Abstract 681: Using a genome-editing approach for the stepwise establishment of zebrafish models of pediatric high-grade gliomas and MPNSTs

Abstract

Abstract By high-throughput sequencing of cancer patient's genomes, driver mutations have recently been described in high-grade pediatric glioma, an aggressive type of brain tumor in children. The most prominent recurrent mutations were found in four genes, namely NF1, TP53, ATRX and H3F3A. NF1 and TP53 are tumor suppressor genes and their loss of function is characteristic for various human malignancies. However, the mechanisms through which precise missense mutations in H3F3A and loss-of-function of ATRX influence tumor biology remain largely unknown. In our project, we employ the genome-editing technology CRISPR-Cas9 to develop a zebrafish model of pediatric high-grade glioma, in order to examine the impact of these mutations on tumor onset and progression. We previously created a zebrafish line that is deficient for tp53 (tp53-/-) and has a loss-of-function mutation in 3 of 4 alleles of nf1 (nf1a+/-, nf1b-/-), which is duplicated in zebrafish. These fish develop high-grade gliomas with low penetrance and malignant peripheral nerve sheath tumors (MPNSTs) with high penetrance. By CRISPR-Cas9 we further incorporated early frameshift mutations of atrx in the background of tp53-/-, nf1a+/-, nf1b-/-. All analyzed fish injected with Cas9-mRNA and CRISPR guide-RNAs (gRNAs) targeting atrx coding sequences showed germline transmission of mutant alleles, of which one third were frameshift mutations. Primary CRISPR-Cas9 atrx-gRNA injected tp53-/-, nf1a+/-, nf1b-/- fertilized eggs developed as mosaics and exhibited increased tumor penetrance and faster onset of MPNSTs and these tumors harbored atrx mutations in 8/10 analyzed samples. This indicates that atrx loss-of-function mutations cooperate with loss of tp53 and nf1 tumor suppressors. Gliomas were not observed in mosaic atrx mutated fish, and these will be reanalyzed in stable atrx mutant lines. Next, we will add previously described missense mutations of h3f3a. To unravel the synergy between oncogenic mutations in atrx and h3f3a in tumor progression, mutant stable lines for both genes will be investigated independently and in a combined manner. The zebrafish model of pediatric high-grade gliomas and MPNSTs that we are developing will be useful to dissect the mechanisms underlying cooperation among driver mutations and for small molecule screens to identify specific inhibitors of cell growth and survival in these malignancies. Citation Format: Felix Oppel, Shuning He, Ting Tao, Mark W. Zimmerman, Adam D. Durbin, Nina Weichert, Dong H. Ki, A Thomas Look. Using a genome-editing approach for the stepwise establishment of zebrafish models of pediatric high-grade gliomas and MPNSTs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 681.