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The efficient removal of replication and recombination intermediates is essential for the maintenance of genome stability. Resolution of these potentially toxic structures requires the MUS81-EME1 endonuclease, which is activated at prometaphase by formation of the SMX tri-nuclease containing three DNA repair structure-selective endonucleases: SLX1-SLX4, MUS81-EME1, and XPF-ERCC1. Here we show that SMX tri-nuclease is more active than the three individual nucleases, efficiently cleaving replication forks and recombination intermediates. Within SMX, SLX4 co-ordinates the SLX1 and MUS81-EME1 nucleases for Holliday junction resolution, in a reaction stimulated by XPF-ERCC1. SMX formation activates MUS81-EME1 for replication fork and flap structure cleavage by relaxing substrate specificity. Activation involves MUS81's conserved N-terminal HhH domain, which mediates incision site selection and SLX4 binding. Cell cycle-dependent formation and activation of this tri-nuclease complex provides a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.
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Reparación del ADN , Replicación del ADN , ADN/biosíntesis , Endonucleasas/metabolismo , Inestabilidad Genómica , Ciclo Celular , ADN/química , ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Endonucleasas/química , Endonucleasas/genética , Activación Enzimática , Humanos , Complejos Multienzimáticos , Conformación de Ácido Nucleico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Recombinasas/genética , Recombinasas/metabolismo , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Despite selective HDAC3 inhibition showing promise in a subset of lymphomas with CREBBP mutations, wild-type tumors generally exhibit resistance. Here, using unbiased genome-wide CRISPR screening, we identify GNAS knockout (KO) as a sensitizer of resistant lymphoma cells to HDAC3 inhibition. Mechanistically, GNAS KO-induced sensitization is independent of the canonical G-protein activities but unexpectedly mediated by viral mimicry-related interferon (IFN) responses, characterized by TBK1 and IRF3 activation, double-stranded RNA formation, and transposable element (TE) expression. GNAS KO additionally synergizes with HDAC3 inhibition to enhance CD8+ T cell-induced cytotoxicity. Moreover, we observe in human lymphoma patients that low GNAS expression is associated with high baseline TE expression and upregulated IFN signaling and shares common disrupted biological activities with GNAS KO in histone modification, mRNA processing, and transcriptional regulation. Collectively, our findings establish an unprecedented link between HDAC3 inhibition and viral mimicry in lymphoma. We suggest low GNAS expression as a potential biomarker that reflects viral mimicry priming for enhanced response to HDAC3 inhibition in the clinical treatment of lymphoma, especially the CREBBP wild-type cases.
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The manipulation of T cell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8+ T cells ex vivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8+ T cells into tumor-bearing mice led to robust tumor control and curative responses. Halo-treated T cells synergized in vivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy. Importantly, treatment of human CD8+ T cells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halo can enhance T cell metabolism and anti-tumor activity.
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Linfocitos T CD8-positivos , Neoplasias , Humanos , Animales , Ratones , Inmunoterapia Adoptiva/métodos , Neoplasias/patología , Inmunoterapia , AminoácidosRESUMEN
Exportin-1 (XPO1) is a key player in the nuclear export pathway and is overexpressed in almost all cancers. This is especially relevant for non-Hodgkin lymphoma (NHL), where high XPO1 expression is associated with poor prognosis due to its oncogenic role in exporting proteins and RNA that are involved in cancer progression and treatment resistance. Here, we discuss the proteins and RNA transcripts that have been identified as XPO1 cargo in NHL lymphoma including tumour suppressors, immune modulators, and transcription factors, and their implications for oncogenesis. We then highlight the research to date on XPO1 inhibitors such as selinexor and other selective inhibitors of nuclear export (SINEs), which are used to treat some cases of non-Hodgkin lymphoma. In vitro, in vivo, and clinical studies investigating the anti-cancer effects of SINEs from bench to bedside, both as a single agent and in combination, are also reported. Finally, we discuss the limitations of the current research landscape and future directions to better understand and improve the clinical utility of SINE compounds in NHL.
