ABSTRACT
CRISPR-based genome-editing technologies, including nuclease editing, base editing, and prime editing, have recently revolutionized the development of therapeutics targeting disease-causing mutations. To advance the assessment and development of genome editing tools, a robust mouse model is valuable, particularly for evaluating in vivo activity and delivery strategies. In this study, we successfully generated a knock-in mouse line carrying the Traffic Light Reporter design known as TLR-multi-Cas variant 1 (TLR-MCV1). We comprehensively validated the functionality of this mouse model for both in vitro and in vivo nuclease and prime editing. The TLR-MCV1 reporter mouse represents a versatile and powerful tool for expediting the development of editing technologies and their therapeutic applications.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , Mice , CRISPR-Cas Systems/genetics , Disease Models, Animal , Endonucleases/genetics , TechnologyABSTRACT
BACKGROUND: Preeclampsia (PE), a pregnancy complication characterized by new-onset hypertension and proteinuria during the second trimester, is the leading cause of neonatal and maternal morbidity and mortality. In the etiology of PE, failure of uterine spiral artery remodeling may be related to functioning abnormally of trophoblast cells, leading to the occurrence and progression of PE. Recently, long noncoding RNAs (lncRNAs) have been reported to play critical roles in PE nowadays. This study aimed to investigate the expression and functions of the TFPI2 pathway-related lncRNA DUXAP8. METHODS: DUXAP8 expression in the placenta from pregnancies was examined using qPCR. Then, the in vitro functions of DUXAP8 were investigated through MTT, EdU, colony, transwell, and flow cytometry experiments. The downstream gene expression profiles were assessed using RNA transcriptome sequencing analysis and verified using qPCR and western blot. Furthermore, Immunoprecipitation (RIP), chromatin immunoprecipitation (CHIP) and fluorescence in situ hybridization (FISH) were used to detect the interaction between lncDUXAP8/EZH2/TFPI2. RESULTS: The expression of lncRNA DUXAP8 in placenta of patients with eclampsia was significantly decreased. After knockout of DUXAP8, the proliferation and migration of trophoblasts were significantly decreased, and the percentage of apoptosis was increased. Flow cytometry showed that low expression of DUXAP8 increased the accumulation of cells in G2/M phase, while overexpression of DUXAP8 had the opposite effect. We also proved that DUXAP8 epigenetically inhibited TFPI2 expression by recruiting EZH2 and mediating H3K27me3 modification. CONCLUSION: Together, these resulting data clarify that aberrant expression of DUXAP8 is involved in the potential PE development and progress. Unraveling the role of DUXAP8 will provide novel insights into the pathogenesis of PE.
Subject(s)
MicroRNAs , Pre-Eclampsia , RNA, Long Noncoding , Female , Humans , Pregnancy , Cell Movement/genetics , Cell Proliferation/genetics , In Situ Hybridization, Fluorescence , MicroRNAs/genetics , Placenta/metabolism , Pre-Eclampsia/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Trophoblasts/metabolismABSTRACT
Surface modification and functionalization is typically required to engineer upconversion nanoparticles (UCNPs) for biosensing and bioimaging applications. Nevertheless, despite various antibody conjugation methods having been applied to UCNPs, no consensus has been reached on the best choice, as the results from individual studies are largely unable to be compared due to inadequate assessment of the properties of the conjugated products. Here, we introduce a systematic approach to quantitatively evaluate the biological activity of antibody-conjugated UCNPs. We determine that the optimal antibody conjugation efficiency to our colominic acid polysaccharide-coated UCNPs via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide (EDC/NHS) coupling is approximately 70%, corresponding to 16 antibodies per nanoparticle of 63 nm hydrodynamic diameter, with on average 12 of the 16 antibodies maintaining their affinity to the target antigens. The binding ability of the antibody-conjugated UCNPs to the antigen was well preserved, as verified by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and cellular imaging. This is the first study to quantitate the active antibody binding capacity of polysaccharide coated UCNP nanoparticles, offering a practical guideline for benchmarking functionalised UCNPs in future studies.
