Epigenetic-focused CRISPR/Cas9 screen identifies (absent, small, or homeotic)2-like protein (ASH2L) as a regulator of glioblastoma cell survival

Abstract

Background: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers.

Method: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers.

Results: 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 greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family 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.

Conclusion: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.

Keywords: ASH2L; CRISPR/Cas9 screen; Chromatin modifiers; Epigenetic; Glioblastoma; sgRNA library.

Fig. 1

Epigenetic Domain-specific Knock Out Library (EpiDoKOL) screen identified common essentiality genes in T98G and U373 cells. A Composition of EpiDoKOL including target molecules, their functions and related epigenetic modifications. B Library generation procedure. Figure created with BioRender.com. C Cas9 activity assay of U373 and T98G cells. Microscopic images of T98G and U373 cells transduced with indicated sgRNAs 18 days post-transduction. D Flow cytometric analysis of U373 and T98G cells transduced with indicated sgRNAs (g-NT1, g-NT2: non-targeting sgRNAs; g-T1 and g-T2: sgRNAs targeting GFP), up to 12 days post-transduction. E Schematic of EpiDoKOL screening procedure. Figure created with BioRender.com. F sgRNA density plots from cells transduced with pLentiCRISPRv2 plasmid containing EpiDoKOL. Cell pellets collected before puromycin selection. G Log2 counts of sgRNAs at initial and final time points in T98G and U373 cells. H Waterfall plots for Log2fold changes of genes after screening T98G cells with EpiDoKOL for a month. Domain and gene-based analysis revealed overlapping essentiality hits. I Waterfall plots for Log2fold changes of genes after screening U373 cells with EpiDoKOL for a month. Domain and gene-based analysis revealed overlapping essentiality hits. Mageck p values were calculated with negative binomial model fitting. Other p values were determined by two-tailed Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Effects of candidate genes on glioblastoma cell fitness were validated with functional assays in vitro. A Scheme of validation experiments for novel EpiDoKOL essentiality hits. TD: post-transduction day. B Representative images of long-term clonogenic assay of cells infected with sgRNAs against selected hits and statistical analysis. Quantification of colonies was performed by ImageJ software. C Results of GFP competition flow cytometric assay for selected hits. Day 0 refers to the day of the cell seeding at TD 7. D-F Cell viability (D), Caspase 3/7 activity (E) and AnnexinV analysis (F) conducted on U373 cells upon depletion of ASH2L, RBX1 and SSRP1 genes. G Western blot analysis of ASH2L protein levels upon transduction of U373 cells with g-ASH2L or g-NT. H Western Blot analysis for cleaved Caspase3 and PARP in U373 cells at day 6 and 14 post-transduction with g-NT control or g-ASH2L. I Western Blot analysis for H3K4 mono and trimethylation levels in U373 cells 6 days post-transduction with g-NT control or g-ASH2L. P values determined by two-tailed Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

ASH2L transcriptionally regulates cell cycle via direct promoter interactions with G2/M-checkpoint and E2F target gene sets. A Schematic of RNAseq and greenCUT&RUN experiments. B Volcano plot of RNAseq data showing differentially expressed genes (DEGs) in ASH2L depleted U373 cells compared to controls on the 14 day post-transduction. C Normalized enrichment score results of gene set enrichment analysis (GSEA) for all gene sets available from MSigDB v7.5. Top 5 biological processes enriched in downregulated or upregulated genes upon ASH2L knockout are shown. P values were calculated by hypergeometric test. D greenCUT&RUN analysis in U373 cells revealing genomic localization of ASH2L at differentially expressed gene promoters. E Venn diagram and volcano plot of overlapped RNAseq and greenCUT&RUN data to show proportion of differentially expressed genes bound by ASH2L in U373 cells. Genes bound by ASH2L and differentially expressed upon ASH2L depletion are now called as critical genes, denoted by blue (for downregulated) and yellow (for upregulated) dots on volcano plot. F Normalized enrichment score and FDR-qval results of GSEA on critical genes for all gene sets available from MSigDB v7.5. Some of the negatively enriched pathways related to cell cycle are highlighted. G Top 10 biological processes enriched in regulatory genes upon ASH2L knockout. H RNAseq heatmaps of critical genes. I greenCUT&RUN representative igv plots of critical genes. J Flow cytometric cell cycle analysis of ASH2L-depleted U373 by PI staining on post-transduction day 14 and its statistical analysis. P values determined by two-tailed Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Glioblastoma cells have differential dependency to SET1/MLL family of transcription factors interacting with ASH2L. A Scheme of tandem mass spectrometry experiments performed on glioblastoma cells. B, C Proteomic analyses of GFP-tagged ASH2L in U373 (B) and U87MG (C) cells. Volcano plots of significant interactors of GFP-ASH2L isolated from nuclear extracts are shown. Stoichiometry plots of bound SET1/MLL family members (MLL family specific: yellow, SETD1A/B specific: red, MLL & SETD1A/B common members: blue) are depicted. All interactors are normalized to the GFP-ASH2L bait. Results shown represent Intensity Based Absolute Quantification with standard deviations. D Distribution of ASH2L common interactors in terms of subcellular localization in U373 and U87MG cells. E qRT-PCR analysis for mRNA levels upon siRNA mediated knockdown of WDR5 and KMT2A genes in U373 and U87MG cells. F Representative images of long-term clonogenic assay upon siRNA mediated knockdown of WDR5 and KMT2A genes and statistical analysis. Quantification of colonies was performed by ImageJ software. P values determined by two-tailed Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001

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