ABSTRACT
Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell's sensitivity to Hsp90 inhibition.
Subject(s)
Gene Expression Regulation, Neoplastic/physiology , HSP90 Heat-Shock Proteins/metabolism , Neoplasms/metabolism , Proteomics/methods , Animals , Antineoplastic Agents/pharmacology , Benzodioxoles/pharmacology , Cell Line, Tumor , Computational Biology , Drug Discovery , Gene Expression Regulation, Neoplastic/drug effects , HSP90 Heat-Shock Proteins/genetics , Humans , Neoplasms/genetics , Purines/pharmacology , Signal TransductionABSTRACT
Alterations in protein-protein interaction networks are at the core of malignant transformation but have yet to be translated into appropriate diagnostic tools. We make use of the kinetic selectivity properties of an imaging probe to visualize and measure the epichaperome, a pathologic protein-protein interaction network. We are able to assay and image epichaperome networks in cancer and their engagement by inhibitor in patients' tumors at single-lesion resolution in real time, and demonstrate that quantitative evaluation at the level of individual tumors can be used to optimize dose and schedule selection. We thus provide preclinical and clinical evidence in the use of this theranostic platform for precision medicine targeting of the aberrant properties of protein networks.
Subject(s)
Antineoplastic Agents/administration & dosage , Molecular Chaperones/antagonists & inhibitors , Neoplasms/drug therapy , Protein Interaction Maps/drug effects , Animals , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Administration Schedule , Epigenesis, Genetic , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Molecular Chaperones/metabolism , Molecular Imaging , Neoplasms/diagnostic imaging , Neoplasms/genetics , Neoplasms/pathology , Precision Medicine/methods , Protein Interaction Mapping/methods , Protein Interaction Maps/genetics , Theranostic Nanomedicine/methods , Xenograft Model Antitumor AssaysABSTRACT
UNLABELLED: Because of deficiencies in l-arginine biosynthesis, some cancers are susceptible to therapeutic intervention with arginine deiminase (ADI), an enzyme responsible for consuming the dietary supply of l-arginine to deprive the disease of an essential nutrient. ADI is currently being evaluated in several clinical trials, and fully realizing the drug's potential will depend on invoking the appropriate metrics to judge clinical response. Without a clear biologic mandate, PET/CT with (18)F-FDG is currently used to monitor patients treated with ADI. However, it is unclear if it can be expected that (18)F-FDG responses will indicate (or predict) clinical benefit. METHODS: (18)F-FDG responses to ADI therapy were studied in preclinical models of melanoma in vitro and in vivo. The molecular mechanism of response to ADI therapy was also studied, with a particular emphasis on biologic pathways known to regulate (18)F-FDG avidity. RESULTS: Although proliferation of SK-MEL 28 was potently inhibited by ADI treatment in vitro and in vivo, no clear declines in (18)F-FDG uptake were observed. Further investigation showed that ADI treatment induces the posttranslational degradation of phosphatase and tensin homolog and the activation of the PI3K signaling pathway, an event known to enhance glycolysis and (18)F-FDG avidity. A more thorough mechanistic study showed that ADI triggered a complex mechanism of cell death, involving apoptosis via poly (ADP-ribose) polymerase cleavage-independent of caspase 3. CONCLUSION: These findings suggest that some unexpected pharmacologic properties of ADI preclude using (18)F-FDG to evaluate clinical response in melanoma and, more generally, argue for further studies to explore the use of PET tracers that target apoptotic pathway activation or cell death.
