DBeQ derivative targets vacuolar protein sorting 4 functions in cancer cells and suppresses tumor growth in mice.

Vacuolar protein sorting 4 (VPS4) is an AAA-ATPase that catalyzes the endosomal sorting complex required for transport-III disassembly, mediating various cellular membrane-remodeling processes including endolysosomal membrane repair and autophagosome closure. Humans have 2 VPS4 paralogs, VPS4A and VPS4B, and the loss of either paralog has been identified in a significant proportion of cancers, rendering them dependent on the remaining paralog for survival. In this study, we explored VPS4 inhibition as an anticancer strategy by investigating the mechanisms of VPS4 inhibition-induced cell death and developing small-molecule compounds that target VPS4 functions. We found that genetic inhibition of VPS4 triggered both caspase-8 (CASP8)-dependent apoptosis and caspase-independent cell death in osteosarcoma cells. We synthesized approximately 100 derivatives of the VPS4 and related AAA-ATPase valosin-containing protein inhibitor DBeQ and screened for their inhibitory effects on VPS4 ATPase activity using the EnzChek phosphate assay and a high-content assay monitoring GFP-CHMP4B puncta formation. In cells, the lead compound 4-107 caused endolysosomal damage, disrupted subsequent membrane repair, inhibited autophagy, and led to the accumulation of the endosomal sorting complex required for transport on membranes. These effects were accompanied by the stabilization of CASP8 on autophagosomal membranes, leading to the induction of CASP8-mediated apoptosis. Notably, the CASP8-mediated cell death induced by 4-107 was further enhanced by the loss of either VPS4 paralog. Moreover, 4-107 exhibited antitumor activity in a syngeneic mouse model of neuroblastoma. Our findings provide an important step for targeting VPS4 in cancer and developing VPS4 inhibitors as a cancer treatment strategy. SIGNIFICANCE STATEMENT: VPS4A and VPS4B, paralogs of the AAA-ATPase VPS4, are critical for cancer cell survival. This study reports that 4-107, a DBeQ derivative, inhibits VPS4 ATPase activity, induces CASP8-mediated apoptosis, and suppresses tumor growth in mice. This study supports the further development of VPS4A/B inhibitors as a promising anticancer treatment strategy.