A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals major role of BAX in BH3 mimetics response [1]

In order to establish a strict p53-dependent gene expression profile, p53 negative clones were derived from two TP53+/+ and two TP53-/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the 6 clones from the two TP53+/+ myeloma cell lines, we established a 13-gene functional p53 score, involving genes downregulated on p53-silencing. This functional score allowed us to segregate clones, myeloma cell lines and patients’ samples according to the presence or absence of a single or double hit in TP53 gene i.e., chromosomal deletion and/or mutation. The score also distinguished 1,162 cell lines from hematological and solid cancers according to hits in TP53. Moreover, the 13-gene score was predictive of overall survival in two independent cohorts of patients. Among the 13 genes, we showed that p53-regulated BAX expression correlated to, and directly impacted, the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by modulating the amount of MCL1-BAX complexes. Resistance to cell death induced by p53-silencing was overcome by combining S63845 with venetoclax, although the synergy was significantly lower in p53-deficient versus -proficient clones and patients’ samples. Finally, by using scRNAseq analysis, we further showed in patients’ bone marrow samples that myeloma cells surviving to the BH3 mimetic combination had an about 3-fold lower p53 score. This data established a functional p53 score and showed that p53-mediating BAX expression greatly impacts mitochondria-mediated cell death in myeloma cells. CD138+ cells from patients’ samples were purified and gene expression profile was performed using RNA-Seq (3'SRP)(MM: multiple myeloma, sPCL: secondary plasma cell leukemia, pPCL: primary plasma cell leukemia)

为构建严谨的TP53依赖型基因表达谱，本研究借助CRISPR/Cas9基因编辑技术，从2株TP53野生型（TP53+/+）及2株携带t(4;14)染色体易位的TP53突变/缺失型（TP53-/mut）人骨髓瘤细胞系（HMCLs）中获得p53阴性克隆。从来自2株TP53野生型骨髓瘤细胞系的6个克隆中共有的17个差异表达基因中，我们筛选得到13个基因构成功能性p53评分系统，该评分包含p53沉默后表达下调的基因。该功能性评分可依据TP53基因是否存在单打击或双打击突变（即染色体缺失和/或基因突变），对克隆、骨髓瘤细胞系及患者样本进行分类。该评分还可依据TP53突变状态，区分1162株血液系统恶性肿瘤及实体瘤细胞系。此外，该13基因评分系统可在两个独立的多发性骨髓瘤患者队列中预测总生存期。在这13个基因中，我们证实p53调控的BAX表达与骨髓瘤细胞对MCL1 BH3模拟物S63845的敏感性相关，并可通过调节MCL1-BAX复合物的数量直接影响该敏感性。将S63845与维奈克拉（venetoclax）联合使用，可克服p53沉默诱导的细胞死亡抵抗，但在p53缺陷型与p53功能正常的克隆及患者样本中，该协同效应显著减弱。最后，通过单细胞RNA测序（scRNAseq）分析，我们在患者骨髓样本中进一步发现，对BH3模拟物联合疗法产生耐受的骨髓瘤细胞，其p53评分约降低3倍。本研究构建了功能性p53评分系统，并证实p53介导的BAX表达可显著影响骨髓瘤细胞中线粒体介导的细胞死亡过程。本研究对患者样本中的CD138阳性细胞进行了纯化，并通过RNA测序（3'SRP）完成基因表达谱分析（MM：多发性骨髓瘤，sPCL：继发性浆细胞白血病，pPCL：原发性浆细胞白血病）