Investigation of the Molecular Action of Bortezomib Treatment In Multiple Myeloma

Abstract 5038

Bortezomib (Velcade®) is a 26S proteasome inhibitor currently used for the treatment of relapsed multiple myeloma (MM) and more recently mantle cell lymphoma patients. Clinical trials are under way to evaluate its efficacy in other hematological malignancies diseases including diffuse large B cell lymphoma (DLBCL). Although it was widely accepted that bortezomib acts as a NF-κB inhibitor, this view has recently been challenged by work by Hideshima et al. (Blood 2009) who found that treated myeloma cells actually displayed down-regulation of NF-κB inhibitors (IκBα), increased activity of pro-NF-κB molecules (RIP2 and IKKβ) as well as increasing levels of NF-κB DNA binding. Additionally, although bortezomib has a potent anti-tumor activity, not all MM patients are responsive. The identification and understanding of the molecular mechanisms leading to bortezomib-resistance will help with the development of new approaches and will improve the treatment of these patients. In order to investigate the direct effect of bortezomib on lymphomas, we treated different cell lines with this drug and levels of cell proliferation and apoptosis were measured. Bortezomib was shown to significantly inhibit proliferation and promote apoptosis in MM cell lines (RPMI-8226, NCI-H929, JJN-3 and Thiel) and DLBCL cell lines (SU-DHL-4 and SU-DHL-10, HLY-1, HLB-1 and RIVA). Since MM cell lines were shown to be the most sensitive to the bortezomib, we selected them to carry out further studies. We next employed whole genome expression arrays (Affymetrix U113plus2.0) and microRNA arrays to identify molecules associated with treatment in these cell lines. Using unsupervised cluster analysis, we observed that the cells treated with bortezomib have a distinct microRNA and gene expression profile from their counterpart controls. Additionally, we used a shRNA whole-genome library to carry out a loss-of-function screen for cells resistant to bortezomib-induced apoptosis. The RPMI-8226 cell line was transduced with the shRNA library. One half of the cells was then treated with bortezomib. Relative gene expression levels were measured by hybridizing samples to custom-made arrays. Unsupervised cluster analysis identified a number of shRNAs which might be involved in mechanisms of resistance to bortezomib. Further functional studies will be necessary to investigate the role of these microRNAs in the mechanism of action of bortezomib.