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.
Highlights
Recycling, and facilitation of protein degradation by removal of the sterically bulky ubiquitin chains[5,6]
We recently showed that the small molecule b-AP15 interferes with the ubiquitin proteasome system (UPS) by inhibiting the enzymatic activities of the proteasomal DUBs USP14 and UCHL512
USP14 activity was strongly inhibited at 2.5 μM VLX1570 whereas UCHL5 inhibition required higher drug concentrations (Fig. 1b), suggesting that USP14 is sensitive to VLX1570
Summary
The small molecule RA190, with a similar structure to b-AP15/VLX1570 (Supplementary Fig. 1a), was shown to induce accumulation of polyubiquitin in cells[29] This drug was found to bind and block the ubiquitin receptor ADRM1 (Rpn[13]). Both USP14 and UCHL5 were present in proteasome preparations from these cells and exposure to VLX1570 did not alter the yield of these proteins (Supplementary Fig. 4e) We conclude from these experiments that VLX1570 does not inhibit binding of polyubiquitin to proteasomes and does not induce gross alterations in proteasome structure. Overexpression of BCL2A1 did not significantly affect cell survival over 72 hours following drug exposure (Fig. 5f) This observation suggested that cell death was apoptosis-independent and we examined whether inhibition of total caspase activity using the pharmacological inhibitor z-VAD-fmk would affect survival of HCT116 cells exposed to VLX1570. Inhibition of ERK phosphorylation was observed in cells exposed to VLX1570 in vitro and was paralleled by decreased activation of RAS (Supplementary Fig. 7b)
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