ABSTRACT
The development of targeted therapies offers new hope for patients affected by incurable cancer. However, multiple challenges persist, notably in controlling tumor cell plasticity in patients with refractory and metastatic illness. Neuroblastoma (NB) is an aggressive pediatric malignancy originating from defective differentiation of neural crest-derived progenitors with oncogenic activity due to genetic and epigenetic alterations and remains a clinical challenge for high-risk patients. To identify critical genes driving NB aggressiveness, we performed combined chromatin and transcriptome analyses on matched patient-derived xenografts (PDXs), spheroids, and differentiated adherent cultures derived from metastatic MYCN nonamplified tumors. Bone marrow kinase on chromosome X (BMX) was identified among the most differentially regulated genes in PDXs and spheroids versus adherent models. BMX expression correlated with high tumor stage and poor patient survival and was crucial to the maintenance of the self-renewal and tumorigenic potential of NB spheroids. Moreover, BMX expression positively correlated with the mesenchymal NB cell phenotype, previously associated with increased chemoresistance. Finally, BMX inhibitors readily reversed this cellular state, increased the sensitivity of NB spheroids toward chemotherapy, and partially reduced tumor growth in a preclinical NB model. Altogether, our study identifies BMX as a promising innovative therapeutic target for patients with high-risk MYCN nonamplified NB.
Subject(s)
N-Myc Proto-Oncogene Protein , Neuroblastoma , Spheroids, Cellular , Neuroblastoma/genetics , Neuroblastoma/pathology , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Humans , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Animals , Spheroids, Cellular/pathology , Spheroids, Cellular/metabolism , Spheroids, Cellular/drug effects , Mice , Xenograft Model Antitumor Assays , Cell Line, Tumor , Gene Expression Regulation, NeoplasticABSTRACT
Structural variation heavily influences the molecular landscape of cancer, in part by impacting DNA methylation-mediated transcriptional regulation. Here, using multi-omic datasets involving >2400 pediatric brain and central nervous system tumors of diverse histologies from the Children's Brain Tumor Network, we report hundreds of genes and associated CpG islands (CGIs) for which the nearby presence of somatic structural variant (SV) breakpoints is recurrently associated with altered expression or DNA methylation, respectively, including tumor suppressor genes ATRX and CDKN2A. Altered DNA methylation near enhancers associates with nearby somatic SV breakpoints, including MYC and MYCN. A subset of genes with SV-CGI methylation associations also have expression associations with patient survival, including BCOR, TERT, RCOR2, and PDLIM4. DNA methylation changes in recurrent or progressive tumors compared to the initial tumor within the same patient can predict survival in pediatric and adult cancers. Our comprehensive and pan-histology genomic analyses reveal mechanisms of noncoding alterations impacting cancer genes.
Subject(s)
Brain Neoplasms , CpG Islands , DNA Methylation , Gene Expression Regulation, Neoplastic , Humans , DNA Methylation/genetics , Brain Neoplasms/genetics , Brain Neoplasms/mortality , Brain Neoplasms/pathology , CpG Islands/genetics , Child , X-linked Nuclear Protein/genetics , X-linked Nuclear Protein/metabolism , Epigenome , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Male , Telomerase/genetics , FemaleABSTRACT
N-Myc is a key driver of neuroblastoma and neuroendocrine prostate cancer (NEPC). One potential way to circumvent the challenge of undruggable N-Myc is to target the protein homeostasis (proteostasis) system that maintains N-Myc levels. Here, we identify heat shock protein 70 (HSP70) as a top partner of N-Myc, which binds a conserved "SELILKR" motif and prevents the access of E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), possibly through steric hindrance. When HSP70's dwell time on N-Myc is increased by treatment with the HSP70 allosteric inhibitor, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites and forms polyubiquitination chains linked by the K11 and K63 sites. Notably, HSP70 inhibition significantly suppressed NEPC tumor growth, increased the efficacy of aurora kinase A (AURKA) inhibitors, and limited the expression of neuroendocrine-related pathways.
