Search

Supplementary Data from Somatic Mutations Enriched in Cis-Regulatory Elements Affect Genes Involved in Embryonic Development and Immune System Response in Neuroblastoma

Supplementary Figures. Figure S1: Percentage of DHS regions shared by NB cell lines and other Encode cell lines; Figure S2: Descriptive statistics of Whole Genome Sequencing (WGS) of 14 normal-primary NB sample pairs; Figure S3: Somatic variants filtering summary; Figure S4: Results from mutational enrichment analysis; Figure S5: K-means clustering of NB samples; Figure S6: ADPRHL1 and MCFL1 genomic interactions with the mutated TFBS; Figure S7: TH deregulation by somatic regulatory variants.

The contribution of coding mutations to oncogenesis has been largely clarified, whereas little is known about somatic mutations in noncoding DNA and their role in driving tumors remains controversial. Here, we used an alternative approach to interpret the functional significance of noncoding somatic mutations in promoting tumorigenesis. Noncoding somatic mutations of 151 neuroblastomas were integrated with ENCODE data to locate somatic mutations in regulatory elements specifically active in neuroblastoma cells, nonspecifically active in neuroblastoma cells, and nonactive. Within these types of elements, transcription factors (TF) were identified whose binding sites were enriched or depleted in mutations. For these TFs, a gene expression signature was built to assess their implication in neuroblastoma. DNA- and RNA-sequencing data were integrated to assess the effects of those mutations on mRNA levels. The pathogenicity of mutations was significantly higher in transcription factor binding site (TFBS) of regulatory elements specifically active in neuroblastoma cells, as compared with the others. Within these elements, there were 18 over-represented TFs involved mainly in cell-cycle phase transitions and 15 under-represented TFs primarily regulating cell differentiation. A gene expression signature based on over-represented TFs correlated with poor survival and unfavorable prognostic markers. Moreover, recurrent mutations in TFBS of over-represented TFs such as EZH2 affected MCF2L and ADP-ribosylhydrolase like 1 expression, among the others. We propose a novel approach to study the involvement of regulatory variants in neuroblastoma that could be extended to other cancers and provide further evidence that alterations of gene expression may have relevant effects in neuroblastoma development.Significance:These findings propose a novel approach to study regulatory variants in neuroblastoma and suggest that noncoding somatic mutations have relevant implications in neuroblastoma development.

Noncoding cis-regulatory variants have gained interest as cancer drivers, yet progress in understanding their significance is hindered by the numerous challenges and limitations of variant prioritization. To overcome these limitations, we focused on active cis-regulatory elements (aCRE) to design a customized panel for the deep sequencing of 56 neuroblastoma tumor and normal DNA sample pairs. To search for driver mutations, aCREs were defined by reanalysis of H3K27ac chromatin immunoprecipitation sequencing peaks in 25 neuroblastoma cell lines. These regulatory genomic regions were tested for an excess of somatic mutations and assessed for statistical significance using a global approach that accounted for chromatin accessibility and replication timing. Additional validation was provided by whole genome sequence analysis of 151 neuroblastomas. Analysis of HiC data determined the presence of candidate target genes interacting with mutated regions. An excess of somatic mutations in aCREs of diverse genes were identified, including IPO7, HAND2, and ARID3A. CRISPR-Cas9 editing was utilized to assess the functional consequences of mutations in the IPO7-aCRE. Patients with noncoding mutations in aCREs showed inferior overall and event-free survival independent of age at diagnosis, stage, risk stratification, and MYCN status. Expression of aCRE-interacting genes correlated strongly with negative prognostic markers and low survival rates. Moreover, a convergence between the biological functions of aCRE target genes and transcription factors with mutated binding motifs was associated with embryonic development and immune system response. Overall, this strategy enabled the identification of somatic mutations in regulatory elements that collectively promote neuroblastoma tumorigenesis.Significance:Assessment of noncoding cis-regulatory variants and long-range interaction data highlight the combined effect of somatic mutations in regulatory elements in driving neuroblastoma.

Supplementary Figure from Somatic Mutations Enriched in Cis-Regulatory Elements Affect Genes Involved in Embryonic Development and Immune System Response in Neuroblastoma

Supplementary Tables. Table S1. Clinical features of In-house collected NB samples; Table S2. Results of mutational enrichment analysis; Table S3. Results of Cox regression analysis; Table S4. Variant clusterization and expression levels of candidate target genes.

