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

<div><p>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.</p></div

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

<p>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.</p

Pathway analysis of genes in JVM-2 cells transfected with control shRNA or p53-specific shRNA consistently altered by KPT185.

<p>The significance of the association between the data set and the canonical pathway was determined based on a ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway and a <i>p</i>-value calculated using Fischer’s exact test determining the probability that the association between the genes in the data set and the canonical pathway is due to chance alone.</p><p>Pathway analysis of genes in JVM-2 cells transfected with control shRNA or p53-specific shRNA consistently altered by KPT185.</p

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

<p>JVM2 cells stably transfected with control shRNA (shC) or <i>p53</i>-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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137210#pone.0137210.g002" target="_blank">Fig 2</a>. The results are representative of two independent experiments.</p