Discovery and validation of the nuclear export protein CRM1 (XPO1) as a new target for the treatment of renal cell carcinoma (RCC).

Abstract

411 Background: The currently available targeted therapies for RCC have had limited success, so it is imperative to discover new therapeutic approaches for metastatic RCC. In a search for new such targets, we have identified inhibitors of CRM1 (XPO1, exportin1), the major karyopherin that mediates the export of most tumor suppressor proteins from the nucleus. To expand our initial work on the first generation of CRM1 inhibitors, we now evaluate KPT-330, an orally available Selective Inhibitor of Nuclear Export (SINE), the best tolerated among a series of CRM1 inhibitors that is currently in phase I clinical trials. Methods: The RCC cell lines (Caki-1 and 786-O) and a primary normal human kidney (NHK) cell line were treated with KPT-330, and MTT assays were performed. The cells were subjected to immunofluorescence and immunoblotting for appropriate proteins. Caki-1 xenograft mice were treated with KPT-330 for 15 days, and tumor volume was assessed. Results: KPT-330 selectively attenuated CRM1 levels and caused dose-dependent toxicity (EC50 < 1 µM) through apoptosis in all RCC cells. In untreated RCC cells, p21 was localized in the nucleus, where it likely functions as a cell cycle arrest protein, and in the cytosol, where it functions as an anti-apoptotic protein. However, in NHK cells, p21 was confined to the nucleus. KPT-330 increased p53 and p21 and confined them to the nucleus in both NHK and RCC cells. KPT-330 given orally inhibited RCC growth in xenograft mice (85.3 % inhibition, p < 0.001). Moreover, KPT-330 showed synergism with a Bcl-2 inhibitor ABT-737 in vitro, indicating the potential for combination therapy with a CRM1 inhibitor and Bcl-2 inhibitor. In vivo studies to test the combination of KPT-330 with Bcl-2 inhibitors are ongoing. Conclusions: We introduce a new therapeutic approach for RCC treatment based on the inhibition of the nuclear export of key tumor suppressors. Inhibition of CRM1 causes forced nuclear retention, and thereby activation, of several key p53-pathway proteins, leading to apoptosis in RCC cell lines in vitro and tumor growth inhibition in vivo.