Immune Microenvironment Contributes to Dysregulation of Aid/Apobec Expression and Genomic Instability in Multiple Myeloma

Abstract

Multiple myeloma (MM) is a plasma cell malignancy with marked genomic instability. Using whole genome sequencing, we have previously demonstrated that myeloma cells display heterogeneity in mutational spectrum and diverse patterns of clonal evolution. We have also identified AID/APOBEC-driven signature as one of 2 biologically distinct mutational signatures responsible for the majority of observed mutations. Our data show that several members of APOBEC family (including 3A, B, C, and G) are up regulated in patient MM cells and cell lines. Using SNP array and whole genome sequencing, we have also confirmed that elevated expression of APOBECs (3B, C, and G) contributes to genomic instability in MM cells. Since genomic instability and associated clonal evolution could also be driven by pressures from microenvironment, in this study we investigated the role of microenvironment in dysregulation of AID/APOBECs in MM cells.In normal cellular environment, AID and APOBECs are expressed by B and T cells and play a key role in adaptive and innate immunity. However, their expression is tightly regulated in order to prevent nonspecific damage to cellular DNA. We hypothesized that abnormal immune components and soluble factors in the bone marrow microenvironment can induce APOBEC expression in MM cells and can contribute to genomic instability. As we have previously shown that pDCs, MDSCs, Th17cells and B1/B2 cell ratio were significantly elevated in peripheral blood and bone marrow of MM patients, we evaluated the role of these immune cells (iDC, pDC, B, B1, and T cells), and soluble components (IL6, IL-17, IL-27, IFNa, and IFNb) on APOBEC expression and activity in myeloma cells. We used purified immune cells from healthy donors and co-cultured them with MM cells. Following a 3 day co-culture, MM cells were purified and analyzed. APOBEC expression by RT-qPCR. Co-culture with immune cells caused increased expression (range: 2-100 fold) of several APOBEC family genes in myeloma cells. Among different immune cells, iDC and pDC induced the expression of AID, A3A, A3G, and A3H; TLR9 activated B cells induced the expression of AID and A3G; B1 cells induced expression of AID, A3A and A3G; and, IL-17 activated T cells induced the expression of AID, A3A and A3G. Among soluble components of immune microenvironment, interferon a and β induced APOBEC expression (A3A, A3G, and A3H; range: 4-15 fold) in myeloma cells after treatment. Interestingly, none of the interleukins tested showed any impact on APOBEC gene expression in these cells. We then tested if IRF9, a downstream effector of type-1 interferons is involved in the interferon-mediated induction of APOBECs. IRF9-siRNA significantly inhibited interferon-mediated induction of APOBECs (by 50%) in myeloma cells suggesting that interferon mediated up-regulation of APOBECs in MM cells is mediated at least in part by IRF9. We also used a functional assay and demonstrated that increased expression of APOBEC genes was associated with increased APOBEC deaminase activity in MM cells. To investigate the genomic impact of interferon induced APOBEC activity, we treated MM cells with IFN-β and evaluated its impact on abasic sites (that could directly result from APOBEC deaminase activity) and micronuclei assay (a marker of genomic instability). The number of abasic sites and micronuclei were increased by 2 and 4 fold respectively, in cells treated with IFN-β. Overall, these results suggest that BM microenvironment can induce APOBEC expression/activity and associated genomic instability in MM cells. DisclosuresNo relevant conflicts of interest to declare.