LncRNAs: Novel therapeutic targets to treat Multiple Myeloma with RNA-based therapies

Abstract

Multiple Myeloma (MM) is a hematologic neoplasm caused by the clonal proliferation of aberrant plasma cells in the bone marrow (BM). Hyperdiploidy and IGH translocations are the primary genetic events in MM, each affecting approximately 50% of patients. However, there are cases where the translocation partner of IGH is still unknown. Moreover, beyond genetics, epigenomic and transcriptomic studies have uncovered the extensive chromatin activation of regulatory elements in MM, from where thousands of long non-coding RNAs (lncRNAs) are expressed. We hypothesize that these lncRNAs could be involved in translocations occurring in MM. Furthermore, the inhibition of one of these lncRNA, named SMILO, triggers MM cell death, suggesting the potential of lncRNAs as novel therapeutic targets in MM.

To unravel the contribution of lncRNAs to translocations in MM, we identified fusion transcripts (FTs) from strand specific RNA-seq data in samples from different B cell subpopulations and MM patient samples. After a stringent computational filtering, we detected FTs in all studied subpopulations, implying that FTs present in healthy samples may be contributing to physiological processes. Nevertheless, FTs present in healthy samples were filtered to detect MM-specific FTs. As hypothesized, the 30% of MM-specific FTs occurred with lncRNAs (lncFTs), and interestingly, an important percentage of MM-specific FTs were transcription readthroughs (RT) between adjacent genes, therefore leading to the identification of novel RNA transcript classes with unknown functional and prognostic potential in this disease. Finally, the contribution of lncFTs to the prognosis of MM patients was assessed by multivariate survival studies performed in the CoMMpass dataset including 599 MM patient samples, demonstrating that the combination of the presence of certain lncFTs with classical genomic high-risk markers can improve the prognostic stratification of MM patients.

On the other hand, we propose the generation of aptamer-siRNA chimeras as RNA-based therapies that will enable the targeted silencing of oncogenic coding and non-coding genes, including lncRNAs and FTs, specifically in the pathological plasma cells of MM. The silencing is achieved with therapeutic siRNA molecules which are delivered specifically to pathological plasma cells by MM-specific RNA aptamers. To identify the MM-specific aptamers we applied a Cell-SELEX approach, where a random RNA aptamer library is subjected to iterative binding rounds with MM cell lines to enrich sequences able to bind target cells specifically. After 10 selection rounds, we identified 10 enriched aptamer clusters, from which aptamer 1 (APT1), aptamer 1 (APT3) and aptamer 1 (APT9) were selected for validation studies. Flow cytometry and confocal microscopy studies showed that all aptamers bind preferentially and internalize into MM cell lines, besides, APT3 and APT9 were minimally toxic to MM cell lines up to 5 M, suggesting they could be used as siRNAs carriers for targeted delivery without inherent toxic effects. Likewise, we identified effective siRNAs able to inhibit both SMILO lncRNA and RRM1 coding gene with known oncogenic effects in MM, so they were selected for chimera generation with the APT3 to obtain the therapeutic silencing of these genes in the MM cell.

In conclusion, this doctoral thesis aimed at elucidating the involvement and the prognostic significance of lncRNAs in fusion transcripts occurring in MM, as well as to develop RNA-based therapeutic approaches that will enable the targeted silencing of these novel transcript classes together with coding genes in a cell-specific fashion.