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1.
Nat Cancer ; 4(9): 1326-1344, 2023 09.
Article in English | MEDLINE | ID: mdl-37640930

ABSTRACT

The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.


Subject(s)
Pancreatic Diseases , Pancreatic Neoplasms , Humans , Gemcitabine , Protein-Lysine 6-Oxidase , Pancreatic Neoplasms/drug therapy
2.
J Med Chem ; 66(16): 11216-11236, 2023 08 24.
Article in English | MEDLINE | ID: mdl-37535857

ABSTRACT

Mixed Lineage Kinase domain-Like pseudokinase (MLKL) is implicated in a broad range of diseases due to its role as the ultimate effector of necroptosis and has therefore emerged as an attractive drug target. Here, we describe the development of PROteolysis TArgeting Chimeras (PROTACs) as a novel approach to knock down MLKL through chemical means. A series of candidate degraders were synthesized from a high-affinity pyrazole carboxamide-based MLKL ligand leading to the identification of a PROTAC molecule that effectively degraded MLKL and completely abrogated cell death in a TSZ model of necroptosis. By leveraging the innate ability of these PROTACs to degrade MLKL in a dose-dependent manner, the quantitative relationship between MLKL levels and necroptosis was interrogated. This work demonstrates the feasibility of targeting MLKL using a PROTAC approach and provides a powerful tool to further our understanding of the role of MLKL within the necroptotic pathway.


Subject(s)
Necroptosis , Protein Kinases , Proteolysis Targeting Chimera , Apoptosis , Cell Death , Necroptosis/drug effects , Protein Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Proteolysis Targeting Chimera/chemistry , Proteolysis Targeting Chimera/pharmacology
4.
Cancers (Basel) ; 14(4)2022 Feb 15.
Article in English | MEDLINE | ID: mdl-35205728

ABSTRACT

Immunotherapies (such as checkpoint inhibitors) and standard chemotherapies (such as cisplatin) have limitations in the successful treatment of malignant pleural mesothelioma (MPM). Fibrosis is the accumulation of collagen in the extracellular matrix (ECM) of tissues, making them denser than that of healthy tissues and thereby affecting drug delivery and immune cell infiltration. Moreover, fibrosis severely affects the patient's breathing and quality of life. The production of collagen and its assembly is highly regulated by various enzymes such as lysyl oxidases. Many solid tumors aberrantly express the family of lysyl oxidases (LOX/LOXL). This review examines how LOX/LOXLs were found to be dysregulated in noncancerous and cancerous settings, discusses their roles in solid tumor fibrosis and pathogenesis and explores the role of fibrosis in the development and poor clinical outcomes of patients with MPM. We examine the current preclinical status of drugs targeting LOX/LOXLs and how the incorporation of such drugs may have therapeutic benefits in the treatment and management of patients with MPM.

6.
J Cell Mol Med ; 23(3): 1759-1770, 2019 03.
Article in English | MEDLINE | ID: mdl-30536539

ABSTRACT

Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε-amino group of lysine or hydroxylysine on collagen side-chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS-5153A, a novel mechanism based, fast-acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS-5153A dose-dependently reduced LOXL2-mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet-induced, PXS-5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS-5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.


Subject(s)
Amino Acid Oxidoreductases/antagonists & inhibitors , Collagen/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Fibrosis/prevention & control , Myocardial Infarction/prevention & control , Non-alcoholic Fatty Liver Disease/prevention & control , Animals , Carbon Tetrachloride/toxicity , Collagen/drug effects , Collagen/metabolism , Cross-Linking Reagents/chemistry , Elastin/antagonists & inhibitors , Elastin/drug effects , Elastin/metabolism , Extracellular Matrix/drug effects , Fibrosis/chemically induced , Fibrosis/enzymology , Fibrosis/pathology , Male , Mice , Mice, Inbred C57BL , Myocardial Infarction/enzymology , Myocardial Infarction/pathology , Non-alcoholic Fatty Liver Disease/enzymology , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/pathology , Rats , Rats, Wistar
7.
Neoplasia ; 19(9): 684-694, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28780387

