Elevated Translation Initiation Factor eIF4E Is an Attractive Therapeutic Target in Multiple Myeloma

eIF4E plays a central role in protein synthesis and has a key role in the control of cell growth, proliferation, differentiation, and metabolism in eukaryotic cells (1). It recognizes and binds to the 7-methylguanosine cap in the 5′ untranslated regions (5′UTR) of mRNAs, transporting these mRNAs to the eIF4F translation initiation complex, which includes eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A. An increase in eIF4E level or activity does not lead to increased rates of global translation, but instead results in increased translation of mRNAs with highly complex 5′UTRs (2). Several genes, including MYC, Cyclin D1, CCAAT/enhancer-binding protein beta (C/EBPβ), and VEGF, involved in tumorigenesis are regulated at the translational level by eIF4E (3–6). Further, eIF4E competitive inhibitors, such as Exportin1 (XPO1) inhibitors, abrogate its prosurvival function by decreasing export and translation of target mRNAs (7). In acute myeloid leukemia (AML), the XPO1 inhibitor KPT-330 (Karyopharm Therapeutics) induced decreased levels of proteins derived from capped mRNAs associated with eIF4E mRNA, which are dependent upon XPO1 for nuclear export further suggesting a critical role for eIF4E (8). Overexpression and/or activation of eIF4E has been also associated with tumor formation and progression including lymphoma, and cancers of the breast, colon, lung, and prostate (9), but the role of eIF4E in multiple myeloma is largely unknown.

Multiple myeloma is a plasma cell disorder, associated with an accumulation of monoclonal terminally differentiated plasma cells within the bone marrow, and usually the presence of a monoclonal immunoglobulin (10). In 2012, approximately 21,700 new cases were diagnosed in the United States and estimated 10,710 deaths occurred from this disease (11). Even with the introduction of novel and more potent treatment regimens for multiple myeloma, the disease remains an incurable plasma cell malignancy (12), and novel treatments especially for relapsed/refractory patients are urgently needed. In multiple myeloma, ribavirin (RBV) is a physical mimic of the m⁷G cap, and thus blocks eIF4E resulting in a potentially effective anticancer agent. Combination of RBV and velcade showed synergistic anti–multiple myeloma effect (13). 4EGI-1 behaves as a functional 4E-BP1 mimetic inhibiting the interaction between eIF4E and eIF4G and decreases the expression of eIF4E-regulated proteins in myeloma cells (14). More recently, Attar-Schneider and colleagues investigated by high-throughput assay of mRNA microarrays the significance of eIF4E/eIF4GI silencing to transcription factors, miRNAs, and phenotype. They showed that different imprints for eIF4E and eIF4GI affect the expression of cellular proteome, transcription factors, miRNAs, and phenotype in multiple myeloma (15).

Taken together, because tumor cell growth is more contingent on cap-dependent protein translation than normal tissues (16), the role of eIF4E in tumorigenesis and cancer progression has generated increasing interest as a therapeutic target in cancer (13, 16–21). Frequent mutations in genes involved in mRNA translation resulting in increased protein translation support the role of translational control in the pathogenesis of multiple myeloma (22). Here, we provide novel insights into the role of the translation initiation factor eIF4E in multiple myeloma tumor growth. By overexpressing and knockdown of eIF4E as well as using an inducible eIF4E-shRNA in human multiple myeloma xenograft mouse models, we found that expression of critical transcription factors and subsequently multiple myeloma tumor growth is highly dependent on eIF4E expression. Therefore, direct or functional inhibition of eIF4E by competitive selective inhibitors of nuclear export (XPO1 inhibitors), such as KPT-330, abrogates its prosurvival function by decreasing export and translation of target mRNAs and represents a promising approach.

Cell culture media, sera, and penicillin–streptomycin were purchased from Gibco BRL. Antibodies and inhibitors were purchased from the following vendors: anti-C/EBPβ (C-19) from Santa Cruz Biotechnology; anti-IRF4, anti–c-MYC, and anti-eIF4E antibody from Cell Signaling Technology; anti–β-actin antibody, and DMSO from Sigma-Aldrich; and anti–l-MYC antibody from Abcam Inc.

