Valosin-containing Protein Inhibitors Research Articles

Abstract PO3-26-07: Epigenetic approaches to deliver a targeted radiotherapy for triple negative breast cancer

Abstract The sodium iodide symporter (NIS, encoded by SLC5A5) is the sole transporter of iodide, and successfully exploited in thyroid cancer to uptake radioiodide (RAI). NIS is essentially undetected in normal mammary epithelial cells but elevated during lactation, and has a heterogeneous expression profile in breast cancer (BRCA), as evidenced by TCGA cohort and also in a ~120 sample TMA we have profiled. Furthermore, pre-clinical studies support an epigenetic mechanisms that involves Retinoic Acid Receptor alpha (RARa) to regulate SLC5A5 expression. Therefore, we have applied an orthogonal approach to both elucidate and target mechanisms that control NIS expression and function to deliver a therapeutic strategy that targets this transporter as a novel radiotherapy in BRCA. To understand the epigenetic control of expression we segregated samples in the TCGA-BRCA cohort into lower and upper quartile SLC5A5 expression and identified ~600 significant differentially expressed of genes (DEG). Interestingly, restricting analyses to either triple negative (TN) or tumors from African American (AA) patients doubled the number of DEGs. These DEGs were significantly associated with several transcription factors including LHX3, a pituitary development transcription factor regulated by thyroid signaling. The most significantly altered ~70 DEGs significantly clustered tumors by genomic ancestry and ERBB2/HER2 IHC status. The DEGs also supported a footprint of RARa signaling include PRAME, a known RARa co-repressor associated with histone deacetylase, and reflects the finding that NIS expression is highly responsive to SAHA treatment. To investigate if PRAME expression suppresses RARa regulation of SLC5A5 we have established a panel of BRCA cell lines (AU565, SKB3 SUM52 and MDA-MB-231) stably transfected with either CRISPR-VP64 (activator) and CRISPR-KRAB (repressor) to modulate PRAME and measure the epigenomic impact, the expression of SLC5A5 and the ability to transport RAI. In parallel we have applied a drug screen approach that targets non-canonical approaches to govern NIS expression, for example by targeting valosin-containing protein (VCP) that induces NIS proteolysis. In the first instance we have developed a therapeutic strategy to target NIS by inhibiting VCP. Specifically, we are screening a range of VCP inhibitors in combination with the HDAC inhibitor SAHA or the transcriptional potentiator, disulfiram metabolite Cu(DDC)2 in BRCA cell lines by testing the ability to augment RAI uptake. Remarkably, in MDA MB 231 cells, Cu(DDC)2 combined with the VCP inhibitor CB5339, resulted in a significant additive increase in RAI uptake and a prominent decrease in cell proliferation and survival. These studies combined with future murine and PDX testing of CRISPR-edited tumors and drug exposures will develop a comprehensive analyses of the mechanisms that control SLC5A5 expression across breast cancer cell types, how this can be most effectively targeted to restore NIS expression, and exploited to promote RAI as a novel targeted BRCA radiotherapy. Citation Format: Katie Brookes, Martin Read, Sajad Wani, Sissy Jhiang, Daniel Stover, Mathew Ringel, Christopher McCabe, Moray Campbell. Epigenetic approaches to deliver a targeted radiotherapy for triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-07.

Akt enhances the vulnerability of cancer cells to VCP/p97 inhibition-mediated paraptosis

Valosin-containing protein (VCP)/p97, an AAA+ ATPase critical for maintaining proteostasis, emerges as a promising target for cancer therapy. This study reveals that targeting VCP selectively eliminates breast cancer cells while sparing non-transformed cells by inducing paraptosis, a non-apoptotic cell death mechanism characterized by endoplasmic reticulum and mitochondria dilation. Intriguingly, oncogenic HRas sensitizes non-transformed cells to VCP inhibition-mediated paraptosis. The susceptibility of cancer cells to VCP inhibition is attributed to the non-attenuation and recovery of protein synthesis under proteotoxic stress. Mechanistically, mTORC2/Akt activation and eIF3d-dependent translation contribute to translational rebound and amplification of proteotoxic stress. Furthermore, the ATF4/DDIT4 axis augments VCP inhibition-mediated paraptosis by activating Akt. Given that hyperactive Akt counteracts chemotherapeutic-induced apoptosis, VCP inhibition presents a promising therapeutic avenue to exploit Akt-associated vulnerabilities in cancer cells by triggering paraptosis while safeguarding normal cells.

