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Supplementary Table S1. Genomic characteristic of ALL PDXs including ALL subtype and key genomic alterations. Supplementary Table S2. Top 5 enriched signaling pathways from the 52 query proteins, as analysed by GO and KEGG pathway analysis platforms. Supplementary Table S3. In vitro combination effects of BMS-754807 and ponatinib. Supplementary Table S4. Differentially expressed genes between Combination and Control (FDR < 0.05). Supplementary Table S5. Differentially expressed genes between BMS-754807 and Control (FDR < 0.05). Supplementary Table S6. Differentially expressed genes between Ponatinib and Control (FDR < 0.05). Supplementary Table S7. KEGG pathway analysis of the differentially expressed genes caused by each treatment condition against control (FDR < 0.25). Supplementary Table S8. Plasma concentration of BMS-754807 and ponatinib as measured by LC-MS/MS.

S1. In vitro effects of TSLP on target cells as assessed by Alamar Blue assay. S2. Demographics of phosphosites identified from MHH-CALL-4 and MUTZ-5 cells in global P-Tyr profiling. S3. In vitro single agent efficacy of BMS-754807 and ponatinib against CRLF2r cell lines and PDXs. S4. siRNA knockdown of IGF1R and FGFR1 in MHH-CALL-4 and MUTZ-5 cell lines. S5. Tolerability testing of ponatinib in combination with BMS754807 in naïve NSG mice as assessed by percentage of body weight change. S6. Ex vivo combination effect of BMS-754807 and ponatinib over time against CRLF2r Ph-like ALL PDXs. S7. Plasma concentrations of BMS-754807 and ponatinib at specific timepoints.

Purpose: To characterize the clinical significance of promoter methylation in a cohort of primary neuroblastoma tumors and investigate the association between DNA methylation and clinical outcome.Experimental Design: A customized Illumina GoldenGate methylation assay was used to assess methylation status of 96 CpG sites within 48 candidate genes in primary neuroblastoma tumors obtained from 131 children diagnosed in Australia. Genes were selected on the basis of previous reports of altered DNA methylation in embryonal cancers. Levels of DNA methylation were validated in a subset of 48 patient samples using combined bisulfite restriction analysis (CoBRA) and bisulfite sequencing. A Cox proportional hazards model was used to investigate the association between promoter hypermethylation and the risk of relapse/death within 5 years of diagnosis, while adjusting for known prognostic factors including MYCN amplification, age, and stage at diagnosis.Results: Levels of promoter methylation of DNAJC15, neurotrophic tyrosine kinase receptor 1 or TrkA (NTRK1), and tumor necrosis factor receptor superfamily, member 10D (TNFRSF10D), were higher in older patients at diagnosis (P < 0.01), whereas higher levels of methylation of DNAJC15, NTRK1, and PYCARD were observed in patients with MYCN amplification (P < 0.001). In multivariate analysis, hypermethylation of folate hydrolase (FOLH1), myogenic differentiation-1 (MYOD1), and thrombospondin-1 (THBS1) remained significant independent predictors of poorer clinical outcome after adjusting for known prognostic factors (P ≤ 0.017). Moreover, more than 30% of patients displayed hypermethylation in 2 genes or more and were at least 2 times more likely to relapse or die (HR = 2.72, 95% confidence interval = 1.55–4.78, P = 0.001), independent of MYCN status, age, and stage at diagnosis.Conclusions: Our findings highlight the potential use of methylation profiling to identify additional prognostic markers and detect new therapeutic targets for selected patient subsets. Clin Cancer Res; 18(20); 5690–700. ©2012 AACR.

Primers used for PCR-analysis

Combination therapy with JQ1 and panobinostat synergistically reduces neuroblastoma cell viability.

