IMiD immunomodulatory compounds block C/EBP{beta} translation through eIF4E down-regulation resulting in inhibition of MM

Abstract

Immunomodulatory derivatives of thalidomide (IMiD compounds), such as pomalidomide and lenalidomide, are highly active in multiple myeloma (MM) treatment. However, the precise mechanisms of action and resistance in MM are unresolved. Here we show that IMiD compounds down-regulate CCAAT/enhancer-binding protein-β (C/EBPβ) resulting in abrogation of cell proliferation. Overexpression of C/EBPβ rescued MM cells from IMiD-induced inhibition of proliferation, indicating that C/EBPβ is critical in mediating antiproliferative effects. IMiD-induced decrease of C/EBPβ protein led to impaired transcription of interferon regulatory factor 4 (IRF4). Down-regulation of IRF4 by lenalidomide was confirmed by longitudinal studies of bone marrow samples from 23 patients obtained before and during lenalidomide treatment using CD138⁺/IRF4⁺ double labeling. In contrast to down-regulation of C/EBPβ protein, IMiD compounds did not alter C/EBPβ mRNA levels or protein stability, suggesting translational regulation of C/EBPβ. We could demonstrate that C/EBPβ protein expression is under eIF4E-translational control in MM. Furthermore, inhibition of the eIF4E-C/EBPβ axis by IMiD compounds was not observed in IMiD-resistant MM cells. However, targeting translation at a different level by inhibiting eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation overcame resistance, suggesting that this pathway is critical and might be a target to overcome drug resistance.

Figure 1

IMiD compounds down-regulate C/EBPβ in MM cells in a time- and dose-dependent manner. (A) MM.1S and OPM2 cells (6 × 10⁴/well) were cultured with lenalidomide or pomalidomide at different concentrations for 2 days. DMSO (0.01%) was used as the control treatment. DNA synthesis was measured by ³H-thymidine incorporation, and the amount of DNA synthesis in treated cells relative to the amount in control cells (as a percentage) was plotted. Results are shown as triplicates of mean ± SD. (B) MM.1S, H929, OPM2, or primary myeloma cells were cultured with DMSO, pomalidomide, or lenalidomide at the indicated concentrations for 3 days. Representative results from 3 independent experiments are shown. Cells were then lysed, and cell lysates were analyzed for C/EBPβ expression by Western blotting. β-actin expression was probed for loading control. The ratio of LAP/LIP is measured using ImageJ 1.44 software. (C-D) MM.1S cells were incubated with lenalidomide (C) or pomalidomide (D) at different concentrations with fixed time period of 4 days, or for different time periods with fixed concentration of 10μM, or with DMSO 0.01% as control. Cells were lysed and cell lysates were analyzed for C/EBPβ expression by Western blotting. β-Actin expression was probed for loading control.

Figure 2

IMiD compounds down-regulate IRF4 in MM cells in vitro and in lenalidomide-treated patients. (A) MM.1S, H929, OPM2, or primary myeloma cells were cultured with DMSO, pomalidomide, or lenalidomide at the indicated concentrations for 3 days. (B-C) MM.1S cells were incubated with lenalidomide (B) or pomalidomide (C) at different concentrations with a fixed time period of 4 days, or for different time periods with fixed concentration of 10μM, or with DMSO 0.01% as control. Cells were lysed, and cell lysates were analyzed for IRF-4 expression by Western blotting. β-actin expression was probed for loading control. (D) Double staining immunohistochemistry on paraffin-embedded marrow biopsy sections for IRF4 (black nuclear staining) and CD138⁺ (red staining) was performed before and during treatment with lenalidomide. Cells were rated for IRF4 reactivity as follows: 3 indicates strong; 2, moderate; and 1, negative/weak. The score for a sample is the sum of 100 cells (original magnification ×1000). (E) IRF4 scores of bone marrow biopsy samples, before and during treatment with lenalidomide. Lines connect scores from the same patients. (F) MM.1S cells were incubated with lenalidomide or pomalidomide for 2 or 3 days at 100μM. BLIMP1 and XBP1 expression was analyzed by Western blotting. β-actin expression was probed for loading control.