Subject(s)
HSP70 Heat-Shock Proteins , Prostatic Neoplasms , Proteostasis , Ubiquitin-Protein Ligases , Ubiquitination , Male , Humans , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Prostatic Neoplasms/genetics , HSP70 Heat-Shock Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitination/drug effects , Cell Line, Tumor , Animals , Aurora Kinase A/metabolism , Aurora Kinase A/genetics , Aurora Kinase A/antagonists & inhibitors , N-Myc Proto-Oncogene Protein/metabolism , N-Myc Proto-Oncogene Protein/genetics , Mice , Carcinoma, Neuroendocrine/metabolism , Carcinoma, Neuroendocrine/genetics , Carcinoma, Neuroendocrine/drug therapy , Carcinoma, Neuroendocrine/pathology , Neuroendocrine Tumors/metabolism , Neuroendocrine Tumors/drug therapy , Neuroendocrine Tumors/genetics , Neuroendocrine Tumors/pathologyABSTRACT
Neuroblastoma is the most common extracranial solid tumor in children. Amplification of the MYCN gene has been observed in approximately 20%-30% of tumors. It is strongly correlated with advanced-stage disease, rapid tumor progression, resistance to chemotherapy and poor outcomes independent of patient age and stage of advanced disease. MYCN amplification identifies high-risk patients. To assess neuroblastoma tumors with MYCN amplification we used paraffin-embedded tissue sections in 57 patients and intraoperative tumor imprints in 10 patients by fluorescence in situ hybridization (FISH). Positive results for MYCN amplification have been observed in twelve patients' paraffin-embedded tissue sections and in three patients' intraoperative tumor imprints, which represents 22.4% of all patients tested in the analysis. Fluorescence in situ hybridization is a highly sensitive and useful technique for detecting MYCN amplification on paraffin-embedded tissue sections of neuroblastoma tumors and intraoperative tumor imprints thus facilitating therapeutic decisions based on the presence or absence of this important biologic marker. The presence of structural changes, regardless of MYCN gene amplification status, influences the clinical behavior of neuroblastoma. High-Density SNP Arrays have emerged as the perfect tools for detecting these changes due to their exceptional accuracy, sensitivity and ability to analyze copy number and allele information. Consequently, they are proven to be highly valuable in the genomic diagnosis of immature neuroectodermal tumors.
Subject(s)
Gene Amplification , In Situ Hybridization, Fluorescence , N-Myc Proto-Oncogene Protein , Neuroblastoma , Polymorphism, Single Nucleotide , Humans , Neuroblastoma/genetics , Neuroblastoma/pathology , In Situ Hybridization, Fluorescence/methods , N-Myc Proto-Oncogene Protein/genetics , Child, Preschool , Child , Infant , Female , MaleABSTRACT
Retinoblastoma are childhood eye tumors arising from retinal precursor cells. Two distinct retinoblastoma subtypes with different clinical behavior have been described based on gene expression and methylation profiling. Using consensus clustering of DNA methylation analysis from 61 retinoblastomas, we identify a MYCN-driven cluster of subtype 2 retinoblastomas characterized by DNA hypomethylation and high expression of genes involved in protein synthesis. Subtype 2 retinoblastomas outside the MYCN-driven cluster are characterized by high expression of genes from mesodermal development, including NKX2-5. Knockdown of MYCN expression in retinoblastoma cell models causes growth arrest and reactivates a subtype 1-specific photoreceptor signature. These molecular changes suggest that removing the driving force of MYCN oncogenic activity rescues molecular circuitry driving subtype 1 biology. The MYCN-RB gene signature generated from the cell models better identifies MYCN-driven retinoblastoma than MYCN amplification and can identify cases that may benefit from MYCN-targeted therapy. MYCN drives tumor progression in a molecularly defined retinoblastoma subgroup, and inhibiting MYCN activity could restore a more differentiated and less aggressive tumor biology.
