Identification of a Novel Long Intergenic Noncoding RNA - Linc00936, with Significant Impact on Multiple Myeloma Cell Growth Via mTOR Pathway Inhibition

Recent advances in high-throughput whole transcriptome analysis of human genome has allowed for the identifications of more than 13,000 long intergenic non-coding RNA transcripts (lincRNAs), with potential central roles in gene regulation and tumorigenesis. Using RNA-seq data from newly-diagnosed multiple myeloma (MM) patients, we identified linc00936 (chr 12) to be highly downregulated in CD138+ MM cells from 320 MM patients compared to 16 normal bone marrow plasma cells. Decreased expression of lincRNA was also observed in a panel of MM cells lines when compared with normal PBMC by both quantitative RT-PCR (qRT-PCR) and RNA-seq (N=66).

Interestingly, linc00936 overexpression significantly inhibited MM cell line growth (N=7), associated with G1 cell cycle arrest, decreased S phase, and induction of apoptotis via caspase 3 activation. Enforced expression of linc00936 was associated with induction of various gene sets, as measured by gene expression profiling. Importantly, linc00936 overexpression had a negative impact on genes involved in the mTOR pathway. WB analysis confirmed reduction in p-mTOR, along with a decrease in p-p70 S6 kinase and p-4E BP1, following linc00936 overexpression. Due to the role of mTOR pathway regulating autophagy, we next analyzed the effect of linc00936 on the autophagic response: autophagy was induced, evidenced by autophagic vacuoles and associated with decreased p62 and increased LC3A/B protein levels. Chloroquine, an autophagy inhibitor, partially abrogated the anti-MM activity of linc00936. RNA pull-down assay identified spleen tyrosine kinase (Syk), an E3 ubiquitin ligase (Rnf2), and serine/threonine-protein kinase (Taok3) as partner proteins. These interactions were validated by WB analysis of the lincRNA-pull-down proteins. Of note, the strongest interaction was with Syk, a well known modulator of mTOR activity. RNA-immunoprecipitation assay (RIP) followed by RT-PCR confirmed an increase in linc00936 levels in RNA-Syk complex compared to immunoglobulin G (IgG)-bound sample. Finally, treatment with demethylating agent 5-aza-deoxycitidine significantly increases the expression of linc00936 in RPMI-8226, MM1S and H929 MM cells, implicating promoter methylation as a mechanism of lincRNA suppression in MM. Based on these data, we analyzed the prognostic significance of linc00936 expression in MM patients enrolled on IFM/DFCI 2009 clinical study (N=296): low linc00936 expression was associated with significantly shorter event free survival compared to higher expression (p= 0.018). Collectively, our data provide the first evidence of a differentially expressed lincRNA in MM with a significant impact on tumor cell growth and survival via inhibition of mTOR pathway, with potential biological, prognostic and therapeutic implications.