Neuroblastoma is a pediatric cancer of the sympathetic nervous system where MYCN amplification is a key indicator of poor prognosis. However, mechanisms by which MYCN promotes neuroblastoma tumorigenesis are not fully understood. In this study, we analyzed global miRNA and mRNA expression profiles of tissues at different stages of tumorigenesis from TH-MYCN transgenic mice, a model of MYCN-driven neuroblastoma. On the basis of a Bayesian learning network model in which we compared pretumor ganglia from TH-MYCN+/+ mice to age-matched wild-type controls, we devised a predicted miRNA–mRNA interaction network. Among the miRNA–mRNA interactions operating during human neuroblastoma tumorigenesis, we identified miR-204 as a tumor suppressor miRNA that inhibited a subnetwork of oncogenes strongly associated with MYCN-amplified neuroblastoma and poor patient outcome. MYCN bound to the miR-204 promoter and repressed miR-204 transcription. Conversely, miR-204 directly bound MYCN mRNA and repressed MYCN expression. miR-204 overexpression significantly inhibited neuroblastoma cell proliferation in vitro and tumorigenesis in vivo. Together, these findings identify novel tumorigenic miRNA gene networks and miR-204 as a tumor suppressor that regulates MYCN expression in neuroblastoma tumorigenesis.Significance: Network modeling of miRNA–mRNA regulatory interactions in a mouse model of neuroblastoma identifies miR-204 as a tumor suppressor and negative regulator of MYCN. Cancer Res; 78(12); 3122–34. ©2018 AACR.

Supplementary Figure S1. PA2G4 is a MYCN transactivation target gene. A, Quantification of protein expression using anti-PA2G4 and anti-MYCN antibodies against whole cell protein lysates from BE(2)-C and Kelly cells, following MYCN siRNA knockdown for 48 hours. B, mRNA expression of PA2G4 and MYCN in SH-EP MYCN3 overexpression cells treated with 1µg/ml doxycycline for 24-96 hr. C, The effect of doxycycline-induced MYCN overexpression in SHEP-TRE-MYCN cells on c-MYC and PA2G4 protein expression. D, Chromatin immunoprecipitation (ChIP) assay in Kelly cells using an anti-MYCN antibody, and real-time PCR analysis with primers identifying the MYCN DNA binding sites in the PA2G4 gene promoter (500bP upstream of transcription start site [TSS]) or Intron 1a & 1b regions of the PA2G4 gene, with and without MYCN siRNA knockdown. ChIP and real-time PCR analysis using primers against a region 1200bp upstream of TSS was used as a negative control for MYCN chromatin binding. ChIP and real-time PCR analysis using primers against the ornithine decarboxylase (ODC1) gene promoter region was used as a positive control for MYCN chromatin binding. E, Immunoblot analysis of PA2G4, MYCN and MYC protein levels in a panel of human MYCN amplified and non-amplified neuroblastoma, and normal fibroblast, cell lines using antibodies recognising PA2G4, MYCN and MYC.

Supplementary Figure S1. F, Confocal microscopy of SHEP Tet21N cells using anti-PA2G4 (red) and anti-MYCN (green) antibodies. G, Immunoblot analysis of cytoplasmic and nuclear fractions from BE(2)-C and Kelly human neuroblastoma cell lines with antibodies recognizing PA2G4 and MYCN, or GAPDH and topoisomerase as loading controls. H, Confocal microscopy of neuroblastoma (BE(2)-C and Kelly) cells using anti-PA2G4 (red) and anti-MYCN (green) antibodies. Alexafluor 555 anti-rabbit (to detect PA2G4) and Alexafluor 488 anti-mouse (to detect MYCN) were used as the secondary antibodies. I, Real-time PCR mRNA expression of MYCN and PA2G4 in ganglia from homozygote TH-MYCN+/+ mice, compared to wild type littermate control mice, obtained at different postnatal age (weeks).

JQ1 and panobinostat do not cooperatively regulate GSK3β and Aurora A expression, and LIN28B does not regulate Aurora A expression.

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.

Supplementary Figure S4. Combination of the BET bromodomain inhibitor I-BET762 and chemotherapy agents, but not proteasome inhibitors, induces cytotoxicity to normal cells.

Supplementary Figure S6. OTX015 and carfilzomib exert synergistic anticancer effects partly by inducing oxidative stress and endoplasmic reticulum stress.

The BET bromodomain proteins BRD3 and BRD4 induce N-Myc expression, but do not function synergistically.

MYCN and HIF-1α do not cooperate in inducing key enzymes of the Warburg effect

This file contains supplemental methods on the PPTP scoring method.

