Evon Poon

The ALK(F1174L) mutation potentiates the oncogenic activity of MYCN in neuroblastoma.

The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.

Small Molecule Inhibitors of Aurora-A Induce Proteasomal Degradation of N-Myc in Childhood Neuroblastoma.

Amplification of MYCN is a driver mutation in a subset of human neuroendocrine tumors, including neuroblastoma. No small molecules that target N-Myc, the protein encoded by MYCN, are clinically available. N-Myc forms a complex with the Aurora-A kinase, which protects N-Myc from proteasomal degradation. Although stabilization of N-Myc does not require the catalytic activity of Aurora-A, we show here that two Aurora-A inhibitors, MLN8054 and MLN8237, disrupt the Aurora-A/N-Myc complex and promote degradation of N-Myc mediated by the Fbxw7 ubiquitin ligase. Disruption of the Aurora-A/N-Myc complex inhibits N-Myc-dependent transcription, correlating with tumor regression and prolonged survival in a mouse model of MYCN-driven neuroblastoma. We conclude that Aurora-A is an accessible target that makes destabilization of N-Myc a viable therapeutic strategy.

Small-Molecule Activation of p53 Blocks Hypoxia-Inducible Factor 1 alpha and Vascular Endothelial Growth Factor Expression In Vivo and Leads to Tumor Cell Apoptosis in Normoxia and Hypoxia

ABSTRACT The p53 tumor suppressor protein negatively regulates hypoxia-inducible factor 1α (HIF-1α). Here, we show that induction of p53 by the small-molecule RITA (reactivation of p53 and induction of tumor cell apoptosis) [2,5-bis(5-hydroxymethyl-2-thienyl) furan] (NSC-652287) inhibits HIF-1α and vascular endothelial growth factor expression in vivo and induces significant tumor cell apoptosis in normoxia and hypoxia in p53-positive cells. RITA has been proposed to stabilize p53 by inhibiting the p53-HDM2 interaction. However, induction of p53 alone was insufficient to block HIF-1α induced in hypoxia and has previously been shown to require additional stimuli, such as DNA damage. Here, we identify a new mechanism of action for RITA: RITA activates a DNA damage response, resulting in phosphorylation of p53 and γH2AX in vivo. Unlike other DNA damage response-inducing agents, RITA treatment of cells induced a p53-dependent increase in phosphorylation of the α subunit of eukaryotic initiation factor 2, requiring PKR-like endoplasmic reticulum kinase activity, and led to the subsequent downregulation of HIF-1α and p53 target proteins, including HDM2 and p21. Through the identification of a new mechanism of action for RITA, our study uncovers a novel link between the DNA damage response-p53 pathway and the protein translational machinery.

Combined MYC and P53 defects emerge at medulloblastoma relapse and define rapidly progressive, therapeutically targetable disease

Summary We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.

Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression

Increased expression of the regulatory subunit of HIFs (HIF-1α or HIF-2α) is associated with metabolic adaptation, angiogenesis, and tumor progression. Understanding how HIFs are regulated is of intense interest. Intriguingly, the molecular mechanisms that link mitochondrial function with the HIF-regulated response to hypoxia remain to be unraveled. Here we describe what we believe to be novel functions of the human gene CHCHD4 in this context. We found that CHCHD4 encodes 2 alternatively spliced, differentially expressed isoforms (CHCHD4.1 and CHCHD4.2). CHCHD4.1 is identical to MIA40, the homolog of yeast Mia40, a key component of the mitochondrial disulfide relay system that regulates electron transfer to cytochrome c. Further analysis revealed that CHCHD4 proteins contain an evolutionarily conserved coiled-coil-helix-coiled-coil-helix (CHCH) domain important for mitochondrial localization. Modulation of CHCHD4 protein expression in tumor cells regulated cellular oxygen consumption rate and metabolism. Targeting CHCHD4 expression blocked HIF-1α induction and function in hypoxia and resulted in inhibition of tumor growth and angiogenesis in vivo. Overexpression of CHCHD4 proteins in tumor cells enhanced HIF-1α protein stabilization in hypoxic conditions, an effect insensitive to antioxidant treatment. In human cancers, increased CHCHD4 expression was found to correlate with the hypoxia gene expression signature, increasing tumor grade, and reduced patient survival. Thus, our study identifies a mitochondrial mechanism that is critical for regulating the hypoxic response in tumors.

