Remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as promising anticancer strategy. Here, we identify a novel synergistic interaction of the BET inhibitor JQ1 with the PI3Kα-specific inhibitor BYL719 to trigger mitochondrial apoptosis and to suppress tumor growth in models of rhabdomyosarcoma (RMS). RNA-Seq revealed that JQ1/BYL719 co-treatment shifts the overall balance of BCL-2 family gene expression towards apoptosis and upregulates expression of BMF, BCL2L11 (BIM), and PMAIP1 (NOXA) while downregulating BCL2L1 (BCL-xL). These changes were confirmed by qRT-PCR and western blot analysis. Ingenuity pathway analysis (IPA) of RNA-Seq data followed by validation qRT-PCR and western blot identified MYC and FOXO3a as potential transcription factors (TFs) upstream of the observed gene expression pattern. Immunoprecipitation (IP) studies showed that JQ1/BYL719-stimulated increase in BIM expression enhances the neutralization of antiapoptotic BCL-2, BCL-xL, and MCL-1. This promotes the activation of BAK and BAX and caspase-dependent apoptosis, as (1) individual silencing of BMF, BIM, NOXA, BAK, or BAX, (2) overexpression of BCL-2 or MCL-1 or (3) the caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk) all rescue JQ1/BYL719-induced cell death. In conclusion, co-inhibition of BET proteins and PI3Kα cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins. This discovery opens exciting perspectives for therapeutic exploitation of BET inhibitors in RMS.

中文翻译：

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BET蛋白和PI3Kα的共同抑制触发横纹肌肉瘤细胞中线粒体的凋亡。

通过靶向溴结构域和末端外（BET）蛋白重塑转录已成为有前途的抗癌策略。在这里，我们确定了横纹肌肉瘤（RMS）模型中BET抑制剂JQ1与PI3Kα特异性抑制剂BYL719的新型协同相互作用，可触发线粒体凋亡并抑制肿瘤生长。RNA-Seq显示，JQ1 / BYL719共同治疗可将BCL-2家族基因表达的总体平衡移向凋亡，并上调BMF，BCL2L11（BIM）和PMAIP1（NOXA）的表达，同时下调BCL2L1（BCL-xL）。通过qRT-PCR和蛋白质印迹分析证实了这些变化。RNA-Seq数据的独创性途径分析（IPA），然后通过验证qRT-PCR和Western blot鉴定，MYC和FOXO3a是观察到的基因表达模式上游的潜在转录因子（TFs）。免疫沉淀（IP）研究表明，JQ1 / BYL719刺激的BIM表达增加可增强抗凋亡BCL-2，BCL-xL和MCL-1的中和作用。这促进了BAK和BAX的激活以及caspase依赖性凋亡，因为（1）BMF，BIM，NOXA，BAK或BAX各自沉默，（2）BCL-2或MCL-1的过表达或（3）caspase抑制剂N-苄氧基羰基-Val-Ala-Asp（O-Me）氟甲基酮（zVAD.fmk）均可挽救JQ1 / BYL719诱导的细胞死亡。总之，BET蛋白和PI3Kα的共同抑制通过BCL-2家族蛋白的凋亡再平衡协同诱导线粒体凋亡。这一发现为RMS中BET抑制剂的治疗开发开辟了令人兴奋的前景。