The Eukaryotic Translation Initiation Factor eIF4E, a Recently Identified Therapeutic Target In AML, Is a Direct Transcriptional Target of NFkB

Abstract

AbstractAbstract 3146It has been well observed that NFκB activity is elevated in a wide range of hematopoietic malignancy including acute myeloid leukemia (AML). However, the downstream effectors that mediate the biological effects of NFκB are not well described. In the present study, we demonstrate that the eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NFκB. Elevated levels of eIF4E correlate with oncogenic transformation in in-vitro and in-vivo models and are associated with poor prognosis in several cancer types including AML. In a Phase II clinical trial in poor prognosis M4 and M5 AML patients, we demonstrated that the anti-viral drug, ribavirin, targeted eIF4E activity in patients correlating with clinical benefit including remissions and blast responses. Below, we describe the first steps in understanding the dysregulation of eIF4E expression in hematological malignancy.Examination of the human eIF4E promoter proximal region revealed the presence of putative κB sites proximal to the transcriptional start region. Given this, we hypothesized that eIF4E was an inducible target of NFκB. First, we tested whether eIF4E expression was modulated in response to NFκB stimulation by several agents including TNFα and PMA. We carried these studies out in multiple cell lines including BJAB, Jurkat and fibroblasts. We found that PMA or TNFα treatment led to substantial increases in eIF4E levels versus untreated controls. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated p65 loading on the eIF4E promoter. This binding correlated with a transcriptionally active complex. Importantly, eIF4E levels were not elevated upon stimulation in p65-/- fibroblasts whereas elevation was observed in control fibroblasts. Further, expression of a dominant negative form of the inhibitor of NFκB, IκB (IκB2NΔ4), substantially increased the expression of eIF4E. Luciferase reporter assays confirm the element is necessary and sufficient for activation of eIF4E. Importantly, this p65 binding element in the eIF4E gene is evolutionarily conserved in mammals. Finally, using EMSA, we observed that this eIF4E promoter element is in active NFκB complexes in M4 and M5 primary AML specimens.We examined the physiological relevance of these findings in primary M4/M5 AML specimens and AML cell lines. The use of certain demethylase inhibitors such as azacytidine have been reported to have undesirable off target effects, including the activation of NFκB. Consistent with these reports, we observe that treatment with 3 uM azacytidine leads to elevated p65 levels and furthermore, elevated eIF4E levels. Consistently, this led to the upregulation of several eIF4E target genes including myeloid cell leukemia protein Mcl-1 and anti-apoptotic proteins such as mdm2. The addition of the eIF4E inhibitor ribavirin led to reduction in these eIF4E targets with no change in eIF4E levels. Consistently, in primary colony assays, azacytidine (3 uM) alone did not substantially reduce colonies in M4/M5 AML specimens relative to untreated cells, whereas the addition of a combination of ribavirin and azacytidine reduced growth 2 to 10 fold. These findings strongly suggest a physiological link between NFκB and eIF4E and furthermore, have implications for the design of novel therapeutic modalities. For instance, ribavirin could substantially dampen unwanted proliferative effects of azacytidine; thus improving its efficacy.This report is the first direct link ever observed between the NFκB pathway and eIF4E and thus, between NFkB and protein translation & mRNA export. Remarkably, most of the genes regulated at the level of mRNA export or translation by eIF4E are targets at the transcriptional level by NFκB. These include VEGF, cyclin D1, mdm2 and Mcl-1. In this way, the NFκB pathway is powerfully positioned to amplify the effects of eIF4E on post-transcriptional gene expression by both promoting the expression of eIF4E and its downstream effectors. Thus, NFκB can amplify the proliferation and survival pathways governed by eIF4E at multiple levels. Disclosures:No relevant conflicts of interest to declare.