Neuroblastoma (NB), a disease of trunk neural crest cells (NCC), represents the most common extracranial solid tumor in childhood. While amplification of MYCN is the strongest marker of poor prognosis in NB patients, no clinical inhibitors exist to directly target MYCN. Instead, targeting a cooperating genetic partner of MYCN represents an alternative strategy to treat these patients. Sequencing NB patient samples revealed that although recurrent single nucleotide variations is rare in NB, chromosomal aberrations are robust, particularly deletions of chromosomes 1p. Since loss of heterozygosity of chromosome 1p correlates with amplification of MYCN (strongest marker of poor prognosis) in NB, I hypothesize that amplification of MYCN cooperates with loss of tumor suppressors within chromosome 1p to transform human NCC to NB. As a postdoc in the William Weiss laboratory, I spent the majority of my time developing the necessary protocols (NCC differentiation) and tools (cloning overexpression vectors and genome editing plasmids) to demonstrate a proof of concept that I can transform human NCC to NB. I have, thus far, been successful at producing tumors driven by MYCN (10% penetrance) and MYCN/ALK F1174L (60% penetrance). For the K99 portion, I will examine other candidate NB drivers in cooperating with MYCN in accelerating NB tumorigenesis. I will also compare the resulting human stem cell (hSC)-derived NB tumors with genetically engineered mouse models of NB via whole genome sequencing and RNA-seq analysis. Thus, part of my training during the K99 will focus on bioinformatics analysis, which will be accomplished with the guidance of Hanlee Ji (Stanford) and a bioinformatics workshop at Cold Spring Harbor. After publication of the hSC-derived model of NB, my goal in the R00 phase is to identify tumor suppressors within chromosome 1p that, when deleted, cooperate with amplified-MYCN to accelerate tumorigenesis. Using the CRISPR/Cas9 system, I can narrow down the region within chromosome 1p that harbors the critical tumor suppressors. Subsequently, I can screen for individual candidate tumor suppressors by performing a CRISPR interference (CRISPRi) knockdown for each gene in that region. To bring CRISPRi to my independent laboratory for the R00 phase, I will work with Jonathan Weissman's (UCSF) group, which has pioneered CRISPRi technology, during the K99 phase. Successful completion of these studies will establish and characterize the first hSC-based model of NB, which can be used to rapidly test candidate driver genes and chromosome copy number alterations in NB tumorigenesis, as well as provide a preclinical model to evaluate the efficacy of various therapeutic strategies. My long term goal and vision for my independent lab is to investigate the significance of different chromosomal aberrations in NB and to utilize CRISPRi to not only identify key drivers of NB tumorigenesis, but also potential therapeutic vulnerabilities in a synthetic lethal screen.

Public Health Relevance

MYCN has been known as a genetic driver of neuroblastoma for the past three decades, but no clinically relevant inhibitors have been developed to target MYCN directly due to its biochemical structure. To address this issue, we propose to develop the first human stem-cell based model of neuroblastoma to test candidate cooperating partners of MYCN in promoting neuroblastoma tumorigenesis, which will provide clues of possible druggable targets.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Career Transition Award (K99)
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Study Section
Subcommittee I - Transistion to Independence (NCI)
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Schmidt, Michael K
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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