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.

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. .

A heritable polymorphism within regulatory sequences of the LMO1 gene is associated with its elevated expression and increased susceptibility to develop neuroblastoma, but the oncogenic pathways downstream of the LMO1 transcriptional co-regulatory protein are unknown. Our ChIP-seq and RNA-seq analyses reveal that a key gene directly regulated by LMO1 and MYCN is ASCL1, which encodes a basic helix-loop-helix transcription factor. Regulatory elements controlling ASCL1 expression are bound by LMO1, MYCN and the transcription factors GATA3, HAND2, PHOX2B, TBX2 and ISL1—all members of the adrenergic (ADRN) neuroblastoma core regulatory circuitry (CRC). ASCL1 is required for neuroblastoma cell growth and arrest of differentiation. ASCL1 and LMO1 directly regulate the expression of CRC genes, indicating that ASCL1 is a member and LMO1 is a coregulator of the ADRN neuroblastoma CRC.

Highlights d Phenothiazine analog iHAP1 activates PP2A-B56ε and potently kills malignant cells d iHAP1 does not inhibit dopamine signaling or cause prohibitive neurologic toxicity d PP2A-B56ε dephosphorylates MYBL2-Ser241, causing prometaphase arrest with apoptosis d Other PP2A activators, SMAPs, activate PP2A-B55a and target different substrates

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Summary A genome-wide association study identified LMO1, which encodes a LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3 and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.

Ptp4a3 (commonly known as PRL-3) is an enigmatic member of the Ptp4a family of prenylated protein tyrosine phosphatases that are highly expressed in many human cancers. Despite strong correlations with tumor metastasis and poor patient prognosis, there is very limited understanding of this gene family's role in malignancy. Therefore, we created a gene-targeted murine knockout model for Ptp4a3, the most widely studied Ptp4a family member. Mice deficient for Ptp4a3 were grossly normal. Fewer homozygous-null males were observed at weaning, however, and they maintained a decreased body mass. Although Ptp4a3 is normally associated with late-stage cancer and metastasis, we observed increased Ptp4a3 expression in the colon of wildtype mice immediately following treatment with the carcinogen azoxymethane. To investigate the role of Ptp4a3 in malignancy, we used the most commonly studied murine colitis-associated colon cancer model. Wildtype mice treated with azoxymethane and dextran sodium sulfate developed approximately 7–10 tumors per mouse in the distal colon. The resulting tumor tissue had 4-fold more Ptp4a3 mRNA relative to normal colon epithelium and increased PTP4A3 protein. Ptp4a3-null mice developed 50% fewer colon tumors than wildtype mice after exposure to azoxymethane and dextran sodium sulfate. Tumors from the Ptp4a3-null mice had elevated levels of both IGF1Rβ and c-MYC compared to tumors replete with Ptp4a3, suggesting an enhanced cell signaling pathway engagement in the absence of the phosphatase. These results provide the first definitive evidence implicating Ptp4a3 in colon tumorigenesis and highlight the potential value of the phosphatase as a therapeutic target for early stage malignant disease.

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2β, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells. Significance: EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2β. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner. This article is highlighted in the In This Issue feature, p. 587