Results of a high-throughput screen to identify compounds that modulate VHL proteostasis.

2012 ◽  
Vol 30 (5_suppl) ◽  
pp. 369-369
Author(s):  
Zhiyong Ding ◽  
Peter German ◽  
Shanshan Bai ◽  
Meng Gao ◽  
Mary M. Sobieski ◽  
...  
Keyword(s):  
Clinical Effect ◽  
Fluorescent Protein ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Full Length ◽  
Inhibitory Effects ◽  
Von Hippel Lindau ◽  
Vhl Protein ◽  
A Cell ◽  
Vhl Disease

369 Background: Mutated von Hippel Lindau (VHL) protein causes disease in sporadic renal cell carcinoma (RCC), and in the eponymous hereditary VHL disease. We developed and tested a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point mutated VHL, and potentially reverse the disease phenotype. Methods: The 786-0 cell line was infected with full-length W117A mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against the known proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of downstream functional readouts, including HIF and GLUT1 levels, were performed. Results: The proteasome inhibitors bortezomib and MG132, and the Prestwick compounds 8-azaguanine, thiostrepton and thioguanosine were found to reliably upregulate VHL-W117A-Venus in 786-0 cells. Thiostrepton has also been recognized as having proteasome inhibitory effects. 8-azaguanine was found to downregulate HIF2α levels, and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate post-translational processing. In addition, nuclear-cytoplasmic localization of pVHL varied amongst the different compounds, additionally suggesting unique effects of each of the agents on VHL homeostasis. Conclusions: We have successfully identified compounds that stabilize mutated VHL, including several proteasome inhibitors. It is possible that proteasome inhibition will have a specific clinical effect on patients with point-mutated pVHL. Further work is currently underway to further test this hypothesis in animal xenograft studies using cell lines containing various mutated VHL isoforms, and clinical trials using next-generation proteasome inhibitors are being explored.

scholarly journals Agents That Stabilize Mutated von Hippel–Lindau (VHL) Protein

2012 ◽  
Vol 17 (5) ◽  
pp. 572-580 ◽  
Author(s):  
Zhiyong Ding ◽  
Peter German ◽  
Shanshan Bai ◽  
Zhehui Feng ◽  
Meng Gao ◽  
...  
Keyword(s):  
Cell Line ◽  
Glucose Transporter ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Full Length ◽  
Posttranslational Processing ◽  
Autosomal Dominant Disorder ◽  
Glucose Transporter 1 ◽  
Von Hippel Lindau ◽  
Vhl Protein

Von Hippel–Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.

scholarly journals von Hippel-Lindau Disease: an Update

2019 ◽  
Vol 7 (4) ◽  
pp. 227-235 ◽  
Author(s):  
Eamonn R Maher ◽  
Richard N Sandford
Keyword(s):  
Natural History ◽  
Patient Outcomes ◽  
Molecular Characterisation ◽  
Von Hippel Lindau ◽  
Functional Characterisation ◽  
History Of ◽  
Vhl Protein ◽  
Von Hippel Lindau Disease ◽  
Vhl Disease ◽  
Improve Patient

Abstract Purpose of Review In this review, we discuss the key molecular and clinical developments in VHL disease that have the potential to impact on the natural history of the disease and improve patient outcomes. Recent Findings Identifiable mutations in VHL underlie most cases of VHL and define clear genotype-phenotype correlations. Detailed clinical and molecular characterisation has allowed the implementation of lifelong screening programmes that have improved clinical outcomes. Functional characterisation of the VHL protein complex has revealed its role in oxygen sensing and the mechanisms of tumourigenesis that are now being exploited to develop novel therapies for VHL and renal cancer. Summary The molecular and cellular landscape of VHL-associated tumours is revealing new opportunities to modify the natural history of the disease and develop therapies. Drugs are now entering clinical trials and combined with improved clinical and molecular diagnosis, and lifelong surveillance programmes, further progress towards reducing the morbidity and mortality associated with VHL disease is anticipated.

