Abstract
Background: Proteasome inhibitors (PI) are remarkably effective in relapsed and refractory myeloma but the origin of this peculiar sensitivity remains unclear. Myeloma is dependent on the unfolded protein response (UPR) and its regulator, transcription factor XBP-1. PI perturbs the unfolded protein response (UPR) by inhibition of the 26S proteasome-the main pathway for protein degradation. We hypothesize that the dependence on the UPR and XBP-1 mediates sensitivity to PI and the level of XBP-1 correlates with sensitivity to PI. The aim of this study is to correlate Bortezomib sensitivity with XBP-1 in vitro and in myeloma patients; to check the effect of manipulating XBP-1 on Bortezomib sensitivity and develop Bortezomib-resistant myeloma cell lines to ascertain the effects on XBP-1 and the UPR.Methods and Results: Sensitivity to Bortezomib was measured by growth inhibition assay. XBP-1 mRNA levels and its isoforms were measured by a two-step quantitative QPCR assay, in 6 myeloma cell lines and 17 other cancer cell lines. There is a strong inverse correlation in myeloma cell lines between total or unspliced XBP-1 with Bortezomib sensitivity (r = −0.9) but not in other cancer cell lines. 23 marrow biopsies from 11 Bortezomib-treated myeloma patients were analysed for XBP-1 expression. Myeloma cells (CD38 hi, CD14 lo, kappa or lambda light chain +ve) were purified by flow cytometry. XBP-1 levels in myeloma cell lines were manipulated by shRNA-mediated knockdown and overexpression by retroviral transduction and had little effect on Bortezomib sensitivity. Bortezomib-resistant myeloma lines were developed. The mechanism of resistance was elucidated (XBP-1, ATF6, P-EIF2a, P58 INK and immunogloblin production). Marked downregulation of XBP-1 was demonstrated.Conclusion: XBP-1 is a surrogate marker of Bortezomib sensitivity and its clinical utility is being tested now. Sensitivity to PI is related to the dependence on the UPR, reflected in the level of XBP-1. Bortezomib resistance is mediated by downregulation of the UPR.
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