Department of Urology, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, 81675, München, Germany.
Center for Translational Cancer Research (TranslaTUM), Klinikum rechts der Isar, Technical University of Munich, Einsteinstrasse 25, 81675, Munich, Germany.
J Exp Clin Cancer Res. 2019 Jul 22;38(1):322. doi: 10.1186/s13046-019-1322-9.
CDK4/6 inhibitors are a promising treatment strategy in tumor therapy but are hampered by resistance mechanisms. This study was performed to reveal predictive markers, mechanisms of resistance and to develop rational combination therapies for a personalized therapy approach in bladder cancer.
A genome-scale CRISPR-dCas9 activation screen for resistance to the CDK4/6 inhibitor Palbociclib was performed in the bladder cancer derived cell line T24. sgRNA counts were analyzed using next generation sequencing and MAGeCK-VISPR. Significantly enriched sgRNAs were cloned and validated on a molecular and functional level for mediating resistance to Palbociclib treatment. Analysis was done in vitro and in vivo in the chorioallantois membrane model of the chicken embryo. Comparison of screen hits to signaling pathways and clinically relevant molecular alterations was performed using DAVID, Reactome, DGIdb and cBioPortal.
In the screen, 1024 sgRNAs encoding for 995 genes were significantly enriched indicative of mediators of resistance. 8 random sgRNAs were validated, revealing partial rescue to Palbociclib treatment. Within this gene panel, members of Receptor-Tyrosine Kinases, PI3K-Akt, Ras/MAPK, JAK/STAT or Wnt signaling pathways were identified. Combination of Palbociclib with inhibitors against these signaling pathways revealed beneficial effects in vitro and in in vivo xenografts.
Identification of potential predictive markers, resistance mechanisms and rational combination therapies could be achieved by applying a CRISPR-dCas9 screening approach in bladder cancer.
CDK4/6 抑制剂是肿瘤治疗中一种很有前途的治疗策略，但受到耐药机制的阻碍。本研究旨在揭示预测标志物、耐药机制，并为膀胱癌的个体化治疗方法开发合理的联合治疗策略。
在膀胱癌衍生细胞系 T24 中，进行了针对 CDK4/6 抑制剂 Palbociclib 耐药的全基因组 CRISPR-dCas9 激活筛选。使用下一代测序和 MAGeCK-VISPR 对 sgRNA 计数进行分析。对显著富集的 sgRNA 进行克隆，并在分子和功能水平上进行验证，以介导对 Palbociclib 治疗的耐药性。在鸡胚绒毛尿囊膜模型中进行体外和体内分析。使用 DAVID、Reactome、DGIdb 和 cBioPortal 对筛选命中与信号通路和临床相关分子改变进行比较。
在筛选中，1024 个 sgRNA 编码 995 个基因显著富集，提示存在耐药的介导物。验证了 8 个随机 sgRNA，发现对 Palbociclib 治疗有部分挽救作用。在这个基因面板中，鉴定出受体酪氨酸激酶、PI3K-Akt、Ras/MAPK、JAK/STAT 或 Wnt 信号通路的成员。Palbociclib 与这些信号通路抑制剂的联合使用在体外和体内异种移植中显示出有益的效果。
通过在膀胱癌中应用 CRISPR-dCas9 筛选方法，可以鉴定出潜在的预测标志物、耐药机制和合理的联合治疗策略。
