Neuroblastoma is one of the most common childhood cancers and is responsible for about 15% of childhood deaths due to cancer. The neuroblastoma tumors arise in cells that develop into and form part of the body’s nervous system. Many researchers have studied the genetics of this disease and have recognised common patterns. In particular, neuroblastomas can occur when a protein called MYCN is over-produced and a tumor suppressor protein called NF1 is lost.

NF1 is a large protein with several distinct parts or domains. The most studied domain of NF1 is called the GRD, and it is mainly responsible for dampening down signals that cause cells to grow, specialize and survive. However, experiments in mice have revealed that this protein uses its other domains to control the normal development of part of the nervous system.

He et al. wanted to know which domains of NF1 are important for suppressing the growth of neuroblastomas. The experiments were conducted in zebrafish that had been engineered to produce an excess of the human version of MYCN. When He et al. also deleted the gene for the zebrafish’s version of NF1, the fish quickly developed neuroblastomas. Supplying the zebrafish with just the GRD of NF1 was enough to supress the growth of the tumors. These experiments show that NF1 uses different domains and signalling pathways to regulate the normal development of part of the nervous system and to prevent formation of neuroblastoma.

These engineered zebrafish represent an animal model of neuroblastoma that mimics the human disease in many ways. This model will make it possible to test new drug combinations and to find more effective treatments for neuroblastoma patients.

Neuroblastoma, a malignant embryonic tumor of childhood, arises in neural crest-derived dopaminergic neuroblasts that generate the peripheral sympathetic nervous system (PSNS). This cancer is the most common noncranial solid tumor in childhood, accounting for 8–10% of all childhood malignancies and 15% of all cancer deaths in children (Maris and Matthay, 1999). Neuroblastoma is genetically heterogeneous, with multiple interacting genetic mutations required to generate fully transformed malignant tumors. As in many other pediatric cancers, resequencing studies have documented a very low frequency of somatic mutations in neuroblastoma (Cheung et al., 2012; Molenaar et al., 2012; Pugh et al., 2013), including ALK (~9.2% of cases), PTPN11 (~2.9%), ATRX (~2.5%) and NF1 (~1%). Amplification and overexpression of the MYCN oncogene occurs in about one-third of patients and is a principal indicator of a poor prognosis (Brodeur et al., 1984; Hansford et al., 2004). Genomic aberrations that inactivate the NF1 tumor suppressor gene, including loss-of-function mutations and deletions, as well as decreased expression levels of the gene, have been identified in 6% of primary neuroblastomas and are predictive of a poor outcome (Hölzel et al., 2010), suggesting an important role for NF1 loss in neuroblastoma tumorigenesis.

The NF1 gene encodes neurofibromin, a 2818 amino acid protein whose main functional domain is the ~330 amino acid GTPase-activating protein-related domain (GRD), which negatively regulates RAS signaling by catalyzing the hydrolysis of RAS-GTP into RAS-GDP (Nur-E-Kamal et al., 1993); thus, one consequence of NF1 loss is the aberrant activation of RAS signaling (Maertens and Cichowski, 2014). Loss of NF1 in neuroblastoma cells has been shown to mediate resistance to retinoic acid via hyperactive RAS signaling, which can be abolished by enforced expression of NF1-GRD (Hölzel et al., 2010). However, it is known that NF1 has other functions in PSNS development besides the downmodulation of RAS signaling, because Nf1 mutant mice die at birth with evidence of massive overgrowth of neural crest tissues, including the sympathetic ganglia, while overexpression of the GRD domain is unable to reverse this overgrowth (Ismat et al., 2006). In addition, studies showed identical or only modestly elevated RAS-GTP levels in NF1-deficient human neuroblastoma cells, in contrast to highly elevated RAS-GTP levels in NF1-deficient Schwannoma tumor cells (Johnson et al., 1993; The et al., 1993). These results, coupled with the numerous mutations of NF1 that cause the disease neurofibromatosis type 1, but do not appear to affect protein stability or GAP function (Abernathy et al., 1997; Fahsold et al., 2000), argue that functional domains outside the GRD may mediate important aspects of neurofibromin function in neuroblastoma tumor suppression.

