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2.
Blood Cancer J ; 12(1): 5, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017466

RESUMO

Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1.


Assuntos
Antineoplásicos/farmacologia , Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Rearranjo Gênico/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Mutação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Sulfonamidas/farmacologia
3.
Pharmacol Rev ; 74(1): 18-47, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34987087

RESUMO

ERBB4 (HER4) is a member of the ERBB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). EGFR and ERBB2 are oncoproteins and validated targets for therapeutic intervention in a variety of solid tumors. In contrast, the role that ERBB4 plays in human malignancies is ambiguous. Thus, here we review the literature regarding ERBB4 function in human malignancies. We review the mechanisms of ERBB4 signaling with an emphasis on mechanisms of signaling specificity. In the context of this signaling specificity, we discuss the hypothesis that ERBB4 appears to function as a tumor suppressor protein and as an oncoprotein. Next, we review the literature that describes the role of ERBB4 in tumors of the bladder, liver, prostate, brain, colon, stomach, lung, bone, ovary, thyroid, hematopoietic tissues, pancreas, breast, skin, head, and neck. Whenever possible, we discuss the possibility that ERBB4 mutants function as biomarkers in these tumors. Finally, we discuss the potential roles of ERBB4 mutants in the staging of human tumors and how ERBB4 function may dictate the treatment of human tumors. SIGNIFICANCE STATEMENT: This articles reviews ERBB4 function in the context of the mechanistic model that ERBB4 homodimers function as tumor suppressors, whereas ERBB4-EGFR or ERBB4-ERBB2 heterodimers act as oncogenes. Thus, this review serves as a mechanistic framework for clinicians and scientists to consider the role of ERBB4 and ERBB4 mutants in staging and treating human tumors.


Assuntos
Neoplasias , Receptor ErbB-4 , Transdução de Sinais , Humanos , Neoplasias/genética , Receptor ErbB-4/genética
4.
Blood ; 139(6): 907-921, 2022 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-34601571

RESUMO

The majority of RUNX1 mutations in acute myeloid leukemia (AML) are missense or deletion-truncation and behave as loss-of-function mutations. Following standard therapy, AML patients expressing mtRUNX1 exhibit inferior clinical outcome than those without mutant RUNX1. Studies presented here demonstrate that as compared with AML cells lacking mtRUNX1, their isogenic counterparts harboring mtRUNX1 display impaired ribosomal biogenesis and differentiation, as well as exhibit reduced levels of wild-type RUNX1, PU.1, and c-Myc. Compared with AML cells with only wild-type RUNX1, AML cells expressing mtRUNX1 were also more sensitive to the protein translation inhibitor homoharringtonine (omacetaxine) and BCL2 inhibitor venetoclax. Homoharringtonine treatment repressed enhancers and their BRD4 occupancy and was associated with reduced levels of c-Myc, c-Myb, MCL1, and Bcl-xL. Consistent with this, cotreatment with omacetaxine and venetoclax or BET inhibitor induced synergistic in vitro lethality in AML expressing mtRUNX1. Compared with each agent alone, cotreatment with omacetaxine and venetoclax or BET inhibitor also displayed improved in vivo anti-AML efficacy, associated with improved survival of immune-depleted mice engrafted with AML cells harboring mtRUNX1. These findings highlight superior efficacy of omacetaxine-based combination therapies for AML harboring mtRUNX1.


Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Mepesuccinato de Omacetaxina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores da Síntese de Proteínas/farmacologia , Sulfonamidas/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Leucemia Mieloide Aguda/genética , Mutação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores
5.
Blood Cancer J ; 11(5): 98, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016956

RESUMO

There is an unmet need to overcome nongenetic therapy-resistance to improve outcomes in AML, especially post-myeloproliferative neoplasm (MPN) secondary (s) AML. Studies presented describe effects of genetic knockout, degradation or small molecule targeted-inhibition of GFI1/LSD1 on active enhancers, altering gene-expressions and inducing differentiation and lethality in AML and (MPN) sAML cells. A protein domain-focused CRISPR screen in LSD1 (KDM1A) inhibitor (i) treated AML cells, identified BRD4, MOZ, HDAC3 and DOT1L among the codependencies. Our findings demonstrate that co-targeting LSD1 and one of these co-dependencies exerted synergistic in vitro lethality in AML and post-MPN sAML cells. Co-treatment with LSD1i and the JAKi ruxolitinib was also synergistically lethal against post-MPN sAML cells. LSD1i pre-treatment induced GFI1, PU.1 and CEBPα but depleted c-Myc, overcoming nongenetic resistance to ruxolitinib, or to BETi in post-MPN sAML cells. Co-treatment with LSD1i and BETi or ruxolitinib exerted superior in vivo efficacy against post-MPN sAML cells. These findings highlight LSD1i-based combinations that merit testing for clinical efficacy, especially to overcome nongenetic therapy-resistance in AML and post-MPN sAML.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Histona Desmetilases/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Transtornos Mieloproliferativos/tratamento farmacológico , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Inativação Gênica/efeitos dos fármacos , Histona Desmetilases/genética , Humanos , Leucemia Mieloide Aguda/genética , Terapia de Alvo Molecular , Transtornos Mieloproliferativos/genética , Fatores de Transcrição/genética , Transcriptoma/efeitos dos fármacos
6.
Leukemia ; 35(9): 2621-2634, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33654205

