Abstract 1540: Creating faithful genetic zebrafish models of pediatric high grade gliomas and MPNSTs
Authors: Oppel Felix , Tao Ting , He Shuning , Zimmerman Mark W. , Ki Dong H. , Weichert Nina , Look A Thomas 
Published: July 2017

Pediatric high-grade gliomas (HGGs) are the leading cause of cancer-related death in children. Despite a slight improvement of patient prognosis over the past decades pediatric HGGs remain largely incurable. Thus, new experimental models are needed to understand the mechanisms of the disease and find more effective treatment options. We previously reported a model of HGGs and malignant peripheral nerve sheath tumors (MPNSTs) which is based on the combined deficiencies in the tumor suppressor genes tp53 and nf1. However, HGG penetrance is very low in this line and most fish develop MPNSTs starting at about 3 months of age. On top of the existing model we used CRISPR/Cas9 to incorporate knock-out mutations in the tumor suppressor genes atrx or suz12 which are described to be involved in pediatric HGG biology. Heterozygous atrx loss-of-function (lof) did not impact tumor onset or penetrance of neither HGGs nor MPNSTs. Since a total loss of atrx was lethal in development, we re-injected effective atrx targeting gRNAs and Cas9 mRNA into the atrx+/- line to create a mosaic atrx-/- genotype. Surprisingly, despite a high mutation efficiency of the remaining atrx allele the re-injection strategy still did not alter tumor onset and penetrance in that model. This suggests that loss of atrx is only effective in HGGs in cooperation with additional hits other than tp53 and nf1. In contrast, loss of suz12 cooperated well with the tp53/nf1-deficient background. As nf1, suz12 is duplicated in zebrafish (suz12a and suz12b) resulting in 4 alleles of each gene per cell. When at least 2 out of 4 alleles of either suz12a or suz12b were lost, MPNST onset was accelerated. This effect was much stronger in tp53-/-, nf1a+/-, nf1b-/- fish (5-7 weeks) compared to tp53-/-, nf1a+/+, nf1b-/- siblings (3-4 weeks). This indicates that the tumor supporting effect of suz12 lof increases the more nf1 levels decrease. However, HGG onset still remained unchanged. We hypothesize that efficient onset of HGGs in our model requires the presence of an activated oncogene. Specific missense mutations in H3F3A are reported to be implicated in HGG progression in children and young adults. Thus, we overexpressed zebrafish h3f3a-K27M or -G34R mutant sequences in the tp53/nf1/atrx/suz12-deficient line and are currently investigating possible changes in tumor biology. Our zebrafish models of pediatric HGGs and MPNSTs will be useful to dissect the mechanisms underlying the 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, Ting Tao, Shuning He, Mark W. Zimmerman, Dong H. Ki, Nina Weichert, A Thomas Look. Creating faithful genetic zebrafish models of pediatric high grade gliomas and MPNSTs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1540. doi:10.1158/1538-7445.AM2017-1540