MAF and NF-κB Signaling Pathways Regulate Expression of APOBEC3B in Multiple Myeloma

Introduction

APOBEC mutagenesis is a source of mutations in ~50% of human cancers, resulting from the over-expression of APOBEC proteins, including APOBEC3B (A3B). High expression of A3B is associated with poor prognosis and drug resistance in many cancers. In multiple myeloma, the chromosomal translocation t(14;16) leads to the over-expression of the transcription factor MAF through juxtaposition with the IGH super-enhancer. We have shown that in this poor prognosis sub-group A3B is also over-expressed and there is an enrichment for APOBEC signature mutations and a higher mutational load. It is not clear whether over-expression of A3B results from direct activation by MAF or indirectly through downstream pathways. In other cancers, mitosis, cell-cycle related gene expression, and the PKC/NF-κB signaling pathway may also affect expression of A3B.

Methods

Gene expression profile (GEP, U133 Plus 2.0) of 50 human myeloma cell lines (HMCLs) and 1310 myeloma patients was performed. mRNA levels of MAF and the AID /APOBEC family was investigated by qPCR in HMCLs. Western blotting and a DNA cytosine deaminase activity assay were used to detect A3B protein and analyze A3B enzymatic activity which results in mutagenesis, respectively. The effect of MAF on expression of A3B was investigated in inducible TRIPZ lentiviral shRNA c-MAF knockdown in HMCLs with high expression of MAF. Bortezomib, a proteasome inhibitor, and phorbol 12-myristate 13-acetate (PMA), a PKC dependent activator of the NF-κB signaling pathway, was used to investigate A3B expression in TRIPZ lentiviral shRNA MAF (MF KO) and A3B (A3B KO) knockdown HMCLs. Localization of A3B in regular HMCLs and cell lines with over-expression of A3B (generated using ViraPower T-Rex lentiviral expression system) was investigated by immunofluorescent analysis (IFA) using laser confocal and high resolution microscopy.

Results

By microarray, high levels (log₂>10) of A3B mRNA were found in approximately half of multiple myeloma patients. The majority of t(14;16) MAF patients had high levels of A3B. Increased expression of A3B was also detected in 72% of HMCLs. In t(14;16), MAF, and t(14;20), MAFB, HMCLs there was a strong positive correlation between MAF and A3B mRNA expression (r²= 0.51 and r²=0.43, respectively). On the basis of levels of MAF expression two groups of HMCLs were selected. Group 1 with high expression of MAF (MF) with the presence of a t(14;16) ARP1, CAG, JJN3, OCMY5, MM1S, and RPMI8226. Group 2 had low expression of MAF (non-MF) and was represented by cell lines with t(11;14) - U266, XG1 and KMS12BM; t(4;14) - KMS28PE and H929; or t(14;20) SACHI. We found a strong positive correlation (r²=0.68) between MAF and A3B mRNA expression in MF cell lines and no such correlation in the non-MF group (r²=0.09). Upon down-regulation of MAF by shRNA, JJN3 and RPMI8226 showed reduced expression of A3B protein (-33% and -29%, respectively) by 72 hours with accompanying reduction in cytosine deaminase activity (-23% and -26%, respectively). There was a positive correlation between expression of MAF protein and A3B protein in JJN3 MAF KO r²= 0.21, in RPMI8226 MAF KO r²=0.50; and a positive correlation between MAF protein and DNA cytosine deaminase activity in JJN3 MAF KO r²=0.21 and in RPMI8226 MAF KO r²=0.34. Bortezomib reduced expression of MAF and A3B and DNA cytosine deaminase activity in JJN3 and RPMI8226 cell lines. PMA increased the expression of A3B mRNA in HMCLs within 6 hours of treatment and lasted for 7 days after treatment. The highest sensitivity of A3B to PMA was detected in JJN3 and U266 cell lines, with increases of 4.4 and 8.2-fold, respectively, after 6 hours of treatment. Protein levels of A3B were increased 2.4-fold in both cell lines but DNA cytosine deaminase activity was increased (27%) only in JJN3 cells after PMA treatment.

Conclusions

Knock down of MAF results in decreased expression and activity of A3B, indicating that MAF is a regulator of A3B expression in multiple myeloma. Expression of A3B in t(14;16) cases is dependent upon MAF expression but can also be increased through activation of the PKC/NF-κB signaling pathway, resulting in increased DNA damage. Understanding the mechanisms responsible for mutagenesis in the t(14;16) subgroup through A3B, which results in new clone formation and drug resistance in multiple myeloma will be highly beneficial in designing new therapeutic strategies.