Subject(s)
N-Myc Proto-Oncogene Protein , Retinoblastoma , Humans , Retinoblastoma/genetics , Retinoblastoma/pathology , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , DNA Methylation , Retinal Neoplasms/genetics , Retinal Neoplasms/pathology , Retinal Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Cell Line, Tumor , Cell Dedifferentiation/genetics , Female , Male , Child, PreschoolABSTRACT
Neuroblastoma (NB) is a heterogeneous childhood cancer with a slightly higher incidence in boys than girls, with the reason for this gender disparity unknown. Given the growing evidence for the involvement of loss of the Y chromosome (LoY) in male diseases including cancer, we investigated Y chromosome status in NB. Male NB tumor samples from a Swedish cohort, analyzed using Cytoscan HD SNP-microarray, were selected. Seventy NB tumors were analyzed for aneuploidy of the Y chromosome, and these data were correlated with other genetic, biological, and clinical parameters. LoY was found in 21% of the male NB tumors and it was almost exclusively found in those with high-risk genomic profiles. Furthermore, LoY was associated with increased age at diagnosis and enriched in tumors with 11q-deletion and activated telomere maintenance mechanisms. In contrast, tumors with an MYCN-amplified genomic profile retained their Y chromosome. The understanding of LoY in cancer is limited, making it difficult to conclude whether LoY is a driving event in NB or function of increased genomic instability. Gene expression analysis of Y chromosome genes in male NB tumors showed low expression of certain genes correlating with worse overall survival. KDM5D, encoding a histone demethylase stands out as an interesting candidate for further studies. LoY has been shown to impact the epigenomic layer of autosomal loci in nonreproductive tissues, and KDM5D has been reported as downregulated and/or associated with poor survival in different malignancies. Further studies are needed to explore the mechanisms and functional consequences of LoY in NB.
Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 11 , Chromosomes, Human, Y , Neuroblastoma , Humans , Neuroblastoma/genetics , Neuroblastoma/pathology , Male , Chromosomes, Human, Y/genetics , Chromosomes, Human, Pair 11/genetics , Infant , Child, Preschool , Female , Telomere Homeostasis/genetics , Child , Histone Demethylases/genetics , Telomere/genetics , N-Myc Proto-Oncogene Protein/genetics , Sweden/epidemiologyABSTRACT
Although MYCN has been considered an undruggable target, MYCN alterations confer poor prognosis in many pediatric and adult cancers. The novel MYCN-speciï¬c inhibitor BGA002 is an antigene peptide nucleic acid oligonucleotide covalently bound to a nuclear localization signal peptide. In the present study, we characterized the pharmacokinetics (PK) of BGA002 after single and repeated administration to mice using a novel specific enzyme-linked immunosorbent assay. BGA002 concentrations in plasma showed linear PK, with dose proportional increase across the tested dose levels and similar exposure between male and female and between intravenous and subcutaneous route of administration. Repeated dosing resulted in no accumulation in plasma. Biodistribution up to 7 days after single subcutaneous administration of [14C]-radiolabeled BGA002 showed broad tissues and organ distribution (suggesting a potential capability to reach primary tumor and metastasis in several body sites), with high concentrations in kidney, liver, spleen, lymph nodes, adrenals, and bone marrow. Remarkably, we demonstrated that BGA002 concentrates in tumors after repeated systemic administrations in three mouse models with MYCN amplification (neuroblastoma, rhabdomyosarcoma, and small-cell lung cancer), leading to a significant reduction in tumor weight. Taking into account the available safety profile of BGA002, these data support further evaluation of BGA002 in patients with MYCN-positive tumors.
Subject(s)
N-Myc Proto-Oncogene Protein , Neuroblastoma , Peptide Nucleic Acids , Animals , Mice , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/antagonists & inhibitors , Female , Humans , Male , Peptide Nucleic Acids/pharmacokinetics , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/administration & dosage , Peptide Nucleic Acids/genetics , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Neuroblastoma/genetics , Tissue Distribution , Cell Line, Tumor , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/drug therapy , Rhabdomyosarcoma/pathology , Nuclear Proteins/genetics , Nuclear Proteins/antagonists & inhibitors , Organic ChemicalsABSTRACT
MYCN oncogene amplification is frequently observed in aggressive childhood neuroblastoma. Using an unbiased large-scale mutagenesis screen in neuroblastoma-prone transgenic mice, we identify a single germline point mutation in the transcriptional corepressor Runx1t1, which abolishes MYCN-driven tumorigenesis. This loss-of-function mutation disrupts a highly conserved zinc finger domain within Runx1t1. Deletion of one Runx1t1 allele in an independent Runx1t1 knockout mouse model is also sufficient to prevent MYCN-driven neuroblastoma development, and reverse ganglia hyperplasia, a known pre-requisite for tumorigenesis. Silencing RUNX1T1 in human neuroblastoma cells decreases colony formation in vitro, and inhibits tumor growth in vivo. Moreover, RUNX1T1 knockdown inhibits the viability of PAX3-FOXO1 fusion-driven rhabdomyosarcoma and MYC-driven small cell lung cancer cells. Despite the role of Runx1t1 in MYCN-driven tumorigenesis neither gene directly regulates the other. We show RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex recruited by HAND2 to enhancer regions to regulate chromatin accessibility and cell-fate pathway genes.
