Unraveling the role of translation factor EIF5A in the ER-stress response in B cell malignancy cell lines
Impairments in protein folding in the endoplasmic reticulum (ER) lead to a condition called ER-stress. This activates the unfolded protein response (UPR) to restore homeostasis under manageable stress conditions, while committing to cell death when stress is chronic or unresolvable. ER-stress is clinically targeted in B cell malignancies, such as Multiple Myeloma (MM) and Mantle Cell Lymphoma (MCL). These cells have high immunoglobulin production, which makes them sensitive to activation of the UPR by proteasome inhibitors. The use of the ER stressor Bortezomib as treatment for MM is effective, but is not curative and often leads to serious side effects and resistance. Moreover, the exact mechanism by which UPR signaling in B cell malignancies is wired is still unknown. By deciphering the underlying signaling pathways, we aim to find new unique targets for treatment.
A targeted CRISPR-Cas9 screen containing approximately 500 genes involved in different cell death and stress pathways was performed in MM (RPMI8226 and LP-1) and MCL (JeKo-1) cell lines treated with Thapsigargin. To verify the hits, single-gene KOs were generated using an inducible CRISPR-Cas9 system in these cell lines. These KO cells were further used to perform competition assays, polysome profiling and functional assays.
The CRISPR-Cas9 screen in RPMI8226 cells under Thapsigargin selection showed enrichment of known ER stress regulators XBP1, IRE1 and Calreticulin as well as the translation factor EIF5A, whose role in ER stress remains poorly defined. A second screen in JeKo-1 cells also enriched for this translation factor. Intriguingly, we found that loss of EIF5A was lethal under basal conditions, but paradoxically conferred resistance to Thapsigargin-induced cell death in multiple cell lines. This was confirmed in single EIF5A KOs which showed selective protection against Thapsigargin. Polysome profiling revealed that EIF5A KO cells displayed a muted UPR and decreased apoptotic protein expression in response to Thapsigargin, suggesting a regulatory role of EIF5A in fine-tuning the cellular stress response in these cells. Interestingly, EIF5A is the only protein in the cell that undergoes the posttranslational modification hypusination, making it an potential therapeutic target. Inhibition of this hypusination by the specific drug GC7, mirrored the effects of its KO, further implicating EIF5A activity in ER stress pathways.
In summary, our findings reveal that loss of EIF5A expression protects B cell malignancy cells to Thapsigargin induced cell death by altering the translation of UPR and apoptotic proteins. These results reveal a novel link between the translational machinery and ER stress-induced cell death, with potential implications for therapeutic targeting.