Review
Genetically engineered murine models – Contribution to our understanding of the genetics, molecular pathology and therapeutic targeting of neuroblastoma

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Abstract

Genetically engineered mouse models (GEMM) have made major contributions to a molecular understanding of several adult cancers and these results are increasingly being translated into the pre-clinical setting where GEMM will very likely make a major impact on the development of targeted therapeutics in the near future. The relationship of pediatric cancers to altered developmental programs, and their genetic simplicity relative to adult cancers provides unique opportunities for the application of new advances in GEMM technology. In neuroblastoma the well-characterized TH-MYCN GEMM is increasingly used for a variety of molecular-genetic, developmental and pre-clinical therapeutics applications. We discuss: the present and historical application of GEMM to neuroblastoma research, future opportunities, and relevant targets suitable for new GEMM strategies in neuroblastoma. We review the potential of these models to contribute both to an understanding of the developmental nature of neuroblastoma and to improved therapy for this disease.

Introduction

Recent advances in genetically engineered mouse modeling (GEMM) technology have provided unique opportunities to address fundamental questions about the developmental origin, molecular pathology and therapeutic susceptibility of pediatric cancers. As expertise grows in understanding of neural crest development and specification, and in the function of oncogenic drivers of the childhood cancer neuroblastoma, GEMM approaches will provide the means to validate these drivers and to generate important future models. This article reviews current models and GEMM strategies aimed at elucidating genes that alter neural crest fate specification and neuroblast function, and that thereby contribute to the formation of NB tumors.

In general, GEMM are most commonly developed to provide mechanistic data elucidating causal roles for particular genes in developmental fate specification or oncogenesis. Developmentally focused approaches examine whether an observed phenotype is associated with tissue- and/or time-specific mis-expression of a gene that regulates differentiation program of the target tissues. For this reason, targeted knock-in strategies incorporating some form of imaging, and the ability to regulate expression of the gene of interest, are most commonly used. The ability to control expression of the target gene in a time-dependent manner and to visualize expression are equally critical to the elucidation of molecular oncogenic mechanisms and to efforts to develop novel therapeutics, respectively.

Cancer focused GEMM strategies seek to determine more specifically: (1) whether altered expression of a cancer-associated gene or mutation can initiate and sustain tumorigenesis, (2) what clinical phenotype if any, is recapitulated by the model, (3) whether the oncoprotein target itself or changes in molecular signaling pathways induced by its mis-expression represent therapeutic targets, and (4) whether secondary genomic alterations evolve and are required for oncogenesis, and 5, whether these validated genes also be therapeutic targets. In neuroblastoma in particular, the sequence of genetic events underlying normal maturation of neural crest precursors is now understood in some detail. Manipulated expression of such genes (PHOX2B, Sox10, Hand2, and TH) promises to provide great insights into where these initiating defects occur, and how they impact the ultimate biology of neural crest-derived malignancy.

Given that many, if not most, pediatric (embryonal) cancers represent aberrations of normal developmental programs, GEMM approaches are ideal in that they combine elements of both controlled and visualized expression. In pediatric cancer the question of whether a particular gene is oncogenic often relates to its role in development, and this by nature demands the ability to manipulate gene expression, ideally in a fully reversible manner, and requires the capacity to confirm gene expression using a surrogate marker. Additional imaging techniques that serve as rapid and practical screens for presence of tumor, and are capable of quantitating volume or bulk responses in the setting of a pre-clinical drug trial are equally critical. Appropriate credentialing of a murine model for therapeutic use is vital and challenging. Below, we review some of the technologies and approaches available in these areas.

Section snippets

How murine genetic models could contribute to a complete understanding of NB

Major differences exist between pediatric and adult cancers in the genesis of the most common non-germline cancers. Spontaneous adult malignancies largely result from stochastic accumulation of somatic genetic mutations and epigenetic changes over a relatively long timescale [1], [2], [3]. In contrast, transformation in many pediatric cancers is initiated by comparably few “driver” mutations that occur during a short window of programmed differentiation [4]. Clones that acquire a survival

Existing GEMM models of NB–TH-MYCN

The crucial role of MYC genes in development of the central and peripheral nervous systems and their derived cancers has been recognized since the identification of MYC and MYCN as cellular homologues of v-MYC (avian myelocytomatosis inducing virus derived oncogene) [5], [6], [7], [8], [9], [10]. Initial attempts using mice to model the role of MYC genes in specification of brain and neural crest derived structures used targeted knock-in approaches to ablate endogenous expression of MYC or

Modeling NB genetics – MYCN modifiers

One of the obvious strengths of GEMM is in the identification of genetic modifiers through forward genetic screens. Cellular expression of MYCN induces a potent oncogenic stimulus that requires significant remodeling of metabolism, cell cycle checkpoint function, apoptotic potential and DNA damage repair mechanisms [51], [52], [53]. A critical question for understanding the role of MYCN as a target is how overexpression of MYCN, and MYC genes in general, is tolerated by cells [54], [55], [56].

