PDF(Pubmed)
Abstract：
The recurrence of diffuse large B-cell lymphoma (DLBCL) has been observed in 40% of cases. The standard of care for refractory/relapsed DLBCL (RR-DLBCL) is platinum-based treatment prior to autologous stem cell transplantation; however, the prognosis for RR-DLBCL patients remains poor. Thus, to identify genes affecting the cisplatin response in DLBCL, cisplatin-based whole-genome CRISPR-Cas9 knockout screens were performed in this study. We discovered DNA damage response (DDR) pathways as enriched among identified sensitizing CRISPR-mediated gene knockouts. In line, the knockout of the nucleotide excision repair genes XPA and ERCC6 sensitized DLBCL cells to platinum drugs irrespective of proliferation rate, thus documenting DDR as essential for cisplatin sensitivity in DLBCL. Functional analysis revealed that the loss of XPA and ERCC6 increased DNA damage levels and altered cell cycle distribution. Interestingly, we also identified BTK, which is involved in B-cell receptor signaling, to affect cisplatin response. The knockout of BTK increased cisplatin sensitivity in DLBCL cells, and combinatory drug screens revealed a synergistic effect of the BTK inhibitor, ibrutinib, with platinum drugs at low concentrations. Applying local and external DLBCL cohorts, we addressed the clinical relevance of the genes identified in the CRISPR screens. BTK was among the most frequently mutated genes with a frequency of 3-5%, and XPA and ERCC6 were also mutated, albeit at lower frequencies. Furthermore, 27-54% of diagnostic DLBCL samples had mutations in pathways that can sensitize cells to cisplatin. In conclusion, this study shows that XPA and ERCC6, in addition to BTK, are essential for the response to platinum-based drugs in DLBCL.
摘要：
在40％的病例中观察到弥漫性大B细胞淋巴瘤（DLBCL）的复发。治疗难治性/复发性DLBCL（RR-DLBCL）的标准是自体干细胞移植前的铂类治疗；然而，RR-DLBCL患者的预后仍然较差.因此，为了鉴定影响DLBCL中顺铂反应的基因，在这项研究中进行了基于顺铂的全基因组CRISPR-Cas9基因敲除筛选。我们发现了DNA损伤反应（DDR）途径，丰富了鉴定的敏感CRISPR介导的基因敲除。在一条线上，核苷酸切除修复基因XPA和ERCC6的敲除使DLBCL细胞对铂类药物敏感，而与增殖率无关，因此证明DDR对于DLBCL中顺铂敏感性至关重要。功能分析显示，XPA和ERCC6的缺失增加了DNA损伤水平并改变了细胞周期分布。有趣的是,我们还确定了BTK，参与B细胞受体信号传导，影响顺铂反应。敲除BTK可提高DLBCL细胞的顺铂敏感性,和组合药物筛选显示BTK抑制剂的协同作用，伊布替尼,低浓度的铂类药物。应用本地和外部DLBCL队列，我们讨论了在CRISPR筛选中鉴定的基因的临床相关性.BTK是最常见的突变基因之一，频率为3-5%，XPA和ERCC6也发生了突变，尽管频率较低。此外，27-54%的诊断性DLBCL样品在可使细胞对顺铂敏感的途径中具有突变。总之,这项研究表明，除了BTK之外，XPA和ERCC6，对DLBCL中铂类药物的反应至关重要。