Targeting VCP potentiates immune checkpoint therapy for colorectal cancer

Immune checkpoint blockade therapies are still ineffective for most patients with colorectal cancer (CRC). Immunogenic cell death (ICD) enables the release of key immunostimulatory signals to drive efficient anti-tumor immunity, which could be used to potentiate the effects of immune checkpoint inhibitors. Here, we showed that inhibition of valosin-containing protein (VCP) elicits ICD in CRC. Meanwhile, VCP inhibitor upregulates PD-L1 expression and compromises anti-tumor immunity invivo. Mechanistically, VCP transcriptionally regulates PD-L1 expression in a JAK1-dependent manner. Combining VCP inhibitor with anti-PD1 remodels tumor immune microenvironment and reduces tumor growth in mouse models of CRC. Addition of oncolytic virus further augments the therapeutic activity of the combination regimen. Our study shows the molecular mechanism for regulating PD-L1 expression by VCP and suggests that inhibition of VCP has the potential to increase the efficacy of immunotherapy in CRC.

Nuclear VCP drives colorectal cancer progression by promoting fatty acid oxidation

Fatty acid oxidation (FAO) fuels many cancers. However, knowledge of pathways that drive FAO in cancer remains unclear. Here, we revealed that valosin-containing protein (VCP) upregulates FAO to promote colorectal cancer growth. Mechanistically, nuclear VCP binds to histone deacetylase 1 (HDAC1) and facilitates its degradation, thus promoting the transcription of FAO genes, including the rate-limiting enzyme carnitine palmitoyltransferase 1A (CPT1A). FAO is an alternative fuel for cancer cells in environments exhibiting limited glucose availability. We observed that a VCP inhibitor blocked the upregulation of FAO activity and CPT1A expression triggered by metformin in colorectal cancer (CRC) cells. Combined VCP inhibitor and metformin prove more effective than either agent alone in culture and in vivo. Our study illustrates the molecular mechanism underlying the regulation of FAO by nuclear VCP and demonstrates the potential therapeutic utility of VCP inhibitor and metformin combination treatment for colorectal cancer.

Inhibition of VCP modulates NF-κB signaling pathway to suppress multiple myeloma cell proliferation and osteoclast differentiation.

Multiple myeloma (MM) is the second most common hematological malignancy, in which the dysfunction of the ubiquitin-proteasome pathway is associated with the pathogenesis. The valosin containing protein (VCP)/p97, a member of the AAA+ ATPase family, possesses multiple functions to regulate the protein quality control including ubiquitin-proteasome system and molecular chaperone. VCP is involved in the occurrence and development of various tumors while still elusive in MM. VCP inhibitors have gradually shown great potential for cancer treatment. This study aims to identify if VCP is a therapeutic target in MM and confirm the effect of a novel inhibitor of VCP (VCP20) on MM. We found that VCP was elevated in MM patients and correlated with shorter survival in clinical TT2 cohort. Silencing VCP using siRNA resulted in decreased MM cell proliferation via NF-κB signaling pathway. VCP20 evidently inhibited MM cell proliferation and osteoclast differentiation. Moreover, exosomes containing VCP derived from MM cells partially alleviated the inhibitory effect of VCP20 on cell proliferation and osteoclast differentiation. Mechanism study revealed that VCP20 inactivated the NF-κB signaling pathway by inhibiting ubiquitination degradation of IκBα. Furthermore, VCP20 suppressed MM cell proliferation, prolonged the survival of MM model mice and improved bone destruction in vivo. Collectively, our findings suggest that VCP is a novel target in MM progression. Targeting VCP with VCP20 suppresses malignancy progression of MM via inhibition of NF-κB signaling pathway.

Elucidating cellular interactome of chikungunya virus identifies host dependency factors

Chikungunya virus (CHIKV) is a re-emerging mosquito-transmitted RNA virus causing joint and muscle pain. To better understand how CHIKV rewires the host cell and usurps host cell functions, we generated a systematic CHIKV-human protein-protein interaction map and revealed several novel connections that will inform further mechanistic studies. One of these novel interactions, between the viral protein E1 and STIP1 homology and U-box containing protein 1 (STUB1), was found to mediate ubiquitination of E1 and degrade E1 through the proteasome. Capsid associated with G3BP1, G3BP2 and AAA+ ​ATPase valosin-containing protein (VCP). Furthermore, VCP inhibitors blocked CHIKV infection, suggesting VCP could serve as a therapeutic target. Further work is required to fully understand the functional consequences of these interactions. Given that CHIKV proteins are conserved across alphaviruses, many virus-host protein-protein interactions identified in this study might also exist in other alphaviruses. Construction of interactome of CHIKV provides the basis for further studying the function of alphavirus biology.

