Abstract
BackgroundMultiple myeloma (MM) patients with t(14;20) have a poor prognosis and their outcome has not improved following the introduction of bortezomib (Bzb). The mechanism underlying the resistance to proteasome inhibitors (PIs) for this subset of patients is unknown.MethodsIC50 of Bzb and carfilzomib (CFZ) in human myeloma cell lines (HMCLs) were established by MTT assay. Gene Expression profile (GEP) analysis was used to determine gene expression in primary myeloma cells. Immunoblotting analysis was performed for MAFb and caspase family proteins. Immunofluorescence staining was used to detect the location of MAFb protein in MM cells. Lentiviral infections were used to knock-down MAFb expression in two lines. Apoptosis detection by flow cytometry and western blot analysis was performed to determine the molecular mechanism MAFb confers resistance to proteasome inhibitors.ResultsWe found high levels of MAFb protein in cell lines with t(14;20), in one line with t(6;20), in one with Igλ insertion into MAFb locus, and in primary plasma cells from MM patients with t(14;20). High MAFb protein levels correlated with higher IC50s of PIs in MM cells. Inhibition of GSK3β activity or treatment with Bzb or CFZ prevented MAFb protein degradation without affecting the corresponding mRNA level indicating a role for GSK3 and proteasome inhibitors in regulation of MAFb stability. Silencing MAFb restored sensitivity to Bzb and CFZ, and enhanced PIs-induced apoptosis and activation of caspase-3, − 8, − 9, PARP and lamin A/C suggesting that high expression of MAFb protein leads to insensitivity to proteasome inhibitors.ConclusionThese results highlight the role of post-translational modification of MAFb in maintaining its protein level, and identify a mechanism by which proteasome inhibitors induced stabilization of MAFb confers resistance to proteasome inhibitors, and provide a rationale for the development of targeted therapeutic strategies for this subset of patients.
Highlights
Multiple myeloma (MM) patients with t(14;20) have a poor prognosis and their outcome has not improved following the introduction of bortezomib (Bzb)
The highest level of MAFb mRNA was seen in SACHI and EJM, both harboring t(14;20); intermediate levels of MAFb were seen in three cell lines; OPM-2 with t(4;14), XG2 with Eμ enhancer of immunoglobulin light chain (IGL) inserted near MAFb in 20q11 [10] and L363 with t(6;20) [7]
In this study, we demonstrate that cell lines with high levels of MAFb protein due to t(14;20) or Igλ insertion of 20q11 are resistant to Bzb and CFZ, and the lack of sensitivity of these cell lines to Bzb or CFZ is similar to human myeloma cell lines (HMCLs) harboring a t(14;16) which have high C-MAF levels [16]
Summary
Multiple myeloma (MM) patients with t(14;20) have a poor prognosis and their outcome has not improved following the introduction of bortezomib (Bzb). The MF molecular subgroup defined by Gene Expression Profiling (GEP) at the University of Arkansas for Medical Sciences (UAMS) includes cases with overexpression of MAFb resulting from t(14;20) and C-MAF from t(14;16) [1]. Translocation t(14;20) occurs in approximately 1 to 2% of MM [2,3,4], and is associated with poor prognosis [3, 4] with the development of extramedullary disease [5] and primary plasma cell leukemia [6]. Our previous work showed that t(4;14) and MF subgroup [including cases with a t(14:16) and t(14;20)] have an inferior survival [12, 13], and that the addition of Bortezomib (Bzb) to high dose melphalan
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