Abbreviations: AAA-ATPase, ATPase associated with diverse cellular activities; BRCA1/2, breast cancer 1/2; PARP1, poly ADP-ribose polymerase-1; DBeQ, N 2 ,N 4 -dibenzylquinazoline-2,4-diamine; PBS, phosphate buffered saline; EGF, epidermal growth factor; TBS, tris-buffered saline; PERK, protein kinase R-like endoplasmic reticulum kinase; TCGA, the Cancer Genome Atlas; PVDF, polyvinylidene difluoride; IRE1a, inositol-requiring enzyme 1 alpha; CHOP, CCAAT/enhancer-binding protein homologous protein; ATF4, activating transcription factor 4; eIF2a, eukaryotic initiation factor 2 alpha; BiP, binding immunoglobulin protein; Grp78, glucose regulated protein 78; GADD34, growth arrest And DNA-damage-inducible 34.
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.
Enhancing the efficacy of proteasome inhibitors (PI) is a central goal in myeloma therapy. We proposed that signaling-level responses after PI may reveal new mechanisms of action that can be therapeutically exploited. Unbiased phosphoproteomics after treatment with the PI carfilzomib surprisingly demonstrates the most prominent phosphorylation changes on splicing related proteins. Spliceosome modulation is invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrates broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings lead us to evaluate direct spliceosome inhibition in myeloma, which synergizes with carfilzomib and shows potent anti-tumor activity. Functional genomics and exome sequencing further support the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma.
According to the World Health Organization, corneal diseases are the fourth leading cause of blindness worldwide accounting for 5.1% of all ocular deficiencies. Current therapies for corneal diseases, which include eye drops, oral medications, corrective surgeries, and corneal transplantation are largely inadequate, have undesirable side effects including blindness, and can require life-long applications. Adeno-associated virus (AAV) mediated gene therapy is an optimistic strategy that involves the delivery of genetic material to target human diseases through gene augmentation, gene deletion, and/or gene editing. With two therapies already approved by the United States Food and Drug Administration and 200 ongoing clinical trials, recombinant AAV (rAAV) has emerged as the in vivo viral vector-of-choice to deliver genetic material to target human diseases. Likewise, the relative ease of applications through targeted delivery and its compartmental nature makes the cornea an enticing tissue for AAV mediated gene therapy applications. This current review seeks to summarize the development of AAV gene therapy, highlight preclinical efficacy studies, and discuss potential applications and challenges of this technology for targeting corneal diseases.
Genomic aberrations inside malignant cells through copy number alterations, aneuploidy, and mutations can exacerbate misfolded and unfolded protein burden resulting in increased proteotoxic stress. Increased proteotoxic stress can be deleterious to malignant cells; therefore, these cells rely heavily on the protein quality control mechanisms for survival and proliferation. Components of the protein quality control, such as the unfolded protein response, heat shock proteins, autophagy, and the ubiquitin proteasome system, orchestrate a cascade of downstream events that allow the mitigation of the proteotoxic stress. This dependency makes components of the protein quality control mechanisms attractive targets in cancer therapeutics. In this review, we explore the components of the protein homeostasis especially focusing on the emerging cancer therapeutic agents/targets that are being actively pursued actively.
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