Subject(s)
Carcinogenesis , N-Myc Proto-Oncogene Protein , Neuroblastoma , Animals , Humans , Mice , Carcinogenesis/genetics , Cell Line, Tumor , Co-Repressor Proteins/metabolism , Co-Repressor Proteins/genetics , Gene Expression Regulation, Neoplastic , Histone Demethylases/metabolism , Histone Demethylases/genetics , Mice, Knockout , Mice, Transgenic , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/genetics , Neuroblastoma/metabolism , Neuroblastoma/pathology , Transcription Factors/metabolism , Transcription Factors/geneticsABSTRACT
Retinoblastoma (RB) is a pediatric malignancy, typically diagnosed at birth or during early childhood. The pathogenesis of RB is marked by the amplification of the Basic Helix-Loop-Helix (BHLH) Transcription Factor MYCN, which serves as a transcriptional regulator capable of binding to Dickkopf 3 (DKK3). However, the precise role of DKK3 in the malignant progression of RB cells caused by MYCN remains elusive. In the present study, the expression of MYCN was either overexpressed or interfered in RB cells. Subsequently, the expression level of DKK3 was assessed through quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation was evaluated using the Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine staining, while cell cycle progression and apoptosis were analyzed by flow cytometry and western blot analysis, respectively. Additionally, the expression of proteins involved in the Wnt/ß-catenin/Fra-1/p53 signaling pathway was evaluated via western blot analysis. To gain further insights, Wnt agonists and the P53 inhibitor PFT-α were introduced into exploration. The current investigation revealed a negative correlation between the expression levels of MYCN and DKK3 in RB cells. Additionally, DKK3 overexpression inhibited cell proliferation, promoted cell apoptosis, and arrested cell cycle in RB cells with high expression of MYCN. Moreover, enhanced DKK3 expression inhibited proliferation, promoted cell cycle arrest and apoptosis of RB cells by modulating the wnt/ßcatenin/Fra-1/p53 signaling pathway. Furthermore, in vivo experiments revealed that overexpression of DKK3 inhibits the growth of RB tumors. Collectively, our findings elucidate that MYCN stimulates the Wnt/ß-catenin/Fra-1 pathway by suppressing DKK3 expression, ultimately suppressing p53 activity and contributing to malignant progression of RB.
