ABSTRACT
Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Mutation , Neuroblastoma/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/antagonists & inhibitors , Anaplastic Lymphoma Kinase , Animals , Cell Line, Tumor , Crizotinib , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Gene Amplification , Humans , Mechanistic Target of Rapamycin Complex 1 , Mice, Inbred NOD , Mice, SCID , Molecular Targeted Therapy , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/metabolism , N-Myc Proto-Oncogene Protein , Neuroblastoma/enzymology , Neuroblastoma/genetics , Neuroblastoma/pathology , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Phosphatidylinositol 3-Kinase/metabolism , Phosphorylation , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins c-akt/metabolism , Pyrazoles/administration & dosage , Pyridines/administration & dosage , RNA Interference , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Ribosomal Protein S6/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , Time Factors , Transfection , Xenograft Model Antitumor AssaysABSTRACT
Since the original descriptions of gain-of function mutations in anaplastic lymphoma kinase (ALK), interest in the role of this receptor tyrosine kinase in neuroblastoma development and as a potential therapeutic target has escalated. As a group, the activating point mutations in full-length ALK, found in approximately 8% of all neuroblastoma tumors, are distributed evenly across different clinical stages. However, the most frequent somatic mutation, F1174L, is associated with amplification of the MYCN oncogene. This combination of features appears to confer a worse prognosis than MYCN amplification alone, suggesting a cooperative effect on neuroblastoma formation by these two proteins. Indeed, F1174L has shown more potent transforming activity in vivo than the second most common activating mutation, R1275Q, and is responsible for innate and acquired resistance to crizotinib, a clinically relevant ALK inhibitor that will soon be commercially available. These advances cast ALK as a bona fide oncoprotein in neuroblastoma and emphasize the need to understand ALK-mediated signaling in this tumor. This review addresses many of the current issues surrounding the role of ALK in normal development and neuroblastoma pathogenesis, and discusses the prospects for clinically effective targeted treatments based on ALK inhibition.
Subject(s)
Neuroblastoma/enzymology , Neuroblastoma/genetics , Receptor Protein-Tyrosine Kinases/genetics , Anaplastic Lymphoma Kinase , Animals , Genes, myc/genetics , Humans , Point MutationABSTRACT
Genistein is a bioflavonoid enriched in soy products. However, high levels of maternal soy consumption have been linked to the development of infant leukemia ALL and AML. The majority of infant leukemia is linked to mixed lineage leukemia gene (MLL) translocations. Previous studies have implicated topoisomerase II (Top2) in genistein-induced infant leukemia. In order to understand the roles of the two Top2 isozymes in and the molecular mechanism for genistein-induced infant leukemia, we carried out studies in vitro using purified recombinant human Top2 isozymes, as well as studies in cultured mouse myeloid progenitor cells (32Dc13) and Top2beta knockout mouse embryonic fibroblasts (MEFs). First, we showed that genistein efficiently induced both Top2alpha and Top2beta cleavage complexes in the purified system as well as in cultured mouse cells. Second, genistein induced proteasomal degradation of Top2beta in 32Dc13 cells. Third, the genistein-induced DNA double-strand break (DSB) signal, gamma-H2AX, was dependent on the Top2beta isozyme and proteasome activity. Fourth, the requirement for Top2beta and proteasome activity was mirrored in genistein-induced DNA sequence rearrangements, as monitored by a DNA integration assay. Together, our results suggest a model in which genistein-induced Top2beta cleavage complexes are processed by proteasome, leading to the exposure of otherwise Top2beta-concealed DSBs and subsequent chromosome rearrangements, and implicate a major role of Top2beta and proteasome in genistein-induced infant leukemia.
Subject(s)
DNA Topoisomerases, Type I/metabolism , Genistein/adverse effects , Isoenzymes/metabolism , Leukemia, Myeloid, Acute/chemically induced , Proteasome Endopeptidase Complex/metabolism , Recombination, Genetic/drug effects , Animals , Cell Line, Tumor , DNA/drug effects , DNA Breaks, Double-Stranded , Humans , Infant , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/genetics , MiceABSTRACT
Doxorubicin is among the most effective and widely used anticancer drugs in the clinic. However, cardiotoxicity is one of the life-threatening side effects of doxorubicin-based therapy. Dexrazoxane (Zinecard, also known as ICRF-187) has been used in the clinic as a cardioprotectant against doxorubicin cardiotoxicity. The molecular basis for doxorubicin cardiotoxicity and the cardioprotective effect of dexrazoxane, however, is not fully understood. In the present study, we showed that dexrazoxane specifically abolished the DNA damage signal gamma-H2AX induced by doxorubicin, but not camptothecin or hydrogen peroxide, in H9C2 cardiomyocytes. Doxorubicin-induced DNA damage was also specifically abolished by the proteasome inhibitors bortezomib and MG132 and much reduced in top2beta(-/-) mouse embryonic fibroblasts (MEF) compared with TOP2beta(+/+) MEFs, suggesting the involvement of proteasome and DNA topoisomerase IIbeta (Top2beta). Furthermore, in addition to antagonizing Top2 cleavage complex formation, dexrazoxane also induced rapid degradation of Top2beta, which paralleled the reduction of doxorubicin-induced DNA damage. Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced DNA damage through its interference with Top2beta, which could implicate Top2beta in doxorubicin cardiotoxicity. The specific involvement of proteasome and Top2beta in doxorubicin-induced DNA damage is consistent with a model in which proteasomal processing of doxorubicin-induced Top2beta-DNA covalent complexes exposes the Top2beta-concealed DNA double-strand breaks.
