ABSTRACT
PR-104, a phosphate ester of the nitrogen mustard prodrug PR-104A, has shown evidence of efficacy in adult leukemia clinical trials. Originally designed to target hypoxic cells, PR-104A is independently activated by aldo-keto-reductase 1C3 (AKR1C3). The aim of this study was to test whether AKR1C3 is a predictive biomarker of in vivo PR-104 sensitivity. In a panel of 7 patient-derived pediatric acute lymphoblastic leukemia (ALL) xenografts, PR-104 showed significantly greater efficacy against T-lineage ALL (T-ALL) than B-cell-precursor ALL (BCP-ALL) xenografts. Single-agent PR-104 was more efficacious against T-ALL xenografts compared with a combination regimen of vincristine, dexamethasone, and l-asparaginase. Expression of AKR1C3 was significantly higher in T-ALL xenografts compared with BCP-ALL, and correlated with PR-104/PR-104A sensitivity in vivo and in vitro. Overexpression of AKR1C3 in a resistant BCP-ALL xenograft resulted in dramatic sensitization to PR-104 in vivo. Testing leukemic blasts from 11 patients confirmed that T-ALL cells were more sensitive than BCP-ALL to PR-104A in vitro, and that sensitivity correlated with AKR1C3 expression. Collectively, these results indicate that PR-104 shows promise as a novel therapy for relapsed/refractory T-ALL, and that AKR1C3 expression could be used as a biomarker to select patients most likely to benefit from such treatment in prospective clinical trials.
Subject(s)
3-Hydroxysteroid Dehydrogenases/biosynthesis , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/analysis , Hydroxyprostaglandin Dehydrogenases/biosynthesis , Nitrogen Mustard Compounds/pharmacology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Adolescent , Aldo-Keto Reductase Family 1 Member C3 , Animals , Cell Survival/drug effects , Child , Child, Preschool , Female , Humans , Immunoblotting , Male , Mice , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Real-Time Polymerase Chain Reaction , Xenograft Model Antitumor AssaysABSTRACT
There is emerging evidence for reduced muscle function in children with neurofibromatosis type 1 (NF1). We have examined three murine models featuring NF1 deficiency in muscle to study the effect on muscle function as well as any underlying pathophysiology. The Nf1(+/-) mouse exhibited no differences in overall weight, lean tissue mass, fiber size, muscle weakness as measured by grip strength or muscle atrophy-recovery with limb disuse, although this model lacks many other characteristic features of the human disease. Next, muscle-specific knockout mice (Nf1muscle(-/-)) were generated and they exhibited a failure to thrive leading to neonatal lethality. Intramyocellular lipid accumulations were observed by electron microscopy and Oil Red O staining. More mature muscle specimens lacking Nf1 expression taken from the limb-specific Nf1Prx1(-/-) conditional knockout line showed a 10-fold increase in muscle triglyceride content. Enzyme assays revealed a significant increase in the activities of oxidative metabolism enzymes in the Nf1Prx1(-/-) mice. Western analyses showed increases in the expression of fatty acid synthase and the hormone leptin, as well as decreased expression of a number of fatty acid transporters in this mouse line. These data support the hypothesis that NF1 is essential for normal muscle function and survival and are the first to suggest a direct link between NF1 and mitochondrial fatty acid metabolism.
