RESUMO
While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Terapia de Alvo Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Diferenciação Celular , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Ensaios de Triagem em Larga Escala , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Células Mieloides/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Pirimidinas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.
Assuntos
Inibidores Enzimáticos/farmacologia , Nitrilas/química , Nitrilas/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Ferroptose/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos SCID , Sondas Moleculares/química , Terapia de Alvo Molecular , Óxidos/química , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/química , Pró-Fármacos/química , Ratos Wistar , Selenocisteína/química , Selenocisteína/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
UNLABELLED: Hepatic stellate cell (HSC) transdifferentiation from a quiescent, adipocyte-like cell to a highly secretory and contractile myofibroblast-like phenotype contributes to negative pathological consequences, including fibrosis/cirrhosis with portal hypertension (PH). Antiadipogenic mechanisms have been shown to underlie activation of HSCs. We examined the role of heme-sensing nuclear receptor Rev-erbα, a transcriptional repressor involved in metabolic and circadian regulation known to promote adipogenesis in preadipocytes, in HSC transdifferentiation. We discovered that Rev-erbα protein was up-regulated in activated HSCs and injured livers; however, transcriptional repressor activity was not affected by fibrogenic treatments. Surprisingly, increased protein expression was accompanied with increased cytoplasmic accumulation of Rev-erbα, which demonstrated distributions similar to myosin, the major cellular motor protein. Cells overexpressing a cytoplasm-localized Rev-erbα exhibited enhanced contractility. Ectopically expressed Rev-erbα responded to both adipogenic ligand and fibrogenic transforming growth factor beta treatment. Rev-erb ligand SR6452 down-regulated cytoplasmic expression of Rev-erbα, decreased expression of fibrogenic markers and the activated phenotype in HSCs, and ameliorated fibrosis and PH in rodent models. CONCLUSIONS: Up-regulation of Rev-erbα is an intrinsic fibrogenic response characterized by cytoplasmic accumulation of the protein in activated HSCs. Cytoplasmic expression of Rev-erbα promotes a contractile phenotype. Rev-erbα acts as a bifunctional regulator promoting either anti- or profibrogenic response, depending on milieu. Rev-erb ligand SR6452 functions by a previously undescribed mechanism, targeting both nuclear activity and cytoplasmic expression of Rev-erbα. Our studies identify Rev-erbα as a novel regulator of HSC transdifferentiation and offers exciting new insights on the therapeutic potential of Rev-erb ligands.
Assuntos
Transdiferenciação Celular , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/etiologia , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Animais , Linhagem Celular , Citoplasma/metabolismo , Humanos , Fígado/lesões , Fígado/metabolismo , Cirrose Hepática/metabolismo , Masculino , Miosinas/antagonistas & inibidores , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/agonistas , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
BACKGROUND: Hepatic regeneration requires coordinated signal transduction for efficient restoration of functional liver mass. This study sought to determine changes in lysophosphatidic acid (LPA) and LPA receptor (LPAR) 1-6 expression in regenerating liver following two-thirds partial hepatectomy (PHx). METHODS: Liver tissue and blood were collected from male C57BL/6 mice following PHx. Circulating LPA was measured by enzyme-linked immunosorbent assay (ELISA) and hepatic LPAR mRNA and protein expression were determined. RESULTS: Circulating LPA increased 72 h after PHx and remained significantly elevated for up to 7 days post-PHx. Analysis of LPAR expression after PHx demonstrated significant increases in LPAR1, LPAR3 and LPAR6 mRNA and protein in a time-dependent manner for up to 7 days post-PHx. Conversely, LPAR2, LPAR4 and LPAR5 mRNA were barely detected in normal liver and did not significantly change after PHx. Changes in LPAR1 expression were confined to non-parenchymal cells following PHx. CONCLUSIONS: Liver regeneration following PHx is associated with significant changes in circulating LPA and hepatic LPAR1, LPAR3 and LPAR6 expression in a time- and cell-dependent manner. Furthermore, changes in LPA-LPAR post-PHx occur after the first round of hepatocyte division is complete.
