Abstract 3285: Importance of Ras signaling in EphB2-mediated ependymoma development

Abstract

Abstract Ependymoma account for 6-12% of pediatric brain tumors and have a mean age of diagnosis of 4 years, a 5-year survival rate ranging from 39%-64%, and a progression free survival rate of 23-45%. General use of chemotherapeutics remains highly ineffective for this disease therefore standard of care consists of surgery followed by radiation with total gross resection (TGR) having the best outcome (60-89% 5-year overall survival for TGR vs. 21-46% 5-year overall survival for subtotal resection (STR)) [6]. Patients having had STR have an extremely high incidence of disease reoccurrence (43% to 72%) requiring additional surgeries and radiation with a median 2-year overall survival after relapse ranging from 20% to 49% [7]. These statistics highlight the need for better adjuvant therapies. In an effort to identify new targets for treating ependymoma we have developed a mouse model that relies on the intracranial implantation of mouse supratentorial (forebrain) embryonic neuronal stem cells (STeNSC) overexpressing the tyrosine kinase receptor ephrin B2 (EphB2). Tumors develop within 4-8 months post-implantation, require receptor activation and are consistently found to be histologically, ultrastructurally and molecularly similar to the human disease. Given the requirement for EphB2 activation in tumor development, it became necessary to identify the EphB2 initiated signaling pathways involved in this process. Among the many pathways initiated by EphB2 activation are those of the small GTPases of the Ras family (H-Ras, K-Ras, N-Ras) that regulate cellular processes including cell proliferation and transformation. Receptor activation is a multistep process involving receptor autophosphorylation of two critical tyrosines positions 604 and 610 within the juxtamembrane (Y604, Y610), receptor/ligand complex dimerization, release of juxtamembrane mediated kinase domain inhibition and finally kinase domain phosphorylation and activation. While EphB2-mediated activation of Ras involves the kinase domain of the receptor, Ras inhibition (RasGTP conversion to RasGDP) is achieved by activating the Ras GTPase activating protein p120RasGAP via its juxtamembrane. We took advantage of a previously identified EphB2 juxtamembrane mutation that inhibits p120RasGAP activation by converting the tyrosines at positions 604 and 610 to glutamic acids (Y604/610E) thus rendering Ras constitutively active. Ligand-mediated activation of this mutant resulted in an absence of p120RasGAP activation and an increase in Ras signaling (elevated levels of phosphorylated C-Raf, MEK1/2 and Erk1/2) in vitro as predicted. More importantly, overexpression of the EphB2(Y604/610E) protein in our model system showed no change in tumor penetrance (100% penetrance); however, there was a dramatic shortening of tumor latency from 207 days with EphB2 to 40 days with the Y604/610E mutation, supporting the role for Ras signaling in EphB2 mediated transformation. Citation Format: Robert A. Johnson, Phylip Chen, Robert Lyons, Samuel Priddy. Importance of Ras signaling in EphB2-mediated ependymoma development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3285. doi:10.1158/1538-7445.AM2015-3285