MAFA Expression Promotes Proliferation of Myeloma Cells and Its Protein Stability Is Regulated By Proteasome Inhibitors

Introduction

Multiple myeloma (MM) patients with a t(14;16) or t(14;20) which result in high expression of MAF and MAFB, respectively, have a poor prognosis, and have not benefitted from the recent advances in MM therapy. Our previous work showed that post-translational modifications (PTM) further up-regulated MAF (Blood, 2016) and MAFB proteins (ASH Abstract #2091, 2014) conferring innate resistance to proteasome inhibitors (PI). The large MAF family of proteins contains a third member, MAFA. Little is known about its biological role in MM pathogenesis, as a probeset for MAFA is not present in gene expression profiling (GEP) analysis of molecular subgroups, and MAFA probes are not routinely used in fluorescence in situ hybridization (FISH) analysis. In the present study we therefore investigated the biological role of MAFA in MM and its effect on PI resistance.

Methods

The MAFA translocation in primary MM cells was determined by FISH analysis in 19 newly diagnosed MM patients. MAFA protein expression and protein level in response to PIs were assessed by immunoblotting and immunofluorescence staining (IFS). The effect of MAFA on proliferation of MM cells was measured by MTT assay. Annexin V staining and BrdU incorporation, analyzed by FACS analysis, was performed to determine the molecular action of MAFA on the survival of MM cells. MAFA expression was down-regulated in human MM cell lines (HMCLs). HMCLs using a lentiviral shRNA expression system. qRT-PCR analysis was used to determine MAFA mRNA expression in 32 HMCL and primary CD138+ plasma cells from 14 MM patients, and MAFA levels after shRNA treatment.

Results

Four levels of MAFA mRNA expression (relative to GAPDH) by RT-PCR analysis were determined: high levels were detected in 5 HMCLs, intermediate levels (100 to 500 ) in 10, low levels (10<100) in 7 and negative (<10) in 10 HMCLs. Among primary CD138+ patient cells (n=14), high levels were found in one patient, intermediate levels in 3, low levels in 4 and negative in 6 patients. FISH analysis found MAFA /t(8;14)(q24;q32) translocation in one out of 19 patients, which was also in the MF group by GEP analysis suggesting common downstream targets of MAFA with MAF and MAFB . MAFA protein was detected in the 6 HMCLs which expressed the highest MAFA mRNA levels by RT-PCR but not in the others.

To determine if MAFA is required for MM cell survival, we knocked-down MAFA expression in three HMCLs that expressed high levels of MAFA . sh MAFA lentiviral supernatant infected HMCLs had 75% lower levels of MAFA mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, bioinformatically defined MAFA target genes were significantly decreased, including TLR4 (2.4-fold, P<0.01), NUAK1 (2.3-fold, p<0.01) and SPP1 (osteopontin, 2.2-fold, P<0.01), compared with HMCLs infected with control viral vector. Silencing MAFA expression significantly decreased the proliferation of HMCLs (62.5% decrease, P=2.1x10^-4). Among 6 high MAFA expressing HMCLs, 3 co-expressed MAF and 2 co-expressed MAFB . To determine if MAFA synergized with MAF in promoting MM proliferation, we silenced MAFA and MAFB genes in cells co-expressing these genes. Simultaneously silencing MAFA and MAFB did not further decrease proliferation compared to either silencing MAFA or MAFB alone. These results indicate that MAFA promotes MM cell proliferation and that there is no synergetic effect between MAFA and MAFB in regulating MM proliferation.

To determine if MAFA protein is regulated by PTM, we treated MM cells with PIs including bortezomib (Bzb) and carfilzomib (CFZ). Bzb induced stabilization of MAFA protein in a dose-dependent manner. Similar effects were seen with CFZ indicating that MAFA protein may be degraded by the ubiquitin proteasome system.

Conclusion

Taken together, our results indicate that increased levels of MAFA are seen in MM including the MAFA /t(8;14) translocation. High expression of MAFA mRNA and protein occurs in 18.7 % of MM cell lines. MAFA promotes myeloma proliferation and regulates TLR4, NUAK1 and SPP1 gene expression. The stability of MAF protein is regulated by PIs. These results provide new insights in understanding the role of the MAF family and, in particular, MAFA in MM pathogenesis. Experiments are ongoing to determine the function of MAFA in PI-resistance and to confirm MAFA targets in order to identify potential alternate approaches for the treatment of this poor performing molecular subgroup.