Targeted Disruption of USP14 and UCHL5 with the Novel Deubiquitinase Enzyme (DUB) Inhibitor, VLX1570, Induces Immense Proteotoxicity and Cell Death in Malignant Plasma Cells
Authors: Paulus Aneel , Akhtar Sharoon , Kuranz-Blake Maja , Novak Anne J. , Ansell Stephen , Gertz Morie A , Kyle Robert A , Martin Peter , Coleman Morton , Ailawadhi Sikander , Linder Stig , Chanan-Khan Asher , Chitta Kasyapa S. 
Published: November 2019

Abstract
Background: Deubiquitinating enzyme (DUB) inhibitors are an emerging class of compounds, which are increasingly being regarded for their anti-cancer activity and therapeutic potential. The 19S regulatory particle of the proteasome contains two DUBs, USP14 and UCHL5, which are critical for the proper unfolding and deubiquitination of proteins prior to their entry into the 20S proteasome β-catalytic core. In B/plasma cell malignancies such as Waldenströms macroglobulinemia (WM) and multiple myeloma (MM), protein homeostasis and optimal proteasome functionality are paramount for tumor survival. This is evidenced by the success of 20S proteasome inhibition with bortezomib and carfilzomib, which have demonstrated remarkable clinical benefit in patients with WM or MM. As such, the proteasome degradation pathway represents a highly attractive and clinically validated, yet still largely unexplored therapeutic target. Here, we provide first evidence of the cytotoxic effects and molecular sequelae associated with a novel 19S proteasome DUB inhibitor, VLX1570, which selectively targets USP14 and UCHL5, in preclinical models of B/plasma cell cancers and their drug resistant tumor subclones.
Materials: VLX1570 was obtained from Vivolux AB, Sweden. Bortezomib was purchased from Sellekchem, Houston TX. Human myeloma cell lines (OPM2, U266, KMS11), human WM cell lines (BCWM.1, MWCL-1 and RPCI-WM1), their respective bortezomib-resistant (BR) subclones as well as ibrutinib-resistant (IR) derivatives of BCWM.1 and RPCI-WM1 were used in experiments (total n=12). Peripheral blood mononuclear cells (PBMCs) from healthy donors were used to assess for cytotoxicity of VLX1570 in non-tumor cells.
Results: A 72hr MTS assay first established sensitivity of plasma cell cancer models towards VLX1570, including drug resistant subclones (EC50 range, 20 – 90nM). Next we assessed whether loss of viability was due to apoptosis and observed dose-dependent annexin-V staining in ~50 – 70% of wild-type MM and WM cells, ~45 – 55% in corresponding BR models and ~50% in IR subclones when exposed to VLX1570 (100nm – 500nM) for 24hrs. VLX1570 was tested at similar concentrations in PBMCs and induced minimal cell death. To understand if VLX1570-induced apoptosis was mitochondrial mediated, we assessed mitochondrial outer membrane permeability (MOMP) in all plasma cell cancer models treated with the DUB inhibitor (100 – 500nM) and found a dose-dependent increase in MOMP (p<0.00025 – 0.000001). Activation of the mitochondrial/intrinsic apoptotic pathway was also evidenced by cleavage of caspase-9 and 3 on immunoblot analysis. As inhibition of protein deubiquitination results in accumulation of polyubiquitinated protein conjugates, we examined global ubiquitinated protein levels and found increasing amounts of high molecular weight poly-ubiquitinated protein in VLX1570 treated tumor cells in a dose-dependent manner. This prompted us to further delineate the potential cellular response mechanisms that relay the antitumor activity of VLX1570 in malignant plasma cells. Immunoblot analysis of drug sensitive and (bortezomib or ibrutinib) resistant MM and WM cell lines revealed several changes in ER stress response proteins, notably induction of HSP70 and splicing of XBP1; the latter event which was less prominent in BR cell lines treated with bortezomib. This suggested the ability of VLX1570 to overcome resistance through a rapid and potent proteotoxic effect, which overwhelmed the tumor cells stress response mechanisms beyond its capacity for survival. Finally, as abnormalities in the NFkB signaling pathway are implicated in malignant plasma cell survival, we found markedly decreased nuclear translocation of NFkB (p-P65) protein in tumor cells treated with VLX1570.
Conclusions: The DUBs of the 19S proteasome cap offer an attractive target within the proteasome degradation system and whose inhibition by VLX1570 is able to circumvent mechanisms that support resistance to b-catalytic PI (bortezomib) or BTK inhibitor (ibrutinib) therapy. The studies undertaken thus far, coupled with more rigorous mechanistic and in vivo efficacy analyses currently underway, provides rational for the clinical development of DUB inhibitors for use in malignant B/plasma cell malignancies.

Disclosures
Martin: Janssen: Honoraria.