Abstract
Besides the well described function of RNA to produce proteins, a large volume of transcribed product has non-coding function. A recent analysis of RNA repertoire has identified a family of non-coding transcripts with sequence longer than 200 nucleotides, the long intergenic non-coding RNAs (lincRNAs). Although lincRNAs have been considered to provide regulatory functions, their precise role in cellular biology remains unclear. Using our RNA-seq data from 360 newly-diagnosed patients and 18 normal plasma cells, we have recently described the landscape of lincRNAs in multiple myeloma (MM) and reported their role as independent risk predictors for survival outcome.
We have now studied the functional role of a lincRNA, the miR-17-92 primary precursor linc-MIR17HG, present in our lincRNA profile and highly correlated with overall survival in MM. We observe that inhibition of linc-MIR17HG by antisense LNA GapmeRs (n=2) leads to apoptosis in 12 genotypically distinct MM cell lines as well as in 13 primary patient MM cells. These effects are not fully rescued by expression of miR-17-92 microRNAs, suggesting a distinct biological function for linc-MIR17HG in MM. We therefore performed gene expression profile in 2 MM cell lines (AMO1 and NCI-H929) and in 2 primary patient MM cells after short-term suppression of linc-MIR17HG; and, at these early time points (18-36h), we found significant downregulation of a subset of genes (FC>2; p<0.05) including ACC1, EXT1, EPT1, ANO6, CCDC91 and KIA1109, but not miR-17-92 microRNAs. These transcriptional changes were validated by qRT-PCR in MM cell lines and primary MM cells exposed to different LNA GapmeRs targeting linc-MIR17HG with a non-overlapping spectrum of off-target effects. Importantly, our RNA-seq analysis of 360 newly-diagnosed MM patients from IFM/DFCI 2009 clinical study showed that expression of linc-MIR17HG strongly correlated with the expression of each of these genes (R2>0.4; p<0.01) in MM patients, further suggesting a regulatory function by linc-MIR17HG at transcriptional level. Using CRISPR interference (CRISPRi), we have also identified that the linc-MIR17HG with transcriptional regulatory functions is not produced from the canonical transcript isoforms MIR17HG-201/-203; these isoforms, rather, appear to be involved in production of microRNAs, leaving an alternative transcription start site usage as possible source for the transcriptional regulator isoform(s).
We next investigated whether regulation of these early targets may contribute to the activity of linc-MIR17HG. We performed a RNAi-based screening in 2 MM cells lines (AMO1 and NCI-H929) by silencing each of the linc-MIR17HG downstream target genes with at least 2 different highly-specific siRNAs. This approach revealed that silencing of acetyl-CoA carboxylase 1 (ACC1, also known as ACACA), a gene encoding the limiting enzyme in the biosynthesis of fatty acids, significantly affects MM cell growth and viability. These results were validated using stable knock-down via shRNAs, confirming ACC1 as a novel vulnerability in MM. These results provide a molecular basis for reported role of fatty acid metabolism in MM cell growth and survival. We have now evaluated two orally available inhibitors of ACC1 activity, ND-630 and ND-646, in a panel of 10 MM cell lines, an report a potent time- and dose-dependent anti-proliferative effect. The activity of these inhibitors and linc-MIR17HG on fatty acid biosynthesis in MM cells is under investigation and will be presented.
We have also begun to investigate molecular pathway used by linc-MIR17HG to modulate ACC1 function. Our preliminary data suggest that linc-MIR17HG may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity.
In conclusion, we highlight a transcriptional regulatory activity of a lincRNA in MM with significant functional impact that can be therapeutically translated.
Disclosures
Anderson: Bristol Myers Squibb: Consultancy; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.
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