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

Multiple Myeloma (MM) is an incurable malignancy characterised by alterations of coding and non-coding genes that promote tumour growth and drug resistance. Despite the crucial role of long non-coding-RNAs (lncRNAs) in myeloma genesis is established, the functional role of the non-coding RNAome in MM remains largely unknown. We performed an unbiased CRISPR-Cas9 recessive screen targeting 671 lncRNAs in MM cell lines unvealing and prioritising a list of novel onco-lncRNAs essential for MM cell fitness and associated with high expression and poor prognosis in MM patients. Among them, RP11-350G8.5 emerged as the most promising vulnerability for MM cells, irrespective of their resistance to the standard-of-care bortezomib. We i) validated the oncogenic role of RP11-350G8.5 in vitro and in vivo; ii) characterised its function in relation to the unfolded protein stress response and induction of immunogenic cell death, and iii) shed light on its sub-cellular localisation, structural and chemical predictions of RNA-G-quadruplex-forming regions to pave the way to the development of novel therapeutics. To reveal the molecular changes induced by RP11-350G8.5 KO in MM cells resistant to bortezomib (ABZB), we profiled the cell line, 7 days post transduction with SCRAMBLE or RP11-350G8.5 KO CRISPR-Cas9 vectors, via RNA-sequencing from two independent biological replicates.

多发性骨髓瘤（Multiple Myeloma, MM）是一类不可治愈的恶性肿瘤，其特征为编码基因与非编码基因发生变异，进而促进肿瘤生长与耐药性产生。尽管长链非编码RNA（long non-coding RNAs, lncRNAs）在骨髓瘤发生过程中的关键作用已得到证实，但非编码转录组在多发性骨髓瘤中的功能仍未被充分阐明。本研究针对多发性骨髓瘤细胞系中的671个lncRNAs开展了无偏倚CRISPR-Cas9隐性筛选，发掘并优先排序出一批新型致癌lncRNAs，这些RNA对于多发性骨髓瘤细胞的适合度至关重要，且与多发性骨髓瘤患者的高表达水平及不良预后密切相关。其中，RP11-350G8.5成为多发性骨髓瘤细胞最具潜力的治疗靶点之一，且其作用不受细胞对临床标准治疗药物硼替佐米耐药性的影响。本研究①在体外与体内验证了RP11-350G8.5的致癌功能；②阐明了其与未折叠蛋白应激反应（unfolded protein stress response）及免疫原性细胞死亡（immunogenic cell death）诱导相关的作用机制；③解析了其亚细胞定位，并对RNA G-四链体（RNA G-quadruplex）形成区域的结构与化学特征进行了预测，为新型治疗策略的开发铺平了道路。为揭示RP11-350G8.5基因敲除（KO）对硼替佐米耐药（ABZB）多发性骨髓瘤细胞所诱导的分子改变，本研究在转导乱序对照（SCRAMBLE）或RP11-350G8.5敲除CRISPR-Cas9载体7天后，通过两次独立生物学重复的RNA测序（RNA-sequencing）完成了该细胞系的转录组分析。