VCP/p97, a pleiotropic protein regulator of the DNA damage response and proteostasis, is a potential therapeutic target in KRAS-mutant pancreatic cancer.

We and others have recently shown that proteins involved in the DNA damage response (DDR) are critical for KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell growth in vitro. However, the CRISPR-Cas9 library that enabled us to identify these key proteins had limited representation of DDR-related genes. To further investigate the DDR in this context, we performed a comprehensive, DDR-focused CRISPR-Cas9 loss-of-function screen. This screen identified valosin-containing protein (VCP) as an essential gene in KRAS-mutant PDAC cell lines. We observed that genetic and pharmacologic inhibition of VCP limited cell growth and induced apoptotic death. Addressing the basis for VCP-dependent growth, we first evaluated the contribution of VCP to the DDR and found that loss of VCP resulted in accumulation of DNA double-strand breaks. We next addressed its role in proteostasis and found that loss of VCP caused accumulation of polyubiquitinated proteins. We also found that loss of VCP increased autophagy. Therefore, we reasoned that inhibiting both VCP and autophagy could be an effective combination. Accordingly, we found that VCP inhibition synergized with the autophagy inhibitor chloroquine. We conclude that concurrent targeting of autophagy can enhance the efficacy of VCP inhibitors in KRAS-mutant PDAC.

TOLLIP-mediated autophagic degradation pathway links the VCP-TMEM63A-DERL1 signaling axis to triple-negative breast cancer progression

ABSTRACT Triple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat due to the lack of effective targeted therapies. Transmembrane (TMEM) proteins represent attractive drug targets for cancer therapy, but biological functions of most members of the TMEM family remain unknown. Here, we report for the first time that TMEM63A (transmembrane protein 63A), a poorly characterized TMEM protein with unknown functions in human cancer, functions as a novel oncogene to promote TNBC cell proliferation, migration, and invasion in vitro and xenograft tumor growth and lung metastasis in vivo. Mechanistic investigations revealed that TMEM63A localizes in endoplasmic reticulum (ER) and lysosome membranes, and interacts with VCP (valosin-containing protein) and its cofactor DERL1 (derlin 1). Furthermore, TMEM63A undergoes autophagy receptor TOLLIP-mediated autophagic degradation and is stabilized by VCP through blocking its lysosomal degradation. Strikingly, TMEM63A in turn stabilizes oncoprotein DERL1 through preventing TOLLIP-mediated autophagic degradation. Notably, pharmacological inhibition of VCP by CB-5083 or knockdown of DERL1 partially abolishes the oncogenic effects of TMEM63A on TNBC progression both in vitro and in vivo. Collectively, these findings uncover a previously unknown functional and mechanistic role for TMEM63A in TNBC progression and provide a new clue for targeting TMEM63A-driven TNBC tumors by using a VCP inhibitor. Abbreviations: ATG16L1, autophagy related 16 like 1; ATG5, autophagy related 5; ATP5F1B/ATP5B, ATP synthase F1 subunit beta; Baf-A1, bafilomycin A1; CALCOCO2/NDP52, calcium binding and coiled-coil domain 2; CANX, calnexin; DERL1, derlin 1; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; HSPA8, heat shock protein family A (Hsp70) member 8; IP, immunoprecipitation; LAMP2A, lysosomal associated membrane protein 2; NBR1, NBR1 autophagy cargo receptor; OPTN, optineurin; RT-qPCR, reverse transcription-quantitative PCR; SQSTM1/p62, sequestosome 1; TAX1BP1, Tax1 binding protein 1; TMEM63A, transmembrane protein 63A; TNBC, triple-negative breast cancer; TOLLIP, toll interacting protein; VCP, valosin containing protein

Multiple Components of Protein Homeostasis Pathway Can Be Targeted to Produce Drug Synergies with VCP Inhibitors in Ovarian Cancer.