In earlier work, we identified two separate duplicated nf1 zebrafish genes, nf1a and nf1b, and generated multiple loss-of-function nf1 mutant zebrafish lines affecting both of these alleles (Lee et al., 2010; Padmanabhan et al., 2009; Shin et al., 2012). Mutant larvae carrying at least one wild-type nf1a or nf1b allele are viable, fertile, and show no obvious phenotypes during early development. To gain insight into the cellular and molecular consequences of NF1 loss in neuroblastoma, we used transgenic zebrafish models of neuroblastoma that overexpresses human MYCN in the PSNS (Zhu et al., 2012).

Here, we report that loss of the nf1a orthologue, which is much more highly expressed than nf1b during early PSNS development, greatly accelerates the onset of neuroblastoma induced by MYCN overexpression, with nearly complete penetrance by 5 weeks of age in nf1-deficient zebrafish. Loss of nf1 led to the aberrant activation of RAS signaling in MYCN-induced neuroblastoma, promoting both tumor cell survival and proliferation. We also show that the very aggressive growth properties of MYCN-induced neuroblastomas with loss of nf1 are due to aberrant activation of RAS signaling, because the increased penetrance and rapid growth could be suppressed by overexpressing the intact NF1 GRD domain. These findings establish nf1-deficient zebrafish that overexpress MYCN as an ideal animal model system for investigating new strategies to improve treatment of very high-risk neuroblastomas with aberrant RAS-MAPK activation.

In vivo structure-function analysis with both the wild-type and inactive GRD domain of NF1 revealed that the GAP activity of the GRD domain is required for the tumor suppressor function of NF1 in neuroblastoma. By contrast, the wild-type GRD domain failed to rescue the hypertrophy of sympathoadrenal cells in nf1 mutant embryos, indicating that the role of NF1 in suppressing neuroblastoma tumorigenesis differs from the mechanism that prevents PSNS hyperplasia during normal development.

In this study, we use a zebrafish model to demonstrate that NF1 loss can potentiate the tumorigenic effects of MYCN-overexpression in high-risk neuroblastoma. We show that nf1 deficiency in the fish leads to aberrant activation of RAS-MAPK signaling in MYCN-induced neuroblastoma, which promotes both tumor cell survival and proliferation, leading to marked acceleration of tumor onset and increased tumor penetrance. To establish the underlying mechanism of these observations, we performed in vivo structure-function analyses to show that the tumor suppression function of NF1 is mediated through the GAP activity of its GRD domain. The same rescue construct does not suppress the PSNS overgrowth that accompanies nf1 loss during development, supporting earlier work implicating a different and still-to-be-identified activity of NF1 in the restriction of the normal growth of PSNS neuroblasts during embryogenesis.

We and others have shown that inactivation of the NF1 gene in human neuroblastoma and melanoma cell lines does not result in increased levels of the GTP-bound form of Ras compared to similar lines that express normal levels of the NF1 protein (Johnson et al., 1993; The et al., 1993). Ostensibly, this would seem inconsistent with our current findings in the zebrafish model implicating downregulation of the RAS pathway in NF1-mediated tumor suppression. However, a recent whole-genome sequencing study revealed that loss of NF1 is one of a strikingly high frequency of diverse mutations that hyperactivate the RAS-MAPK signaling pathway in relapsed neuroblastoma (Eleveld et al., 2015). Thus, our inability to demonstrate increased GTP on RAS in NF1-deficient neuroblastoma cell lines may reflect the strong activation of the RAS-MAPK signaling pathway in essentially all high-risk neuroblastomas, if not by NF1 loss then by direct mutation of RAS itself or other types of mutations. This interpretation becomes more appealing when one realizes that neuroblastomas able to grow in vitro as continuous cell lines are invariably from patients with the very highest risk of treatment failure, and often are obtained from tumor biopsies at the time of relapse. The same relationship likely applies to melanoma cell lines as well, since most of these tumors harbor activating mutations in the RAS-MAPK pathway, including BRAF V600E and NRAS Q61L/R, as well as the loss of NF1 in a subset of cases (Cancer Genome Atlas Network, 2015; Hodis et al., 2012).