Subject(s)
Muscle Development/genetics , Muscles/metabolism , Neurofibromatosis 1/genetics , Neurofibromatosis 1/metabolism , Neurofibromin 1/genetics , Neurofibromin 1/metabolism , Animals , Body Weight , Bone Resorption/genetics , Bone and Bones/metabolism , Bone and Bones/pathology , Disease Models, Animal , Genes, Lethal , Heterozygote , Homozygote , Humans , Mice , Mice, Knockout , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscle Fibers, Skeletal/ultrastructure , Muscle Strength/genetics , Muscles/pathology , Muscles/ultrastructure , Neurofibromin 1/deficiency , Organ SizeABSTRACT
PURPOSE: Improvement in long-term outcomes through innovative, cost-effective medical technologies is a focus for endovascular procedures aimed at treating symptomatic lower-limb peripheral arterial disease (PAD). The advent of drug-eluting stents (DES) has improved symptomatic PAD treatment via a reduction in high rates of target lesion revascularisation (TLR). The present study aimed to compare the 5-year financial impact of treatment with Eluvia, a new paclitaxel-eluting stent, versus treatment with Zilver PTX, a drug-coated stent, among patients in Australia by developing a budget impact model (BIM). METHODS: A BIM was developed from an Australian public hospital payer perspective using Australian national cost weights (AUD), published literature, and public hospital audit data. Clinical outcomes, including clinically driven TLRs (CD-TLRs), adverse events, and length of stay, were based on the 2-year results of the IMPERIAL trial, which compared Eluvia DES to Zilver PTX. RESULTS: Assuming EVP eligibility rate of 80% and DES uses rate ranging from 10 to 28% (superficial femoral artery lesions only), the 5-year model forecasted a treatment population between 14,428 and 40,399 patients. The model estimated 1499-4198 fewer CD-TLRs and 16,515-46,243 fewer hospital days with Eluvia DES use. This translated to 5-year potential savings of $4.3-$12.1 million to the Australian public hospital payer attributable to reduced CD-TLRs for Eluvia DES and $33.1-$92.6 million to Australian public hospitals owing to reduced adverse events and hospital bed days. CONCLUSION: Eluvia DES use as treatment for symptomatic lower-limb PAD could lead to potential savings for the Australian public healthcare system based on improved patient outcomes.
Subject(s)
Drug-Eluting Stents , Peripheral Arterial Disease , Australia , Femoral Artery , Humans , Paclitaxel , Peripheral Arterial Disease/therapy , Popliteal Artery , Prosthesis Design , Treatment Outcome , Vascular PatencyABSTRACT
The ATF4 transcription factor is a key regulator of osteoblast differentiation that controls osteocalcin gene transcription and type I collagen protein synthesis. We have characterized factor-inhibiting ATF4-mediated transcription (FIAT), a leucine zipper protein that dimerizes with ATF4 to form inactive dimers that cannot bind DNA. Overexpression of FIAT in osteoblasts of transgenic mice inhibited osteocalcin gene transcription and reduced osteoblastic activity, leading to osteopenia (Yu et al. [2005] J Cell Biol 169:591-601). We therefore hypothesized that inhibition of FIAT would enhance ATF4 activity, leading to increased osteocalcin transcription, type I collagen synthesis, and mineralization. We used small interfering RNAs (siRNA) to knockdown FIAT in pools of MC3T3-E1 cells stably transfected with 1.3 kb of the mouse osteocalcin gene promoter driving expression of luciferase. Stable expression of the FIAT siRNA sequence inhibited FIAT expression without significantly affecting the level of total or Ribosomal S6 Kinase-2-phosphorylated ATF4 protein. Occupancy of the osteocalcin proximal promoter by ATF4 was increased and transcription of the osteocalcin-promoter-dependent luciferase reporter showed earlier onset and increased levels. Similarly, endogenous osteocalcin gene expression was enhanced in primary osteoblasts transfected with the FIAT siRNA. FIAT knockdown cells also displayed higher expression of bone sialoprotein, increased type I collagen protein synthesis, and enhanced mineralization. These data suggest that inhibition of FIAT expression increases ATF4 activity and confirm the important role of FIAT in osteoblast function.
Subject(s)
Activating Transcription Factor 4/metabolism , Carrier Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Osteoblasts/metabolism , Animals , Co-Repressor Proteins , Mice , Osteocalcin/genetics , Osteocalcin/metabolism , Promoter Regions, Genetic , RNA, Messenger/metabolism , RNA, Small Interfering/metabolismABSTRACT
Patient derived xenografts (PDXs) have become a vital, frequently used, component of anti-cancer drug development. PDXs can be serially passaged in vivo for years, and shared across laboratories. As a consequence, the potential for mis-identification and cross-contamination is possible, yet authentication of PDXs appears limited. We present a PDX Authentication System (PAS), by combining a commercially available OpenArray assay of single nucleotide polymorphisms (SNPs) with in-house R studio programs, to validate PDXs established in individual mice from acute lymphoblastic leukemia biopsies. The PAS is sufficiently robust to identify contamination at levels as low as 3%, similar to the gold standard of short tandem repeat (STR) profiling. We have surveyed a panel of PDXs established from 73 individual leukemia patients, and found that the PAS provided sufficient discriminatory power to identify each xenograft. The identified SNP-discrepant PDXs demonstrated distinct gene expression profiles, indicating a risk of contamination for PDXs at high passage number. The PAS also allows for the authentication of tumor cells with complex karyotypes from solid tumors including prostate cancer and Ewing's sarcoma. This study highlights the demands of authenticating PDXs for cancer research, and evaluates a reliable authentication platform that utilizes a commercially available and cost-effective system.
