MYCN Amplicon Research Articles

A MYCN-independent mechanism mediating secretome reprogramming and metastasis in MYCN-amplified neuroblastoma.

MYCN amplification (MNA) is a defining feature of high-risk neuroblastoma (NB) and predicts poor prognosis. However, whether genes within or in close proximity to the MYCN amplicon also contribute to MNA+ NB remains poorly understood. Here, we identify that GREB1, a transcription factor encoding gene neighboring the MYCN locus, is frequently coexpressed with MYCN and promotes cell survival in MNA+ NB. GREB1 controls gene expression independently of MYCN, among which we uncover myosin 1B (MYO1B) as being highly expressed in MNA+ NB and, using a chick chorioallantoic membrane (CAM) model, as a crucial regulator of invasion and metastasis. Global secretome and proteome profiling further delineates MYO1B in regulating secretome reprogramming in MNA+ NB cells, and the cytokine MIF as an important pro-invasive and pro-metastatic mediator of MYO1B activity. Together, we have identified a putative GREB1-MYO1B-MIF axis as an unconventional mechanism promoting aggressive behavior in MNA+ NB and independently of MYCN.

Abstract 2593: A MYCN-independent mechanism mediating secretome reprogramming and metastasis in MYCN-amplified neuroblastoma

Abstract MYCN amplification (MNA) is a defining feature of high-risk neuroblastoma (NB) that predicts poor prognosis. However, whether genes within or in close proximity to the MYCN amplicon also contribute to aggressiveness in MNA+ NB remains poorly understood. Here we identify that GREB1, a transcription factor encoding gene neighboring the MYCN locus, is frequently co-expressed with MYCN, and promotes gene expression independently of MYCN in MNA+ NB. We found that GREB1, as a crucial estrogen receptor (ER) regulatory factor, is highly expressed in MNA+ NB comparable to ER+ breast cancer. Among GREB1-regulated genes altered in MNA+ NB, we uncover that Myosin 1B (MYO1B) promotes aggressive features in MNA+ NB, including invasive capacity in vitro, as well as extravasation and distant metastasis in vivo. Global secretome and proteome profiling uncover MYO1B as a major regulator of secretome reprogramming in MNA+ NB cells. Moreover, we identify the cytokine MIF as an important pro-invasive and pro-metastatic mediator of MYO1B activity. Together, we have identified a putative GREB1-MYO1B-MIF axis as an unconventional mechanism that promotes the aggressiveness of MNA+ NB, and independently of MYCN. Furthermore, we found that MYO1B is also upregulated in association with other oncoproteins during cellular transformation, and is dramatically increased in multiple human cancer types, suggesting a crucial role of MYO1B in cancers in addition to MNA+ NB. Citation Format: Hai-Feng Zhang, Alberto Delaidelli, Sumreen Javed, Busra Turgu, Christopher S. Hughes, Xiaqiu Yang, Michael Lizardo, Daniel Martinez, John M. Maris, Kristopher R. Bosse, Karla C. Williams, Poul H. Sorensen. A MYCN-independent mechanism mediating secretome reprogramming and metastasis in MYCN-amplified neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2593.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in neuroblastoma

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.

Abstract SY24-01: Targeting master transcription factor dependency in pediatric cancer

