Matthew R. Grimmer

Pleiotropic role for MYCN in medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in approximately 5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.

Distinct Neural Stem Cell Populations Give Rise to Disparate Brain Tumors in Response to N-MYC

The proto-oncogene MYCN is mis-expressed in various types of human brain tumors. To clarify how developmental and regional differences influence transformation, we transduced wild-type or mutationally stabilized murine N-myc(T58A) into neural stem cells (NSCs) from perinatal murine cerebellum, brain stem, and forebrain. Transplantation of N-myc(WT) NSCs was insufficient for tumor formation. N-myc(T58A) cerebellar and brain stem NSCs generated medulloblastoma/primitive neuroectodermal tumors, whereas forebrain NSCs developed diffuse glioma. Expression analyses distinguished tumors generated from these different regions, with tumors from embryonic versus postnatal cerebellar NSCs demonstrating Sonic Hedgehog (SHH) dependence and SHH independence, respectively. These differences were regulated in part by the transcription factor SOX9, activated in the SHH subclass of human medulloblastoma. Our results demonstrate context-dependent transformation of NSCs in response to a common oncogenic signal.

Childhood tumors of the nervous system as disorders of normal development

Purpose of review Advances in tumor genetics have increasingly linked pediatric neoplasms with disordered mechanisms of normal development, supporting the model of embryonal tumorigenesis. We provide a detailed discussion of two pediatric neural tumors, medulloblastoma and neuroblastoma, addressing tumorigenic causality and similarities within a pharmacological context. Recent findings Expression profiling, elegant murine models, and chemical blockades of oncogenic signaling pathways have encouraged a new generation of therapeutic approaches for tumor treatment. Recent data have further clarified regulation of neural developmental and factors triggering malignancy. Summary Medulloblastoma and neuroblastoma exemplify the current embryonal tumor model. Sonic hedgehog signaling is required for cerebellar development and its dysregulation is implicated in formation of medulloblastoma. The transcription factor Mycn orchestrates proliferation and differentiation of the developing peripheral neural crest. Amplification of the MYCN gene is the predominant marker for aggressive neuroblastoma, and correlates with poor prognosis. Current evidence suggests that Mycn is also the primary executor of Sonic hedgehog signaling in the cerebellum and that the Sonic hedgehog pathway regulates levels of both MYCN mRNA and Mycn protein product independently. Destabilization of Myc through inhibition of phosphoinositide 3-kinase signaling exhibits promise not only in medulloblastoma and neuroblastoma, but in a wide range of Myc-driven tumors.

Malignant Progression and Blockade of Angiogenesis in a Murine Transgenic Model of Neuroblastoma

Targeted expression of MYCN to the neural crest [under control of the rat tyrosine hydroxylase (TH) promoter] causes neuroblastoma in transgenic mice (TH-MYCN) and is a well-established model for this disease. Because high levels of MYCN are associated with enhanced tumor angiogenesis and poor clinical outcome in neuroblastoma, we serially characterized malignant progression, angiogenesis, and sensitivity to angiogenic blockade in tumors from these animals. Tumor cells were proliferative, secreted high levels of the angiogenic ligand vascular endothelial growth factor (VEGF), and recruited a complex vasculature expressing the angiogenic markers VEGF-R2, alpha-SMA, and matrix metalloproteinases MMP-2 and MMP-9, all of which are also expressed in human disease. Treatment of established murine tumors with the angiogenesis inhibitor TNP-470 caused near-complete ablation, with reduced proliferation, enhanced apoptosis, and vasculature disruption. Because TNP-470 has been associated with neurotoxicity, we tested the recently described water-soluble HPMA copolymer-TNP-470 conjugate (caplostatin), which showed comparable efficacy and was well tolerated without weight loss or neurotoxicity as measured by rotarod testing. This study highlights the importance of angiogenesis inhibition in a spontaneous murine tumor with native tumor-microenvironment interactions, validates the use of mice transgenic for TH-MYCN as a model for therapy in this common pediatric tumor, and supports further clinical development of caplostatin as an antiangiogenic therapy in childhood neuroblastoma.

Paracrine signaling through MYCN enhances tumor-vascular interactions in neuroblastoma.

PI3K/mTOR inhibitors inhibit angiogenesis by blocking MYCN-dependent paracrine signaling between tumor and endothelial cells. Childhood Cancers—and MYCN Therapy—Strike a Nerve It’s easy to become jaded by the current celebrity-advocate overload. But appeals for children suffering from lethal cancers never fail to tug at the heartstrings. Consider the disease neuroblastoma, one of the most common childhood cancers: Nearly half of the cases occur in children under 2 years of age, and treatments for the so-called high-risk version of the disease often fail. But mouse models of neuroblastoma provide a clue: Overexpression of the MYCN proto-oncogene foreshadows treatment failure. Now, Chanthery et al. demonstrate that a drug currently being tested in clinical trials for solid tumors improves survival by targeting the MYCN protein. This promising drug, NVP-BEZ235, inhibits the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway, which is frequently activated in cancers. NVP-BEZ235 blocked new blood vessel formation (angiogenesis) by tumors and improved survival in two mouse models of neuroblastoma, a xenograft variety developed with primary tumor from patients and a genetic model in which the MYCN gene drives spontaneous tumor formation in mice. The antiangiogenic effect depended in part on function of the MYCN protein, a transcriptional regulator: NVP-BEZ235 therapy spurred degradation of MYCN in tumor cells, which, in turn, produced local (paracrine) effects that inhibited angiogenesis, a process required for tumor growth and metastasis. Future clinical trials will reveal whether NVP-BEZ235 represents a new therapeutic option for MYCN-amplified neuroblastoma—and a chance for sick toddlers to thrive. Neuroblastoma, a tumor of peripheral neural crest origin, numbers among the most common childhood cancers. Both amplification of the proto-oncogene MYCN and increased neoangiogenesis mark high-risk disease. Because angiogenesis is regulated by phosphatidylinositol 3-kinase (PI3K), we tested a clinical PI3K inhibitor, NVP-BEZ235, in MYCN-dependent neuroblastoma. NVP-BEZ235 decreased angiogenesis and improved survival in both primary human (highly pretreated recurrent MYCN-amplified orthotopic xenograft) and transgenic mouse models for MYCN-driven neuroblastoma. Using both gain- and loss-of-function approaches, we demonstrated that the antiangiogenic efficacy of NVP-BEZ235 depended critically on MYCN in vitro and in vivo. Thus, clinical PI3K/mammalian target of rapamycin inhibitors drove degradation of MYCN in tumor cells, with secondary paracrine blockade of angiogenesis. Our data demonstrated significantly improved survival in treated animals and suggest that NVP-BEZ235 should be tested in children with high-risk, MYCN-amplified neuroblastoma.

Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation

Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consisting of multiple zinc finger domains are currently being developed for clinical applications. However, no genome-wide investigations into their binding specificity have been performed. We have created six-finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB domain (SKD) that interacts with a complex containing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼5-fold, with a majority of the new sites located outside of promoters. De novo motif analyses suggest that the lack of binding specificity is due to subsets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger binding sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications.

BMPs oppose Math1 in cerebellar development and in medulloblastoma

 Bone morphogenetic proteins (BMPs) are soluble effectors of differentiation and are central in orchestrating development of organs including the cerebellum. The transcription factor and BMP target Math1 (mouse atonal homolog 1) is a critical regulator of neuronal progenitors destined to form the cerebellar cortex. Signaling networks controlled by BMPs, Math1, and Sonic Hedgehog (Shh) together regulate the proliferation and differentiation of cerebellar progenitor cells. Defects in these pathways are also implicated in medulloblastoma, the most common pediatric brain tumor. In this issue of Genes & Development, Zhao et al. (2008) perform an elegant set of experiments that clarify the role of BMPs and of Math1 in medulloblastoma. Destabilization of Math1 by treatment with BMP-2 or BMP-4 induced neuronal differentiation in cells from mouse models of Shh-driven medulloblastoma and reduced proliferation in established tumors in vivo. These studies provide important new insights into the vulnerabilities of medulloblastoma and present future therapeutic promise.

Effects on the transcriptome upon deletion of a distal element cannot be predicted by the size of the H3K27Ac peak in human cells

Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with increased risk for colorectal cancer (CRC). A molecular understanding of the functional consequences of this genetic variation is complicated because most GWAS SNPs are located in non-coding regions. We used epigenomic information to identify H3K27Ac peaks in HCT116 colon cancer cells that harbor SNPs associated with an increased risk for CRC. Employing CRISPR/Cas9 nucleases, we deleted a CRC risk-associated H3K27Ac peak from HCT116 cells and observed large-scale changes in gene expression, resulting in decreased expression of many nearby genes. As a comparison, we showed that deletion of a robust H3K27Ac peak not associated with CRC had minimal effects on the transcriptome. Interestingly, although there is no H3K27Ac peak in HEK293 cells in the E7 region, deletion of this region in HEK293 cells decreased expression of several of the same genes that were downregulated in HCT116 cells, including the MYC oncogene. Accordingly, deletion of E7 causes changes in cell culture assays in HCT116 and HEK293 cells. In summary, we show that effects on the transcriptome upon deletion of a distal regulatory element cannot be predicted by the size or presence of an H3K27Ac peak.

Can genome engineering be used to target cancer-associated enhancers?

Transcriptional misregulation is involved in the development of many diseases, especially neoplastic transformation. Distal regulatory elements, such as enhancers, play a major role in specifying cell-specific transcription patterns in both normal and diseased tissues, suggesting that enhancers may be prime targets for therapeutic intervention. By focusing on modulating gene regulation mediated by cell type-specific enhancers, there is hope that normal epigenetic patterning in an affected tissue could be restored with fewer side effects than observed with treatments employing relatively nonspecific inhibitors such as epigenetic drugs. New methods employing genomic nucleases and site-specific epigenetic regulators targeted to specific genomic regions, using either artificial DNA-binding proteins or RNA-DNA interactions, may allow precise genome engineering at enhancers. However, this field is still in its infancy and further refinements that increase specificity and efficiency are clearly required.

Frequent lack of repressive capacity of promoter DNA methylation identified through genome-wide epigenomic manipulation

It is widely assumed that the addition of DNA methylation at CpG rich gene promoters silences gene transcription. However, this conclusion is largely drawn from the observation that promoter DNA methylation inversely correlates with gene expression in natural conditions. The effect of induced DNA methylation on endogenous promoters has yet to be comprehensively assessed. Here, we induced the simultaneous methylation of thousands of promoters in the genome of human cells using an engineered zinc finger-DNMT3A fusion protein, enabling assessment of the effect of forced DNA methylation upon transcription, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that DNA methylation is frequently insufficient to transcriptionally repress promoters. Furthermore, DNA methylation deposited at promoter regions associated with H3K4me3 is rapidly erased after removal of the zinc finger-DNMT3A fusion protein. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. These findings suggest that promoter DNA methylation is not generally sufficient for transcriptional inactivation, with implications for the emerging field of epigenome engineering. One Sentence Summary Genome-wide epigenomic manipulation of thousands of human promoters reveals that induced promoter DNA methylation is unstable and frequently does not function as a primary instructive biochemical signal for gene silencing and chromatin reconfiguration.