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Purpose Radiation dose calculated on tumors for radiopharmaceutical therapy varies significantly from tumor to tumor and from patient to patient. Accurate estimation of radiation dose requires multiple time point measurements using radionuclide imaging modalities such as SPECT or PET. In this report, we show our technical development of reducing the number of scans needed for reasonable estimation of tumor and normal organ dose in our pretherapy imaging and dosimetry platform of 124 I-metaiodobenzylguanidine (MIBG) positron emission tomography/computed tomography (PET/CT) for 131 I-MIBG therapy of neuroblastoma. Methods We analyzed the simplest kinetic data, areas of two-time point data for five patients with neuroblastoma who underwent 3 or 4 times of 124 I-MIBG PET/CT scan prior to 131 I-MIBG therapy. The data for which we derived areas were percent of injected activity (%IA) and standardized uptake value of tumors. These areas were correlated with time-integrated activity coefficients (TIACs) from full data (3 or 4 time points). TIACs are direct correlates with radiation dose as long as the volume and the radionuclide are known. Results The areas of %IAs between data obtained from all the two-time points with time points 1 and 2 (day 0 and day 1), time points 2 and 3 (day 1 and day 2), and time points 1 and 3 (day 0 and day 2) showed reasonable correlation (Pearson's correlation coefficient |r| > 0.5) with not only tumor and organ TIACs but also tumor and organ absorbed doses. The tumor and organ doses calculated using %IA areas of time point 1 and time point 2 were our best fits at about 20% individual percent difference compared to doses calculated using 3 or 4 time points. Conclusions We could achieve reasonable accuracy of estimating tumor doses for subsequent radiopharmaceutical therapy using only the two-time point imaging sessions. Images obtained from these time points (within the 48-h after administration of radiopharmaceutical) were also viewed as useful for diagnostic reading. Although our analysis was specific to 124 I-MIBG PET/CT pretherapy imaging data for 131 I-MIBG therapy of neuroblastoma and the number of imaging datasets was not large, this feasible methodology would generally be applicable to other imaging and therapeutic radionuclides with an appropriate data analysis similar to our analysis to other imaging and therapeutic radiopharmaceuticals.

Amplification of MYCN is a poor prognostic feature in neuroblastoma (NBL) indicating aggressive disease. We and others have shown BET bromodomain inhibitors (BETi) target MYCN indirectly by downregulating its transcription. Here we sought to identify agents that synergize with BETi and to identify biomarkers of resistance. We previously performed a viability screen of ∼1,900 oncology-focused compounds combined with BET bromodomain inhibitors against MYCN-amplified NBL cell lines. Reanalysis of our screening results prominently identified inhibitors of aurora kinase A (AURKAi) to be highly synergistic with BETi. We confirmed the anti-proliferative effects of several BETi+AURKAi combinations in MYCN-amplified NBL cell lines. Compared to single agents, these combinations cooperated to decrease levels of N-myc. We treated both TP53-wild type and mutant, MYCN-amplified cell lines with the BETi JQ1 and the AURKAi Alisertib. The combination had improved efficacy in the TP53-WT context, notably driving apoptosis in both genetic backgrounds. JQ1+Alisertib combination treatment of a MYCN-amplified, TP53-null or TP53-restored genetically engineered mouse model of NBL prolonged survival better than either single agent. This was most profound with TP53 restored, with marked tumor shrinkage and apoptosis induction in response to combination JQ1+Alisertib. BETi+AURKAi in MYCN-amplified NBL, particularly in the context of functional TP53, provided anti-tumor benefits in preclinical models. This combination should be studied more closely in a pediatric clinical trial.