Assuntos
Hepatectomia/métodos , Regeneração Hepática , Fígado/cirurgia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Animais , Proliferação de Células , Regulação da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Lisofosfolipídeos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Animais , RNA Mensageiro/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Transdução de Sinais , Fatores de TempoRESUMO
BACKGROUND: Lysophosphatidic acid (LPA) is a ubiquitously expressed phospholipid that regulates diverse cellular functions. Previously identified LPA receptor subtypes (LPAR1-5) are weakly expressed or absent in the liver. This study sought to determine LPAR expression, including the newly identified LPAR6, in normal human liver (NL), hepatocellular carcinoma (HCC), and non-tumor liver tissue (NTL), and LPAR expression and function in human hepatoma cells in vitro. METHODS: We determined LPAR1-6 expression by quantitative reverse transcriptase polymerase chain reaction, Western blot, or immunohistochemistry in NL, NTL, and HCC, and HuH7, and HepG2 cells. Hepatoma cells were treated with LPA in the absence or presence of LPAR1-3 (Ki16425) or pan-LPAR (α-bromomethylene phosphonate) antagonists and proliferation and motility were measured. RESULTS: We report HCC-associated changes in LPAR1, 3, and 6 mRNA and protein expression, with significantly increased LPAR6 in HCC versus NL and NTL. Analysis of human hepatoma cells demonstrated significantly higher LPAR1, 3, and 6 mRNA and protein expression in HuH7 versus HepG2 cells. Treatment with LPA (0.05-10 µg/mL) led to dose-dependent HuH7 growth and increased motility. In HepG2 cells, LPA led to moderate, although significant, increases in proliferation but not motility. Pretreatment with α-bromomethylene phosphonate inhibited LPA-dependent proliferation and motility to a greater degree than Ki16425. CONCLUSIONS: Multiple LPAR forms are expressed in human HCC, including the recently described LPAR6. Inhibition of LPA-LPAR signaling inhibits HCC cell proliferation and motility, the extent of which depends on LPAR subtype expression.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Receptores de Ácidos Lisofosfatídicos/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/química , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Neoplasias Hepáticas/química , Lisofosfolipídeos/farmacologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Receptores de Ácidos Lisofosfatídicos/análise , Receptores de Ácidos Lisofosfatídicos/genéticaRESUMO
Inhibiting the interaction of menin with the histone methyltransferase MLL1 (KMT2A) has recently emerged as a novel therapeutic strategy. Beneficial therapeutic effects have been postulated in leukemia, prostate, breast, liver and in synovial sarcoma models. In those indications, MLL1 recruitment by menin was described to critically regulate the expression of disease associated genes. However, most findings so far rely on single study reports. Here we independently evaluated the pathogenic functions of the menin-MLL interaction in a large set of different cancer models with a potent and selective probe inhibitor BAY-155. We characterized the inhibition of the menin-MLL interaction for anti-proliferation, gene transcription effects, and for efficacy in several in vivo xenografted tumor models. We found a specific therapeutic activity of BAY-155 primarily in AML/ALL models. In solid tumors, we observed anti-proliferative effects of BAY-155 in a surprisingly limited fraction of cell line models. These findings were further validated in vivo. Overall, our study using a novel, highly selective and potent inhibitor, shows that the menin-MLL interaction is not essential for the survival of most solid cancer models. We can confirm that disrupting the menin-MLL complex has a selective therapeutic benefit in MLL-fused leukemia. In solid cancers, effects are restricted to single models and more limited than previously claimed.
