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1.
Theranostics ; 11(16): 7844-7868, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335968

RESUMO

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.


Assuntos
Glutamina/metabolismo , Neoplasias da Próstata/metabolismo , Tolerância a Radiação/genética , Animais , Autofagia , Proteína 5 Relacionada à Autofagia/metabolismo , Biomarcadores Farmacológicos , Linhagem Celular Tumoral , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Glutaminase/metabolismo , Humanos , Masculino , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Science ; 373(6556): 760-767, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385390

RESUMO

The origin of human metaplastic states and their propensity for cancer is poorly understood. Barrett's esophagus is a common metaplastic condition that increases the risk for esophageal adenocarcinoma, and its cellular origin is enigmatic. To address this, we harvested tissues spanning the gastroesophageal junction from healthy and diseased donors, including isolation of esophageal submucosal glands. A combination of single-cell transcriptomic profiling, in silico lineage tracing from methylation, open chromatin and somatic mutation analyses, and functional studies in organoid models showed that Barrett's esophagus originates from gastric cardia through c-MYC and HNF4A-driven transcriptional programs. Furthermore, our data indicate that esophageal adenocarcinoma likely arises from undifferentiated Barrett's esophagus cell types even in the absence of a pathologically identifiable metaplastic precursor, illuminating early detection strategies.


Assuntos
Adenocarcinoma/patologia , Esôfago de Barrett/patologia , Cárdia/citologia , Neoplasias Esofágicas/patologia , Esôfago/patologia , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Cárdia/química , Diferenciação Celular , Linhagem da Célula , Transformação Celular Neoplásica , Epigênese Genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Esôfago/citologia , Esôfago/metabolismo , Glândulas Exócrinas/química , Glândulas Exócrinas/citologia , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Queratina-7/análise , Metaplasia , Fenótipo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA-Seq , Análise de Célula Única , Transcrição Genética , Transcriptoma
3.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299194

RESUMO

A heterogeneous genetic subtype of B-cell precursor acute lymphoblastic leukemia is driven by constitutive kinase-activation, including patients with JAK2 fusions. In our study, we model the impact of a novel JAK2 fusion protein on hematopoietic development in human induced pluripotent stem cells (hiPSCs). We insert the RUNX1-JAK2 fusion into one endogenous RUNX1 allele through employing in trans paired nicking genome editing. Tagging of the fusion with a degron facilitates protein depletion using the heterobifunctional compound dTAG-13. Throughout in vitro hematopoietic differentiation, the expression of RUNX1-JAK2 is driven by endogenous RUNX1 regulatory elements at physiological levels. Functional analysis reveals that RUNX1-JAK2 knock-in cell lines yield fewer hematopoietic progenitors, due to RUNX1 haploinsufficiency. Nevertheless, these progenitors further differentiate toward myeloid lineages to a similar extent as wild-type cells. The expression of the RUNX1-JAK2 fusion protein only elicits subtle effects on myeloid differentiation, and is unable to transform early hematopoietic progenitors. However, phosphoprotein and transcriptome analyses reveal that RUNX1-JAK2 constitutively activates JAK-STAT signaling in differentiating hiPSCs and at the same time upregulates MYC targets-confirming the interaction between these pathways. This proof-of-principle study indicates that conditional expression of oncogenic fusion proteins in combination with hematopoietic differentiation of hiPSCs may be applicable to leukemia-relevant disease modeling.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Janus Quinase 2/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição STAT/metabolismo , Diferenciação Celular , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Perfilação da Expressão Gênica/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Janus Quinase 2/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transdução de Sinais
4.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198626

RESUMO

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a') tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.


