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INFORM is a prospective, multinational registry gathering clinical and molecular data of relapsed, progressive, or high-risk pediatric patients with cancer. This report describes long-term follow-up of 519 patients in whom molecular alterations were evaluated according to a predefined seven-scale target prioritization algorithm. Mean turnaround time from sample receipt to report was 25.4 days. The highest target priority level was observed in 42 patients (8.1%). Of these, 20 patients received matched targeted treatment with a median progression-free survival of 204 days [95% confidence interval (CI), 99–not applicable], compared with 117 days (95% CI, 106–143; P = 0.011) in all other patients. The respective molecular targets were shown to be predictive for matched treatment response and not prognostic surrogates for improved outcome. Hereditary cancer predisposition syndromes were identified in 7.5% of patients, half of which were newly identified through the study. Integrated molecular analyses resulted in a change or refinement of diagnoses in 8.2% of cases.Significance:The pediatric precision oncology INFORM registry prospectively tested a target prioritization algorithm in a real-world, multinational setting and identified subgroups of patients benefiting from matched targeted treatment with improved progression-free survival, refinement of diagnosis, and identification of hereditary cancer predisposition syndromes.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659

Supplementary Table from The Pediatric Precision Oncology INFORM Registry: Clinical Outcome and Benefit for Patients with Very High-Evidence Targets

Supplementary Figure from The Pediatric Precision Oncology INFORM Registry: Clinical Outcome and Benefit for Patients with Very High-Evidence Targets

Supplementary Table 2 from Accurate Outcome Prediction in Neuroblastoma across Independent Data Sets Using a Multigene Signature

Differentially expressed genes in IMR5 cells after OTX015 and JQ1 treatment respectively

The quassinoid analogue NBT-272 has been reported to inhibit MYC, thus warranting a further effort 7to better understand its preclinical properties in models of embryonal tumors (ET), a family of childhood malignancies sharing relevant biological and genetic features such as deregulated expression of MYC oncogenes. In our study, NBT-272 displayed a strong antiproliferative activity in vitro that resulted from the combination of diverse biological effects, ranging from G1/S arrest of the cell cycle to apoptosis and autophagy. The compound prevented the full activation of both eukaryotic translation initiation factor 4E (eIF4E) and its binding protein 4EBP-1, regulating cap-dependent protein translation. Interestingly, all responses induced by NBT-272 in ET could be attributed to interference with 2 main proproliferative signaling pathways, that is, the AKT and the MEK/extracellular signal-regulated kinase pathways. These findings also suggested that the depleting effect of NBT-272 on MYC protein expression occurred via indirect mechanisms, rather than selective inhibition. Finally, the ability of NBT-272 to arrest tumor growth in a xenograft model of neuroblastoma plays a role in the strong antitumor activity of this compound, both in vitro and in vivo, with its potential to target cell-survival pathways that are relevant for the development and progression of ET. Mol Cancer Ther; 9(12); 3145–57. ©2010 AACR.

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Mechanisms of reduced cell viability induced by OTX015 are cell line dependent. OTX015 reduces cell proliferation over time and induces cell cycle changes as well as apoptosis. (a) Representative photomicrographs of neuroblastoma cell lines treated with OTX015 for 72 h. Scale bar=200 µm. (b) The slope (1/h) of cell proliferation over time measured using xCELLigence of cell lines treated with OTX015. Significance was measured with the Student's t test. (c) Cell proliferation monitored using the xCELLigence system over time in neuroblastoma cell lines treated with 500 nM OTX015 (black) or DMSO control (grey). (d) Cell lines were treated 72 h with 500 nM OTX015 or DMSO, then fixed and stained with propidium iodide for flow cytometry. The distribution of OTX015-treated cells in different stages of the cell cycle is shown. (e) Fraction of apoptotic cells measured in the cell death ELISA assay after 72 h treatment with 250 nM or 500 nM OTX015 or DMSO control. Significant differences between treated cells versus controls were calculated by Student's t test. * p < 0.05, ** p < 0.01. (f) Cell proliferation measured in the BrdU ELISA after 72 h of treatment with 250 nM or 500 nM OTX015 or DMSO. Significant differences between treatment groups and the controls were calculated by Student's t test.

