Richter Günther H S, Hensel Tim, Schmidt Oxana, Saratov Vadim, von Heyking Kristina, Becker-Dettling Fiona, Prexler Carolin, Yen Hsi-Yu, Steiger Katja, Fulda Simone, Dirksen Uta, Weichert Wilko, Wang Shudong, Burdach Stefan, Schäfer Beat W
Children's Cancer Research Centre and Department of Pediatrics, Klinikum rechts der Isar, Technische Universität München, 80804 Munich, Germany.
German Cancer Research Center (DKFZ), partner site Munich, 81377 Munich, Germany.
Cancers (Basel). 2020 Jan 28;12(2):304. doi: 10.3390/cancers12020304.
Previously, we used inhibitors blocking BET bromodomain binding proteins (BRDs) in Ewing sarcoma (EwS) and observed that long term treatment resulted in the development of resistance. Here, we analyze the possible interaction of BRD4 with cyclin-dependent kinase (CDK) 9.
Co-immunoprecipitation experiments (CoIP) to characterize BRD4 interaction and functional consequences of inhibiting transcriptional elongation were assessed using drugs targeting of BRD4 or CDK9, either alone or in combination.
CoIP revealed an interaction of BRD4 with EWS-FLI1 and CDK9 in EwS. Treatment of EwS cells with CDKI-73, a specific CDK9 inhibitor (CDK9i), induced a rapid downregulation of EWS-FLI1 expression and block of contact-dependent growth. CDKI-73 induced apoptosis in EwS, as depicted by cleavage of Caspase 7 (CASP7), PARP and increased CASP3 activity, similar to JQ1. Microarray analysis following CDKI-73 treatment uncovered a transcriptional program that was only partially comparable to BRD inhibition. Strikingly, combined treatment of EwS with BRD- and CDK9-inhibitors re-sensitized cells, and was overall more effective than individual drugs not only in vitro but also in a preclinical mouse model in vivo.
Treatment with BRD inhibitors in combination with CDK9i offers a new treatment option that significantly blocks the pathognomonic EWS-ETS transcriptional program and malignant phenotype of EwS.
此前，我们在尤因肉瘤（EwS）中使用了阻断BET溴结构域结合蛋白（BRD）的抑制剂，并观察到长期治疗会导致耐药性的产生。在此，我们分析BRD4与细胞周期蛋白依赖性激酶（CDK）9之间可能的相互作用。
使用单独或联合靶向BRD4或CDK9的药物，通过免疫共沉淀实验（CoIP）来表征BRD4的相互作用以及抑制转录延伸的功能后果。
CoIP显示在EwS中BRD4与EWS-FLI1和CDK9存在相互作用。用特异性CDK9抑制剂（CDK9i）CDKI-73处理EwS细胞，可诱导EWS-FLI1表达迅速下调并阻断接触依赖性生长。CDKI-73诱导EwS细胞凋亡，如半胱天冬酶7（CASP7）、聚（ADP-核糖）聚合酶（PARP）的裂解以及CASP3活性增加所示，这与JQ1类似。CDKI-73处理后的微阵列分析揭示了一个转录程序，该程序仅部分与BRD抑制相当。引人注目的是，BRD抑制剂和CDK9抑制剂联合处理EwS可使细胞重新敏感，并且不仅在体外而且在体内临床前小鼠模型中总体上比单一药物更有效。
BRD抑制剂与CDK9i联合治疗提供了一种新的治疗选择，可显著阻断EwS特征性的EWS-ETS转录程序和恶性表型。
