Nucleoli-localized KANSL2 as an epigenetic regulator of ribosome biogenesis in glioblastoma cells.
KANSL2 is a subunit of the non-specific lethal (NSL) chromatin-modifying complex involved in epigenetic reprogramming and plays a critical role in tumorigenesis of glioblastoma (GBM), by enriching the GBM stem-like cell population. Analysis of KANSL2 expression in TCGA-GBM tumor samples and GTEx normal brain tissue confirmed that KANSL2 is highly expressed in GBM and positively correlates with both the stemness index score and ribosomal protein mRNA expression. KANSL2 localized to nucleoli, depending on the cell cycle phase, suggesting its dynamic shifting during the cell cycle. Ectopic overexpression of KANSL2-RFP upregulated 45S pre-rRNA and 28S rRNA expression, concomitant with higher cell proliferation. In contrast, KANSL2 downregulation decreased proliferation, 45S pre-RNA and 28S rRNA expression levels, and acetylated levels of H4 at lysine 5 and 8 in the rDNA promoter region, suggesting KANSL2 is necessary for proper rRNA gene expression potentially via histone acetylation. In support, RNA-seq analysis of patient-derived GBM spheroids silenced for KANSL2 showed significant downregulation of ribosomal biogenesis-related genes. Collectively, these findings revealed an unprecedented role of KANSL2 as a positive regulator of rRNA expression in GBM, suggesting it to be a critical mediator of cell stemness and proliferation, making it an attractive target for potential therapy. Overall design: F18-1 and F2-4 cells used for the RNA-seq assay were obtained from the University of Colorado, Colorado, USA, through the Neurosurgery Nervous System Biorepository (protocol COMIRB #13–3007), with no expiration date. According to the WHO CNS5 (2021) classification, F18-1 is a grade 4 glioblastoma (GBM), IDH-wildtype, with O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation. F2-4 is a grade 4 IDH-mutant astrocytoma (positive for the IDH1 mutation by immunohistochemistry), negative for epidermal growth factor receptor (EGFR) amplification, but positive for the loss of phosphatase and tensin homolog (PTEN) sequences relative to chromosome 10 centromere sequences. F18-1 was derived from a primary tumor, whereas F2-4 was obtained from a resection of a recurrent tumor.  Briefly, stable cells expressing small hairpin RNA of a non-targeting control and two KANSL2-targeting sequences were cultured in serum-free medium supplemented with B27, N2, 20 ng/ml bFGF, 20 ng/ml EGF, 2 mM L-glutamine, 2 mM non-essential amino acids, 50 U/ml penicillin/streptomycin, and 75 µg/ml low-endotoxin bovine serum albumin (Sigma, USA). Cells were plated onto Geltrex (A1413202)-coated plates for three days before being harvested for sequencing.