Multiple myeloma cell lines RPMI-8226, MM.1S, H929, and U266 were purchased from the ATCC in the last 5 years. OPM2 was provided by Dr. Klaus Podar (Dana Farber Cancer Institute, Boston, MA) in 2002. Cells were tested for mycoplasma and shown to be contamination free, and authentication was not performed. The stocks of the cell lines were stored frozen in liquid nitrogen, cell are thawed at need and maintained for no more than 3 months. The multiple myeloma cell lines MM.1S, RPMI-8226, U266, H929, and OPM2 were cultured in RPMI-1640 plus 10% FBS and 100 U/mL penicillin/streptomycin at 37°C and 5% CO₂ as described before (23). Human multiple myeloma cells were isolated from patient bone marrow samples as described before (24). Mononuclear cells were isolated by Ficoll (Invitrogen) followed by magnetic separation using CD138⁺ antibody–specific micro beads according to the manufacturer's protocol (Miltenyi Biotech). The negative population was considered as CD138⁻. The purity of the myeloma cells was assessed by hCD138⁺/hCD45⁺ staining.

Normal plasma cells were obtained from pilot vials of mobilized peripheral blood from healthy donors. In brief, mononuclear cells were isolated by Ficoll (Invitrogen) followed by magnetic separation using CD138⁺ antibody–specific micro beads (Miltenyi Biotech). All samples were obtained after informed consent was given. All studies were approved by the Institutional Review Board of Columbia University Medical Center, New York.

Human bone marrow stromal cells (BMSC) were isolated from patient bone marrow samples as described previously (25). Briefly, isolated mononuclear cells were cultured with 10% (vol/vol) heat-inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L sodium pyruvate, and 2 mmol/L glutamine at 37°C and 5% CO₂ overnight. Nonadherent cells were removed and adherent cells were cultured for expanding for 7 to 10 days. The purity of the BMSCs was assessed by hCD29⁺/hCD90⁺/hCD45⁻ staining.

Briefly, U266, RPMI-8226, or primary stromal cells (5 × 10⁴/well) were incubated in 96-well for 72 hours. The WST-1 reagent (Clontech) was added to react for 3 hours. Measurement of absorbance of the samples at 450 nm (reference wavelength 690 nm) against the background control was performed using the Synergy HT Multi-Detection Microplate Reader (Biotek Instruments, Inc.).

Briefly, protein was extracted from cells using RIPA buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Cell lysates were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinyl di-fluoride membranes (Bio-Rad Laboratories). The blots were incubated with the appropriate antibodies to detect the protein level of interest, and the immune complexes were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) as described before (26).

For the determination of mRNA levels of eIF4E and c-MYC, total RNAs were isolated from cells using Trizol reagent (Invitrogen) following the manufacturer's instructions. Total RNA was converted into cDNA using Superscript III RT (Invitrogen). Quantitative RT-PCR was performed as described before (23). The following primer sets were used:

• eIF4E: 5′-ACAAGTCAGTCTGAAACCATCGAAC-3′ and 5′-CTTCATCCTCTTCGGCCACTCCTCC -3′;

• c-MYC: 5′- TCTCCGTCCTCGGATTCTCT-3′ and 5′-TCTGACCTTTTGCCAGGAGC-3′;

• β-actin: 5′-GGACTTCGAGCAAGAGATGG-3′ and 5′-AGCACTGTGTTGGCGTACAG-3′.

Expression vectors for the full-length wild-type eIF4E were generated by inserting the respective coding regions into pCDH-GFP vector. In brief, full-length human eIF4E cDNA was cut out from pHA-eIF4E (Addgene; ref. 27) by HindIII/XhoI double digestion followed by filling-in of 5′ overhangs by DNA polymerase I large (Klenow) fragment to form blunt ends. Similarly, pCDH-CMV-MCS-EF1-COPGFP (System Biosciences) was digested by EcoRI followed by filling-in of 5′ overhangs then ligated with eIF4E fragment by T4 ligase (NEB). Lentivirus was packaged and concentrated by PEG-it (System Biosciences) from 293TN cells supernatant after cotransfection of pCDH-CMV-eIF4E-EF1-COPGFP with pPACKH1-packaging plasmids (System Biosciences). Myeloma cells (2 × 10⁶) were transduced with an empty vector lentiviral control pCDH-GFP(EV) or pCDH-GFP-eIF4E construct. The efficiency of virus transduction was determined by GFP coexpression. The transduction rate of U266 and RPMI-8266 cells was greater than 70% and 90%, respectively, at 72 hours after transduction. Transduced cells were selected by Influx cell sorter (BD Bioscience) and analyzed by Western blotting or cell proliferation assay.