Abstract Master transcription factors are regulatory proteins that bind to the genome and establish cell-type specific gene expression programs. They are required for proper multicellular development and are often co-opted to drive oncogenic processes in cancer. In pediatric cancers, master transcription factor deregulation and dependency are hallmarks of these otherwise genetically quiet tumor. Nowhere is this more evident than in neuroblastoma where MYCN amplification singularly defines high risk disease and poor prognosis. Targeting of oncogenic master transcription factors is conceptually attractive as their activity is essential in tumor cells and dispensable in most normal cell types. However as most transcription factors are not considered classically druggable, novel approaches are required to target their activity either directly or indirectly. In this symposia, we will explore mechanisms of master transcription factor deregulation and dependency in pediatric cancers. We and others have shown that almost all oncogenic master transcription factors are driven by super enhancers, large non-coding enhancer elements, and that these master transcription factors are themselves responsible for establishing the tumor cell enhancer and super enhancer landscapes. We will demonstrate how these features of transcriptional regulatory circuitry can be exploited to identify new master transcription factors and tumor dependencies in medulloblastoma, neuroblastoma and other tumor types. We will highlight new chemical and genetic approaches to model and target oncogenic transcription factor regulation including chromatin mediated silencing of non-coding regulatory elements, and degradation based strategies for target perturbation. Finally, we will explore in depth how targeting components of the general transcriptional apparatus such as transcriptional kinases can have surprisingly selective effects on oncogenic transcription factor expression. In our research, we find that CDK9/2 inhibition potently and selectively targets the proliferation of MYCN amplified neuroblastoma with little effect on non MYCN amplified neuroblastoma or normal cells. Selectivity for CDK9 inhibition emerges from its ability to preferentially down-regulate nascent MYCN transcription emanating from the MYCN amplicon. MYCN depletion down regulates gene expression programs associated with neuroblastoma adrenergic/noradrenergic identity and combined with CDK2 inhibition, it strongly induces tumor cell apoptosis in vitro and in vivo. In contrast, MYCN overexpression in non-MYCN amplified neuroblastoma reprograms cells to a more adrenergic state and in turn sensitizes them to CDK9/2 inhibition. These data suggest that MYCN dependent reprogramming of neuroblastoma cell state can be selectively targeted. They also provide a rationale for future clinical exploration of CDK9/2 inhibition in high risk relapse neuroblastoma. From these emerging insights in pediatric cancer as well as from recent pan cancer genetic screening data (e.g. Novartis Project Drive and Broad Achilles) it has become increasingly clear that master transcription factors make for some of the most potent and selective tumor dependencies across a variety of cancers. Transcriptional inhibitors represent an attractive first strategy to indirectly target their expression in a variety of tumor contexts. Although these inhibitors exhibit promising selectivity in pre-clinical models, in most cases they also have pleiotropic effects on transcription in both tumor and normal cells. Ultimately, given the urgent unmet need for therapeutics in aggressive tumors like MYCN amplified neuroblastoma, new strategies will be required to more directly target transcription factor addiction. Citation Format: Charles Lin. Targeting master transcription factor dependency in pediatric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY24-01.

Inactive extra-chromosomal MYCN amplicons co-localize to the nuclear lamina in therapy-induced senescent neuroblastoma cells

Inactive extra-chromosomal MYCN amplicons co-localize to the nuclear lamina in therapy-induced senescent neuroblastoma cells