ABSTRACT

Vascular endothelial growth factor A (VEGF-A) is considered one of the most important factors in tumor angiogenesis, and consequently, a number of therapeutics have been developed to inhibit VEGF signaling. Therapeutic strategies to target brain malignancies, both primary brain tumors, particularly in pediatric patients, and metastases, are lacking, but targeting angiogenesis may be a promising approach. Multiparametric MRI was used to investigate the response of orthotopic SF188luc pediatric glioblastoma xenografts to small molecule pan-VEGFR inhibitor cediranib and the effects of both cediranib and cross-reactive human/mouse anti-VEGF-A antibody B20-4.1.1 in intracranial MDA-MB-231 LM2-4 breast cancer xenografts over 48 hours. All therapeutic regimens resulted in significant tumor growth delay. In cediranib-treated SF188luc tumors, this was associated with lower Ktrans (compound biomarker of perfusion and vascular permeability) than in vehicle-treated controls. Cediranib also induced significant reductions in both Ktrans and apparent diffusion coefficient (ADC) in MDA-MB-231 LM2-4 tumors associated with decreased histologically assessed perfusion. B20-4.1.1 treatment resulted in decreased Ktrans, but in the absence of a change in perfusion; a non-significant reduction in vascular permeability, assessed by Evans blue extravasation, was observed in treated tumors. The imaging responses of intracranial MDA-MB-231 LM2-4 tumors to VEGF/VEGFR pathway inhibitors with differing mechanisms of action are subtly different. We show that VEGF pathway blockade resulted in tumor growth retardation and inhibition of tumor vasculature in preclinical models of pediatric glioblastoma and breast cancer brain metastases, suggesting that multiparametric MRI can provide a powerful adjunct to accelerate the development of antiangiogenic therapies for use in these patient populations.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/metabolism , Magnetic Resonance Imaging , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/metabolism , Animals , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Cell Line, Tumor , Diffusion Magnetic Resonance Imaging , Disease Models, Animal , Humans , Image Enhancement , Luminescent Measurements/methods , Magnetic Resonance Imaging/methods , Mice , Molecular Imaging , Molecular Targeted Therapy , Neovascularization, Pathologic/diagnosis , Neovascularization, Pathologic/diagnostic imaging , Neovascularization, Pathologic/drug therapy , Treatment Outcome , Xenograft Model Antitumor Assays
8.
NMR Biomed ; 29(11): 1608-1617, 2016 11.
Article in English | MEDLINE | ID: mdl-27671990

ABSTRACT

High grade and metastatic brain tumours exhibit considerable spatial variations in proliferation, angiogenesis, invasion, necrosis and oedema. Vascular heterogeneity arising from vascular co-option in regions of invasive growth (in which the blood-brain barrier remains intact) and neoangiogenesis is a major challenge faced in the assessment of brain tumours by conventional MRI. A multiparametric MRI approach, incorporating native measurements and both Gd-DTPA (Magnevist) and ultrasmall superparamagnetic iron oxide (P904)-enhanced imaging, was used in combination with histogram and unsupervised cluster analysis using a k-means algorithm to examine the spatial distribution of vascular parameters, water diffusion characteristics and invasion in intracranially propagated rat RG2 gliomas and human MDA-MB-231 LM2-4 breast adenocarcinomas in mice. Both tumour models presented with higher ΔR1 (the change in transverse relaxation rate R1 induced by Gd-DTPA), fractional blood volume (fBV) and apparent diffusion coefficient than uninvolved regions of the brain. MDA-MB-231 LM2-4 tumours were less densely cellular than RG2 tumours and exhibited substantial local invasion, associated with oedema, whereas invasion in RG2 tumours was minimal. These additional features were reflected in the more heterogeneous appearance of MDA-MB-231 LM2-4 tumours on T2 -weighted images and maps of functional MRI parameters. Unsupervised cluster analysis separated subregions with distinct functional properties; areas with a low fBV and relatively impermeable blood vessels (low ΔR1 ) were predominantly located at the tumour margins, regions of MDA-MB-231 LM2-4 tumours with relatively high levels of water diffusion and low vascular permeability and/or fBV corresponded to histologically identified regions of invasion and oedema, and areas of mismatch between vascular permeability and blood volume were identified. We demonstrate that dual contrast MRI and evaluation of tissue diffusion properties, coupled with cluster analysis, allows for the assessment of heterogeneity within invasive brain tumours and the designation of functionally diverse subregions that may provide more informative predictive biomarkers.


