Disruption of NEAT1-transcriptional network identifies targetable non-genetic dependencies in multiple myeloma

Abstract

Abstract Long non-coding RNAs (lncRNAs) represent an important layer of gene regulation and have emerged as significant contributors to multiple myeloma (MM) progression (Malard, F., Neri, P., Bahlis, N.J. et al.,2024). The lncRNA NEAT1 is over-expressed in MM patients, supporting malignant plasma cells proliferation and promoting therapy resistance (Taiana E.et al., 2020). Besides its conventional function in the assembly of paraspeckles (PSs), we recently demonstrated that NEAT1 is directly involved in transcriptional control (Puccio N. et al., 2024). In this study, we deepen our understanding of NEAT1's function in shaping the activity of transcriptional bodies, providing the rationale for targeted therapeutic intervention. Starting from a transcriptomic approach in NEAT1-silenced (KD) and CRISPRa NEAT1-overexpressing MM cells, we derived a list of 378 targets, primarily involved in mitosis. 52% of these present the cell cycle homology region motif (CHR) in their promoter region, which synchronizes their activation during late cell cycle phases. We confirmed the clinical relevance of NEAT1's transcriptional program in the coMMpass cohort of almost 900 MM patients. Unsupervised clustering analysis, based on the expression of 378 genes NEAT1's target genes, identified two different groups of patients characterized by high or low NEAT1's expression program and different clinical outcomes. Notably, the cluster with the high NEAT1-CHR program showed reduced overall survival. To explore the mechanistic contribution of NEAT1 to mitotic gene expression, we performed computational analysis through the binding model of analysis of transcription (BART) and query of the ENCODE database. These analyses revealed that FOXM1 and CDK9 as upstream regulators of NEAT1 program. Notably, FOXM1 resulted to be highly expressed in R2-ISS stages II-III of myeloma disease as compared to stage I, and in secondary plasma cell leukemia (sPCL) of myeloma origin, based on coMMpass and GSE66293 datasets analysis. We supported these observations by performing in vitro assays. We described a co-localization of NEAT1 and FOXM1 condensates in MM cells, through combined RNA-FISH/immunofluorescence, and direct binding of NEAT1-FOXM1/NEAT1-CDK9 by targeted RNA-immunoprecipitation (RIP). Additionally, with Chromatin immunoprecipitation experiments (ChIP), we demonstrated that NEAT1 KD results in a reduced occupancy of FOXM1 at the promoters of essential CHR genes. Finally, by performing high-throughput drug screening, we identified CDK9 inhibitors to have a strong synergistic effect with NEAT1 KD, consistent with a synthetic lethality effect. These results were confirmed by live cell imaging approach, with two selective CDK9 inhibitors (CDK9i) on a panel of MM cell lines. Consistently, NEAT1 overexpression confers an increased resistance to CDK9i, confirming the participation of NEAT1 to the transcriptional complex that synchronizes mitotic genes and executes the cell division program. These data are in line with our recent findings that demonstrate that NEAT1 controls and cooperates with Aurora kinase A (AURKA) in ensuring proficient chromosomal segregation(Puccio N. et al., 2024). In conclusion, our data demonstrated that NEAT1 contributes to overt phases of MM and to the clinical aggressive behaviour of the disease, by orchestrating a mitotic transcriptional program. Furthermore, through the application of a multi-dimensional approach, we revealed that the dual inhibition of NEAT1 and transcriptional regulators as CDK9 may offer a promising strategy for novel combinatorial anti-MM therapies.