Repression Of Cap-Dependent Translation With a Next-Generation mTOR Inhibitor Attenuates c-Myc Transcription and Survival In Lymphoma Cells

The mammalian target of rapamycin (mTOR) exists in two complexes, mTORC1 and mTORC2. Most mTOR inhibitors used for hematologic diseases such as rapamycin, everolimus, and temsirolimus are considered mTORC1 inhibitors and do not target mTORC2. The central role of the mTOR kinase in controlling cell growth has sparked interest in discovering mTOR inhibitors that bind to the ATP active site and therefore target both mTORC2 and mTORC1. mTOR also regulates protein synthesis by phosphorylating the P70S6-Kinase and the eIF4E-binding-protein, 4EBP1. The effect of active site dual mTOR inhibitors on cap dependent translation has not been investigated in lymphomas.

In this study we moved beyond the effect of the dual mTOR inhibitor CC214-1 (Celgene Pharmceuticals) on the mTORC2/AKT pathway to focus on its effects on cap dependent protein translation. Cap dependent translation is regulated by the translation initiation complex eIF4F (eIF4E-eIF4G-eIF4A). First, we assessed the formation of the active eIF4F (m7GTP-eIF4E-eIF4G) translation complex by a pull down assay using agarose-immobilized m7GTP cap analog to capture eIF4E and its binding partner in lymphoma cell lines (Jeko, Mino, Granta and JVM2, Ly3, Ly1, DHL6) and in primary cells (n=5). Our data demonstrate that the cap bound fraction from normal B cells contain very little eIF4G and eIF4A; however, lymphoma cells were enriched with eIF4G, eIF4E and eIF4A. Increased mRNA of eIF4E and eIF4G was observed in all lymphoma cell lines tested as compared to normal B-cells. 150 untreated diffuse large B cell lymphoma samples were stained for eIF4E by immunohistochemistry and 76% (135/150) were found eIF4E positive. Taken together, these data suggest that lymphoma cells exist in a translational activated state compared to normal B-cells. Next, we examined the effect of the dual mTOR inhibitor CC214-1 on the integrity of the eIF4F complex. In the CC214-1 treated cells the quantity of the eIF4E bound to 4EBP1 was increased whereas the amount of the eIF4E bound to eIF4G was decreased in the CC214-1 treated lymphoma cells but not in those treated with a selective mTORC1 agent (everolimus). We also found that the phosphorylation of 4EBP1 that controls protein synthesis is partially resistant to everolimus/rapamycin but fully suppressed with CC214-1. CC214-1 had a dose-dependent inhibitory effect on lymphoma cell survival; however, everolimus and rapamycin were only cytostatic. Moreover, CC-214 was much more potent than rapamycin and everolimus in blocking protein translation measured by the leucine incorporation assay. To elucidate the role of the eIF4E complex in the response to CC214-1, we transiently expressed WT-eIF4E plasmid in Jeko cells and then treated with CC214-1. Ectopic expression of wt-eIF4E led to a loss in the CC-214-1 dependent decrease in the eIF4E bound eIF4G as compared with the control plasmid. Moreover, overexpression of eIF4E also protected against CC214-1-induced apoptosis. To evaluate the efficiency of cap-dependent translation in the presence of CC214-1 and rapamycin, we used the bicistronic reporter assay where translation initiation of the first cistron is dependent on the 5′ cap, whereas initiation of the second cistron depends on a viral internal ribosome entry site. Transfection of this plasmid in CC214-1 treated cells caused a significant decrease in cap-dependent, but not IRES-dependent, translation. We next looked the effect of CC214-1 on the eIF4E regulated translationally active mRNA targets. We isolated eIF4E known mRNA targets (Myc, Cylin D1, Cyclin D2 and MCL-1) by immunoprecipitation assays using eIF4E antibody (mRNA-IP). CC214-1 treated RNA-IP data showed decreased expression of Myc but not the other mRNA targets. Myc is highly expressed in the DLBCL tumors and would be a good prognostic or predictive biomarker for CC214-1 therapy.

Translation deregulation is an important mechanism that causes aberrant cell growth in lymphoma cells. Currently, there is a need to identify additional agents that target this pathway. These studies provide a mechanistic basis of targeting cap dependent translation in lymphoma through next generation dual mTOR inhibitors. This study also suggests eIF4E (indicative of activated eIF4F complex) as a potential biomarker for the selection of patients for next generation mTOR inhibitors clinical trials in lymphoma patients.