Highlights from Recent Cancer Literature

Kleinman CL, Gerges N, Papillon-Cavanagh S, Sin-Chan P, Pramatarova A, Quang DA, et al. Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR. Nat Genet 2014;46:39–44.Kleinman and colleagues identify a recurrent fusion linking the promoter of a brain-specific gene, TTYH1, with the microRNA (miRNA) cluster C19MC in a rare childhood brain tumor, embryonal tumor with multilayered rosettes. Present in all 12 tumors analyzed, the fusion showed C19MC miRNA levels 150 to 1,000 times higher than in other supratentorial primitive neuroectodermal tumors. Based on seed sequence similarity between miRNAs in the C19MC and miR-290–295 cluster, analysis of 5,445 diverse tumor and normal samples revealed that C19MC miRNAs targeted retinoblastoma-like 2 (RBL2) transcripts, resulting in derepression of the de novo DNA methyltransferase DNMT3B and altered DNA methylation. These data identify abnormal regulation of a fetal neural developmental program as a potential tumorigenic mechanism.Handschick K, Beuerlein K, Jurida L, Bartkuhn M, Müller H, Soelch J, et al. Cyclin-dependent kinase 6 is a chromatin-bound cofactor for NF-κB-dependent gene expression. Mol Cell 2014;53:193–208.Handschick and colleagues demonstrate cell-cycle dysregulation as a driver of cytokine-triggered inflammatory gene expression in cancer. IL-1 or TNF-α stimulation induced a cell cycle–dependent increase in inflammatory gene expression, with knockdown of CDK6 decreasing IL-1–induced gene expression. Using ChIP-seq, they demonstrate that CDK6 could either stabilize NF-κB p65 (RELA) binding to DNA or regulate recruitment of p65 to the nucleus. CDK6 knockdown suppressed IL-1–induced p65 recruitment to the IL-8 promoter, decreased RNA-Pol II loading, and reduced IL8 mRNA expression. TAK1 (MAP3K7), an activator of MAPK and NF-κB, regulated cytokine-induced nuclear translocation and chromatin association of CDK6. These data link alterations between cell cycle and expression of inflammatory genes in cancer.Brastianos PK, Taylor-Weiner A, Manley PE, Jones RT, Dias-Santagata D5, Thorner AR, et al. Exome sequencing identifies BRAF mutations in papillary craniopharyngiomas. Nat Genet 2014;46:161–5.Craniopharyngiomas are termed “the most formidable of intracranial tumors” due to their suprasellar, surgically challenging location. Recurrent mutations in CTNBB1 drive adamantinomatous variants. To identify molecular mechanisms in more commonly occurring papillary variants, Brastianos and colleagues performed whole-exome sequencing (n = 15) followed by targeted genotyping (n = 95). Mutations in BRAF (3 of 3 papillary craniopharyngiomas in discovery set; 34 of 36 samples in validation set) were exclusive to papillary tumors and uniformly present throughout neoplastic epithelial cells, confirming clonality for these mutations. The high prevalence and mutually exclusive nature of mutations in CTNBB1 and BRAF in two prevalent subtypes of craniopharyngiomas support their roles as drivers, with BRAF inhibitors immediately relevant to patients.Cheung KJ, Gabrielson E, Werb Z, Ewald AJ. Collective invasion in breast cancer requires a conserved Basal epithelial program. Cell 2013;155:1639–51.Cheung and colleagues used organoid cultures to model collective invasion, mediated by a cohesive multicellular unit. Epithelial cells with a K14 (KRT14)/p63high basal phenotype led the invasion out of organoids and were enriched at metastatic sites in vivo. Besides the K14/p63 requirement, the authors indicate that collective invasion proceeded without epithelial-to-mesenchymal transition (EMT), suggesting that different subpopulations in a tumor adopt either EMT or K14/p63high programs for dissemination. K14/p63high cells may produce factors that recruit macrophages in vivo, as collagen-I fibrils, previously shown to promote dissemination, stimulated collective invasion. The results from these studies suggest that plastic interconversion between epithelial and mesenchymal states facilitates invasion. If K14/p63high tumor cells are required to maintain metastases, then targeting these subpopulations might prove efficacious.Shern JF, Chen L Chmielecki J, Wei JS, Patidar R, Rosenberg M, et al. Comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion-positive and fusion-negative tumors. Cancer Discov 2014;4:216–31.Rhabdomyosarcoma is the most common soft-tissue sarcoma of childhood. Alveolar rhabdomyosarcoma shows a common fusion between PAX3 or PAX7 and FOXO1 that is absent in embryonal rhabdomyosarcoma. These authors sequenced 147 tumors and found that tumors with PAX fusions (50/147) showed increased mutational load. Fusion-negative tumors showed increased aneuploidy and mutations in receptor tyrosine kinase and RAS/PIK3CA. LOH of 11p15 occurred in 50% of cases, with 12q and MYCN amplicons in fusion-positive cases. Mutations in FGFR signaling occurred in 88% of fusion-negative samples. A PAX fusion cDNA upregulated genes corresponding to fusion-negative tumors FGFR4, CCND2, and IGF2 and upregulated PAX3–FOXO1 targets. Rhabdomyosarcoma tumors thus utilize a common RTK/RAS/PIK3CA pathway driven via PAX fusion (alveolar tumors) or mutations that are downstream targets of the PAX fusion (embryonal tumors).Note: Breaking Advances are written by Cancer Research editors. Readers are encouraged to consult the articles referred to in each item for full details on the findings described.

