OAB-009: Genome-wide CRISPR interference screen identifies RNA Regulator of Lipogenesis (RROL) as a leading LncRNA dependency in Multiple Myeloma

Introduction Long noncoding RNAs (lncRNAs) are profoundly dysregulated in multiple myeloma (MM), yet their tumor-promoting features remain to be defined. Here, we have coupled extensive transcriptomic profiling of patient samples (n=360) with a genome-wide CRISPR interference (CRISPRi) viability screen (4 cell lines) to generate a map of lncRNA "dependencies" in MM; moreover, we describe the molecular and functional role – as well as its therapeutic potential – of the screen top hit RNA Regulator of Lipogenesis (RROL), a nuclear-retained lncRNA generated by alternative splicing of MIR17HG. Methods Loss-of-function (LOF) studies using antisense oligonucleotides (ASO) confirmed the RROL dependency in a large panel of MM cell lines and CD138+ patient cells, in vitro and in vivo in animal models. This effect was not antagonized in DROSHA KO MM cells or by ectopic expression of MIR17HG-derived miR-17-92, indicating a microRNA-independent role of RROL. Results Transcriptomic analysis after RROL depletion indicated a significant impact on the de novo lipogenesis (DNL) gene networks. RROL occupancy at DNL gene loci was detected by chromatin isolation by RNA precipitation followed by qRT-PCR (ChIRP-qPCR) and confirmed by DUAL RNA FISH analysis. At functional level, we demonstrated that RROL depletion reduces DNL in MM cells using an unbiased lipidomic profiling as well as by measuring the incorporation of C14-radiolabeled glucose into lipids. Importantly, we also proved that exogenous palmitate, the downstream product of DNL pathway, can significantly rescue the growth inhibitory effect of RROL depletion in MM cells; thus implicating a role for DNL in the growth promoting effect of this lncRNA. RNA-Protein Pull Down (RPPD) and in vivo RNA yeast three-hybrid (Y3H) assays led to identify c-MYC (MYC) as a relevant protein interactor of RROL; a finding validated by RIP-qPCR. Coupling RNA FISH with immunofluorescence, we co-detected RROL and MYC at the ACC1 promoter. Importantly, neither RROL or MYC could occupy ACC1 promoter and exert regulatory control in the absence of the other factor. Moreover, using in vitro (Co-IP/MS) and in vivo (BioID) assays, we identified WDR82 as an RROL-dependent MYC partner implicated in the transcriptional control of ACC1 expression. Finally, to therapeutically antagonize RROL, we have screened >100 ASOs and provided the basis to develop a first-in-class RROL inhibitor. This inhibitor has shown a very strong anti-MM activity in vitro (IC50 Conclusion In conclusion, we here report a unique regulatory function of a novel lncRNA supporting MM cell growth via its control of the lipogenic metabolic axis. The ongoing development of RROL inhibitors may allow clinical application of this unique targeted therapy in MM.