Simple SummaryThe increased dependency of cancer cells on protein homeostasis creates a vulnerability that can be exploited using protein homeostasis inhibiting drugs. Recently, valosin-containing protein has emerged as a key component of the pathway and a novel therapeutic target for cancer treatment. The aim of our research is to explore the potential synergies between VCP inhibitors and other agents that disrupt protein homeostasis and to provide mechanistic understanding of drug synergies targeting multiple components of protein homeostasis. Our results indicate that VCP inhibitors can be combined with other endoplasmic reticulum stress-inducing agents to produce synergistic cytotoxicity in ovarian cancer cells.Protein quality control mechanisms play an important role in cancer progression by providing adaptive responses and morphologic stability against genome-wide copy number alterations, aneuploidy, and conformation-altering somatic mutations. This dependency on protein quality control mechanisms creates a vulnerability that may be exploited for therapeutic benefits by targeting components of the protein quality control mechanism. Recently, valosin-containing protein (VCP), also known at p97 AAA-ATPase, has emerged as a druggable target in cancer cells to affect their dependency on protein quality control. Here, we show that VCP inhibitors induce cytotoxicity in several ovarian cancer cell lines and these compounds act synergistically with mifepristone, a drug previously shown to induce an atypical unfolded protein response. Although mifepristone at a clinically achievable dose induces a weak unfolded protein response, it enhances the cytotoxic effects of VCP inhibitor CB-5083. Mechanistically, mifepristone blocks the cytoprotective effect of ATF6 in response to endoplasmic reticulum (ER) stress while activating the cytotoxic effects of ATF4 and CHOP through the HRI (EIF2AK1)-mediated signal transduction pathway. In contrast, CB-5083 activates ATF4 and CHOP through the PERK (EIF2AK3)-mediated signaling pathway. This combination activates ATF4 and CHOP while blocking the adaptive response provided by ATF6, resulting in increased cytotoxic effects and synergistic drug interaction.

Abstract 1035: VCP/p97 promotes pancreatic cancer growth by enhancing mutant p53 activity

Abstract Pancreatic cancer is the most lethal malignant neoplasms across the world with the lowest overall five-year survival rate (9%). TP53 is mutated in ~70% of pancreatic ductal adenocarcinomas (PDACs). Hotspot p53 mutants promote cancer cell proliferation, metastasis and metabolism through their gain-of-function (GOF). Among the p53 “hotspot” mutations, R273 is the most frequent mutation position (~12%) with R273H as the most commonly mutated codon (~8%) in PDACs. However, the mechanisms underlying regulation of mutant p53s’ GOF in PDACs remain incompletely understood. In our attempt to address this question, we recently identified the ATPase valosin-containing protein (VCP)/p97 as a novel mutant p53-binding protein by performing immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) analysis. VCP bound to p53-R273H via the DNA-binding domain. VCP inhibition either by genetic depletion or pharmacological inhibition by CB-5083 resulted in the increase of MDM2-mediated ubiquitination and degradation of p53-R273H. VCP did so by binding to MDM2 and disturbing its interaction with mutant p53. As a result, VCP protected mutant p53 from degradation and enhanced its GOF in cancer development. Ablation of VCP retarded PDAC cell growth in vitro and in vivo. Our further studies also unveiled the negative regulation of wt p53 by VCP in cancer cells. Together, these results demonstrate that VCP can strengthen mutant p53’s oncogenic function by stabilizing it and negating wt p53 function, suggesting VCP as a resistant factor for chemotherapy against PDACs and thus a potential therapeutic target of the mutant p53-harboring pancreatic cancers as well as of the wt p53-containing cancer cells. Citation Format: Jieqiong Wang. VCP/p97 promotes pancreatic cancer growth by enhancing mutant p53 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1035.

AAA-ATPase valosin-containing protein binds the transcription factor SREBP1 and promotes its proteolytic activation by rhomboid protease RHBDL4

Valosin-containing protein (VCP) is a member of AAA-ATPase superfamily involved in various cellular functions. To investigate the pathophysiological role of VCP in metabolic disorders, we generated knock-in mice bearing an A232E mutation in VCP, a known human VCP pathogenic variant. When heterozygous mutant mice (A232E/+) were fed a high-fat diet, we observed that fatty liver was ameliorated and the proteolytic processing of the transcription factor sterol regulatory element-binding protein 1 (SREBP1) was impaired. Further co-immunoprecipitation analysis in wildtype mice revealed interactions of VCP with SREBP1 and a rhomboid protease, RHBDL4, in the liver, and these interactions were attenuated in A232E/+ mice. Consistent with these results, we show that knockdown or chemical inhibition of VCP or RHBDL4 in human hepatocytes impaired the proteolytic processing of SREBP1. Finally, we found that knockdown of E3 ligases such as glycoprotein 78 and HMG-CoA reductase degradation protein 1 disrupted the interaction of VCP with SREBP1 and impaired the proteolytic processing of SREBP1. These results suggest that VCP recognizes ubiquitinylated SREBP1 and recruits it to RHBDL4 to promote its proteolytic processing. The present study reveals a novel proteolytic processing pathway of SREBP1 and may lead to development of new therapeutic strategies to treat fatty liver diseases.

