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

我们合成了新型、高选择性、高效的组蛋白脱乙酰酶-6抑制剂(HDAC6i)，其中Marbostat-100是我们的先导化合物。虽然抑制HDAC6会增加HDAC6和其他被蛋白酶体降解的蛋白质的水平，但抑制热休克蛋白-90(HSP90)及其新型的临床测试的HSP90抑制剂(HSP90i)Onalespib会破坏这些蛋白质的稳定性。HDAC6和HSP90的联合抑制导致多泛素化蛋白的增加，蛋白酶体的酶活性更高，蛋白酶体失去了抗凋亡蛋白。我们想要确定HDAC6和HSP90如何调节控制蛋白质动态平衡的分子平衡。HDAC6参与侵袭体的形成和运输，侵袭体通过自噬和溶酶体降解来解毒。因此，联合抑制HDAC6和HSP90并不会增加侵袭体的形成或诱导自噬。相反，自噬的分解和细胞的凋亡被诱导。这些数据表明，HDAC6作为一个主开关，防止蛋白酶体活性的过度激活和蛋白毒性应激诱导的晚期细胞凋亡。到目前为止，还没有报道通过HDAC6将细胞凋亡和蛋白酶体活性整合在一起。这些机制可能被忽视了，因为许多细胞生物学研究分析了HDAC6与蛋白酶体抑制剂在诱导蛋白毒性应激中的作用。这并不是对已公布数据的批评，而只是对实验环境的描述。有了Marbostat-100，我们拥有一种非常特异和无毒的工具来抑制HDAC6。我们想要证明并扩展我们的假设，即HDAC6作为蛋白质稳定性和蛋白酶体活性的守门人。我们希望在这些实验中使用遗传和药理学策略。我们将用CRISPR-Cas9方法确定HDAC6的催化和泛素结合结构域与蛋白质和细胞在蛋白毒性应激中命运的相关性。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检测HDAC6和HSP90对细胞凋亡、蛋白酶体活性、侵袭体形成、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，我们想要研究USP10、P62、HSP90β、HSP70、HSP72、与HSP70相关的E3泛素连接酶芯片、GRP78/BIP、BAG1/BAG3、c-MYC以及其他调节蛋白的潜在作用。同样，我们想要阐明为什么Flt3-ITD阳性的白血病细胞对HSP90i以及HDAC6i和HSP90i的组合敏感。为了确定Marbostat-100和Onalespib的治疗相关性，我们将分析原代Flt3-ITD阳性白血病细胞和斑马鱼模型。

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.