Lentiviral shRNAs were used to knock down eIF4E expression in multiple myeloma cells. In brief, lentiviral pLKO.1-sh-eIF4E (Sigma) target to the sequence is as follows: 5′-ACTCTGTAATAGTTCAGTA-3′. The shRNA stable transfectants were obtained by puromycin (5 μg/mL) selection and analyzed by Western blotting or cell proliferation assay. For tet-on–inducible eIF4E knockdown multiple myeloma cells, tet-on-pLKO-puro (Addgene; ref. 28) was used to generate inducible sh-eIF4E lentiviral construct with the same target sequence as above, and the correct constructs were confirmed by DNA sequencing. The viruses containing empty vector sequences were used as a control. RPMI-8266 or U266 cells were incubated with lentivirus particle and polybrene (8 μg/mL) for 16 hours, and then washed with media. Transduced multiple myeloma cells were maintained in 10% tetracycline-free FBS (Clontech). Cells were selected for 3 days in puromycin (5 μg/mL). The efficiency of virus transduction was determined by puromycin resistance, and the transduction rate of U266 or RPMI-8266 was greater than 60% or 70%, respectively. Selected cells were treated with or without doxycycline (200 ng/mL) for 3 days to induce knockdown eIF4E.

Multiple myeloma cells (1 × 10⁶ cells/mL) were cultured for 72 hours at 37°C, harvested, and washed with ice-cold PBS, fixed with 70% ethanol for 1 hour at 4°C, and pretreated with RNase (Worthington) for 30 minutes at 37°C. Cells were stained with propidium iodide (PI; 20 μg/mL; Sigma Aldrich). Analyses were performed on a BD FACSCalibur flow cytometer and analyzed using ModFit LT2.0 and Cellquest software (BD Biosciences).

Myeloma cells (5 × 10³) per well were seeded in a 24-well plate in methylcellulose medium (Stem Cell Technologies). Methylcellulose (1.1 %) was diluted to a final concentration of 1% by the addition of cell suspension. Colonies were stained with crystal violet (Sigma Aldrich) after 14 days, and colony numbers were counted using a Leica DM IL LED inverted phase contrast microscope.

Female severe combined immunodeficient (SCID) Beige (CB17.Cg-Prkdc^scidLyst^bg-J/Crl) mice were purchased from Charles River Laboratories at age 6 to 8 weeks with a weight between 20 and 25 g. All animal procedures were approved by the Institutional Animal Care and Use Committee of Columbia University, New York. Sample size was chosen according to our experience (29). For human tumor xenograft studies, U266 myeloma cells (5 × 10⁶) in 100 μL PBS together with an equal volume of Matrigel basement membrane matrix (BD Biosciences) were injected s.c. Mice were weighed twice weekly and observed daily for diarrhea or any changes in behavior and condition. Tumor sizes were measured twice weekly in a no blinded manner. After mice were sacrificed, the tumors were excised, weighed, and pictures were taken. Tumor weights are reported as mean ± SEM (n = 5). CT-tet-on shRNA or eIF4E-tet-on shRNA cells were injected s.c. into SCID/bg mice. Eleven days after implantation, animals were randomized to receive either vehicle (5% sucrose) or doxycycline (1 mg/mL in 5% sucrose) via drinking water for duration of study.

Each experiment was repeated at least 3 times, and all quantitative data are presented as mean ± SEM. Statistical differences were determined by the Student t test (two tailed). The results were considered statistically significant if P < 0.05.

