Ribosomal Biogenesis and Translational Flux Inhibition by the Selective Inhibitor of Nuclear Export (SINE) XPO1 Antagonist KPT-185

Abstract

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the aberrant expression of several growth-regulating, oncogenic effectors. Exportin 1 (XPO1) mediates the nucleocytoplasmic transport of numerous molecules including oncogenic growth-regulating factors, RNAs, and ribosomal subunits. In MCL cells, the small molecule KPT-185 blocks XPO1 function and exerts anti-proliferative effects. In this study, we investigated the molecular mechanisms of this putative anti-tumor effect on MCL cells using cell growth/viability assays, immunoblotting, gene expression analysis, and absolute quantification proteomics. KPT-185 exhibited a p53-independent anti-lymphoma effect on MCL cells, by suppression of oncogenic mediators (e.g., XPO1, cyclin D1, c-Myc, PIM1, and Bcl-2 family members), repression of ribosomal biogenesis, and downregulation of translation/chaperone proteins (e.g., PIM2, EEF1A1, EEF2, and HSP70) that are part of the translational/transcriptional network regulated by heat shock factor 1. These results elucidate a novel mechanism in which ribosomal biogenesis appears to be a key component through which XPO1 contributes to tumor cell survival. Thus, we propose that the blockade of XPO1 could be a promising, novel strategy for the treatment of MCL and other malignancies overexpressing XPO1.

Fig 1. KPT-185 induced cell growth inhibition, apoptosis, and cell cycle arrest in MCL.

Cells of wt-p53 lines Z138 and JVM-2 or the mt-p53 lines MINO and Jeko-1 were plated at a density of 2 x 10⁵ cells per mL and treated with the indicated concentrations of KPT-185. After 72 h, the effect on cell growth was assessed by the MTS test. Inhibition of cell growth is displayed as percent absorbance of untreated control cells. The concentrations of KPT-185 at which cell growth is inhibited by 50% (i.e., the IC50 concentration) was18 nM for Z138, 141 nM for JVM-2, 132 nM for MINO, and 144 nM for Jeko-1 (A). The percentage of dead cells was quantified by the tTypan blue dye exclusion method. The effective dose for cell killing of approximately 50% of the population (i.e., the ED50 concentration) after a 72-h exposure to KPT-185 was 57 nM for Z138 cells, 770 nM for JVM-2 cells, 917 nM for MINO cells, and 511 nM for Jeko-1 cells (B). Z138 cells were exposed to 50, 100, and 200nM KPT-185 for 24, 48, 72 and 96 h and assessed for cell growth as described in A. (C) The percentage of G₀-G₁, S, and G₂-M phase cells in the viable cell population was assessed at 48 h by PI flow cytometry (histograms for representative samples are shown). Graphs show the mean ± SD of results of three independent experiments (D). The percentage of apoptotic MCL cells was quantified by annexin V/PI staining 72 h following the KPT-185 treatment as described above. The effective dose for cell killing of approximately 50% of the population (i.e., the ED50 dose) after a 72-h exposure to KPT-185 was 62 nM for Z138 cells, 910 nM for JVM-2 cells, 67 nM for MINO cells, and 618 nM for Jeko-1 cells (E). Graphs show the mean ± SD of results of three independent experiments. *p<0.05, **p<0.01.

Fig 2. KPT-185 modulates XPO1 and Bcl-2 family members in MCL cells.

After an 18-h treatment with KPT-185 (50 nM for Z138, 200 nM for JVM2, MINO and Jeko-1), cells were lysed and analyzed by immunoblot. The results are representative of three independent experiments, and the intensity, compared to that of β-actin, of the immunoblot signals was quantified using ImageJ software.

Fig 3. KPT-185 represses cell viability and cell cycle progression independent of p53-status.

JVM2 cells stably transfected with control shRNA (shC) or p53-specific shRNA (shp53) were treated with 100 nM KPT-185. (A) The cell viability was assessed by the Trypan blue dye exclusion method and displayed as percent of untreated control cells at 72 h. The percentage of G₀-G₁, S, and G₂-M phase cells in the viable cell population was assessed at 48 h by PI flow cytometry (histograms for representative samples are shown). Graphs show the mean ± SD of results of three independent experiments. (B) After an 18-h treatment of KPT-185, protein expression levels of CDC25C, BRCA1, CDK1 were analyzed by immunoblot as described in Fig 2. The results are representative of two independent experiments.

Fig 4. KPT-185 diminishes HSP70, HSF1 and p-HSF1^Ser326 expression.

After an 18-h treatment of KPT-185 (i.e., 50 nM for Z138, 100 nM for JVM2, MINO, and Jeko-1), the cells were lysed and analyzed HSP70 (A) and HSF1 and p-HSF1Ser326 expression (B) by immunoblot as described in Fig 2. α-tubulin was used as a loading control. The intensity, compared to that of α-tubulin or p-HSF1 / HSF1 levels after background subtraction were obtained using ImageJ software. The results are representative of three independent experiments.α.

Fig 5. KPT-185 targets multiple signaling pathways in MCL cells.

(A) After an 18-h treatment of KPT-185 (i.e., 50nM for Z138 and MINO, 100 nM for JVM2 and Jeko-1), Cyclin D1 and its downstream target phosphorylated Rb expression were analyzed by immunoblot. (B, C) After an 18-h treatment of KPT-185 (i.e., 50nM for Z138 and 100 nM for JVM2, MINO, and Jeko-1), PIM1 and p27KIP (B), phospho-S6, and phospho-4EBP1 (C) were analyzed by immunoblot. For c-Myc expression analysis, cells were treated with 500 nM KPT-185. α-tubulin was used as a loading control. The intensity, compared to that of α-tubulin or p-S6K / S6K, p-4E-BP1 / 4E-BP1 levels after background subtraction were obtained using ImageJ software. The results are representative of three independent experiments.

Fig 6. XPO1 inhibition by KPT-185 in MCL.

XPO1 inhibition by KPT-185 impairs ribosomal biogenesis, in addition to blocking p53 degradation and inhibiting CyclinD1, c-Myc, and PIM1 translation in MCL. Please refer to the discussion for further details.