Apobec Family of Proteins Mediate Genomic Changes in Multiple Myeloma By Inducing DNA Damage and C>T Mutations

Multiple myeloma (MM) is a heterogeneous disease characterized by unstable genome. Spontaneous DNA damage coupled with dysregulated repair accentuates the genomic instability seen in MM. The resulting clonal heterogeneity plays a critical role in resistance to treatment and relapse. Understanding the molecular factors that contribute to genomic instability in myeloma is therefore of great importance in developing better treatment strategies. Recently, a role for the AID/APOBEC family of cytidine deaminases in generating somatic mutations has been described with in MM as well as number of other cancers. We and others have previously shown that significant number of mutations bear APOBEC signature and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in MM. We have now further explored role of APOBEC in MM with special focus on its ability to induce clonal diversity.

We observed that APOBECs 3A, 3B, 3C, and 3G are highly expressed in MM. Using a fluorescent oligonucleotide based APOBEC deaminase assay to measure deaminase activity, we also observed significantly increased APOBEC activity in MM cell lines as well as primary patient MM cells. Using shRNA-mediated knockdown, we report that depletion of APOBECs in MM cell lines results in decreased DNA double strand breaks. We next investigated the impact of overexpression of APOBEC genes on genome integrity in MM cells. Over expression of APOBECs 3B or 3G in U266 and LP1 MM cell lines led to 3-5 fold increase in deaminase activity in MM cell extracts measured by APOBEC deaminase assay. Furthermore, APOBEC overexpression resulted in elevated double strand breaks as evidenced by increased gH2AX nuclear foci and by western blot. In order to determine the functional impact of APOBEC-induced mutations, we co-transfected EGFP plasmid along with APOBEC expression vectors in myeloma cells. 72-hrs after transfection, we isolated the EGFP plasmid, transformed bacteria, and sequenced the plasmid DNA isolated from 5 colonies from each group. Overexpression of APOBECs resulted in increased number of C>T mutations (3-5 fold) in the EGFP plasmid co-transfected into these cells. Interestingly, APOBEC over expression also resulted in the loss of GFP fluorescence in 20-30% of the cells compared to control cells, suggesting that overexpression of APOBECs can cause deleterious mutations leading to loss of function.

APOBEC-mediated deamination of cytosine to uracil leads to generation of abasic sites that are cleaved by abasic endonuclease (APE1), a regulator of homologous recombination pathway dysregulated in MM contributing to genomic instability. Therefore, we investigated how modulating APOBEC activity affects downstream DNA damage repair pathways in the gain-of and loss-of-function cells. Using in vitro activity assays, we measured APE1 and HR activity in extracts collected from APOBEC overexpression and knockdown cells. Extracts from APOBEC overexpressing cells showed elevated APE1 and HR activity (>50%), whereas depletion of APOBEC proteins resulted in up to 80% reduction in HR and APE1 activity in the extracts. Interestingly, using a small molecule HR inhibitor we found that inhibiting HR pathway can reduce the DNA breaks induced by APOBEC overexpression. Furthermore, we investigated the acquisition of genomic changes such as amplifications, deletions, and SNVs over time, in control and APOBEC knockdown cells, using SNP arrays and whole genome sequencing (WGS). Our results show that knockdown of APOBECs reduces the accumulation of copy number changes, structural variants and mutations over time. This suggests that elevated expression of APOBECs may be contributing to genomic instability in MM cells.

In summary, our results suggest that APOBEC mediated DNA damage could contribute to increased dysregulation of these important repair pathways and genome stability in myeloma. Inhibiting APOBEC activity could reduce the rate of accumulation of the ongoing genomic changes.