We examined eIF4E expression in normal plasma cells, primary CD138⁺ multiple myeloma cells, multiple myeloma cell lines, and CD138⁻ mononucleated cells. Analysis of eIF4E protein and mRNA levels by Western blotting and real-time PCR revealed significantly (P < 0.01) higher expression in primary CD138⁺ multiple myeloma cells and multiple myeloma cell lines compared with normal plasma cells and CD138⁻ cells (Fig. 1A and B). This was confirmed by patient bone marrow cells immunofluorescence staining showing higher expression of eIF4E in primary multiple myeloma cells (red arrow) compared with CD138⁻ mono-nuclear cells (MNC; green arrow, Fig. 1C).

To determine whether eIF4E protein is required for multiple myeloma cell growth, we used shRNA to knock down eIF4E in multiple myeloma cells by lentiviral-mediated transduction. The human multiple myeloma cell lines RPMI-8226 and U266 were infected with shRNAs lentivirus generated from pLKO.1-puro vectors containing either control non-targeting shRNA (CT-shRNA) or eIF4E targeting shRNA (eIF4E-shRNA), and stably transfected cells were obtained after puromycin selection. eIF4E-shRNA but not the CT-shRNA led to significant reduction of eIF4E expression at protein (Fig. 2A) and mRNA (Fig. 2B) levels. Silencing of eIF4E in myeloma cells significantly inhibited cell growth (66% in RPMI-8226, 76% in U266, P < 0.01; Fig. 2C). In accordance with that, cell-cycle analysis revealed that knockdown of eIF4E decreased the cell numbers in S-phase with concomitant increase of cells in G₀–G₁ growth arrest (63% vs. 75% in U266 cells; 37% vs. 50% in RPMI-8226; Fig. 2D). Importantly, eIF4E knockdown resulted in significant reduction (P < 0.001) of clonogenic tumor growth reflected by decreased colony numbers (mean ± SD: CT-shRNA 27.6 ± 4.2 vs. eIF4E-shRNA 5.3 ± 3.4) in U266 and (CT-shRNA 21.3 ± 1.2 vs. eIF4E-shRNA 3.7 ± 1.2) in RPMI-8226 cells (Fig. 2E). Next, we established an inducible eIF4E knockdown in our multiple myeloma cells. We stably infected U266 and RPMI-8226 with shRNA lentivirus generated from a robust inducible knockdown vector pLKO-tet-On. Doxycycline-induced eIF4E-tet-on-shRNA expression resulted in significant decrease of eIF4E protein (Fig. 2F) and significantly inhibited (RPMI by 72% and U266 by 46%, P < 0.01) cell growth (Fig. 2G). To determine whether eIF4E is specifically critical for multiple myeloma cells growth, we tested its role in primary stromal cells from multiple myeloma patients, which exhibit a low eIF4E expression (Supplementary Fig. S1). Primary stromal cells were similarly infected with eIF4E-shRNA or CT-shRNA lentivirus, and stably transfected cells were obtained after puromycin selection (Fig. 2H). Interestingly, silencing of eIF4E in stromal cells did not affect proliferation (Fig. 2I), indicating that eIF4E is specifically critical for malignant cells growth in multiple myeloma cells.

To examine the effects of overexpression of eIF4E on multiple myeloma, we transduced RPMI-8226 and U266 cells with lentiviral particles encoding human eIF4E with GFP as selection marker. eIF4E overexpression was confirmed by Western blotting and RT-PCR (Fig. 3A and B). Overexpression of eIF4E resulted in significant (P < 0.001) increase of multiple myeloma cell growth compared with EV control cells (Fig. 3C). Cell-cycle analysis revealed decreased population of U266 cells in G₀–G₁ (62% vs. 49%) and RPMI-8226 cells in G₀–G₁ (40% vs. 31%; Fig. 3D), and without significant effects on cell apoptosis in U266 (Supplementary Fig. S2). Overexpression of eIF4E further led to the significant increase (P = 0.004) of clonogenic multiple myeloma tumor growth with expansion of clonogenic colony numbers (22.3 ± 2.5 vs. 40.3 ± 2.1 in U266 and 23 ± 1 vs. 39.3 ± 5.7 in RPMI-8226; Fig. 3E).

