Specific mutations in the D1\u2013D2 linker region of VCP/p97 enhance ATPase activity and confer resistance to VCP inhibitors

Prabhakar Bastola,Taiping Gan,Matthew A Schaich,Tsui-Fen Chou,Feng Wang,Jeremy Chien,Bret D Freudenthal

doi:10.1038/cddiscovery.2017.65

Abstract

Valosin-containing protein (VCP), together with several partner proteins, extracts ubiquitinated client proteins from E3 ligase complex and facilitates their degradation through ubiquitin–proteasome system. Therefore, it plays an important role in regulating protein quality control and various cellular pathways. Recent studies also identified VCP as a lineage-specific essential gene in ovarian cancer. An orally bioavailable VCP inhibitor, CB-5083, is currently in Phase I clinical trials because it shows therapeutic effects in multiple tumor xenograft models. However, the mechanism of resistance to CB-5083 is unknown. Here, we characterized molecular mechanism of resistance to CB-5083. Using incremental exposure to CB-5083, we established CB-5083-resistant ovarian cancer cells that showed five- to six-fold resistance in vitro compared with parental cells. Genomic and complementary DNA sequencing of the VCP coding region revealed a pattern of co-selected mutations: (1) missense mutations at codon 470 in one copy resulting in increased ATPase activity and (2) nonsense or frameshift mutations at codon 606 or codon 616 in another copy causing the loss of allele-specific expression. Unbiased molecular docking studies showed codon 470 as a putative binding site for CB-5083. Furthermore, the analysis of somatic mutations in cancer genomes from the Cancer Genome Atlas (TCGA) indicated that codon 616 contains hotspot mutations in VCP. Thus, identification of these mutations associated with in vitro resistance to VCP inhibitors may be useful as potential theranostic markers while screening for patients to enroll in clinical trials. VCP has emerged as a viable therapeutic target for several cancer types, and therefore targeting such hyperactive VCP mutants should aid in improving the therapeutic outcome in cancer patients.

Highlights

Valosin-containing protein belongs to AAA (ATPase Associated with diverse cellular Activities) family of ATPase
The analysis of somatic mutations in cancer genomes from the Cancer Genome Atlas (TCGA) indicated that codon 616 contains hotspot mutations in Valosin-containing protein (VCP). Identification of these mutations associated with in vitro resistance to VCP inhibitors may be useful as potential theranostic markers while screening for patients to enroll in clinical trials
The structure revealed that binding of ATP to the D2 domain brings about a conformational change to D2 and subsequently to D1–D2 linker region

Summary

INTRODUCTION

Valosin-containing protein ( known as p97 or Cdc[48] in yeast) belongs to AAA (ATPase Associated with diverse cellular Activities) family of ATPase. VCP, while the ATPase activity in D1 domain provides a supportive function.[6] Recently solved high-resolution cryoelectron microscopy structure of VCP depicted a two-step ATP-driven conformational changes associated with VCP activity.[2] The structure revealed that binding of ATP to the D2 domain brings about a conformational change to D2 and subsequently to D1–D2 linker region. This allows for ATP binding at D1 domain resulting in the movement of N-terminal region. Our studies highlight the relevance for the further development of novel VCP inhibitors that can overcome resistance to existing VCP inhibitors

RESULTS

35 KDa ― VCP

DISCUSSION