Mutant-specific NRAS targeting via an RNA-based therapeutic in multiple myeloma

Multiple myeloma (MM) is a haematologic malignancy characterized by the abnormal proliferation of plasma cells in the bone marrow. Despite therapeutic advances over the past 15 years, it is still considered incurable. Therefore, a deeper understanding of the molecular mechanisms underlying the disease is essential for identifying new therapeutic targets for MM treatment. NRAS mutations are considered high-risk factors in MM patients, contributing to disease progression, relapse, and drug resistance. Among these, NRAS c.181C>A and c.182A>G are the most frequent mutations in MM. It is known that these mutations are associated with poorer prognosis and drug resistance in MM patients, but only few studies have explored their molecular role. The aim of this study was to assess whether these mutations could represent promising therapeutic targets in MM. To achieve this, we investigated their molecular and biological roles in MM in vitro models by allele-specific NRAS silencing using small interfering RNAs (siRNAs). Mutation-specific siRNAs targeting NRAS c.181C>A (Q61K) and c.182A>G (Q61R) were designed and screened. Among these, siNRAS A4 and siNRAS G2, targeting NRAS c.181C>A (Q61K) and c.182A>G (Q61R), respectively, were selected as efficient siRNAs. In particular, siNRAS A4 emerged as a potential candidate for future clinical application due to its high specificity and silencing efficiency. Silencing of mutant NRAS alleles determines a downregulation of the MAPK pathway, indicated by decreased ERK phosphorylation, resulting in reduced cell viability, impaired cell cycle progression, increased caspase 3/7 activity, diminished migration and colony-forming ability in vitro. These effects were not observed upon wild-type NRAS silencing. Furthermore, our in vitro findings were validated in vivo using a zebrafish xenograft model, where mutant NRAS inhibition significantly reduced tumour growth within the yolk sac three days post-injection. Overall, these results demonstrate that NRAS mutations modulate various biological processes, mainly via MAPK pathway regulation, confirming NRAS mutations as promising therapeutic targets in MM treatment. Finally, the high specificity and efficiency of siNRAS A4 in silencing mutant NRAS c.181C>A (Q61K) and exerting anti-tumoral activities identify this siRNA as a potential RNA interference tool for the treatment of NRAS-mutated MM.