Effective treatment of pediatric solid tumors has been hampered by the predominance of currently “undruggable” driver transcription factors. Improving outcomes while decreasing the toxicity of treatment necessitates the development of novel agents that can directly inhibit or degrade these elusive targets. MYCN in pediatric neural-derived tumors, including neuroblastoma and medulloblastoma, is a paradigmatic example of this problem. Attempts to directly and specifically target MYCN have failed due to its similarity to MYC, the unstructured nature of MYC family proteins in their monomeric form, the lack of an understanding of MYCN-interacting proteins and ability to test their relevance in vivo, the inability to obtain structural information on MYCN protein complexes, and the challenges of using traditional small molecules to inhibit protein–protein or protein–DNA interactions. However, there is now promise for directly targeting MYCN based on scientific and technological advances on all of these fronts. Here, we discuss prior challenges and the reasons for renewed optimism in directly targeting this “undruggable” transcription factor, which we hope will lead to improved outcomes for patients with pediatric cancer and create a framework for targeting driver oncoproteins regulating gene transcription.

The metaiodobenzylguanidine (MIBG) scan is one of the most sensitive noninvasive lesion detection modalities for neuroblastoma. Unlike (123)I-MIBG, (124)I-MIBG allows high-resolution PET. We evaluated (124)I-MIBG PET/CT for its diagnostic performance as directly compared with paired (123)I-MIBG scans. Methods: Before (131)I-MIBG therapy, standard (123)I-MIBG imaging (5.2 MBq/kg) was performed on 7 patients, including whole-body (anterior–posterior) planar imaging, focused-field-of-view SPECT/CT, and whole-body (124)I-MIBG PET/CT (1.05 MBq/kg). After therapy, 2 of 7 patients also completed (124)I-MIBG PET/CT as well as paired (123)I-MIBG planar imaging and SPECT/CT. One patient underwent (124)I-MIBG PET/CT only after therapy. We evaluated all 8 patients who showed at least 1 (123)I-MIBG–positive lesion with a total of 10 scans. In 8 pairs, (123)I-MIBG and (124)I-MIBG were performed within 1 mo of each other. The locations of identified lesions, the number of total lesions, and the curie scores were recorded for the (123)I-MIBG and (124)I-MIBG scans. Finally, for 5 patients who completed at least 3 PET/CT scans after administration of (124)I-MIBG, we estimated the effective dose of (124)I-MIBG. Results: (123)I-MIBG whole-body planar scans, focused-field-of-view SPECT/CT scans, and whole-body (124)I-MIBG PET scans found 25, 32, and 87 total lesions, respectively. There was a statistically significant difference in lesion detection for (124)I-MIBG PET/CT versus (123)I-MIBG planar imaging (P < 0.0001) and (123)I-MIBG SPECT/CT (P < 0.0001). The curie scores were also higher for (124)I-MIBG PET/CT than for (123)I-MIBG planar imaging and SPECT/CT in 6 of 10 patients. (124)I-MIBG PET/CT demonstrated better detection of lesions throughout the body, including the chest, spine, head and neck, and extremities. The effective dose estimated for patient-specific (124)I-MIBG was approximately 10 times that of (123)I-MIBG; however, given that we administered a very low activity of (124)I-MIBG (1.05 MBq/kg), the effective dose was only approximately twice that of (123)I-MIBG despite the large difference in half-lives (100 vs. 13.2 h). Conclusion: The first-in-humans use of low-dose (124)I-MIBG PET for monitoring disease burden demonstrated tumor detection capability superior to that of (123)I-MIBG planar imaging and SPECT/CT.

MYCN is a major driver for the childhood cancer, neuroblastoma, however, there are no inhibitors of this target. Enhanced MYCN protein stability is a key component of MYCN oncogenesis and is maintained by multiple feedforward expression loops involving MYCN transactivation target genes. Here, we reveal the oncogenic role of a novel MYCN target and binding protein, proliferation-associated 2AG4 (PA2G4). Chromatin immunoprecipitation studies demonstrated that MYCN occupies the PA2G4 gene promoter, stimulating transcription. Direct binding of PA2G4 to MYCN protein blocked proteolysis of MYCN and enhanced colony formation in a MYCN-dependent manner. Using molecular modeling, surface plasmon resonance, and mutagenesis studies, we mapped the MYCN–PA2G4 interaction site to a 14 amino acid MYCN sequence and a surface crevice of PA2G4. Competitive chemical inhibition of the MYCN–PA2G4 protein–protein interface had potent inhibitory effects on neuroblastoma tumorigenesis in vivo. Treated tumors showed reduced levels of both MYCN and PA2G4. Our findings demonstrate a critical role for PA2G4 as a cofactor in MYCN-driven neuroblastoma and highlight competitive inhibition of the PA2G4-MYCN protein binding as a novel therapeutic strategy in the disease. Significance: Competitive chemical inhibition of the PA2G4–MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCN-binding partners in malignancies driven by MYC family oncoproteins.

