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BACKGROUND: In clear cell renal cell carcinoma (ccRCC), first-line treatment combines nivolumab (anti-PD-1) and ipilimumab (anti-CTLA4), yielding long-term remissions but with only a 40% success rate. Our study explored the potential of enhancing ccRCC treatment by concurrently using CXCR2 inhibitors alongside immunotherapies. METHODS: We analyzed ELR + CXCL levels and their correlation with patient survival during immunotherapy. RCT001, a unique CXCR2 inhibitor, was examined for its mechanism of action, particularly its effects on human primary macrophages. We tested the synergistic impact of RCT001 in combination with immunotherapies in both mouse models of ccRCC and human ccRCC in the presence of human PBMC. RESUTS: Elevated ELR + CXCL cytokine levels were found to correlate with reduced overall survival during immunotherapy. RCT001, our optimized compound, acted as an inverse agonist, effectively inhibiting angiogenesis and reducing viability of primary ccRCC cells. It redirected M2-like macrophages without affecting M1-like macrophage polarization directed against the tumor. In mouse models, RCT001 enhanced the efficacy of anti-CTLA4 + anti-PD1 by inhibiting tumor-associated M2 macrophages and tumor-associated neutrophils. It also impacted the activation of CD4 T lymphocytes, reducing immune-tolerant lymphocytes while increasing activated natural killer and dendritic cells. Similar effectiveness was observed in human RCC tumors when RCT001 was combined with anti-PD-1 treatment. CONCLUSIONS: RCT001, by inhibiting CXCR2 through its unique mechanism, effectively suppresses ccRCC cell proliferation, angiogenesis, and M2 macrophage polarization. This optimization potentiates the efficacy of immunotherapy and holds promise for significantly improving the survival prospects of metastatic ccRCC patients.
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Carcinoma de Células Renais , Neoplasias Renais , Animais , Camundongos , Humanos , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Agonismo Inverso de Drogas , Leucócitos Mononucleares/patologia , ImunoterapiaRESUMO
Local or metastatic relapse following surgery, radiotherapy, and cisplatin is the leading cause of death in patients with head and neck squamous cell carcinoma (HNSCC). Our study shows overexpression of c-MET and AXL in HNSCC cells and patients resistant to radiotherapy and cisplatin. We demonstrate that cabozantinib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), c-MET, and AXL, decreases migration, invasion, and proliferation and induces mitotic catastrophe and apoptotic cell death of naive and radiotherapy- and cisplatin-resistant HNSCC cells. Cabozantinib inhibits the growth and metastatic spread of experimental HNSCC in zebrafish and the growth of experimental HNSCC in mice by blocking tumor cell proliferation and angiogenesis. The efficacy of cabozantinib is also confirmed on viable sections of surgically removed specimens of human HNSCC and on a patient who relapses after five lines of treatment. These results suggest that cabozantinib is relevant for the treatment of patients with HNSCC after relapse under radiotherapy and cisplatin.
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Carcinoma , Neoplasias de Cabeça e Pescoço , Anilidas , Animais , Linhagem Celular Tumoral , Cisplatino/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Camundongos , Recidiva Local de Neoplasia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridinas , Receptores Proteína Tirosina Quinases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Many cancers can be cured by combining surgery with healthy margins, radiation therapy and chemotherapies. However, when the pathology becomes metastatic, cancers can be incurable. The best situation involves "chronicization" of the pathology even for several years. However, most of the time, patients die within a few months. To disseminate throughout the body, cancer cells must enter the vascular network and seed in another organ. However, during the initiation of cancer processes, the tumor is avascular. Later, the production of angiogenic factors causes tumor neovascularization and subsequent growth and spread, and the presence of blood and/or lymphatic vessels is associated with high grade tumors. Moreover, during tumor development, cancer cells enter lymphatic vessels and disseminate via the lymphatic network. Hence, blood and lymphatic vessels are considered as main routes of metastatic dissemination and cancer aggressiveness. Therefore, anti-angiogenic drugs entered in the therapeutic arsenal from 2004. Despite undeniable effects however, they are far from curative and only prolong survival by a few months.Recently, the concepts of angio/lymphangiogenesis were revisited by analyzing the role of blood and lymphatic vessels at the initiation steps of tumor development. During this period, cancer cells enter lymphatic vessels and activate immune cells within lymph nodes to initiate an antitumor immune response. Moreover, the presence of blood vessels at the proximity of the initial nodule allows immune cells to reach the tumor and eliminate cancer cells. Therefore, blood and lymphatic networks have a beneficial role during a defined time window. Considering only their detrimental effects is a concern. Hence, administration of anti-angio/lymphangiogenic therapies should be revisited to avoid the destruction of networks involved in antitumor immune response. This review mainly focuses on one of the main drivers of lymphangiogenesis, the VEGFC and its beneficial and pejorative roles according to the grade of aggressive tumors.