Figure 3

Overexpression of C/EBPβ induces resistance to IMiD compounds. (A) MM.1S cells (2 × 10⁶) were transfected by electroporation with either empty vector pcDNA3.1 or WT-C/EBPβ plasmids. After selection, cells were treated with pomalidomide (10μM) for 2 days. Cells were then lysed, and whole cell lysates were analyzed for C/EBPβ expression by Western blotting. β-actin expression was probed for loading control. (B) MM.1S cells overexpressing WT-C/EBPβ were treated with pomalidomide (10μM) for 2 days, and DNA synthesis was measured by ³H-thymidine incorporation. The amount of DNA synthesis in treated cells relative to the amount in control cells (as a percentage) was graphed. Results are shown as triplicates of mean ± SD.

Figure 4

IMiD compounds down-regulate C/EBPβ by targeting its protein translation. (A) MM.1S cells were cultured with pomalidomide, lenalidomide, or 0.01% DMSO (control). Total RNA was extracted from 12-hour cultures, reverse transcribed to cDNA, and used for quantitative real-time PCR. Data were analyzed according to the ΔΔC_t method. Results are shown as mRNA expression relative to control (DMSO). mRNA levels were normalized with β-actin mRNA expression as control. (B) MM.1S cells were incubated with pomalidomide (10μM) with and without cycloheximide (20 μg/mL) for 12, 24, or 36 hours. Cells were lysed, and whole cell lysates were analyzed by Western blotting for C/EBPβ. (C) MM.1S, H929, OPM2, or primary myeloma cells were cultured with lenalidomide, pomalidomide, or 0.01% DMSO as control for 3 days. Cells were lysed, and whole cell lysates were analyzed by Western blotting for eIF4E. β-Actin expression was probed for loading control. (D-E) MM.1S cells (2 × 10⁶) were incubated with lenalidomide (D) or pomalidomide (E) at different concentrations with a fixed time period of 4 days, or for different time periods with fixed concentration of 10μM, or with DMSO 0.01% as control. Cells were lysed, and whole cell lysates were analyzed for eIF4E expression by Western blotting. β-Actin expression was probed for loading control. (F) MM.1S cells were incubated with lenalidomide or pomalidomide for 12 hours, and total RNA was extracted by Trizol and followed by real-time PCR. Data were analyzed according to the ΔΔC_t method. Results are shown as mRNA fold compared with control (DMSO). (G) eIF4E knockdown cell lines were generated by lentiviral infection of MM.1S cells. 1# and 2# indicate different eIF4E shRNA sequences. Green fluorescence protein was used as control for eIF4E shRN-expressing cells. Cell lysates were analyzed by Western blotting to compare the levels of eIF4E, C/EBPβ, and IRF4. β-actin expression was probed for loading control.

Figure 5

Protein translation is not affected in IMiD-resistant MM cells. (A) RPMI 8226 cells (3 × 10⁴) were cultured with different concentrations of lenalidomide, pomalidomide, or DMSO as control for 2 days. DNA synthesis was measured by ³H-thymidine incorporation. Results are shown as triplicates of mean ± SD. (B) RPMI 8226 cells were cultured with lenalidomide, pomalidomide, or DMSO for 2 or 3 days. Cells were lysed, and whole cell lysates were analyzed for eIF4E, C/EBPβ, and IRF4 by Western blotting. β-actin expression was probed for a loading control.

Figure 6

Combination of mTOR inhibitors and IMiD compounds might overcome resistance to IMiD compounds. (A) IMiD compounds inhibit the translation of C/EBPβ by down-regulation of eIF4E protein. Rapamycin targets the mTOR complex by inhibiting 4EBP1 phosphorylation, resulting in sequestering of eIF4E. This blocks the assembly of the translational complex and leads to decreased C/EBPβ translation. (B) MM.1S cells were incubated with rapamycin or DMSO 0.01% as control for 2 days. Cells were lysed, and lysates were analyzed for both total-and phospho-4EBP1 as well as for eIF4E by Western blot. β-actin expression was probed for loading control. (C) RPMI 8226 cells (3 × 10⁴) were cultured with rapamycin or pomalidomide alone or in combination for 2 days. DMSO 0.01% treatment was used as a control. DNA synthesis was measured by ³H-thymidine incorporation. Results are shown as triplicates of mean ± SD.