SummaryThe development of targeted therapeutics for neuroblastoma, the third most common tumor in children, has been limited by a poor understanding of growth signaling mechanisms unique to the peripheral nerve precursors from which tumors arise. In this study, we combined genetics with gene-expression analysis in the peripheral sympathetic nervous system to implicate arginase 1 and GABA signaling in tumor formation in vivo. In human neuroblastoma cells, either blockade of ARG1 or benzodiazepine-mediated activation of GABA-A receptors induced apoptosis and inhibited mitogenic signaling through AKT and MAPK. These results suggest that ARG1 and GABA influence both neural development and neuroblastoma and that benzodiazepines in clinical use may have potential applications for neuroblastoma therapy.

Combining anti-GD2 ganglioside monoclonal antibody with immune-activating cytokines improves survival in high-risk neuroblastoma, a breakthrough for immunotherapy in this disease. However, approaches such as immune checkpoint blockade, anti-GD2 CAR-T cells, and tumor vaccines have thus far shown limited success. While a better understanding of the tumor microenvironment (TME) promises to improve the success of immunotherapy strategies, few immunocompetent models exist for high-risk neuroblastoma. We used a 30-parameter mouse-specific CyTOF mass cytometetry panel to characterize the TME of neuroblastoma arising in TH-MYCN transgenic mice. The TME was dominated by tumor-associated macrophages (TAMs) and devoid of T, B or NK cells; recapitulating the TME of human high-risk neuroblastoma. The TH-MYCN model is highly penetrant only in the 129/SvJ strain however, a challenge to leveraging immunological tools available mainly in the C57/BL6 background. We therefore created a non-germline GEM model in which primary trunk neural crest cells isolated from C57BL6/J embryos were modified genetically by transducing MYCN (MYCN-nGEMM). Orthotopic transplantation in renal capsules resulted in neuroblastoma with 50% penetrance. Co-expression of ALK(F1174L)with MYCN resulted in 80% penetrance and decreased latency. Cell lines could be derived robustly from these tumors and serially transplanted with 100% penetrance. CyTOF analysis of MYCN-nGEMM tumors also revealed a highly immunosuppressive TME similar to TH-MYCN tumors, with 80% of immune cells being TAMs, and only 5% T-cells. MYCN-nGEMM tumors were then treated with various immunotherapies, including anti-GD2 antibody, checkpoint inhibitors, and a combination of both, and changes in the TME were analyzed by CyTOF. In summary, we have characterized an immune-intact model for MYCN-driven neuroblastoma that can leverage modern methods to rapidly modify the murine genome, enabling customization of this mouse model for immunological research. We propose this model to understand the immunosuppressive mechanisms of MYCN-driven neuroblastoma tumors, and evaluate the potential of combining MYCN-directed therapies with immunotherapy.

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.