Supplementary Table 1. Functional prioritization of SNPs in linkage disequilibrium (r2=0.6) with significant rs4673067 at SCG2 locus. Supplementary Table 2. SNPs in LD (r2>0.60) with the SNP rs118727. Supplementary Table 3. SNPs in LD (r2>0.60) with the SNP rs196830. Supplementary Table 4. SNPs in LD (r2>0.60) with the SNP rs169061. Supplementary Table 5. SNPs in LD (r2>0.60) with the SNP rs11994014. Supplementary Table 6. SNPs in LD (r2>0.60) with the SNP rs17830286. Supplementary Table 7. Functional prioritization of SNPs in 3'UTR region of NEFL and in linkage disequilibrium (r2=0.6) with significant typed SNPs rs118727, rs196830, rs17830286, rs11994014. Supplementary Table 8. Prediction of mircoRNAs whose binding is affected by the SNPs rs1059111, rs2979704, rs3761 by mrSNP web tool. Supplementary Table 9. Prediction of transcriptional factors whose binding is affected by the SNPs rs1059111, rs2979704, rs3761 by HaploReg V2. Supplementary Table 10. List of UTR motifs analyzed for the SNPs rs3761, rs2979704, and rs1059111. Supplementary Table 11. Imputation results of SNPs at SCG2 and NEFL loci (+/- 1Kb) by using 1000 Genomes data. Supplementary Table 12. Association of NEFL SNP genotypes with pathologic characteristics of neuroblastoma in European American cohort. Supplementary Table 13. Association of NEFL SNP genotypes with pathologic characteristics of neuroblastoma in Italian cohort. Supplementary Figure 1. Linkage disequilibrium (LD) plot of NEFL gene (chr8: positions 24,845,004 to 24,887,674) by Haploview 4.2 for HapMap CEU subjects. Supplementary Figure 2. REST gene silencing in neuroblastoma cells. The efficiency of gene silencing mediated by lentiviral delivery of hairpin RNA directed against REST (shREST) in SH-SY5Y and SK-N-BE2c cell lines was assessed by western blotting (a-b). The bar graphs show integral optical density (OD) value for each band, normalized respect to β-Actin expression. The results are shown as mean of three experiments and are represented as fold respect to shCTR cells which are infected by lentivirus-mediated delivery of non-silencing hairpin RNA. Induction of NEFL and REST gene expression levels after REST silencing in SH-SY5Y (c) and SK-N-BE2c (d). *P

All-trans-retinoic acid (ATRA), the most biologically active metabolite of vitamin A, controls cell proliferation, apoptosis, and differentiation depending on the cellular context. These activities point to ATRA as a candidate for cancer therapy. A pivotal effect of the molecule is the modulation of p27Kip1, a cyclin-dependent kinase (CDK) inhibitor (CDKI). Here, we investigate the mechanisms by which ATRA regulates p27Kip1 level in LAN-5, a neuroblastoma cell line. When added to the cells, ATRA causes a rapid nuclear increase of p27Kip1, which clearly precedes growth arrest. The early buildup is not due to impairment of the CDKI degradation, in contrast to previous observations. Particularly, we did not detect the down-regulation of Skp2 and Cks1, two proteins involved in the nuclear ubiquitin-dependent p27Kip1 removal. Moreover, the morphogen does not impair the CDKI nuclear export and does not cause CDK2 relocalization. The characterization of CDKI isoforms by two-dimensional PAGE/immunoblotting showed that ATRA induces an early nuclear up-regulation of monophosphorylated p27Kip1. Immunologic studies established that this isoform corresponds to p27Kip1 phosphorylated on S10. The buildup of phospho(S10)p27Kip1 precedes the CDKI accumulation and increases its half-life. Finally, ATRA-treated nuclear LAN-5 extracts showed an enhanced capability of phosphorylating p27Kip1 on S10, thus explaining the nuclear up-regulation of the isoform. In conclusion, our data suggest a novel mechanism of ATRA antiproliferative activity, in which the morphogen rapidly up-regulates a nuclear kinase activity that phosphorylates p27Kip1 on S10. In turn, this event causes the stabilization of p27Kip1 and its accumulation in the nuclear compartment. (Cancer Res 2006; 66(8): 4240-8)

Supplementary Materials and Methods from Retinoic Acid Induces p27Kip1 Nuclear Accumulation by Modulating Its Phosphorylation

Noncoding cis-regulatory variants have gained interest as cancer drivers, yet progress in understanding their significance is hindered by the numerous challenges and limitations of variant prioritization. To overcome these limitations, we focused on active cis-regulatory elements (aCRE) to design a customized panel for the deep sequencing of 56 neuroblastoma tumor and normal DNA sample pairs. To search for driver mutations, aCREs were defined by reanalysis of H3K27ac chromatin immunoprecipitation sequencing peaks in 25 neuroblastoma cell lines. These regulatory genomic regions were tested for an excess of somatic mutations and assessed for statistical significance using a global approach that accounted for chromatin accessibility and replication timing. Additional validation was provided by whole genome sequence analysis of 151 neuroblastomas. Analysis of HiC data determined the presence of candidate target genes interacting with mutated regions. An excess of somatic mutations in aCREs of diverse genes were identified, including IPO7, HAND2, and ARID3A. CRISPR-Cas9 editing was utilized to assess the functional consequences of mutations in the IPO7-aCRE. Patients with noncoding mutations in aCREs showed inferior overall and event-free survival independent of age at diagnosis, stage, risk stratification, and MYCN status. Expression of aCRE-interacting genes correlated strongly with negative prognostic markers and low survival rates. Moreover, a convergence between the biological functions of aCRE target genes and transcription factors with mutated binding motifs was associated with embryonic development and immune system response. Overall, this strategy enabled the identification of somatic mutations in regulatory elements that collectively promote neuroblastoma tumorigenesis. Significance: Assessment of noncoding cis-regulatory variants and long-range interaction data highlight the combined effect of somatic mutations in regulatory elements in driving neuroblastoma.

