Long Noncoding RNA LINC01410 Interacts with the Minichromosome Maintenance (MCM) Complex to Promote Tumor Cell Growth in Multiple Myeloma

The human genome contains thousands of loci that produce long non-protein coding RNAs (lncRNAs) that play critical roles in healthy cell function as well as tumorigenesis. We have recently described the lncRNA landscape in newly diagnosed multiple myeloma (MM), identified their role as independent risk predictors for clinical outcome, and validated the functional contribution of specific lncRNAs to MM cell growth and viability. Although these examples suggest the significance of lncRNAs in MM pathobiology, the vast majority of lncRNA genes have not been functionally tested.

To identify lncRNAs that promote MM cell growth, we performed a large-scale cell viability screen of lncRNAs using CRISPR interference (CRISPRi) to inhibit gene expression. To find lncRNA targets, we first interrogated our large RNA-seq dataset from 360 newly diagnosed MM patients and identified 913 lncRNA transcripts expressed in primary MM cells. We designed the CRISPRi library to target the transcription start site of these 913 lncRNA genes, each with 7 sgRNAs. We screened 3 MM cell lines (H929, KMS12BM, KMS11) and identified 32 lncRNA loci that modified cell growth upon CRISPRi targeting, with 7 lncRNAs impacting all three cell lines tested.

We then evaluated the differential expression of these lncRNAs in primary MM cells compared to normal plasma cells (PC). We observed that LINC01410 was significantly overexpressed in MM patients compared to normal PC, especially in MM patients with t(4;14) translocations, and was an independent risk predictor that highly correlated with overall survival in newly diagnosed MM patients. We next knocked down LINC01410 using gapmeR antisense oligonucleotides (ASO) in a panel of MM cell lines and observed time-dependent inhibition of MM cell viability, including in the t(4;14) cell lines. As LINC01410 appears to localize to both the cytosol and nucleus, we also utilized siRNAs to specifically target the cytosolic isoform and observed a robust reduction of cell growth, suggesting that both cellular localizations are important for the activity of LINC01410.

Transcriptomic analysis indicated a set of genes modulated in response to LINC01410 depletion, with an enrichment in genes involved in the cell cycle and DNA replication. An RNA-protein pull down (RPPD) assay identified three out of six MCM complex subunits (MCM4, MCM5, MCM7) as relevant protein interactors of LINC01410 and was confirmed using RNA-immunoprecipitation (RIP) assay.

The MCM complex is a DNA helicase essential for genomic DNA replication, and we hypothesize that LINC01410 affects cell proliferation by interacting with this complex.

We are now investigating this functional interaction by looking at the impact of LINC01410 modulation on the assembly of the complex, by using integrated mass spectrometry analysis, and on its ability to load onto chromatin by using chromatin immunoprecipitation sequencing (ChIP-Seq).

In conclusion, we report the identification of LINC01410 as a novel lncRNA dependancy that supports MM cell growth via interacting with the MCM complex and controlling the cell cycle/DNA replication axis. Promising developments in the field of lncRNA inhibitors may allow forthcoming clinical applications.

Roccaro:Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Fondazione AIRC: Research Funding; Transcan2/Era-NET/FRRB: Research Funding. Munshi:Pfizer: Consultancy; Adaptive Biotechnology: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Takeda Oncology: Consultancy; GSK: Consultancy; Karyopharm: Consultancy; Legend: Consultancy; Celgene: Consultancy.