Subject(s)
Brain Neoplasms/diagnostic imaging , Brain Neoplasms/pathology , Dextrans , Gadolinium DTPA , Magnetic Resonance Imaging/methods , Magnetite Nanoparticles , Neovascularization, Pathologic/diagnostic imaging , Neovascularization, Pathologic/pathology , Animals , Cell Line, Tumor , Cell Proliferation , Contrast Media , Female , Image Enhancement/methods , Mice , Mice, Nude , Multimodal Imaging/methods , Neoplasm Invasiveness , Rats , Reproducibility of Results , Sensitivity and Specificity
9.
Nucl Med Biol ; 43(3): 198-205, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26924500

ABSTRACT

BACKGROUND: The radiolabeled amino acid O-(2-(18)F-fluoroethyl)-L-tyrosine (FET) and thymidine analogue 3'-deoxy-3'-(18)F-fluorothymidine (FLT) are widely used for positron emission tomography (PET) brain tumor imaging; however, comparative studies are scarce. The aim of this study therefore was to compare FLT and FET PET for the assessment of anti-VEGF response in glioblastoma xenografts. METHODS: Xenografts with confirmed intracranial glioblastoma were treated with anti-VEGF therapy (B20-4.1) or saline as control. Weekly bioluminescence imaging (BLI), FLT and FET PET/CT were used to follow treatment response. Tracer uptake of FLT and FET was quantified using maximum standardized uptake (SUVmax) values and tumor-to-background ratios (TBRs). Survival, the Ki67 proliferation index and micro-vessel density (MVD) were evaluated. RESULTS: In contrast to FLT TBRs, FET TBRs were significantly lower as early as one week after treatment initiation in the anti-VEGF group as compared to the control group. Following two weeks of treatment, both FLT and FET TBRs were significantly lower in the anti-VEGF group. In contrast, no significant difference between the treatment groups was detected using BLI. Furthermore, we found a significantly lower MVD in the anti-VEGF group as compared to the control group. However, we found no difference in the Ki67 proliferation index or mean survival time. CONCLUSION: FET appears to be a more sensitive tracer than FLT to measure early response to anti-VEGF therapy with PET. Advances in knowledge and implications for patient care FET PET appears to be an early predictor of anti-VEGF efficacy. Confirmation of these results in clinical studies is needed.


Subject(s)
Cell Transformation, Neoplastic , Dideoxynucleosides , Glioblastoma/diagnostic imaging , Glioblastoma/therapy , Positron-Emission Tomography/methods , Tyrosine/analogs & derivatives , Vascular Endothelial Growth Factor A/immunology , Animals , Bevacizumab/immunology , Bevacizumab/therapeutic use , Brain Neoplasms/blood supply , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/pathology , Brain Neoplasms/therapy , Female , Glioblastoma/blood supply , Glioblastoma/pathology , HEK293 Cells , Humans , Mice , Microvessels/metabolism , Survival Analysis
10.
Cell ; 163(1): 202-17, 2015 Sep 24.
Article in English | MEDLINE | ID: mdl-26388441

ABSTRACT

Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks.


Subject(s)
Ovarian Neoplasms/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Signal Transduction , Female , Humans , Information Storage and Retrieval , Models, Molecular , Point Mutation , Protein Kinases/chemistry , Software
11.
Nature ; 522(7554): 106-110, 2015 Jun 04.
Article in English | MEDLINE | ID: mdl-26017313

ABSTRACT

Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in patients with oestrogen-receptor negative breast cancer. Global quantitative analysis of the hypoxic secretome identified lysyl oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis, independent of RANK ligand, which disrupts normal bone homeostasis leading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to colonize and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications.