scholarly journals Clear cell chondrosarcoma in Von Hippel-Lindau disease

2019 ◽  
Vol 19 (1) ◽  
pp. 41-45 ◽  
Author(s):  
Koen M. A. Dreijerink ◽  
Rachel S. van Leeuwaarde ◽  
Wenzel M. Hackeng ◽  
Rachel H. Giles ◽  
Wendy W. J. de Leng ◽  
...  
Keyword(s):  
Loss Of Heterozygosity ◽  
Clear Cell ◽  
Immunohistochemical Analysis ◽  
Tumor Grade ◽  
Suppressor Gene ◽  
Clear Cell Chondrosarcoma ◽  
Von Hippel Lindau ◽  
Vhl Protein ◽  
Von Hippel Lindau Disease ◽  
A Cell

Abstract A diagnosis of clear cell chondrosarcoma of the ulna was made in a patient with Von Hippel-Lindau disease (VHL). After surgery, genetic analysis of the tumor tissue showed loss of heterozygosity at the VHL gene locus. Immunohistochemical analysis confirmed loss of expression of the VHL protein in the tumor cells. In addition, abundant Cyclin D1 expression in the tumor was observed. Chondrosarcoma has been described before in a VHL patient and VHL protein expression has been correlated to tumor grade in a series of sporadic chondrosarcomas. In this report, we show that clear cell chondrosarcoma may be a rare but canonical VHL manifestation through a cell-autonomous mechanism involving somatic loss-of-heterozygosity of the VHL tumor suppressor gene. We discuss the relevance of this observation with regard to the pathogenesis of clear cell chondrosarcoma in the context of VHL.

scholarly journals Bilateral Pheochromocytomas in a Patient with Y175C Von Hippel-Lindau Mutation

2018 ◽  
Vol 2018 ◽  
pp. 1-4
Author(s):  
Olga Astapova ◽  
Anindita Biswas ◽  
Alessandra DiMauro ◽  
Jacob Moalem ◽  
Stephen R. Hammes
Keyword(s):  
Tumor Development ◽  
Screening Guidelines ◽  
Von Hippel Lindau ◽  
Hypoxic Conditions ◽  
Oxygen Conditions ◽  
Vhl Protein ◽  
Function Relationship ◽  
Vhl Disease ◽  
Relationship Of ◽  
Dependent Mechanism

Von Hippel-Lindau (VHL) disease, caused by germline mutations in the VHL gene, is characterized by metachronously occurring tumors including pheochromocytoma, renal cell carcinoma (RCC), and hemangioblastoma. Although VHL disease leads to reduced life expectancy, its diagnosis is often missed and tumor screening guidelines are sparse. VHL protein acts as a tumor suppressor by targeting hypoxia-inducible factors (HIFs) for degradation through an oxygen-dependent mechanism. VHL mutants with more severely reduced HIF degrading function carry a high risk of RCC, while mutants with preserved HIF degrading capacity do not cause RCC but still lead to other tumors. VHL disease is classified into clinical types (1 and 2A-2C) based on this genotype-phenotype relationship. We report a case of bilateral pheochromocytomas and no other VHL-related tumors in a patient with Y175C VHL and show that this mutant preserves the ability to degrade HIF in normal oxygen conditions but, similar to the wild-type VHL protein, loses its ability to degrade HIF under hypoxic conditions. This study adds to the current understanding of the structure-function relationship of VHL mutations, which is important for risk stratification of future tumor development in the patients.

scholarly journals Tubulin binding potentially clears up Bortezomib and Carfilzomib differential neurotoxic effect

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. Malacrida ◽  
S. Semperboni ◽  
A. Di Domizio ◽  
A. Palmioli ◽  
L. Broggi ◽  
...  
Keyword(s):  
Proteasome Inhibitors ◽  
Direct Interaction ◽  
Proteasome Inhibition ◽  
Binding Studies ◽  
Gtp Hydrolysis ◽  
First Line Therapy ◽  
Adult Mice ◽  
Free Model ◽  
A Cell ◽  
Wide Scale