Abstract 4107: Targeted re-sequencing of neuroblastoma tumors reveals chromosomal rearrangements that involve the Anaplastic Lymphoma Kinase (ALK) gene.

Abstract Neuroblastoma (NBL) is a cancer of early childhood arising from the developing sympathetic nervous system. NBL tumors display a broad clinical and biological heterogeneity, ranging from highly aggressive tumors with fatal outcome to tumors with spontaneous regression. Recurrent mutations are mainly only observed in Anaplastic Lymphoma Kinase (ALK), which is involved in the pathogenesis of both familiar and sporadic NBL. ALK encodes a tyrosine kinase receptor with importance in neuronal development and was initially characterized in anaplastic large cell lymphoma from a translocation leading to the NPM-ALK fusion protein. Subsequent studies show that additional ALK chimeras have been generated with different fusion partners in various cancers. To date no ALK-containing rearrangement has been reported in primary NBL tumors although one study has described a NBL cell line with an ALK intragenic deletion leading to constitutive ALK kinase activity. This led us to screen for and further investigate possible ALK containing translocations in our NBL material. Genomic profiling of 350 NBL samples though SNP microarrays indicated ALK-rearrangement in four primary tumors and two cell lines (IMR32 and CLB-BAR) which were further analyzed through targeted re-sequencing of the common MYCN amplicon region (Chr2;14-18Mb) and the entire ALK gene. Briefly, detection of rearrangements was performed through solution-based enrichment followed pair-end sequencing of 400-500bp libraries. Junction sites were identified by manual sequence viewing on IGV and validated through PCR followed by Sanger sequencing. With this strategy we were able to determine translocation sites for all but one sample. In one tumor ALK was found to be translocated to GAB2 (chr11q14) causing loss of the first exon of respective gene. However, ALK and GAB2 were placed head-to-head with respect to transcription direction and this novel translocation is therefore unlikely to cause a chimeric protein. Another tumor showed translocation between ALK intron 4 and chr 4 subtelomeric region without known genes. Two NBL cell lines and one tumor had both MYCN and ALK amplification and sequencing revealed high level complexity of respective amplicon with multiple rearrangements. IMR32 show involvement of at least six different 2p-regions including MYCN and ALK exon 3-4. CLB-BAR has also multiple rearrangements including an ALK intragenic translocation causing loss of exon 4-11, ultimately resulting in an ALK protein with truncated extracellular domain. Here we show that ALK translocation sites could be deduced from targeted re-sequencing without previous knowledge about fusion partner. However, no translocations resulting in chimeric protein were detected. We were also able to detect MYCN amplicon junctions, which are highly unique for each patient and could be used for detection of minimal residual disease. Citation Format: Susanne M. Fransson, Anna Djos, Niloufar Javanmardi, Rosa Noguera, Ana Berbegall, Magnus Hansson, Per Kogner, Kristina Ruuth, Ruth Palmer, Bengt Hallberg, Tommy Martinsson. Targeted re-sequencing of neuroblastoma tumors reveals chromosomal rearrangements that involve the Anaplastic Lymphoma Kinase (ALK) gene. [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 4107. doi:10.1158/1538-7445.AM2013-4107

Atlas of Cytogenomics in Oncology and Hematology: a Platform-Neutral Clinical Cancer Genomics Database

