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In acute myeloid leukemia (AML), chemotherapy resistance remains prevalent and poorly understood. Using functional proteomics of patient AML specimens, we identified MEF2C S222 phosphorylation as a specific marker of primary chemoresistance. We found that Mef2cS222A/S222A knock-in mutant mice engineered to block MEF2C phosphorylation exhibited normal hematopoiesis, but were resistant to leukemogenesis induced by MLL-AF9 MEF2C phosphorylation was required for leukemia stem cell maintenance and induced by MARK kinases in cells. Treatment with the selective MARK/SIK inhibitor MRT199665 caused apoptosis and conferred chemosensitivity in MEF2C-activated human AML cell lines and primary patient specimens, but not those lacking MEF2C phosphorylation. These findings identify kinase-dependent dysregulation of transcription factor control as a determinant of therapy response in AML, with immediate potential for improved diagnosis and therapy for this disease.Significance: Functional proteomics identifies phosphorylation of MEF2C in the majority of primary chemotherapy-resistant AML. Kinase-dependent dysregulation of this transcription factor confers susceptibility to MARK/SIK kinase inhibition in preclinical models, substantiating its clinical investigation for improved diagnosis and therapy of AML. Cancer Discov; 8(4); 478-97. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.
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Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/tratamiento farmacológico , Factores de Transcripción MEF2/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Línea Celular , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Factores de Transcripción MEF2/química , Ratones , Ratones Transgénicos , Fosforilación , ProteómicaRESUMEN
Aberrant gene expression is a hallmark of acute leukemias. MYB-driven transcriptional coactivation with CREB-binding protein (CBP)/P300 is required for acute lymphoblastic and myeloid leukemias, including refractory MLL-rearranged leukemias. Using structure-guided molecular design, we developed a peptidomimetic inhibitor MYBMIM that interferes with the assembly of the molecular MYB:CBP/P300 complex and rapidly accumulates in the nuclei of AML cells. Treatment of AML cells with MYBMIM led to the dissociation of the MYB:CBP/P300 complex in cells, its displacement from oncogenic enhancers enriched for MYB binding sites, and downregulation of MYB-dependent gene expression, including of MYC and BCL2 oncogenes. AML cells underwent mitochondrial apoptosis in response to MYBMIM, which was partially rescued by ectopic expression of BCL2. MYBMIM impeded leukemia growth and extended survival of immunodeficient mice engrafted with primary patient-derived MLL-rearranged leukemia cells. These findings elucidate the dependence of human AML on aberrant transcriptional coactivation, and establish a pharmacologic approach for its therapeutic blockade.
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Materiales Biomiméticos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Peptidomiméticos/farmacología , Proteínas Proto-Oncogénicas c-myb/genética , Activación Transcripcional/genética , Factores de Transcripción p300-CBP/genética , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión/fisiología , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Femenino , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Simulación de Dinámica Molecular , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-myb/biosíntesis , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Factores de Transcripción p300-CBP/biosíntesisRESUMEN
Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as encoding an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints and recurrently inactivated tumor-suppressor genes. PGBD5 was physically associated with genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. Ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote cell transformation in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain as well as end-joining DNA repair and induced structural rearrangements with PSS breakpoints. These results define PGBD5 as an oncogenic mutator and provide a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.
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Transformación Celular Neoplásica/genética , Tumor Rabdoide/genética , Transposasas/fisiología , Adulto , Animales , Dominio Catalítico , Línea Celular , Niño , Preescolar , Aberraciones Cromosómicas , Puntos de Rotura del Cromosoma , Reparación del ADN por Unión de Extremidades/genética , ADN de Neoplasias/genética , Reordenamiento Génico/genética , Genes Supresores de Tumor , Humanos , Lactante , Ratones , Ratones Desnudos , Mutagénesis Sitio-Dirigida , Interferencia de ARN , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Secuencias Repetidas Terminales/genética , Transposasas/química , Transposasas/genéticaRESUMEN
Cure rates of children and adults with acute myeloid leukaemia (AML) remain unsatisfactory partly due to chemotherapy resistance. We investigated the genetic basis of AML in 107 primary cases by sequencing 670 genes mutated in haematological malignancies. SETBP1, ASXL1 and RELN mutations were significantly associated with primary chemoresistance. We identified genomic alterations not previously described in AML, together with distinct genes that were significantly overexpressed in therapy-resistant AML. Defined gene mutations were sufficient to explain primary induction failure in only a minority of cases. Thus, additional genetic or molecular mechanisms must cause primary chemoresistance in paediatric and adult AML.
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Resistencia a Antineoplásicos/genética , Genómica/métodos , Leucemia Mieloide Aguda/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Portadoras/genética , Moléculas de Adhesión Celular Neuronal/genética , Niño , Preescolar , Proteínas de la Matriz Extracelular/genética , Femenino , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Lactante , Masculino , Persona de Mediana Edad , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Embarazo , Proteína Reelina , Inducción de Remisión/métodos , Proteínas Represoras/genética , Serina Endopeptidasas/genética , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
Transposons are mobile genetic elements that are found in nearly all organisms, including humans. Mobilization of DNA transposons by transposase enzymes can cause genomic rearrangements, but our knowledge of human genes derived from transposases is limited. In this study, we find that the protein encoded by human PGBD5, the most evolutionarily conserved transposable element-derived gene in vertebrates, can induce stereotypical cut-and-paste DNA transposition in human cells. Genomic integration activity of PGBD5 requires distinct aspartic acid residues in its transposase domain, and specific DNA sequences containing inverted terminal repeats with similarity to piggyBac transposons. DNA transposition catalyzed by PGBD5 in human cells occurs genome-wide, with precise transposon excision and preference for insertion at TTAA sites. The apparent conservation of DNA transposition activity by PGBD5 suggests that genomic remodeling contributes to its biological function.