AbstractProteasome inhibitors (PIs) represent the gold standard in the treatment of multiple myeloma. Among PIs, Bortezomib (BTZ) is frequently used as first line therapy, but peripheral neuropathy (PN), occurring approximately in 50% of patients, impairs their life, representing a dose-limiting toxicity. Carfilzomib (CFZ), a second-generation PI, induces a significantly less severe PN. We investigated possible BTZ and CFZ off-targets able to explain their different neurotoxicity profiles. In order to identify the possible PIs off-targets we used the SPILLO-PBSS software that performs a structure-based in silico screening on a proteome-wide scale. Among the top-ranked off-targets of BTZ identified by SPILLO-PBSS we focused on tubulin which, by contrast, did not turn out to be an off-target of CFZ. We tested the hypothesis that the direct interaction between BTZ and microtubules would inhibit the tubulin alfa GTPase activity, thus reducing the microtubule catastrophe and consequently furthering the microtubules polymerization. This hypothesis was validated in a cell-free model, since BTZ (but not CFZ) reduces the concentration of the free phosphate released during GTP hydrolysis. Moreover, NMR binding studies clearly demonstrated that BTZ, unlike CFZ, is able to interact with both tubulin dimers and polymerized form. Our data suggest that different BTZ and CFZ neurotoxicity profiles are independent from their proteasome inhibition, as demonstrated in adult mice dorsal root ganglia primary sensory neurons, and, first, we demonstrate, in a cell free model, that BTZ is able to directly bind and perturb microtubules.

Evaluation of the Specificity and Cytotoxicity of Three Proteasome Inhibitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3366-3366
Author(s):  
Lisa J.A. Crawford ◽  
Huib Ovaa ◽  
Brian Walker ◽  
Dharminder Chauhan ◽  
Kenneth C. Anderson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Active Site ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Rate Limiting Step ◽  
Inhibitory Compounds ◽  
Gene And Protein Expression ◽  
A Cell ◽  
Proteolytic Activities ◽  
Active Site Probe

Abstract The proteasome is a mutlicatalytic protease with three main catalytic activities - chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolising (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma (MM). The CT-L activity, considered to be the rate-limiting step in protein degradation, is the primary target of many proteasome inhibitors. We have compared the specificity and potency of the novel proteasome inhibitor BzLLLCOCHO to the previously characterised inhibitors PS-341 (Velcade, bortezomib) and MG-132. Specific fluorogenic substrates were used to measure proteasome proteolytic activity in the presence and absence of the inhibitory compounds. An active site directed probe with a dansyl-sulfonamidohexanoyl hapten tag was used in conjuction with immunoblotting to determine the subunit specificity of the proteasome inhibitors (Nature Methods2005;2:357–362). MM cell lines (U266, OPM-2, KMS-11, KMS-18) were incubated with 10 μM BzLLLCOCHO, 5 nM PS-341 or 1 μM MG-132 for 24 hrs and proteasome activity was measured. Addition of BzLLLCOCHO reduced CT-L activity by 83 ± 13 % in the fluorogenic assay, and T-L and PGPH activities were reduced by 93 ± 6 % and 92 ± 2 % respectively. Immunoblot results revealed a similar pattern, the T-L and PGPH subunits were completely inhibited by BzLLLCOCHO and there was only weak labeling of the CT-L subunit with the active site probe. In contrast, treatment with PS-341 completely inhibited the CT-L and PGPH activities and incubation with MG-132 resulted in weak inhibition of the CT-L and PGPH activities, neither inihibitor significantly affected T-L activity. The ability of the different inihibitors to induce apoptosis in MM cell lines was then evaluated. All three inhibitors were demonstrated to act through both the caspase-8 and caspase-9 signalling pathways. Using Mitosensor™ and Hoescht/Propidium Iodide staining we found that MM cells were more sensitive to the induction of apoptosis by PS-341 and MG-132 than BzLLLCOCHO (U266 cells treated for 72 hrs with BzLLLCOCHO 51 % apoptosis, PS-341 79 % apoptosis and MG-132 84 % apoptosis). BzLLLCOCHO is a cell permeable and potent inhibitor of all three proteolytic activities of the proteasome. PS-341 and MG-132 inhibited only two of the three proteasome activities but were more efficient than BzLLLCOCHO at inducing apoptosis in MM cell lines. MG-132 is known to inhibit non proteasomal proteases such as Cathepsin B and Calpain 1 which may contribute to its potency. Further investigation on the effects of these inhibitors on gene and protein expression in the cell may lead to the development of more specific and targeted inhibitors.