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Vasos Linfáticos , Fator C de Crescimento do Endotélio Vascular , Humanos , Linfonodos/metabolismo , Linfangiogênese , Metástase Linfática/patologia , Vasos Linfáticos/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Rationale: Head and neck squamous cell carcinoma (HNSCC) represent the 4th most aggressive cancer. 50% of patients relapse to the current treatments combining surgery, radiotherapy and cisplatin and die two years after the diagnosis. Elevated expression of the polo-like kinase 1 (Plk1) correlated to a poor prognosis in epidermoid carcinomas. Methods: The molecular links between Plk1 and resistance to cisplatin/radiotherapy were investigated in patients and cell lines resistant to cisplatin and/or to radiotherapy. The therapeutic relevance of the Plk1 inhibitor onvansertib, alone or combined with cisplatin/radiotherapy, was evaluated on the proliferation/migration on HNSCC cell lines, in experimental HNSCC in mice, in a zebrafish metastasis model and on patient-derived 3D tumor sections. Results: Plk1 expression correlated to a bad prognosis in HNSCC and increased after relapse on cisplatin/radiotherapy. Onvansertib induced mitotic arrest, chromosomic abnormalities and polyploidy leading to apoptosis of sensitive and resistant HNSCC cells at nanomolar concentrations without any effects on normal cells. Onvansertib inhibited the growth of experimental HNSCC in mice and metastatic dissemination in zebrafishes. Moreover, onvansertib combined to cisplatin and/or radiotherapy resulted in a synergic induction of tumor cell death. The efficacy of onvansertib alone and in combination with reference treatments was confirmed on 3D viable sections of HNSCC surgical specimens. Conclusions: Targeting Plk1 by onvansertib represents a new strategy for HNSCC patients at the diagnosis in combination with reference treatments, or alone as a second line treatment for HNCSCC patients experiencing relapses.
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Piperazinas/uso terapêutico , Pirazóis/uso terapêutico , Quinazolinas/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/uso terapêutico , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Camundongos , Camundongos Nus , Recidiva Local de Neoplasia/tratamento farmacológico , Piperazinas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis/metabolismo , Quinazolinas/metabolismo , Radioterapia/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Peixe-Zebra , Quinase 1 Polo-LikeRESUMO
Overcoming acquired drug resistance is a primary challenge in cancer treatment. Notably, more than 50% of patients with BRAFV600E cutaneous metastatic melanoma (CMM) eventually develop resistance to BRAF inhibitors. Resistant cells undergo metabolic reprogramming that profoundly influences therapeutic response and promotes tumor progression. Uncovering metabolic vulnerabilities could help suppress CMM tumor growth and overcome drug resistance. Here we identified a drug, HA344, that concomitantly targets two distinct metabolic hubs in cancer cells. HA344 inhibited the final and rate-limiting step of glycolysis through its covalent binding to the pyruvate kinase M2 (PKM2) enzyme, and it concurrently blocked the activity of inosine monophosphate dehydrogenase, the rate-limiting enzyme of de novo guanylate synthesis. As a consequence, HA344 efficiently targeted vemurafenib-sensitive and vemurafenib-resistant CMM cells and impaired CMM xenograft tumor growth in mice. In addition, HA344 acted synergistically with BRAF inhibitors on CMM cell lines in vitro. Thus, the mechanism of action of HA344 provides potential therapeutic avenues for patients with CMM and a broad range of different cancers. SIGNIFICANCE: Glycolytic and purine synthesis pathways are often deregulated in therapy-resistant tumors and can be targeted by the covalent inhibitor described in this study, suggesting its broad application for overcoming resistance in cancer.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Proteínas de Transporte/antagonistas & inibidores , IMP Desidrogenase/antagonistas & inibidores , Melanoma/tratamento farmacológico , Proteínas de Membrana/antagonistas & inibidores , Ribonucleotídeos/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Idoso , Aminoimidazol Carboxamida/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Melanoma/enzimologia , Melanoma/patologia , Camundongos , Camundongos Nus , Distribuição Aleatória , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Hormônios Tireóideos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Ligação a Hormônio da Tireoide , Melanoma Maligno CutâneoRESUMO
The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.