Subject(s)
Nanoparticles , Antibodies , Nanoparticles/chemistry , PolysaccharidesABSTRACT
Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.
Subject(s)
CRISPR-Associated Protein 9/metabolism , Dependovirus/genetics , Gene Editing/methods , Genetic Vectors/genetics , Mucopolysaccharidosis II/genetics , Recombinational DNA Repair , Tyrosinemias/genetics , Animals , CRISPR-Associated Protein 9/genetics , Dependovirus/metabolism , Female , Genetic Therapy , Genetic Vectors/metabolism , Humans , Male , Mice , Mucopolysaccharidosis II/therapy , Tyrosinemias/therapyABSTRACT
BACKGROUND AND AIMS: Despite surgical and chemotherapeutic advances, the 5-year survival rate for stage IV hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. Yes-associated protein 1 (YAP1) and ß-catenin co-activation occurs in 80% of children's HB; however, a lack of conditional genetic models precludes tumor maintenance exploration. Thus, the need for a targeted therapy remains unmet. Given the predominance of YAP1 and ß-catenin activation in HB, we sought to evaluate YAP1 as a therapeutic target in HB. APPROACH AND RESULTS: We engineered the conditional HB murine model using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A , constitutive ß-cateninDelN90 , and a luciferase reporter to murine liver. Tumor regression was evaluated using bioluminescent imaging, tumor landscape characterized using RNA and ATAC sequencing, and DNA footprinting. Here we show that YAP1S127A withdrawal mediates more than 90% tumor regression with survival for 230+ days in mice. YAP1S127A withdrawal promotes apoptosis in a subset of tumor cells, and in remaining cells induces a cell fate switch that drives therapeutic differentiation of HB tumors into Ki-67-negative hepatocyte-like HB cells ("HbHeps") with hepatocyte-like morphology and mature hepatocyte gene expression. YAP1S127A withdrawal drives the formation of hbHeps by modulating liver differentiation transcription factor occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and rescue liver damage in mice. CONCLUSIONS: YAP1S127A withdrawal, without silencing oncogenic ß-catenin, significantly regresses hepatoblastoma, providing in vivo data to support YAP1 as a therapeutic target for HB. YAP1S127A withdrawal alone sufficiently drives long-term regression in HB, as it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Hepatoblastoma/metabolism , Hepatocytes/metabolism , Liver Neoplasms/metabolism , Transcription Factors/metabolism , Animals , Cell Differentiation , Chromatin/metabolism , Genetic Engineering , Hepatoblastoma/therapy , Humans , Liver Neoplasms/therapy , Mice , YAP-Signaling ProteinsABSTRACT
Upconversion nanoparticles (UCNPs) exhibit unique optical properties such as photo-emission stability, large anti-Stokes shift, and long excited-state lifetimes, allowing significant advances in a broad range of applications from biomedical sensing to super-resolution microscopy. In recent years, progress on nanoparticle synthesis led to the development of many strategies for enhancing their upconversion luminescence, focused in particular on heavy doping of lanthanide ions and core-shell structures. In this article, we investigate the non-linear emission properties of fully Yb-based core-shell UCNPs and their impact on the super-resolution performance of stimulated excitation-depletion (STED) microscopy and super-linear excitation-emission (uSEE) microscopy. Controlling the power-dependent emission curve enables us to relax constraints on the doping concentrations and to reduce the excitation power required for accessing sub-diffraction regimes. We take advantage of this feature to implement multiplexed super-resolution imaging of a two-sample mixture.