Subject(s)
Fluorodeoxyglucose F18 , Hydrolases/pharmacology , Melanoma/drug therapy , Animals , Cell Line, Tumor , Female , Humans , Hydrolases/therapeutic use , Melanoma/diagnostic imaging , Melanoma/metabolism , Melanoma/pathology , Mice , Positron-Emission Tomography , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
INTRODUCTION: The present investigation focuses on the chemical and biological fate of (89)Zr in mice. Electrophoreses of (89)Zr solvated or chelated in different conditions are here presented. The biological fate of mice injected with [(89)Zr]Zr-oxalate, [(89)Zr]Zr-chloride, [(89)Zr]Zr-phosphate, [(89)Zr]Zr-desferrioxamine and [(89)Zr]Zr-citrate is studied with the biodistribution, the clearances and positron emission tomography images. A special focus is also given regarding the quality of (89)Zr bone accumulation. METHODS: Electrophoreses were carried out on chromatography paper and read by gamma counting. Then, the solutions were intravenously injected in mice, imaged at different time points and sacrificed. The bones, the epiphysis and the marrow substance were separated and evaluated with gamma counts. RESULTS: The clearances of [(89)Zr]Zr-chloride and [(89)Zr]Zr-oxalate reached 20% of injected dose (ID) after 6 days whereas [(89)Zr]Zr-phosphate was only 5% of ID. [(89)Zr]Zr-citrate and [(89)Zr]Zr-DFO were noticeably excreted after the first day postinjection (p.i.). [(89)Zr]Zr-chloride and [(89)Zr]Zr-oxalate resulted in a respective bone uptake of â¼15% ID/g andâ¼20% ID/g at 8 h p.i. with minor losses after 6 days. [(89)Zr]Zr-citrate bone uptake was also observed, but [(89)Zr]Zr-phosphate was absorbed in high amounts in the liver and the spleen. The marrow cells were insignificantly radioactive in comparison to the calcified tissues. CONCLUSION: Despite the complexity of Zr coordination, the electrophoretic analyses provided detailed evidences of Zr charges either as salts or as complexes. This study also shows that weakly chelated, (89)Zr is a bone seeker and has a strong affinity for phosphate.
Subject(s)
Radioisotopes/pharmacokinetics , Zirconium/pharmacokinetics , Animals , Bone Marrow/diagnostic imaging , Bone Marrow/metabolism , Bone and Bones/diagnostic imaging , Bone and Bones/metabolism , Buffers , Electrophoresis , Female , Injections , Metabolic Clearance Rate , Mice , Positron-Emission Tomography , Radioisotopes/isolation & purification , Radioisotopes/metabolism , Solutions , Solvents/chemistry , Zirconium/isolation & purification , Zirconium/metabolismABSTRACT
UNLABELLED: The human somatostatin receptor subtype 2 (hSSTr2)-68Ga-DOTATOC reporter system has several attractive features for potential translation to human studies. These include a low expression of hSSTr2 in most organs, a rapid internalized accumulation of 68Ga-DOTATOC in the SSTr2-expressing cells, and a rapid excretion of unbound radioligand by the renal system. We performed a series of in vitro and in vivo validation studies of this reporter system. METHODS: A retroviral vector containing a dual reporter, pQCXhSSTr2-IRES-GFP (IRES: internal ribosome entry site; GFP: green fluorescent protein), was constructed and transduced into Jurkat, C6, and U87 cells. Stably transduced reporter cells were characterized in vitro using optical and radiometric methods. Multiple tumor-bearing mice were evaluated with 68Ga-DOTATOC PET studies. RESULTS: The dual-reporter genes were incorporated into all tumor cell lines, and their expression levels were confirmed by fluorescence-activated cell sorting (FACS), GFP visualization, and reverse-transcriptase polymerase chain reaction (RT-PCR) analysis for hSSTr2. In vitro, hSSTr2 cell membrane expression was 36,000, 280,000, and 1,250,000 copies per cell for the SSTR2-transfected Jurkat, U87, and C6 cell lines. Small-animal PET of 68Ga-DOTATOC in tumor-bearing mice demonstrated that the in vivo uptake of this radioligand was directly proportional to the in vitro expression of hSSTr2. The in vivo uptake of 68Ga-DOTATOC, at 2 h after injection, was low in all organs except the kidneys (7.8 percentage of injected dose per gram [%ID/g]) and as high as 15.2 %ID/g in transduced C6 tumors. The corresponding transduced-to-nontransduced tumor uptake ratio was 64, and the tumor-to-muscle uptake ratio was around 500. CONCLUSION: 68Ga-DOTATOC is an excellent specific ligand for this hSSTr2 reporter system and for hSSTr2 reporter gene PET. Because DOTATOC has undergone extensive clinical testing, this human reporter system has the potential for translation to human studies.