Assuntos
Fator de Células-Tronco/biossíntese , Sequência de Aminoácidos , Ciclo Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Fator de Células-Tronco/química
5.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201047

RESUMO

MYC is a transcription factor that controls the expression of a large fraction of cellular genes linked to cell cycle progression, metabolism and differentiation. MYC deregulation in tumors leads to its pervasive genome-wide binding of both promoters and distal regulatory regions, associated with selective transcriptional control of a large fraction of cellular genes. This pairs with alterations of cell cycle control which drive anticipated S-phase entry and reshape the DNA-replication landscape. Under these circumstances, the fine tuning of DNA replication and transcription becomes critical and may pose an intrinsic liability in MYC-overexpressing cancer cells. Here, we will review the current understanding of how MYC controls DNA and RNA synthesis, discuss evidence of replicative and transcriptional stress induced by MYC and summarize preclinical data supporting the therapeutic potential of triggering replicative stress in MYC-driven tumors.


Assuntos
Replicação do DNA , Regulação da Expressão Gênica , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Dano ao DNA , Humanos , Neoplasias/genética , Neoplasias/metabolismo
6.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201061

RESUMO

BRAFV600E mutations are found in approximately 10% of colorectal cancer patients and are associated with worse prognosis and poor outcomes with systemic therapies. The aim of this study was to identify novel druggable features of BRAFV600E-mutated colon cancer (CC) cells associated with the response and resistance to BRAFV600E inhibitor vemurafenib. Towards this aim, we carried out global proteomic profiling of BRAFV600E mutant vs. KRAS mutant/BRAF wild-type and double wild-type KRAS/BRAF CC cells followed by bioinformatics analyses. Validation of selected proteomic features was performed by immunohistochemistry and in silico using the TCGA database. We reveal an increased abundance and activity of nucleophosmin (NPM1) in BRAFV600E-mutated CC in vitro, in silico and in tumor tissues from colon adenocarcinoma patients and demonstrate the roles of NPM1 and its interaction partner c-Myc in conveying the resistance to vemurafenib. Pharmacological inhibition of NPM1 effectively restored the sensitivity of vemurafenib-resistant BRAF-mutated CC cells by down-regulating c-Myc expression and activity and consequently suppressing its transcriptional targets RanBP1 and phosphoserine phosphatase that regulate centrosome duplication and serine biosynthesis, respectively. Altogether, findings from this study suggest that the NPM1/c-Myc axis could represent a promising therapeutic target to thwart resistance to vemurafenib in BRAF-mutated CC.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mutação , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Vemurafenib/farmacologia , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Proteoma/análise , Células Tumorais Cultivadas
7.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070449

RESUMO

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that are responsible for immunosuppression in tumor microenvironment. Here we report the impact of mucin 1 (MUC1), a transmembrane glycoprotein, on proliferation and functional activity of MDSCs. To determine the role of MUC1 in MDSC phenotype, we analyzed MDSCs derived from wild type (WT) and MUC1-knockout (MUC1KO) mice bearing syngeneic pancreatic (KCKO) or breast (C57MG) tumors. We observed enhanced tumor growth of pancreatic and breast tumors in the MUC1KO mice compared to the WT mice. Enhanced tumor growth in the MUC1KO mice was associated with increased numbers of suppressive MDSCs and T regulatory (Tregs) cells in the tumor microenvironment. Compared to the WT host, MUC1KO host showed higher levels of iNOS, ARG1, and TGF-ß, thus promoting proliferation of MDSCs with an immature and immune suppressive phenotype. When co-cultured with effector T cells, MDSCs from MUC1KO mice led to higher repression of IL-2 and IFN-γ production by T cells as compared to MDSCs from WT mice. Lastly, MDSCs from MUC1KO mice showed higher levels of c-Myc and activated pSTAT3 as compared to MDSCs from WT mice, suggesting increased survival, proliferation, and prevention of maturation of MDSCs in the MUC1KO host. We report diminished T cell function in the KO versus WT mice. In summary, the data suggest that MUC1 may regulate signaling pathways that are critical to maintain the immunosuppressive properties of MDSCs.


