Abstract A42: EphB2 a potential therapeutic target for pediatric medulloblastoma

Abstract

Abstract Aim: Medulloblastoma (MB) is the most frequent malignant brain tumor to occur in children and remains the leading cause of cancer-related mortality in childhood. The current standard of care for patients with MB includes surgical resection and radiation therapy followed by high-dose chemotherapy. Overall survival rates have reached 70%, but the outcome for young children, especially infants, is worse. Those who do survive suffer from long-term therapy-induced side effects. Thus, more effective and less toxic therapies are needed. Eph family receptors predominantly function during embryonic development and are typically either not expressed or expressed at very low levels in normal healthy tissues. However, it is now established that many Eph receptors are re-expressed and functional in human cancers, making them attractive, relatively tumor-specific targets. Thus, in MB anti-Eph therapy has the potential to target tumor cells and not normal brain, reducing off-target toxicity and potentially increasing response rates. We have generated compelling preliminary data showing that EphB2 is functional and overexpressed in pediatric MB. We now propose to extend these studies and validate antibody-based strategies to target this receptor. Methods: 1. EphB receptor expression was characterized using a number of approaches: RNA (RNASeq and QPCR), protein (flow cytometry and Western blot) and receptor localization by immunofluorescence in MB ATCC lines, primary lines, and patient tissue. 2. To better understand the function of the EphB2, gene expression was downregulated using shRNA in primary MB cell line models. Following EphB downregulation, we assessed in vitro growth and cell death induction and tumor-forming capacity using orthotopic MB intracranial xenografts. Moreover, following EphB activation, using soluble ligand we assessed in vitro growth via direct cell count and MTT assay, cell migration via scratch assay, and cell death induction via Annexin V staining. Following EphB inhibition, using peptide strategies we assessed in vitro growth, cell migration, and cell death induction. Results: Our study shows that EphB2 is both functional and highly expressed in MB. Furthermore, EphB2 appears to be expressed in a subtype-specific manner. We further confirmed our expression studies using the MB Advanced Genomics International Consortium (MAGIC) dataset. The EphB2 high-affinity ligands ephrinB1/B2-Fc were used to induce kinase activation. Following receptor activation, we observed reduced tumor cell proliferation and invasion in vitro. EphB2 receptor blocking studies, using the SNEW blocking peptide, effectively inhibited kinase phosphorylation and subsequently FAK phosphorylation. Most importantly, the small peptide rescues MB cell growth upon ephrin B ligand stimulation, indicating the specific mitogenic capacity of this receptor. Conclusion: In summary, this study furthers our understanding of EphB2 expression and function in MB and paves the way for future receptor-targeting studies in MB. Citation Format: Yuchen Li, Carolin Offenhauser, Rochelle D’Souza, Seckin Akgul, Bryan W. Day. EphB2 a potential therapeutic target for pediatric medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A42.