Structural Basis of N-Myc Binding by Aurora-A and its Destabilization by Kinase Inhibitors

Significance Elevated levels of N-Myc protein (the product of the MYCN oncogene) drive cancers such as neuroblastoma. Accumulation of N-Myc in these cancer cells depends upon the formation of a complex with the protein kinase Aurora-A in which the N-Myc is not properly degraded. We mapped the region of N-Myc that interacts with Aurora-A and determined the molecular structure of the complex. Because this region also interacts with cellular machinery that targets N-Myc for degradation, we sought to understand the mechanism by which N-Myc stabilizes Aurora-A. The structure explains how compounds that induce distorted conformations of Aurora-A are able to disrupt the interaction with N-Myc. This understanding may provide a basis for designing better compounds that work in this way for the treatment of neuroblastoma. Myc family proteins promote cancer by inducing widespread changes in gene expression. Their rapid turnover by the ubiquitin–proteasome pathway is regulated through phosphorylation of Myc Box I and ubiquitination by the E3 ubiquitin ligase SCFFbxW7. However, N-Myc protein (the product of the MYCN oncogene) is stabilized in neuroblastoma by the protein kinase Aurora-A in a manner that is sensitive to certain Aurora-A–selective inhibitors. Here we identify a direct interaction between the catalytic domain of Aurora-A and a site flanking Myc Box I that also binds SCFFbxW7. We determined the crystal structure of the complex between Aurora-A and this region of N-Myc to 1.72-Å resolution. The structure indicates that the conformation of Aurora-A induced by compounds such as alisertib and CD532 is not compatible with the binding of N-Myc, explaining the activity of these compounds in neuroblastoma cells and providing a rational basis for the design of cancer therapeutics optimized for destabilization of the complex. We also propose a model for the stabilization mechanism in which binding to Aurora-A alters how N-Myc interacts with SCFFbxW7 to disfavor the generation of Lys48-linked polyubiquitin chains.

Evaluation of Clinically Translatable MR Imaging Biomarkers of Therapeutic Response in the TH-MYCN Transgenic Mouse Model of Neuroblastoma

PURPOSE To evaluate noninvasive and clinically translatable magnetic resonance (MR) imaging biomarkers of therapeutic response in the TH-MYCN transgenic mouse model of aggressive, MYCN-amplified neuroblastoma. MATERIALS AND METHODS All experiments were performed in accordance with the local ethical review panel and the UK Home Office Animals Scientific Procedures Act 1986 and with the UK National Cancer Research Institute guidelines for the welfare of animals in cancer research. Multiparametric MR imaging was performed of abdominal tumors found in the TH-MYCN model. T2-weighted MR imaging, quantitation of native relaxation times T1 and T2, the relaxation rate R2*, and dynamic contrast-enhanced MR imaging were used to monitor tumor response to cyclophosphamide (25 mg/kg), the vascular disrupting agent ZD6126 (200 mg/kg), or the antiangiogenic agent cediranib (6 mg/kg, daily). Any significant changes in the measured parameters, and in the magnitude of the changes after treatment between treated and control cohorts, were identified by using Student two-tailed paired and unpaired t test, respectively, with a 5% level of significance. RESULTS Treatment with cyclophosphamide or cediranib induced a 54% or 20% reduction in tumor volume at 48 hours, respectively (P < .005 and P < .005, respectively; P < .005 and P < .005 versus control, respectively). Treatment with ZD6126 induced a 45% reduction in mean tumor volume 24 hours after treatment (P < .005; P < .005 versus control). The antitumor activity of cyclophosphamide, cediranib, and ZD6126 was consistently associated with a decrease in tumor T1 (P < .005, P < .005, and P < .005, respectively; P < .005, P < .005, and P < .005 versus control, respectively) and with a correlation between therapy-induced changes in native T1 and changes in tumor volume (r = 0.56; P < .005). Tumor response to cediranib was also associated with a decrease in the dynamic contrast-enhanced MR imaging-derived volume transfer constant (P = .07; P < .05 versus control) and enhancing fraction (P < .05; P < .01 versus control), and an increase in R2* (P < .005; P < .05 versus control). CONCLUSION The T1 relaxation time is a robust noninvasive imaging biomarker of response to therapy in tumors in TH-MYCN mice, which emulate high-risk neuroblastoma in children. T1 measurements can be readily implemented on clinical MR systems and should be investigated in translational clinical trials of new targeted therapies for pediatric neuroblastoma. SUPPLEMENTAL MATERIAL http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12120128/-/DC1.

