Mechanistic studies of a novel inhibitor of the ubiquitin-proteasome system

The ubiquitin-proteasome system (UPS) not only maintains cellular proteostasis, but plays an essential role in multiple vital cellular processes, including survival and growth. Proteasomal inhibitors, such as bortezomib, have been shown to have effective anti-cancer properties, verifying the UPS as a viable drug target in cancer treatment. Yet, these compounds have encountered problems regarding toxicity, and inevitably development of resistance. The search for alternative targets within the UPS has revealed the 19S regulatory particle-associated deubiquitinases USP14 and UCHL5. Their inhibition blocks the deubiquitinating activity necessary for protein degradation by the proteasome. This has been shown to have cytotoxic effects in a range of cancer cells lines, as well as inhibiting tumor growth in several in vivo models. The small molecule inhibitor b-AP15 and its optimized lead VLX1570 were first discovered and characterized by the Linder research group at Karolinska Institute. Though thought to be highly promiscuous due to its α,βunsaturated ketone motif, b-AP15 was demonstrated to selectively bind and inhibit the proteasomal deubiquitinases USP14 and UCHL5, with preferential binding to USP14. Inhibition of USP14 by b-AP15 results in a strong proteotoxic stress characterized by elevated levels of poly-ubiquitin, activation of the ER stress response, and oxidative stress, followed by apoptosis. We show here that the mechanism of b-AP15-induced apoptosis is characteristic of proteasome inhibition, but significantly differs from the effects of catalytic proteasome inhibitors. The results of b-AP15 treatment manifest as: severe proteotoxicity, mitochondrial damage without mitophagy induction, and lack of cytoprotective aggresome formation. Available evidence supports that the cellular response to b-AP15 is primarily dependent on USP14. Additionally, we use a drug screen of compounds that share a reactive unsaturated ketone motif with b-AP15, to show their potential pharmacological applications, relative selectivity for USP14, and ability to inhibit the UPS. This thesis describes in detail the proteotoxic effects induced by b-AP15 and its derivative VLX1570, and shows that despite its potential reactivity, b-AP15 selectively targets USP14. Similarly reactive compounds are shown to also display selectivity for the 19S deubiquitinases, indicating a potential for phamacological application in cancer therapy. List of Publications I. Xin Wang, Magdalena Mazurkiewicz, Ellin-Kristina Hillert, Maria Hägg Olofsson, Stefan Pierrou, Per Hillertz, Joachim Gullbo, Karthik Selvaraju, Aneel Paulus, Sharoon Akhtar, Felicitas Bossler, Asher Chanan Khan, Stig Linder, and Padraig D’Arcy The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitinspecific protease-14 and induces apoptosis of multiple myeloma cells Scientific Reports 2016; 6:269–279. II. Xiaonan Zhang, Paola Pellegrini, Amir Ata Saei,Ellin-Kristina Hillert, Magdalena Mazurkiewicz, Maria Hägg Olofsson, Roman A Zubarev, Padraig B D’Arcy and Stig Linder The deubiquitinase inhibitor b-AP15 induces strong proteotoxic stress and mitochondrial damage Biochemical Phamacology 2018; 156:291–301. III. Ellin-Kristina Hillert, Slavica Brnjic, Xiaonan Zhang, Magdalena Mazurkiewicz, Karthik Selvaraju, Arjan Mofers, Amir Ata Saei, Roman Zubarev, Stig Linder and Padraig D’Arcy Proteasome inhibitor b-AP15 induces enhanced proteotoxicity by inhibiting cytoprotective aggresome formation Cancer Letters 2019; 448:70–83 IV. Karthik Selvaraju, Arjan Mofers, Paola Pellegrini, Johannes Salomonsson, Alexandra Ahlner, Vivian Morad,Ellin-Kristina Hillert, Belen Espinosa, Elias S.J. Arnér, Lasse Jensen, Jonas Malmström, Maria V. Turkina, Padraig D’Arcy, Michael A. Walters, Maria Sunnerhagen, and Stig Linder Cytotoxic unsaturated electrophilic compounds commonly target the ubiquitin proteasome system Manuscript submitted for publication.Scientific Reports, under revision V. Ellin-Kristina Hillert, Karthik Selvaraju, Arjan Mofers, Johannes Gubat, Stig Linder and Padraig D’Arcy, Studies on the specificity of the deubiquitinase inhibitor b-AP15 Manuscript The articles will be referred to in the text by their Roman numerals, and are reproduced in full at the end of the thesis. Related publications • Magdalena Mazurkiewicz,Ellin-Kristina Hillert, Xin Wang, Paola Pellegrini, Maria Hägg Olofsson, Karthik Selvaraju, Padraig D’Arcy, and Stig Linder Acute lymphoblastic leukemia cells are sensitive to disturbances in protein homeostasis induced by proteasome deubiquitinase inhibition Oncotarget 2017; 8(13):21115–21127. • Xin Wang, Magdalena Mazurkiewicz, Ellin-Kristina Hillert, Maria Hägg Olofsson, Stefan Pierrou, Per Hillertz, Joachim Gullbo, Karthik Selvaraju, Aneel Paulus, Sharoon Akhtar, Felicitas Bossler, Asher Chanan Khan, Stig Linder, and Padraig D’Arcy Corrigendum: The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells Scientific Reports 2016; 6:30667.