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Carioferinas , Linfoma no Hodgkin , Humanos , Transporte Activo de Núcleo Celular , Carioferinas/genética , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/patología , ARN/metabolismo , Línea Celular TumoralRESUMEN
Cutaneous melanoma (CM) patients respond better to immune checkpoint inhibitors (ICI) than mucosal and uveal melanoma patients (MM/UM). Aiming to explore these differences and understand the distinct response to ICI, we evaluated the serum metabolome of advanced CM, MM, and UM patients. Levels of 115 metabolites were analyzed in samples collected before ICI, using a targeted metabolomics platform. In our analysis, molecules involved in the tryptophan-kynurenine axis distinguished UM/MM from CM. UM/MM patients had higher levels of 3-hydroxykynurenine (3-HKyn), whilst patients with CM were found to have higher levels of kynurenic acid (KA). The KA/3-HKyn ratio was significantly higher in CM versus the other subtypes. UM, the most ICI-resistant subtype, was also associated with higher levels of sphingomyelin-d18:1/22:1 and the polyamine spermine (SPM). Overall survival was prolonged in a cohort of CM patients with lower SPM levels, suggesting there are also conserved metabolic factors promoting ICI resistance across melanoma subtypes. Our study revealed a distinct metabolomic profile between the most resistant melanoma subtypes, UM and MM, compared to CM. Alterations within the kynurenine pathway, polyamine metabolism, and sphingolipid metabolic pathway may contribute to the poor response to ICI. Understanding the different metabolomic profiles introduces opportunities for novel therapies with potential synergic activity to ICI, to improve responses of UM/MM.
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Tumour-associated macrophages are linked with poor prognosis and resistance to therapy in Hodgkin lymphoma; however, there are no suitable preclinical models to identify macrophage-targeting therapeutics. We used primary human tumours to guide the development of a mimetic cryogel, wherein Hodgkin (but not Non-Hodgkin) lymphoma cells promoted primary human macrophage invasion. In an invasion inhibitor screen, we identified five drug hits that significantly reduced tumour-associated macrophage invasion: marimastat, batimastat, AS1517499, ruxolitinib, and PD-169316. Importantly, ruxolitinib has demonstrated recent success in Hodgkin lymphoma clinical trials. Both ruxolitinib and PD-169316 (a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor) decreased the percent of M2-like macrophages; however, only PD-169316 enhanced the percentage of M1-like macrophages. We validated p38 MAPK as an anti-invasion drug target with five additional drugs using a high-content imaging platform. With our biomimetic cryogel, we modeled macrophage invasion in Hodgkin lymphoma and then used it for target discovery and drug screening, ultimately identifying potential future therapeutics.
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Enfermedad de Hodgkin , Macrófagos Asociados a Tumores , Humanos , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/patología , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/patología , Criogeles , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Matriz Extracelular/metabolismoRESUMEN
CCM3 mutations give rise to cerebral cavernous malformations (CCMs) of the vasculature through a mechanism that remains unclear. Interaction of CCM3 with the germinal center kinase III (GCKIII) subfamily of Sterile 20 protein kinases, MST4, STK24, and STK25, has been implicated in cardiovascular development in the zebrafish, raising the possibility that dysregulated GCKIII function may contribute to the etiology of CCM disease. Here, we show that the amino-terminal region of CCM3 is necessary and sufficient to bind directly to the C-terminal tail region of GCKIII proteins. This same region of CCM3 was shown previously to mediate homodimerization through the formation of an interdigitated α-helical domain. Sequence conservation and binding studies suggest that CCM3 may preferentially heterodimerize with GCKIII proteins through a manner structurally analogous to that employed for CCM3 homodimerization.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de la Membrana/metabolismo , Multimerización de Proteína/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Sistema Cardiovascular/embriología , Quinasas del Centro Germinal , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Organogénesis/fisiología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Pez Cebra/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
INTRODUCTION: Pembrolizumab is an immune checkpoint inhibitor (ICI) targeted against the programmed death 1 (PD-1) pathway, a key pathway in the biology of Classical Hodgkin lymphoma (cHL). Anti-PD-1 antibodies are approved for use in relapsed/refractory cHL but ongoing studies continue to optimize the use of this treatment. AREAS COVERED: This review highlights recent and established data regarding pembrolizumab in the management of relapsed/refractory cHL and emerging areas of study including translational biology, combinations with chemotherapy and trials earlier in the disease course. EXPERT OPINION: Pembrolizumab provides superior progression-free survival for patients with cHL who relapse post-autologous stem cell transplant or who have chemotherapy refractory disease and should be used in these high-risk populations. A key challenge remains the development of predictive biomarkers for anti-PD1 antibodies. There is promising evidence of the improved efficacy of salvage chemotherapy regimens and frontline regimens incorporating pembrolizumab but larger randomized studies are needed to demonstrate clear patient benefit.