ABSTRACT
Lanthanide-based upconversion nanoparticles (UCNPs) generally require high power laser excitation. Here, we report wide-field upconversion microscopy at single-nanoparticle sensitivity using incoherent excitation of a 970 nm light-emitting diode (LED). We show that due to its broad emission spectrum, LED excitation is about 3 times less effective for UCNPs and generates high background compared to laser illumination. To counter this, we use time-gated luminescence detection to eliminate the residual background from the LED source, so that individual UCNPs with high sensitizer (Yb3+) doping and inert shell protection become clearly identified under LED excitation at 1.18 W cm-2, as confirmed by correlated electron microscopy images. Hydrophilic UCNPs are obtained by polysaccharide coating via a facile ligand exchange protocol to demonstrate imaging of cellular uptake using LED excitation. These results suggest a viable approach to bypassing the limitations associated with high-power lasers when applying UCNPs and upconversion microscopy to life science research.
ABSTRACT
BACKGROUND: Preeclampsia (PE) is a widespread progressive condition that can occur pregnancy and is related to high maternal morbidity and fetal mortality in the perinatal period. However, the exact mechanism responsible has not been specific. Accumulating evidence has highlighted the prominent role of the epithelial-mesenchymal transition (EMT) in the biological behaviors of PE. METHODS: We explored the role of a lincRNA in extravillous trophoblast (EVTs) cell viability, migration, invasion and apoptosis in vitro, along with the use of linc00468 knockdown or overexpression. Clinically, we discovered that the expression of linc00468 was frequently correlated with adverse clinical features and poor prognosis of PE patients. RESULTS: We uncovered that linc00468 was downregulated in PE samples compared to in healthy tissues and in trophoblast cells. Functionally, gain and loss-of-function experiments demonstrated that linc00468 inhibited cell proliferation, migration, invasion and linc00468 accelerated apoptosis of the trophoblast phenotype in cell lines. Moreover, we demonstrated that downregulation of linc00468 promoted the expression of E-cadherin and ß-catenin but reduced the expression of N-cadherin, Vimentin and Snail, resulting in progression of EMT. CONCLUSIONS: In conclusion, linc00468 promoted EMT and a consequent increase in invasiveness in HTR-8/Svneo and JAR EVT cell lines. Our study provides the first evidence that linc00468 has a pivotal role in cell invasion and promotes intrinsic and extrinsic EMT ability of PE.
ABSTRACT
CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.
Subject(s)
Adenine/chemistry , CRISPR-Cas Systems , RNA, Guide, Kinetoplastida/chemistry , RNA, Messenger/chemistry , Alleles , Animals , Cell Line , Codon , Codon, Nonsense , Cystic Fibrosis/pathology , Gene Editing , HEK293 Cells , Humans , Mice , Mutation , Nucleotides , Phenotype , Plasmids , Transfection , Uridine/analogs & derivatives , Uridine/chemistryABSTRACT
Motor Unit Number Index (MUNIX) is a technique that provides a susceptive biomarker for monitoring innervation conditions in patients with neurodegenerative diseases. A satisfactory repeatability is essential for the interpretation of MUNIX results. This study aims to examine the effect of channel number and location on the repeatability of MUNIX. In this study, 128 channels of high-density surface electromyography (EMG) signals were recorded from the biceps brachii muscles of eight healthy participants, at 10, 20, 30, 40, 50, 60, 70, 80, and 100% of maximal voluntary contraction. The repeatability was defined by the coefficient of variation (CV) of MUNIX estimated from three experiment trials. Single-channel MUNIX (sMUNIX) was calculated on a channel-specific basis and a multi-channel MUNIX (mMUNIX) approach as the weighted average of multiple sMUNIX results. Results have shown (1) significantly improved repeatability with the proposed mMUNIX approach; (2) a higher variability of sMUNIX when the recording channel is positioned away from the innervation zone. Our results have demonstrated that (1) increasing the number of EMG channels and (2) placing recording channels close to the innervation zone (IZ) are effective methods to improve the repeatability of MUNIX. This study investigated two potential causes of MUNIX variations and provided novel perspectives to improve the repeatability, using high-density surface EMG. The mMUNIX technique proposed can serve as a promising tool for reliable neurodegeneration evaluation.
