Martin, Sarah E.

Abstract Medulloblastoma (MB) is a pediatric brain tumor comprising of four molecular subgroups, with Group 3 and 4 tumors characterized by particularly poor prognosis. While research has advanced our understanding of MB, there is still a pressing need for new knowledge of the functional processes underlying its progression due to a scarcity of therapeutics. Recent studies have identified Orthodenticle homeobox 2 (Otx2), a cerebellar transcription factor, to be highly expressed in MB Group 3/4 subtypes. Datamining studies from Northcott and Robinson datasets revealed co-expression of Otx2 and c-Myc in Group 3/4 tumors. DAB staining conducted in human MB specimens showed increased expression of Otx2 and c-Myc. Colocalization studies confirmed datamining results. Next, we used a clinical Otx2 inhibitor, all-trans retinoic acid (ATRA), in a PDX cell line, P9, and in UW228 cells. Western blot analysis demonstrated reduced levels of c-Myc along with Otx2. In another experiment, we used the c-Myc/Max inhibitor 10058-F4 and observed comparable results to Otx2 inhibition. Real time PCR and immunocytochemistry experiments confirmed the aforementioned results. Further, treatment with ATRA and 10058-F4 showed reduced proliferation and migratory abilities in p9 and UW228 when compared to their respective controls. Mass spectrometry conducted on immune-precipitated Otx2 in P9 cells revealed a plethora of novel interaction partners that drive tumor growth and progression. In addition, western blot analysis in ATRA treated cells showed reduced expression of Max, suggestive of the existence of a feedback loop that link with Otx2 and c-Myc. Collectively our studies highlight Otx2 as a potential contributor to the malignant phenotype and describe its role in mediating the aggressiveness of Group 3/4 medulloblastoma. Citation Format: Yining Lu, Maheedhara R. Guda, Swapna Asuthkar, Ian Purvis, Collin M. Labak, Neha Jain, David J. Daniels, Sarah E. Martin, Andrew J. Tsung, Kiran K. Velpula. Otx2 and c-Myc association drives tumor progression in medulloblastoma molecular subtypes 3/4 [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 3537. doi:10.1158/1538-7445.AM2017-3537

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children and accounts for 20% of pediatric CNS tumors. Following surgery and treatment with chemotherapy and radiation, MBs recur in a significant proportion of patients. Moreover, survivors experience comorbidities related to the tumor and/or its treatment. New insights into MB tumor pathogenesis are needed for improving the survivorship and outcome of MB. Epigenetic silencing, including histone modification and DNA methylation, is an important tumorigenic mechanism in MB. However, its role in cancer immunopathology is poorly understood. Using datamining and immunohistochemistry analysis, we show that B7-H3 (CD276), an important immune checkpoint member of the B7 and CD28 families, is highly upregulated in all four molecular subtypes of MB compared to normal tissue. Based on the clinical success of inhibitory immune checkpoint blockades (CTLA-4, PD-1, and PD-L1), CD276 appears to be a promising therapeutic target for cancer immunotherapy. Moreover, we observed that increased expression of CD276 correlates significantly with the expression of Enhancer of Zeste Homologue 2 (EZH2), a histone H3-methylating (H3K27me3) catalytic subunit in the human MB specimens. We hypothesize that combined targeting of CD276 and EZH2 may have greater impact on tumor progression and immune cell response in MB. Simultaneously, we performed LC/MS analysis to identify novel interaction partners of CD276 in MB tumor. These findings taken together suggest that CD276 is strongly associated with several epigenetic markers, indicating a strong relationship between cancer immune evasion and epigenetic mechanisms. Citation Format: Maheedhara R. Guda, Jose R. Castellanos, Collin M. Labak, Ramadevi Sanam, David J. Daniels, Sarah E. Martin, Julian J. Lin, Andrew J. Tsung, Kiran K. Velpula, Swapna Asuthkar. Epigenetic targeting of CD276 expression in medullolbastoma [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 2004. doi:10.1158/1538-7445.AM2017-2004