CRISPR Screen of Venetoclax Response-Associated Genes Identifies Transcription Factor ZNF740 as a Key Functional Regulator [CUT&Run]. CRISPR Screen of Venetoclax Response-Associated Genes Identifies Transcription Factor ZNF740 as a Key Functional Regulator [CUT&Run]

BCL-2 inhibitors such as venetoclax offer therapeutic promise in acute myeloid leukemia (AML) and other cancers, but drug resistance poses a significant challenge. It is crucial to understand the mechanisms that regulate venetoclax response. While correlative studies have identified numerous genes linked to venetoclax sensitivity, their direct impact on the drug response remains unclear. In this study, we targeted around 1,400 genes upregulated in venetoclax-sensitive primary AML samples and carried out a CRISPR knockout screen to evaluate their direct effects on venetoclax response. Our screen identified the transcription factor ZNF740 as a critical regulator, with its expression consistently predicting venetoclax sensitivity across subtypes of the FAB classification. ZNF740 depletion leads to increased resistance to venetoclax, while its overexpression enhances sensitivity to the drug. Mechanistically, our integrative transcriptomic and genomic analysis identifies NOXA as a direct target of ZNF740, which negatively regulates MCL-1 protein stability. Loss of ZNF740 downregulates NOXA and increases the steady state protein levels of MCL-1 in AML cells. Restoring NOXA expression in ZNF740-depleted cells re-sensitizes AML cells to venetoclax treatment. Furthermore, we demonstrated that dual targeting of MCL-1 and BCL-2 effectively treats ZNF740-deficient AML in vivo. Together, our work systematically elucidates the causal relationship between venetoclax response signature genes and establishes ZNF740 as a novel transcription factor regulating venetoclax sensitivity. Overall design: We performed Cut&Run-seq to profile ZNF740 binding sites in Cas9-positive OCI-AML2 and MOLM-13 cells expressing guide RNAs targeting either Luciferase or ZNF740.

以维奈克拉（venetoclax）为代表的BCL-2抑制剂（BCL-2 inhibitor）在急性髓系白血病（AML）及其他癌症中展现出治疗潜力，但耐药性仍是其临床转化的重大挑战。阐明调控维奈克拉应答的分子机制具有重要意义。既往关联研究已鉴定出众多与维奈克拉敏感性相关的基因，但这些基因对药物应答的直接作用仍不明确。
本研究针对维奈克拉敏感的原代AML样本中上调的约1400个基因，开展CRISPR敲除筛选（CRISPR knockout screen）以评估其对维奈克拉应答的直接影响。筛选结果鉴定出转录因子ZNF740作为关键调控因子，其表达水平可稳定预测法美英（FAB）分型各亚型AML细胞对维奈克拉的敏感性。
敲低ZNF740会增强AML细胞对维奈克拉的耐药性，而过表达ZNF740则可提升细胞对药物的敏感性。
机制层面，本研究通过整合转录组与基因组分析，确定NOXA为ZNF740的直接靶标，且NOXA负调控MCL-1蛋白的稳定性。敲除ZNF740会下调NOXA的表达，并提升AML细胞中MCL-1蛋白的稳态水平。在ZNF740敲低的细胞中恢复NOXA的表达，可重新使AML细胞对维奈克拉治疗产生敏感性。
此外，本研究证实同时靶向MCL-1与BCL-2可在体内有效治疗ZNF740缺陷型AML。
综上，本研究系统阐明了维奈克拉应答特征基因与药物敏感性之间的因果关系，确立ZNF740作为调控维奈克拉敏感性的新型转录因子。
总体实验设计：我们在分别转染靶向荧光素酶（Luciferase）与ZNF740的向导RNA（guide RNA）的Cas9阳性OCI-AML2及MOLM-13细胞中，开展Cut&Run-seq（切割与运行测序）以解析ZNF740的全基因组结合位点。