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Enfermedad de Hodgkin , Anticuerpos Monoclonales Humanizados/uso terapéutico , Humanos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Trasplante AutólogoRESUMEN
This scoping review was designed to synthesize the extant literature on associations between subjective and/or objective measures of cancer-related cognitive impairment (CRCI) and blood-based biomarkers in adults with non-central nervous system cancers. The literature search was done for studies published from the start of each database searched (i.e., MEDLINE, Embase, PsycINFO, Cumulative Index to Nursing and Allied Health Literature, Cochrane Central Register of Controlled Trials, grey literature) through to October 20, 2021. A total of 95 studies are included in this review. Of note, a wide variety of biomarkers were evaluated. Most studies evaluated patients with breast cancer. A variety of cognitive assessment measures were used. The most consistent significant findings were with various subjective and objective measures of CRCI and levels of interleukin-6 and tumor necrosis factor. Overall, biomarker research is in an exploratory phase. However, this review synthesizes findings and proposes directions for future research.
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Neoplasias de la Mama , Disfunción Cognitiva , Adulto , Humanos , Femenino , Disfunción Cognitiva/diagnóstico , Disfunción Cognitiva/etiología , Biomarcadores , Sistema NerviosoRESUMEN
DNA cruciforms play an important role in the regulation of natural processes involving DNA. These structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling. Cruciform structures are fundamentally important for a wide range of biological processes, including replication, regulation of gene expression, nucleosome structure and recombination. They also have been implicated in the evolution and development of diseases including cancer, Werner's syndrome and others.Cruciform structures are targets for many architectural and regulatory proteins, such as histones H1 and H5, topoisomerase IIß, HMG proteins, HU, p53, the proto-oncogene protein DEK and others. A number of DNA-binding proteins, such as the HMGB-box family members, Rad54, BRCA1 protein, as well as PARP-1 polymerase, possess weak sequence specific DNA binding yet bind preferentially to cruciform structures. Some of these proteins are, in fact, capable of inducing the formation of cruciform structures upon DNA binding. In this article, we review the protein families that are involved in interacting with and regulating cruciform structures, including (a) the junction-resolving enzymes, (b) DNA repair proteins and transcription factors, (c) proteins involved in replication and (d) chromatin-associated proteins. The prevalence of cruciform structures and their roles in protein interactions, epigenetic regulation and the maintenance of cell homeostasis are also discussed.
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Replicación del ADN , ADN/ultraestructura , Regulación de la Expresión Génica , Conformación de Ácido Nucleico , Animales , Secuencia de Bases , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Metabolic programming is intricately linked to the anti-tumor properties of T cells. To study the metabolic pathways associated with increased anti-tumor T cell function, we utilized a metabolomics approach to characterize three different CD8+ T cell subsets with varying degrees of anti-tumor activity in murine models, of which IL-22-producing Tc22 cells displayed the most robust anti-tumor activity. Tc22s demonstrated upregulation of the pantothenate/coenzyme A (CoA) pathway and a requirement for oxidative phosphorylation (OXPHOS) for differentiation. Exogenous administration of CoA reprogrammed T cells to increase OXPHOS and adopt the CD8+ Tc22 phenotype independent of polarizing conditions via the transcription factors HIF-1α and the aryl hydrocarbon receptor (AhR). In murine tumor models, treatment of mice with the CoA precursor pantothenate enhanced the efficacy of anti-PDL1 antibody therapy. In patients with melanoma, pre-treatment plasma pantothenic acid levels were positively correlated with the response to anti-PD1 therapy. Collectively, our data demonstrate that pantothenate and its metabolite CoA drive T cell polarization, bioenergetics, and anti-tumor immunity.