RESUMO

Richter Transformation (RT) develops in CLL as an aggressive, therapy-resistant, diffuse large B cell lymphoma (RT-DLBCL), commonly clonally-related (CLR) to the concomitant CLL. Lack of available pre-clinical human models has hampered the development of novel therapies for RT-DLBCL. Here, we report the profiles of genetic alterations, chromatin accessibility and active enhancers, gene-expressions and anti-lymphoma drug-sensitivity of three newly established, patient-derived, xenograft (PDX) models of RT-DLBCLs, including CLR and clonally-unrelated (CLUR) to concomitant CLL. The CLR and CLUR RT-DLBCL cells display active enhancers, higher single-cell RNA-Seq-determined mRNA, and protein expressions of IRF4, TCF4, and BCL2, as well as increased sensitivity to BET protein inhibitors. CRISPR knockout of IRF4 attenuated c-Myc levels and increased sensitivity to a BET protein inhibitor. Co-treatment with BET inhibitor or BET-PROTAC and ibrutinib or venetoclax exerted synergistic in vitro lethality in the RT-DLBCL cells. Finally, as compared to each agent alone, combination therapy with BET-PROTAC and venetoclax significantly reduced lymphoma burden and improved survival of immune-depleted mice engrafted with CLR-RT-DLBCL. These findings highlight a novel, potentially effective therapy for RT-DLBCL.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteínas/metabolismo , Proteólise , Adenina/administração & dosagem , Adenina/análogos & derivados , Animais , Apoptose , Biomarcadores Tumorais/genética , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Piperidinas/administração & dosagem , Proteínas/genética , Sulfonamidas/administração & dosagem , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Blood Cancer J ; 11(3): 64, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33753715

RESUMO

Ecotropic viral integration site 1 (Evi1) was discovered in 1988 as a common site of ecotropic viral integration resulting in myeloid malignancies in mice. EVI1 is an oncogenic zinc-finger transcription factor whose overexpression contributes to disease progression and an aggressive phenotype, correlating with poor clinical outcome in myeloid malignancies. Despite progress in understanding the biology of EVI1 dysregulation, significant improvements in therapeutic outcome remain elusive. Here, we highlight advances in understanding EVI1 biology and discuss how this new knowledge informs development of novel therapeutic interventions. EVI1 is overexpression is correlated with poor outcome in some epithelial cancers. However, the focus of this review is the genetic lesions, biology, and current therapeutics of myeloid malignancies overexpressing EVI1.


Assuntos
Leucemia Mieloide/genética , Proteína do Locus do Complexo MDS1 e EVI1/genética , Animais , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Humanos , Leucemia Mieloide/patologia , Leucemia Mieloide/terapia , Mutação , Processamento de Proteína Pós-Traducional , Ativação Transcricional
8.
PLoS One ; 15(12): e0243901, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33378376

RESUMO

Whereas recent clinical studies report metastatic melanoma survival rates high as 30-50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that is based on the observation that the Q43L mutant of the naturally occurring ErbB4 agonist Neuregulin-2beta (NRG2ß) functions as a partial agonist at ErbB4. NRG2ß/Q43L stimulates tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z' > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.


Assuntos
Proliferação de Células/genética , Melanoma/genética , Fatores de Crescimento Neural/genética , Receptor ErbB-4/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Mutação com Ganho de Função/genética , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Fosforilação/genética , Receptor ErbB-4/agonistas , Receptor ErbB-4/antagonistas & inibidores , Transdução de Sinais/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
9.
Blood ; 135(15): 1255-1269, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32068780

RESUMO

The promising activity of BET protein inhibitors (BETi's) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear ß-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the ß-catenin-TCF7L2-JMJD6-c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear ß-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the ß-catenin-TCF7L2-JMJD6-c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Antineoplásicos/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo
10.
Blood ; 134(1): 59-73, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31023702