Subject(s)
Bone Neoplasms/pathology , Bone Neoplasms/secondary , Breast Neoplasms/enzymology , Breast Neoplasms/metabolism , Neoplasm Metastasis , Protein-Lysine 6-Oxidase/metabolism , Animals , Bone Neoplasms/metabolism , Bone Neoplasms/prevention & control , Breast Neoplasms/pathology , Cell Hypoxia , Cell Line, Tumor , Cell Movement , Female , Humans , Mice , NFATC Transcription Factors/metabolism , Neoplasm Metastasis/pathology , Neoplastic Cells, Circulating/pathology , Osteoblasts/metabolism , Osteoblasts/pathology , Osteoclasts/metabolism , Osteoclasts/pathology , Protein-Lysine 6-Oxidase/antagonists & inhibitors , Receptors, Estrogen/deficiency , Receptors, Estrogen/genetics
12.
Sci Transl Med ; 6(247): 247fs28, 2014 Jul 30.
Article in English | MEDLINE | ID: mdl-25080473

ABSTRACT

The discovery that ~20% of patients with brain cancer have circulating tumor cells breaks the dogma that these cells are confined to the brain and has important clinical implications (Müller et al., this issue).


Subject(s)
Brain Neoplasms/pathology , Glioblastoma/secondary , Neoplastic Cells, Circulating/pathology , Female , Humans , Male
13.
Future Oncol ; 10(9): 1709-17, 2014.
Article in English | MEDLINE | ID: mdl-25145437

ABSTRACT

Metastasis is the main reason for cancer-associated deaths and therapies are desperately needed to target the progression of cancer. Lysyl oxidase (LOX) plays a pivotal role in cancer progression, including metastasis, and is therefore is an attractive therapeutic target. In this review we will breakdown the process of cancer progression and the various roles that LOX plays has in the advancement of cancer. We will highlight why LOX is an exciting therapeutic target for the future.


Subject(s)
Neoplasms/enzymology , Protein-Lysine 6-Oxidase/physiology , Animals , Biomedical Research , Humans , Neoplasm Metastasis , Neoplasms/pathology , Protein Processing, Post-Translational , Tumor Suppressor Proteins/physiology
14.
Nat Rev Clin Oncol ; 9(7): 400-13, 2012 May 29.
Article in English | MEDLINE | ID: mdl-22641364

ABSTRACT

Gliomas in children differ from their adult counterparts by their distribution of histological grade, site of presentation and rate of malignant transformation. Although rare in the paediatric population, patients with high-grade gliomas have, for the most part, a comparably dismal clinical outcome to older patients with morphologically similar lesions. Molecular profiling data have begun to reveal the major genetic alterations underpinning these malignant tumours in children. Indeed, the accumulation of large datasets on adult high-grade glioma has revealed key biological differences between the adult and paediatric disease. Furthermore, subclassifications within the childhood age group can be made depending on age at diagnosis and tumour site. However, challenges remain on how to reconcile clinical data from adult patients to tailor novel treatment strategies specifically for paediatric patients.


Subject(s)
Brain Neoplasms/pathology , Glioma/pathology , Adult , Age Factors , Brain Neoplasms/therapy , Child , Glioma/therapy , Humans , Prognosis
15.
Proc Natl Acad Sci U S A ; 109(20): E1267-76, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22529373

ABSTRACT

We have previously demonstrated an increased DNA copy number and expression of IGF1R to be associated with poor outcome in Wilms tumors. We have now tested whether inhibiting this receptor may be a useful therapeutic strategy by using a panel of Wilms tumor cell lines. Both genetic and pharmacological targeting resulted in inhibition of downstream signaling through PI3 and MAP kinases, G(1) cell cycle arrest, and cell death, with drug efficacy dependent on the levels of phosphorylated IGF1R. These effects were further associated with specific gene expression signatures reflecting pathway inhibition, and conferred synergistic chemosensitisation to doxorubicin and topotecan. In the in vivo setting, s.c. xenografts of WiT49 cells resembled malignant rhabdoid tumors rather than Wilms tumors. Treatment with an IGF1R inhibitor (NVP-AEW541) showed no discernable antitumor activity and no downstream pathway inactivation. By contrast, Wilms tumor cells established orthotopically within the kidney were histologically accurate and exhibited significantly elevated insulin-like growth factor-mediated signaling, and growth was significantly reduced on treatment with NVP-AEW541 in parallel with signaling pathway ablation. As a result of the paracrine effects of enhanced IGF2 expression in Wilms tumor, this disease may be acutely dependent on signaling through the IGF1 receptor, and thus treatment strategies aimed at its inhibition may be useful in the clinic. Such efficacy may be missed if only standard ectopic models are considered as a result of an imperfect recapitulation of the specific tumor microenvironment.