All H3K27Ac peaks called in non-promoter regions in IMR5

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Purpose: Neuroblastoma is a heterogeneous childhood tumor with poor survival outcome for the aggressive type despite intensive multimodal therapies. In this study, we aimed to identify new treatment options for neuroblastoma based on integrative genomic analysis.Experimental Design: The Connectivity Map is a database comprising expression profiles in response to known therapeutic compounds. This renders it a useful tool in the search for potential therapeutic compounds based on comparison of gene expression profiles of diseased cells and a database of profiles in response to known therapeutic compounds. We have used this strategy in the search for new therapeutic molecules for neuroblastoma based on data of an integrative meta-analysis of gene copy number and expression profiles from 146 primary neuroblastoma tumors and normal fetal neuroblasts.Results: In a first step, a 132-gene classifier was established that discriminates three major genomic neuroblastoma subgroups, reflecting inherent differences in gene expression between these subgroups. Subsequently, we screened the Connectivity Map database using gene lists generated by comparing expression profiles of fetal adrenal neuroblasts and the genomic subgroups of neuroblastomas. A putative therapeutic effect was predicted for several compounds of which six were empirically tested. A significant reduction in cell viability was shown for five of these molecules: 17-allylamino-geldanamycin, monorden, fluphenazine, trichostatin, and rapamycin.Conclusions: This proof-of-principle study indicates that an integrative genomic meta-analysis approach with inclusion of neuroblast data enables the identification of promising compounds for treatment of children with neuroblastoma. Further studies are warranted to explore in detail the therapeutic potential of these compounds.

Purpose: The phosphoinositide 3-kinase (PI3K)/Akt pathway is frequently activated in human cancer and plays a crucial role in neuroblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K/Akt signaling as a novel antiproliferative approach in neuroblastoma.Experimental Design: The expression pattern and functions of class IA PI3K isoforms were investigated in tumor samples and cell lines. Effects on cell survival and downstream signaling were analyzed following down-regulation of p110α or p110δ in SH-SY5Y and LA-N-1 cells by means of RNA interference.Results: Overexpression of the catalytic p110δ and regulatory p85α isoforms was detected in a panel of primary neuroblastoma samples and cell lines, compared with normal adrenal gland tissue. Although down-regulation of either p110α or p110δ led to impaired cell growth, reduced expression of p110δ also had a selective effect on the survival of SH-SY5Y cells. Decreased levels of p110δ were found to induce apoptosis and lead to lower expression levels of antiapoptotic Bcl-2 family proteins. SH-SY5Y cells with decreased p110δ levels also displayed reduced activation of ribosomal protein S6 kinase in response to stimulation with epidermal growth factor and insulin-like growth factor-I.Conclusions: Together, our data reveal a novel function of p110δ in neuroblastoma growth and survival.

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OTX015 has no significant effect on non-malignant human fibroblasts. (a) Representative pictures of human fibroblasts treated for 72 h with 500 nM OTX015 or DMSO control. Scale bar=100 µm. (b) Growth of fibroblasts treated with 500 nM OTX015 (black) or DMSO control (grey) monitored over 96 h using the xCELLigence system. The slope (1/h) of both curves was calculated, and did not show a significant difference (right) using the Student's t test.

Supplementary Data from The Quassinoid Derivative NBT-272 Targets Both the AKT and ERK Signaling Pathways in Embryonal Tumors

Purpose: Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression.Experimental Design: The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis.Results: ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival.Conclusions: ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants. Clin Cancer Res; 16(17); 4353–62. ©2010 AACR.

