Fig. 5.

ASH2L is essential for glioblastoma tumor growth in vivo. A qRT-PCR analysis of ASH2L expression levels upon transduction of GBM4 primary glioblastoma cells with g-ASH2L or g-NT. B Representative images of GBM4 primary neurospheres 16 days post-transduction. C Cell viability analysis of GBM4 cells 1, 3 and 5 days after seeding. D Scheme for in vivo validation of ASH2L essentiality for GBM via intracranial injection of g-ASH2L or g-NT transduced U87MG cells (n = 5 per group) and bioluminescence detection. E Representative images of 3 mice taken during bioluminescence measurements at days 0-12-33 were illustrated. Bioluminescence signal of tumors formed by g-ASH2L or g-NT transduced U87MG cells were compared 33 days post-injection. F The boxplots displaying the ASH2L gene expression ratio (RSEM normalization values) in low grade gliomas (LGG, grade 2 and grade 3) and GBM based on TCGA. G The boxplots displaying the ASH2L gene expression ratio in GBM subtypes based on TCGA. H Representative core images from Brain Glioblastoma tissue microarray (TMA) stained with anti-ASH2L antibody. Scale bar 500 μm. I Percentage of ASH2L-positive cores were shown (n = 80 cores). P values determined by two-tailed Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001. J Model of ASH2L essentiality for glioblastoma cell survival. ASH2L together with DPY30, RBBP5 and WDR5 forms WRAD module, which acts as cofactor of SET1/MLL family transcription factors (MLL1/2, MLL3/4, SETD1A, SETD1B) to bind promoters of target genes and induce methylation of H3K4, a mark for euchromatin state. Active RNA polymerase binds to open chromatin to initiate transcription of genes involved in cell cycle progression, regulation of mitotic spindles and survival of cells (e.g. TRA2B, BARD1, KIF20B, ARID4A, SMARCC1). Deregulation of ASH2L levels interferes with cell cycle and leads to cell cycle arrest and apoptosis