Targeting translation by disrupting the newly identified prohibitin-eif4f complex as a novel therapeutic strategy in chronic lymphocytic leukemia

Background: CLL is the most common type of leukemia in adult, and despite great advance in the standard of care in the last decades, there is still no cure available. CLL cells are dependent on their microenvironment for proliferation and survival. Microenvironmental stimuli are associated with an increase in translation globally but also at the level of specific transcripts, including Myc (Yeomans et al., 2016, Blood).
Aims: Here, we tested the targeting of translation initiation in CLL as a novel therapeutic strategy and identified prohibitin as partner of the translation initiation machinery.
Methods: In order to target translation, we used the FL3 molecule, a synthetic flavagline, which is a known inhibitor of translation initiation in other types of cancers (Boussemart et al., 2014, Nature). This drug binds to the scaffold proteins prohibitins (PHBs), but the mechanism for translation inhibition is still unclear. PHBs are crucial partners for the activation of the Ras-Raf-MEK-ERK pathway, ultimately leading to eIF4E (a factor of the eIF4F translation initiation machinery) phosphorylation and to activation of translation.
Results: We confirmed the increase in translation in human primary CLL cells upon stimulation (A). In addition, we showed that in the Eµ-TCL1 murine model, CLL cells have a higher translation rate compared to normal B cells and T cells. In vitro treatment of human CLL cells (either cell lines or primary patient samples) with FL3 leads to a decrease in translation rate. It is associated with a strong decrease of cell viability (B) and apoptosis induction, even at a low nanomolar dose. By western blot, we showed that in CLL, contrary to other cancer types, FL3 does not prevent RAF1 and ERK phosphorylation. However, eIF4e phosphorylation is impaired. This observation strongly suggests a direct impact of the drug through its molecular target PHBs on the translation initiation machinery. By co-immunoprecipitation, proximity-ligation assay (C) and Nanoluciferase experiments, we demonstrated the interaction between PHBs and the members of the eIF4F complex. In addition, gene silencing of PHB by shRNA leads to a decrease in translation. This indicates for the first time a direct role of PHBs in translation, and allows a better understanding of FL3’s mechanism of action. We thus propose that PHB is necessary for the correct assembly of the eIF4F complex. Interestingly, we showed that the loss of eIF4e phosphorylation is neither responsible for the impairment of translation nor for the decrease in cell proliferation. By pulse SILAC experiments, we determined the proteins that are subjected to increased translation upon TLR stimulation, and to decreased translation upon FL3 treatment. Among others, oncogenes such as c-MYC or ETS-1 (D) have been identified. In vivo treatment of mice with FL3 after TCL1 adoptive transfer leads to a significant delay in CLL progression and an increased survival (E-F), demonstrating the relevance and possible impact of this molecule. Finally, high expression of translation initiation-related genes correlated with poor survival and unfavorable clinical parameters in CLL patients (G).