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GSEA of gene sets describing general cellular processes after treatment with OTX015 and/or JQ1(GSEA, Broad institute)

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Supplementary Figure from Targeted Analysis of Cell-free Circulating Tumor DNA is Suitable for Early Relapse and Actionable Target Detection in Patients with Neuroblastoma

Supplementary Data from Targeted Analysis of Cell-free Circulating Tumor DNA is Suitable for Early Relapse and Actionable Target Detection in Patients with Neuroblastoma

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BET inhibition preferentially disrupts expression of genes associated with super-enhancers. (a) ChIP-seq profiles for H3K27Ac binding at representative super-enhancer-associated gene loci, including MYCN, NCOR2, BCOR and GLI2, in MYCN-amplified IMR-5 cells. The x-axis shows genomic position and the y-axis the histone mark signal in units of reads per million per base pair (rpm/bp). The gene model is depicted below and scale bars above the binding profiles. (b) The Fisher exact test shows enrichment of super-enhancer associated genes among the downregulated genes (logFC < 0 and q < 0.05) by OTX015 or JQ1 treatment. (c) Cumulative distribution plots of the top 200 super-enhancer-associated genes and their fold-change measured using genes which are differentially regulated in IMR-5 cells by OTX015 treatment, JQ1 treatment or the overlap between these datasets (left to right). Plots show a shift towards negative logFC for super-enhancer associated genes (blue curves) compared to genes not associated with super-enhancers (black curves). (d) Box plots of log2-fold changes in the top 200 genes associated with super-enhancers (SE) and regular enhancers (non-SE) in genes differentially regulated (compared to DMSO-treated controls) in IMR-5 cells treated with OTX015, JQ1 or the overlap from both datasets (left to right). Statistical significance was determined using a two-sided Mann-Whitney test.

GSEA of cancer gene sets after treatment with OTX015 and/or JQ1(GSEA, Broad institute)

BET bromodomain inhibition leads to disruption of MYCN driven gene-expression programs even at high levels of MYCN. (a) Enrichment of the published gene expression signature for JQ1-treated human MYCN-amplified neuroblastoma cell lines (11) in differentially expressed genes in IMR-5 cells after treatment with OTX015, JQ1 or DMSO control. (b) Enrichment of the published gene expression signature for MYC target genes (16) in differentially expressed genes in IMR-5 cells after treatment with OTX015, JQ1 or DMSO control. (c) Enrichment of the published gene expression signature for JQ1-treated human MYCN-amplified neuroblastoma cell lines (11) in differentially expressed genes in mNB-A1 cells after treatment of with OTX015, JQ1 or DMSO control. (d) Enrichment of the published gene expression signature for MYC target genes (16) in differentially expressed genes in mNB-A1 cells after treatment with OTX015, JQ1 or DMSO control (* p < 0.05, ** p < 0.01, *** p < 0.001). (e) GSEA (version 2.0, Broad Institute) of differentially expressed genes in IMR5 cells after treatment with OTX015 or JQ1 using gene sets representing signatures of JQ1 as well as MYC target genes (11, 16).

Purpose: More accurate assessment of prognosis is important to further improve the choice of risk-related therapy in neuroblastoma (NB) patients. In this study, we aimed to establish and validate a prognostic miRNA signature for children with NB and tested it in both fresh frozen and archived formalin-fixed paraffin-embedded (FFPE) samples.Experimental Design: Four hundred-thirty human mature miRNAs were profiled in two patient subgroups with maximally divergent clinical courses. Univariate logistic regression analysis was used to select miRNAs correlating with NB patient survival. A 25-miRNA gene signature was built using 51 training samples, tested on 179 test samples, and validated on an independent set of 304 fresh frozen tumor samples and 75 archived FFPE samples.Results: The 25-miRNA signature significantly discriminates the test patients with respect to progression-free and overall survival (P < 0.0001), both in the overall population and in the cohort of high-risk patients. Multivariate analysis indicates that the miRNA signature is an independent predictor of patient survival after controlling for current risk factors. The results were confirmed in an external validation set. In contrast to a previously published mRNA classifier, the 25-miRNA signature was found to be predictive for patient survival in a set of 75 FFPE neuroblastoma samples.Conclusions: In this study, we present the largest NB miRNA expression study so far, including more than 500 NB patients. We established and validated a robust miRNA classifier, able to identify a cohort of high-risk NB patients at greater risk for adverse outcome using both fresh frozen and archived material. Clin Cancer Res; 17(24); 7684–92. ©2011 AACR.

