Abstract 3477: ADGRB3 is epigenetically silenced in WNT-medulloblastoma and inhibits WNT signaling

Abstract

Abstract Medulloblastoma (MB), is the most aggressive primary malignant brain tumor in children and are classified into four molecular subgroups. While some subtypes of MB show a favorable prognosis with treatment, still one third of patients succumb to this disease and the children who survive after therapy suffer from long-term neurocognitive and endocrine side effects of the conventional treatments. ADGRB3 (formerly called BAI3) is a member of the ADGRB1-3 subfamily of adhesion GPCR transmembrane proteins, which are highly expressed in the brain specially in cerebellum and hippocampal neurons. Our recent analysis of RNA-seq data from a published panel of medulloblastoma tumor samples and RT-PCR experiments with MB tumor samples showed that ADGRB3 mRNA expression was selectively repressed in WNT-MB tumor tissue compared to other three molecular subgroups and normal human cerebellar tissue. Using bisulfite sequencing and MS-PCR we have detected hypermethylation of the ADGRB3 promoter region exclusively in WNT-MB subgroup of MB tissues but not in the other three molecular subgroups and normal human cerebellar tissue. ChIP assays revealed enrichment of repressive methyl CpG binding protein MBD2 and the trimethylated histone H3K9me3 in the ADGRB3 promoter region of UW288-1 cells. Collectively, these indicates epigenetic silencing of ADGRB3 in WNT-MB via promoter hypermethylation and repressive histone modifications. We found that knockdown of ATP dependent chromatin remodeler protein Brg1 in MB cells can silence ADGRB3 expression through epigenetic reprogramming at the gene promoter. Lentiviral reconstitution of ADGRB3 in silent MB cells (UW288-1, PFSK-1) inhibited growth of these cells in culture and inhibited WNT signaling targets. ADGRB3 reconstituted UW288-1 MB cells when xenografted yielded significantly reduced tumor in immunocompromised mice compared to the parental cells. Pharmacological reactivation of ADGRB3 expression in silent WNT MB cells using our recently established MBD2 antagonist and an EZH2 inhibitor significantly reduced cell growth in vitro and inhibits some specific WNT signaling targets. We further identified a novel mechanism underlying ADGRB3 mediated regulation of WNT signaling by performing co-immunoprecipitation experiments. Altogether, our findings define an epigenetic mechanism for ADGRB3 silencing in WNT-MB and demonstrates a mechanism through which ADGRB3 restrains activation of WNT signaling involved in cerebellar transformation. Our findings highlight the potential of epigenetic reactivation of ADGRB3 as a less toxic therapeutic intervention for the children suffering from WNT-MB. Citation Format: Debanjan Bhattacharya, Dan Zhu, Satoru Osuka, Saroja Narra Devi, Erwin G. Van Meir. ADGRB3 is epigenetically silenced in WNT-medulloblastoma and inhibits WNT signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3477.