Subject(s)
Genotype , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prostatic Neoplasms/genetics , Sarcoma, Ewing/genetics , Animals , Cell Line, Tumor , Chimerism , Chromosome Aberrations , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Mice , Mice, SCID , Polymorphism, Single Nucleotide , Tissue Array Analysis , Xenograft Model Antitumor AssaysABSTRACT
In osteoblasts, Integrin-Linked Kinase (ILK)-dependent phosphorylation of the cJUN transcriptional coactivator, αNAC, induces the nuclear accumulation of the coactivator and potentiates cJUN-dependent transcription. Mutation of the ILK phosphoacceptor site within the αNAC protein leads to cytoplasmic retention of the coactivator and cell-autonomous increases in osteoblastic activity. In order to gain further insight into the ILK-αNAC signaling cascade, we inactivated ILK using RNA knockdown in osteoblastic cells and engineered mice with specific ablation of ILK in osteoblasts. ILK knockdown in MC3T3-E1 osteoblast-like cells reduced phosphorylation of its downstream target glycogen synthase kinase 3ß (GSK3ß), which led to cytoplasmic retention of αNAC and increased mineralization with augmented expression of the osteoblastic differentiation markers, pro-α1(I) collagen (col1A1), Bone Sialoprotein (Bsp) and Osteocalcin (Ocn). Cultured ILK-deficient primary osteoblasts also showed increased cytoplasmic αNAC levels, and augmented mineralization with higher Runx2, Col1a1 and Bsp expression. Histomorphometric analysis of bones from mutant mice with ILK-deficient osteoblasts (Col1-Cre;Ilk(-/fl)) revealed transient changes, with increased bone volume in newborn animals that was corrected by two weeks of age. Our data suggest that the ILK-αNAC cascade acts to reduce the pace of osteoblast maturation. We propose that in vivo, functional redundancy is able to compensate for the loss of ILK activity, leading to the absence of an obvious phenotype when osteoblast-specific Ilk-deficient mice reach puberty.
Subject(s)
Calcification, Physiologic , Osteoblasts/enzymology , Protein Serine-Threonine Kinases/antagonists & inhibitors , 3T3 Cells , Animals , Gene Knockdown Techniques , Mice , RNA/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.
Subject(s)
3-Hydroxysteroid Dehydrogenases/metabolism , Antineoplastic Agents/metabolism , Fluorometry/methods , Hydroxyprostaglandin Dehydrogenases/metabolism , Nitrogen Mustard Compounds/metabolism , Prodrugs/metabolism , 3-Hydroxysteroid Dehydrogenases/antagonists & inhibitors , 3-Hydroxysteroid Dehydrogenases/genetics , Aerobiosis , Aldo-Keto Reductase Family 1 Member C3 , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Bone Marrow/enzymology , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , HCT116 Cells , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/metabolism , Humans , Hydroxyprostaglandin Dehydrogenases/antagonists & inhibitors , Hydroxyprostaglandin Dehydrogenases/genetics , Leukocytes/enzymology , Morpholines/chemistry , Morpholines/metabolism , Nitrogen Mustard Compounds/pharmacokinetics , Nitrogen Mustard Compounds/pharmacology , Oxidation-Reduction , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Substrate Specificity , Time Factors , Urea/analogs & derivatives , Urea/chemistry , Urea/metabolismABSTRACT
Neurofibromatosis type 1 (NF1) is a common genetic condition caused by mutations in the NF1 gene. Patients often suffer from tissue-specific lesions associated with local double-inactivation of NF1. In this study, we generated a novel fracture model to investigate the mechanism underlying congenital pseudarthrosis of the tibia (CPT) associated with NF1. We used a Cre-expressing adenovirus (AdCre) to inactivate Nf1 in vitro in cultured osteoprogenitors and osteoblasts, and in vivo in the fracture callus of Nf1(flox/flox) and Nf1(flox/-) mice. The effects of the presence of Nf1(null) cells were extensively examined. Cultured Nf1(null)-committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with