Subject(s)
Adaptor Proteins, Signal Transducing , Cell Proliferation , N-Myc Proto-Oncogene Protein , Retinoblastoma , Tumor Suppressor Protein p53 , Wnt Signaling Pathway , Humans , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Retinoblastoma/metabolism , Retinoblastoma/genetics , Retinoblastoma/pathology , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cell Line, Tumor , Animals , Mice , Apoptosis , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Mice, Nude , beta Catenin/metabolismABSTRACT
Acquiring a telomere maintenance mechanism is a hallmark of high-risk neuroblastoma and commonly occurs by expressing telomerase (TERT). Telomerase-negative neuroblastoma has long telomeres and utilizes the telomerase-independent alternative lengthening of telomeres (ALT) mechanism. Conversely, no discernable telomere maintenance mechanism is detected in a fraction of neuroblastoma with long telomeres. Here, we show, unlike most cancers, DNA of the TERT promoter is broadly hypomethylated in neuroblastoma. In telomerase-positive neuroblastoma cells, the hypomethylated DNA promoter is approximately 1.5 kb. The TERT locus shows active chromatin marks with low enrichment for the repressive mark, H3K27me3. MYCN, a commonly amplified oncogene in neuroblstoma, binds to the promoter and induces TERT expression. Strikingly, in neuroblastoma with long telomeres, the hypomethylated region spans the entire TERT locus, including multiple nearby genes with enrichment for the repressive H3K27me3 chromatin mark. Furthermore, subtelomeric regions showed enrichment of repressive chromatin marks in neuroblastomas with long telomeres relative to those with short telomeres. These repressive marks were even more evident at the genic loci, suggesting a telomere position effect (TPE). Inhibiting H3K27 methylation by three different EZH2 inhibitors induced the expression of TERT in cell lines with long telomeres and H3K27me3 marks in the promoter region. EZH2 inhibition facilitated MYCN binding to the TERT promoter in neuroblastoma cells with long telomeres. Taken together, these data suggest that epigenetic regulation of TERT expression differs in neuroblastoma depending on the telomere maintenance status, and H3K27 methylation is important in repressing TERT expression in neuroblastoma with long telomeres. SIGNIFICANCE: The epigenetic landscape of the TERT locus is unique in neuroblastoma. The DNA at the TERT locus, unlike other cancer cells and similar to normal cells, are hypomethylated in telomerase-positive neuroblastoma cells. The TERT locus is repressed by polycomb repressive complex-2 complex in neuroblastoma cells that have long telomeres and do not express TERT. Long telomeres in neuroblastoma cells are also associated with repressive chromatin states at the chromosomal termini, suggesting TPE.
Subject(s)
Neuroblastoma , Promoter Regions, Genetic , Telomerase , Telomere , Neuroblastoma/genetics , Neuroblastoma/pathology , Neuroblastoma/metabolism , Telomerase/genetics , Telomerase/metabolism , Humans , Promoter Regions, Genetic/genetics , Telomere/metabolism , Telomere/genetics , Cell Line, Tumor , DNA Methylation/genetics , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Gene Expression Regulation, Neoplastic , Polycomb-Group Proteins/genetics , Polycomb-Group Proteins/metabolismABSTRACT
Neuroblastoma is the most common solid extracranial tumour in children. Despite major advances in available therapies, children with drug-resistant and/or recurrent neuroblastoma have a dismal outlook with 5-year survival rates of less than 20%. Therefore, tackling relapsed tumour biology by developing and characterising clinically relevant models is a priority in finding targetable vulnerability in neuroblastoma. Using matched cisplatin-sensitive KellyLuc and resistant KellyCis83Luc cell lines, we developed a cisplatin-resistant metastatic MYCN-amplified neuroblastoma model. The average number of metastases per mouse was significantly higher in the KellyCis83Luc group than in the KellyLuc group. The vast majority of sites were confirmed as having lymph node metastasis. Their stiffness characteristics of lymph node metastasis values were within the range reported for the patient samples. Targeted transcriptomic profiling of immuno-oncology genes identified tumour necrosis factor receptor superfamily member 4 (TNFRSF4) as a significantly dysregulated MYCN-independent gene. Importantly, differential TNFRSF4 expression was identified in tumour cells rather than lymphocytes. Low TNFRSF4 expression correlated with poor prognostic indicators in neuroblastoma, such as age at diagnosis, stage, and risk stratification and significantly associated with reduced probability of both event-free and overall survival in neuroblastoma. Therefore, TNFRSF4 Low expression is an independent prognostic factor of survival in neuroblastoma.
Subject(s)
Cisplatin , Drug Resistance, Neoplasm , Neuroblastoma , Neuroblastoma/genetics , Neuroblastoma/pathology , Neuroblastoma/drug therapy , Neuroblastoma/mortality , Neuroblastoma/metabolism , Humans , Drug Resistance, Neoplasm/genetics , Animals , Cisplatin/therapeutic use , Cisplatin/pharmacology , Mice , Cell Line, Tumor , Prognosis , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Gene Expression Regulation, Neoplastic , Female , Lymphatic MetastasisABSTRACT
The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.
