Bioprocessing of MIR17HG Results in Long and Short Noncoding RNAs with Targetable Tumor-Promoting Activity in Multiple Myeloma

Abstract

Our recent studies demonstrated that multiple myeloma (MM) cells can become significantly addicted to and in turn therapeutically susceptible to the modulation of oncogenic noncoding RNAs (ncRNAs). Through large-scale CRISPR interference (CRISPRi) and CRISPR-Cas13d viability screens, we identified MIR17HG as a leading cell growth-dependency in 6 MM cell lines. MIR17HG is mainly known for producing the miRNA cluster miR-17-92, which includes six mature oncogenic miRNAs (-17, -18a, -19a, -20a, -19b, -92a). However, we observed that depleting MIR17HG pre-RNA with sgRNAs or antisense oligonucleotides (ASO) took >3 days to downregulate the miR-17-92 members, yet appreciable transcriptional changes were immediately produced followed by cell cycle arrest in G0-G1 in 10 MM cell lines and CD138+ cells from 13 MM patients. Moreover, antagonism of miR-17-92 using anti-miRs did not phenocopy the anti-MM activity of targeting the MIR17HG pre-RNA, and this activity was only partially rescued by ectopic expression of miR-17-92 mimics. Therefore, we reasoned that alternative transcripts generated at this locus could provide cell growth dependency. We analyzed our RNA-seq dataset of CD138+ cells from >300 MM patients and 70 MM cell lines for these transcripts and observed reads from regions of MIR17HG that are not included in miR-17-92. Using qRT-PCR and single molecule RNA FISH, we identified the expression of a nuclear-enriched, ~5k-nt long, polyA(+) lncRNA. This lncRNA, named lnc-17-92 was quickly downregulated within a few hours of exposure to ASOs targeting the MIR17HG pre-RNA. Using DROSHA-knockout MM cells, in which MIR17HG produces lnc-17-92 but not miR-17-92, we found that targeting the MIR17HG pre-RNA effectively antagonized MM cell growth. Using easy-to-manipulate cellular models (e.g., HCT-116), we observed stronger rescue by ectopic expression of lnc-17-92, as compared to miR-17-92, after targeting MIR17HG pre-RNA. Using RNA-protein pull down (RPPD) and RNA yeast-3-hybrid (Y3H) assays, we found that the transcription factor c-MYC and the epigenetic modulator WDR82 were binding partners for lnc-17-92. In fact, we found that lnc-17-92 mediated their protein-protein interaction by providing the chromatin scaffold for the assembly of a MYC-WDR82 complex at the promoter region of ACC1. This gene encodes the limiting enzyme for de novo lipogenesis, a metabolic pathway that we show is controlled by lnc-17-92 to promote MM cell growth. Using three different cellular models (P493-6, HMECMYC, and U226MYC), we found that targeting MIR17HG pre-RNA preferentially killed MYC+ cells. This could be explained both by lnc-17-92 acting as a chromatin scaffold for MYC and also the known role of miR-17-92 in fine-tuning the downstream transcriptional program of MYC. However, we also observed that lnc-17-92 protected MYC from proteasomal degradation and found that the miR-17-92 member miR-92a negatively regulated MYC protein expression via an, as yet, unknown mechanism. The concerted action of lnc-17-92 and miR-17-92 on MYC protein stability is under investigation and updates will also be presented. Finally, we explored MIR17HG as a therapeutic target, which includes targeting both its lncRNA and miRNAs. To develop clinically applicable inhibitors, we screened >80 fully phosphorothioated (PS), 2'-O-methoxyethyl (2'-MOE)-modified, lipid-conjugated ASOs that could either trigger RNase H-mediated degradation of MIR17HG pre-RNA (gapmeRs) or exert function via an RNase H-independent mechanism (blockmeRs). We identified an 18-mer tocopherol (T)-conjugated gapmeR G2-15b-T ("G") and an 18-mer tocopherol (T)-conjugated steric blocker SB9-19-T ("B"). We found they had potent anti-tumor effects both in vitro and in vivo in three pre-clinical animal models, including a clinically relevant PDX-NSG mouse model. Tumor growth inhibition ranged from 100% (regression) to 50% in these models. Inhibitors did not cause overt toxicity in the mice, as shown by blood cell count, clinical, and body weight analysis. These inhibitors are currently being developed for translation to clinical trials. Altogether, our studies characterize a novel oncogenic mechanism for MIR17HG and provide clinically applicable inhibitors.