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1.  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
2.  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
3.  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
4.  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
5.  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

Results 1-5 (5)