Interleukin-6 Adversely Impacts Genomic Stability Via Targeting Multiple Pathways in Multiple Myeloma

In our previous investigation, using whole exome sequencing, we have shown that multiple myeloma (MM) patients display a complex dynamic of clonal evolution and the number of mutations in patient samples correlate with overall and relapse free survival. These results highlight the importance of understanding the mechanisms driving genomic instability in MM. Investigating mechanisms underlying genomic instability, we have shown that dysregulated homologous recombination (HR), nuclease (especially apurinic/apyrimidinic related) and APOBEC deaminase activities contribute to genomic instability in MM. We have also demonstrated that bone marrow microenvironment (BMM) also contributes to genomic instability in MM. So here we have further investigated the soluble factors in BMM that may impact genomic integrity. We treated RPMI8226 MM cells with IL-6, IL-17 and TGFb and evaluated their impact on DNA breaks by monitoring the levels of gH2AX (a DNA break marker). Treatment with all 3 cytokines (IL-6, IL-17 and TGFb) caused DNA breaks, as demonstrated by increase in gH2AX, in MM as well as a solid tumor (esophageal cancer) cell lines. Importantly, among these cytokines, the exposure to IL-6 was associated with the highest induction of gH2AX expression. These observations were confirmed in additional MM cell lines (MM1S, H929, OPM2 and U266) treated with IL-6.

Since we have previously demonstrated that elevated HR is a key mechanism of genomic instability in MM, we investigated the role of IL-6 in dysregulation of HR pathway by evaluating its impact on p-RPA32 (a marker of DNA end resection which is a decisive step in the initiation of HR), recombinase (RAD51) expression and HR activity (as assessed by a functional assay). Treatment of MM cells with IL-6 led to increased expression of p-RPA32 and RAD51 (as detected by Western blotting) as well as increased HR activity in MM cells. Increased HR activity was also observed following exposure of MM cells to IL-17, TGFb and IFNb, with the highest induction caused by IL-6. However, the combination of cytokines (IL-6, IL-17 and TGFb) led to a further (> 2-fold) increase in HR activity in these cells, indicating that these soluble factors may interact in inducing mechanisms underlying genomic instability in MM.

Based on our data showing important role of apurinic/apyrimidinic (AP) nuclease in regulation of HR and genome stability in MM, we also evaluated the impact of IL-6 on abasic sites, the substrate of AP nuclease activity. An increase in the number of abasic sites (ranging from 1.7- to 2.3-fold increase) was observed with increasing concentration of IL-6 in MM1S cells. To further investigated the impact of IL-6 on number of micronuclei, used as marker of genomic instability. The treatment of MM cell lines with IL-6 for 48 hrs led to a dose-dependent increase (ranging from 2- to > 2.5-fold increase) in the number of micronuclei in MM1S and RPMI cells, indicating increased genomic instability. We have previously shown that inhibition of HR by RAD51 knockdown or ABL kinase inhibitor, nilotinib, significantly reduces genomic instability, as assessed by micronuclei assay as well as direct evaluation of impact on genomewide acquisition of copy number changes over time using SNP arrays. To investigate if HR inhibition can reverse IL-6-induced genomic instability, we treated MM cells with IL-6, nilotinib and combination of both and observed that inhibition of HR by nilotinib can reverse IL-6-induced increase in number of micronuclei (by 48±17%) in MM cells. Similarly, addition of anti-IL-6 antibody also reversed the IL6-induced DNA breaks (as assessed from gH2AX expression) in MM1S cells. Moreover, combination of nilotinib and anti-IL6 antibody resulted in maximum inhibition of IL6-induced DNA breaks in MM1S cells.

Taken together, these data suggest that IL-6 significantly contributes to dysregulation of HR and genome stability in MM, and agents targeting HR and/or other mechanisms of genomic instability including anti-IL-6 antibody, have potential to reduce/delay genomic evolution and disease progression.