当前，Velcade作为治疗多发性骨髓瘤（MM）和套细胞淋巴瘤（MCL）的前沿药物，在临床上取得了良好的治疗效果；然而，如同其他的化疗药物一样，Velcade也面临部分病人对其无应答和药物耐受问题。这里，我们主要运用CRISPR/Cas9基因编辑体系工具对MM细胞进行全基因组的基因表达的修饰，通过敲除或激活目标基因，进而引起MM细胞对Velcade耐受。我们已经通过运用CRISPRi（基因敲除）技术，初步完成了全基因组基因的筛选，并针对性选择了６６３个基因，其被敲除后引起MM对Velcade耐受，建立了其对应的sgRNA亚文库（FL01），被亚文库编辑后的MM细胞明显对Velcade不敏感。我们将对FL01文库进行进一步的筛选；同时，从初步筛选的结果，我们挑选了四个抑癌基因（ATG9A,BIRC2,PMAIP1和TRAF2）进行具体的功能验证及耐药机理研究。我们亦将运用CRISPRa（基因激活）技术，通过对对Velcade耐受机理进行全面的研究。筛选出的基因将进行功能验证。

Velcade (Bortezomib) is an effective therapy for multiple myeloma (MM) and refractory mantle cell lymphoma (MCL). Although it has been successfully used in the clinic for over a decade and achieved dramatically therapeutic effects on MM, resistance impedes its wide applications. The molecular mechanism of the resistance remains under investigation. Here, we employ unbiased screening methods to understand the genetic mechanism of the resistance. We used CRISPRi gene editing system to knockout whole genomic genes in a MM line, RPMI8226. Edited cells were treated with Velcade, and survival cells were collected for sgRNAs enrichment analysis. Totally, we picked up 663 genes which response sgRNAs were significantly enriched for further validation. For the validation, we generated a sgRNA sub-library (FL01) to target the selected 663 genes. We transfected RPMI8226 cells with FL01 and treated the cells with Velcade for dose response. We found that cells edited by Fl01 showed significant resistance to Velcade compared to unedited cells. At same time, based on sgRNA enrichment analysis and previous studies, we selected four tumor suppressor genes (ATG9A, BIRC2, PMAIP1, and TRAF2) for individual studies to investigate the mechanism of causing Velcade resistance in MM. In this proposal, we will also use CRISPRa system to activate whole genomic gene expression for Velcade resistance studies, and enriched genes will be performed functional studies.

主要研究内：本课题主要研究内容是揭示多发性骨髓瘤（MM）细胞对临床一线药物蛋白水解酶复合体 （proteasome）抑制剂Velcade的耐药机理，探索鉴定研究目标是鉴定可以和Velcade联合应用于MM的治疗的可能潜在靶点。重要结果：通过利用CRISPR基因编辑技术， 我们发现在MM细胞上，敲出某些基因的表达导致MM细胞对Velcade耐受；针对这些基因，我们构建了CRISPR亚文库，进一步验证了这些基因的缺失确实可以引起MM细胞对Velcade耐受。我们计划进一步研究该类基因引起的Velcade耐药机理。在临床上,对Velcade治疗的MM病人预后进行预测，提出预防Velcade耐药的方案及替代治疗策略。科学意义：发现引起MM细胞对Velcade耐受的基因，并阐明引起耐药机理，将有助于阐明Velcade抗肿瘤机制，鉴定可以与Velcade协同作用的新靶点，进而开发可以Velcade联合用于MM治疗的新药物。