Supplementary Figures S1-S2 from Targeting the Phosphoinositide 3-Kinase Isoform p110δ Impairs Growth and Survival in Neuroblastoma Cells

Purpose: Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma.Experimental Design: The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control.Results: OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes.Conclusions: We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. Clin Cancer Res; 22(10); 2470–81. ©2015 AACR.

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List of all Super-enhancers identified in IMR5 cells

OTX015 effect on cell viability positively correlates with MYCN status. (a) IC50 of the IMR 5, Chp 134, Chp 212, SK N-BE(2), IMR-32, SK-N-BE, NB69, SK N AS and GI-M-EN neuroblastoma cell lines measured using MTT assays. (b) Maximum reduction of viability in IMR 5, Chp 134, Chp 212, SK N-BE(2), IMR-32, SK-N-BE, NB69, SK N AS and GI-M-EN cells treated for 72 h with 6 µM OTX015 and measured using MTT assays. (c) Relative MYCN mRNA (top) and protein (bottom) expression in all analyzed neuroblastoma cell lines. (d/e) The IC50 values of the cell lines shown in (a) were plotted against MYCN mRNA expression (d) or MYCN protein expression (e), which is shown in (c). Both graphs show weak anti-correlative tendencies in both cell lines (MYCN mRNA p=0.22, MYCN protein p=0.16).

Purpose: The purpose of this study was to further define the biology of the 11q− neuroblastoma tumor subgroup by the integration of array-based comparative genomic hybridization with microRNA (miRNA) expression profiling data to determine if improved patient stratification is possible.Experimental Design: A set of primary neuroblastoma (n = 160), which was broadly representative of all genetic subtypes, was analyzed by array-based comparative genomic hybridization and for the expression of 430 miRNAs. A 15-miRNA expression signature previously shown to be predictive of clinical outcome was used to analyze an independent cohort of 11q− tumors (n = 37).Results: Loss of 4p and gain of 7q occurred at a significantly higher frequency in the 11q− tumors, further defining the genetic characteristics of this subtype. The 11q− tumors could be split into two subgroups using a miRNA expression survival signature that differed significantly in clinical outcome and the overall frequency of large-scale genomic imbalances, with the poor survival subgroup having significantly more imbalances. miRNAs from the expression signature, which were upregulated in unfavorable tumors, were predicted to target downregulated genes from a published mRNA expression classifier of clinical outcome at a higher-than-expected frequency, indicating the miRNAs might contribute to the regulation of genes within the signature.Conclusion: We show that two distinct biological subtypes of neuroblastoma with loss of 11q occur, which differ in their miRNA expression profiles, frequency of segmental imbalances, and clinical outcome. A miRNA expression signature, combined with an analysis of segmental imbalances, provides greater prediction of event-free survival and overall survival outcomes than 11q status by itself, improving patient stratification. Clin Cancer Res; 16(11); 2971–8. ©2010 AACR.

Supplementary Figures S1-S2; Supplementary Tables S1 and S3-S5.