RESUMO

RUNX1 transcription factor regulates normal and malignant hematopoiesis. Somatic or germline mutant RUNX1 (mtRUNX1) is associated with poorer outcome in acute myeloid leukemia (AML). Knockdown or inhibition of RUNX1 induced more apoptosis of AML expressing mtRUNX1 versus wild-type RUNX1 and improved survival of mice engrafted with mtRUNX1-expressing AML. CRISPR/Cas9-mediated editing-out of RUNX1 enhancer (eR1) within its intragenic super-enhancer, or BET protein BRD4 depletion by short hairpin RNA, repressed RUNX1, inhibited cell growth, and induced cell lethality in AML cells expressing mtRUNX1. Moreover, treatment with BET protein inhibitor or degrader (BET-proteolysis targeting chimera) repressed RUNX1 and its targets, inducing apoptosis and improving survival of mice engrafted with AML expressing mtRUNX1. Library of Integrated Network-based Cellular Signatures 1000-connectivity mapping data sets queried with messenger RNA signature of RUNX1 knockdown identified novel expression-mimickers (EMs), which repressed RUNX1 and exerted in vitro and in vivo efficacy against AML cells expressing mtRUNX1. In addition, the EMs cinobufagin, anisomycin, and narciclasine induced more lethality in hematopoietic progenitor cells (HPCs) expressing germline mtRUNX1 from patients with AML compared with HPCs from patients with familial platelet disorder (FPD), or normal untransformed HPCs. These findings highlight novel therapeutic agents for AML expressing somatic or germline mtRUNX1.


Assuntos
Antineoplásicos/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Leucemia Mieloide Aguda/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Técnicas de Silenciamento de Genes , Mutação em Linhagem Germinativa , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Camundongos
11.
Blood Cancer J ; 9(2): 4, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30647404

RESUMO

First-generation bromodomain extra-terminal protein (BETP) inhibitors (BETi) (e.g., OTX015) that disrupt binding of BETP BRD4 to chromatin transcriptionally attenuate AML-relevant progrowth and prosurvival oncoproteins. BETi treatment induces apoptosis of AML BPCs, reduces in vivo AML burden and induces clinical remissions in a minority of AML patients. Clinical efficacy of more potent BETis, e.g., ABBV-075 (AbbVie, Inc.), is being evaluated. Venetoclax and A-1210477 bind and inhibit the antiapoptotic activity of BCL2 and MCL1, respectively, lowering the threshold for apoptosis. BETi treatment is shown here to perturb accessible chromatin and activity of enhancers/promoters, attenuating MYC, CDK6, MCL1 and BCL2, while inducing BIM, HEXIM1, CDKN1A expressions and apoptosis of AML cells. Treatment with venetoclax increased MCL1 protein levels, but cotreatment with ABBV-075 reduced MCL1 and Bcl-xL levels. ABBV-075 cotreatment synergistically induced apoptosis with venetoclax or A-1210477 in patient-derived, CD34+ AML cells. Compared to treatment with either agent alone, cotreatment with ABBV-075 and venetoclax was significantly more effective in reducing AML cell-burden and improving survival, without inducing toxicity, in AML-engrafted immune-depleted mice. These findings highlight the basis of superior activity and support interrogation of clinical efficacy and safety of cotreatment with BETi and BCL2 or MCL1 inhibitor in AML.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Sítios de Ligação , Biomarcadores Tumorais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Humanos , Indóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Ligação Proteica , Piridonas/farmacologia , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Leukemia ; 33(6): 1373-1386, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30575820

RESUMO

Transformation of post-myeloproliferative neoplasms into secondary (s) AML exhibit poor clinical outcome. In addition to increased JAK-STAT and PI3K-AKT signaling, post-MPN sAML blast progenitor cells (BPCs) demonstrate increased nuclear ß-catenin levels and TCF7L2 (TCF4) transcriptional activity. Knockdown of ß-catenin or treatment with BC2059 that disrupts binding of ß-catenin to TBL1X (TBL1) depleted nuclear ß-catenin levels. This induced apoptosis of not only JAKi-sensitive but also JAKi-persister/resistant post-MPN sAML BPCs, associated with attenuation of TCF4 transcriptional targets MYC, BCL-2, and Survivin. Co-targeting of ß-catenin and JAK1/2 inhibitor ruxolitinib (rux) synergistically induced lethality in post-MPN sAML BPCs and improved survival of mice engrafted with human sAML BPCs. Notably, co-treatment with BET protein degrader ARV-771 and BC2059 also synergistically induced apoptosis and improved survival of mice engrafted with JAKi-sensitive or JAKi-persister/resistant post-MPN sAML cells. These preclinical findings highlight potentially promising anti-post-MPN sAML activity of the combination of ß-catenin and BETP antagonists against post-MPN sAML BPCs.