Assuntos
Neoplasias da Mama/metabolismo , Mucina-1/metabolismo , Células Supressoras Mieloides/imunologia , Neoplasias Pancreáticas/metabolismo , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Interferon gama/metabolismo , Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-1/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Baço/citologia , Baço/metabolismo , Fator de Crescimento Transformador beta/sangue , Microambiente Tumoral/genética
8.
J Biol Chem ; 297(1): 100903, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34157284

RESUMO

c-Myc is a transcription factor that plays a crucial role in cellular homeostasis, and its deregulation is associated with highly aggressive and chemotherapy-resistant cancers. After binding with partner MAX, the c-Myc-MAX heterodimer regulates the expression of several genes, leading to an oncogenic phenotype. Although considered a crucial therapeutic target, no clinically approved c-Myc-targeted therapy has yet been discovered. Here, we report the discovery via computer-aided drug discovery of a small molecule, L755507, which functions as a c-Myc inhibitor to efficiently restrict the growth of diverse Myc-expressing cells with low micromolar IC50 values. L755507 successfully disrupts the c-Myc-MAX heterodimer, resulting in decreased expression of c-Myc target genes. Spectroscopic and computational experiments demonstrated that L755507 binds to the c-Myc peptide and thereby stabilizes the helix-loop-helix conformation of the c-Myc transcription factor. Taken together, this study suggests that L755507 effectively inhibits the c-Myc-MAX heterodimerization and may be used for further optimization to develop a c-Myc-targeted antineoplastic drug.


Assuntos
Antineoplásicos/química , Apoptose/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/química , Antineoplásicos/farmacologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação , Descoberta de Drogas , Células HT29 , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
9.
Nucleic Acids Res ; 49(13): 7361-7374, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181729

RESUMO

N6-methyladenosine (m6A) is a common modification on endogenous RNA transcripts in mammalian cells. Technologies to precisely modify the RNA m6A levels at specific transcriptomic loci empower interrogation of biological functions of epitranscriptomic modifications. Here, we developed a bidirectional dCasRx epitranscriptome editing platform composed of a nuclear-localized dCasRx conjugated with either a methyltransferase, METTL3, or a demethylase, ALKBH5, to manipulate methylation events at targeted m6A sites. Leveraging this platform, we specifically and efficiently edited m6A modifications at targeted sites, reflected in gene expression and cell proliferation. We employed the dCasRx epitranscriptomic editor system to elucidate the molecular function of m6A-binding proteins YTHDF paralogs (YTHDF1, YTHDF2 and YTHDF3), revealing that YTHDFs promote m6A-mediated mRNA degradation. Collectively, our dCasRx epitranscriptome perturbation platform permits site-specific m6A editing for delineating of functional roles of individual m6A modifications in the mammalian epitranscriptome.


Assuntos
Adenosina/análogos & derivados , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Metiltransferases/metabolismo , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Proteínas Associadas a CRISPR/genética , Proliferação de Células , Células Cultivadas , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Metiltransferases/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcriptoma
10.
J Med Chem ; 64(13): 8992-9009, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34132534

RESUMO

Glycine-N-methyl transferase (GNMT) downregulation results in spontaneous hepatocellular carcinoma (HCC). Overexpression of GNMT inhibits the proliferation of liver cancer cell lines and prevents carcinogen-induced HCC, suggesting that GNMT induction is a potential approach for anti-HCC therapy. Herein, we used Huh7 GNMT promoter-driven screening to identify a GNMT inducer. Compound K78 was identified and validated for its induction of GNMT and inhibition of Huh7 cell growth. Subsequently, we employed structure-activity relationship analysis and found a potent GNMT inducer, K117. K117 inhibited Huh7 cell growth in vitro and xenograft in vivo. Oral administration of a dosage of K117 at 10 mpk (milligrams per kilogram) can inhibit Huh7 xenograft in a manner equivalent to the effect of sorafenib at a dosage of 25 mpk. A mechanistic study revealed that K117 is an MYC inhibitor. Ectopic expression of MYC using CMV promoter blocked K117-mediated MYC inhibition and GNMT induction. Overall, K117 is a potential lead compound for HCC- and MYC-dependent cancers.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Glicina N-Metiltransferase/genética , Ensaios de Triagem em Larga Escala , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Glicina N-Metiltransferase/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
11.
J Med Chem ; 64(11): 7312-7330, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34009981

