In certain human cancers, the expression of critical oncogenes is driven from large regulatory elements, called super-enhancers, which recruit much of the cell’s transcriptional apparatus and are defined by extensive acetylation of histone H3 lysine 27 (H3K27ac). In a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases, we found that heterozygous somatic mutations are acquired that introduce binding motifs for the MYB transcription factor in a precise noncoding site, which creates a super-enhancer upstream of the TAL1 oncogene. MYB binds to this new site and recruits it’s H3K27 acetylase binding partner CBP, as well as core components of a major leukemogenic transcriptional complex that contains RUNX1, GATA-3, and TAL1 itself. Additionally, most endogenous super-enhancers found in T-ALL cells are occupied by MYB and CBP, suggesting a general role for MYB in super-enhancer initiation. Thus, this study identifies a genetic mechanism responsible for the generation of oncogenic super-enhancers in malignant cells.
Childhood high-risk neuroblastomas with MYCN gene amplification are difficult to treat effectively. This has focused attention on tumor-specific gene dependencies that underlie tumorigenesis and thus provide valuable targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR-Cas9 approaches to detect genes involved in tumor cell growth and survival, we identified 147 candidate gene dependencies selective for MYCN-amplified neuroblastoma cell lines, compared to over 300 other human cancer cell lines. We then used genome-wide chromatin-immunoprecipitation coupled to high-throughput sequencing analysis to demonstrate that a small number of essential transcription factors-MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2-are members of the transcriptional core regulatory circuitry (CRC) that maintains cell state in MYCN-amplified neuroblastoma. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which act synergistically, in vitro and in vivo, with rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma that are essential for cell state and survival in this tumor.
Summary Neuroblastoma is a pediatric malignancy that typically arises in early childhood and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumors with excellent outcomes to widely metastatic disease where long-term survival is approximately 40% despite intensive therapy1. A previous genome-wide association study (GWAS) identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumor cells2. Here we sought to discover the causal DNA variant at this locus and the mechanism by which it leads to neuroblastoma tumorigenesis. We first imputed all possible genotypes across the LMO1 locus and then mapped highly associated single nucleotide polymorphism (SNPs) to areas of chromatin accessibility, evolutionary conservation, and transcription factor binding sites. SNP rs2168101 G>T was the most highly associated variant (combined P=7.47×10-29, Odds Ratio 0.65, 95% CI: 0.60-0.70) and resided in a super-enhancer defined by extensive acetylation of histone H3 lysine 27 within the first intron of LMO1. The ancestral G-allele that is associated with tumor formation resides in a conserved GATA transcription factor binding motif. We show that the newly evolved protective TATA allele is associated with decreased total LMO1 expression (P=0.028) in neuroblastoma primary tumors and ablates GATA3 binding (P<0.0001). We demonstrate allelic imbalance favoring the G-containing strand in tumors heterozygous for this SNP as demonstrated both by RNA sequencing (P<0.0001) and reporter assays (P=0.002). These findings show that a recently evolved polymorphism within a super-enhancer element in the first intron of LMO1 influences neuroblastoma susceptibility through differential GATA transcription factor binding and direct modulation of LMO1 expression in cis, and this leads to an oncogenic dependency in tumor cells.
The amplified gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here, we show that the family member is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (∼10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic activation include focally amplified distal enhancers and translocation of highly active enhancers from other genes to within topologically associating domains containing the gene locus. The clinical outcome for patients with high levels of expression is virtually identical to that of patients with amplification of the gene, a known high-risk feature of this disease. Together, these findings establish as a bona fide oncogene in a clinically significant group of high-risk childhood neuroblastomas. Amplification of the oncogene is a recognized hallmark of high-risk pediatric neuroblastoma. Here, we demonstrate that is also activated as a potent oncogene in a distinct subset of neuroblastoma cases through either focal amplification of distal enhancers or enhancer hijacking mediated by chromosomal translocation. .
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