Assuntos
Núcleo Celular/efeitos dos fármacos , Sinergismo Farmacológico , Leucemia Mieloide Aguda/tratamento farmacológico , Transtornos Mieloproliferativos/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , beta Catenina/antagonistas & inibidores , Acetanilidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transtornos Mieloproliferativos/complicações , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Nitrilas , Pirazóis/farmacologia , Pirimidinas , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética , beta Catenina/metabolismo
13.
Mol Cell ; 69(5): 729-743.e7, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29499131

RESUMO

MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid ß-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1Matrix) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1Matrix alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Metabolismo dos Lipídeos/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Ácido Palmítico/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/genética , Animais , Linhagem Celular , Camundongos , Camundongos Knockout , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Oxirredução , Estrutura Secundária de Proteína
14.
J Vet Sci ; 18(2): 149-158, 2017 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-27515268

RESUMO

Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer.


Assuntos
Doenças do Cão/metabolismo , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/metabolismo , Neoplasias Mamárias Animais/metabolismo , Receptores de Progesterona/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Cães , Feminino , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária
15.
Leukemia ; 31(3): 678-687, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27677740

RESUMO

Myeloproliferative neoplasms with myelofibrosis (MPN-MF) demonstrate constitutive activation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling that responds to treatment with the JAK1 and 2 kinase inhibitor (JAKi) ruxolitinib. However, MPN-MF often progresses (~20%) to secondary acute myeloid leukemia (sAML), where standard induction chemotherapy or ruxolitinib is relatively ineffective, necessitating the development of novel therapeutic approaches. In the present studies, we demonstrate that treatment with BET (bromodomain and extraterminal) protein inhibitor (BETi), for example, JQ1, inhibits growth and induces apoptosis of cultured and primary, patient-derived (PD), post-MPN sAML blast progenitor cells. Reverse-phase protein array, mass-cytometry and Western analyses revealed that BETi treatment attenuated the protein expressions of c-MYC, p-STAT5, Bcl-xL, CDK4/6, PIM1 and IL-7R, whereas it concomitantly induced the levels of HEXIM1, p21 and BIM in the sAML cells. Co-treatment with BETi and ruxolitinib synergistically induced apoptosis of cultured and PD sAML cells, as well as significantly improved survival of immune-depleted mice engrafted with human sAML cells. Although BETi or heat shock protein 90 inhibitor (HSP90i) alone exerted lethal activity, cotreatment with BETi and HSP90i was synergistically lethal against the ruxolitinib-persister or ruxolitinib-resistant sAML cells. Collectively, these findings further support in vivo testing of BETi-based combinations with JAKi and HSP90i against post-MPN sAML cells.


Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Transtornos Mieloproliferativos/complicações , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores , Caspases/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Genes myc , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Camundongos , Domínios e Motivos de Interação entre Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Receptores de Interleucina-7/metabolismo , Fator de Transcrição STAT5/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cancer Res ; 76(18): 5467-78, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27503926

RESUMO

SIRT2 is a protein deacetylase with tumor suppressor activity in breast and liver tumors where it is mutated; however, the critical substrates mediating its antitumor activity are not fully defined. Here we demonstrate that SIRT2 binds, deacetylates, and inhibits the peroxidase activity of the antioxidant protein peroxiredoxin (Prdx-1) in breast cancer cells. Ectopic overexpression of SIRT2, but not its catalytically dead mutant, increased intracellular levels of reactive oxygen species (ROS) induced by hydrogen peroxide, which led to increased levels of an overoxidized and multimeric form of Prdx-1 with activity as a molecular chaperone. Elevated levels of SIRT2 sensitized breast cancer cells to intracellular DNA damage and cell death induced by oxidative stress, as associated with increased levels of nuclear FOXO3A and the proapoptotic BIM protein. In addition, elevated levels of SIRT2 sensitized breast cancer cells to arsenic trioxide, an approved therapeutic agent, along with other intracellular ROS-inducing agents. Conversely, antisense RNA-mediated attenuation of SIRT2 reversed ROS-induced toxicity as demonstrated in a zebrafish embryo model system. Collectively, our findings suggest that the tumor suppressor activity of SIRT2 requires its ability to restrict the antioxidant activity of Prdx-1, thereby sensitizing breast cancer cells to ROS-induced DNA damage and cell cytotoxicity. Cancer Res; 76(18); 5467-78. ©2016 AACR.


