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An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma
Blood ( IF 21.0 ) Pub Date : 2024-08-20 , DOI: 10.1182/blood.2023021991
Katia Grillone 1 , Serena Ascrizzi 2 , Paolo Cremaschi 3 , Jussara Amato 4 , Nicoletta Polerà 1 , Ottavio Croci 5 , Roberta Rocca 1 , Caterina Riillo 1 , Francesco Conforti 6 , Raffaele Graziano 4 , Diego Brancaccio 4 , Daniele Caracciolo 1 , Stefano Alcaro 7 , Bruno Pagano 4 , Antonio Randazzo 4 , Pierosandro Tagliaferri 8 , Francesco Iorio 9 , Pierfrancesco Tassone 8
Affiliation  

Multiple myeloma (MM) is an incurable malignancy characterized by altered expression of coding and noncoding genes promoting tumor growth and drug resistance. Although the crucial role of long noncoding RNAs (lncRNAs) in MM is clearly established, the function of the noncoding 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 prioritization pipeline combining functional data from cellular screens with prognostic and transcriptional data from patients with MM. With this approach, we unveiled and prioritized 8 onco-lncRNAs essential for MM cell fitness, associated with high expression and poor prognosis in patients with MM. The previously uncharacterized RP11-350G8.5 emerged as the most promising target, irrespective of BZB resistance. We (1) demonstrated the anti-tumoral effect obtained by RP11-350G8.5 inhibition in vitro and in vivo; (2) highlighted a modulation of the unfolded protein response and the induction of immunogenic cell death triggered by the RP11-350G8.5 knockout, via RNA sequencing and molecular studies; (3) characterized its cytoplasmic homing through RNA fluorescence in situ hybridization; and (4) predicted its 2-dimensional structure and identified 2 G-quadruplex and 3 hairpin-forming regions by biophysical assays, including thioflavin T, 1H nuclear magnetic resonance, 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 patients with MM.

中文翻译:


无偏倚的 lncRNA 缺失 CRISPR-Cas9 筛选显示 RP11-350G8.5 是多发性骨髓瘤的新型治疗靶点



多发性骨髓瘤 (MM) 是一种无法治愈的恶性肿瘤,其特征是编码和非编码基因的表达发生改变,促进肿瘤生长和耐药性。尽管长链非编码 RNA (lncRNA) 在 MM 中的关键作用已明确确定,但可能允许设计新型疗法的非编码 RNAome 的功能在很大程度上是未知的。我们对 MM 细胞及其硼替佐米 (BZB) 抗性衍生物中的 671 个 lncRNAs 进行了无偏倚的 CRISPR-Cas9 功能丧失筛选。为了对功能和临床相关的候选者进行排名,我们设计并使用了一个生物信息学优先流程,将来自细胞筛选的功能数据与 MM 患者的预后和转录数据相结合。通过这种方法,我们揭示了 8 种对 MM 细胞适应性至关重要的 onco-lncRNAs,这些 RNA 与 MM 患者的高表达和不良预后相关。无论 BZB 耐药性如何,先前未表征的 RP11-350G8.5 都成为最有希望的靶标。我们 (1) 证明了 RP11-350G8.5 抑制在体外和体内获得的抗肿瘤作用;(2) 通过 RNA 测序和分子研究,强调了 RP11-350G8.5 敲除触发的未折叠蛋白反应的调节和免疫原性细胞死亡的诱导;(3) 通过 RNA 荧光原位杂交表征其细胞质归巢;(4) 预测其二维结构,并通过生物物理测定鉴定 2 个 G 四链体和 3 个发夹形成区域,包括硫黄素 T、1H 核磁共振和圆二色谱,为新型靶向治疗剂的开发铺平道路。总体而言,我们提供了有关 MM 中未探索的 lncRNAs 的创新见解,并确定了 RP11-350G8。5 作为初治和 BZB 耐药 MM 患者的致癌靶点。
更新日期:2024-08-20
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