Inhibitors of Histone Deacetylases; evaluation and target identification of a cardioprotective combination therapy for cancer

心肌收缩功能的恶化是癌症治疗成功的主要限制因素。此外，心脏功能与癌症相关的发病率和死亡率密切相关。蒽环类药物是久负盛名的抗癌药物，也是许多经典治疗方案的一部分，它们对心脏有深刻的副作用。尽管它与临床相关，但人们对共同治疗的下游靶点和潜在的心脏保护机制的了解非常有限。组蛋白脱乙酰酶(HDAC)作为表观遗传抑制因子发挥作用。最近的工作表明，HDAC抑制剂(HDACi)不仅在各种癌症实体中成功地作为治疗药物，而且在心力衰竭的临床前模型中也对心脏有益。尽管HDACi的有益潜力已被证明，但其心脏保护作用的具体下游机制迄今尚不清楚。在我们正在进行的工作中，我们确定了伴侣14-3-3在用第一个FDA批准的HDACi伏立诺治疗后在心肌细胞中被高乙酰化。我们可以证明，14-3-3的超乙酰化导致与II类HDACs的结合减少，即HDAC4和5。核HDAC4/5是转录因子(TF)心肌细胞增强因子2(MEF2)的抑制因子，MEF2是导致病理性心脏重构和功能障碍的基因上调所必需的。此外，我们还发现，恒涡肌抑制HDAC4/5的核输出。相反，恒涡恒对新生大鼠心肌细胞MEF2有明显的抑制作用，而敲除HDAC4和HDAC5则减弱了依从性抑制MEF2的作用。因此，我们发现心肌细胞特异性的HDAC4基因缺失增加了在用蒽环类药物阿霉素治疗时心脏功能障碍的易感性，并上调了MEF2靶基因myh7的表达。进一步鉴定myh7启动子以阿霉素依赖的方式与拓扑异构酶IIb结合。基于我们未发表的工作，我们提出了依赖HDAC的MEF2抑制在阿霉素诱导的心脏毒性中的关键作用。这种效应可以通过与HDACi涡流调节器共处理来放大。然而，涡旋器和其他潜在的独立于TFMEF2的下游靶标引起的表观遗传学变化仍然是未知的，这就是为什么它们需要进一步研究的原因。具体目标是：(A)鉴定心脏中依赖HDACi的全球表观遗传学变化，以及(B)确定心肌细胞中HDACi靶向的基因组区域。2：探讨MEF2在阿霉素治疗病理基因调控中的作用。3.通过体外功能得失实验，研究安非他明对大鼠心脏的潜在保护作用，并探讨其可能的作用机制。

Deterioration of myocardial contractile function is a major limitation for successful cancer therapies. Moreover, cardiac function is strongly associated with cancer-related morbidity and mortality. Anthracyclines, which are well-established anti-cancer drugs and part of many classical therapeutic regimes share profound cardiac side effects. Despite its clinical relevance, there is only very limited knowledge about the downstream targets involved and potential cardioprotective mechanisms of co-therapies. Histone deacetylases (HDACs) work as epigenetic repressors. Recent work has shown that HDAC inhibitors (HDACi) not only successful as therapeutics in various cancer entities but also beneficial for the heart in preclinical models of heart failure. Even though its beneficial potential has been shown, the specific downstream mechanisms of the cardioprotective effects of HDACi are not clarified so far. In our ongoing work, we identified the chaperone 14-3-3 to be hyperacetylated in cardiomyocytes upon treatment with vorinostat, the first FDA approved HDACi. We could show, that hyperacetylation of 14-3-3 leads to reduced binding to class II HDACs, namely HDAC4 and 5. Nuclear HDAC4/5 act as repressors of the transcription factor (TF) myocyte enhancer factor 2 (MEF2), which is required for upregulation of genes that are causative for pathological cardiac remodeling and dysfunction. In addition, we found that vorinostat inhibits the nuclear export of HDAC4/5. Conversely, vorinostat resulted in remarkable suppression of MEF2 in neonatal rat ventricular cardiomyocytes (NRMVs) while knockdown of HDAC4 and HDAC5 attenuated the vorinostat-dependent MEF2 inhibition. These results suggest that the protective effects of vorinostat are partially transmitted by HDAC4/5. Accordingly, we found that cardiomyocyte-specific deletion of HDAC4 increases the susceptibility for cardiac dysfunction upon treatment with the anthracycline ‘doxorubicin’ and upregulates the MEF2 target gene myh7. The myh7-promotor was further identified to be bound to topoisomerase IIb in a doxorubicin-dependent manner. Based on our unpublished work, we propose a crucial role of HDAC-dependent MEF2 inhibition in doxorubicin-induced cardiotoxicity. This effect can be amplified by co-treatment with the HDACi vorinostat. Still, the epigenetic changes caused by vorinostat and potential other downstream targets independent of the TF MEF2 are widely unknown, which is why they require further investigations. The specific aims are: 1: (a) Identification of global HDACi-dependent epigenetic changes in the heart and (b) determination of HDACi-targeted genomic regions in cardiomyocytes. 2: To determine the role of MEF2 in the control of pathological gene regulation upon doxorubicin treatment. 3: To determine potential cardioprotective effects of vorinostat upon co-treatment with anthracyclines in vivo and to elucidate of the downstream mechanism via gain and loss of function experiments in vitro.