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Antineoplásicos , Leucemia Mieloide Aguda , Adenosina Trifosfatases/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Reparo do DNA , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Proteína com ValosinaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
GAPDH is emerging as a key player in T cell development and function. To investigate the role of GAPDH in T cells, we generated a transgenic mouse model overexpressing GAPDH in the T cell lineage. Aged mice developed a peripheral Tfh-like lymphoma that recapitulated key molecular, pathological, and immunophenotypic features of human angioimmunoblastic T cell lymphoma (AITL). GAPDH induced non-canonical NF-κB pathway activation in mouse T cells, which was strongly activated in human AITL. We developed a NIK inhibitor to reveal that targeting the NF-κB pathway prolonged AITL-bearing mouse survival alone and in combination with anti-PD-1. These findings suggest the therapeutic potential of targeting NF-κB signaling in AITL and provide a model for future AITL therapeutic investigations.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Linfadenopatia Imunoblástica/patologia , Linfoma de Células T/patologia , NF-kappa B/metabolismo , Linfócitos T/imunologia , Idoso , Animais , Linhagem Celular Tumoral , Linhagem da Célula/imunologia , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Células HEK293 , Humanos , Linfadenopatia Imunoblástica/genética , Linfoma de Células T/tratamento farmacológico , Linfoma de Células T/genética , Linfoma de Células T/imunologia , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , NF-kappa B/genética , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Chaperone-mediated autophagy (CMA) is a highly selective form of autophagy. During CMA, the HSC70 chaperone carries target proteins endowed with a KFERQ-like motif to the lysosomal receptor LAMP2A, which then translocate them into lysosomes for degradation. In the present study, we scrutinized the mechanisms underlying the response and resistance to Azacytidine (Aza) in MDS/AML cell lines and bone marrow CD34+ blasts from MDS/AML patients. In engineered Aza-resistant MDS cell lines and some AML cell lines, we identified a profound defect in CMA linked to the absence of LAMP2A. LAMP2 deficiency was responsible for Aza resistance and hypersensitivity to lysosome and autophagy inhibitors. Accordingly, gain of function of LAMP2 in deficient cells or loss of function in LAMP2-expressing cells rendered them sensitive or resistant to Aza, respectively. A strict correlation was observed between the absence of LAMP2, resistance to Aza and sensitivity to lysosome inhibitors. Low levels of LAMP2 expression in CD34+ blasts from MDS/AML patients correlated with lack of sensitivity to Aza and were predictive of poor overall survival. We propose that CD34+/LAMP2Low patients at diagnosis or who become CD34+/LAMP2Low during the course of treatment with Aza might benefit from a lysosome inhibitor already used in the clinic.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Seguimentos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Inflammatory caspases, activated within the inflammasome, are responsible for the maturation and secretion of IL-1ß/IL-18. Although their expression in psoriasis was shown several years ago, little is known about the role of inflammatory caspases in the context of psoriasis. Here, we confirmed that caspases 1, 4, and 5 are activated in lesional skin from psoriasis patients. We showed in three psoriasis-like models that inflammatory caspases are activated, and accordingly, caspase 1/11 invalidation or pharmacological inhibition by Ac-YVAD-CMK (i.e., Ac-Tyr-Val-Ala-Asp-chloromethylketone) injection induced a decrease in ear thickness, erythema, scaling, inflammatory cytokine expression, and immune cell infiltration in mice. We observed that keratinocytes were primed to secrete IL-1ß when cultured in conditions mimicking psoriasis. Generation of chimeric mice by bone marrow transplantation was carried out to decipher the respective contribution of keratinocytes and/or immune cells in the activation of inflammatory caspases during psoriasis-like inflammatory response. Our data showed that the presence of caspase 1/11 in the immune system is sufficient for a fully inflammatory response, whereas the absence of caspase 1/11 in keratinocytes/fibroblasts had no impact. In summary, our study indicates that inflammatory caspases activated in immune cells are implicated in psoriasis pathogenesis.
