PO.ET02.07 · 实验与分子治疗
一种携带VEXAS综合征中常见UBA1突变的工程化人髓系细胞系对蛋白酶体抑制剂硼替佐米敏感
An engineered human myeloid cell line with a UBA1 mutation frequently detected in VEXAS syndrome is sensitive to the proteasome inhibitor bortezomib
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摘要 Abstract
中文摘要
VEXAS综合征是一种由造血祖细胞中UBA1基因体细胞点突变引起的全身性炎症性疾病。骨髓增生异常综合征(MDS)是一类造血系统恶性肿瘤,其造血干细胞携带基因突变,尚无可治愈药物。与VEXAS综合征中检出的相同类型UBA1基因点突变偶尔可在MDS中检出。靶向突变型UBA1蛋白的试剂或许能够清除携带UBA1点突变的异常细胞。为验证这一假设,我们建立了一个携带UBA1突变的模型细胞系,并分析了突变细胞的药物敏感性。首先,我们采用基于AI的预测分析,以寻找将突变导入UBA1基因最可靠的靶点位点和酶。其次,我们使用改良的CRISPR/Cas9方法、腺嘌呤碱基编辑(ABE)酶和一个靶向sgRNA构建体,将VEXAS综合征中常检出的点突变导入UBA1基因的第二个起始密码子。第三,我们使用聚乙烯亚胺将该点突变导入293T细胞,并确认了靶点位点的碱基改变以及靶点周围无非特异性碱基改变。第四,我们还使用电穿孔法将UBA1突变导入人髓系细胞系K562,发现导入细胞在硼替佐米处理后出现大量细胞死亡(野生型细胞的ED50为15.7nM,导入细胞的ED50为3.8nM),硼替佐米可抑制蛋白酶体活性。UBA1在泛素/蛋白酶体通路中发挥重要作用,用以降解待处理蛋白。UBA1基因突变导致异常细胞中降解蛋白的累积。通过硼替佐米抑制泛素/蛋白酶体通路使降解蛋白进一步累积,可能杀伤了突变细胞。值得注意的是,由于该方法仅使30-40%的K562细胞发生碱基改变,发生碱基转换的细胞可能分泌了细胞毒性细胞因子/趋化因子,杀伤了未发生碱基转换的邻近细胞,从而导致大量细胞死亡。我们成功分离了发生碱基转换的单细胞,并评估了硼替佐米(一种在临床上常用于治疗多发性骨髓瘤的药物)对携带UBA1基因突变细胞的作用。我们所建立的工程化细胞系将有助于临床医生为携带UBA1突变的MDS患者寻找有效的新药。
查看英文原文 English abstract
VEXAS syndrome is a systemic inflammatory disorder caused by somatic point mutations ofthe UBA1 gene in hematopoietic progenitor cells. Myelodysplastic syndrome (MDS) is oneof hematologic malignancies, in which hematopoietic stem cells have genetic mutations,without curable drugs. The same types of point mutations of the UBA1 gene as detected inVEXAS syndrome are occasionally detected in MDS. Reagents targeting mutated UBA1proteins may be able to eradicate the abnormal cells with the UBA1 point mutations.To examine this hypothesis, we have established a model cell line with the UBA1 mutationand analyzed drug-sensitivity of the mutated cells. First, we employed AI-based predictionanalyses to find the most reliable target sites and enzymes for introduction of the mutationinto the UBA1 gene. Second, we introduced the point mutation into the second start codonof the UBA1 gene, which is often detected in VEXAS syndrome, using a modifiedCRISPR/Cas9 method, adenosine-base-editing (ABE) enzyme and a targeting sgRNAconstruct. Third, we introduced the mutation into 293T cells using polyethyleneimine andconfirmed the base-change of the target site and absence of non-specific base-changes aroundthe target site. Fourth, we also introduced the UBA1 mutation into a human myeloid cell line,K562, using electroporation and found massive cell death of the introduced cells afterbortezomib treatment (ED₅₀ of wild-type cells was 15.7nM and ED₅₀ of the introduced cellswas 3.8nM), which inhibits proteasome activity.UBA1 plays an important role in ubiquitin/proteasome pathway to destroy degraded proteins.The mutation of the UBA1 gene leads to accumulation of degraded proteins in the abnormalcells. Further accumulation of degraded proteins by inhibition of the ubiquitin/proteasomepathway with bortezomib may have killed the mutated cells.Of note, since only 30-40% of K562 cells had the base-change by this method, cells with thebase-conversion may have secreted cytotoxic cytokines/chemokines, killing neighboring cellswithout the base-conversion, causing the massive cell death.We successfully isolated single cells with the base-conversion and evaluated effects of a drug,
bortezomib, which is frequently used at bedside for treatments of multiple myeloma, for thecells with the mutation of UBA1 gene.The engineered cell line we have established would help clinicians to find new drugs effectivefor MDS patients with the UBA1 mutations.
利益披露 Disclosure
H. Yazawa, None.