Subject(s)
N-Myc Proto-Oncogene Protein , Nuclear Proteins , Oncogene Proteins , RNA-Binding Proteins , N-Myc Proto-Oncogene Protein/metabolism , N-Myc Proto-Oncogene Protein/genetics , Humans , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Oncogene Proteins/metabolism , Oncogene Proteins/genetics , Promoter Regions, Genetic , Cell Line, Tumor , Neuroblastoma/metabolism , Neuroblastoma/genetics , Neuroblastoma/pathology , Exosomes/metabolism , Exosomes/genetics , Introns , Protein Binding , Cell Nucleus/metabolism , Exosome Multienzyme Ribonuclease Complex/metabolism , Exosome Multienzyme Ribonuclease Complex/genetics , Gene Expression Regulation, Neoplastic , RNA/metabolism , RNA/genetics , Repressor Proteins/metabolism , Repressor Proteins/genetics , Cell ProliferationABSTRACT
Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.
Subject(s)
Cell Differentiation , DNA Copy Number Variations , N-Myc Proto-Oncogene Protein , Neural Crest , Neuroblastoma , Humans , Neuroblastoma/genetics , Neuroblastoma/pathology , Neural Crest/metabolism , Neural Crest/pathology , Female , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Chromosome Aberrations , Human Embryonic Stem Cells/metabolism , Transcriptome , Cell Line, Tumor , Gene Expression Regulation, NeoplasticABSTRACT
Pediatric cancers are frequently driven by genomic alterations that result in aberrant transcription factor activity. Here, we used functional genomic screens to identify multiple genes within the transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex as selective dependencies for MYCN-amplified neuroblastoma, a disease of dysregulated development driven by an aberrant oncogenic transcriptional program. We characterized the DNA recruitment sites of the SAGA complex in neuroblastoma and the consequences of loss of SAGA complex lysine acetyltransferase (KAT) activity on histone acetylation and gene expression. We demonstrate that loss of SAGA complex KAT activity is associated with reduced MYCN binding on chromatin, suppression of MYC/MYCN gene expression programs, and impaired cell cycle progression. Further, we showed that the SAGA complex is pharmacologically targetable in vitro and in vivo with a KAT2A/KAT2B proteolysis targeting chimeric. Our findings expand our understanding of the histone-modifying complexes that maintain the oncogenic transcriptional state in this disease and suggest therapeutic potential for inhibitors of SAGA KAT activity in MYCN-amplified neuroblastoma.
Subject(s)
Gene Expression Regulation, Neoplastic , N-Myc Proto-Oncogene Protein , Neuroblastoma , Neuroblastoma/genetics , Neuroblastoma/metabolism , Neuroblastoma/pathology , Humans , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Cell Line, Tumor , Histone Acetyltransferases/metabolism , Histone Acetyltransferases/genetics , Acetylation , Histones/metabolism , Animals , Gene Amplification , Chromatin/metabolism , Chromatin/genetics , MiceABSTRACT
BACKGROUND: Neuroblastoma (NB) patients with amplified MYCN often face a grim prognosis and are resistant to existing therapies, yet MYCN protein is considered undruggable. KAP1 (also named TRIM28) plays a crucial role in multiple biological activities. This study aimed to investigate the relationship between KAP1 and MYCN in NB. METHODS: Transcriptome analyses and luciferase reporter assay identified that KAP1 was a downstream target of MYCN. The effects of KAP1 on cancer cell proliferation and colony formation were explored using the loss-of-function assays in vitro and in vivo. RNA stability detection was used to examine the influence of KAP1 on MYCN expression. The mechanisms of KAP1 to maintain MYCN mRNA stabilization were mainly investigated by mass spectrum, immunoprecipitation, RIP-qPCR, and western blotting. In addition, a xenograft mouse model was used to reveal the antitumor effect of STM2457 on NB. RESULTS: Here we identified KAP1 as a critical regulator of MYCN mRNA stability by protecting the RNA N6-methyladenosine (m6A) reader YTHDC1 protein degradation. KAP1 was highly expressed in clinical MYCN-amplified NB and was upregulated by MYCN. Reciprocally, KAP1 knockdown reduced MYCN mRNA stability and inhibited MYCN-amplified NB progression. Mechanistically, KAP1 regulated the stability of MYCN mRNA in an m6A-dependent manner. KAP1 formed a complex with YTHDC1 and RNA m6A writer METTL3 to regulate m6A-modified MYCN mRNA stability. KAP1 depletion decreased YTHDC1 protein stability and promoted MYCN mRNA degradation. Inhibiting MYCN mRNA m6A modification synergized with chemotherapy to restrain tumor progression in MYCN-amplified NB. CONCLUSIONS: Our research demonstrates that KAP1, transcriptionally activated by MYCN, forms a complex with YTHDC1 and METTL3, which in turn maintain the stabilization of MYCN mRNA in an m6A-dependent manner. Targeting m6A modification by STM2457, a small-molecule inhibitor of METTL3, could downregulate MYCN expression and attenuate tumor proliferation. This finding provides a new alternative putative therapeutic strategy for MYCN-amplified NB.
