Thursday, June 1, 2023
6/1/2023
Project Summary Neuroblastoma (NB) remains one of the deadliest childhood cancers. NB exhibits a paucity of recurrent protein coding mutations and few targetable mutations (2-5), providing the rationale for this proposal. Noncoding variants can disrupt regulatory and/or structural DNA leading to dysregulated transcriptional programs that promote tumorigenesis. Our objective here is to identify noncoding variants and mechanisms that drive NB. Our central hypothesis is that germline variants and somatic mutations within noncoding regulatory regions of DNA potently influence NB initiation, progression and/or disease relapse. We will test our hypothesis in three specific aims: 1) Define and evaluate differences in the epigenomic landscape of NB and NB precursor cells. First, a panel of genetically diverse human-derived NB cell lines and neural crest cells (NCC; NB precursor cells) will be characterized. 3D chromatin architecture at all promoters will be ascertained using an ultra-high-resolution promoter-focused Capture C approach. Cells will be further profiled by whole genome sequencing (WGS), RNA- seq, ATAC-Seq, and ChIP-seq for histone marks and key structural proteins. Evolution of the epigenomic landscape from NB precursor to NB cells will be assessed. Data will be integrated with transcription factor binding site functional predictions to provide a comprehensive resource for the interpretation of noncoding variants. 2) Identify germline noncoding variants influencing NB tumorigenesis. We will perform variant-to-gene mapping at NB susceptibility loci identified by GWAS and identify putative causal variants mapping to open chromatin and involved in chromatin interactions in NB precursor or NB cells. Next, rare germline noncoding mutations from WGS will be assessed in a similar manner to identify variants interacting with known cancer predisposition genes. Further in silico prioritization will be accomplished via clinical correlative and integrative host-tumor analyses. The mechanism by which top prioritized noncoding variants promote NB will be determined using genetic manipulation in cell models in combination with Capture C, ChIP-seq, RNA-seq and/or functional studies. 3) Discover and assess biological relevance of somatic noncoding drivers of NB. We will integrate WGS of diagnostic and relapsed NB tumors to identify noncoding mutations affecting regulatory DNA and chromatin interactions in NB genomes. Recurrent variants will be further characterized through integration with matched RNA-seq (n=443), DNA methylation (n=223) and clinical correlative studies. We will elucidate biological relevance of prioritized mutations via massively parallel reporter assay (MPRA) coupled with CRISPR-based genetic manipulation in cell models in combination with Capture C, ChIP-seq, RNA-seq and/or functional assays. This work will have a sustained and positive impact on the field by providing substantial insights into the role of the noncoding genome in this important childhood cancer, and has the potential to inform development of clinical biomarkers and/or evidence-based therapies to improve outcomes of children with NB.
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