Assuntos
Caspase 1/deficiência , Inibidores de Caspase/administração & dosagem , Caspases Iniciadoras/deficiência , Psoríase/tratamento farmacológico , Clorometilcetonas de Aminoácidos/administração & dosagem , Animais , Biópsia , Transplante de Medula Óssea , Caspase 1/genética , Caspase 1/imunologia , Caspases Iniciadoras/genética , Caspases Iniciadoras/imunologia , Caspases Iniciadoras/metabolismo , Células Cultivadas , Ensaios Clínicos como Assunto , Feminino , Humanos , Injeções Intraperitoneais , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Queratinócitos , Masculino , Camundongos , Camundongos Knockout , Cultura Primária de Células , Psoríase/imunologia , Psoríase/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/imunologia , Pele/patologia , Quimeras de TransplanteRESUMO
AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase consisting of the arrangement of various αï¬ ß, and γï isoforms that are expressed differently depending on the tissue or the cell lineage. AMPK is one of the major sensors of energy status in mammalian cells and as such plays essential roles in the regulation of cellular homeostasis, metabolism, cell growth, differentiation, apoptosis, and autophagy. AMPK is activated by two upstream kinases, the tumor suppressor liver kinase B1 (LKB1) and the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) through phosphorylation of the kinase on Thr172, leading to its activation. In addition, AMPK inhibits the mTOR pathway through phosphorylation and activation of tuberous sclerosis protein 2 (TSC2) and causes direct activation of unc-51-like autophagy activating kinase 1 (ULK1) via phosphorylation of Ser555, thus promoting initiation of autophagy. Although it is well established that AMPK can control the differentiation of different cell lineages, including hematopoietic stem cells (HSCs), progenitors, and mature hematopoietic cells, the role of AMPK regarding myeloid cell differentiation is less documented. The differentiation of monocytes into macrophages triggered by colony stimulating factor 1 (CSF-1), a process during which both caspase activation (independently of apoptosis induction) and AMPK-dependent stimulation of autophagy are necessary, is one noticeable example of the involvement of AMPK in the physiological differentiation of myeloid cells. The present review focuses on the role of AMPK in the regulation of the physiological and pathological differentiation of myeloid cells. The mechanisms of autophagy induction by AMPK will also be addressed, as autophagy has been shown to be important for differentiation of hematopoietic cells. In addition, myeloid malignancies (myeloid leukemia or dysplasia) are characterized by profound defects in the establishment of proper differentiation programs. Reinduction of a normal differentiation process in myeloid malignancies has thus emerged as a valuable and promising therapeutic strategy. As AMPK seems to exert a key role in the differentiation of myeloid cells, notably through induction of autophagy, we will also discuss the potential to target this pathway as a pro-differentiating and anti-leukemic strategy in myeloid malignancies.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diferenciação Celular , Células Mieloides/enzimologia , Células Mieloides/patologia , Proteínas Quinases Ativadas por AMP/química , Animais , Ativação Enzimática , Neoplasias Hematológicas/patologia , Células-Tronco Hematopoéticas/patologia , HumanosRESUMO
Advanced cutaneous melanoma is one of the most challenging cancers to treat because of its high plasticity, metastatic potential, and resistance to treatment. New targeted therapies and immunotherapies have shown remarkable clinical efficacy. However, such treatments are limited to a subset of patients and relapses often occur, warranting validation of novel targeted therapies. Posttranslational modification of proteins by ubiquitin coordinates essential cellular functions, including ubiquitin-proteasome system (UPS) function and protein homeostasis. Deubiquitinating enzymes (DUB) have been associated to multiple diseases, including cancer. However, their exact involvement in melanoma development and therapeutic resistance remains poorly understood. Using a DUB trap assay to label cellular active DUBs, we have observed an increased activity of the proteasome-associated DUB, USP14 (Ubiquitin-specific peptidase 14) in melanoma cells compared with melanocytes. Our survey of public gene expression databases indicates that high expression of USP14 correlates with melanoma progression and with a poorer survival rate in metastatic melanoma patients. Knockdown or pharmacologic inhibition of USP14 dramatically impairs viability of melanoma cells irrespective of the mutational status of BRAF, NRAS, or TP53 and their transcriptional cell state, and overcomes resistance to MAPK-targeting therapies both in vitro and in human melanoma xenografted mice. At the molecular level, we find that inhibition of USP14 rapidly triggers accumulation of poly-ubiquitinated proteins and chaperones, mitochondrial dysfunction, ER stress, and a ROS production leading to a caspase-independent cell death. Our results provide a rationale for targeting the proteasome-associated DUB USP14 to treat and combat melanomas. Mol Cancer Ther; 17(7); 1416-29. ©2018 AACR.
