The Cap-Translation Inhibitor 4EGI-1 Induces Apoptosis in Multiple Myeloma Through Noxa Induction and Synergizes with ABT-737

Abstract 1832

Cancer cells are frequently addicted to deregulated oncogenic protein translation. The interaction between the eukaryotic initiation factors eIF4E and eIF4G is tightly regulated and is limiting for the formation of the translation initiating complex eIF4F. The small molecule 4EG-I selectively inhibits the cap-dependent translation of mRNAs by competing for the eIF4E/eIF4G interaction. Since Multiple Myeloma is an incurable disease that requires new therapeutic approaches, we investigated whether targeting the translation initiation pathway using 4EGI-1 could be a target for myeloma therapy. In this study, we performed pull down assays on myeloma cells demonstrating that 4EGI-1 inhibits the eIF4E/eIF4G interaction. The biological effects of 4EGI-1 were then investigated in 6 myeloma cell lines (U266, L363, LP1, OPM-2, NCI-H929, XG-6) and primary samples. 4EGI-I decreased the cellular expression of cyclin D eIF4E-regulated proteins in the six cell lines studied. We next demonstrated that 50μM 4EGI-1 treatment induced apoptosis in 5 out of 6 myeloma cell lines while U266 was resistant to 4EGI-1 at the doses and time point investigated. The percentage of apoptotic cells ranged from 30% to 78% at 50 μM4EGI-1. In a similar manner, 4EGI-1 induced apoptosis in two purified myeloma cell samples at 50 μM 4EGI-1. Apoptosis was associated with Bax activation and caspase 9 and 3 cleavage, indicating the activation of the intrinsic mitochondrial pathway. Bak or Bax knockdown was not sufficient to inhibit 4EGI-1-induced apoptosis, indicating that they played a redundant role in this process. In contrast the simultaneous silencing of Bax and Bax significantly decreased the 4EGI-1-induced apoptosis. To decipher the events that trigger apoptosis through the mitochondrial pathway, Bcl-2 family protein expression was analyzed by Western blot analysis after 4EGI-1 treatment. Among anti-apoptotic proteins, the expression of both Mcl-1 and Bcl-x_L was consistently decreased in every cell line studied, whereas Bcl-2 expression was not affected. Puma was up-regulated in every cell line that showed endogenous expression of Puma, whereas Noxa was only strongly increased in myeloma cell lines that underwent 4EGI-1-induced apoptosis. Furthermore, Noxa silencing but not Puma silencing prevented myeloma cells from 4EGI-1-induced apoptosis demonstrating that Noxa induction was required. Finally, Noxa induction led to a disruption of Mcl-1/Bim complexes and the generation of free Noxa that was not bound to Mcl-1. Because the combination of several drugs usually appears to be the most efficient chemotherapeutic regimen we sought to combine ABT-737 that targeted Bcl-2 with 4EGI-1 giving the fact that Mcl-1 is the major resistance factor for Bcl-2 targeted therapy. We demonstrated that a low dose of 4EGI-1 in combination with ABT-737 resulted in a synergistic apoptotic effect in ABT-737 resistant cell lines. Our results suggest that the use of inhibitors that directly target the translation initiation complex eIF4F could represent a potential novel approach for multiple myeloma therapy.