Analysis of molecular mechanisms that are regulated through HDAC6and heat shock proteins in leukemic cells

白血病细胞中HDAC6和热休克蛋白调控的分子机制分析

• 批准号: 427404172
• 负责人: Professor Dr. Oliver Holger Krämer
• 金额: --
• 依托单位: Institut für Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/427404172?language=en
• 关键词: Analysis; molecular; mechanisms; regulated; through

We have synthesized novel, highly selective, and efficient histone deacetylase-6 inhibitors (HDAC6i), of which Marbostat-100 is our lead compound. While inhibition of HDAC6 increases the levels of HDAC6 and other proteins that are degraded by the proteasome, the inhibition of heat shock protein-90 (HSP90) with its novel, clinically tested HSP90 inhibitor (HSP90i) Onalespib destabilizes such proteins. A combined inhibition of HDAC6 and HSP90 leads to an increase of polyubiquitinated proteins, a higher enzymatic activity of the proteasome, and a loss of anti-apoptotic proteins by the proteasome. We want to determine how HDAC6 and HSP90 regulate a molecular equilibrium that controls protein homeostasis. HDAC6 is involved in the formation and transport of aggresomes, which are detoxified by autophagy and lysosomal degradation. Accordingly, a combinatorial inhibition of HDAC6 and HSP90 does not lead to an increase of aggresome formation or induction of autophagy. In contrast, there is a breakdown of autophagy and apoptosis is induced. These data suggest that HDAC6 acts as a master switch that prevents the hyperactivation of proteasomal activity and the induction of late apoptosis upon proteotoxic stress. Such an integration of apoptosis and proteasomal activity through HDAC6 has not been reported so far. These mechanisms might have been overlooked, because a lot of cell biological studies analyzed the role of HDAC6 with proteasomal inhibitors to induce proteotoxic stress. This is no criticism of the published data, but merely a description of experimental settings. With Marbostat-100 we hold a very specific and non-toxic tool to inhibit HDAC6. We want to proof and expand our hypothesis that HDAC6 acts as a gatekeeper for protein stability and proteasomal activity. We want to use genetic and pharmacological strategies for these experiments. We will determine the relevance of the catalytic and ubiquitin binding domains of HDAC6 on protein and cell fate during proteotoxic stress with the CRISPR-Cas9 method. We aim to examine the influence of HDAC6 and HSP90 on apoptosis, proteasomal activity, aggresome formation, autophagy, stress granules, endoplasmic reticulum-associated stress, and protein hyperacetylation with analyses that are driven by hypotheses and with unbiased global tests. Within this context, we want to investigate potential roles of USP10, p62, HSP90β, HSP70, HSP72, the HSP70-associated E3 ubiquitin ligase CHIP, GRP78/BIP, BAG1/BAG3, c-MYC, and further regulatory proteins. Likewise, we want to clarify why FLT3-ITD positive leukemic cells are hypersensitive to HSP90i and combinations of HDAC6i and HSP90i. To identify a therapeutic relevance of Marbostat-100 and Onalespib, we will analyze primary FLT3-ITD positive leukemic cells and a zebrafish model.

我们合成了新型、高选择性和有效的组蛋白脱乙酰酶-6抑制剂（HDAC 6 i），其中Marbostat-100是我们的先导化合物。虽然HDAC 6的抑制增加了HDAC 6和其他被蛋白酶体降解的蛋白质的水平，但热休克蛋白-90（HSP 90）的抑制及其新的临床测试的HSP 90抑制剂（HSP 90 i）Onalb使这些蛋白质不稳定。HDAC 6和HSP 90的组合抑制导致多聚泛素化蛋白的增加、蛋白酶体的更高酶活性和蛋白酶体的抗凋亡蛋白的损失。我们想确定HDAC 6和HSP 90如何调节控制蛋白质稳态的分子平衡。HDAC 6参与侵袭体的形成和运输，侵袭体通过自噬和溶酶体降解解毒。因此，HDAC 6和HSP 90的组合抑制不会导致攻击组形成的增加或自噬的诱导。相反，自噬被破坏并诱导细胞凋亡。这些数据表明，HDAC 6作为一个主开关，防止蛋白酶体活性的过度激活和诱导晚期细胞凋亡后蛋白毒性应激。这种通过HDAC 6整合细胞凋亡和蛋白酶体活性的研究至今未见报道。这些机制可能被忽视了，因为许多细胞生物学研究分析了HDAC 6与蛋白酶体抑制剂诱导蛋白毒性应激的作用。这并不是对已发表数据的批评，而仅仅是对实验设置的描述。Marbostat-100是一种非常特异且无毒的抑制HDAC 6的工具。我们希望证明并扩展我们的假设，即HDAC 6作为蛋白质稳定性和蛋白酶体活性的看门人。我们希望在这些实验中使用遗传和药理学策略。我们将使用CRISPR-Cas9方法确定HDAC 6的催化和泛素结合结构域在蛋白毒性应激期间对蛋白质和细胞命运的相关性。我们的目标是通过假设驱动的分析和无偏见的全球测试来研究HDAC 6和HSP 90对细胞凋亡、蛋白酶体活性、攻击体形成、自噬、应激颗粒、内质网相关应激和蛋白质超乙酰化的影响。在此背景下，我们希望研究USP 10，p62，HSP 90 β，HSP 70，HSP 72，HSP 70相关的E3泛素连接酶CHIP，GRP 78/BIP，BAG 1/BAG 3，c-MYC和其他调控蛋白的潜在作用。同样地，我们想要阐明为什么FLT 3-ITD阳性白血病细胞对HSP 90 i以及HDAC 6 i和HSP 90 i的组合超敏感。为了确定Marbostat-100和Onalbib的治疗相关性，我们将分析原代FLT 3-ITD阳性白血病细胞和斑马鱼模型。