PurposeVesicular monoamine transporters 1 and 2 (VMAT1 and VMAT2) are thought to mediate MIBG uptake in adult neuroendocrine tumors. In neuroblastoma, the norepinephrine transporter (NET) has been investigated as the principal MIBG uptake protein, though some tumors without NET expression concentrate MIBG. We investigated VMAT expression in neuroblastoma and correlated expression with MIBG uptake and clinical features.MethodsWe evaluated VMAT1 and VMAT2 expression by immunohistochemistry (IHC) in neuroblastoma tumors from 76 patients with high-risk metastatic disease treated in a uniform cooperative group trial (COG A3973). All patients had baseline MIBG diagnostic scans centrally reviewed. IHC results were scored as the product of intensity grading (0 - 3+) and percent of tumor cells expressing the protein of interest. The association between VMAT1 and VMAT2 scores and clinical and biological features was tested using Wilcoxon rank-sum tests.ResultsPatient characteristics were typical of high-risk neuroblastoma, though the cohort was intentionally enriched in patients with MIBG-nonavid tumors (n = 20). VMAT1 and VMAT2 were expressed in 62% and 75% of neuroblastoma tumors, respectively. VMAT1 and VMAT2 scores were both significantly lower in MYCN amplified tumors and in tumors with high mitotic karyorrhectic index. MIBG-avid tumors had significantly higher VMAT2 scores than MIBG-nonavid tumors (median 216 vs. 45; p = 0.04). VMAT1 expression did not correlate with MIBG avidity.ConclusionVMAT1 and VMAT2 are expressed in the majority of neuroblastomas. Expression correlates with other biological features. The expression level of VMAT2 but not that of VMAT1 correlates with avidity for MIBG.

Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genomic data and exon-level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from the peripheral neural crest. Here, we describe splicing quantitative trait loci associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma. Significance: Somatic mutations with predictable downstream effects are largely relegated to coding regions, which comprise less than 2% of the human genome. Using an unbiased in vivo analysis of a mouse model of neuroblastoma, we have identified intronic splicing motifs that translate into sites for recurrent somatic mutations in human cancers. Cancer Discov; 5(4); 380–95. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 333

[(124)I]m-iodobenzylguanidine ((124)I-mIBG) provides a quantitative tool for pretherapy tumor imaging and dosimetry when performed before [(131)I]m-iodobenzylguanidine ((131)I-mIBG) targeted radionuclide therapy of neuroblastoma. (124)I (T (1/2) = 4.2 days) has a comparable half-life to that of (131)I (T (1/2) = 8.02 days) and can be imaged by positron emission tomography (PET) for accurate quantification of the radiotracer distribution. We estimated expected radiation dose in tumors from (131)I-mIBG therapy using (124)I-mIBG microPET/CT imaging data in a murine xenograft model of neuroblastoma transduced to express high levels of the human norepinephrine transporter (hNET).In order to enhance mIBG uptake for in vivo imaging and therapy, NB 1691-luciferase (NB1691) human neuroblastoma cells were engineered to express high levels of hNET protein by lentiviral transduction (NB1691-hNET). Both NB1691 and NB1691-hNET cells were implanted subcutaneously and into renal capsules in athymic mice. (124)I-mIBG (4.2-6.5 MBq) was administered intravenously for microPET/CT imaging at 5 time points over 95 h (0.5, 3-5, 24, 48, and 93-95 h median time points). In vivo biodistribution data in normal organs, tumors, and whole-body were collected from reconstructed PET images corrected for photon attenuation using the CT-based attenuation map. Organ and tumor dosimetry were determined for (124)I-mIBG. Dose estimates for (131)I-mIBG were made, assuming the same in vivo biodistribution as (124)I-mIBG.All NB1691-hNET tumors had significant uptake and retention of (124)I-mIBG, whereas unmodified NB1691 tumors did not demonstrate quantifiable mIBG uptake in vivo, despite in vitro uptake. (124)I-mIBG with microPET/CT provided an accurate three-dimensional tool for estimating the radiation dose that would be delivered with (131)I-mIBG therapy. For example, in our model system, we estimated that the administration of (131)I-mIBG in the range of 52.8-206 MBq would deliver 20 Gy to tumors.The overexpression of hNET was found to be critical for (124)I-mIBG uptake and retention in vivo. The quantitative (124)I-mIBG PET/CT is a promising new tool to predict tumor radiation doses with (131)I-mIBG therapy of neuroblastoma. This methodology may be applied to tumor dosimetry of (131)I-mIBG therapy in human subjects using (124)I-mIBG pretherapy PET/CT data.

