Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Although combined treatment with gemcitabine and albumin-bound paclitaxel has improved the prognosis of PDAC, both intrinsic and acquired chemoresistance remain as severe hurtles towards improved prognosis. Thus, new therapeutic targets and innovative strategies are urgently needed. In this study, we used the KPC mouse model-derived PDAC cell line TB32047 to perform kinome-wide CRISPR-Cas9 loss-of-function screening. Next-generation sequencing and MAGeCK-VISPR analysis were performed to identify candidate genes. We then conducted cell viability, clonogenic, and apoptosis assays and evaluated the synergistic therapeutic effects of cyclin-dependent kinase 7 (CDK7) depletion or inhibition with gemcitabine (GEM) and paclitaxel (PTX) in a murine orthotopic pancreatic cancer model. For mechanistic studies, we performed genome enrichment analysis (GSEA) and Western blotting to identify and verify the pathways that render PDAC sensitive to GEM/PTX therapy. We identified several cell cycle checkpoint kinases and DNA damage-related kinases as targets for overcoming chemoresistance. Among them, CDK7 ranked highly in both screenings. We demonstrated that both gene knockout and pharmacological inhibition of CDK7 by THZ1 result in cell cycle arrest, apoptosis induction, and DNA damage at least predominantly through the STAT3-MCL1-CHK1 axis. Furthermore, THZ1 synergized with GEM and PTX in vitro and in vivo, resulting in enhanced antitumor effects. Our findings support the application of CRISPR-Cas9 screening in identifying novel therapeutic targets and suggest new strategies for overcoming chemoresistance in pancreatic cancer.

中文翻译：

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CDK7抑制增强了胰腺癌对多药化疗的反应

胰腺导管腺癌 (PDAC) 是一种侵袭性癌症，预后不佳。尽管吉西他滨和白蛋白结合紫杉醇的联合治疗改善了 PDAC 的预后，但内在和获得性化学抗性仍然是改善预后的严重障碍。因此，迫切需要新的治疗靶点和创新策略。在本研究中，我们使用 KPC 小鼠模型衍生的 PDAC 细胞系 TB32047 进行了整个激酶组的 CRISPR-Cas9 功能丧失筛选。进行了下一代测序和 MAGeCK-VISPR 分析以鉴定候选基因。然后我们进行了细胞活力、克隆形成、和细胞凋亡测定，并评估了细胞周期蛋白依赖性激酶 7 (CDK7) 消耗或吉西他滨 (GEM) 和紫杉​​醇 (PTX) 在小鼠原位胰腺癌模型中抑制的协同治疗效果。对于机制研究，我们进行了基因组富集分析 (GSEA) 和蛋白质印迹，以识别和验证使 PDAC 对 GEM/PTX 治疗敏感的途径。我们确定了几种细胞周期检查点激酶和 DNA 损伤相关激酶作为克服化学抗性的目标。其中，CDK7在两次放映中均名列前茅。我们证明了 THZ1 对 CDK7 的基因敲除和药理学抑制至少主要通过 STAT3-MCL1-CHK1 轴导致细胞周期停滞、细胞凋亡诱导和 DNA 损伤。此外，THZ1 在体外和体内与 GEM 和 PTX 协同作用，从而增强抗肿瘤作用。我们的研究结果支持 CRISPR-Cas9 筛选在识别新的治疗靶点中的应用，并提出了克服胰腺癌化疗耐药性的新策略。