Subject(s)
Insulin-Like Growth Factor I/metabolism , Kidney Neoplasms/physiopathology , Signal Transduction/physiology , Wilms Tumor/physiopathology , Analysis of Variance , Animals , Cell Line, Tumor , Electrochemistry , Gene Expression Profiling , HEK293 Cells , Humans , Magnetic Resonance Imaging , Mice , Paracrine Communication/physiology , Phosphorylation , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Signal Transduction/drug effects , Transplantation, Heterologous
16.
Mol Cancer Ther ; 10(8): 1407-18, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21659463

ABSTRACT

Pediatric glioblastoma (pGBM), although rare, is one of the leading causes of cancer-related deaths in children, with tumors essentially refractory to existing treatments. We have identified IGF1R to be a potential therapeutic target in pGBM due to gene amplification and high levels of IGF2 expression in some tumor samples, as well as constitutive receptor activation in pGBM cell lines. To evaluate the therapeutic potential of strategies targeting the receptor, we have carried out in vitro and in vivo preclinical studies using the specific IGF1R inhibitor NVP-AEW541. A modest inhibitory effect was seen in vitro, with GI(50) values of 5 to 6 µmol/L, and concurrent inhibition of receptor phosphorylation. Specific targeting of IGF1R with short interfering RNA decreased cell viability, diminished downstream signaling through phosphoinositide 3-kinase (PI3K), and induced G(1) arrest, effects mimicked by NVP-AEW541, both in the absence and presence of IGF2. Hallmarks of PI3K inhibition were observed after treatment with NVP-AEW541 by expression profiling and Western blot analysis. Phospho-receptor tyrosine kinase (RTK) arrays showed phosphorylation of platelet-derived growth factor receptor (PDGFR) α/ß in pGBM cells, suggesting coactivation of an alternative RTK pathway. Treatment of KNS42 with the PDGFR inhibitor imatinib showed additional effects targeting the mitogen-activated protein kinase pathway, and cotreatment of the PDGFR inhibitor imatinib with NVP-AEW541 resulted in a highly synergistic interaction in vitro and increased efficacy after 14 days therapy in vivo compared with either agent alone. These data provide evidence that inhibition of IGF1R, in combination with other targeted agents, may be a useful and novel therapeutic strategy in pGBM.


Subject(s)
Antineoplastic Agents/pharmacology , Glioblastoma/metabolism , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, Platelet-Derived Growth Factor alpha/antagonists & inhibitors , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Autophagy/drug effects , Cell Line, Tumor , Child , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Mice , Mice, Nude , Neoplasm Staging , Phosphatidylinositol 3-Kinases/metabolism , Pyrimidines/chemistry , Pyrroles/chemistry , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Receptor, Platelet-Derived Growth Factor beta/metabolism , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
17.
Cancer Res ; 70(22): 9243-52, 2010 Nov 15.
Article in English | MEDLINE | ID: mdl-20935218

ABSTRACT

Sensitivity to temozolomide is restricted to a subset of glioblastoma patients, with the major determinant of resistance being a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT, although other mechanisms are thought to be active. There are, however, limited preclinical data in model systems derived from pediatric glioma patients. We screened a series of cell lines for temozolomide efficacy in vitro, and investigated the differential mechanisms of resistance involved. In the majority of cell lines, a lack of MGMT promoter methylation and subsequent protein overexpression were linked to temozolomide resistance. An exception was the pediatric glioblastoma line KNS42. Expression profiling data revealed a coordinated upregulation of HOX gene expression in resistant lines, especially KNS42, which was reversed by phosphoinositide 3-kinase pathway inhibition. High levels of HOXA9/HOXA10 gene expression were associated with a shorter survival in pediatric high-grade glioma patient samples. Combination treatment in vitro of pathway inhibition and temozolomide resulted in a highly synergistic interaction in KNS42 cells. The resistance gene signature further included contiguous genes within the 12q13-q14 amplicon, including the Akt enhancer PIKE, significantly overexpressed in the KNS42 line. These cells were also highly enriched for CD133 and other stem cell markers. We have thus shown an in vitro link between phosphoinositide 3-kinase-mediated HOXA9/HOXA10 expression, and a drug-resistant, progenitor cell phenotype in MGMT-independent pediatric glioblastoma.