RESUMO

The A-type Aurora kinase is upregulated in many human cancers, and it stabilizes MYC-family oncoproteins, which have long been considered an undruggable target. Here, we describe the design and synthesis of a series of pyrimidine-based derivatives able to inhibit Aurora A kinase activity and reduce levels of cMYC and MYCN. Through structure-based drug design of a small molecule that induces the DFG-out conformation of Aurora A kinase, lead compound 13 was identified, which potently (IC50 < 200 nM) inhibited the proliferation of high-MYC expressing small-cell lung cancer (SCLC) cell lines. Pharmacokinetic optimization of 13 by prodrug strategies resulted in orally bioavailable 25, which demonstrated an 8-fold higher oral AUC (F = 62.3%). Pharmacodynamic studies of 25 showed it to effectively reduce cMYC protein levels, leading to >80% tumor regression of NCI-H446 SCLC xenograft tumors in mice. These results support the potential of 25 for the treatment of MYC-amplified cancers including SCLC.


Assuntos
Aurora Quinase A/antagonistas & inibidores , Desenho de Fármacos , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirimidinas/química , Animais , Aurora Quinase A/metabolismo , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/metabolismo , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Nat Chem Biol ; 17(9): 954-963, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33972797

RESUMO

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
13.
Methods Mol Biol ; 2318: 13-19, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019284

RESUMO

The b-HLH-LZ domain of c-Myc is a key target for the development of cancer therapies by blunting its binding to DNA with cell penetrant b-HLH-LZs and/or by stabilizing it into a state that cannot recognize Max to activate and amplify transcription of oncogenic genes. Although recent milestones have been reached with DNA binding blunting of c-Myc with the cell penetrant b-HLH-LZ Omomyc, the targeting of its b-HLH-LZ with small molecules, peptides, or proteins is lagging. As reviewed recently, the main problem relies in the intrinsically disordered nature of the b-HLH-LZ of c-Myc. This greatly complicates the classical approach of targeting a docking site with inhibitors. The solution state methods such as NMR are progressing towards the characterization of the ensembles of structures or states the b-HLH-LZ can adopt. However, the delicate balance that dictates the population of these dynamically interchanging states relies on its primary structure and the weak polar, electrostatic and hydrophobic interactions allowed. In this context, it is of the utmost importance to study the b-HLH-LZ of c-Myc in its WT background and avoid the use of tags such as His-tags. These tags could disrupt the balance of forces which could alter the conformational and physical transitions and states it can undergo and adopt. Here, we describe a robust protocol to express the WT b-HLH-LZ in E. coli and purify it, without the need of tags, to obtain the required quantities for solution state biophysical characterization such as NMR.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/isolamento & purificação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/isolamento & purificação , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , DNA/química , DNA/genética , Dimerização , Escherichia coli/genética , Expressão Gênica , Genes myc , Sequências Hélice-Alça-Hélice , Humanos , Zíper de Leucina/genética , Zíper de Leucina/fisiologia , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo
14.
Methods Mol Biol ; 2318: 1-11, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019283

RESUMO

The MYC transcription factor coordinates a wide range of intra- and extracellular processes associated with tissue proliferation and regeneration. While these processes are typically tightly regulated in physiological conditions, they become deregulated in cancer, where MYC is oncogenically activated.The last decade has seen MYC progress from a renowned undruggable target to a hot topic in the cancer therapy field, as proof emerged from mouse models that its inhibition constitutes an effective and broadly applicable approach to fight cancer. However, there are several aspects of MYC biology that still appear to be elusive and maintain the interest in further studying this intriguing protein. Since MYC's discovery, more than four decades ago, multiple strategies have been developed to study it, related to the many and varied facets of its biology. This new version of The Myc gene: Methods and Protocols provides valuable tips from key "inhabitants of the MYC world," which significantly increase the reach of our investigative tools to shed light on the mysteries still surrounding MYC.