scholarly journals RNAi screen of the druggable genome identifies modulators of proteasome inhibitor sensitivity in myeloma including CDK5

Blood ◽  
2011 ◽  
Vol 117 (14) ◽  
pp. 3847-3857 ◽  
Author(s):  
Yuan Xiao Zhu ◽  
Rodger Tiedemann ◽  
Chang-Xin Shi ◽  
Holly Yin ◽  
Jessica E. Schmidt ◽  
...  
Keyword(s):  
Cell Lines ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Myeloma Cell ◽  
Small Interfering Rnas ◽  
Inhibitory Effects ◽  
Internal Validation ◽  
Growth Inhibitory ◽  
Proteasome Subunits ◽  
Druggable Genome

Abstract The molecular target(s) cooperating with proteasome inhibition in multiple myeloma (MM) remain unknown. We therefore measured proliferation in MM cells transfected with 13 984 small interfering RNAs in the absence or presence of increasing concentrations of bortezomib. We identified 37 genes, which when silenced, are not directly cytotoxic but do synergistically potentiate the growth inhibitory effects of bortezomib. To focus on bortezomib sensitizers, genes that also sensitized MM to melphalan were excluded. When suppressed, the strongest bortezomib sensitizers were the proteasome subunits PSMA5, PSMB2, PSMB3, and PSMB7 providing internal validation, but others included BAZ1B, CDK5, CDC42SE2, MDM4, NME7, RAB8B, TFE3, TNFAIP3, TNK1, TOP1, VAMP2, and YY1. The strongest hit CDK5 also featured prominently in pathway analysis of primary screen data. Cyclin-dependent kinase 5 (CDK5) is expressed at high levels in MM and neural tissues with relatively low expression in other organs. Viral shRNA knockdown of CDK5 consistently sensitized 5 genetically variable MM cell lines to proteasome inhibitors (bortezomib and carfilzomib). Small-molecule CDK5 inhibitors were demonstrated to synergize with bortezomib to induce cytotoxicity of primary myeloma cells and myeloma cell lines. CDK5 regulation of proteasome subunit PSMB5 was identified as a probable route to sensitization.

scholarly journals Cerebellar hemangioblastomas: A study of the immunoprofile of neoplastic stromal component

2004 ◽  
Vol 61 (3) ◽  
pp. 273-282
Author(s):  
Desanka Tasic ◽  
Dragan Dimov ◽  
Milos Kostov ◽  
Srbislav Ilic ◽  
Dojcin Dojcinov ◽  
...  
Keyword(s):  
Mast Cells ◽  
Stromal Cells ◽  
Protein Function ◽  
Neuron Specific Enolase ◽  
Factor Xiiia ◽  
Von Hippel Lindau ◽  
Stromal Component ◽  
S 100 ◽  
Vhl Protein ◽  
Vhl Disease

Background. Central nervous system hemangioblastomas (HBs) are uncommon highly vascularized tumors that are predominantly found in the cerebellum. They occur sporadically or in association with von Hippel-Lindau (VHL) disease. HBs are of unknown histogenesis, and the origin of stromal cells is still a subject of debate. The aim of this study was to investigate the immunoprofile of neoplastic stromal component, and to determine whether the profile of the expression of immunomarkers used can contribute to the elucidation of the histogenesis of HBs. Methods. A series of eight cerebellar HBs were histochemically examined for the detection of mast cells and immunohistochemically for the expression of factor VIII-related antigen (FVIII-RAg), CD34, vimentin, factor XIIIa (FXIIIa), S-100 protein, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE) neurofilaments (NF), synaptophysin, chromogranin, and somatostatin. Results. Mast cells were present in all hemangioblastomas, and were particularly abundant in one tumor. Immunohistochemically, intense reactivity for vimentin and NSE in the stromal cells was constantly seen. Immunoreactivity with S-100 protein and FXIIIa was variable, but generally many HBs stromal cells were negative for these markers. However, stromal cells were uniformly negative for FVIII-RAg in all HBs investigated. They were negative for CD34 GFAP, NF, synaptophysin, chromogranin, as well as somatostatin. GFAP-positivity of the occasional stromal type cells, located only peripherally, was interpreted as "pseudopositivity". Conclusion. The immunoprofile of neoplastic stromal component in this study suggested a possible origin from undifferentiated multipotential mesenchymal cells. High expression of NSE (glycolytic and hypoxia-inducible enzyme) in the HBs stromal cells might be related to the loss of the VHL protein function.