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Coenzima A , Subgrupos de Linfocitos T , Animales , Linfocitos T CD8-positivos , Diferenciación Celular , Coenzima A/metabolismo , Humanos , Activación de Linfocitos , Ratones , Subgrupos de Linfocitos T/metabolismoRESUMEN
Cancer cells display metabolic plasticity to survive stresses in the tumor microenvironment. Cellular adaptation to energetic stress is coordinated in part by signaling through the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Here, we demonstrate that miRNA-mediated silencing of LKB1 confers sensitivity of lymphoma cells to mitochondrial inhibition by biguanides. Using both classic (phenformin) and newly developed (IM156) biguanides, we demonstrate that elevated miR-17â¼92 expression in Myc+ lymphoma cells promotes increased apoptosis to biguanide treatment in vitro and in vivo. This effect is driven by the miR-17-dependent silencing of LKB1, which reduces AMPK activation in response to complex I inhibition. Mechanistically, biguanide treatment induces metabolic stress in Myc+ lymphoma cells by inhibiting TCA cycle metabolism and mitochondrial respiration, exposing metabolic vulnerability. Finally, we demonstrate a direct correlation between miR-17â¼92 expression and biguanide sensitivity in human cancer cells. Our results identify miR-17â¼92 expression as a potential biomarker for biguanide sensitivity in malignancies.
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Quinasas de la Proteína-Quinasa Activada por el AMP/genética , Biguanidas/uso terapéutico , Linfoma/tratamiento farmacológico , ARN Largo no Codificante/fisiología , Quinasas de la Proteína-Quinasa Activada por el AMP/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Células HEK293 , Humanos , Linfoma/genética , Linfoma/patología , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-myc/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: Cancer-related cognitive impairment (CRCI) can have detrimental effects on quality of life, even among patients with non-central nervous system (CNS) cancers. Several studies have been conducted to explore different markers associated with CRCI to understand its pathobiology. It is proposed that the underlying mechanisms of CRCI are related to a cascade of physiological adaptive events in response to cancer and/or treatment. Hence, peripheral blood would be a logical source to observe and identify these physiological events. This paper outlines the protocol for a scoping review being conducted to summarise the extant literature regarding blood-based biomarkers of CRCI among patients with non-CNS cancer. METHODS/ANALYSIS: Methods will be informed by the updated guidelines of Arksey and O'Malley. The systematic search for literature will include electronic databases, handsearching of key journals and reference lists, forward citation tracking and consultation with content experts. Study selection will be confirmed by duplicate review and calculation of inter-rater reliability. Data to be charted will include study design, sample size, cancer and treatment characteristics, demographic characteristics, cognitive variable/s and biomarkers assessed, associations between cognitive functioning and biomarkers (including statistics used), and rigour in biomarker sample collection and processing. Results will be presented through: (1) a descriptive numerical summary of studies, including a flow diagram based on the Preferred Reporting Items for Systematic Reviews and Meta-analyses statement, (2) a list of blood-based biomarkers associated with CRCI and (3) a narrative overview developed through collaboration among the research team and consultation with content experts. DISSEMINATION: The findings of this review will highlight current directions and gaps in the current body of evidence that may lead to improved rigour in future CRCI investigations. The dissemination of this work will be facilitated through the involvement of clinicians and researchers on the research team, an external consultation process and the presentation of the results through scholarly publication and presentation.