Array-based technology has been showing great impact in clinical cancer cytogenetics. This study was to test the feasibility of single nucleotide polymorphism (SNP) microarrays in the clinical diagnosis of pediatric cancers. A total of 74 cases, including 39 acute leukemias (27B-cell precursor acute lymphoblastic leukemia [BCPALL], 6 T-cell lymphoblastic leukemia, 5 acute myeloid leukemia, and 1 mix-phenotypic acute leukemia), 15 solid tumors, 5 myelodysplastic disorders, 6 non-malignant hematologic disorders, 5 lymphomas, 1 chronic myeloid leukemia cell line, as well as 3 normal remissionmarrows were tested using Illumina Cyto 12 and/ or 1M quad SNP arrays. Overall the SNP array data showed good concordance with cytogenetic/FISH data. SNP arrays revealed most cytogenetically unidentifiable abnormalities including copy neutral loss of heterozygosity (CN-LOH). In addition, SNP arrays provided prognostic information in 10 acute leukemia cases previously reported with normal cytogenetics and/or FISH and detected deletion of cancer genes (PAX5, MLL, PTEN and EVI1, etc.) at the exon level. Importantly, in 1 case with thrombocytopenia and a known constitutional RUNX1 gene deletion, SNP array revealed a low level (w20%)ofmosaicCN-LOH for chromosome21; and in2 BCP-ALL cases reported as hyperdiploid by chromosome /FISH analyses, SNP array detected CN-LOH in all disomic chromosomes, indicating that the hyperdiploidy in fact resulted from doubling of a hypodiploid clone. Finally, SNParray detected 3 novel amplificationsat 2p25.2 ina neuroblastomacaseoriginally reported with MYCN amplification and amplicons at 2p25.1wp24.3 encompassing 16 genes in a high-grade glioma originally reported with double minute chromosomes. In conclusion, SNP arrays could not only confirm most karyotypic/FISH data but could also detect additional genomic aberrations, some with significant clinical implications. This pilot study shows that SNP array has great potential as a diagnostic tool in pediatric cancer cytogenetics and can be integrated into routine clinical cancer cytogenetic testing.

Clinical Utility of Single Nucleotide Polymorphism (SNP) Microarrays in Pediatric Cancer and Non-Malignant Hematologic Disorders

Array-based technology has been showing great impact in clinical cancer cytogenetics. This study was to test the feasibility of single nucleotide polymorphism (SNP) microarrays in the clinical diagnosis of pediatric cancers. A total of 74 cases, including 39 acute leukemias (27 B-cell precursor acute lymphoblastic leukemia [BCP-ALL], 6 T-cell lymphoblastic leukemia, 5 acute myeloid leukemia, and 1 mix-phenotypic acute leukemia), 15 solid tumors, 5 myelodysplastic disorders, 6 non-malignant hematologic disorders, 5 lymphomas, 1 chronic myeloid leukemia cell line, as well as 3 normal remission marrows were tested using Illumina Cyto 12 and/or 1M quad SNP arrays. Overall the SNP array data showed good concordance with cytogenetic/FISH data. SNP arrays revealed most cytogenetically unidentifiable abnormalities including copy neutral loss of heterozygosity (CN-LOH). In addition, SNP arrays provided prognostic information in 10 acute leukemia cases previously reported with normal cytogenetics and/or FISH and detected deletion of cancer genes (PAX5, MLL, PTEN and EVI1, etc.) at the exon level. Importantly, in 1 case with thrombocytopenia and a known constitutional RUNX1 gene deletion, SNP array revealed a low level (∼20%) of mosaic CN-LOH for chromosome 21; and in 2 BCP-ALL cases reported as hyperdiploid by chromosome /FISH analyses, SNP array detected CN-LOH in all disomic chromosomes, indicating that the hyperdiploidy in fact resulted from doubling of a hypodiploid clone. Finally, SNP array detected 3 novel amplifications at 2p25.2 in a neuroblastoma case originally reported with MYCN amplification and amplicons at 2p25.1∼p24.3 encompassing 16 genes in a high-grade glioma originally reported with double minute chromosomes. In conclusion, SNP arrays could not only confirm most karyotypic/FISH data but could also detect additional genomic aberrations, some with significant clinical implications. This pilot study shows that SNP array has great potential as a diagnostic tool in pediatric cancer cytogenetics and can be integrated into routine clinical cancer cytogenetic testing.

