P-194: Non-coding RNA LINC01410 interacts with the minichromosome maintenance (MCM) complex and is a dependency in Multiple Myeloma

Introduction: Our initial characterization of the non-coding transcriptome identified long non-coding RNAs (lncRNAs) as key players in multiple myeloma (MM) pathobiology, with 14 lncRNA transcript signature being a strong predictor of progression-free survival.

Methods: We performed a large CRISPR interference screen to evaluate lncRNA dependency in MM and identified LINC01410 as a top dependency in 3 MM cell lines (H929, KMS12BM, KMS11).

Results: We further observed that LINC01410 was significantly overexpressed in MM patient cells compared to normal PC [P=0.047], with a slightly higher expression in the cytogenetic high-risk t(4;14) subgroup [ t(4,14) vs other MM subgroups, [P=0.076], and higher expression was correlated with inferior overall survival in newly diagnosed MM patients [P=0.014].
We verified functional impact of LINC01410 using siRNA-mediated silencing in a larger panel of MM cell lines (n=9) covering all the genetic subtypes. We also used gapmer antisense oligonucleotides (ASO) in a subset of MM cells. Both methods found time-dependent inhibition of MM cell viability [P < 0.0001] after LINC01410 knockdown. In cell culture, LINC01410 knockdown caused a decline in the population of S-phase cells and induced apoptotic cell death, as shown by flow cytometer analysis after BrdU and Annexin-PI staining respectively. LINC01410 knockdown negatively impacted key MM cell survival pathways including cell cycle control, unfolded protein response (UPR) and oxidative stress response. Moreover, cell cycle-dependent cyclins, which are important in MM (i.e., CCND1, CCND2, CCND3), were downregulated at the RNA and protein level.
To determine LINC01410’s interaction partners, we used an RNA-protein pull down (RPPD) assay and found that, among others, three minichromosome maintenance proteins (MCM4, MCM5, MCM7) interacted with LINC01410, and this was confirmed by RNA immunoprecipitation using an anti-MCM5 antibody. These proteins are part of the MCM complex, which is a helicase important for DNA replication and mRNA transcription. Importantly, a Co-Immunoprecipitation of MCM5 subunit followed by Mass spectrometry analysis after LINC01410 knockdown showed the loss of interaction of MCM proteins with RNA polymerase 1 (POLR1), which transcribes ribosomal rRNA. MCM proteins have long been associated with the activity of RNA polymerase II. However, our results suggest a novel role of the MCM complex in the transcription of rRNA, mediated by LINC01410.

Conclusions: In conclusion, we propose a model where, in MM, LINC01410 binds to the MCM complex to assist DNA replication and cell cycle progression, with a potential role also in the transcription of rRNA. These results suggest important role for LINC01410 in MM pathobiology and using small molecule inhibitors and/or Gapmers a potential therapeutic target.