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Cognición , Disfunción Cognitiva/etiología , Neoplasias/complicaciones , Biomarcadores/sangre , Disfunción Cognitiva/sangre , Humanos , Neoplasias/sangre , Neoplasias/fisiopatología , Sistema Nervioso , Proyectos de InvestigaciónRESUMEN
Elucidation of activation mechanisms governing protein fusions is essential for therapeutic development. MLL undergoes rearrangement with numerous partners, including a recurrent translocation fusing the epigenetic regulator to a cytoplasmic RAS effector, AF6/afadin. We show here that AF6 employs a non-canonical, evolutionarily conserved α-helix to bind RAS, unique to AF6 and the classical RASSF effectors. Further, all patients with MLL-AF6 translocations express fusion proteins missing only this helix from AF6, resulting in exposure of hydrophobic residues that induce dimerization. We provide evidence that oligomerization is the dominant mechanism driving oncogenesis from rare MLL translocation partners and employ our mechanistic understanding of MLL-AF6 to examine how dimers induce leukemia. Proteomic data resolve association of dimerized MLL with gene expression modulators, and inhibiting dimerization disrupts formation of these complexes while completely abrogating leukemogenesis in mice. Oncogenic gene translocations are thus selected under pressure from protein structure/function, underscoring the complex nature of chromosomal rearrangements.
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Evolución Molecular , Cinesinas/metabolismo , Leucemia/genética , Miosinas/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Secuencia de Aminoácidos , Dimerización , Humanos , Cinesinas/química , Cinesinas/genética , Leucemia/enzimología , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Miosinas/química , Miosinas/genética , Proteína Oncogénica p21(ras)/química , Proteína Oncogénica p21(ras)/genética , Unión Proteica , Dominios Proteicos , Translocación GenéticaRESUMEN
AT7519M is a small molecule inhibitor of cyclin-dependent kinases 1, 2, 4, 5, and 9 with in vitro activity against lymphoid malignancies. In two concurrent Phase II trials, we evaluated AT7519M in relapsed or refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) using the recommended Phase II dosing of 27 mg/m2 twice weekly for 2 of every 3 weeks. Primary objective was objective response rate (ORR). Nineteen patients were accrued (7 CLL, 12 MCL). Four CLL patients achieved stable disease (SD). Two MCL patients achieved partial response (PR), and 6 had SD. One additional MCL patient with SD subsequently achieved PR 9 months after completion of AT7519M. Tumor lysis syndrome was not reported. In conclusion, AT7519M was safely administered to patients with relapsed/refractory CLL and MCL. In CLL, some patients had tumor reductions, but the ORR was low. In MCL, activity was noted with ORR of 27%.
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Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores , Canadá , Aberraciones Cromosómicas , Terapia Combinada , Quinasas Ciclina-Dependientes/genética , Resistencia a Antineoplásicos , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/mortalidad , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/mortalidad , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/farmacocinética , Recurrencia , RetratamientoRESUMEN
The N terminus of the c-Myc oncoprotein interacts with Bin1, a ubiquitously expressed nucleocytoplasmic protein with features of a tumor suppressor. The c-Myc/Bin1 interaction is dependent on the highly conserved Myc Box 1 (MB1) sequence of c-Myc. The c-Myc/Bin1 interaction has potential regulatory significance as c-Myc-mediated transformation and apoptosis can be modulated by the expression of Bin1. Multiple splicing of the Bin1 transcript results in ubiquitous, tissue-specific and tumor-specific populations of Bin1 proteins in vivo. We report on the structural features of the interaction between c-Myc and Bin1, and describe two mechanisms by which the binding of different Bin1 isoforms to c-Myc may be regulated in cells. Our findings identify a consensus class II SH3-binding motif in c-Myc and the C-terminal SH3 domain of Bin1 as the primary structure determinants of their interaction. We present biochemical and structural evidence that tumor-specific isoforms of Bin1 are precluded from interaction with c-Myc through an intramolecular polyproline-SH3 domain interaction that inhibits the Bin1 SH3 domain from binding to c-Myc. Furthermore, c-Myc/Bin1 interaction can be inhibited by phosphorylation of c-Myc at Ser62, a functionally important residue found within the c-Myc SH3-binding motif. Our data provide a structure-based model of the c-Myc/Bin1 interaction and suggest a mode of regulation that may be important for c-Myc function as a regulator of gene transcription.
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Empalme Alternativo , Proteínas Portadoras/metabolismo , Modelos Moleculares , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilación , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Dominios Homologos srcRESUMEN
We herein report the design and synthesis of the first nanomolar binding inhibitor of STAT5 protein. Lead compound 13a, possessing a phosphotyrosyl-mimicking salicylic acid group, potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain complexation events in vitro, silences activated STAT5 in leukemic cells, as well as STAT5's downstream transcriptional targets, including MYC and MCL1, and, as a result, leads to apoptosis. We believe 13a represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials.
