S141: inhibition of myc translation through targeting of the newly identified phb-eif4f complex as therapeutic strategy inchronic lymphocytic leukemia (cll)

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 the MYC oncogene. Interestingly, translation was recognized as one “Achilles’ heel” of cancer cells and increased translation seems to be a common feature of a large variety of tumors. Several inhibitors of translation are available. We used FL3, a synthetic flavagline that was shown to bind prohibitins (PHBs). These proteins are found in several cellular localizations that dictate their activity. At the membrane, they are required for the RAF activation by RAS in a large variety of cancers, leading to the phosphorylation of eukaryotic initiation factor 4E (eIF4E) through the MAPK pathway, and ultimately resulting in increased translation. By binding to PHBs, FL3 was shown to prevent the activation of RAF and therefore decreases the translation. Aims: Here, we tested the targeting of translation initiation in CLL as a novel therapeutic strategy and aimed to dissect the mechanism of action of FL3, a translation inhibitor. Methods: We used CLL cells from patients, human and murine cell lines. We performed O-propargyl-puromycin (OPP) and L-homopropargylglycine (HPG) incorporation assays; Proximity Ligation Assay (PLA) of the interaction between translation initiation factors eIF4E and eIF4G; pulsed SILAC and polysome profiling to assess translation rate. We carried-out metabolomics analysis using [U-13C]-glucose and [U-13C]-glutamine tracing. Co-immunoprecipitation, PLA, Nano-BRET and cap-pull down were used to validate protein interactions. In vivo validation were done by adoptive transfer of splenocytes from sick Eµ-TCL1 mice. Finally, we performed qPCR of 6 genes from the translation initiation machinery and of PHBs in a cohort of 144 patients to determine correlation between expression of these genes and clinical parameters. Results: We showed that CLL cells display a high translation rate (A), which can be inhibited by FL3 (B). A multiomics analysis consisting of pulsed SILAC, RNA sequencing and polysome profiling performed in CLL patient samples and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene (C). Furthermore, inhibition of translation was associated with a block of proliferation (D) and a profound rewiring of MYC-driven metabolism. Interestingly, contrary to other models, in CLL, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation. We rather showed that PHBs are directly associated with the translation initiation complex (E). Knock-down of PHBs resembled FL3 treatment (F), confirming the direct involvement of PHBs in translation initiation. Importantly, inhibition of translation was efficient in controlling CLL development in vivo (G). Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in CLL patients (H). Summary/Conclusion: We demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for CLL patients.Keywords: Chronic lymphocytic leukemia, Targeted therapy