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Elementos Transponibles de ADN , Recombinación Genética , Transposasas/metabolismo , Humanos , Especificidad por SustratoRESUMEN
The PI3K/AKT/mTOR pathway has frequently been found activated in human tumors. We show that in addition to Wnt signaling dysfunction, the PI3K/AKT/mTOR pathway is often upregulated in mouse Msh2(-/-) initiated intestinal tumors. NVP-BEZ235 is a dual PI3K/mTOR inhibitor toxic to many cancer cell lines and currently involved in clinical trials. We have treated two mouse models involving Msh2 that develop small intestinal and/or colonic tumors with NVP-BEZ235, and a subset of animals with NVP-BEZ235 and MEK inhibitor ADZ4266. The disease phenotype has been followed with pathology, (18)F FDG PET imaging, and endoscopy. Intestinal adenocarcinomas are significantly decreased in multiplicity by both drug regimens. The majority of tumors treated with combined therapy regress significantly, while a small number of highly progressed tumors persist. We have examined PTEN, AKT, MEK 1&2, MAPK, S6K, mTOR, PDPK1, and Cyclin D1 and find variable alterations that include downregulation of PTEN, upregulation of AKT and changes in its phosphorylated forms, upregulation of pMEK 1&2, p42p44MAPK, pS6K, and Cyclin D1. Apoptosis has been found intact in some tumors and not in others. Our data indicate that NVP-BEZ235 alone and in combination with ADZ4266 are effective in treating a proportion of colorectal cancers, but that highly progressed resistant tumors grow in the presence of the drugs. Pathways upregulated in some resistant tumors also include PDPK1, suggesting that metabolic inhibitors may also be useful in treating these tumors.
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Reparación de la Incompatibilidad de ADN/genética , Neoplasias/genética , Neoplasias/terapia , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Reordenamiento Génico/efectos de los fármacos , Genotipo , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Integrasas/metabolismo , Neoplasias Intestinales/tratamiento farmacológico , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteína 2 Homóloga a MutS/deficiencia , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Tomografía de Emisión de Positrones , Quinolinas/farmacología , Quinolinas/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteínas Wnt/metabolismoRESUMEN
Exogenous cell-permeable C6 ceramide has been demonstrated to act synergistically with chemotherapeutic drugs, including paclitaxel, cisplatin, doxorubicin and the histone deacetylase inhibitor, trichostatin A, to induce cell death in a variety of cancer cells. We previously demonstrated that C6 ceramide and paclitaxel function synergistically to induce ovarian cancer cell death via modulation of the PI3/AKT cell survival pathway. In the present study, the entry pattern of C6 ceramide into ovarian cancer cells was investigated using fluorescent short chain C6-NBD sphingomyelin (C6-NBD). Confocal microscopy revealed that C6-NBD enters the cells in a polarized pattern, characterized by marked signals at one cellular end, representing a likely mitosis initiation site. Pretreatment of the cells with filipin, an inhibitor of the lipid raft/caveolae endocytosis pathway, decreases C6-NBD entry into the cells. A pretreatment with the water channel inhibitor, CuSO4, was also found to reduce the entry of C6-NBD. Notably, the pretreatment with paclitaxel was shown to disrupt the polarized entry of C6-NBD into the cells, resulting in an even distribution of C6-NBD in the cytoplasm. In addition, the pretreatment of the cells with paclitaxel destabilized the cytoskeletal proteins, releasing an increased number of short tubulin fragments. The results of the present study indicate that C6 ceramide preferentially enters the cells via a predetermined initiation site of mitosis. In addition to diffusion, short chain C6 ceramide may also enter cells via water channels and caveolae-mediated endocytosis. Paclitaxel disrupts the cell cytoskeleton and induces an even distribution of C6 ceramide in the cytoplasm resulting in synergistic ovarian cancer cell death.
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Doxorubicin has been used clinically to treat various types of cancer, and yet the molecular mode of actions of doxorubicin remains to be fully unraveled. In this study, we investigated the effect of doxorubicin on cultured ovarian cancer cells (CaOV3). MTT assay data showed that doxorubicin inhibits cell proliferation in a time- and dose-dependent manner. Phagokinetic cell motility assay data indicated that doxorubicin inhibits both basal level and EGF-induced cell migration in CaOV3 cells. Confocal microscopic data revealed that doxorubicin induces reorganization of cytoskeletal proteins including actin, tubulin and vimentin. Doxorubicin induces phosphorylation of p53 at Ser15 and 20, acetylation of p53 and ATM activation. Doxorubicin also induces phosphorylation of histone H2AX at Ser139. Interestingly, doxorubicin also inhibits mTOR activity, measured by phosphorylation of S6 ribosomal protein. Pretreatment of CaOV3 cells with antioxidant N-acetylcysteine (NAC), but not pyrrolidine dithiocarbamate (PDTC) potentiates doxorubicin-induced phosphorylation of p53 and ATM. Collectively, we conclude that doxorubicin induces ATM/p53 activation leading to reorganization of cytoskeletal networks, inhibition of mTOR activity, and inhibition of cell proliferation and migration. Our data also suggest that removal of oxidants by antioxidants such as NAC may enhance the efficacy of doxorubicin in vivo.