RSK1 modulates the translatome of glioblastoma cells in an isoform-specific manner

The p90 ribosomal S6 kinase (RSK) family, downstream targets of Ras/ERK signaling, encompass four human isoforms (RSK1-4). Glioblastoma (GBM) predominantly expresses RSK1 and RSK2. Notably, RSK1 is markedly upregulated in a subset of GBMs associated with dismal prognosis and immune infiltration, whereas RSK2 expression remains consistent across cases. We verified that GBM-derived cell lines recapitulate RSK isoform expression profiles and estimated their stoichiometry. Through the generation of RSK1 and RSK2 knockout (RSK1KO and RSK2KO) as well as double knockout (DKO) GBM cells, we investigated isoform-specific functions of RSK. Surprisingly, contrary to conventional belief, we found that mTORC1 is not activated by RSK isoforms. Instead, eIF4B phosphorylation at S422 was RSK-dependent rather than mTORC1/S6K-dependent, with RSK1 exerting a more pronounced effect. Intriguingly, RSK1 and RSK2 displayed differential effects on translation, with RSK1 maintaining translation of specific mRNAs under conditions of reduced overall translation due to mTORC1 inhibition, unlike RSK2. Finally, we verified that while the translatome of RSK1KO cells show a downregulation of mRNAs affecting cell cycle, DNA replication and repair, RSK2 depletion impacted mitochondrial and respiratory genes. Notably, DKO cells exhibited compounded phenotypes, underscoring the existence of isoform-specific gene regulation programs orchestrated by RSK. Collectively, our findings offer mechanistic insights crucial for unraveling the role of RSK1 in GBMs. In order to define RSK-isoform specific functions, we applied the CRISPR/Cas9 technique to edit the genome of glioblastome-derived LN18 cells (LN18CRISPR) and thus generate knockout cells for RSK1 (RSK1KO) or RSK2 (RSK2KO), and also double-knockout cells for both RSK1 and RSK2 (DKO). Cells were pre-treated with Torin1 (or DMSO) before serum stimulation for 6 hours. Total and polysome-associated (poly) mRNA were analyzed by microarray.