ABSTRACT
In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH+ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.
Subject(s)
Adenine/metabolism , Gene Editing/methods , Tyrosinemias/genetics , Animals , Disease Models, Animal , Female , HEK293 Cells , Hepatocytes/metabolism , Humans , Hydrolases/genetics , Liver/metabolism , Point Mutation , RNA/administration & dosageABSTRACT
Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with few effective treatments, and the underlying mechanisms that drive HCC pathogenesis remain poorly characterized. Identifying genes and pathways essential for HCC cell growth will aid the development of new targeted therapies for HCC. Using a kinome CRISPR screen in three human HCC cell lines, we identified transformation/transcription domain-associated protein (TRRAP) as an essential gene for HCC cell proliferation. TRRAP has been implicated in oncogenic transformation, but how it functions in cancer cell proliferation is not established. Here, we show that depletion of TRRAP or its co-factor, histone acetyltransferase KAT5, inhibits HCC cell growth through induction of p53-independent and p21-independent senescence. Integrated cancer genomics analyses using patient data and RNA sequencing identified mitotic genes as key TRRAP/KAT5 targets in HCC, and subsequent cell cycle analyses revealed that TRRAP-depleted and KAT5-depleted cells are arrested at the G2/M phase. Depletion of topoisomerase II alpha (TOP2A), a mitotic gene and TRRAP/KAT5 target, was sufficient to recapitulate the senescent phenotype of TRRAP/KAT5 knockdown. Conclusion: Our results uncover a role for TRRAP/KAT5 in promoting HCC cell proliferation by activating mitotic genes. Targeting the TRRAP/KAT5 complex is a potential therapeutic strategy for HCC.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Nuclear Proteins/genetics , Tumor Suppressor Protein p53/physiology , Cell Line, Tumor , Cell Proliferation , Cellular Senescence , Down-Regulation , Humans , Mitosis/geneticsABSTRACT
CRISPR/Cas9 has revolutionized cancer mouse models. Although loss-of-function genetics by CRISPR/Cas9 is well-established, generating gain-of-function alleles in somatic cancer models is still challenging because of the low efficiency of gene knock-in. Here we developed CRISPR-based Somatic Oncogene kNock-In for Cancer Modeling (CRISPR-SONIC), a method for rapid in vivo cancer modeling using homology-independent repair to integrate oncogenes at a targeted genomic locus. Using a dual guide RNA strategy, we integrated a plasmid donor in the 3'-UTR of mouse ß-actin, allowing co-expression of reporter genes or oncogenes from the ß-actin promoter. We showed that knock-in of oncogenic Ras and loss of p53 efficiently induced intrahepatic cholangiocarcinoma in mice. Further, our strategy can generate bioluminescent liver cancer to facilitate tumor imaging. This method simplifies in vivo gain-of-function genetics by facilitating targeted integration of oncogenes.
Subject(s)
Bile Duct Neoplasms/genetics , CRISPR-Cas Systems , Cholangiocarcinoma/genetics , Gene Knock-In Techniques/methods , Genes, ras , Actins/genetics , Animals , Bile Duct Neoplasms/diagnostic imaging , Bile Duct Neoplasms/pathology , Cell Line, Tumor , Cholangiocarcinoma/diagnostic imaging , Cholangiocarcinoma/pathology , Genes, Reporter , Genes, p53 , Humans , MiceABSTRACT
CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (N4CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for in vivo genome editing applications.
Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , DNA/genetics , Gene Editing/methods , Liver/enzymology , Neisseria meningitidis/enzymology , Proprotein Convertase 9/genetics , Animals , CRISPR-Associated Protein 9/metabolism , DNA/metabolism , Dependovirus/genetics , Embryo Transfer , Female , Genetic Vectors , HEK293 Cells , Humans , K562 Cells , Mice, Inbred C57BL , Nucleotide Motifs , Proprotein Convertase 9/metabolism , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , Substrate Specificity , Zygote/metabolismABSTRACT
Structural and morphological regulation is a distinctly important topic in peptide self-assembly, and is also regarded as the fundamental point in peptide-based biomaterials development. In this paper, we showed that adding anionic surfactant SDS to a bola amphiphilic peptide KI4K could result in the reconstruction of ß-sheet secondary structure besides the changes in self-assembly morphologies from nanotubes to helical ribbons, nanofibers, or straight nanotapes according to the negatively stained transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, and Fourier transform infrared spectroscopy results. The inducing effect of SDS was observed at both above and below its CMC but with different transformation rates. Through comparison to other surfactants, including CTAB, C12EO4, and AOT, we proposed that the transitions of KI4K self-assemblies induced by anionic surfactants could be mainly attributed to the effect of hydrophobic interaction and electrostatic attraction between surfactants and peptide molecules. Rheological property measurement and dye adsorption experiments were also carried out to evaluate the properties of hydrogels formed by the peptide/surfactant hybrids. The samples formed self-supporting hydrogels at proper SDS or AOT concentrations, and the charges of hydrogel could be regulated by peptide to surfactant ratio.
Subject(s)
Peptides/chemistry , Circular Dichroism , Hydrogels , Hydrophobic and Hydrophilic Interactions , Protein Structure, Secondary , Spectroscopy, Fourier Transform Infrared , Surface-Active AgentsABSTRACT
A smart lyotropic liquid crystal (LLC) system was prepared to control the diffusion rate of hydrophilic and hydrophobic molecules. The LLC system is composed of a nonionic surfactant (tetraethylene glycol monododecylether; C12 EO4 ) and an anionic azobenzene surfactant (Azo-surfactant). C12 EO4 was the main component of the LLC system. The Azo-surfactant, which can undergo photo-isomerization, played the role of trigger in this system. LLC gels formed in a solution comprised of Azo-surfactant (10â mm) and C12 EO4 (300â mm). The LLC gels became broken when more Azo-surfactant was added (e.g., up to 15â mm) and the viscoelasticity was lost. Surprisingly, when we used UV light to irradiate the 300â mm C12 EO4 /15â mm Azo-surfactant sample, the gel was recovered and high viscoelasticity was observed. However, under visible-light irradiation, the gel became broken again. The gel formation could also be triggered by heating the sample. On heating the 300â mm C12 EO4 /15â mm Azo-surfactant sample, the system thickened to a point at which typical gel behavior was registered. When the sample was cooled, the gel broke again. The LLC could be used for controlled release of hydrophilic and hydrophobic molecules, and could be considered as a versatile vehicle for the delivery of actives in systems of practical importance.
ABSTRACT
The proteasome was validated as an oncology target following the clinical success of VELCADE (bortezomib) for injection for the treatment of multiple myeloma and recurring mantle cell lymphoma. Consequently, several groups are pursuing the development of additional small-molecule proteasome inhibitors for both hematologic and solid tumor indications. Here, we describe MLN9708, a selective, orally bioavailable, second-generation proteasome inhibitor that is in phase I clinical development. MLN9708 has a shorter proteasome dissociation half-life and improved pharmacokinetics, pharmacodynamics, and antitumor activity compared with bortezomib. MLN9708 has a larger blood volume distribution at steady state, and analysis of 20S proteasome inhibition and markers of the unfolded protein response confirmed that MLN9708 has greater pharmacodynamic effects in tissues than bortezomib. MLN9708 showed activity in both solid tumor and hematologic preclinical xenograft models, and we found a correlation between greater pharmacodynamic responses and improved antitumor activity. Moreover, antitumor activity was shown via multiple dosing routes, including oral gavage. Taken together, these data support the clinical development of MLN9708 for both hematologic and solid tumor indications.