OTX015 reduces cell proliferation and induces apoptosis in MYCN amplified human xenografts. (a) Representative pictures of IMR5 xenograft tumor sections stained with hematoxylin/eosin, and immunohistochemical staining for apoptotic cells (cleaved caspase 3) and proliferating cells (Mib-1/Ki 67) are shown. Scale bar=250 µm. The relative fraction of positively stained cells for cleaved caspase 3 (b) and Mib-1 (c) were calculated from three representative images from each xenograft tumor and are shown as box plots. Statistical difference between groups was assessed by Student's t test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Westermann et al. as well as Valentijn et al. MYCN target gene list and expression changes after OTX015 treatment as well as BRD4 binding

List of all genes from hg19 with changes in BRD4 binding using BRD4- ChIP-seq in IMR5 cells with changes after treatment with OTX015

Supplementary Data from Meta-mining of Neuroblastoma and Neuroblast Gene Expression Profiles Reveals Candidate Therapeutic Compounds

Purpose: Genomic alterations of the anaplastic lymphoma kinase (ALK) gene have been postulated to contribute to neuroblastoma pathogenesis. This study aimed to determine the interrelation of ALK mutations, ALK expression levels, and clinical phenotype in primary neuroblastoma.Experimental Design: The genomic ALK status and global gene expression patterns were examined in 263 primary neuroblastomas. Allele-specific ALK expression was determined by cDNA cloning and sequencing. Associations of genomic ALK alterations and ALK expression levels with clinical phenotypes and transcriptomic profiles were compared.Results: Nonsynonymous point mutations of ALK were detected in 21 of 263 neuroblastomas (8%). Tumors with ALK mutations exhibited about 2-fold elevated median ALK mRNA levels in comparison with tumors with wild-type (WT) ALK. Unexpectedly, the WT allele was preferentially expressed in 12 of 21 mutated tumors. Whereas survival of patients with ALK mutated tumors was significantly worse as compared with the entire cohort of WT ALK patients, it was similarly poor in patients with WT ALK tumors in which ALK expression was as high as in ALK mutated neuroblastomas. Global gene expression patterns of tumors with ALK mutations or with high-level WT ALK expression were highly similar, and suggested that ALK may be involved in cellular proliferation in primary neuroblastoma.Conclusions: Primary neuroblastomas with mutated ALK exhibit high ALK expression levels and strongly resemble neuroblastomas with elevated WT ALK expression levels in both their clinical and molecular phenotypes. These data suggest that high levels of mutated and WT ALK mediate similar molecular functions that may contribute to a malignant phenotype in primary neuroblastoma. Clin Cancer Res; 17(15); 5082–92. ©2011 AACR.

Purpose: Reliable prognostic stratification remains a challenge for cancer patients, especially for diseases with variable clinical course such as neuroblastoma. Although numerous studies have shown that outcome might be predicted using gene expression signatures, independent cross-platform validation is often lacking.Experimental Design: Using eight independent studies comprising 933 neuroblastoma patients, a prognostic gene expression classifier was developed, trained, tested, and validated. The classifier was established based on reanalysis of four published studies with updated clinical information, reannotation of the probe sequences, common risk definition for training cases, and a single method for gene selection (prediction analysis of microarray) and classification (correlation analysis).Results: Based on 250 training samples from four published microarray data sets, a correlation signature was built using 42 robust prognostic genes. The resulting classifier was validated on 351 patients from four independent and unpublished data sets and on 129 remaining test samples from the published studies. Patients with divergent outcome in the total cohort, as well as in the different risk groups, were accurately classified (log-rank P < 0.001 for overall and progression-free survival in the four independent data sets). Moreover, the 42-gene classifier was shown to be an independent predictor for survival (odds ratio, >5).Conclusion: The strength of this 42-gene classifier is its small number of genes and its cross-platform validity in which it outperforms other published prognostic signatures. The robustness and accuracy of the classifier enables prospective assessment of neuroblastoma patient outcome. Most importantly, this gene selection procedure might be an example for development and validation of robust gene expression signatures in other cancer entities. Clin Cancer Res; 16(5); 1532–41

Supplementary Data from Chromosomal and MicroRNA Expression Patterns Reveal Biologically Distinct Subgroups of 11q− Neuroblastoma

Supplementary Table 4 from High ALK Receptor Tyrosine Kinase Expression Supersedes ALK Mutation as a Determining Factor of an Unfavorable Phenotype in Primary Neuroblastoma