RESUMO
Structural mutants of p53 induce global p53 protein destabilization and misfolding, followed by p53 protein aggregation. First evidence indicates that p53 can be part of protein condensates and that p53 aggregation potentially transitions through a condensate-like state. We show condensate-like states of fluorescently labeled structural mutant p53 in the nucleus of living cancer cells. We furthermore identified small molecule compounds that interact with the p53 protein and lead to dissolution of p53 structural mutant condensates. The same compounds lead to condensation of a fluorescently tagged p53 DNA-binding mutant, indicating that the identified compounds differentially alter p53 condensation behavior depending on the type of p53 mutation. In contrast to p53 aggregation inhibitors, these compounds are active on p53 condensates and do not lead to mutant p53 reactivation. Taken together our study provides evidence for structural mutant p53 condensation in living cells and tools to modulate this process.
RESUMO
INTRODUCTION: The chromosomal rearrangements of the mixed-lineage leukemia gene MLL (KMT2A) have been extensively characterized as a potent oncogenic driver in leukemia. For its oncogenic function, most MLL-fusion proteins exploit the multienzyme super elongation complex leading to elevated expression of MLL target genes. High expression of MLL target genes overwrites the normal hematopoietic differentiation program, resulting in undifferentiated blasts characterized by the capacity to self-renew. Although extensive resources devoted to increased understanding of therapeutic targets to overcome de-differentiation in ALL/AML, the inter-dependencies of targets are still not well described. The majority of inhibitors potentially interfering with MLL-fusion protein driven transformation have been characterized in individual studies, which so far hindered their direct cross-comparison. METHODS: In our study, we characterized head-to-head clinical stage inhibitors for BET, DHODH, DOT1L as well as two novel inhibitors for CDK9 and the Menin-MLL interaction with a focus on differentiation induction. We profiled those inhibitors for global gene expression effects in a large cell line panel and examined cellular responses such as inhibition of proliferation, apoptosis induction, cell cycle arrest, surface marker expression, morphological phenotype changes, and phagocytosis as functional differentiation readout. We also verified the combination potential of those inhibitors on proliferation and differentiation level. RESULTS: Our analysis revealed significant differences in differentiation induction and in modulating MLL-fusion target gene expression. We observed Menin-MLL and DOT1L inhibitors act very specifically on MLL-fused leukemia cell lines, whereas inhibitors of BET, DHODH and P-TEFb have strong effects beyond MLL-fusions. Significant differentiation effects were detected for Menin-MLL, DOT1L, and DHODH inhibitors, whereas BET and CDK9 inhibitors primarily induced apoptosis in AML/ALL cancer models. For the first time, we explored combination potential of the abovementioned inhibitors with regards to overcoming the differentiation blockage. CONCLUSION: Our findings show substantial diversity in the molecular activities of those inhibitors and provide valuable insights into the further developmental potential as single agents or in combinations in MLL-fused leukemia.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Histona-Lisina N-Metiltransferase/genética , Leucemia/tratamento farmacológico , Proteína de Leucina Linfoide-Mieloide/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Rearranjo Gênico/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia/genética , Leucemia/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.
Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Di-Hidro-Orotato Desidrogenase , Feminino , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pirimidinas/metabolismo , Células THP-1 , Translocação Genética/efeitos dos fármacosRESUMO
The lactate transporter SLC16A1/monocarboxylate transporter 1 (MCT1) plays a central role in tumor cell energy homeostasis. In a cell-based screen, we identified a novel class of MCT1 inhibitors, including BAY-8002, which potently suppress bidirectional lactate transport. We investigated the antiproliferative activity of BAY-8002 in a panel of 246 cancer cell lines and show that hematopoietic tumor cells, in particular diffuse large B-cell lymphoma cell lines, and subsets of solid tumor models are particularly sensitive to MCT1 inhibition. Associated markers of sensitivity were, among others, lack of MCT4 expression, low pleckstrin homology like domain family A member 2, and high pellino E3 ubiquitin protein ligase 1 expression. The antitumor effect of MCT1 inhibition was less pronounced on tumor xenografts, with tumor stasis being the maximal response. BAY-8002 significantly increased intratumor lactate levels and transiently modulated pyruvate levels. In order to address potential acquired resistance mechanisms to MCT1 inhibition, we generated MCT1 inhibitor-resistant cell lines and show that resistance can occur by upregulation of MCT4 even in the presence of sufficient oxygen, as well as by shifting energy generation toward oxidative phosphorylation. These findings provide insight into novel aspects of tumor response to MCT1 modulation and offer further rationale for patient selection in the clinical development of MCT1 inhibitors. Mol Cancer Ther; 17(11); 2285-96. ©2018 AACR.
