Abstract
In recent years, multiple studies including ours have reported on the mechanism of resistance towards valosin-containing protein (VCP) inhibitors. While all these studies reported target alterations via mutations in VCP as the primary mechanism of resistance, discrepancies persist to date regarding the zygosity of these mutations responsible for the resistance. In addition, the extent to which resistant cells harbor additional mutations in other genes is not well described. In this study, we performed global transcript analysis of the parental and previously reported VCP inhibitor (CB-5083) resistant cells and found additional mutations in the resistant cells. However, our CRISPR-Cas9 gene editing studies indicate that specific mutations in VCP are sufficient to produce resistance to CB-5083 suggesting the importance of on-target mutations in VCP for resistance. Strikingly, our analysis indicates a preexisting heterozygous frameshift mutation at codon 616 (N616fs*) in one of the VCP alleles in HCT116 cells, and we showed that this mutant allele is subjected to the nonsense-mediated decay (NMD). Accordingly, we identified a heterozygous mutation at codon 526 (L526S) in genomic DNA sequencing but a homozygous L526S mutation in complementary DNA sequencing in our independently generated CB-5083 resistant HCT116 cells, implying that the L526S mutation occurs in the allele that does not harbor the frameshift N616fs* mutation. Our results suggest the NMD as a possible mechanism for achieving the homozygosity of VCP mutant responsible for the resistance to VCP inhibitors while resolving the discrepancies among previous studies. Our results also underscore the importance of performing simultaneous genomic and complementary DNA sequencing when attributing mutational effects on the functionality particularly for an oligomer protein like VCP.
Highlights
Valosin-containing protein (VCP), known as p97, has been shown to be involved in a myriad of cellular functions associated with the protein quality control mechanisms including the endoplasmic reticulum-associated degradation (ERAD)[1], mitochondria-associated degradation[2], the ubiquitin proteasome system (UPS)[3], chromatin-associated degradation[4] and autophagy[5,6,7]
To investigate potential molecular mechanisms contributing to CB-5083 resistance, we characterized the transcriptomes perturbed by CB-5083 in previously reported OVSAHO parental and OVSAHO resistant cells (OVSAHO-R1)[21]
The Metascape analysis identified autophagy and apoptosis signaling pathways to be upregulated (Fig. 1A). Such pathway activation can be attributed to the induction of the unfolded protein response (UPR) mediated apoptosis[19] and autophagy[16]
Summary
Valosin-containing protein (VCP), known as p97, has been shown to be involved in a myriad of cellular functions associated with the protein quality control mechanisms including the endoplasmic reticulum-associated degradation (ERAD)[1], mitochondria-associated degradation[2], the ubiquitin proteasome system (UPS)[3], chromatin-associated degradation[4] and autophagy[5,6,7]. Non-oncogenes, including components of the protein quality control pathway, have been proposed as therapeutic targets for cancer therapy[11,12] This led to an interest in the inhibition of VCP, a common essential protein in cancer, since such inhibition would result in increased proteotoxic stress and subsequent cell death in cancer[13]. Initial studies by Anderson et al reported that homozygous missense mutations in VCP are sufficient to produce resistance to the VCP inhibitor CB-5083 in HCT116 cells[16]. Her et al reported that a single heterozygous missense mutation in VCP (A530T) is sufficient to produce resistance to the VCP inhibitor NMS-873 in HCT116 cells[20] Her et al speculated that such a difference in zygosity may reflect the potential differences in the mechanisms of action between CB-5083 and NMS-873. In our previous study, we successfully generated OVSAHO cells with resistance to both CB-5083 and NMS-873 inhibitors that possess unique patterns of co-selected mutations with heterozygous missense mutations in one VCP allele and heterozygous nonsense mutations in the other VCP allele[21,22]
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