Abstract 3919: Transformation of mouse embryonic neuronal stem cells by EphB2 requires receptor mediated activation of the MAPK pathway

Abstract

Abstract Our ability to offer patients more personalized options for cancer treatment relies on the deep understanding of the genetic and genomic changes driving tumor development, growth and maintenance gained thru the use of both human and mouse cell lines and model systems. Unfortunately, such systems are largely unavailable for the study of ependymoma, the third most common pediatric central nervous system (CNS) tumor, resulting in little change in the current standard of care (surgery followed by radiation) and dismal cure rate of 60% that currently exists. However, with our development of the first mouse model of ependymoma we hope to improve this statistic. Our current model was developed by the intracranial implantation of cultured mouse supratentorial (forebrain) embryonic neuronal stem cells (eNSC) overexpressing the tyrosine kinase receptor ephrin B2 (EphB2), one of the more common alterations found within ependymomas developing in the supratentorium. Tumors develop within 4-8 months post-implantation exclusively in eNSCs overexpressing EphB2 and are consistently found to be histologically, ultrastructurally and molecularly similar to the human disease. The current study was designed to uncover the mechanism underlying EphB2 mediate transformation of eNSCs. Our model clearly demonstrates the requirement for EphB2 overexpression in eNSCs transformation; however, the requirement for receptor activation was not known. Using eNSCs overexpressing EphB2 inactive mutants (kinase domain mutation and ligand binding domain deletion) in similar intracranial implantation experiments we were able to show that tumor development required receptor activity, as neither EphB2 mutant receptor was capable of tumor formation. A detailed analysis of known EphB2 initiated pathways identified the MAPK pathway as potentially being involved in EphB2 mediated transformation. First, we observed a protein concentration independent increase in the levels of the active phosphorylated form of Erk1 and Erk2 in our EphB2 driven mouse tumors (EphB2MT) relative to untransformed EphB2 overexpressing eNSCs (EphB2eNSCs). Second, quantitative PCR (qPCR) shows an increase in p38γ level in tumor samples relative to their untransformed counterpart. Interestingly, EphB2eNSCs and EphB2MT cells respond differently when their receptor is activated in vitro by ligand (ephrin-B2 or ephrin-B3) in both differentiation and neurosphere formation assays. We find that EphB2MT cells show an increase in neurosphere formation and decrease in differentiation potential in the presence of ephrin-B2 and to a lesser extent ephrin-B3, while EphB2eNSCs show no change in either assay when ligand is present. Taken together this data suggesting that EphB2 mediated transformation is the result of the sensitization of EphB2 overexpressing eNSCs to the effects of receptor activation and the triggering of increased MAPK signaling. Citation Format: Samuel Priddy, Phylip Chen, Nathan Rossi, Robert A. Johnson. Transformation of mouse embryonic neuronal stem cells by EphB2 requires receptor mediated activation of the MAPK pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3919. doi:10.1158/1538-7445.AM2014-3919