Assuntos
Aminobenzoatos/farmacologia , Benzoatos/farmacologia , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Sulfonas/farmacologia , Simportadores/antagonistas & inibidores , Aminobenzoatos/química , Animais , Benzoatos/química , Transporte Biológico/efeitos dos fármacos , Radioisótopos de Carbono , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Camundongos SCID , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Pirimidinonas/farmacologia , Ácido Pirúvico/metabolismo , Sulfonas/química , Simportadores/metabolismo , Tiofenos/farmacologia , Resultado do Tratamento , Xenopus laevisRESUMO
MTH1 is a hydrolase responsible for sanitization of oxidized purine nucleoside triphosphates to prevent their incorporation into replicating DNA. Early tool compounds published in the literature inhibited the enzymatic activity of MTH1 and subsequently induced cancer cell death; however recent studies have questioned the reported link between these two events. Therefore, it is important to validate MTH1 as a cancer dependency with high quality chemical probes. Here, we present BAY-707, a substrate-competitive, highly potent and selective inhibitor of MTH1, chemically distinct compared to those previously published. Despite superior cellular target engagement and pharmacokinetic properties, inhibition of MTH1 with BAY-707 resulted in a clear lack of in vitro or in vivo anticancer efficacy either in mono- or in combination therapies. Therefore, we conclude that MTH1 is dispensable for cancer cell survival.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Sistemas de Liberação de Medicamentos , Morfolinas/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Células CACO-2 , Células Cultivadas , Enzimas Reparadoras do DNA/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Hepatócitos/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Morfolinas/química , Neoplasias/fisiopatologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacologia , RatosRESUMO
Throughout evolution, effective nutrient sensing and control of systemic energy homeostasis have relied on a close physical and functional interaction between immune and metabolically active cells. However, in today's obesogenic environment, this fine-tuned immunometabolic interface is perturbed. As a consequence, chronic inflammatory conditions and aberrant activation of immune cells have emerged as key features of obesity-related metabolic disorders, including insulin resistance, cardiovascular complications, and type 2 diabetes, whereas a major research focus has been placed on the adipocyte-macrophage interaction in the context of metabolic dysfunction; recent studies have not only expanded the scope of relevant immune cells in this setting but also highlight the impact of distinct metabolic organs, including the liver, on immunometabolic control, metabolic disease development, and potential anti-inflammatory therapeutic options in obesity-driven pathologies. This review will thus summarize recent progress in this emerging area of metabolic research.
Assuntos
Síndrome Metabólica/imunologia , Síndrome Metabólica/metabolismo , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Animais , Humanos , Imunidade Inata/fisiologia , Inflamação/imunologia , Inflamação/metabolismo , Resistência à Insulina , Fígado/citologia , Fígado/imunologia , Fígado/metabolismo , Obesidade/imunologia , Obesidade/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a global health burden with limited treatment options and poor prognosis. Silibinin, an antioxidant derived from the Milk Thistle plant (Silybum marianum), is reported to exert hepatoprotective and antitumorigenic effects in vitro and in vivo by suppressing oxidative stress and proliferation. Using a DEN-initiated mouse model of HCC, this study examined the effects of dietary silibinin supplementation alone, or in combination with chronic ethanol consumption on HCC progression. Our data demonstrate silibinin exerted marginal hepatoprotective effects in early stages of hepatocarcinogenesis but, when co-administered with ethanol, exacerbated the promotional effects of ethanol in HCC bearing mice, but only in males.