Subject(s)
Gallium Radioisotopes , Octreotide/analogs & derivatives , Radiopharmaceuticals , Receptors, Somatostatin/genetics , Animals , Genetic Therapy , Humans , Isotope Labeling , Male , Mice , Octreotide/metabolism , Receptors, Somatostatin/physiology , Signal Transduction , Tissue DistributionABSTRACT
BACKGROUND: Carbonic anhydrase IX (CAIX) is a membrane spanning protein involved in the enzymatic regulation of tumor acid-base balance. CAIX has been shown to be elevated in a number of hypoxic tumor types. The purpose of this study was to determine the efficiency of intact and IgG fragments of cG250 to target CAIX in vivo in a hypoxic tumor model. METHODOLOGY/PRINCIPAL FINDINGS: Conventional biodistribution studies were performed with (111)In-DO3A-cG250, (111)In-DO3A-F(ab')(2)-cG250 and (111)In-DO3A-Fab-cG250. Additional ex vivo analysis of the tumor was performed with markers for tumor hypoxia, blood perfusion and endogenous CAIX expression. All four data sets were digitally correlated to determine the optimal agent for determining hypoxia in a HT29 colon cancer xenograft. The HT29 human colorectal tumor xenografts show strong CAIX expression in hypoxic areas of poor blood perfusion. The intact IgG had an initial high focal uptake at the periphery of these hypoxic regions and penetration into the areas of highest CAIX expression over the 7-day study period. The lower molecular weight antibody fragments had a faster uptake into areas of high CAIX expression, but had a much lower absolute uptake at the optimal imaging times. CONCLUSIONS/SIGNIFICANCE: For the clinical detection of hypoxia induced CAIX using cG250 antibody based agents, imaging with the intact IgG at 7 days post injection would allow for the most sensitive and accurate detection of CAIX.
Subject(s)
Carbonic Anhydrases/metabolism , Colorectal Neoplasms/pathology , Molecular Imaging/methods , Xenograft Model Antitumor Assays , Animals , Antibodies, Monoclonal/pharmacokinetics , Carbonic Anhydrase IX , Cell Hypoxia , Colorectal Neoplasms/blood supply , HT29 Cells , Humans , Mice , Mice, NudeABSTRACT
The SAR development is described for a series of N-acyl pyrrolidine inhibitors of the Hepatitis C virus RNA-dependent RNA polymerase, NS5B, from tractable Delta21 enzyme inhibitors to an example with antiviral activity in a cellular assay (HCV replicon).
Subject(s)
Antiviral Agents/pharmacology , Chemistry, Pharmaceutical/methods , Hepacivirus/chemistry , Hepacivirus/genetics , Pyrrolidines/antagonists & inhibitors , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Replicon/genetics , Viral Nonstructural Proteins/pharmacology , Antiviral Agents/chemistry , Drug Design , Enzyme Inhibitors/pharmacology , Inhibitory Concentration 50 , Models, Chemical , Molecular Conformation , RNA, Viral/chemistry , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effectsABSTRACT
HTS of the compound collection for inhibition of the HCV RNA dependent RNA polymerase identified two 168 member N-acyl pyrrolidine combinatorial mixture hits. Deconvolution and expansion of these mixtures by solid phase synthesis to establish initial SAR and identify a potent inhibitor is reported.
Subject(s)
Hepacivirus/enzymology , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Combinatorial Chemistry Techniques , Molecular StructureABSTRACT
Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A biosynthetic pathway. Here we report the identification of the Staphylococcus aureus coaA gene and characterization of the enzyme. We have also identified a series of low-molecular-weight compounds which are effective inhibitors of S. aureus CoaA.