MAF Protein Elicits Innate Resistance To Bortezomib In Multiple Myeloma

Multiple myeloma (MM) is a malignancy of terminally differentiated clonal plasma cells displaying significant molecular heterogeneity with 7 subgroups defined by gene expression profiling (GEP). Our previous work showed that MS and MF subgroups have been associated with inferior survival (Zhan et al, blood 2006). Furthermore, clinical studies have demonstrated that the addition of the proteasome inhibitor (PI) bortezomib (Bzb) to high dose melphalan based regimens provided a major advantage to patients in MS subgroup (Barlogie NJE 2006, Blood 2008; Pineda-Roman et al BJH 2008), while patients in the MF subgroups did not benefit from Bzb (Nair, Blood 2010). These findings led us to hypothesize that overexpression of MAF protein confers innate resistance to Bzb. In the present study, we assessed the ability of MAF to influence the innate resistance to Bzb and identify the molecular mechanism underlying the resistance of Bzb in high MAF-expressing patients.

To investigate association of the limited therapeutic effect of Bzb with molecular subgroup of myeloma, we established the IC50 of Bzb in 24 myeloma cell lines (MMCL) belonging to different GEP-based molecular subgroups. IC50 concentration were higher (>25nM) in 7 of 9 MAF and in all 5 MAFb MMCL, and >40 nM in 5 MAF and one MAFb MMCL, which expressed the highest levels of MAF protein, as determined by immunoblotting analysis. In contrast, Bzb IC50 levels were lower (7.5-20 nM) for the MMCL belonging to the other molecular subgroups. These results indicate that high MAF expression in myeloma cells may contribute to primary resistance to Bzb. Mechanistically, immunoblotting analysis demonstrated that exposure to Bzb resulted in increased MAF protein levels. These results suggested that Bzb prevents the degradation of MAF protein in myeloma cells.

To further confirm that Bzb-induced stabilization of MAF protein confers resistance to Bzb, we overexpressed MAF cDNA in myeloma cells lacking MAF expression, and silenced MAF expression in myeloma cells expressing high level of MAF mRNA and protein. MM cells were infected with Lentiviral vector containing MAF cDNA or with empty vector, and stable clones selected with puromycin, designated as MM^maf and MM^EV, respectively. qPCR and immunoblotting analysis showed that expression of MAF mRNA and protein in MM^maf cells were significantly higher (1.8x10⁵-fold) than in MM^EV cells. The functionality of ectopic MAF protein was confirmed by qRT-PCR analysis of downstream target genes; the mRNA level of E-cadherin was higher in MM^maf cells than those of MM^EV (1.42-fold, p<0.01). MTT assay showed that the proliferation rate of MM^maf cells was 53% higher than that of MM^EV cells (p <0.001). Similar results were observed in other two MM cell lines that transiently ectopic expressed MAF. Moreover, MM^maf showed higher IC50 of Bzb than that of MM^EV , indicating that increase MAF protein in myeloma cells reduces sensitivity to Bzb.

We further generated loss of functional MAF by silencing MAF expression in MAF positive myeloma cells using shRNA specific to maf mRNA (shMAF) by lentiviral expressing system. shMAF infected myeloma cells had 75% lower levels of MAF mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, significantly decreased integrin E-cadherin (9.1-fold), cyclin D2 (4.99x10⁵-fold), and CCR1 (4.9-fold) levels were observed in these cells, compared with the cells infected with control viral vector. Silencing MAF expression significantly decreased proliferation of myeloma cells (81.9% decrease, p=2.5E-6). Moreover, Bzb treatment of the cells infected with shMAF lead to 53.1%, inhibition (P=3.3E-8) of proliferation compared with control cells. Taken together, our results indicate that high expression of MAF protein confers myeloma primary resistance to Bzb, and Bzb induces stabilization of MAF protein further increases resistance to Bzb, providing the molecular rational for therapeutic strategy for high-MAF expressing myeloma patients.