To determine the role of high expression of eIF4E in multiple myeloma tumor growth in vivo, we generated subcutaneous multiple myeloma xenografts in SCID/bg mice using the U266-CT-shRNA and U266-eIF4E-shRNA cells. Mice injected with U266-eIF4E-shRNA cells showed a significant (P < 0.001) and up to 90% decreased tumor size after 23 days (Fig. 4A). To determine whether overexpressed eIF4E induces multiple myeloma tumor growth in vivo, we injected EV-U266 or eIF4E-OE-U266 cells subcutaneously. In contrast with EV-U266 tumors, animals bearing eIF4E-OE-U266 xenografts showed a significant increase (P < 0.001) of tumor growth (180%) after 13 days (Fig. 4B). Next, we wanted to explore whether the further growth of already established tumors depends on eIF4E by using a tet-on–inducible knockdown system. We generated subcutaneous multiple myeloma xenografts in SCID/bg mice using the inducible U266-tet-CT-shRNA and U266-tet-eIF4E-shRNA cells. Doxycycline or vehicle treatment was started after the tumor was established on day 11. In contrast with vehicle-treated U266-tet-on-eIF4E-shRNA or doxycycline-treated U266-control-tet-on-shRNA tumors, doxycycline-treated animals bearing U266-tet-eIF4E-shRNA xenografts showed a significant inhibition (P < 0.001) of tumor growth by 80% after 19 days. The inhibition of tumor growth correlated with the doxycycline-induced eIF4E knockdown, further confirming the critical role of eIF4E in multiple myeloma tumorigenesis (Fig. 4C). Immunohistochemical staining of tumors confirmed the decrease of eIF4E expression in doxycycline-treated mice bearing U266-tet-eIF4E-shRNA tumors compared with tumors of vehicle-treated or non–doxycyclin-treated mice (Fig. 4D).

In multiple myeloma, several transcription factors, such as c-MYC, IRF4, and C/EBPβ, are essential for malignant tumor growth and involved in tumorigenesis (30–32). The expression of c-MYC and C/EBPβ is highly regulated at the translational level (2, 6, 33). c-MYC is activated in multiple myeloma cells (30, 34), and targeting MYC by shRNA induces cell death in myeloma cell lines (31). We analyzed whether eIF4E regulates C/EBPβ, c-MYC, and IRF4, all critical for multiple myeloma cell proliferation. Indeed as shown by Western blotting, eIF4E KD and overexpression in multiple myeloma cells up regulated and down regulated expression of eIF4E, respectively (Fig. 5A). The regulation at the translational level is further supported by quantitative RT-PCR, showing that c-MYC mRNA level is not affected by eIF4E knockdown when c-MYC protein level is downregulated (Fig. 5B).

It is known that the expressions of proteins, such as c-MYC, Cyclin D1, C/EBPβ, and VEGF, are regulated at the translational level by eIF4E (3–6). Because these factors are critically involved in multiple myeloma cell growth and survival, eIF4E might be an attractive target for antimyeloma treatment (17). Indeed, we found significantly elevated eIF4E levels in myeloma cell lines, including H929, U266, MM.1S, RPMI 8226, OPM2, and primary myeloma cells compared with CD138⁻ cells on both protein and mRNA levels. In contrast, normal plasma cells, which usually do not actively proliferate and exhibited limited protein translation activity, showed undetectable eIF4E protein levels (Fig. 1), suggesting that eIF4E might be a specific target to inhibit protein translation in multiple myeloma. In accordance with that, we found that eIF4E knockdown inhibits multiple myeloma cell growth and decreases colony formation in U266 and RPMI-8226 cells. In contrast, knockdown of eIF4E in BMSCs did not affect proliferation, suggesting that nonmalignant cells are less dependent on protein translation. Interestingly Attar-Schneider and colleagues reported that bone marrow mesenchymal stem cells from patients promoted eIF4E/eIF4GI expression in multiple myeloma cells, causing elevated translational activity, enhanced expression of oncogenic signals, and resulted in increase of proliferation and death (35). On the other hand, introduction of ectopic eIF4E significantly increased the in vitro growth as well as colony formation of multiple myeloma cells. The role of eIF4E in multiple myeloma tumorigenesis was confirmed in a human multiple myeloma xenograft mouse model, showing that knockdown of eIF4E inhibits multiple myeloma tumor growth, whereas overexpression of eIF4E accelerated tumor progression. In addition, we established an inducible shRNA human multiple myeloma xenograft mouse model in which eIF4E knockdown could be precisely turned-on or -off upon doxycycline treatment or withdrawn. In this model, the tumor growth curves separate with knockdown of eIF4E, reflecting a significant tumor growth inhibition associated with knockdown of eIF4E. As expected, eIF4E did not significantly affect apoptosis because protein translation is primarily required for cell proliferation. Taken together, these results strongly suggest that eIF4E is essential for the maintenance of the transformed phenotype of myeloma cells both in vitro and in vivo.

