MECHANISMS OF DRUG-ESCAPE AND RELAPSE IN MULTIPLE MYELOMA: INTERPLAY BETWEEN ADAR1 AND NOTCH IN ABERRANT RNA EDITING

Multiple myeloma (MM) is a plasma cell malignancy that accounts for more than 10% of all blood cancers. Despite a wide variety of available lines of treatment, virtually all patients experience cycles of remission/relapse or become unresponsive to treatment. A growing body of evidence highlights the contribution of clonal heterogeneity to disease progression and resistance to therapy. In recent years the importance of post-transcriptional regulatory mechanisms (such as RNA editing) in cancer has emerged. RNA editing is particularly intriguing as a potential source of genetic diversity, as can affect several mRNA features, including stability, localization, nuclear retention, and alternative splicing. In mammals, most RNA editing is carried out by Adenosine Deaminases acting on dsRNA (ADAR) that catalyze the hydrolytic deamination of adenosines (A) to inosines (I). In particular, ADAR1 has been associated with disease progression and cancer stem cell maintenance in both solid tumors and hematopoietic malignancies. The central aim of this work was to investigate ADAR1 as a mechanism of clonal heterogeneity and drug resistance in MM. We postulated that ADAR1-dependent aberrant A-to-I RNA editing in MM cells could drive transcriptome “reprogramming” in MM tumor cells, thus contributing to disease relapse and drug resistance. We also sought to identify cell-intrinsic and microenvironment-derived mechanisms that promote aberrant ADAR1-mediated RNA editing. We hypothesized that BM inflammatory signals, promoted by MM deregulated pathways such as Notch, sustain ADAR1-mediated reprogramming. We observed significantly increased ADAR1 expression in plasma cell leukemia (PCL), the advanced, highly drug-resistant stage of MM, and detected aberrant RNA editing in GLI1 and APOBEC3D transcripts by a novel RNA editing site-specific qPCR assay (RESSqPCR) that we developed. Furthermore, we established successful MM xenografts by intrahepatic transplantation of ADAR1-enriched PCL samples, thus providing a robust new in vivo model and platform for testing new therapeutic strategies aimed at treating drug-resistant forms of MM. We showed that continuous in vitro exposure to the anti-MM agent and immunomodulatory drug (IMiD), lenalidomide, induced ADAR1 expression and widespread aberrant RNA editing activity in MM cells, coupled with increased self-renewal capacity and a cancer stem cell phenotype. Furthermore, we observed that pro-inflammatory IL-6 promoted RNA editing, suggesting that MM-associated microenvironmental factors may play a key role in triggering ADAR1-associated malignant transcriptome recoding. Notably, IL-6 production from human BM stromal cells and from MM cells can be inhibited by silencing the overexpressed Notch ligands Jagged1 and Jagged2 in MM cells. In keeping with these findings, ADAR1 overexpression and deregulated RNA editing represents a unique source of RNA and protein diversity, and may endow survival advantages to MM cells in selective environments, such as the BM niche or under the pressure of chemotherapy. This work therefore identifies ADAR1 as a potential new diagnostic and therapeutic target in MM, and inhibition of this pathway, or its regulators and editing substrates, could provide a dynamic avenue to prevent disease relapse and disease progression and achieve long-term survival.