All-trans-retinoic acid (ATRA), the most biologically active metabolite of vitamin A, controls cell proliferation, apoptosis, and differentiation depending on the cellular context. These activities point to ATRA as a candidate for cancer therapy. A pivotal effect of the molecule is the modulation of p27Kip1, a cyclin-dependent kinase (CDK) inhibitor (CDKI). Here, we investigate the mechanisms by which ATRA regulates p27Kip1 level in LAN-5, a neuroblastoma cell line. When added to the cells, ATRA causes a rapid nuclear increase of p27Kip1, which clearly precedes growth arrest. The early buildup is not due to impairment of the CDKI degradation, in contrast to previous observations. Particularly, we did not detect the down-regulation of Skp2 and Cks1, two proteins involved in the nuclear ubiquitin-dependent p27Kip1 removal. Moreover, the morphogen does not impair the CDKI nuclear export and does not cause CDK2 relocalization. The characterization of CDKI isoforms by two-dimensional PAGE/immunoblotting showed that ATRA induces an early nuclear up-regulation of monophosphorylated p27Kip1. Immunologic studies established that this isoform corresponds to p27Kip1 phosphorylated on S10. The buildup of phospho(S10)p27Kip1 precedes the CDKI accumulation and increases its half-life. Finally, ATRA-treated nuclear LAN-5 extracts showed an enhanced capability of phosphorylating p27Kip1 on S10, thus explaining the nuclear up-regulation of the isoform. In conclusion, our data suggest a novel mechanism of ATRA antiproliferative activity, in which the morphogen rapidly up-regulates a nuclear kinase activity that phosphorylates p27Kip1 on S10. In turn, this event causes the stabilization of p27Kip1 and its accumulation in the nuclear compartment. (Cancer Res 2006; 66(8): 4240-8)

Medulloblastoma (MB) is the most common Central nervous system (CNS) neoplasia accounting for 25% of all childhood brain tumors. The Wnt, Sonic and Notch developmental cascades all regulate CNS cell dynamics, and are mutationally activated in a subset of MBs. So far the main limitation of the current treatment of MB is the lack of drug specificity, we aim to identify new specific impairment pathway drugs to offer new therapeutic tools in order to improve the clinical management of this neoplasia. The Wnt signaling pathway ends in the regulation of the levels of β-catenin which accumulates in the nucleus and interacts with the LEF/TCF family of transcription factors; in the absence of Wnt stimulation, β-catenin is phosphorylated by several kinases, as a complex comprising GSK3-β, Axin and APC, and it is targeted for degradation in the cytoplasm via the ubiquitin-proteasome pathway. Wnt activation includes key regulators of proliferation, among which c-Myc and cyclin D1 play a crucial role. We screened a drug library including almost 1200 compounds which are not well characterized on their activity against cancer cells. The library contains a repository of Natural Products, that derive from marine and plant sources collected worldwide, and a Synthetic library Products. We tested the efficacy of the Natural Products to interfere with Wnt pathway, using the TOP/FOP luciferase assay. From this large screening, we focused our attention on three natural compounds (Cantharidin, Parthenolide, Homoharringotnine) which positively inhibit Wnt pathway in three independent MB cell lines: Daoy (a desmoplastic medulloblastoma), ONS-76 and UW288(classic medulloblastoma cell lines). We determined their ability to turn-off Wnt signaling by western blotting detecting β-catenin activated protein and by RT-PCR for controlling of mRNA levels of WNT target genes. Cantharidin (CTD) has an anticancer activity in leukemic cells and melanoma cells, but it is still to be identified which is the basis of its mechanism. CTD is a potent inhibitor of phosphatase PP1 and PP2A which play a positive role in regulating Wnt signalling since it dephosphorylates AXIN, by impairing APC complex. We screened CTD as the most effective drug to inhibit Wnt pathway in MB cells. By immunofluorescence, we observed that the nuclear amount of β-catenin was found reduced when the cells were exposed to CTD. We found its effectiveness in vitro and in vivo on tumor growth assays in nude and strengthen those with additional orthotopic cerebellum xenografs assays to further assign its molecular mechanism of action in MB thus suggesting its use for future therapeutic applications in mouse MB genetic models. Altogether these results are suggesting a new therapeutic strategy to treat MB to be further applied to other solid tumors which are mainly Wnt driven. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 763.