In vitro efficacy of a first-generation valosin-containing protein inhibitor (CB-5083) against canine lymphoma.

ABSTRACT

Valosin-containing protein (VCP), through its critical role in the maintenance of protein homeostasis, is a promising target for the treatment of several malignancies, including canine lymphoma. CB-5083, a first-in-class VCP inhibitor, exerts cytotoxicity through the induction of irreversible proteotoxic stress and possesses a broad spectrum of anticancer activity. Here, we determined the cytotoxicity CB-5083 in canine lymphoma cells and its mechanism of action in vitro. Canine lymphoma cell lines were treated with varying concentrations of CB-5083 and assessed for viability by trypan blue exclusion and apoptosis by caspase activity assays. The mechanism of CB-5083 action was determined by immunoblotting and RT-qPCR analyses of Lys48 ubiquitination and markers of ER stress (DDIT3), autophagy (SQSTM1, MAP1LC3A) and DNA damage (γH2AX). Unfolded protein response markers were also evaluated by immunoblotting (eIF2α, P-eIF2α) and RT-qPCR (ATF4). CB-5083 treatment resulted in preferential cytotoxicity in canine lymphoma cell lines over control peripheral blood mononuclear cells. CB-5083 rapidly disrupted the ubiquitin-dependent protein degradation system, inducing sustained ER stress as indicated by a dramatic increase in DDIT3. Activation of the unfolded protein response occurred through the increase eIF2α phosphorylation and increased transcription of ATF4, but did not re-establish protein homeostasis. Cells rapidly underwent apoptosis through activation of the caspase cascade. These results further validate VCP as an attractive target for the treatment of canine lymphoma and identify CB-5083 as a novel therapy with clinical potential for this malignancy.