Subject(s)
Boron Compounds/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Glycine/analogs & derivatives , Neoplasms/drug therapy , Neoplasms/enzymology , Proteasome Inhibitors , Animals , Boron Compounds/pharmacokinetics , Boronic Acids/pharmacology , Bortezomib , Cysteine Proteinase Inhibitors/pharmacokinetics , Drug Screening Assays, Antitumor , Female , Glycine/pharmacokinetics , Glycine/pharmacology , HCT116 Cells , HT29 Cells , Humans , Lymphoma/drug therapy , Lymphoma/enzymology , Mice , Mice, SCID , Proteasome Endopeptidase Complex/blood , Pyrazines/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Understanding a compound's preclinical pharmacokinetic, pharmacodynamic, and efficacy relationship can greatly facilitate its clinical development. Bortezomib is a first-in-class proteasome inhibitor whose pharmacokinetic/pharmacodynamic parameters are poorly understood in terms of their relationship with efficacy. Here we characterized the bortezomib pharmacokinetic/pharmacodynamic/efficacy relationship in the CWR22 and H460 xenograft models. These studies allowed us to specifically address the question of whether the lack of broad bortezomib activity in solid tumor xenografts was due to insufficient tumor penetration. In vivo studies showed that bortezomib treatment resulted in tumor growth inhibition in CWR22 xenografts, but not in H460 xenografts. Using 20S proteasome inhibition as a pharmacodynamic marker and analyzing bortezomib tumor exposures, we show that efficacy was achieved only when suitable drug exposures drove proteasome inhibition that was sustained over time. This suggested that both the magnitude and duration of proteasome inhibition were important drivers of efficacy. Using dynamic contrast-enhanced magnetic resonance imaging and high-resolution computed tomographic imaging of vascular casts, we characterized the vasculature of CWR22 and H460 xenograft tumors and identified prominent differences in vessel perfusion, permeability, and architecture that ultimately resulted in variations in bortezomib tumor exposure. Comparing and contrasting the differences between a bortezomib-responsive and a bortezomib-resistant model with these techniques allowed us to establish a relationship among tumor perfusion, drug exposure, pharmacodynamic response and efficacy, and provided an explanation for why some solid tumor models do not respond to bortezomib treatment.
Subject(s)
Boronic Acids/therapeutic use , Neoplasms/drug therapy , Pyrazines/therapeutic use , Xenograft Model Antitumor Assays , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Area Under Curve , Boronic Acids/pharmacokinetics , Bortezomib , Cell Line, Tumor , Cell Survival/drug effects , Humans , Magnetic Resonance Imaging/methods , Male , Metabolic Clearance Rate , Mice , Mice, SCID , Neoplasms/metabolism , Neoplasms/pathology , Neovascularization, Pathologic/diagnostic imaging , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Pyrazines/pharmacokinetics , Treatment Outcome , Tumor Burden/drug effects , X-Ray Microtomography/methodsABSTRACT
Strains within the genus Salinospora have been shown to produce complex natural products having antibiotic and antiproliferative activities. The biochemical basis for the cytotoxic effects of salinosporamide A has been linked to its ability to inhibit the proteasome. Synthetically accessible salinosporamide A (ML858) was used to determine its biochemical and biological activities and to compare its effects with those of bortezomib. ML858 and bortezomib show time- and concentration-dependent inhibition of the proteasome in vitro. However, unlike bortezomib, which is a reversible inhibitor, ML858 covalently binds to the proteasome, resulting in the irreversible inhibition of 20S proteasome activity. ML858 was equipotent to bortezomib in cell-based reporter stabilization assays, but due to intramolecular instability is less potent in long-term assays. ML858 failed to maintain levels of proteasome inhibition necessary to achieve efficacy in tumor models responsive to bortezomib. Our results show that ML858 and bortezomib exhibit different kinetic and pharmacologic profiles and suggest that additional characterization of ML858 is warranted before its therapeutic potential can be fully appreciated.