MDB-36. REST-dependent alternative transcripts control CXCR4-SDF1 signaling in SHH-driven medulloblastoma

Elevated expression and activity of the RE-1 Silencing Transcription Factor (REST), a canonical repressor of neuronal differentiation genes, is seen in subsets of patients with sonic hedgehog (SHH)-driven medulloblastomas and is associated with poor patient survival. In mice, the context of hyperactive SHH signaling and conditional REST elevation in cerebellar granule neuron progenitors (CGNPs), the cells of origin of SHH-medulloblastomas, promotes leptomeningeal dissemination of tumor cells. Using RESTTg mice and xenograft models, we discovered that REST elevation increased the levels of and signaling through the C-X-C motif receptor 4 (CXCR4) G-protein coupled receptor, a pathway previously implicated in SHH medulloblastoma metastasis. Further, we identified the gene encoding the G-protein coupled receptor 6 (Grk6), a negative regulator of CXCR4 stability, as a novel REST target. REST elevation promoted the expression of Grk6 splice variants in CGNPs, and specifically one that lacked a portion of the protein kinase domain known to phosphorylate CXCR4 protein, to allow constitutive activation of CXCR4-mediated cell migration. Interestingly, the genomic locus corresponding to this domain houses a REST-binding RE1 motif. Further, in contrast to WT cells, chromatin decompaction and accumulation of Brg1 - a component of the SWI/SNF chromatin remodeling complex was observed at the Grk6 RE1 motif in RESTTg CGNPs. This was accompanied by a significant reduction in REST binding, enrichment of phosphorylated and total RNA Polymerase II (PolII) and loss of transcription through the RE1 motif, suggesting exclusion of this exonic region in Grk6 in the context of REST elevation. Surprisingly, integrated RNAseq-ATACseq analyses and chromatin immunoprecipitation assays identified a role for the polycomb repressive complex 2 (PRC2) in modulating REST occupancy at the Grk6 RE1 motif and expression of the SDF1-independent Grk6 variant. Thus, our data suggest REST elevation drives a Brg1- and PRC2-dependent exon exclusion/inclusion program, which influences cell migration, and potentially metastasis in SHH medulloblastoma.