Novel CRM1 Inhibitor Prevent the Nuclear Export of Beclin-1 Blocks Autophagy in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin’s lymphoma (NHL-B) with poor response to conventional chemotherapy and short survival which prompt the urgent need for novel therapeutic agents. Chromosomal Region Maintenance 1 (CRM1, Exportin 1, Xpo 1), the major mammalian export protein, plays a critical role in nuclear-cytosolic transport of multiple tumor suppressors which lose their normal function once they have exited the nucleus. We had found that CRM1 expression was higher in MCL cell lines and primary MCL cells than in normal B lymphocytes. Inhibiting CRM1 with small interfering RNA inhibited MCL cell growth. Our findings suggest that CRM1 plays a key role in the pathophysiology of MCL cells and that targeting CRM1 in MCL could have therapeutic value. Small molecule selective inhibitors of nuclear export (SINE) block XPO1-mediated nuclear export, inducing cancer cell death and sensitizing cancer cells to other cytotoxic drugs. Although the cytotoxic effects of SINE on MCL cells have now been established, the mechanism of cell death is still not fully understood. The objective of our study was to elucidate the mechanism of CRM1 inhibitor-mediated cytotoxic effects on MCL cells.

We used 8 established MCL cell lines and primary cells from 4 patients with relapsed/refractory MCL. Flow cytometry analysis with fluorescence-labeled Annexin V and propidium iodide showed that KPT-185,a KPT-SINE compound, induced MCL cells apoptosis in both time- and dose-dependent manners. However, specific knockdown of CRM1 has minimal effect on apoptosis induction. We therefore hypothesized that KPT-185 may induce other types of programmed cell death, autophagy than apoptosis. Autophagic features were determined by detecting acidic vesicular organelles (AVO) by MDC staining under fluorescence microscope after flow cytometry analysis. The autophagy related protein expressions of Beclin-1, a well-known key regulator of autophagy, and LC3-II were assessed by Western blotting. The results showed that after KPT-185 treatment autophagy declined in MCL cells and the declination was most obvious at 2 h and at 50nM. the combination of KPT-185 with chloroquine, an autophagy inhibitor that block lysosome acidification and consequent autophagosome fusion, resulted in synergistic cell death (CI 0.2-0.6) characterized by enhanced induction of both apoptosis and autophagy in MCL cells. There is no LC3-II expression being detected by Western blotting in MCL cells with low basal autophagy. As shown in Western blot and confocal microscopy, inhibiting CRM1 activity with KPT185 in MCL cells up-regulated the protein expression of Beclin-1 and trapping Beclin-1 in the nucleus where fail to promote autophagy. The same results were reported by Beth Levine that the nuclear export mutant of Beclin-1 fails to promote nutrient deprivation-induced autophagy in MCF7 cells.

In summary, our studies are the first to suggest that autophagy is a survival pathway used by MCL cells. Targeting autophagy has therapeutic potential in MCL.The CRM1 nuclear export pathway may be important in the functional regulation of autophagic growth control that is enhanced in response to combined treatment with chloroquine. Our data suggest that KPT185 combination with chloroquine provides a potential therapy for patients with MCL.