Assuntos
Enzimas Desubiquitinantes/genética , Melanoma/tratamento farmacológico , Terapia de Alvo Molecular , Ubiquitina Tiolesterase/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Enzimas Desubiquitinantes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase 1/genética , Melanócitos/efeitos dos fármacos , Melanoma/genética , Melanoma/patologia , Proteínas de Membrana/genética , Camundongos , Inibidores de Proteassoma/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Polo-like kinases (Plks) define a highly conserved family of Ser/Thr kinases with crucial roles in the regulation of cell division. Here we show that Plk1 is cleaved by caspase 3, but not by other caspases in different hematopoietic cell lines treated with competitive inhibitors of the ATP-binding pocket of Plk1. Intriguingly, Plk1 was not cleaved in cells treated with Rigosertib, a non-competitive inhibitor of Plk1, suggesting that binding of the inhibitor to the ATP binding pocket of Plk1 triggers a conformational change and unmasks a cryptic caspase 3 cleavage site on the protein. Cleavage occurs after Asp-404 in a DYSD/K sequence and separates the kinase domain from the two PBDs of Plk1. All Plk1 inhibitors triggered G2/M arrest, activation of caspases 2 and 3, polyploidy, multiple nuclei and mitotic catastrophe, albeit at higher concentrations in the case of Rigosertib. Upon BI-2536 treatment, Plk1 cleavage occurred only in the cytosolic fraction and cleaved Plk1 accumulated in this subcellular compartment. Importantly, the cleaved N-Terminal fragment of Plk1 exhibited a higher enzymatic activity than its non-cleaved counterpart and accumulated into the cytoplasm conversely to the full length and the C-Terminal Plk1 fragments that were found essentially into the nucleus. Finally, the DYSD/K cleavage site was highly conserved during evolution from c. elegans to human. In conclusion, we described herein for the first time a specific cleavage of Plk1 by caspase 3 following treatment of cancer cells with ATP-competitive inhibitors of Plk1.
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Phosphorylation of Ser/Thr residues is a well-established modulating mechanism of the pro-apoptotic function of the BH3-only protein Bim. However, nothing is known about the putative tyrosine phosphorylation of this Bcl-2 family member and its potential impact on Bim function and subsequent Bax/Bak-mediated cytochrome c release and apoptosis. As we have previously shown that the tyrosine kinase Lyn could behave as an anti-apoptotic molecule, we investigated whether this Src family member could directly regulate the pro-apoptotic function of Bim. In the present study, we show that Bim is phosphorylated onto tyrosine residues 92 and 161 by Lyn, which results in an inhibition of its pro-apoptotic function. Mechanistically, we show that Lyn-dependent tyrosine phosphorylation of Bim increases its interaction with anti-apoptotic members such as Bcl-xL, therefore limiting mitochondrial outer membrane permeabilization and subsequent apoptosis. Collectively, our data uncover one molecular mechanism through which the oncogenic tyrosine kinase Lyn negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or the chemotherapeutic resistance of cancer cells.
