Abstract
Background
Chromosomal region maintenance (CRM1), also known as exportin 1 (XPO1) plays an important role in the nuclear-cytoplasmic shuttling. The nuclear export receptor, XPO1, is considered as a regulator of subcellular distribution of several proteins involved in the regulation of centrosome duplication such as nucleophosmin (NPM), breast and ovarian cancer susceptibility protein 1 (BRCA1) and many tumor suppressor proteins (p53, p21, FOXO and pRB). Furthermore, XPO1 is required for the export of assembled ribosomal subunits (60S & 40S) from the nucleolus back into the cytoplasm. Inhibition of XPO1 triggers a ribosomal stress response that may result in the death of transformed cells with stressed ribogenesis. Silencing of XPO1 is reported to be synthetically lethal in MM cells, however the mechanisms that mediate this effect are not fully elucidated.
Methods and Results
To determine the effect of XPO1 inhibition in MM, cells were exposed to different doses of KPT330 (Karyopharm), a selective inhibitor of nuclear-cytoplasmic transport by irreversibly binding to the XPO1 cargo recognition site. Nanomolar concentrations of KPT330 (50-150 nM) induced apoptosis (Puma up-regulation and caspase 3 cleavage) and suppressed the proliferation of myeloma cell lines MM1S, OPM2 while KMS11 cells were more resistant. Mechanistically treatment with KPT330 up-regulated the expression of p53, as well as p21, p27 and MDM2 at the protein and RNA levels and significantly decreased the expression of c-Myc and IRF4. Cognizant of the role XPO1 in cytoplasmic-nuclear shuttling of ribosomal subunits, we reasoned that c-Myc downregulation and p53 induction in MM cells exposed to KPT330 results from ribosomal biogenesis stress. Therefore we analyzed the cellular co-localization of ribosomal proteins (RPL5, RPL11), c-Myc and MDM2 in presence of KPT330. A shifting of RPL11 and RPL5 from the nucleolus to the nucleoplasm and cytosol was observed in presence of KPT330 where they accumulated in ribosome-free cellular fractions. Co-immunoprecipitation studies showed that RPL11 and RPL5 released from the nucleolus bind MDM2 and c-Myc. This binding of RPL11 and RPL5 to MDM2 and c-Myc is known to suppress their function and expression. Therefore our data explain the suppression of MDM2 E3 ligase activity with p53 stabilization and reduction of c-Myc at the post-transcriptional levels. Study of ribosome fractions with sucrose gradients showed that in the presence of KPT330, 40S and the polysomes were completely suppressed while 60S and 80S subunits were significantly downregulated in OPM2 and MM1S cell lines. Consistent with the disruption of ribosomal function and the translational machinery, c-Myc mRNA levels were significantly decreased in 40S, 60S and 80S fractions after treatment with KPT330. Confirming the role of the ribosomal stress response in KPT330-mediated MM cells' death, silencing of ribosomal proteins RPL11 or RPL5 fully protected them from KPT330 cytotoxicity. Furthermore silencing RPL11 or RPL5, suppressed the effects of KPT330 on MDM2, p53, p21 and c-MYC.
Conclusion
Inhibition of XPO1 induces a perturbation in the ribosome subunits transfer, disruption of ribosomal assembly and the induction of a ribosomal stress response in MM cells. Perturbation on the nucleolar-cytoplasmic shuttling by KPT330 and the targeting of the translational factory represents a novel therapeutic approach in multiple myeloma.
Disclosures:
No relevant conflicts of interest to declare.
Ribosomal protein S14 unties the MDM2–p53 loop upon ribosomal stress
