| 1. |
- Hillert, Ellin-Kristina
(författare)
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Mechanistic studies of a novel inhibitor of the ubiquitin-proteasome system
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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 , - unsaturated 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.
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| 2. |
- Hillert, Ellin-Kristina, et al.
(författare)
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Proteasome inhibitor b-AP15 induces enhanced proteotoxicity by inhibiting cytoprotective aggresome formation
- 2019
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Ingår i: Cancer Letters. - : ELSEVIER IRELAND LTD. - 0304-3835 .- 1872-7980. ; 448, s. 70-83
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Tidskriftsartikel (refereegranskat)abstract
- Proteasome inhibitors have been shown to induce cell death in cancer cells by triggering an acute proteotoxic stress response characterized by accumulation of poly-ubiquitinated proteins, ER stress and the production of reactive oxygen species. The aggresome pathway has been described as an escape mechanism from proteotoxicity by sequestering toxic cellular aggregates. Here we show that b-AP15, a small-molecule inhibitor of proteasomal deubiquitinase activity, induces poly-ubiquitin accumulation in absence of aggresome formation. b-AP15 was found to affect organelle transport in treated cells, raising the possibility that microtubule-transport of toxic protein aggregates is inhibited, leading to enhanced cytotoxicity. In contrast to the antiproliferative effects of the clinically used proteasome inhibitor bortezomib, the effects of b-AP15 are not further enhanced by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Our results suggest an inhibitory effect of b-AP15 on the transport of misfolded proteins, resulting in a lack of aggresome formation, and a strong proteotoxic stress response.
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| 3. |
- Mazurkiewicz, Magdalena, et al.
(författare)
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Acute lymphoblastic leukemia cells are sensitive to disturbances in protein homeostasis induced by proteasome deubiquitinase inhibition
- 2017
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Ingår i: Oncotarget. - : IMPACT JOURNALS LLC. - 1949-2553. ; 8:13, s. 21115-21127
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Tidskriftsartikel (refereegranskat)abstract
- The non-genotoxic nature of proteasome inhibition makes it an attractive therapeutic option for the treatment of pediatric malignancies. We recently described the small molecule VLX1570 as an inhibitor of proteasome deubiquitinase (DUB) activity that induces proteotoxic stress and apoptosis in cancer cells. Here we show that acute lymphoblastic leukemia (ALL) cells are highly sensitive to treatment with VLX1570, resulting in the accumulation of polyubiquitinated proteasome substrates and loss of cell viability. VLX1570 treatment increased the levels of a number of proteins, including the chaperone HSP70B , the oxidative stress marker heme oxygenase-1 (HO-1) and the cell cycle regulator p21(Cip1). Unexpectedly, polybiquitin accumulation was found to be uncoupled from ER stress in ALL cells. Thus, increased phosphorylation of eIF2a occurred only at supra-pharmacological VLX1570 concentrations and did not correlate with polybiquitin accumulation. Total cellular protein synthesis was found to decrease in the absence of eIF2a phosphorylation. Furthermore, ISRIB (Integrated Stress Response inhibitor) did not overcome the inhibition of protein synthesis. We finally show that VLX1570 can be combined with L-asparaginase for additive or synergistic antiproliferative effects on ALL cells. We conclude that ALL cells are highly sensitive to the proteasome DUB inhibitor VLX1570 suggesting a novel therapeutic option for this disease.
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| 4. |
- Selvaraj, Karthik, et al.
(författare)
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Cytotoxic unsaturated electrophilic compounds commonly target the ubiquitin proteasome system
- 2019
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- A large number of natural products have been advocated as anticancer agents. Many of these compounds contain functional groups characterized by chemical reactivity. It is not clear whether distinct mechanisms of action can be attributed to such compounds. We used a chemical library screening approach to demonstrate that a substantial fraction (similar to 20%) of cytotoxic synthetic compounds containing Michael acceptor groups inhibit proteasome substrate processing and induce a cellular response characteristic of proteasome inhibition. Biochemical and structural analyses showed binding to and inhibition of proteasome-associated cysteine deubiquitinases, in particular ubiquitin specific peptidase 14 (USP14). The results suggested that compounds bind to a crevice close to the USP14 active site with modest affinity, followed by covalent binding. A subset of compounds was identified where cell death induction was closely associated with proteasome inhibition and that showed significant antineoplastic activity in a zebrafish embryo model. These findings suggest that proteasome inhibition is a relatively common mode of action by cytotoxic compounds containing Michael acceptor groups and help to explain previous reports on the antineoplastic effects of natural products containing such functional groups.
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| 5. |
- Wang, Xin, et al.
(författare)
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The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells
- 2016
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 6, s. 1-14
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Tidskriftsartikel (refereegranskat)abstract
- Inhibition of deubiquitinase (DUB) activity is a promising strategy for cancer therapy. VLX1570 is an inhibitor of proteasome DUB activity currently in clinical trials for relapsed multiple myeloma. Here we show that VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). Exposure of multiple myeloma cells to VLX1570 resulted in thermostabilization of USP14 at therapeutically relevant concentrations. Transient knockdown of USP14 or UCHL5 expression by electroporation of siRNA reduced the viability of multiple myeloma cells. Treatment of multiple myeloma cells with VLX1570 induced the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and an apoptotic response. Sensitivity to VLX1570 was moderately affected by altered drug uptake, but was unaffected by overexpression of BCL2-family proteins or inhibitors of caspase activity. Finally, treatment with VLX1570 was found to lead to extended survival in xenograft models of multiple myeloma. Our findings demonstrate promising antiproliferative activity of VLX1570 in multiple myeloma, primarily associated with inhibition of USP14 activity.
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| 6. |
- Zhang, Xiaonan, et al.
(författare)
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The deubiquitinase inhibitor b-AP15 induces strong proteotoxic stress and Check for mitochondrial damage
- 2018
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Ingår i: Biochemical Pharmacology. - : PERGAMON-ELSEVIER SCIENCE LTD. - 0006-2952 .- 1356-1839 .- 1873-2968. ; 156, s. 291-301
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Tidskriftsartikel (refereegranskat)abstract
- Human cancers are characterized by intrinsic or acquired resistance to apoptosis and evasion of apoptosis has been proposed to contribute to treatment resistance. Bis-benzylidine piperidone compounds, containing alpha,beta-unsaturated carbonyl functionalities, have been extensively documented as being effective in killing apoptosis-resistant cells and to display promising antineoplastic activities in a number of tumor models. We here explored the phenotypic response of colon cancer cells to b-AP15, a bis-benzylidine piperidone previously shown to inhibit the proteasome deubiquitinases (DUBs) USP14 and UCHL5. Whereas similar overall mRNA and protein expression profiles were induced by b-AP15 and the clinically available proteasome inhibitor bortezomib, b-APIS induced stronger increases of chaperone expression. b-AP15 also induced a stronger accumulation of polyubiquitinated proteins in exposed cells. These proteins were found to partially colocalize with organelle structures, including mitochondria. Mitochondrial oxidative phosphorylation decreased in cells exposed to b-APIS, a phenomenon enhanced under conditions of severe proteotoxic stress caused by inhibition of the VCP/p97 ATPase and inhibition of protein translocation over the ER. We propose that mitochondrial damage caused by the association of misfolded proteins with mitochondrial membranes may contribute to the atypical cell death mode induced by b-AP15 and related compounds. The robust mode of cell death induction by this class of drugs holds promise for treatment of tumor cells characterized by apoptosis resistance.
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