Assuntos
Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Genes myc , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo
15.
Methods Mol Biol ; 2318: 21-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019285

RESUMO

The C-terminal region of the c-MYC transcription factor consists of approximately 100 amino acids that in its native state does not adopt a stable structure. When this region binds to the obligatory partner MAX via a coupled folding-and-binding mechanism, it forms a basic-helix-loop-helix-leucine zipper (bHLHZip) heterodimeric complex. The C-terminal region of MYC is the target for numerous drug discovery programs for direct MYC inhibition via blocking the dimerization event and/or binding to DNA, and a proper understanding of the partially folded, dynamic nature of the heterodimeric complex is essential to these efforts. The bHLHZip motif also drives protein-protein interactions with cofactors that are crucial for both transcriptional repression and activation of MYC target genes. Targeting these interactions could potentially provide a means of developing alternative approaches to halt MYC functions; however, the molecular mechanism of these regulatory interactions is poorly understood. Herein we provide methods to produce high-quality human c-MYC C-terminal by itself and in complex MAX, and how to study them using Nuclear Magnetic Resonance spectroscopy and X-ray crystallography. Our protein expression and purification protocols have already been used to study interactions with cofactors.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/isolamento & purificação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/isolamento & purificação , Sequência de Aminoácidos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação , Cristalografia por Raios X/métodos , DNA/química , DNA/genética , Dimerização , Genes myc/genética , Genes myc/fisiologia , Sequências Hélice-Alça-Hélice/genética , Sequências Hélice-Alça-Hélice/fisiologia , Humanos , Zíper de Leucina/genética , Zíper de Leucina/fisiologia , Espectroscopia de Ressonância Magnética/métodos , Ligação Proteica , Domínios Proteicos/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo
16.
Methods Mol Biol ; 2318: 45-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019286

RESUMO

By identifying MYC protein-protein interactors, we aim to gain a deeper mechanistic understanding of MYC as a regulator of gene transcription and potent oncoprotein. This information can then be used to devise strategies for disrupting critical MYC protein-protein interactions to inhibit MYC-driven tumorigenesis. In this chapter, we discuss four techniques to identify and validate MYC-interacting partners. First, we highlight BioID, a powerful discovery method used to identify high-confidence proximal interactors in living cells. We also discuss bioinformatic prioritization strategies for the BioID-derived MYC-proximal complexes. Next, we discuss how protein interactions can be validated using techniques such as in vivo-in vitro pull-down assays and the proximity ligation assay (PLA). We conclude with an overview of biolayer interferometry (BLI), a quantitative method used to characterize direct interactions between two proteins in vitro. Overall, we highlight the principles of each assay and provide methodology necessary to conduct these experiments and adapt them to the study of interactors of additional proteins of interest.


Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/isolamento & purificação , Sequência de Aminoácidos/genética , Sítios de Ligação , Biologia Computacional/métodos , DNA/química , DNA/genética , Dimerização , Genes myc/genética , Genes myc/fisiologia , Humanos , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo
17.
Methods Mol Biol ; 2318: 69-85, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019287

RESUMO

Detection of post-translational modifications in c-Myc is an invaluable tool in assessing Myc status, particularly in cancer. However, it can be challenging to detect these modifications. The evaluation of phosphorylation status of c-Myc can also be challenging with the current commercially available phosphorylation sensitive antibodies. Here, we describe protocols for the immunoprecipitation of endogenous c-Myc to probe for phosphorylation status, as well as the detection of ubiquitination and SUMOylation on c-Myc. We will also discuss the challenges of detecting phosphorylated c-Myc in formalin-fixed paraffin-embedded tissues by immunofluorescence and describe a protocol using a new rat monoclonal antibody we have generated suitable for this purpose.