VCP suppresses proteopathic seeding in neurons

BackgroundNeuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery participating in this process is poorly understood. One proteinopathy called multisystem proteinopathy (MSP) is associated with dominant mutations in Valosin Containing Protein (VCP). MSP patients have muscle and neuronal degeneration characterized by aggregate pathology that can include αS, Tau and TDP-43.MethodsWe performed a fluorescent cell sorting based genome-wide CRISPR-Cas9 screen in αS biosensors. αS and TDP-43 seeding activity under varied conditions was assessed using FRET/Flow biosensor cells or immunofluorescence for phosphorylated αS or TDP-43 in primary cultured neurons. We analyzed in vivo seeding activity by immunostaining for phosphorylated αS following intrastriatal injection of αS seeds in control or VCP disease mutation carrying mice.ResultsOne hundred fifty-four genes were identified as suppressors of αS seeding. One suppressor, VCP when chemically or genetically inhibited increased αS seeding in cells and neurons. This was not due to an increase in αS uptake or αS protein levels. MSP-VCP mutation expression increased αS seeding in cells and neurons. Intrastriatal injection of αS preformed fibrils (PFF) into VCP-MSP mutation carrying mice increased phospho αS expression as compared to control mice. Cells stably expressing fluorescently tagged TDP-43 C-terminal fragment FRET pairs (TDP-43 biosensors) generate FRET when seeded with TDP-43 PFF but not monomeric TDP-43. VCP inhibition or MSP-VCP mutant expression increases TDP-43 seeding in TDP-43 biosensors. Similarly, treatment of neurons with TDP-43 PFFs generates high molecular weight insoluble phosphorylated TDP-43 after 5 days. This TDP-43 seed dependent increase in phosphorlyated TDP-43 is further augmented in MSP-VCP mutant expressing neurons.ConclusionUsing an unbiased screen, we identified the multifunctional AAA ATPase VCP as a suppressor of αS and TDP-43 aggregate seeding in cells and neurons. VCP facilitates the clearance of damaged lysosomes via lysophagy. We propose that VCP’s surveillance of permeabilized endosomes may protect against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.

VCP inhibition induces an unfolded protein response and apoptosis in human acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by the accumulation of undifferentiated white blood cells (blasts) in the bone marrow. Valosin-containing protein (VCP) is an abundant molecular chaperone that extracts ubiquitylated substrates from protein complexes and cellular compartments prior to their degradation by the proteasome. We found that treatment of AML cell lines with the VCP inhibitor CB-5083 leads to an accumulation of ubiquitylated proteins, activation of unfolded protein response (UPR) and apoptosis. Using quantitative mass spectrometry-based proteomics we assessed the effects of VCP inhibition on the cellular ubiquitin-modified proteome. We could further show that CB-5083 decreases the survival of the AML cell lines THP-1 and MV4-11 in a concentration-dependent manner, and acts synergistically with the antimetabolite cytarabine and the BH3-mimetic venetoclax. Finally, we showed that prolonged treatment of AML cells with CB-5083 leads to development of resistance mediated by mutations in VCP. Taken together, inhibition of VCP leads to a lethal unfolded protein response in AML cells and might be a relevant therapeutic strategy for treatment of AML, particularly when combined with other drugs. The toxicity and development of resistance possibly limit the utility of VCP inhibitors and have to be further explored in animal models and clinical trials.