Cyclin-dependent kinase inhibitor AT7519 as a potential drug for MYCN-dependent neuroblastoma

Purpose: MYCN-dependent neuroblastomas have low cure rates with current multimodal treatment regimens and novel therapeutic drugs are therefore urgently needed. In previous preclinical studies, we have shown that targeted inhibition of cyclin-dependent kinase 2 (CDK2) resulted in specific killing of MYCN-amplified neuroblastoma cells. This study describes the in vivo preclinical evaluation of the CDK inhibitor AT7519. Experimental Design: Preclinical drug testing was performed using a panel of MYCN-amplified and MYCN single copy neuroblastoma cell lines and different MYCN-dependent mouse models of neuroblastoma. Results: AT7519 killed MYCN-amplified neuroblastoma cell lines more potently than MYCN single copy cell lines with a median LC50 value of 1.7 compared to 8.1 μmol/L (P = 0.0053) and a significantly stronger induction of apoptosis. Preclinical studies in female NMRI homozygous (nu/nu) mice with neuroblastoma patient-derived MYCN-amplified AMC711T xenografts revealed dose-dependent growth inhibition, which correlated with intratumoral AT7519 levels. CDK2 target inhibition by AT7519 was confirmed by significant reductions in levels of phosphorylated retinoblastoma (p-Rb) and nucleophosmin (p-NPM). AT7519 treatment of Th-MYCN transgenic mice resulted in improved survival and clinically significant tumor regression (average tumor size reduction of 86% at day 7 after treatment initiation). The improved efficacy of AT7519 observed in Th-MYCN mice correlated with higher tumor exposure to the drug. Conclusions: This study strongly suggests that AT7519 is a promising drug for the treatment of high-risk neuroblastoma patients with MYCN amplification. Clin Cancer Res; 21(22); 5100–9. ©2015 AACR.

Noninvasive detection of carboxypeptidase G2 activity in vivo

The pseudomonad protein, carboxypeptidase G2 (CPG2), is a prodrug‐activating enzyme utilized in the targeted chemotherapy strategies of antibody‐ and gene‐directed enzyme prodrug therapy (ADEPT and GDEPT). We have developed a noninvasive imaging approach to monitor CPG2 activity in vivo that will facilitate the preclinical and clinical development of CPG2‐based ADEPT and GDEPT strategies. Cleavage of the novel reporter probe, 3,5‐difluorobenzoyl‐L‐glutamic acid (3,5‐DFBGlu), by CPG2, in human colon adenocarcinoma WiDr xenografts engineered to stably express CPG2, was monitored using 19F MRSI. The high signal‐to‐noise ratio afforded by the two MR‐equivalent 19F nuclei of 3,5‐DFBGlu, and the 1.4 ppm 19F chemical shift difference on CPG2‐mediated cleavage, enabled the dynamics and quantification of the apparent pharmacokinetics of 3,5‐DFBGlu and its CPG2‐mediated cleavage in the tumor to be evaluated. In addition, the apparent rate of increase of 3,5‐difluorobenzoic acid concentration could also provide a biomarker of CPG2 activity levels in tumors of patients undergoing CPG2‐based therapies, as well as a biomarker of treatment response. The addition of in vivo reporter probes, such as 3,5‐DFBGlu, to the armamentarium of prodrugs cleaved by CPG2 affords new applications for CPG2 as a gene reporter of transgene expression. Copyright

Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus

Neural crest migration is critical to its physiological function. Mechanisms controlling mammalian neural crest migration are comparatively unknown, due to difficulties accessing this cell population in vivo. Here we report requirements of glycogen synthase kinase 3 (GSK3) in regulating the neural crest in Xenopus and mouse models. We demonstrate that GSK3 is tyrosine phosphorylated (pY) in mouse neural crest cells and that loss of GSK3 leads to increased pFAK and misregulation of Rac1 and lamellipodin, key regulators of cell migration. Genetic reduction of GSK3 results in failure of migration. We find that pY-GSK3 phosphorylation depends on anaplastic lymphoma kinase (ALK), a protein associated with neuroblastoma. Consistent with this, neuroblastoma cells with increased ALK activity express high levels of pY-GSK3, and blockade of GSK3 or ALK can affect migration of these cells. Altogether, this work identifies a role for GSK3 in cell migration during neural crest development and cancer.Defects in neural crest development cause neurocristopathies and cancer, but what regulates this is unclear. Here, the authors show that glycogen synthase kinase 3 (GSK3) regulates migration of neural crest cells, as shown on genetic deletion of GSK3 in the mouse, and that this acts via anaplastic lymphoma kinase.