Prior and current studies of the effects of eIF4E on proteins regulating cell growth and cell cycle revealed that c-MYC, IRF4, and C/EBPβ protein expression level correlates with eIF4E expressions in multiple myeloma cells (23, 32). In contrast, normal plasma and CD138⁻ cells showed very low or no detectable expression levels of eIF4E as well as c-MYC, IRF4, and C/EBPβ. Knockdown and introduction of ectopic eIF4E resulted in down- and upregulation of MYC, IRF4, and C/EBPβ, suggesting that those factors depend on eIF4E-mediated protein translation (Fig. 5A). In multiple myeloma, MYC, IRF4, and C/EBPβ are essential for malignant tumor growth. c-MYC is activated in multiple myeloma cells (30, 34), and targeting MYC by shRNA induces cell death in myeloma cell lines (31). Further, MYC overexpression in tumorigenesis is linked to increased eIF4E activity and upregulated protein synthesis (33, 36). IRF4 was identified as an oncogene associated with the chromosomal translocation t(6;14) (p25;q32) (37) controlling multiple myeloma survival (31), and overexpression of IRF4 is an adverse prognostic survival marker (31, 38). IRF4 is a target of lenalidomide in multiple myeloma (23, 39). Interestingly, in myeloma, IRF4 and c-MYC mutually reinforce the expression of each other (31). Taken together, these data suggest that high expression or at least a critical threshold of eIF4E is required for protein translation of transcription factors c-MYC, IRF4, and C/EBPβ and therefore for malignant growth of multiple myeloma cells. The use of inhibitors that directly target the translation initiation complex eIF4F shows a promise effects in multiple myeloma (13–16). Further, eIF4E also exhibits oncogenic potential that arises from its critical roles in the nuclear export and cytosolic translation of oncogenic transcripts. Therefore, approaches using eIF4E nuclear pore complex inhibitors, such as KPT-330, present a promising approach. In conclusion, our studies show that high or at least a critical threshold level of eIF4E is required for myeloma cell growth in order to maintain a sufficient protein translation of cap-dependent transcription factors. This results in a higher sensitivity and dependence of multiple myeloma cells on protein translation in contrast to nonmalignant cells. Therefore, protein translation provides an ideal selective therapeutic target allowing the simultaneous and selective reduction of numerous potent growth and survival factors critical for malignant growth.

S. Lentzsch reports receiving a commercial research grant from Celgene and other commercial research support from MMP13 ROI; has received honoraria from the speakers bureau from Axiom, BMS, CCO, and Takeda; and is a consultant/advisory board member for Janssen. No potential conflicts of interest were disclosed by the other authors.

Conception and design: S. Li, U. Hengst, S. Lentzsch

Development of methodology: S. Li, S. Lentzsch

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Li, J. Fu, M.Y. Mapara, S. Raza, S. Lentzsch

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Li, J. Fu, C. Lu, M.Y. Mapara, S. Lentzsch

Writing, review, and/or revision of the manuscript: S. Li, M.Y. Mapara, S. Lentzsch

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Lu, S. Lentzsch

S. Li, J. Fu, and S. Lentzsch were supported by NIH grant R01CA175313, C. Lu and M.Y. Mapara were supported by NIH grant R01HL93716, and U. Hengst was supported by NIH grant R01MH096702.

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