recombinant bone morphogenetic protein-2 (rhBMP-2) treatment. Similarly, Nf1(null)-inducible osteoprogenitors obtained from Nf1 MyoDnull mouse muscle were also unresponsive to rhBMP-2. In both closed and open fracture models in Nf1(flox/flox) and Nf1(flox/-) mice, local AdCre injection significantly impaired bone healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1(flox/flox) and Nf1(flox/-) mice. Histological analyses showed invasion of the Nf1(null) fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upward of 10-fold in Nf1(null) fractures and bromodeoxyuridine (BrdU) staining in closed fractures showed increased numbers of proliferating cells. In Nf1(null) fractures, tartrate-resistant acid phosphatase-positive (TRAP+) cells were frequently observed within the fibrous tissue, not lining a bone surface. In summary, we report that local Nf1 deletion in a fracture callus is sufficient to impair bony union and recapitulate histological features of clinical CPT. Cell culture findings support the concept that Nf1 double inactivation impairs early osteoblastic differentiation. This model provides valuable insight into the pathobiology of the disease, and will be helpful for trialing therapeutic compounds.
Subject(s)
Neurofibromatosis 1/complications , Neurofibromatosis 1/pathology , Osteoclasts/pathology , Pseudarthrosis/complications , Pseudarthrosis/pathology , Tibia/pathology , Acid Phosphatase/metabolism , Animals , Bone Morphogenetic Protein 2/pharmacology , Cell Differentiation/drug effects , Cell Lineage/drug effects , Cell Proliferation/drug effects , Disease Models, Animal , Female , Fibrosis , Fracture Healing/drug effects , Gene Deletion , HEK293 Cells , Humans , Integrases/metabolism , Isoenzymes/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscles/pathology , Neurofibromin 1/deficiency , Neurofibromin 1/metabolism , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteoblasts/pathology , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteogenesis/drug effects , Pseudarthrosis/metabolism , Recombinant Proteins/pharmacology , Reproducibility of Results , Tartrate-Resistant Acid Phosphatase , Tibia/drug effects , Tibia/metabolism , Transforming Growth Factor beta/pharmacologyABSTRACT
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder that is associated with a variety of manifestations, including orthopedic complications such as scoliosis and tibial pseudarthrosis. Orthopedic management of these skeletal complications is rendered more challenging due to a lack of standardized adjunctive pharmacotherapies. NF1 leads to disruption of the canonical Ras/Raf-1/MEK/ERK axis, and this has been associated with defects in bone anabolism. The roles of other non-canonical Ras effector pathways, such as the c-Jun N-terminal Kinase (JNK) pathway, are less well understood. In this study we examine the effects of an anthrapyrazolone inhibitor of JNK (SP600125) on inducible osteoprogenitors as well as Nf1-deficient and Nf1-null primary osteoblasts. C2C12 cells, which are highly responsive to rhBMP-2, were examined with exogenous rhBMP-2 and a range of SP600125 doses. Based on the expression of early and late bone markers and matrix mineralization, 10 µM SP600125 was found to be pro-osteogenic whether delivered concurrent with or following 2 days of rhBMP-2 treatment. Aberrant JNK activity was identified in Nf1-deficient osteoprogenitors (increased rhBMP-2 induced phospho-c-Jun) and in Nf1-null mature osteoblasts (increased total c-Jun). Next, SP600125 was used to treat these cells and was found to facilitate osteogenesis in Nf1-deficient osteoprogenitors, and in Nf1-null osteoblasts when given in conjunction with rhBMP-2. Outcome measures included alkaline phosphatase activity, matrix mineralization, and osteogenic gene expression. In summary, JNK inhibitors represent a class of potentially useful adjunctive agents for orthopedic medicine, particularly in the context of NF1.