Critical Role for Apobec and Its Interacting Partners in Mediating Mutations and Cell Growth in Multiple Myeloma (MM)

The APOBEC family of cytidine deaminases include AID (activity induced deaminase) and 10 related APOBEC enzymes (A1,A2,A3A,A3B,A3C,A3D,A3F,A3G,A3H and A4). AID is well studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes. APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Recently, a causal role for the AID/APOBECs in inducing somatic mutations in myeloma has been proposed and we have previously published that APOBEC signature mutations as a frequent event in Myeloma. We have also observed that APOBEC-mediated mutations may account for mutations associated with progression of smoldering myeloma to MM.

We further investigated the role of APOBEC in genomic changes in MM and observed that APOBEC expression and activity is elevated in myeloma cell lines as well as patient samples. Using knockdown and over expression approaches, we showed that depletion of APOBECs in myeloma cell lines reduces genomic instability. Following APOBEC3G knock down we observed decreased DNA damage (by g-H2AX), decrease in acquisition of new copy number events over time, and reduced mutational load, strongly suggesting that inhibiting APOBECs could be a potential approach to reduce genome evolution in MM.

We next investigated the effect of APOBEC inhibition on myeloma cell proliferation. We observed that Sh-RNA-based APOBEC knock down in MM1S and H929 MM cell lines, led to significant inhibition of MM cell proliferation, and induction of apoptotic cell death. Associated with APOBEC knockdown, we also observed increased levels of Cyclin-dependent kinase inhibitor 1B (p27Kip1) at both RNA and protein level. By immunoprecipitation we found that APOBEC3G interacts and inhibits the RNA binding protein DEAD-END 1 (DND1), thereby preventing it from inhibiting miR-221-mediated targeting of p27 transcripts. Knockdown of DND1, or over-expression of miR-221 in APOBEC-depleted cells rescued the cell proliferation defects with concomitant decrease in p27 levels. These results show that APOBCs bind to and sequester DND1, leading to miR-221-mediated depletion of p27. In the absence of APOBEC, DND1 prevents the degradation of p27 mRNA, leading to elevated p27 levels and inhibition of cell cycle, suggesting a role for APOBECs in regulating MM cell proliferation that might be independent of its RNA/DNA mutator function. Taken together, these results indicate a significant functional role for APOBECs both in genome evolution as well as cell growth in myeloma and may constitute an important therapeutic target.