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Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG) was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1ß transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress.
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Resistencia a Antineoplásicos , Complejo IV de Transporte de Electrones/biosíntesis , Leucemia Mieloide Aguda/metabolismo , Proteínas Mitocondriales/biosíntesis , Proteínas de Neoplasias/biosíntesis , Biosíntesis de Proteínas , Antibacterianos/farmacología , Línea Celular Tumoral , Complejo IV de Transporte de Electrones/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/genética , Glucólisis/efectos de los fármacos , Glucólisis/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Minociclina/análogos & derivados , Minociclina/farmacología , Proteínas Mitocondriales/genética , Proteínas de Neoplasias/genética , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/genética , TigeciclinaRESUMEN
Among the diseases of protein misfolding, amyotrophic lateral sclerosis (ALS) is unusual in that the proteinaceous neuronal inclusions that are the hallmark of the disease have neither the classic fibrillar appearance of amyloid by transmission electron microscopy nor the affinity for the dye Congo red that is a defining feature of amyloid. Mutations in the Cu, Zn superoxide dismutase (SOD1) cause the largest subset of inherited ALS cases. The mechanism by which this highly stable enzyme misfolds to form non-amyloid aggregates is currently poorly understood, as are the stresses that initiate misfolding. The oxidative damage hypothesis proposes that SOD1's normal free radical scavenger role puts it at risk of oxidative damage and that it is this damage that triggers the misfolding primed by mutation. Here, we present evidence that hydrogen peroxide treatment, which generates free radical species at the SOD1 active site, causes oxidative damage to active-site histidine residues, leading to major structural changes and non-amyloid aggregation similar to that seen in ALS. Time-resolved measurements of release of bound metal ligands, exposure of hydrophobic surface area, and alterations in the SOD1 proton NMR spectrum have allowed us to model the early structural changes occurring as SOD1 misfolds, prior to aggregation. ALS-causing SOD1 mutations apparently alter this pathway by increasing exposure of buried epitopes in misfolded species populated at endpoint. We have identified a well-populated early misfolding intermediate that could serve as a target for therapies designed to block downstream misfolding and aggregation events and thereby treat SOD1-associated ALS.
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Pliegue de Proteína , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Humanos , Peróxido de Hidrógeno/metabolismo , Modelos Químicos , Modelos Moleculares , Oxidación-Reducción , Desnaturalización Proteica , Multimerización de Proteína , Superóxido Dismutasa-1RESUMEN
A growing body of evidence shows that Signal Transducer and Activator of Transcription 5 (STAT5) protein, a key member of the STAT family of signaling proteins, plays a pivotal role in the progression of many human cancers, including acute myeloid leukemia and prostate cancer. Unlike STAT3, where significant medicinal effort has been expended to identify potent direct inhibitors, Stat5 has been poorly investigated as a molecular therapeutic target. Thus, in an effort to identify direct inhibitors of STAT5 protein, we conducted an in vitro screen of a focused library of SH2 domain binding salicylic acid-containing inhibitors (â¼150) against STAT5, as well as against STAT3 and STAT1 proteins for SH2 domain selectivity. We herein report the identification of several potent (K(i) < 5 µM) and STAT5 selective (>3-fold specificity for STAT5 cf. STAT1 and STAT3) inhibitors, BP-1-107, BP-1-108, SF-1-087, and SF-1-088. Lead agents, evaluated in K562 and MV-4-11 human leukemia cells, showed potent induction of apoptosis (IC(50)'s â¼ 20 µM) which correlated with potent and selective suppression of STAT5 phosphorylation, as well as inhibition of STAT5 target genes cyclin D1, cyclin D2, C-MYC, and MCL-1. Moreover, lead agent BP-1-108 showed negligible cytotoxic effects in normal bone marrow cells not expressing activated STAT5 protein. Inhibitors identified in this study represent some of the most potent direct small molecule, nonphosphorylated inhibitors of STAT5 to date.