Epigenetic-focused CRISPR/Cas9 screen identifies ASH2L as a regulator of glioblastoma cell survival

Glioblastoma is the most common and aggressive primary brain tumor with dismal prognosis, highlighting an urgent need for novel treatment strategies. In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using our customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and Cut&Run sequencing together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A, SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis demonstrated the interaction partners of ASH2L in glioblastoma cell lines as COMPASS family members including SETD1A, SETD1B, MLL1 and MLL2, however glioblastoma cells had differential dependency to COMPASS members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Overall design: For RNA sequencing experiment, gASH2L and gNT transduced U373 cells were collected 14days later as triplicates and RNA isolation was carried out to investigate the differentialiy expressed genes between two groups. For CRISPR screens, U373 and T98G cells were transduced with EpiDoKOL virus with low MOI (MOI=4) as 400 cell/sgRNA representation. Following transduction, initial time point samples were collected as single samples for both cell line. After Puromycin selection, cells were separated into two sets; Set1 was seeded to 6-well plates as 400 cell per guide representation and Set2 was seeded to 15 cm plates with 8000 cells per guide representation. Two sets were passaged for 12 days and at day30, final time point samples were collected as dublicates for each cell line.