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.

Summary Neuroblastoma is a pediatric malignancy that typically arises in early childhood and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumors with excellent outcomes to widely metastatic disease where long-term survival is approximately 40% despite intensive therapy1. A previous genome-wide association study (GWAS) identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumor cells2. Here we sought to discover the causal DNA variant at this locus and the mechanism by which it leads to neuroblastoma tumorigenesis. We first imputed all possible genotypes across the LMO1 locus and then mapped highly associated single nucleotide polymorphism (SNPs) to areas of chromatin accessibility, evolutionary conservation, and transcription factor binding sites. SNP rs2168101 G>T was the most highly associated variant (combined P=7.47×10-29, Odds Ratio 0.65, 95% CI: 0.60-0.70) and resided in a super-enhancer defined by extensive acetylation of histone H3 lysine 27 within the first intron of LMO1. The ancestral G-allele that is associated with tumor formation resides in a conserved GATA transcription factor binding motif. We show that the newly evolved protective TATA allele is associated with decreased total LMO1 expression (P=0.028) in neuroblastoma primary tumors and ablates GATA3 binding (P<0.0001). We demonstrate allelic imbalance favoring the G-containing strand in tumors heterozygous for this SNP as demonstrated both by RNA sequencing (P<0.0001) and reporter assays (P=0.002). These findings show that a recently evolved polymorphism within a super-enhancer element in the first intron of LMO1 influences neuroblastoma susceptibility through differential GATA transcription factor binding and direct modulation of LMO1 expression in cis, and this leads to an oncogenic dependency in tumor cells.

The amplified gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here, we show that the family member is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (∼10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic activation include focally amplified distal enhancers and translocation of highly active enhancers from other genes to within topologically associating domains containing the gene locus. The clinical outcome for patients with high levels of expression is virtually identical to that of patients with amplification of the gene, a known high-risk feature of this disease. Together, these findings establish as a bona fide oncogene in a clinically significant group of high-risk childhood neuroblastomas. Amplification of the oncogene is a recognized hallmark of high-risk pediatric neuroblastoma. Here, we demonstrate that is also activated as a potent oncogene in a distinct subset of neuroblastoma cases through either focal amplification of distal enhancers or enhancer hijacking mediated by chromosomal translocation. .

Loss-of-function mutations in pten genes, or expression of a constitutively active version of Akt2, render T-ALL cell survival and disease progression independent of Myc.

SUMMARY The tyrosine phosphatase SHP2 (PTPN11) regulates cellular proliferation, survival, migration and differentiation during development. Germline mutations in PTPN11 cause Noonan and LEOPARD syndromes, which have overlapping clinical features. Paradoxically, Noonan syndrome mutations increase SHP2 phosphatase activity, while LEOPARD syndrome mutants are catalytically impaired, raising the possibility that SHP2 has phosphatase-independent roles. By comparing shp2-deficient zebrafish embryos with those injected with mRNA encoding LEOPARD syndrome point mutations, we identify a phosphatase- and Erk-dependent role for Shp2 in neural crest specification and migration. We also identify an unexpected phosphatase-and Erk-independent function, mediated through its SH2 domains, which is evolutionarily conserved and prevents p53-mediated apoptosis in the brain and neural crest. Our results indicate that previously enigmatic aspects of LEOPARD syndrome pathogenesis can be explained by the combined effects of loss of Shp2 catalytic function and retention of an SH2 domain-mediated role that is essential for neural crest cell survival.

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Summary A genome-wide association study identified LMO1, which encodes a LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3 and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.

The RAS small GTPases orchestrate multiple cellular processes. Studies on knock-out mice showed the essential and sufficient role of K-RAS, but not N-RAS and H-RAS in embryonic development. However, many physiological functions of K-RAS in vivo remain unclear. Using wild-type and fli1:GFP transgenic zebrafish, we showed that K-ras-knockdown resulted in specific hematopoietic and angiogenic defects, including the impaired expression of erythroid-specific gene gata1 and ße3-hemoglobin, reduced blood circulation and disorganized blood vessels. Expression of either K-rasC40 that links to phosphoinositide 3-kinase (PI3K) activation, or Akt2 that acts downstream of PI3K, could rescue both hematopoietic and angiogenic defects in the K-ras knockdown. Consistently, the functional rescue by k-ras mRNA was significantly suppressed by wortmannin, a PI3K-specific inhibitor. Our results provide direct evidence that PI3K-Akt plays a crucial role in mediating K-ras signaling during hematopoiesis and angiogenesis in vivo, thus offering new targets and alternative vertebrate model for studying these processes and their related diseases.