Subject(s)
N-Myc Proto-Oncogene Protein , Neuroblastoma , Tripartite Motif-Containing Protein 28 , Animals , Humans , Mice , Adenosine/analogs & derivatives , Adenosine/metabolism , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Mice, Nude , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/genetics , Neuroblastoma/metabolism , Neuroblastoma/pathology , RNA Splicing Factors/metabolism , RNA Splicing Factors/genetics , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tripartite Motif-Containing Protein 28/metabolism , Tripartite Motif-Containing Protein 28/geneticsABSTRACT
High-grade serous ovarian cancer (HGSC) is a challenging disease, especially for patients with immunologically "cold" tumors devoid of tumor-infiltrating lymphocytes (TILs). We found that HGSC exhibits among the highest levels of MYCN expression and transcriptional signature across human cancers, which is strongly linked to diminished features of antitumor immunity. N-MYC repressed basal and induced IFN type I signaling in HGSC cell lines, leading to decreased chemokine expression and T cell chemoattraction. N-MYC inhibited the induction of IFN type I by suppressing tumor cell-intrinsic STING signaling via reduced STING oligomerization, and by blunting RIG-I-like receptor signaling through inhibition of MAVS aggregation and localization in the mitochondria. Single-cell RNA sequencing of human clinical HGSC samples revealed a strong negative association between cancer cell-intrinsic MYCN transcriptional program and type I IFN signaling. Thus, N-MYC inhibits tumor cell-intrinsic innate immune signaling in HGSC, making it a compelling target for immunotherapy of cold tumors.
Subject(s)
Immunity, Innate , N-Myc Proto-Oncogene Protein , Ovarian Neoplasms , Signal Transduction , Female , Humans , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Cell Line, Tumor , Cystadenocarcinoma, Serous/pathology , Cystadenocarcinoma, Serous/immunology , Cystadenocarcinoma, Serous/genetics , Cystadenocarcinoma, Serous/metabolism , Gene Expression Regulation, Neoplastic , Immunity, Innate/genetics , Interferon Type I/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Neoplasm Grading , Ovarian Neoplasms/immunology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Ovarian Neoplasms/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolismSubject(s)
Gene Amplification , Mutation , N-Myc Proto-Oncogene Protein , Neuroblastoma , Nuclear Proteins , Oncogene Proteins , Humans , Neuroblastoma/genetics , Neuroblastoma/pathology , Neuroblastoma/complications , N-Myc Proto-Oncogene Protein/genetics , Nuclear Proteins/genetics , Oncogene Proteins/genetics , MaleABSTRACT
Small-cell lung cancer (SCLC) accounts for nearly 15% of all lung cancers. Although patients respond to first-line therapy readily, rapid relapse is inevitable, with few treatment options in the second-line setting. Here, we describe SCLC cell lines harboring amplification of MYC and MYCN but not MYCL1 or non-amplified MYC cell lines exhibit superior sensitivity to treatment with the pan-BET bromodomain protein inhibitor mivebresib (ABBV075). Silencing MYC and MYCN partially rescued SCLC cell lines harboring these respective amplifications from the antiproliferative effects of mivebresib. Further characterization of genome-wide binding of MYC, MYCN, and MYCL1 uncovered unique enhancer and epigenetic preferences. Implications: Our study suggests that chromatin landscapes can establish cell states with unique gene expression programs, conveying sensitivity to epigenetic inhibitors such as mivebresib.
