Inducible Silencing Of eIF4E Using a Tet-On System Results In Myeloma Growth In Vivo That Correlates With eIF4E Expression

Overexpression and/or activation of eukaryotic initiation factor 4E (eIF4E) is critical for oncogenic protein synthesis. Mutations in genes related to mRNA translation are involved in the pathogenesis of multiple myeloma (Chapman, Lawrence et al. 2011). Recently, we found that MM cells express high levels of eIF4E protein compared to normal plasma cells and overexpression of eIF4E induces transcription factors such as c-myc critical for the growth of multiple myeloma cells (Li, Fu et al. 2011,2012). The understanding of the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. Here we show the critical role of eIF4E driven protein synthesis by using an inducible knockdown system to silence eIF4E gene expression and confirm the critical role of eIF4E in multiple myeloma growth in vivo and in vitro.

We stably infected U266, RPMI-8226, IM-9 and MM.1S cells with a robust inducible single-lentiviral knockdown vector pLKO-Tet-On containing either control non-targeting shRNA or eIF4E targeting shRNA sequences. Doxycycline-induced eIF4E shRNA expression resulted in significant decrease of eIF4E mRNA and protein in eIF4E-shRNA but not the control shRNA infected MM cells. To determine the effects of eIF4E knockdown on MM cell growth and viability, stably transfected cell lines were grown in the presence or absence of doxycycline. Silencing of eIF4E by doxycycline induction of eIF4E shRNA in RPMI-8226 cells significantly inhibited (>72%,P<0.01) cell growth accompanied by a decrease of c-myc, cyclin D1, C/EBP beta and IRF4 all critical for myeloma cell growth. Cell cycle analysis revealed increased cells population in G0/G1 phase (62% vs 80%) in doxycycline-induced eIF4E shRNA cells with a significant reduction (P<0.001) of clonogenic tumor growth reflected by a decrease in colony numbers (27.6 ± 4.2 vs 5.3 ± 3.4) and size. To determine the role of high expression of eIF4E in MM tumor growth in vivo, we generated subcutaneous MM xenografts in severe combined immunodeficient x beige (SCID/bg) mice using the inducible U266-Tet-CT-shRNA and U266-Tet-eIF4E-shRNA cells. In contrast to vehicle or doxycycline-treated control shRNA tumors, doxycycline treated animals bearing U266-Tet-eIF4E-shRNA xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 21 days. The transient inhibition of tumor growth correlated with the transient doxycycline-induced eIF4E knockdown further confirming the critical role of eIF4E. Immunohistochemical staining of tumors confirmed the decreased of eIF4E expression in doxycycline-treated mice bearing U266-Tet-eIF4E-shRNA tumors compared with tumors of vehicle-treated or non-doxycyclin treated mice.

Here we show that eIF4E, a key player in the translational machinery, promotes multiple myeloma cell growth. We found that high eIF4E expression is indispensable for the growth of MM cells both in vitro and in vivo. Silencing of eIF4E decreases protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation, and resulted in tumor inhibition. Our study indicated that targeting transcriptional initiating factor eIF4E may represent a novel therapeutic strategy for MM treatment.

Schecter:Seattle Genetics: Honoraria, Research Funding. Lentzsch:Celgene: Research Funding.