An unbiased lncRNAs dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for Multiple Myeloma

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An unbiased lncRNAs dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for Multiple Myeloma

08/2024 - Blood

An unbiased lncRNAs dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for Multiple Myeloma

Authors:

Grillone K.,
Ascrizzi S.,
Cremaschi P.,
Amato J.,
Polerà N.,
Croci O.,
Rocca R.,
Riillo C.,
Conforti F.,
Graziano R.,
Brancaccio D.,
Alcaro S.,
Pagano B.,
Randazzo A.,
Tagliaferri P.,
Iorio F.,
Tassone P.

Abstract:

Key Points

We unveiled 8 lncRNAs essential for Multiple Myeloma (MM) cell fitness and associated with poor prognosis and high expression in MM patients
We identified lncRNA RP11-350G8.5 as a therapeutic target for MM and characterised its oncogenic role, molecular and structural features

Multiple Myeloma (MM) is an incurable malignancy characterised by altered expression of coding and non-coding genes promoting tumour growth and drug resistance. Although the crucial role of long non-coding RNAs (lncRNAs) in MM is clearly established, the function of the non-coding RNAome, which might allow the design of novel therapeutics, is largely unknown. We performed an unbiased CRISPR-Cas9 loss-of-function screen of 671 lncRNAs in MM cells and their Bortezomib (BZB)-resistant derivative. To rank functionally and clinically relevant candidates, we designed and used a bioinformatic prioritisation pipeline combining functional data from cellular screens with prognostic and transcriptional data from MM patients. With this approach, we unveiled and prioritised 8 onco-lncRNAs essential for MM cell fitness, associated with high expression and poor prognosis in MM patients. The previously uncharacterised RP11-350G8.5 emerged as the most promising target, irrespective of BZB resistance. We i) demonstrated the anti-tumoral effect obtained by RP11-350G8.5 inhibition in vitro and in vivo; ii) highlighted a modulation of the unfolded protein response and the induction of immunogenic cell death triggered by the RP11-350G8.5 knock-out, via RNA-sequencing and molecular studies; iii) characterised its cytoplasmic homing through RNA-FISH; iv) predicted its 2D structure and identified 2 G-quadruplex and 3 hairpin-forming regions by biophysical assays, including Thioflavin T, 1H-NMR and circular dichroism to pave the way to the development of novel targeted therapeutics. Overall we provided innovative insights about unexplored lncRNAs in MM and identified RP11-350G8.5 as an oncogenic target for treatment-naïve and BZB-resistant MM patients.