A chemical screen identifies the chemotherapeutic drug topotecan as a specific inhibitor of the B-MYB/MYCN axis in neuroblastoma

The transcription factor MycN is the prototypical neuroblastoma oncogene and a potential therapeutic target. However, its strong expression caused by gene amplification in about 30% of neuroblastoma patients is a considerable obstacle to the development of therapeutic approaches aiming at eliminating its tumourigenic activity. We have previously reported that B-Myb is essentially required for transcription of the MYCN amplicon and have also shown that B-MYB and MYCN are engaged in a feed forward loop promoting the survival/proliferation of neuroblastoma cells. We postulated that pharmacological strategies breaking the B-MYB/MYCN axis should result in clinically desirable effects. Thus, we implemented a high throughput chemical screen, using a curated library of ~1500 compounds from the National Cancer Institute, whose endpoint was the identification of small molecules that inhibited B-Myb. At the end of the screening, we found that the compounds pinafide, ellipticine and camptothecin inhibited B-Myb transcriptional activity in luciferase assays. One of the compounds, the topoisomerase-1 inhibitor camptothecin, is of considerable clinical interest since its derivatives topotecan and irinotecan are currently used as first and second line treatment agents for various types of cancer, including neuroblastoma. We found that neuroblastoma cells with amplification of MYCN are more sensitive than MYCN negative cells to camptothecin and topotecan killing. Campothecin and topotecan caused selective down-regulation of B-Myb and MycN expression in neuroblastoma cells. Notably, forced overexpression of B-Myb could antagonize the killing effect of topotecan and camptothecin, demonstrating that the transcription factor is a key target of the drugs. These results suggest that camptothecin and its analogues should be more effective in patients whose tumours feature amplification of MYCN and/or overexpression of B-MYB.

Outcome of the p53-mediated DNA damage response in neuroblastoma is determined by morphological subtype and MYCN expression

Background: MYCN oncogene amplification occurs in 20-25% of neuroblastoma and is associated with a poor prognosis. We previously reported that MYCN amplified (MNA) p53 wild-type neuroblastoma cell lines failed to G1 arrest in response to irradiation, but this could not be attributed to MYCN alone. Hypothesis: Genes co-amplified with MYCN and/or the predominant cell type, neuronal (N) or substrate adherent (S) phenotypes determine the downstream response to DNA damage in neuroblastoma cell lines. Methods: The MYCN amplicons of five MNA and two non-MNA cell line were mapped using 50K Single Nucleotide Polymorphism (SNP) arrays. One MNA (NBL-W) and one non-MNA neuroblastoma cell line (SKNSH) were sub-cloned into N and S-type cells and the p53 pathway investigated after irradiation induced DNA damage. To determine the role of p53 it was knocked down using siRNA. Results: No genes with a potential role in cell cycle regulation were consistently co-amplified in the MNA cell lines studied. High MYCN expressing NBLW-N cells failed to G1 arrest following irradiation and showed impaired induction of p21 and MDM2, whereas low MYCN expressing NBLW-S cells underwent a G1 arrest with induction of p21 and MDM2. Conversely N type cells underwent higher levels of apoptosis than S type cells. Following p53 knockdown in SHSY5Y N-type cells there was a decrease in apoptosis. Conclusions: The downstream response to DNA damage in p53 wild-type neuroblastoma cell lines is p53 dependent, and determined both by the morphological sub-type and MYCN expression.