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 anti-angiogenic 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.

As a neuroendocrine tumor, neuroblastoma expresses various gastrointestinal (GI) hormones, such as vasoactive intestinal peptide, gastrin-releasing peptide (GRP), neurotensin, and somatostatin, which exert diverse cellular functions in neuroblastoma. In particular, we have recently found that GRP and its cell surface receptor, GRP-R, are abundantly expressed in neuroblastomas. Moreover, more advanced-stage neuroblastomas demonstrated an increased level of GRP-R, suggesting an important role of GRP in aggressive tumor behavior. This review describes the role of several GI hormones commonly expressed in neuroblastoma and discusses in depth the mitogenic actions of GRP in neuroblastoma. In addition, the molecular mechanisms involved in the GRP-induced stimulation of neuroblastoma cell growth are discussed. Our study results demonstrate a role of GRP as an autocrine/paracrine growth factor and elucidate involvement of specific intracellular signaling, the phosphatidylinositol 3-kinase pathway, in the growth regulation of neuroblastoma.

MYC family proteins represent a critical node in oncogenesis across a wide range of pediatric and adult cancers. This is particularly true in neuroblastoma, where amplification of MYCN confers an especially poor prognosis. Proteolytic degradation of MYCN protein is regulated in part by a kinase-independent function of Aurora Kinase A. We describe a class of allosteric inhibitors that disrupts the native conformation of Aurora A and causes degradation of MYCN protein across MYCN-expressing neuroblastoma cell lines. We compare co-crystal structures with structure-activity relationships across multiple inhibitors and chemotypes to delineate an Aurora A conformation-specific effect on proteolytic degradation of MYCN protein, which is driven markedly by conformation disrupting inhibitors, and impacted more modestly by current clinical Aurora Kinase A inhibitors, which disrupt the kinase structure less efficiently. Using functional assays of apoptosis, cell cycle, and proliferation we further define a distinct MYCN-specific effect distinguishing it from simple inhibition of mitosis by conventional Aurora kinase inhibitors. This new class of conformation disrupting inhibitors, which block stabilizing interactions between Aurora A and MYCN, represents a novel strategy to target MYCN-driven cancers. This abstract is also presented as Poster B46. Citation Format: W. Clay Gustafson, Justin G. Meyerowitz, Erin A. Nekritz, Elise Charron, Katherine K. Matthay, Nicholas T. Hertz, Martin Eilers, Kevan M. Shokat, William A. Weiss. Drugging MYCN protein stability through an allosteric transition in Aurora kinase A. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR08.