Assuntos
Imunoprecipitação/métodos , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas c-myc/genética , Imunofluorescência , Genes myc/genética , Genes myc/fisiologia , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sumoilação , Ubiquitinação
18.
Methods Mol Biol ; 2318: 87-117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019288

RESUMO

The MYC oncogene was originally identified as a transduced allele (v-myc) in the genome of the highly oncogenic avian retrovirus MC29. The protein product (MYC) of the cellular MYC (c-myc) protooncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. MYC regulates fundamental cellular processes like growth control, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of MYC, leading to increased levels of the MYC protein, is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to quantify the oncogenic potential of MYC alleles, to test MYC inhibitors, and to monitor MYC-specific protein-protein interactions that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of MYC and to analyze structure, function, and regulation of the ancestral MYC proto-oncogenes.


Assuntos
Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Alelos , Sequência de Aminoácidos , Animais , Carcinogênese , Transformação Celular Neoplásica/genética , Evolução Molecular , Genes myc/genética , Genes myc/fisiologia , Humanos , Mutação , Neoplasias/patologia , Oncogenes , Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética
19.
Methods Mol Biol ; 2318: 161-185, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019290

RESUMO

Here, we present a strategy to map and quantify the interactions between Myc and chromatin using a calibrated Myc ChIP-seq approach. We recommend the use of an internal spike-in control for post-sequencing normalization to enable detection of broad changes in Myc binding as can occur under conditions with varied Myc abundance. We also highlight a range of bioinformatic analyses that can dissect the downstream effects of Myc binding. These methods include peak calling, mapping Myc onto an integrated metagenome, juxtaposing ChIP-seq data with matching RNA-seq data, and identifying gene ontologies enriched for genes with high Myc binding. Our aim is to provide a guided strategy, from cell harvest through to bioinformatic analysis, to elucidate the global effects of Myc on transcription.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Imunoprecipitação da Cromatina/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Sítios de Ligação , Cromatina/genética , Biologia Computacional/métodos , DNA/genética , Proteínas de Ligação a DNA , Genes myc , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Análise de Sequência de DNA/métodos
20.
Methods Mol Biol ; 2318: 119-160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019289

RESUMO

The c-MYC oncogene is activated in ~50% of all tumors and its product, the c-MYC transcription factor, regulates numerous processes, which contribute to tumor initiation and progression. Therefore, the genome-wide characterization of c-MYC targets and their role in different tumor entities is a recurrent theme in cancer research. Recently, next-generation sequencing (NGS) has become a powerful tool to analyze mRNA and miRNA expression, as well as DNA binding of proteins in a genome-wide manner with an extremely high resolution and coverage. Since the c-MYC transcription factor regulates mRNA and miRNA expression by binding to specific DNA elements in the vicinity of promoters, NGS can be used to generate integrated representations of c-MYC-mediated regulations of gene transcription and chromatin modifications. Here, we provide protocols and examples of NGS-based analyses of c-MYC-regulated mRNA and miRNA expression, as well as of DNA binding by c-MYC. Furthermore, we describe the validation of single c-MYC targets identified by NGS . Taken together, these approaches allow an accelerated and comprehensive analysis of c-MYC function in numerous cellular contexts. Ultimately, these analyses will further illuminate the role of this important oncogene.


Assuntos
Estudo de Associação Genômica Ampla/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas Proto-Oncogênicas c-myc/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Genes myc , Humanos , MicroRNAs/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA/genética , RNA Mensageiro/genética , Análise de Sequência de DNA/métodos
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