VCP/p97 inhibitor CB-5083 modulates muscle pathology in a mouse model of VCP inclusion body myopathy

BackgroundPathogenic gain of function variants in Valosin-containing protein (VCP) cause a unique disease characterized by inclusion body myopathy with early-onset Paget disease of bone and frontotemporal dementia (also known as Multisystem proteinopathy (MSP)). Previous studies in drosophila models of VCP disease indicate treatment with VCP inhibitors mitigates disease pathology. Earlier-generation VCP inhibitors display off-target effects and relatively low therapeutic potency. New generation of VCP inhibitors needs to be evaluated in a mouse model of VCP disease. In this study, we tested the safety and efficacy of a novel and potent VCP inhibitor, CB-5083 using VCP patient-derived myoblast cells and an animal model of VCP disease.MethodsFirst, we analyzed the effect of CB-5083 in patient-derived myoblasts on the typical disease autophagy and TDP-43 profile by Western blot. Next, we determined the maximum tolerated dosage of CB-5083 in mice and treated the 2-month-old VCPR155H/R155H mice for 5 months with 15 mg/kg CB-5083. We analyzed motor function monthly by Rotarod; and we assessed the end-point blood toxicology, and the muscle and brain pathology, including autophagy and TDP-43 profile, using Western blot and immunohistochemistry. We also treated 12-month-old VCPR155H/+ mice for 6 months and performed similar analysis. Finally, we assessed the potential side effects of CB-5083 on retinal function, using electroretinography in chronically treated VCPR155H/155H mice.ResultsIn vitro analyses using patient-derived myoblasts confirmed that CB-5083 can modulate expression of the proteins in the autophagy pathways. We found that chronic CB-5083 treatment is well tolerated in the homozygous mice harboring patient-specific VCP variant, R155H, and can ameliorate the muscle pathology characteristic of the disease. VCP-associated pathology biomarkers, such as elevated TDP-43 and p62 levels, were significantly reduced. Finally, to address the potential adverse effect of CB-5083 on visual function observed in a previous oncology clinical trial, we analyzed retinal function in mice treated with moderate doses of CB-5083 for 5 months and documented the absence of permanent ocular toxicity.ConclusionsAltogether, these findings suggest that long-term use of CB-5083 by moderate doses is safe and can improve VCP disease-associated muscle pathology. Our results provide translationally relevant evidence that VCP inhibitors could be beneficial in the treatment of VCP disease.

The degradation of mutant RhodopsinIle256del follows the VCP‐dependent endoplasmic‐reticulum‐associated protein degradation pathway

AbstractPurposeIn Europe, the Rholle256del mutation (deletion of isoleucine at position 255 or 256 in Rho gene) accounts for over 17% of familial ADRP cases (C. F. Inglehearn, 1991). From a clinical perspective, the severity of this mutation can be compared to RhoP23H. In previous studies, we found that the degeneration of misfolded RHOP23H follows the ERAD pathway and the clearance of RHOP23H requires the ERAD effector valosin‐containing protein (VCP). This study aims to characterize the subcellular structural and functional changes in Rholle256del transfected mammalian cells, evaluate VCP inhibition's effects, and understand the pathological similarity between Rholle256del and RhoP23H transfected cells.MethodsHEK293 and COS‐7 cells were transfected with plasmids encoding RhoWT, Rholle256del, or RhoP23H, tagged with GFP. Immunostaining was performed to reveal the localization of transfected RHO‐GFP and the co‐localization between RHO‐GFP with ER, endogenous VCP, ubiquitin, and proteasome subunit (PSMB5). Formation and degree of Rho protein aggregates were analyzed in transfected cells before and after proteasome‐ and VCP‐inhibition.ResultsWe found that RHOWT mainly localizes at the plasma membrane, while mutant RHOlle256del and RHOP23H accumulates in the cytosol forming cytoplasmic aggregates. Co‐staining results suggest that both RHOlle256del and RHOP23H aggregates are retained within the ER, partially co‐localizing with VCP. RHOlle256del and RHOP23H aggregates were ubiquitinated and partially transported to the proteasome system for degradation. VCP inhibition led to enhanced accumulation of high molecular weight (HMW) RHO‐containing oligomers and aggregates.ConclusionsOur results indicate similar clearance characteristics between Rholle256del and RhoP23H in transfected cells, including the excessive formation of misfolded RHO aggregates, retention in the ER, and, at least partially, interaction with VCP. Moreover, VCP inhibition affects the abundance of RHO aggregates. This suggests that the clearance of RHOlle256del also follows the VCP/ERAD/proteasome axis, raising a possibility that modulation of VCP could be considered as a potential therapy for Rholle256del‐related RP as well.

The Biogenesis of Dengue Virus Replication Organelles Requires the ATPase Activity of Valosin-Containing Protein.