MYCN amplicon junctions as tumor-specific targets for minimal residual disease detection in neuroblastoma

The MYCN gene is frequently amplified in unfavorable neuroblastoma tumors. Therefore, this study aimed at characterizing the novel junctions connecting the amplified DNA segments (amplicons) and obtaining tumor-specific PCR fragments for use in detecting minimal residual disease (MRD). High-density SNP arrays were used to map the end-points of the MYCN amplicons in a subset of neuroblastoma tumors. Primers were designed to give rise to a tumor-specific PCR product and were examined for MRD in the blood and bone marrow using quantitative PCR. Tumor-specific junction fragments were detected in all cases, confirming a head-to-tail tandem orientation of the amplicons and revealing microhomology at the amplicon junctions, thus suggesting a rolling circle caused by microhomology-mediated break-induced replication (MMBIR) as a possible mechanism initiating the MYCN amplification. We also evaluated the use of these junctions as tumor-specific targets for detecting MRD and observed that tumor DNA could be readily detected and quantified in either blood or bone marrow at a sensitivity of 1/106 tumor/control DNA. This study provides new information on the mechanisms of oncogene amplification and envisages means of rapidly obtaining highly sensitive PCR-based tools for tumor/patient-specific monitoring of treatment response and the early detection of relapse in patients with neuroblastoma.

Integrated Cytogenetic and High-Resolution Array CGH Analysis of Genomic Alterations Associated with MYCN Amplification

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.

The FRA2C common fragile site maps to the borders of MYCN amplicons in neuroblastoma and is associated with gross chromosomal rearrangements in different cancers

Common fragile sites (cFS) represent chromosomal regions that are prone to breakage after partial inhibition of DNA synthesis. Activation of cFS is associated with various forms of DNA instability in cancer cells, and is thought to be an initiating event in the generation of DNA damage in early-stage tumorigenesis. Only a few cFS have been fully characterized despite the growing interest in cFS instability in cancer genomes. In this study, six-color fluorescence in situ hybridization revealed that FRA2C consists of two cFS spanning 747 kb FRA2Ctel and 746 kb FRA2Ccen at 2p24.3 and 2p24.2, respectively. Both cFS are separated by a 2.8 Mb non-fragile region containing MYCN. Fine-tiling array comparative genomic hybridization of MYCN amplicons from neuroblastoma (NB) cell lines and primary tumors revealed that 56.5% of the amplicons cluster in FRA2C. MYCN amplicons are either organized as double minutes or as homogeneously stained regions in addition to the single copy of MYCN retained at 2p24. We suggest that MYCN amplicons arise from extra replication rounds of unbroken DNA secondary structures that accumulate at FRA2C. This hypothesis implicates cFS in high-level gene amplification in cancer cells. Complex genomic rearrangements, including deletions, duplications and translocations, which originate from double-strand breaks, were detected at FRA2C in different cancers. These data propose a dual role for cFS in the generation of gross chromosomal rearrangements either after DNA breakage or by inducing extra replication rounds, and provide new insights into the highly recombinogenic nature of cFS in the human cancer genome.

Addiction to B-MYB.

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.

Addiction of MYCN amplified tumours to B-MYB underscores a reciprocal regulatory loop.

MYCN is a member of the MYC family of oncoproteins frequently amplified or overexpressed in aggressive, paediatric tumours of the nervous system. In this study we have identified the gene B-MYB, encoding the transcription factor also known as MYBL2, as a downstream target of MYCN. Using multiple in silico databases we show that expression of B-MYB significantly correlates with that of MYCN in neuroblastoma patients. MYCN binds to and activates the B-MYB gene in vivo and in vitro. Blunting B-MYB expression by RNA interference causes reduced proliferation of MYCN amplified, but not MYCN-non amplified, neuroblastoma cell lines, indicating that tumour cells are addicted to B-MYB in a MYCN dependent manner. Notably, B-MYB binds in vivo to the MYCN amplicon and is required for its expression. We conclude that MYCN and B-MYB are engaged in a reciprocal regulatory loop whose pharmacological targeting could be beneficial to patients with the aggressive forms of cancer in which MYCN is amplified.

