Induction of Raf-1 Kinase Inhibitory Protein (RKIP) by the Proteasome Inhibitor NPI-0052 and Reversal of B-NHL Resistance to Apoptosis.

Patients with B-cell malignancies initially respond to conventional cytotoxic therapies. However, many patients experience relapses and recurrences. Novel therapeutics are being explored in the reversal of resistance such as TRAIL. TRAIL has been shown to be selectively cytotoxic to few tumors and both TRAIL and agonist TRAIL-R1 (DR4) and TRAIL-R2 (DR5) antibodies are currently being explored clinically in unresponsive cancer patients. We have recently found that treatment of TRAIL resistant B-NHL cell lines such as Ramos with the novel proteasome inhibitor NPI-0052 (Nereus Pharmaceuticals) sensitizes tumor cells to TRAIL-induced apoptosis. The concentration of NPI-0052 achieving optimal sensitization to TRAIL was in the range of 1–5 nM. In contrast 400 fold more VelcadeTM (bortezomib) was necessary to achieve a similar level of apoptosis. The mechanism of NPI-0052- mediated sensitization to TRAIL was examined. Treatment of Ramos with NPI-0052 inhibited significantly the constitutively activated NF-κB concomitantly with inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) resulting in up-regulation of DR5 expression. The role of YY1 in NPI-0052-induced sensitization to TRAIL was corroborated by the use of YY1siRNA as treatment with the YY1siRNA mimicked NPI-0052 induced DR5 up-regulation and sensitization to TRAIL. Studies examining the inhibition of the NF-κB pathway by NPI-0052 revealed a novel finding demonstrating the induced expression of the metastatic suppressor gene, Raf-1 kinase inhibitory protein (RKIP). RKIP has been reported to inhibit NF-κB activity and, thus, its induction by NPI-0052 contributed to the inhibition of NF-κB activity. The role of RKIP in NPI-0052 induced sensitization to TRAIL was corroborated by the use of RKIPsiRNA and transfectants mimicked NPI-0052 mediated inhibition of YY1, DR5 upregulation and sensitization to TRAIL mediated apoptosis. The above findings suggest that NPI-0052-induced inhibition of NF-κB results at least from two distinct mechanisms, namely, inhibition of p-IκBα degradation by the proteasome and by the induction of RKIP expression. The apoptosis induced by combination of NPI-0052 and TRAIL was the result of the activation of the type II mitochondrial pathway. It has been reported that the RKIP levels are reduced in many tumors (e.g. breast, prostate, ovarian) and the expression levels are of prognostic significance. Hence, the present findings demonstrating NPI-0052 induced RKIP expression may be therapeutically relevant in the prevention of metastasis. In addition, NPI-0052 should facilitate the reversal of tumor resistance when used in combination with subtoxic levels of cytotoxics.