Abstract B003: The OTX2 gene induces tumor growth and triggers leptomeningeal metastasis by regulating the mTOR signaling pathway in medulloblastomas
Authors: Mesias Elisabet Ampudia , Cameron Charles Scott , Anderson Sarah , Abrahante Lloréns Juan E. , Moertel Christopher L. , Odde David J. , Saydam Okay 
Published: March 2024

Abstract
Medulloblastoma (MB) is the most common malignant brain tumor in children, and is classified into four genetic subgroups: WNT, SHH, Group 3 and Group 4. These subgroups are driven by major canonical driver genes (CDG). The OTX2 (Orthodenticle homeobox 2) oncogene is overexpressed or amplified in Group 3 and 4 MBs promoting self-renewal and tumor growth leading to enhanced tumorigenesis. Leptomeningeal disease/metastasis (LMD) is a hallmark of medulloblastomas. Despite several comprehensive studies on MB tumor development through CDG, understanding of the molecular mechanism(s) of LMD and possible contributions of the MB CDG on the development of LMD has been particularly limited. In the present study, we therefore performed gain-of-function experiments to investigate the possible contribution of the OTX2 gene in LMD metastasis. Using a human MB patient-derived Group 3 relevant cell line called D425 and transducing this cell line with an OTX2 gene-carrying lentivirus vector, we found that OTX2OE-D425 cell motility is ~2 fold higher (0.4891 um^2/min, 0.2244 um^2/min; p&lt;0.0001), and displays enhanced polarization compared to control cells on a 4.6 kPa polyacrylamide hydrogel (PAG) coated with collagen type I. In addition, OTX2OE-D425 cells moved faster than control cells on mouse cerebellum slices (0.01981um^2/min vs 0.01489 um^2/min, p&lt;0.00001). We next implanted the human-MB D425 cell line overexpressing the OTX2 gene into the cisterna magna of immune-deficient nude mice and observed that these mice had a lower median survival than that of the vehicle group (20 days vs. 33 days, log-rank test, p&lt;0.00014). Moreover, we found that the mice implanted with OTX2OE-D425 cells also developed spinal cord and brain metastases, whereas almost no metastases were observed in the control cell-implanted mouse group. Taken together, our data indicate that the overexpression of OTX2 gene induces tumor growth and triggers spinal and brain metastases in Group 3 MB. In order to investigate the molecular mechanism by which the OTX2 gene regulates metastasis in MB, we performed bulk RNA sequencing studies and found that the overexpression of OTX2 gene activates the mTOR signaling pathway in D425 cells. Next, we treated MB cells with two mTOR inhibitors, AZ8055 and PQR620, and found a significant reduction in cell viability of D425 cells compared to control solvent (DMSO) treated cells. We are currently investigating the possible effect of PQR620 (that can pass through the Blood Brain Barrier) on our orthotopic MB mouse model.
Citation Format: Elisabet Ampudia Mesias, Charles Scott Cameron, Sarah Anderson, Juan E. Abrahante Lloréns, Christopher L. Moertel, David J. Odde, Okay Saydam. The OTX2 gene induces tumor growth and triggers leptomeningeal metastasis by regulating the mTOR signaling pathway in medulloblastomas [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B003.