This file contains legends to the 4 supplemental figures.

Supplementary Figure S2. PA2G4 increases MYCN protein stability. A and B, Representative immunoblots from cycloheximide (CHX) chase assays measuring the half-life of MYCN protein after PA2G4 knockdown (A) or overexpression (B) in BE(2)-C and Kelly cells for 48 hours. Cells were then treated with 100 µg/µl CHX for up to 60 minutes followed by immunoblotting. C, Co-IP of total protein from BE(2)-C cells using IgG, anti-MYCN, anti-PA2G4 antibodies, followed by immunoblotting with anti-MYCN, anti-PA2G4, anti-AURKA, anti-Fbxw7 or anti-vinculin antibodies.

Supplementary Methods, Tables and Figures

Supplementary Figure S1. TERT induces TERT-rearranged neuroblastoma cell proliferation and cell cycle progression through a telomerase-independent mechanism.

PDF file, 415K, Representative CoBRA and bisulfite sequencing results are shown in Supplementary Figure S1-S2. PCR conditions for CoBRA and bisulfite sequencing are listed in Supplementary Table S1 and S2. Clinical characteristics of the study population is presented in Supplementary Table S3. Representative GoldenGate and CoBRA results are shown in Supplementary Table S4.

Supplementary Figure S4. Characterization of the PA2G4-MYCN protein-protein interface. A, BE(2)-C cells were transiently transfected with EV, wildtype PA2G4 or 6 different PA2G4 point mutants for 48 hours, then treated with 100 µg/µl Cycloheximide (CHX) for up to 60 minutes, followed by immunoblot analysis for MYCN protein half-life. B, Differential Scanning Fluorimetry (DSF) showed both the seven amino acid (DHKALST, aa248-254) and large peptide (GGDHKALSTGEDTL, aa246-259) MYCN oligopeptides, along with the MYCN oligopeptide shown not to bind via SPR (DHAALAT) changed the melting temperature of the PA2G4 protein, relative to baseline (i.e. 0mM), in a dose-response manner. Shown are the means of 3 independent experiments {plus minus} SEM. C, An example of the raw data for DHKALST, with the shift to the left correlating to an increase in concentration. D, Raw SPR data for PA2G4 triple mutant (R271A, R272A and S47A) and single mutants (S47A and R272A). Also, raw SPR data for MYCN oligopeptide mutants (DHAALST, DHAALAT and DHKALAT). For the mutants and triple mutations, no binding was observed, thus analysis could not be conducted and is not shown. E, Root Mead Squared Deviation (RMSD) of the peptide over the time of the simulation. After the first 100 frames the peptide is relatively stable. Analysis was only conducted after this time.

Re - Revised Supplementary Materials & Methods

Supplementary Information

The BET bromodomain protein BRD2 does not regulate N-Myc expression.

Supplementary Figure S6. PA2G4 increases neuroblastoma tumorigenicity. A, Representative images of athymic nude mice inoculated with neuroblastoma (SH-EP) cells stably transfected with either EV control or a PA2G4 expression vector at 10 weeks post-injection. B, Images of tumor formation after mice were culled 12 weeks post-injection. C, Real-time PCR analysis of PA2G4 mRNA expression level in tumors from mice injected with either SH-EP cells overexpressing PA2G4, or SH-EP EV control cells. β2-microglobulin was used as a reference gene for total RNA loading. D, Protein was extracted from SH-EP tumor xenografts overexpressing PA2G4 and control vectors, then analysed for PA2G4-Flag and MYCN expression by immunoblotting using anti-MYCN and anti-PA2G4 antibodies, using a Vinculin loading control. E, Immunoblots of three tumor samples from each siRNA-treated cohort showing the levels of PA2G4 and MYCN protein expression, using a Vinculin loading control. F, Real-time PCR analysis of PA2G4 and MYCN mRNA expression in tumor samples taken from tumour-bearing mice xenografted with BE(2)-C neuroblastoma cells, which had been treated with either nano-particle encapsulated siRNA control, PA2G4 siRNA or PA2G4-p48 siRNA. Data are shown as means and SD derived from 6 mice per group, P-values were calculated by t-test.