Studies of neural crest and PSNS development in Nf1 null mice were the first to implicate a GAP-independent activity of Nf1 in restricting the growth of sympathoadrenal cells, as the GRD domain was not able to rescue the overgrowth of sympathetic neuroblasts in the adrenal medulla (Ismat et al., 2006). Our results support those studies, in that the GRD domain expressed in the same zebrafish line that suppressed neuroblastoma pathogenesis failed to suppress sympathetic neuroblast overgrowth in nf1-deficient zebrafish embryos. Thus, GAP-independent activities of NF1 appear to restrict the growth of normal neural crest-derived tissues during development, as the PSNS overgrowth phenotype was not rescued by the same level of GRD restoration that suppressed the accelerated onset of MYCN-driven neuroblastoma (Figures 1 and 7).

The recent discovery that mutations leading to activated RAS-MAPK signaling occur in nearly 80% of relapsed neuroblastomas has clear implications for the selection of targeted therapy for this tumor (Eleveld et al., 2015). Indeed, it suggests that MEK inhibitors and other agents targeting the RAS-MAPK pathway might suppress the outgrowth of resistant clones and therefore should be investigated as part of the initial combination therapy for high-risk neuroblastoma. Our zebrafish model of MYCN-driven neuroblastoma with nf1 loss is ideally suited for the rapid in vivo analysis of the effects of candidate small-molecule inhibitors selected for this purpose. Neuroblastomas can be detected in most MYCN-transgenic fish with nf1a loss by 3 weeks of age, when the fish are very small, making it feasible to test the effectiveness of many drugs and drug combinations for their ability to kill primary neuroblastoma cells in vivo. This advantage of the model is readily apparent in our demonstration of marked antitumor synergy between the MEK inhibitor trametinib and the retinoid isoretinoin in MYCN-overexpressing fish with loss of nf1 (Figure 8). Studies using our model should be a valuable asset in devising new therapeutic strategies for neuroblastomas with mutations affecting the RAS-MAPK pathway, which appear to be a major cause of relapse in children with this devastating tumor.

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Author details

1. Shuning He

   Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   SH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   shuning_he@dfci.harvard.edu

   Competing interests

   The authors declare that no competing interests exist.

2. Marc R Mansour

   1. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States
   2. Department of Hematology, UCL Cancer Institute, University College London, London, United Kingdom

   Contribution

   MRM, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Mark W Zimmerman

   Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   MWZ, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Dong Hyuk Ki

   Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   DHK, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. Hillary M Layden

   Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   HML, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. Koshi Akahane

   Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   KA, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

7. Evisa Gjini

   Center for Immuno-Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States

   Contribution

   EG, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

8. Eric D de Groh

   1. Department of Cell and Developmental Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States
   2. Penn Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States
   3. Institute for Regenerative Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States

   Contribution

   EDdG, Acquisition of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

9. Antonio R Perez-Atayde

   Department of Pathology, Children's Hospital Boston, Harvard Medical School, Boston, United States

   Contribution

   ARP-A, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

10. Shizhen Zhu

    1. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States
    2. Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, United States
    3. Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, United States

    Contribution

    SZ, Analysis and interpretation of data, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

11. Jonathan A Epstein

    1. Department of Cell and Developmental Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States
    2. Penn Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States
    3. Institute for Regenerative Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States

    Contribution

    JAE, Conception and design, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

12. A Thomas Look

    Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States

    Contribution

    ATL, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    thomas_look@dfci.harvard.edu

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0001-7851-8617

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