The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells

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.A diverse set of cellular processes such as cell cycle progression, DNA repair, metabolism and cell survival are dynamically controlled by the synthesis and degradation of protein regulators. In eukaryotic cells the regulated degradation of proteins is controlled mainly by the ubiquitin proteasome system (UPS)1 . The UPS is composed of a destruction tag in the form of the small protein ubiquitin and the 26S proteasome, a large multi-subunit proteolytic complex that specifically degrades ubiquitin tagged proteins into small peptides. The proteolytic activities of the proteasome reside within the 20S core particle (20S CP), a barrel like structure composed of 4 stacked heptameric rings (α 7 β 7 β 7 α 7 ) associated with one or two 19S regulatory particles (19S RP) 2,3 . Protein degradation begins with the covalent tagging of substrates with multi-ubiquitin chains, an event that initiates traffic to the proteasome and subsequent capture by highly specific ubiquitin receptors located within the 19S RP. Once bound, substrates undergo a sequence of modifications including de-ubiquitination by proteasome associated deubiquitinases (DUBs), unwinding by the 19S RP ATPases and finally translocation into the 20S CP where they are degraded 4 . Several roles for proteasome DUBs have been proposed including a rescue mechanism for improperly or poorly ubiquitinated substrates, maintenance of ubiquitin homeostasis by ubiquitin

Acute lymphoblastic leukemia cells are sensitive to disturbances in protein homeostasis induced by proteasome deubiquitinase inhibition

“…The level of sensitivity of ALL B-cell lines to VLX1570 is similar to that previously found in multiple myeloma cells [19], which are generally classified as sensitive to inhibitors of the ubiquitin-proteasome system (UPS), and higher than that of melanoma and colon cancer cells [21]. Inhibition of proteasome ubiquitin processing following VLX1570 treatment results in the accumulation of high molecular weight polyubiquitinated substrates in cells [19, 21]. We indeed found a dose-dependent increase in high-molecular polyubiquitinated proteins in ALL cells following exposure to VLX1570 (Figure 1B).…”

Induction of apoptosis via proteasome inhibition in leukemia/lymphoma cells by two potent piperidones

“…b-AP15 has been found to inhibit the proteasome by inhibiting the DUBs USP14 and UCHL5 [15]. VLX1570, which is an analog of b-AP15, has been found to inhibit the proteasome by preferentially binding to USP14 [57]. The structural similarities between P1 and P2 and the aforementioned compounds support the argument that P1 and P2 may interact with these DUBs.…”

“…Further DEG analysis led to the identification of two similar compounds, b-AP15 and VLX5710, that induce proteotoxic stress [15, 57]. These compounds act by inhibiting proteasome-related de-ubiquitinases (DUBs).…”

Protein Degradation Systems as Antimalarial Therapeutic Targets

“…The P. falciparum homolog PfUSP14 (PF3D7_0527200) was shown to bind plasmodial proteasomes, and small-molecule inhibition (b-AP15) of this enzyme led to accumulation of proteasomal substrates and P. falciparum death [31]. Inhibitors of the human USP14 are currently in clinical trials to treat multiple myeloma [55, 56]. Recently, multiple natural and synthetic compounds have been identified to inhibit the deubiquitinating activity of the 19S subunit Rpn11 [57–59].…”