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

点击翻译按钮获取中文摘要

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.