The dengue virus (DENV) causes the most prevalent arthropod-borne viral disease worldwide. While its incidence is increasing in many countries, there is no approved antiviral therapy currently available. In infected cells, the DENV induces extensive morphological alterations of the endoplasmic reticulum (ER) to generate viral replication organelles (vRO), which include convoluted membranes (CM) and vesicle packets (VP) hosting viral RNA replication. The viral non-structural protein NS4B localizes to vROs and is absolutely required for viral replication through poorly defined mechanisms, which might involve cellular protein partners. Previous interactomic studies identified the ATPase valosin-containing protein (VCP) as a DENV NS4B-interacting host factor in infected cells. Using both pharmacological and dominant-negative inhibition approaches, we show, in this study, that VCP ATPase activity is required for efficient DENV replication. VCP associates with NS4B when expressed in the absence of other viral proteins while in infected cells, both proteins colocalize within large DENV-induced cytoplasmic structures previously demonstrated to be CMs. Consistently, VCP inhibition dramatically reduces the abundance of DENV CMs in infected cells. Most importantly, using a recently reported replication-independent plasmid-based vRO induction system, we show that de novo VP biogenesis is dependent on VCP ATPase activity. Overall, our data demonstrate that VCP ATPase activity is required for vRO morphogenesis and/or stability. Considering that VCP was shown to be required for the replication of other flaviviruses, our results argue that VCP is a pan-flaviviral host dependency factor. Given that new generation VCP-targeting drugs are currently evaluated in clinical trials for cancer treatment, VCP may constitute an attractive broad-spectrum antiviral target in drug repurposing approaches.

Retinal neuroprotection by controlled release of a VCP inhibitor from self-assembled nanoparticles

Mutations in rhodopsin lead to its misfolding resulting in autosomal dominant retinitis pigmentosa (adRP). Pharmacological inhibition of the ATP-driven chaperone valosin-containing protein (VCP), a molecular checkpoint for protein quality control, slows down retinal degeneration in animal models. However, poor water-solubility of VCP inhibitors poses a challenge to their clinical translation as intravitreal injections for retinal treatment. In order to enable the delivery of VCP inhibitors, we have developed and investigated two formulations for the VCP inhibitor ML240. Nanoformulations of ML240 were obtained by using amphiphilic polymers methoxy-poly (ethylene glycol)5kDa–cholane (mPEG5kDa-cholane) and methoxy-poly (ethylene glycol)5kDa–cholesterol (mPEG5kDa-cholesterol). Both formulations increased the water-solubility of ML240 by two orders of magnitude and prolonged the drug released over ten days. In addition, encapsulation of ML240 in mPEG5kDa-cholane showed superior photoreceptor protection at lower drug concentrations, normalized rhodopsin localization, and alleviated inflammatory microglial responses in an ex vivo rat model of retinal degeneration. The study demonstrates the potential of VCP inhibitor nanoformulations to treat adRP, a pharmacologically orphan disease.

USP7 and VCPFAF1 define the SUMO/Ubiquitin landscape at the DNA replication fork.

SummaryThe AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin.

Valosin-Containing Protein Stabilizes Mutant p53 to Promote Pancreatic Cancer Growth.

Approximately 80% of human pancreatic ductal adenocarcinomas (PDAC) harbor TP53 mutations, among which, R273H is the most frequent. Although p53-R273H is known to possess gain-of-function properties, how it is regulated in PDAC has not been extensively explored. Here we identify valosin-containing protein (VCP) as a regulator of p53-R273H by conducting immunoprecipitation-tandem mass spectrometry analysis. VCP bound p53-R273H at its DNA binding domain. Ectopic or endogenous VCP stabilized p53-R273H by binding to MDM2 and disrupting its association with mutant p53. Inhibition of VCP either by genetic depletion or the pharmacologic inhibitor CB-5083 increased ubiquitination and degradation of p53-R273H, leading to cell death. Consistently, ablation of VCP markedly retarded growth of cultured PDAC cells and xenograft PDAC tumors. Together, these results unveil VCP as a novel partner of p53-R273H in promoting PDAC growth and as a potential target for developing anti-PDAC therapy. SIGNIFICANCE: These findings identify valosin-containing protein (VCP) as a novel regulator of p53-R273H stability and suggest VCP as a potential target for development of pancreatic cancer therapy.

Valosin-containing protein/p97 plays critical roles in the Japanese encephalitis virus life cycle.

Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle - nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses.ImportanceJEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.