The top 35 genes most dramatically down-regulated by JQ1 and panobinostat combination therapy.

JQ1 and panobinostat do not cooperatively induce apoptosis in normal non-malignant cells.

LDHC mRNA expression is very low in NB cell lines and patients

Purpose: Patients with neuroblastoma associated with MYCN oncogene amplification experience a very poor prognosis. BET bromodomain inhibitors are among the most promising novel anticancer agents as they block BRD3 and BRD4 from activating oncogene transcription. However, treatment with BET bromodomain inhibitors alone does not result in cancer remission in many murine models.Experimental Design: MYCN-amplified neuroblastoma cells were treated with vehicle control, the BET bromodomain inhibitor JQ1, the histone deacetylase inhibitor panobinostat, or the combination of JQ1 and panobinostat. Genes modulated by JQ1, panobinostat, or the combination therapy were identified by Affymetrix microarray, and cell proliferation and apoptosis were examined by Alamar blue assays and flow cytometry analysis. Modulation of LIN28B promoter activity by BRD3 and BRD4 was examined by chromatin immunoprecipitation and luciferase assays. In addition, neuroblastoma-bearing mice were treated with vehicle control, JQ1, and/or panobinostat.Results: LIN28B was one of the top genes synergistically reduced by JQ1 and panobinostat. BRD3 and BRD4 directly bound to the LIN28B gene promoter and activated LIN28B gene transcription, and knocking down LIN28B reduced the expression of N-Myc protein, but not N-Myc mRNA. JQ1 and panobinostat synergistically reduced LIN28B gene and N-Myc protein expression, and synergistically induced growth inhibition and apoptosis in neuroblastoma cells, but not normal nonmalignant cells in vitro. In neuroblastoma-bearing mice, JQ1 and panobinostat synergistically and considerably reduced N-Myc protein expression in tumor tissues and blocked tumor progression.Conclusions: Our findings have identified a novel strategy to reduce the N-Myc oncoprotein expression and a novel therapeutic approach for the treatment of aggressive neuroblastoma. Clin Cancer Res; 22(10); 2534–44. ©2016 AACR.

Combination therapy with JQ1 and panobinostat exerts synergistic anticancer effects in neuroblastoma cells.

Supplementary Figure S5 F-H F), at 72 hours post-transfection of EV or a PA2G4 expression vector. G, Cell viability measured by the Alamar Blue assay and immunoblot analyses using an antibody identifying MYC-tagged PA2G4-p42 protein expression in Kelly and SH-SY5Y cells transfected with EV or the MYC-tagged PA2G4-p42 expression vector at 48 and 72 hours post-transfection. Vinculin was used as loading control. H, Colony formation in vitro by Kelly and SH-SY5Y cells following transfection with either EV or MYC-tagged PA2G4-p42.

Supplementary Figure S3 F-G F, Left panel: Histopathologic and immunohistochemical analyses of MYCN;GFP tumors treated with vehicle (left) or WS6 (right). Left Panel, Top to Bottom: Neuroblastoma tumour sections immunohistochemically stained for Haematoxylin & Eosin (H&E), Proliferating Cell Nuclear Antigen (pCNA), Neural Hu protein C (Hu-C), MYCN, PA2G4 and Tyrosine Hydroxylase. Scale bar, 50 μm. Right Panel: Histograms illustrating the staining intensity of cells of vehicle (left) or WS6 treated neuroblastoma tumours expressing either MYCN, PA2G4 or Tyrosine Hydroxylase as measured by Image J software. Sample means (horizontal bars) were compared by students t-test (two-tailed). *** represents p-value

Purpose:TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma.Experimental Design:Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice.Results:The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression.Conclusions:OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

Supplementary Figure S2. BRD4 is required for TERT expression and cell proliferation in TERT-rearranged neuroblastoma cells.

Cox proportional hazard analyses show that LDHA mRNA is independently associated with OS and EFS in NB.

Training set to determine the optimal prognostic LDHA mRNA expression cut-off

JQ1 and panobinostat synergistically modulate gene expression.

Supplementary Figure S3. Screening for Approved Oncology Drugs (AODs) exerting synergistic anticancer effects with BET bromodomain inhibitors against TERT-rearranged neuroblastoma cells.