Subject(s)
DNA Breaks, Double-Stranded , Doxorubicin/antagonists & inhibitors , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/enzymology , Razoxane/pharmacology , Animals , Antibiotics, Antineoplastic/antagonists & inhibitors , Antibiotics, Antineoplastic/toxicity , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Doxorubicin/toxicity , Drug Interactions , Heart Diseases/chemically induced , Heart Diseases/enzymology , Heart Diseases/prevention & control , Histones/metabolism , Humans , Mice , Models, Molecular , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Protein Conformation , Topoisomerase II InhibitorsABSTRACT
Drugs that target DNA topoisomerase II (Top2), including etoposide (VP-16), doxorubicin, and mitoxantrone, are among the most effective anticancer drugs in clinical use. However, Top2-based chemotherapy has been associated with higher incidences of secondary malignancies, notably the development of acute myeloid leukemia in VP-16-treated patients. This association is suggestive of a link between carcinogenesis and Top2-mediated DNA damage. We show here that VP-16-induced carcinogenesis involves mainly the beta rather than the alpha isozyme of Top2. In a mouse skin carcinogenesis model, the incidence of VP-16-induced melanomas in the skin of 7,12-dimethylbenz[a]anthracene-treated mice is found to be significantly higher in TOP2beta(+) than in skin-specific top2beta-knockout mice. Furthermore, VP-16-induced DNA sequence rearrangements and double-strand breaks (DSBs) are found to be Top2beta-dependent and preventable by cotreatment with a proteasome inhibitor, suggesting the importance of proteasomal degradation of the Top2beta-DNA cleavage complexes in VP-16-induced DNA sequence rearrangements. VP-16 cytotoxicity in transformed cells expressing both Top2 isozymes is, however, found to be primarily Top2alpha-dependent. These results point to the importance of developing Top2alpha-specific anticancer drugs for effective chemotherapy without the development of treatment-related secondary malignancies.
Subject(s)
Antineoplastic Agents/adverse effects , DNA Topoisomerases, Type II/physiology , Isoenzymes/physiology , Neoplasms, Second Primary/chemically induced , Animals , DNA Damage , Disease Models, Animal , Drug Design , Etoposide/adverse effects , Isoenzymes/antagonists & inhibitors , Melanoma, Experimental/drug therapy , Mice , Mice, Knockout , Neoplasms, Second Primary/drug therapy , Neoplasms, Second Primary/etiology , Protease Inhibitors/pharmacology , Topoisomerase II InhibitorsABSTRACT
Despite rapid advances in the field of DNA repair, little is known about the repair of protein-DNA adducts. Previous studies have demonstrated that topoisomerase II (TopII)-DNA adducts (TopII-DNA covalent complexes) are rapidly degraded by the proteasome. It has been hypothesized that proteasomal degradation of TopII-DNA covalent adducts exposes TopII-concealed DNA double-strand breaks (DSBs) for repair. To test this hypothesis, the anticancer drug, VP-16 (etoposide), was employed to induce TopII-DNA covalent complexes in mammalian cells, and the involvement of proteasome in processing TopII-DNA covalent complexes into DSBs was investigated. Consistent with the hypothesis, VP-16-induced DSBs as monitored by neutral comet assay, as well as DNA damage signals (e.g. gamma-H2AX) were significantly reduced in the presence of the proteasome inhibitor, MG132. Using both top2beta knock-out mouse embryonic fibroblasts and Top2beta small interfering RNA knockdown PC12 cells, as well as postmitotic neurons in which TopIIalpha was absent, we showed that VP-16-induced DNA damage signals were attenuated upon proteasome inhibition, suggesting the involvement of proteasome in the repair/processing of both TopIIalpha-DNA and TopIIbeta-DNA adducts. By contrast, hydrogen peroxide-induced gamma-H2AX was unaffected upon proteasome inhibition, suggesting a specific requirement of the proteasome pathway in the processing of TopII-DNA covalent complexes into DNA damage.
Subject(s)
DNA Topoisomerases, Type II/chemistry , DNA/chemistry , Peptide Hydrolases/chemistry , Animals , Antineoplastic Agents/pharmacology , Comet Assay , Fibroblasts/metabolism , Histones/metabolism , Mice , Neurons/metabolism , PC12 Cells , Protein Binding , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Mice lacking topoisomerase IIbeta (TopIIbeta) are known to exhibit a perinatal death phenotype. In the current study, transcription profiles of the brains of wild-type and top2beta knockout mouse embryos were generated. Surprisingly, only a small number (1 to 4%) of genes were affected in top2beta knockout embryos. However, the expression of nearly 30% of developmentally regulated genes was either up- or down-regulated. By contrast, the expression of genes encoding general cell growth functions and early differentiation markers was not affected, suggesting that TopIIbeta is not required for early differentiation programming but is specifically required for the expression of developmentally regulated genes at later stages of differentiation. Consistent with this notion, immunohistochemical analysis of brain sections showed that TopIIbeta and histone deacetylase 2, a known TopIIbeta-interacting protein, were preferentially expressed in neurons which are in their later stages of differentiation. Chromatin immunoprecipitation analysis of the developing brains revealed TopIIbeta binding to the 5' region of a number of TopIIbeta-sensitive genes. Further studies of a TopIIbeta-sensitive gene, Kcnd2, revealed the presence of TopIIbeta in the transcription unit with major binding near the promoter region. Together, these results support a role of TopIIbeta in activation/repression of developmentally regulated genes at late stages of neuronal differentiation.