Purpose:Treating refractory or relapsed neuroblastoma remains challenging. Monitoring body fluids for tumor-derived molecular information indicating minimal residual disease supports more frequent diagnostic surveillance and may have the power to detect resistant subclones before they give rise to relapses. If actionable targets are identified from liquid biopsies, targeted treatment options can be considered earlier.Experimental Design:Droplet digital PCR assays assessing MYCN and ALK copy numbers and allelic frequencies of ALK p.F1174L and ALK p.R1275Q mutations were applied to longitudinally collected liquid biopsies and matched tumor tissue samples from 31 patients with high-risk neuroblastoma. Total cell-free DNA (cfDNA) levels and marker detection were compared with data from routine clinical diagnostics.Results:Total cfDNA concentrations in blood plasma from patients with high-risk neuroblastoma were higher than in healthy controls and consistently correlated with neuron-specific enolase levels and lactate dehydrogenase activity but not with 123I-meta-iodobenzylguanidine scores at relapse diagnosis. Targeted cfDNA diagnostics proved superior for early relapse detection to all current diagnostics in 2 patients. Marker analysis in cfDNA indicated intratumor heterogeneity for cell clones harboring MYCN amplifications and druggable ALK alterations that were not detectable in matched tumor tissue samples in 17 patients from our cohort. Proof of concept is provided for molecular target detection in cerebrospinal fluid from patients with isolated central nervous system relapses.Conclusions:Tumor-specific alterations can be identified and monitored during disease course in liquid biopsies from pediatric patients with high-risk neuroblastoma. This approach to cfDNA surveillance warrants further prospective validation and exploitation for diagnostic purposes and to guide therapeutic decisions.

PDF file - 2.6MB, Effects of LBW242 or XIAP silencing on apoptosis induction and caspase activation in SH-EP neuroblastoma cells.

PDF file - 298K, Effect of LBW242 and etoposide treatment on TNF-� expression.

PDF file - 656K, Induction of apoptosis in NXS2 neuroblastoma cells by LBW242 in combination with etoposide or vincristine.

PDF file - 1.7MB, Effect of XIAP silencing by specific antisense oligonucleotide on apoptosis induction in neuroblastoma cells.

Supplementary Tables 1-4 from Lysine-Specific Demethylase 1 Is Strongly Expressed in Poorly Differentiated Neuroblastoma: Implications for Therapy

Aberrant epigenetic changes in DNA methylation and histone acetylation are hallmarks of most cancers, whereas histone methylation was previously considered to be irreversible and less versatile. Recently, several histone demethylases were identified catalyzing the removal of methyl groups from histone H3 lysine residues and thereby influencing gene expression. Neuroblastomas continue to remain a clinical challenge despite advances in multimodal therapy. Here, we address the functional significance of the chromatin-modifying enzyme lysine-specific demethylase 1 (LSD1) in neuroblastoma. LSD1 expression correlated with adverse outcome and was inversely correlated with differentiation in neuroblastic tumors. Differentiation of neuroblastoma cells resulted in down-regulation of LSD1. Small interfering RNA–mediated knockdown of LSD1 decreased cellular growth, induced expression of differentiation-associated genes, and increased target gene–specific H3K4 methylation. Moreover, LSD1 inhibition using monoamine oxidase inhibitors resulted in an increase of global H3K4 methylation and growth inhibition of neuroblastoma cells in vitro. Finally, targeting LSD1 reduced neuroblastoma xenograft growth in vivo. Here, we provide the first evidence that a histone demethylase, LSD1, is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells. We show that inhibition of LSD1 reprograms the transcriptome of neuroblastoma cells and inhibits neuroblastoma xenograft growth. Our results suggest that targeting histone demethylases may provide a novel option for cancer therapy. [Cancer Res 2009;69(5):2065–71]

Supplementary Figure and Table Legends from Lysine-Specific Demethylase 1 Is Strongly Expressed in Poorly Differentiated Neuroblastoma: Implications for Therapy