Purpose: To investigate whether lactate dehydrogenase A (LDHA), an important component of the LDH tetramer crucial for aerobic glycolysis, is associated with patient outcome and constitutes a therapeutic target in neuroblastoma (NB).Experimental Design: Expression of LDHA mRNA and protein was determined in 709 and 110 NB patient samples, respectively, and correlated with survival and risk factors. LDHA and LDHB were depleted in human NB cell lines by CRISPR/Cas9 and shRNA, respectively, and aerobic glycolysis, clonogenicity, and tumorigenicity were determined. Expression of LDHA in relation to MYCN was measured in NB cell lines and in the TH-MYCN NB mouse model.Results: Expression of LDHA, both on the mRNA and the protein level, was significantly and independently associated with decreased patient survival. Predominant cytoplasmic localization of LDHA protein was associated with poor outcome. Amplification and expression of MYCN did not correlate with expression of LDHA in NB cell lines or TH-MYCN mice, respectively. Knockout of LDHA inhibited clonogenicity, tumorigenicity, and tumor growth without abolishing LDH activity or significantly decreasing aerobic glycolysis. Concomitant depletion of LDHA and the isoform LDHB ablated clonogenicity while not abrogating LDH activity or decreasing aerobic glycolysis. The isoform LDHC was not expressed.Conclusions: High expression of LDHA is independently associated with outcome of NB, and NB cells can be inhibited by depletion of LDHA or LDHB. This inhibition appears to be unrelated to LDH activity and aerobic glycolysis. Thus, investigations of inhibitory mechanisms beyond attenuation of aerobic glycolysis are warranted, both in NB and normal cells. Clin Cancer Res; 24(22); 5772–83. ©2018 AACR.

This file contains supplemental figures on in vivo xenograft responses to birinapant, a supplemental figure on mouse tolerability to birinapant, and a supplemental figure on gene expression networks. It also contains a supplemental table on patient demographics, a supplemental table on in vivo xenograft responses to birinapant, a supplemental table on individual mouse responses to birinapant, a supplemental table on in vivo birinapant efficacy in drug combinations, and a supplemental table on differentially expressed genes.

This file contains Supplementary Figure legends and Figures Supplementary Figure 1 - Comparisons between time-dependent gene expression quantification models Supplementary Figure 2 - Comparisons of Piscore for miRNA and mRNA members of interaction hubs Supplementary Figure 3 - Survival metrics for miRNA and miRNA signature in human neuroblastoma tumors Supplementary Figure 4 - Gene set enrichment analysis of predicted miR204 target genes upon MYCN siRNA treatment Supplementary Figure 5 - Data to support the creation of dox-inducible miR204 expressing cells and to support miR204 MYCN relationship Supplementary Figure 6 - Data to support miR204 effects on MYCN expression, neuroblastoma proliferation and xenograft experiments.

Supplementary Material and Methods

Supplementary Figure S6 G-K G, Overall patient survival using Kaplan-Meier survival probability plots from the Cologne data set (http://r2.amc.nl) for PA2G4 mRNA expression subdivided around the median PA2G4 expression level among 477 neuroblastoma patients. H, Kaplan-Meier plot for event-free survival of 649 neuroblastoma patients from the kocak dataset (R2 microarray analysis and visualization platform, http://r2.amc.nl) I, Real-time PCR mRNA expression of PA2G4 among 40 neuroblastoma patient tumors treated at Sydney Children's Hospital, subdivided by MYCN amplification status. J, Multivariate event-free survival analysis using cox regression modelling. The p-values were obtained from the cox-regression analysis. K, Incidence of PA2G4 amplification among a range of different human cancer types within The Cancer Genome Atlas (TCGA). Results were generated using cBioportal for Cancer Genomics (https://cancergenome.nih.gov/). NEPC, neuroendocrine prostate cancer; CS, carcinosarcoma.

mRNA and miRNA expression in 1 week, 2 week and 6 week wildtype or TH-MYCN+/+ ganglia or tumors