Assuntos
Apoptose/genética , Proteína 11 Semelhante a Bcl-2/fisiologia , Quinases da Família src/fisiologia , Animais , Proteína 11 Semelhante a Bcl-2/antagonistas & inibidores , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos/genética , Células HEK293 , Células HeLa , Humanos , Células K562 , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oncogenes/fisiologia , Transdução de Sinais/genética , Quinases da Família src/genéticaRESUMO
Endoplasmic reticulum (ER) stress is activated in nonalcoholic fatty liver disease (NAFLD), raising the possibility that ER stress-dependent metabolic dysfunction, inflammation, and cell death underlie the transition from steatosis to steatohepatitis (nonalcoholic steatohepatitis; NASH). B-cell lymphoma 2 (BCL2)-associated X protein (Bax) inhibitor-1 (BI-1), a negative regulator of the ER stress sensor, inositol-requiring enzyme 1 alpha (IRE1α), has yet to be explored in NAFLD as a hepatoprotective agent. We hypothesized that the genetic ablation of BI-1 would render the liver vulnerable to NASH because of unrestrained IRE1α signaling. ER stress was induced in wild-type and BI-1-/- mice acutely by tunicamycin (TM) injection (1 mg/kg) or chronically by high-fat diet (HFD) feeding to determine NAFLD phenotype. Livers of TM-treated BI-1-/- mice showed IRE1α-dependent NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation, hepatocyte death, fibrosis, and dysregulated lipid homeostasis that led to liver failure within a week. The analysis of human NAFLD liver biopsies revealed BI-1 down-regulation parallel to the up-regulation of IRE1α endoribonuclease (RNase) signaling. In HFD-fed BI-1-/- mice that presented NASH and type 2 diabetes, exaggerated hepatic IRE1α, X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP) expression was linked to activated NLRP3 inflammasome and caspase-1/-11. Rises in interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein 1 (MCP1), chemokine (C-X-C motif) ligand 1 (CXCL1), and alanine transaminase (ALT)/aspartate transaminase (AST) levels revealed significant inflammation and injury, respectively. Pharmacological inhibition of IRE1α RNase activity with the small molecules, STF-083010 or 4µ8c, was evaluated in HFD-induced NAFLD. In BI-1-/- mice, either treatment effectively counteracted IRE1α RNase activity, improving glucose tolerance and rescuing from NASH. The hepatocyte-specific role of IRE1α RNase activity in mediating NLRP3 inflammasome activation and cell death was confirmed in primary mouse hepatocytes by IRE1α axis knockdown or its inhibition with STF-083010 or 4µ8c. CONCLUSION: Targeting IRE1α-dependent NLRP3 inflammasome signaling with pharmacological agents or by BI-1 may represent a tangible therapeutic strategy for NASH. (Hepatology 2018).
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Técnicas de Cultura de Células , Morte Celular , Citocinas/metabolismo , Humanos , Immunoblotting , Inflamassomos/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genéticaRESUMO
CSF-1 and IL-34 share the CSF-1 receptor and no differences have been reported in the signaling pathways triggered by both ligands in human monocytes. IL-34 promotes the differentiation and survival of monocytes, macrophages and osteoclasts, as CSF-1 does. However, IL-34 binds other receptors, suggesting that differences exist in the effect of both cytokines. In the present study, we compared the differentiation and polarization abilities of human primary monocytes in response to CSF-1 or IL-34. CSF-1R engagement by one or the other ligands leads to AKT and caspase activation and autophagy induction through expression and activation of AMPK and ULK1. As no differences were detected on monocyte differentiation, we investigated the effect of CSF-1 and IL-34 on macrophage polarization into the M1 or M2 phenotype. We highlighted a striking increase in IL-10 and CCL17 secretion in M1 and M2 macrophages derived from IL-34 stimulated monocytes, respectively, compared to CSF-1 stimulated monocytes. Variations in the secretome induced by CSF-1 or IL-34 may account for their different ability to polarize naïve T cells into Th1 cells. In conclusion, our findings indicate that CSF-1 and IL-34 exhibit the same ability to induce human monocyte differentiation but may have a different ability to polarize macrophages.