Addiction of MYCN amplified tumours to B-MYB underscores a reciprocal regulatory loop.

MYCN is a member of the MYC family of oncoproteins frequently amplified or overexpressed in aggressive, paediatric tumours of the nervous system. In this study we have identified the gene B-MYB, encoding the transcription factor also known as MYBL2, as a downstream target of MYCN. Using multiple in silico databases we show that expression of B-MYB significantly correlates with that of MYCN in neuroblastoma patients. MYCN binds to and activates the B-MYB gene in vivo and in vitro. Blunting B-MYB expression by RNA interference causes reduced proliferation of MYCN amplified, but not MYCN-non amplified, neuroblastoma cell lines, indicating that tumour cells are addicted to B-MYB in a MYCN dependent manner. Notably, B-MYB binds in vivo to the MYCN amplicon and is required for its expression. We conclude that MYCN and B-MYB are engaged in a reciprocal regulatory loop whose pharmacological targeting could be beneficial to patients with the aggressive forms of cancer in which MYCN is amplified.

Genes Proximal and Distal to MYCN Are Highly Expressed in Human Neuroblastoma as Visualized by Comparative Expressed Sequence Hybridization

MYCN amplification is associated with poor prognosis in neuroblastoma disease. To improve our understanding of the influence of the MYCN amplicon and its corresponding expression, we investigated the 2p expression pattern of MYCN amplified (n = 13) and nonamplified (n = 4) cell lines and corresponding primary tumors (n = 3) using the comparative expressed sequence hybridization technique. All but one MYCN amplified cell line displayed overexpression at 2p. Expression peaks were observed frequently at 2pter and less frequently at 2p24 (MYCN locus), 2p23.3-23.2, and/or 2p23.1. Importantly, cell lines and two corresponding primary tumors displayed expression peaks at similar loci. No significant 2p24 expression level was observed for those cell lines displaying a low amplification rate (n = 3) by comparative genomic hybridization. Only the cell lines with an enhanced peak at 2p23.2-23.3 displayed coamplification of the ALK gene (2p23.2), reported to be associated with unfavorable prognosis. Finally, two of four cell lines without MYCN amplification, both derived from patients with poor outcome, also showed an expression peak at 2p23.2. These data indicate that, besides MYCN, other genes proximal and distal to MYCN are highly expressed in neuroblastoma. The prognostic significance of expression peaks at 2p23.2-23.3, independent of MYCN and ALK status, remains to be investigated.

Identification and characterization of DNA imbalances in neuroblastoma by high-resolution oligonucleotide array comparative genomic hybridization

Neuroblastoma (NB) is a pediatric tumor characterized by high genetic heterogeneity. Although the prognostic significance of some genomic abnormalities (i.e., MYCN amplification, 1p loss, and 17q gain) is recognized, genes that are involved in chromosome rearrangements remain largely unknown. Considerable progress has been made over the last years in characterizing DNA abnormalities by metaphase comparative genomic hybridization (mCGH) and array CGH (aCGH). Here we report a pilot study of 5 localized and 12 disseminated NB by 44,000 and of 4 out of 17 cases by 244,000 oligonucleotide aCGH. Localized tumors were predominantly characterized by losses of whole chromosomes 3, 4, 10, and 16, and gains of 6, 7, 8, 13, 17, 18, and 20, whereas disseminated tumors showed several structural aberrations including 17q gain and 3p and 11q losses. Characterization of chromosome 2p in MYCN-amplified NB revealed several structural rearrangements with regions of gain interspersed among sites of amplification, indicating that the MYCN amplicon may encompass several genes. The high-resolution zooming in chromosomal aberrant regions detected several micro-deletions and micro-amplifications in the NB genome. Our results indicate that the increased sensitivity of the aCGH also allows the identification of DNA aberrations in challenging samples (i.e., NB showing tissue and genetic heterogeneity).