Modeling MYCN-targets and cooperating genes

Simple genetic intercrosses (Fig. 2) provide an additional, candidate-driven means to assess cooperativity between an oncogene and additional gene targets in the genesis of a particular tumor. This approach can also help to clarify the oncogenically relevant targets, which in the case of MYC is particularly useful since the gene-set regulated by this enigmatic family of transcription factors encompasses thousands of targets regulating ribosomal and protein biosynthesis, metabolism and

Modeling the role of p53 pathway

Trp53 is important in NB, since MYC-driven cancers are characterized by coordinate induction of proliferation and apoptosis, with both ARF and p53 pathways implicated [68], [83], [84], [85], [86]. Concurrent deletion of p53 enhances tumorigenesis by MYC in a wide range of tumor models [87], [88]. The advent of GEMM with reactivatable p53 transgenes has provided an additional opportunity to study the impact of p53 modulation in the context of MYCN misexpression. Mutations in p53 occur commonly

Beyond MYCN modeling – ALK and PHOX2B

That relatively few mutations drive the majority of NB cases is clear from recent genome-wide studies. MYCN is a definitive marker of 40% of high-risk NB, however the majority of high-risk cases are not defined by known single gene alterations [89]. The recognition that distinct ALK mutations are differentially present within the germline and somatic tumor tissue of NB patients, and underlie the majority of hereditary NB, led to the elegant validation of Knudson's two-hit hypothesis (describing

Beyond MYCN modeling – additional targets

Since aberrant control of MYCN and ALK accounts for only a minority of high-risk patients, additional models are needed both for MYCN-independent high-risk disease, and for intermediate-risk patients. Genome-wide-association-studies are rapidly defining the spectrum of nucleotide polymorphisms, copy number variations, expression changes and epigenetic events that account for a predisposition to both sporadic and familial NB and which appear to selectively associate with discrete tumor

Modeling of additional expression and genomic alterations

Cross-platform, integrative genomic screens that analyze copy number abnormalities (CNAs) suggest that distinct patient populations are defined by global patterns that cluster alterations in DNA ploidy and focal-segmental copy number (numerical or focal segmental copy number changes) [118], [119], [120]. These include the commonly observed regions of focal gain and loss (1p36, 11q, 13, 14, gain of 17p), which are being incorporated into clinical risk-assignment systems. With the routine

Modeling NB as a deficiency of neural crest development

NB is one of several neurocristopathies associated with disordered development of the autonomic nervous system, including central hypoventilation syndrome, Hirschsprung disease, pheochromocytoma and neurofibromatosis [139]. The high degree of clinical diversity and specificity of age distribution reinforces the hypothesis that specific NB phenotypes are likely to originate from distinct aberrations that occur at specific points within the neural crest fate-specification pathway. The use of GEMM

Technologies for current and future modeling strategies

The TH-MYCN model utilized comparatively simple, non-targeted tissue-specific mis-expression (a conventional transgenic approach, to establish that targeted expression of an initiating oncogene could drive NB (Fig. 4A). Given the unique biology of this disease as a derivative of aberrant neural crest differentiation and its propensity to regress in an age- and time-dependent manner, future modeling strategies should incorporate technologies allowing for inducible or fully reversible expression

GEMM-based pre-clinical drug development

A potential strength of GEMM is their applicability to pre-clinical drug development, but efficient utilization for this purpose has not yet been fully realized. A significant deficiency of tumor implantation modeling has been lack of predictive power for clinical response, most likely related to altered behavior of implanted human cell lines in the subcutaneous murine microenvironment. It is becoming apparent that in certain trial designs, GEMM may provide superior modeling of drug response as

Conclusions

GEMM are continuing to define the role of clinically important mutations in the genesis of the major adult cancers. The use of GEMM-based approaches is increasing in pediatric cancer, and will play an important role in the development of novel therapeutics targeted at specific oncogenes and pathways. NB can be a paradigm for the rational development of GEMM that define all discrete clinical cohorts of this distinctive cancer.

Conflict of interest

None.

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