Subject(s)
Lung Neoplasms , Small Cell Lung Carcinoma , Humans , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/pathology , Small Cell Lung Carcinoma/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Cell Line, Tumor , Gene Amplification , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Bromodomain Containing Proteins , Proteins , Pyridones , SulfonamidesABSTRACT
The purpose of this study was to explore the relationship between the MYCN gene, serum neuron-specific enolase (NSE), urinary vanillylmandelic acid (VMA) levels, and neuroblastoma pathological features and prognosis. Ninety-four children with neuroblastoma treated in the hospital were selected to compare the differences in MYCN gene amplification, serum NSE, and urinary VMA levels in children with different clinicopathological features and prognoses. The proportion of children with MYCN gene copy number ≥10 in INSS stage 3-4 was higher than that of children with INSS stage 1-2 (P < 0.05); the proportion of children with MYCN gene copy number ≥10 in high-risk children in the COG risk stratification was higher than that of children with intermediate and low risk (P < 0.05); the serum NSE of children aged >12 months higher than that of children aged ≤12 months (P < 0.05); serum NSE of children with tumors >500 cm3 higher than that of children with tumors ≤500 cm3 (P < 0.05); serum NSE and urinary VMA of children with INSS staging of stages 3-4 were higher than that of children with stages 1 to 2 (P < 0.05); serum NSE and urinary VMA in children with lymph node metastasis were higher than that of children without lymph node metastasis (P < 0.05); serum NSE of children with MYCN gene copy number ≥10 was higher than that of children without lymph node metastasis (P < 0.05); the proportion of children with MYCN gene copy number ≥10 who died, and the percentages of serum NSE and urinary VMA were higher than those of the surviving children (P < 0.05). MYCN gene amplification and serum NSE and urinary VMA levels were related to the age, tumor size, INSS stage, COG stage, lymph node metastasis, and prognosis of the children with neuroblastoma.
Subject(s)
N-Myc Proto-Oncogene Protein , Neuroblastoma , Phosphopyruvate Hydratase , Vanilmandelic Acid , Humans , Neuroblastoma/genetics , Neuroblastoma/blood , Neuroblastoma/urine , Neuroblastoma/pathology , N-Myc Proto-Oncogene Protein/genetics , Male , Female , Prognosis , Infant , Child, Preschool , Phosphopyruvate Hydratase/blood , Phosphopyruvate Hydratase/genetics , Phosphopyruvate Hydratase/urine , Vanilmandelic Acid/urine , Vanilmandelic Acid/blood , Neoplasm Staging , Gene Dosage , Child , Gene Amplification , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Biomarkers, Tumor/urineABSTRACT
PURPOSE: The aim of this study was to develop a nomogram specially for predicting overall survival (OS) for Chinese patients with neuroblastoma (NB). METHODS: Patients with pathologically confirmed NB who were newly diagnosed and received treatments at our hospital from October 2013 to October 2021 were retrospectively reviewed. The nomogram for OS were built based on Cox regression analysis. The validation of the prognostic model was evaluated by concordance index (C-index), calibration curves, and decision curve analyses (DCAs). RESULTS: A total of 254 patients with NB were included in this study. They were randomly divided into a training cohort (n = 178) and a validation cohort (n = 76) at a ratio of 7:3. Multivariate analyses revealed that prognostic variables significantly related to the OS were age at diagnosis, bone metastasis, hepatic metastasis, INSS stage, MYCN status and DNA ploidy. The nomogram was constructed based on above 6 factors. The C-index values of the nomogram for predicting 3-year and 5-year OS were 0.926 and 0.964, respectively. The calibration curves of the nomogram showed good consistency between nomogram prediction and actual survival. The DCAs showed great clinical usefulness of the nomograms. Furthermore, patients with low-risk identified by our nomogram had much higher OS than those with high-risk (p < 0.001). CONCLUSION: The nomogram we constructed exhibited good predictive performance and could be used to assist clinicians in their decision-making process.