Familial tumour syndromes: von Hippel–Lindau disease

2017 ◽  
Author(s):  
Hiroshi Kanno ◽  
Joachim P. Steinbach
Keyword(s):  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Laboratory Data ◽  
Von Hippel Lindau ◽  
Vhl Protein ◽  
Von Hippel Lindau Disease ◽  
The Central Nervous System ◽  
Microsurgical Techniques ◽  
Vhl Disease ◽  
Hif Pathway

Von Hippel–Lindau (VHL) disease, an autosomal dominant familial tumour syndrome, is often associated with haemangioblastoma of the central nervous system. In the presence of oxygen, VHL protein serves to prevent the accumulation of hypoxia-inducible factor (HIF) protein by targeting it to the proteasomal pathway, while biallelic inactivation of the VHL gene blocks degradation of HIF and leads to constitutive activation of the HIF pathway although oxygen is present. HIF-target genes are involved in angiogenesis, proliferation, and metabolism enabling tumour growth. Haemangioblastoma is a highly vascularized, begin tumour commonly associated with a cyst, but it is linked with neurological morbidity and mortality based on its location and multiplicity. Haemangioblastoma in VHL is diagnosed according to symptoms and signs, past and family histories, laboratory data, neuroradiological findings, pathological findings, and genetic testing. Surgical treatment is usually the most recommended therapy for haemangioblastomas, and using well-defined microsurgical techniques, the majority can be resected safely.

scholarly journals EglN3 hydroxylase stabilizes BIM-EL linking VHL type 2C mutations to pheochromocytoma pathogenesis and chemotherapy resistance

2019 ◽  
Vol 116 (34) ◽  
pp. 16997-17006 ◽  
Author(s):  
Shuijie Li ◽  
Javier Rodriguez ◽  
Wenyu Li ◽  
Petra Bullova ◽  
Stuart M. Fell ◽  
...  
Keyword(s):  
Chemotherapy Resistance ◽  
Von Hippel Lindau ◽  
Extracellular Signal ◽  
Familial Pheochromocytoma ◽  
Oxygen Sensitive ◽  
Vhl Protein ◽  
Sensitive Function ◽  
Induced Apoptosis ◽  
Vhl Disease

Despite the discovery of the oxygen-sensitive regulation of HIFα by the von Hippel–Lindau (VHL) protein, the mechanisms underlying the complex genotype/phenotype correlations in VHL disease remain unknown. Some germline VHL mutations cause familial pheochromocytoma and encode proteins that preserve their ability to down-regulate HIFα. While type 1, 2A, and 2B VHL mutants are defective in regulating HIFα, type 2C mutants encode proteins that preserve their ability to down-regulate HIFα. Here, we identified an oxygen-sensitive function of VHL that is abolished by VHL type 2C mutations. We found that BIM-EL, a proapoptotic BH3-only protein, is hydroxylated by EglN3 and subsequently bound by VHL. VHL mutants fail to bind hydroxylated BIM-EL, regardless of whether they have the ability to bind hydroxylated HIFα or not. VHL binding inhibits BIM-EL phosphorylation by extracellular signal-related kinase (ERK) on serine 69. This causes BIM-EL to escape from proteasomal degradation, allowing it to enhance EglN3-induced apoptosis. BIM-EL was rapidly degraded in cells lacking wild-type VHL or in which EglN3 was inactivated genetically or by lack of oxygen, leading to enhanced cell survival and chemotherapy resistance. Combination therapy using ERK inhibitors, however, resensitizes VHL- and EglN3-deficient cells that are otherwise cisplatin-resistant.

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