In the beginning, Trk was an oncogene. Yet Neurotrophin-Trk signaling came to preeminence in the field of neurobiology. Now it is appreciated that Trks regulate important processes in nonneuronal cells and, in addition to their impact on tumors of neural origin, may contribute to the pathogenesis of carcinomas, myelomas, and prostate and lymphoid tumors. Although mutations and rearrangements of Trk are seen only sporadically in human cancers, such as medullary thyroid carcinoma, a number of recent studies indicate that expression of TrkB contributes to tumor pathology. In neuroblastoma, TrkA expression marks good prognosis which TrkB and Brain-derived neurotrophic factor (BDNF) expression marks poor prognosis. Activation of the BDNF/TrkB signal transduction pathway also stimulates tumor cell survival and angiogenesis and contributes to resistance to cytotoxic drugs and anoikis, enabling cells to acquire many of the characteristic features required for tumorigenesis. Small molecule inhibitors, such as Cephalon's CEP-701, are in phase 1 and 2 clinical trials, and a series of AstraZeneca Trk inhibitors are poised to enter the clinic. As monotherapy, inhibitors may be effective only in tumors with activating Trk mutations. Important clinical follow-up will be the assessment of Trk inhibitors in combination with standard chemo- or radiotherapy or other signal transduction pathway inhibitors. (Clin Cancer Res 2009;15(19):5962–7)

It is well known that Neuroblastoma (NB) patients whose tumors have an undifferentiated histology and a transcriptome enriched in cell cycle genes have a worse prognosis. This contrasts with the good prognoses of patients whose tumors have histologic evidence of differentiation and a transcriptome enriched in differentiation genes. Tumor cell lines from poor prognosis, high-risk patients contain a number of genetic alterations, including amplification of MYCN, 1pLOH, and unbalanced 11q or gains of Chr 17 and 7, and exhibit uncontrolled growth and an undifferentiated phenotype in in vitro culture. Yet treatment of such NB cell lines with retinoic acid results in growth control and induction of differentiation. This indicates that the signaling pathways that regulate cell growth and differentiation are not functionally lost but dysregulated. Agents such as retinoic acid normalize the signaling pathways and impose growth control and induction of differentiation. Recent studies in embryonic stem cells indicate that polycomb repressor complex proteins (PRC1 and PRC2) play a major role in regulating stem cell lineage specification and coordinating the shift from a transcriptome that supports self-renewal or growth to one that specifies lineage and controls growth. We have shown that in NB, the PRC2 complex is elevated in undifferentiated NB tumors and functions to suppress a number of tumor suppressor genes. This study will review the role of MYC genes in regulating the epigenome in normal development and explore how this role may be altered during tumorigenesis.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with an undifferentiated status and generally poor prognosis, but the basis for these characteristics remains unknown. In this study, we show that upregulation of the Polycomb protein histone methyltransferase EZH2, which limits differentiation in many tissues, is critical to maintain the undifferentiated state and poor prognostic status of NB by epigenetic repression of multiple tumor suppressor genes. We identified this role for EZH2 by examining the regulation of CASZ1, a recently identified NB tumor suppressor gene whose ectopic restoration inhibits NB cell growth and induces differentiation. Reducing EZH2 expression by RNA interference–mediated knockdown or pharmacologic inhibiton with 3-deazaneplanocin A increased CASZ1 expression, inhibited NB cell growth, and induced neurite extension. Similarly, EZH2^−/− mouse embryonic fibroblasts (MEF) displayed 3-fold higher levels of CASZ1 mRNA compared with EZH2^+/+ MEFs. In cells with increased expression of CASZ1, treatment with histone deacetylase (HDAC) inhibitor decreased expression of EZH2 and the Polycomb Repressor complex component SUZ12. Under steady-state conditions, H3K27me3 and PRC2 components bound to the CASZ1 gene were enriched, but this enrichment was decreased after HDAC inhibitor treatment. We determined that the tumor suppressors CLU, NGFR, and RUNX3 were also directly repressed by EZH2 like CASZ1 in NB cells. Together, our findings establish that aberrant upregulation of EZH2 in NB cells silences several tumor suppressors, which contribute to the genesis and maintenance of the undifferentiated phenotype of NB tumors. Cancer Res; 72(1); 315–24. ©2011 AACR.

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

Background: Alternative treatment options are needed for advanced neuroblastoma patients because their prognosis remains poor after intensive chemotherapy. Neuroblastoma cells express platelet-derived growth factor (PDGF), stem cell factor (SCF), and vascular endothelial growth factor (VEGF) and their respective receptors, PDGFR, c-Kit, and Flk-1. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the tyrosine kinase activities of c-Kit and PDGFR, on the growth of neuroblastoma cells in vivo and in vitro. Methods: We tested seven human neuroblastoma cell lines for their sensitivity to imatinib. Cell viability was assessed by trypan blue dye exclusion. Apoptosis was evaluated by nuclear staining, flow cytometry, and western blotting. Protein assays included immunoprecipitation, western blotting, enzyme-linked immunosorbent assays, and immunohistochemistry. mRNA expression was assessed by northern blotting. We used a xenograft model in SCID mice (10 mice per group) to evaluate the effects of imatinib oral therapy (50 or 100 mg/kg every 12 hours for 14 days) on neuroblastoma tumor growth. All statistical tests were two-sided. Results: All seven neuroblastoma cell lines treated with imatinib displayed concentration-dependent decreases in cell viability, which coincided with an induction of apoptosis, and with ligand-stimulated phosphorylation of c-Kit and PDGFR. The imatinib concentrations that caused 50% inhibition of growth and 50% inhibition of ligand-induced phosphorylation of these receptors were 9–13 μM and 0.1–0.5 μM, respectively. Expression of VEGF, but not phosphorylation of Flk-1, its receptor, was reduced in neuroblastoma cells treated with imatinib at 10 μM or higher. Mice treated with imatinib at 50 mg/kg or 100 mg/kg had statistically significantly smaller tumors than control mice treated with vehicle (mean tumor volume in mice treated with imatinib at 50 mg/kg = 1546 mm³, in control mice = 2954 mm³; difference = 1408 mm³, 95% confidence interval [CI] = 657 to 2159 mm³; P<.001; mean tumor volume in mice treated with imatinib at 100 mg/kg = 463 mm³; difference = 2491 mm³, 95% CI = 1740 to 3242 mm³; P<.001). Conclusions: Imatinib inhibited the growth of neuroblastoma cells in vitro and in vivo. This inhibition was associated with suppression of PDGFR and c-Kit phosphorylation and inhibition of VEGF expression.