RNA Modifications on Long Non-Coding RNAs in Multiple Myeloma

Multiple myeloma (MM) is a metastatic cancer of plasma cells, primarily forming tumors within the bone marrow. It is estimated in 2024, there will be over 35,000 new cases diagnosed and over 12,000 deaths from this disease with a 5-year survival rate at 60% (Siegel, Giaquinto et al. 2024). MM is still considered incurable and most patients eventually relapse, exhaust all treatment options, and succumb to their disease. Thus, knowledge surrounding biological mechanisms in MM patients is critical. Currently, > 170 RNA modifications have been identified (Saletore, Meyer et al. 2012). Studies have shown that thedysregulation of RNA modifications are involved in the pathogenesis of human disease, including cancer (Sun, Wu et al. 2019, Barbieri and Kouzarides 2020). Long non-coding RNAs (lncRNAs) contain RNA modifications(He, Jiang et al. 2020, Song, Ren et al. 2022) and their expression has been found to contribute to MM (Carrasco-Leon, Amundarain et al. 2021, Mishra, Thunuguntla et al. 2024). Our work has shown that lncRNAs play important regulatory roles in promoting metastasis and therapy resistance (Silva, Perez et al. 2010, Silva, Boczek et al. 2011, Silva and Smith 2012, Silva-Fisher, Dang et al. 2020, Mishra, Thunuguntla et al. 2024), yet no studies have fully characterized lncRNA modifications or how they may contribute to MM disease progression. To assess global RNA modifications in MM, we used a novel method of detecting more than 60 RNA modifications by permethylation of ribonucleosides followed by mass spectrometry (Xie, Janssen et al. 2022). We applied this method using isolated mRNA from five myeloma cell lines including RPMI 8226, MM.1R, MM.1S, U266B1, and U266B1 treated with high dose melphalan to identify 14 different RNA modifications across the samples. Previous studies have shown high abundance of RNA modifications N6-methyladenosine (m6A) and 5-methylcytosine (m5C) on lncRNAs to have potential novel therapeutic targets (Nombela, Miguel-Lopez et al. 2021). Here, we found both modifications to be decreased upon treatment of melphalan (m6A, p = 0.04, m5C p = 0.01). Next, as the protein HNRNPA2B1 binds to m6A sites, we performed an analysis of publicly available methylated RNA immunoprecipitation sequencing from MM cell lines with knockdown of HNRNPA2B1(Jiang, Tang et al. 2021) to detect m6A sites on twenty-five lncRNAs, we term Myeloma Associated m6A-Modified LncRNAs (MAMMLs). We focused our efforts on characterizing MAMML1, due to it containing the most enriched m6A sites. We performed HNRNPA2B1 individual-nucleotide resolution cross-linking immunoprecipitation (iCLIP) with RT-qPCR in RPMI 8226 cells using tiled primers of the predicted sites. We show direct binding of HNRNPA2B1 to all five m6A sites as compared to IgG control and validated m6A sites using a novel BstI RT-QPCR-based assay. Lastly, to show feasibility that m6A modifications play a role in promoting MM, we mutated MAMML1 m6A sites and observed a significant decrease in apoptosis in MM cell lines. Overall, we are the first to demonstrates the importance of RNA modifications in MM and specifically that m6A modifications on lncRNAs may play a role in promoting MM.

DiPersio:Magenta Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; WUGEN: Current equity holder in private company, Research Funding; NeoImmune Tech: Research Funding; hC Bioscience, Inc.: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Research Funding; Bioline Rx: Research Funding; RiverVest Venture Partners: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vertex: Consultancy; SPARC: Consultancy.