Subject(s)
Dacarbazine/analogs & derivatives , Homeodomain Proteins/genetics , O(6)-Methylguanine-DNA Methyltransferase/genetics , Phosphatidylinositol 3-Kinases/genetics , Antineoplastic Agents, Alkylating/pharmacology , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Child , Cluster Analysis , DNA Methylation/drug effects , Dacarbazine/pharmacology , Drug Resistance, Neoplasm/genetics , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Glioblastoma/metabolism , Glioblastoma/pathology , Homeobox A10 Proteins , Homeodomain Proteins/metabolism , Humans , Inhibitory Concentration 50 , Neoplastic Stem Cells/metabolism , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Oligonucleotide Array Sequence Analysis , Phosphatidylinositol 3-Kinases/metabolism , Promoter Regions, Genetic/genetics , Temozolomide
18.
Clin Cancer Res ; 15(18): 5753-61, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19737945

ABSTRACT

PURPOSE: The epidermal growth factor receptor (EGFR) is amplified and overexpressed in adult glioblastoma, with response to targeted inhibition dependent on the underlying biology of the disease. EGFR has thus far been considered to play a less important role in pediatric glioma, although extensive data are lacking. We have sought to clarify the role of EGFR in pediatric high-grade glioma (HGG). EXPERIMENTAL DESIGN: We retrospectively studied a total of 90 archival pediatric HGG specimens for EGFR protein overexpression, gene amplification, and mutation and assessed the in vitro sensitivity of pediatric glioma cell line models to the small-molecule EGFR inhibitor erlotinib. RESULTS: Amplification was detected in 11% of cases, with corresponding overexpression of the receptor. No kinase or extracellular domain mutations were observed; however, 6 of 35 (17%) cases harbored the EGFRvIII deletion, including two anaplastic oligodendrogliomas and a gliosarcoma overexpressing EGFRvIII in the absence of gene amplification and coexpressing platelet-derived growth factor receptor alpha. Pediatric glioblastoma cells transduced with wild-type or deletion mutant EGFRvIII were not rendered more sensitive to erlotinib despite expressing wild-type PTEN. Phosphorylated receptor tyrosine kinase profiling showed a specific activation of platelet-derived growth factor receptor alpha/beta in EGFRvIII-transduced pediatric glioblastoma cells, and targeted coinhibition with erlotinib and imatinib leads to enhanced efficacy in this model. CONCLUSIONS: These data identify an elevated frequency of EGFR gene amplification and EGFRvIII mutation in pediatric HGG than previously recognized and show the likely necessity of targeting multiple genetic alterations in the tumors of these children.


Subject(s)
ErbB Receptors/genetics , ErbB Receptors/metabolism , Glioma/drug therapy , Glioma/genetics , Sequence Deletion , Adolescent , Blotting, Western , Cell Proliferation/drug effects , Child , ErbB Receptors/antagonists & inhibitors , Erlotinib Hydrochloride , Glioma/diagnosis , Humans , Prognosis , Quinazolines/pharmacology , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Tumor Cells, Cultured
19.
PLoS One ; 4(4): e5209, 2009.
Article in English | MEDLINE | ID: mdl-19365568

ABSTRACT

BACKGROUND: Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines. PRINCIPAL FINDINGS: All lines proliferate as adherent monolayers and express glial markers. Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways. Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response. SIGNIFICANCE: These data demonstrate that glioma cell lines derived from paediatric patients show key molecular differences to those from adults, some of which are well known, whilst others may provide novel targets for evaluation in primary tumours. We thus provide the rationale and demonstrate the practicability of using paediatric glioma cell lines for preclinical and mechanistic studies.


Subject(s)
Cell Line, Tumor , Drug Design , Glioma , Phenotype , Adult , Astrocytes/metabolism , Biomarkers/metabolism , Cell Line, Tumor/metabolism , Cell Line, Tumor/pathology , Child , Chromosome Aberrations , Enzyme Activation , Epigenesis, Genetic , Gene Expression Profiling , Glioma/genetics , Glioma/metabolism , Glioma/pathology , Humans , Immunophenotyping , Loss of Heterozygosity , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/physiology , Stem Cells/metabolism
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