Neuroblastoma, the most common and deadly extracranial solid tumor of childhood, has long provided a challenge to both clinicians and basic scientists. Despite its high prevalence among pediatric cancers and extensive efforts over the last several decades, there is still been only marginal improvement in overall survival and almost no improvement in survival for high-risk neuroblastoma [1]. Numerous risk stratification studies have shown that amplification of the MYCN oncogene is the clearest clinical indicator of poor prognosis. Despite the importance of MYCN amplification in neuroblastoma, little work has been done to examine the transcriptional regulation of the MYCN amplicon. In addition to transcriptional regulation, a second primary means through which cells regulate levels of Mycn is at the level of protein stability. Therefore, exploring methods enabling targeting of Mycn protein stability is a major focus of current therapeutic research (reviewed in [2]). If significant transcriptional regulation of the MYCN amplicon in neuroblastoma could be characterized and exploited, these could result in novel strategies to target this deadly disease. What do we know about transcriptional regulation of MYCN in neuroblastoma? In conjunction with binding partners including Max and Miz, Myc family transcription factors (including c-MYC, MYCN, and MYCL) bind to specific “E-box” elements to regulate gene expression. E-boxes are quite common in the genome and are present in the promoter regions of approximately 25% of known genes indicating complex and tissue specific regulation of Myc mediated transcription [3]. While many downstream transcriptional targets of c-Myc have been determined (including the B-MYB gene)[4], detailed analysis and confirmation of MYCN regulated genes is of vital importance to our understanding of MYCN mediated oncogenesis. In this issue of Oncotarget, Gualdrini et al propose the B-MYB transcription factor as one such target, and provide preliminary evidence for a reciprocal regulation between the amplified MYCN and the B-MYB transcription factors [5]. Expression of the B-MYB transcription factor has been previously shown to be a poor prognostic indicator in neuroblastoma, independent of MYCN amplification [6]. In this study, Gualdrini et al show an intriguing correlation between expression of mRNA for MYCN and B-MYB in neuroblastoma patient samples amplified for MYCN, extending this observation to the protein level in several MYCN amplified neuroblastoma cell lines. Importantly, the correlation between MYCN and B-MYB does not hold true in MYCN non-amplified samples. Additionally, using CHIP in established cell lines, the authors provide preliminary evidence that Mycn protein can bind to and induce transcription of B-MYC, consistent with B-MYB as a transcriptional target of MYCN. Perhaps most importantly, the study shows that knockdown of B-MYB by shRNA blocks proliferation in neuroblastoma cell lines with amplified MYCN. However, mechanistic details of this synthetic anti-proliferative effect between knockdown of B-MYB and amplification of MYCN remain to be determined. Also importantly, the authors use a single shRNA against BMYB, and do not extend this observation to primary tumors, thus limiting the impact of this result. Lastly, B-MYB was also shown to bind to the MYCN promoter in chromatin-IP assays of MYCN amplified neuroblastoma cells in culture, and induced a relatively modest transcriptional upregulation of a MYCN promoter element. The authors use these exciting albeit preliminary results to propose a novel model for a reciprocal regulatory loop between these two transcription factors. If significant epigenetic and transcriptional regulation of amplified MYCN (implied by these studies) can be validated in primary tumors, these studies would support BMYB as target for therapeutic intervention in MYCN amplified cancers. However, given myriad difficulties currently in-place in targeting Mycn itself, the ability to target both BMYC and MYCN in neuroblastoma raises its own group of challenges.

Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically-defined cancer cell lines using a prototypical inhibitor of BET (bromodomain and extra-terminal domain) bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis demonstrated downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription, and a BET Bromodomain inhibitor was found to displace BRD4 from the MYCN promoter region in neuroblastoma cell lines. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knock-down phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in three in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma. Citation Format: Alexandre Puissant, Stacey M. Frumm, Gabriela Alexe, Christopher F. Bassil, Jun Qi, Yvan H. Chanthery, Erin A. Nekritz, Rhamy Zeid, W. Clay Gustafson, Patricia Greninger, Matthew J. Garnett, Ultan McDermott, Cyril H. Benes, Andrew L. Kung, William A. Weiss, James E. Bradner, Kimberly Stegmaier. Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4622. doi:10.1158/1538-7445.AM2013-4622

Neuroblastoma, a tumor of peripheral neural crest origin, numbers among the most common childhood cancers. Both amplification of the proto-oncogene MYCN and increased vascular density mark high-risk disease. Since angiogenesis is regulated by phosphatidylinositol-3’ kinase (PI3K), we tested a clinical PI3K inhibitor, NVP-BEZ235, in MYCN-dependent neuroblastoma. NVP-BEZ235 decreased angiogenesis in a primary human neuroblastoma (MYCN-amplified orthotopic xenograft), and improved survival in a transgenic mouse model for MYCN-driven neuroblastoma. Using both gain and loss of function approaches, we demonstrate that the anti-angiogenic efficacy of NVP-BEZ235 was critically dependent on Mycn, in vitro and in-vivo. Thus, clinical PI3K/mTOR inhibitors drive degradation of Mycn in tumor cells, with secondary paracrine blockade of tumor-vascular microenvironment. These observations argue that NVP-BEZ235 should be tested in children with high-risk, MYCN amplified neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1558. doi:10.1158/1538-7445.AM2011-1558