Assuntos
Neoplasias da Mama/patologia , Peroxirredoxinas/metabolismo , Sirtuína 2/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ensaio Cometa , Eletroforese em Gel Bidimensional , Feminino , Humanos , Immunoblotting , Imunoprecipitação , Microscopia Confocal , Oxidantes/farmacologia , Estresse Oxidativo/fisiologia , Peroxidase/metabolismo , Peixe-Zebra
17.
Protein Expr Purif ; 125: 26-33, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-26363121

RESUMO

Overexpression of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu) results in ligand independent activation of kinase signaling and is found in about 30% of human breast cancers, and is correlated with a more aggressive tumor phenotype. The HER2 extracellular domain (ECD) consists of four domains - I, II, III and IV. Although the role of each domain in the dimerization and activation of the receptor has been extensively studied, the role of domain IV (DIV) is not clearly understood yet. In our previous studies, we reported peptidomimetic molecules inhibit HER2:HER3 heterodimerization. In order to study the binding interactions of peptidomimetics with HER2 DIV in detail, properly folded recombinant HER2 protein in pure form is important. We have expressed and purified HER2 ECD and DIV proteins in the Drosophila melanogaster Schneider2 (S2) cell line. Using the commercial Drosophila expression system (DES), we transfected S2 cells with plasmids designed to direct the expression of secreted recombinant HER2 ECD and DIV proteins. The secreted proteins were purified from the conditioned medium by filtration, ultrafiltration, dialysis and nickel affinity chromatography techniques. The purified HER2 proteins were then analyzed using Western blot, mass spectrometry and circular dichroism (CD) spectroscopy.


Assuntos
Receptor ErbB-2 , Animais , Linhagem Celular , Cromatografia de Afinidade , Drosophila melanogaster , Feminino , Humanos , Mapeamento de Peptídeos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
Genome Announc ; 2(3)2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24903876

RESUMO

The genome sequence of Acetobacter aceti 1023, an acetic acid bacterium adapted to traditional vinegar fermentation, comprises 3.0 Mb (chromosome plus plasmids). A. aceti 1023 is closely related to the cocoa fermenter Acetobacter pasteurianus 386B but possesses many additional insertion sequence elements.

19.
Oncol Res ; 20(7): 303-17, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23879171

RESUMO

Epidermal growth factor receptor (EGFR) expression has been linked to progression of basal breast cancers. Many breast cancer cells harbor the EGFR and produce its family of ligands, suggesting they may participate in autocrine and paracrine signaling with cells of the tumor microenvironment. EGFR ligand expression was profiled in the basal breast cancer cell line MDA-231 where AREG, TGF-alpha, and HBEGF were the three ligands most highly expressed. Autocrine signaling was modulated through silencing or overexpression of these three ligands using lentiviral constructs and the impact measured using motility, proliferation, and cytokine expression assays. Changes in receptor phosphorylation and receptor turnover were examined. Knockdown of AREG or TGF-alpha in vitro resulted in decreased motility (p < 0.05) and decreased expression of macrophage chemoattractants. Overexpression of TGF-alpha increased motility and chemoattractant expression, whereas AREG did not. HBEGF modulation had no effect on any cellular behaviors. All the cells with altered ligand production were inoculated into female athymic nude mice to form mammary fat pad tumors, followed by immunohistochemical analysis for necrosis, angiogenesis, and macrophage recruitment. In vivo, knockdown of AREG or TGF-alpha increased survival (p < 0.001) while decreasing angiogenesis (p < 0.001), tumor growth (p < 0.001), and macrophage attraction (p < 0.001). Overexpression of AREG appeared to elicit a greater effect than TGF-alpha on mammary fat pad tumor growth by increasing angiogenesis (p < 0.001) and macrophage attraction to the tumor (p < 0.01). We propose these changes in mammary tumor growth were the result of increased recruitment of macrophages to the tumor by cells with altered autocrine EGFR signaling. We conclude that AREG and TGF-alpha were somewhat interchangeable in their effects on EGFR signaling; however, TGF-alpha had a greater effect in vitro and AREG had a greater effect in vivo.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptores ErbB/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Macrófagos/imunologia , Fator de Crescimento Transformador alfa/metabolismo , Anfirregulina , Animais , Comunicação Autócrina/fisiologia , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Progressão da Doença , Família de Proteínas EGF , Feminino , Técnicas de Silenciamento de Genes , Humanos , Ligantes , Camundongos , Camundongos Nus , Neoplasia de Células Basais/imunologia , Neoplasia de Células Basais/metabolismo , Neoplasia de Células Basais/patologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
J Cancer Res Ther Oncol ; 1(1): 10, 2013 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-24791013

RESUMO

ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and -active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored. Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavage by gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.

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