Identification of Biomarkers Associated with MAF-Mediated Resistance to Proteasome Inhibitors in t(14;16) Multiple Myeloma

Introduction: Multiple myeloma (MM) patients with t(14;16)(p16;q32) leading to high expression of C-MAF mRNA and protein have a poor prognosis and have not benefitted from the recent advances in MM therapy. Our previous work showed that posttranslational modification up-regulated C-MAF (ASH Abstract # 281, ASH 2013) and MAF-B proteins (ASH Abstract #2091, 2014) conferred innate resistance to proteasome inhibitors (PI). In order to identify the molecular mechanism underlying this resistance in high C-MAF and MAFB-expressing patients, the present study investigated the downstream targets regulated by C-MAF in MM cells and analyzed the clinical association of target genes using a C-MAF-loss and gain of function cell line model as well as primary plasma cells from MM patients.

Methods : To elucidate the molecular mechanism underlying the resistance of MAF expressing cells to PIs, we silenced the C-MAF gene in 3 MM cell lines that highly expressed C-MAF mRNA and protein using lentiviral shRNA. Annexin V staining and analyzed by FACS were used to determine the effect of manipulating MAF on PI-induced apoptosis and activation of the caspase family. Gene expression prolife  (GEP) was utilized to determine downstream target genes regulated by MAF. To identify the proteins associated with C-MAF, we used mass spectrometry (MS)-based quantitative proteomics with stable isotope labeling by amino acids in cell culture (SILAC). 

Results:   Our results demonstrated that  apoptotic cell numbers were higher in shMAF cells, compared to shControl cells when cultured in the presence of serial concentrations of bortezomib (BZB) or carfilzomib (CFZ), indicating that silencing MAF expression enhances PI-induced apoptosis. The Annexin V + cells numbers were also higher in shMAF compared to shControl when cells were treated with BZB or CFZ in co-culture with bone marrow stromal cells. Furthermore, knockdown of C-MAF expression augmented the PI-induced activation of the caspase family with an increase in the cleavage fragments of caspases-3, -7, -8, PARP and LaminA/C. In contrast, PI-induced apoptotic numbers were lower in XG1/MAF, a stably induced MAF overexpressing MM cell line, than in XG1/EV, the control cell line carrying an empty vector when cultured in serial concentrations of BZB and CFZ. Similarly, the caspase cleavage fragments, PARP and lamin A/C were decreased in XG1/MAF compared with XG1/EV in response to BZB and CFZ. These results suggest that MAF confers MM resistance to PI by preventing the PI-induction of apoptosis.

To further identify the downstream target genes regulated by C-MAF, we analyzed the gene expression between shMAF and shControl MM cells by microarray analysis  and identified more than 150 genes differentially expressed. Of them, ATP2C1, CCNE2 , CCND2, ELK4 ,  IGFBP3, SUPT16  and TUBA1A were significantly  (p=5.62e-5) lower in shMAF than shControl cells indicating that these genes might be regulated by C-MAF. Furthermore, expression of these genes in primary CD138 plasma cells were significant higher (p<0.05) in the UAMS molecular MF subgroup than other UAMS molecular subgroups. A number of genes were also expressed significantly lower (p=5.62e-5) in shMAF cells compared to shControl cells.  The clinical association of these genes was analyzed and showed that the expression of IL10Ra (p<0.05), PAXBP1PAX3 (p=0.0032), USP1 (p=0.0038), BNIP3 (p<0.05), ACSL (p=0.009) and SP3 (p=0.018) in CD138 plasma cells was associated with poor overall survival in patients from the MF subgroup indicating that these targets may play a central role in MAF regulated proliferation of MM cells.

To identify the proteins associated with C-MAF, we used MS-SILAC and identified more than 20 proteins as differentially expressed between MAF positive cell lines and shMAF cell lines .  Western blotting and flow cytometry analysis are ongoing to confirm the functional role of these proteins in MAF regulated MM cell growth.           

Conclusions: Taken together, these results suggest that MAF confers MM resistance to PI by preventing the induction of apoptosis. Global gene and protein expression profiling along with correlation with clinical data, has identified a number of relevant biologically relevant candidates.  Experiments are ongoing to confirm the function of these MAF regulated candidates in order to identify potential alternate approaches for the treatment of this poor performing molecular subgroup.