Assuntos
Diferenciação Celular , Interleucinas/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Humanos , Interleucinas/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Azacitidine (AZA), the reference treatment for most higher-risk myelodysplastic (MDS) patients can also improve overall survival (OS) in elderly acute myeloid leukemia (AML) patients ineligible for intensive chemotherapy, but reliable biological markers predicting response and OS in patients treated with AZA are lacking. In a preliminary study, we found that an increase of the percentage of BCL2L10, an anti-apoptotic member of the bcl-2 family, was correlated with AZA resistance. In this study, we assessed prospectively by flow cytometry the prognostic value of BCL2L10 positive bone marrow mononuclear cells in 70 patients (42 MDS and 28 AML), prior to AZA treatment.In patients with baseline marrow blasts below 30%, the baseline percentage of bone marrow BCL2L10 positive cells inversely correlated with response to AZA and OS independently of the International Prognostic Scoring System (IPSS) and IPSS-revised (IPSS-R). Specifically, OS was significantly lower in patients with more than 10% BCL2L10 positive cells (median 8.3 vs 22.9 months in patients with less than 10% positivity, p = 0,001). In summary, marrow BCL2L10 positive cells may be a biomarker for azacitidine response and OS, with a potential impact in clinical practice.
Assuntos
Células da Medula Óssea/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/mortalidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Biomarcadores , Células da Medula Óssea/patologia , Feminino , Expressão Gênica , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/tratamento farmacológico , Estadiamento de Neoplasias , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Resultado do TratamentoAssuntos
Autofagia/fisiologia , Neoplasias Hematológicas , Leucemia , Terapia de Alvo Molecular/métodos , Animais , Transformação Celular Neoplásica/patologia , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Leucemia/patologia , Leucemia/terapia , ProteóliseRESUMO
Background: MITF encodes an oncogenic lineage-specific transcription factor in which a germline mutation ( MITFE318K ) was identified in human patients predisposed to both nevus formation and, among other tumor types, melanoma. The molecular mechanisms underlying the oncogenic activity of MITF E318K remained uncharacterized. Methods: Here, we compared the SUMOylation status of endogenous MITF by proximity ligation assay in melanocytes isolated from wild-type (n = 3) or E318K (n = 4) MITF donors. We also used a newly generated Mitf E318K knock-in (KI) mouse model to assess the role of Mitf E318K (n = 7 to 13 mice per group) in tumor development in vivo and performed transcriptomic analysis of the tumors to identify the molecular mechanisms. Finally, using immortalized or normal melanocytes (wild-type or E318K MITF, n = 2 per group), we assessed the role of MITF E318K on the induction of senescence mediated by BRAF V600E . All statistical tests were two-sided. Results: We demonstrated a decrease in endogenous MITF SUMOylation in melanocytes from MITF E318K patients (mean of cells with hypoSUMOylated MITF, MITF E318K vs MITF WT , 94% vs 44%, difference = 50%, 95% CI = 21.8% to 67.2%, P = .004). The Mitf E318K mice were slightly hypopigmented (mean melanin content Mitf WT vs Mitf E318K/+ , 0.54 arbitrary units [AU] vs 0.36 AU, difference = -0.18, 95% CI = -0.36 to -0.007, P = .04). We provided genetic evidence that Mitf E318K enhances BRaf V600E -induced nevus formation in vivo (mean nevus number for Mitf E318K , BRaf V600E vs Mitf WT , BRaf V600E , 68 vs 44, difference = 24, 95% CI = 9.1 to 38.9, P = .006). Importantly, although Mitf E318K was not sufficient to cooperate with BRaf V600E alone in promoting metastatic melanoma, it accelerated tumor formation on a BRaf V600E , Pten-deficient background (median survival, Mitf E318K/+ = 42 days, 95% CI = 31 to 46 vs Mitf WT = 51 days, 95% CI = 50 to 55, P < .001). Transcriptome analysis suggested a decrease in senescence in tumors from Mitf E318K mice. We confirmed this hypothesis by in vitro experiments, demonstrating that Mitf E318K impaired the ability of human melanocytes to undergo BRAF V600E -induced senescence. Conclusions: We characterized the functions of melanoma-associated MITF E318K mutations. Our results demonstrate that MITF E318K reduces the program of senescence to potentially favor melanoma progression in vivo.
Assuntos
Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Nevo/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Senescência Celular/genética , Modelos Animais de Doenças , Mutação em Linhagem Germinativa , Humanos , Melanócitos , Camundongos , Pessoa de Meia-Idade , PTEN Fosfo-Hidrolase/genética , Cultura Primária de Células , Sumoilação , TranscriptomaRESUMO
Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens.