinase), suggesting their pathogenic cooperation. We generated a transgenic zebrafish model of neuroblastoma in which MYCN-induced tumors arise from a subpopulation of neuroblasts that migrate into the adrenal medulla analogue following organogenesis. Coexpression of activated ALK with MYCN in this model triples the disease penetrance and markedly accelerates tumor onset. MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. Coexpression of activated ALK with MYCN provides prosurvival signals that block this apoptotic response and allow continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma.
doi:10.1016/j.ccr.2012.02.010
PMCID: PMC3315700  PMID: 22439933
4.  Emerging importance of ALK in neuroblastoma 
Seminars in cancer biology  2011;21(4):267-275.
Since the original descriptions of gain-of function mutations in anaplastic lymphoma kinase (ALK), interest in the role of this receptor tyrosine kinase in neuroblastoma development and as a potential therapeutic target has escalated. As a group, the activating point mutations in full-length ALK, found in approximately 8% of all neuroblastoma tumors, are distributed evenly across different clinical stages. However, the most frequent somatic mutation, F1174L, is associated with amplification of the MYCN oncogene. This combination of features appears to confer a worse prognosis than MYCN amplification alone, suggesting a cooperative effect on neuroblastoma formation by these two proteins. Indeed, F1174L has shown more potent transforming activity in vivo than the second most common activating mutation, R1275Q, and is responsible for innate and acquired resistance to crizotinib, a clinically relevant ALK inhibitor that will soon be commercially available. These advances cast ALK as a bona fide oncoprotein in neuroblastoma and emphasize the need to understand ALK-mediated signaling in this tumor. This review addresses many of the current issues surrounding the role of ALK in normal development and neuroblastoma pathogenesis, and discusses the prospects for clinically effective targeted treatments based on ALK inhibition.
doi:10.1016/j.semcancer.2011.09.005
PMCID: PMC3242371  PMID: 21945349
neuroblastoma; ALK; tyrosine kinase receptor; targeted therapy; crizotinib; drug resistance; point mutations; small molecule inhibitors; combination treatment
5.  Phase I Study of Decitabine with Doxorubicin and Cyclophosphamide in Children with Neuroblastoma and Other Solid Tumors: A Children’s Oncology Group Study 
Pediatric blood & cancer  2010;55(4):629-638.
Background
Demethylating agents may alter the expression of genes involved in chemotherapy resistance. We conducted a phase I trial to determine the toxicity and molecular effects of the demethylating agent, decitabine, followed by doxorubicin and cyclophosphamide in children with refractory solid tumors.
Procedure
Stratum A included children with any solid tumor; Stratum B included neuroblastoma patients only. Patients received a 1-hour decitabine infusion for 7 days, followed by doxorubicin (45mg/m2) and cyclophosphamide (1g/m2) on day 7. Pharmacokinetic studies were performed after the first dose of decitabine. Biological studies included methylation and gene expression analyses of caspase-8, MAGE-1 and fetal hemoglobin (HbF), and expression profiling of pre- and post-treatment peripheral blood and bone marrow cells.
Results
The maximum-tolerated dose of decitabine was 5 mg/m2/d for 7 days. Dose-limiting toxicities at 10 mg/m2/d were neutropenia and thrombocytopenia. Decitabine exhibited rapid clearance from plasma. Three of 9 patients in Stratum A and 4/12 patients in Stratum B had stable disease for ≥4 months. Sustained MAGE-1 demethylation and increased HbF expression were observed in the majority of patients post-treatment (12/20 and 14/16 respectively). Caspase-8 promoter demethylation and gene expression were seen in 2/7 bone marrow samples. Differentially expressed genes were identified by microarray analysis.
Conclusion
Low-dose decitabine when combined with doxorubicin/cyclophosphamide has tolerable toxicity in children. However, doses of decitabine capable of producing clinically relevant biologic effects were not well tolerated with this combination. Alternative strategies of combining demethylating agents with non-cytotoxic, biologically targeted agents such as histone deactelyase inhibitors should be explored.
doi:10.1002/pbc.22607
PMCID: PMC3025700  PMID: 20589651
demethylation; decitabine; pediatric solid tumor; neuroblastoma
6.  Comparison of Primary Neuroblastoma Tumors and Derivative Early-Passage Cell Lines Using Genome-Wide Single Nucleotide Polymorphism Array Analysis 
Cancer research  2009;69(10):4143-4149.
Stromal contamination is one of the major confounding factors in the analysis of solid tumor samples by single nucleotide polymorphism (SNP) arrays. As we propose to use genome-wide SNP microarray analysis as a diagnostic platform for neuroblastoma, the sensitivity, specificity, and accuracy of these studies must be optimized. To investigate the effects of stromal contamination, we derived early-passage cell lines from nine primary tumors and compared their genomic signature with that of the primary tumors using 100K SNP arrays. The average concordance between tumor and cell line for raw loss of heterozygosity (LOH) calls was 96% (range, 91–99%) and for raw copy number alterations, 71% (range, 43–87%). In general, there were a larger number of LOH events identified in the cell lines compared with the matched tumor samples (mean increase, 3.2% ± 1.9%). We have developed an algorithm that shows that the presence of stroma contributes to under-reporting of LOH and copy number loss. Notable findings in this sample set were uniparental disomy of chromosome arms 11p, 1q, 14q, and 15q and a novel area of amplification on chromosome band 11p15. Our analysis shows that LOH was identified significantly more often in derived cell lines compared with the original tumor samples. Although these may in part be due to clonal selection during adaptation to tissue culture, our study indicates that stromal contamination may be a major contributing factor in underestimation of LOH and copy number loss events.
doi:10.1158/0008-5472.CAN-08-3112
PMCID: PMC2739280  PMID: 19435921
7.  Anaplastic lymphoma kinase: role in cancer pathogenesis and small-molecule inhibitor development for therapy 
Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase in the insulin receptor superfamily, was initially identified in constitutively activated oncogenic fusion forms – the most common being nucleophosmin-ALK – in anaplastic large-cell lymphomas, and subsequent studies have identified ALK fusions in diffuse large B-cell lymphomas, systemic histiocytosis, inflammatory myofibroblastic tumors, esophageal squamous cell carcinomas and non-small-cell lung carcinomas. More recently, genomic DNA amplification and protein overexpression, as well as activating point mutations, of ALK have been described in neuroblastomas. In addition to those cancers for which a causative role for aberrant ALK activity is well validated, more circumstantial links implicate the full-length, normal ALK receptor in the genesis of other malignancies – including glioblastoma and breast cancer – via a mechanism of receptor activation involving autocrine and/or paracrine growth loops with the reported ALK ligands, pleiotrophin and midkine. This review summarizes normal ALK biology, the confirmed and putative roles of ALK in the development of human cancers and efforts to target ALK using small-molecule kinase inhibitors.
doi:10.1586/14737140.9.3.331
PMCID: PMC2780428  PMID: 19275511
anaplastic large-cell lymphoma; anaplastic lymphoma kinase; esophageal squamous cell carcinoma; glioblastoma; inflammatory myofibroblastic tumor; midkine; neuroblastoma; non-small-cell lung carcinoma; pleiotrophin; targeted cancer therapy; tyrosine kinase inhibitor
8.  Activating mutations in ALK provide a therapeutic target in neuroblastoma 
Nature  2008;455(7215):975-978.
Neuroblastoma, an embryonal tumor of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer1. High-risk neuroblastomas, prevalent in the majority of patients, are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal2,3. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germline. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK cDNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed IL-3-dependent murine hematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to a small-molecule inhibitor of ALK, TAE6844. Furthermore, two human neuroblastoma cell lines harboring the F1174L mutation were sensitive to the inhibitor. Cytotoxicity was associated with increased levels of apoptosis as measured by TUNEL-labeling. shRNA-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumors and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.
doi:10.1038/nature07397
PMCID: PMC2587486  PMID: 18923525
9.  Genome-Wide Analysis of Neuroblastomas using High-Density Single Nucleotide Polymorphism Arrays 
PLoS ONE  2007;2(2):e255.
Background
Neuroblastomas are characterized by chromosomal alterations with biological and clinical significance. We analyzed paired blood and primary tumor samples from 22 children with high-risk neuroblastoma for loss of heterozygosity (LOH) and DNA copy number change using the Affymetrix 10K single nucleotide polymorphism (SNP) array.
Findings
Multiple areas of LOH and copy number gain were seen. The most commonly observed area of LOH was on chromosome arm 11q (15/22 samples; 68%). Chromosome 11q LOH was highly associated with occurrence of chromosome 3p LOH: 9 of the 15 samples with 11q LOH had concomitant 3p LOH (P = 0.016). Chromosome 1p LOH was seen in one-third of cases. LOH events on chromosomes 11q and 1p were generally accompanied by copy number loss, indicating hemizygous deletion within these regions. The one exception was on chromosome 11p, where LOH in all four cases was accompanied by normal copy number or diploidy, implying uniparental disomy. Gain of copy number was most frequently observed on chromosome arm 17q (21/22 samples; 95%) and was associated with allelic imbalance in six samples. Amplification of MYCN was also noted, and also amplification of a second gene, ALK, in a single case.