Supplementary Figure S5. PA2G4 expression enhances the malignant neuroblastoma phenotype in vitro. A, Neurite formation in SH-SY5Y and BE(2)-C cells transfected with PA2G4 siRNA and then treated with 2 µM 13-cis-retinoic acid. B, Immunoblots assessing PA2G4-p48 and PA2G4-p42 protein expression levels using an anti-PA2G4 antibody against whole cell protein lysates from a panel of neuroblastoma and non-malignant myofibroblast cell lines. C, Immunoblots assessing the effect of PA2G4-p48 knockdown on PA2G4 and MYCN protein levels in BE(2)-C and CHP-134 cells. D, Immunoblots confirming transfection of PA2G4-p48 siRNA and MYCN expression plasmid DNA for 72 hours. E and F Cell proliferation measured by BrdU incorporation (E), and, cell viability measured by the Alamar Blue assay.

Supplementary Figure S3. Competitive chemical inhibition of MYCN-PA2G4 binding. A, Chemical structure of WS6. B, Cycloheximide chase assay measuring the half-life of MYCN protein in BE(2)-C and Kelly cells following 0.8 µM WS6 treatment for 24 hours, then treatment with 100 µg/µl CHX for up to 60 minutes. C, BE(2)-C and Kelly cells treated with 0.2 µM or 0.4 µM WS6, followed by colony formation assessment, compared to untreated cells. D, Immunoblotting with an anti-MYC-Tag antibody to quantify PA2G4 protein expression from SH-SY5Y and Kelly cells transfected with the MYC-tagged PA2G4 vector. E, Densitometric quantification of MYCN and PA2G4 protein levels of immunoblots using tumor tissues of TH-MYCN mice treated with either DMSO (n=5) or WS6 (n=5), and quantified by Image J software. Sample means were compared by students t-test. * Represents p-value

Purpose: Deregulated MYC drives oncogenesis in many tissues yet direct pharmacologic inhibition has proven difficult. MYC coordinately regulates polyamine homeostasis as these essential cations support MYC functions, and drugs that antagonize polyamine sufficiency have synthetic-lethal interactions with MYC. Neuroblastoma is a lethal tumor in which the MYC homologue MYCN, and ODC1, the rate-limiting enzyme in polyamine synthesis, are frequently deregulated so we tested optimized polyamine depletion regimens for activity against neuroblastoma.Experimental Design: We used complementary transgenic and xenograft-bearing neuroblastoma models to assess polyamine antagonists. We investigated difluoromethylornithine (DFMO; an inhibitor of Odc, the rate-limiting enzyme in polyamine synthesis), SAM486 (an inhibitor of Amd1, the second rate-limiting enzyme), and celecoxib (an inducer of Sat1 and polyamine catabolism) in both the preemptive setting and in the treatment of established tumors. In vitro assays were performed to identify mechanisms of activity.Results: An optimized polyamine antagonist regimen using DFMO and SAM486 to inhibit both rate-limiting enzymes in polyamine synthesis potently blocked neuroblastoma initiation in transgenic mice, underscoring the requirement for polyamines in MYC-driven oncogenesis. Furthermore, the combination of DFMO with celecoxib was found to be highly active, alone, and combined with numerous chemotherapy regimens, in regressing established tumors in both models, including tumors harboring highest risk genetic lesions such as MYCN amplification, ALK mutation, and TP53 mutation with multidrug resistance.Conclusions: Given the broad preclinical activity demonstrated by polyamine antagonist regimens across diverse in vivo models, clinical investigation of such approaches in neuroblastoma and potentially other MYC-driven tumors is warranted. Clin Cancer Res; 22(17); 4391–404. ©2016 AACR.

Supplementary Table 2-1 All miRNA-mRNA interactions detected for network presented in Figure 2A, Supplementary Table 2-2 Expression of miRNAs in human adrenal neuroblasts and MYCN-amplified tumours

Supplementary Figure S5. BET bromodomain inhibitors and proteasome inhibitors exert synergistic anticancer effects against TERT-rearranged neuroblastoma cells.

Supplementary Table S1. Primary screening of the Food and Drug Administration-Approved Oncology Drugs (AODs) Set IV from the US National Cancer Institute for BET bromodomain inhibitor enhancers.