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CDK9 Blockade Exploits Context-Dependent Transcriptional Changes to Improve Activity and Limit Toxicity of Mithramycin for Ewing Sarcoma
Molecular Cancer Therapeutics ( IF 5.3 ) Pub Date : 2020-03-03 , DOI: 10.1158/1535-7163.mct-19-0775
Guillermo Flores 1, 2 , Joel H Everett 3 , Elissa A Boguslawski 1 , Brandon M Oswald 1 , Zachary B Madaj 4 , Ian Beddows 4 , Sergey Dikalov 5 , Marie Adams 4 , Carleen A Klumpp-Thomas 6 , Susan M Kitchen-Goosen 1 , Scott E Martin 6 , Natasha J Caplen 7 , Lee J Helman 8 , Patrick J Grohar 1, 8, 9, 10, 11, 12
Affiliation  

There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor–targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1–targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic “class” effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1–targeted combination therapy for Ewing sarcoma.

中文翻译:

CDK9 封锁利用上下文相关的转录变化来提高活性并限制光神霉素对尤文肉瘤的毒性

需要开发新的方法来改善转录因子靶向治疗的疗效和毒性之间的平衡。在这项研究中,我们利用 RNA 聚合酶 II 持续合成能力的上下文相关差异作为提高活性和限制 EWS-FLI1 靶向小分子光神霉素对尤文肉瘤的毒性的一种方法。光神霉素对尤文肉瘤的临床活性受到脱靶肝毒性的限制,该毒性将血清浓度限制在不足以抑制 EWS-FLI1 的水平。在这项研究中,我们对可成药基因组进行 siRNA 筛选,然后进行基质药物筛选,以确定光神霉素增效剂和细胞周期蛋白依赖性激酶 9 (CDK9) 抑制剂的协同“类”效应。这些 CDK9 抑制剂增强了光神霉素介导的 EWS-FLI1 转录程序的抑制,导致 IC50 的变化和尤文肉瘤异种移植物的显着消退。为了确定这些化合物是否也可能具有肝脏保护作用,我们对 HepG2 细胞中所有已知的肝毒性基因进行了 qPCR 筛选,以确定导致肝毒性的光神霉素驱动的转录变化。光神霉素在 HepG2 而非尤文肉瘤细胞中诱导 BTG2 基因的表达,这导致活性氧 (ROS) 的肝脏特异性积累。BTG2 的 siRNA 沉默挽救了这些细胞中 ROS 的诱导和光神霉素的细胞毒性。此外,CDK9 抑制阻止了 BTG2 的诱导,以限制 HepG2 中的细胞毒性,但不是尤文肉瘤细胞。
更新日期:2020-03-03
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