Conclusions
This analysis demonstrates the power of SNP arrays for high-resolution determination of LOH and DNA copy number change in neuroblastoma, a tumor in which specific allelic changes drive clinical outcome and selection of therapy.
doi:10.1371/journal.pone.0000255
PMCID: PMC1797488  PMID: 17327916
10.  Discovery of 3,5-Diamino-1,2,4-triazole Ureas as Potent Anaplastic Lymphoma Kinase Inhibitors 
ACS Medicinal Chemistry Letters  2011;2(5):379-384.
A series of novel 3,5-diamino-1,2,4-triazole benzyl ureas was identified as having potent anaplastic lymphoma kinase (ALK) inhibition exemplified by 15a, 20a, and 23a, which exhibited antiproliferative IC50 values of 70, 40, and 20 nM in Tel-ALK transformed Ba/F3 cells, respectively. Moreover, 15a and 23a potently inhibited the growth and survival of NPM-ALK positive anaplastic large cell lymphoma cell (SU-DHL-1) and neuroblastoma cell lines (KELLY, SH-SY5Y) containing the F1174L ALK mutation. These compounds provide novel leads for the development of small-molecule ALK inhibitors for cancer therapy.
doi:10.1021/ml200002a
PMCID: PMC3093683  PMID: 21572589
ALK; 3,5-diamino-1,2,4-triazole urea
11.  The ALKF1174L mutation potentiates the oncogenic activity of MYCN in neuroblastoma 
Cancer cell  2012;22(1):117-130.
SUMMARY
The ALKF1174L mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALKF1174L in the neural crest. Compared to ALKF1174L and MYCN alone, coexpression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance and enhanced lethality. ALKF1174L/MYCN tumors exhibited increased MYCN dosage due to ALKF1174L-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2, overcame the resistance of ALKF1174L/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALKF1174L in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.
doi:10.1016/j.ccr.2012.06.001
PMCID: PMC3417812  PMID: 22789543
12.  Expression of the neuron-specific protein CHD5 is an independent marker of outcome in neuroblastoma 
Molecular Cancer  2010;9:277.
Background
The chromodomain, helicase DNA-binding protein 5 (CHD5) is a potential tumor suppressor gene located on chromosome 1p36, a region recurrently deleted in high risk neuroblastoma (NB). Previous data have shown that CHD5 mRNA is present in normal neural tissues and in low risk NB, nevertheless, the distribution of CHD5 protein has not been explored. The aim of this study was to investigate CHD5 protein expression as an immunohistochemical marker of outcome in NB. With this purpose, CHD5 protein expression was analyzed in normal neural tissues and neuroblastic tumors (NTs). CHD5 gene and protein expression was reexamined after induction chemotherapy in a subset of high risk tumors to identify potential changes reflecting tumor response.
Results
We provide evidence that CHD5 is a neuron-specific protein, absent in glial cells, with diverse expression amongst neuron types. Within NTs, CHD5 immunoreactivity was found restricted to differentiating neuroblasts and ganglion-like cells, and absent in undifferentiated neuroblasts and stromal Schwann cells. Correlation between protein and mRNA levels was found, suggesting transcriptional regulation of CHD5. An immunohistochemical analysis of 90 primary NTs highlighted a strong association of CHD5 expression with favorable prognostic variables (age at diagnosis <12 months, low clinical stage, and favorable histology; P < 0.001 for all), overall survival (OS) (P < 0.001) and event-free survival (EFS) (P < 0.001). Multivariate analysis showed that CHD5 prognostic value is independent of other clinical and biologically relevant parameters, and could therefore represent a marker of outcome in NB that can be tested by conventional immunohistochemistry. The prognostic value of CHD5 was confirmed in an independent, blinded set of 32 NB tumors (P < 0.001).
Reactivation of CHD5 expression after induction chemotherapy was observed mainly in those high risk tumors with induced tumor cell differentiation features. Remarkably, these NB tumors showed good clinical response and prolonged patient survival.
Conclusions
The neuron-specific protein CHD5 may represent a marker of outcome in NB that can be tested by conventional immunohistochemistry. Re-establishment of CHD5 expression induced by chemotherapy could be a surrogate marker of treatment response.
doi:10.1186/1476-4598-9-277
PMCID: PMC2992029  PMID: 20950435

Results 1-12 (12)