RESUMO
Hyperactive FMS-like receptor tyrosine kinase-3 mutants with internal tandem duplications (FLT3-ITD) are frequent driver mutations of aggressive acute myeloid leukemia (AML). Inhibitors of FLT3 produce promising results in rationally designed cotreatment schemes. Since FLT3-ITD modulates DNA replication and DNA repair, valid anti-leukemia strategies could rely on a combined inhibition of FLT3-ITD and regulators of cell cycle progression and DNA integrity. These include the WEE1 kinase which controls cell cycle progression, nucleotide synthesis, and DNA replication origin firing. We investigated how pharmacological inhibition of FLT3 and WEE1 affected the survival and genomic integrity of AML cell lines and primary AML cells. We reveal that promising clinical grade and preclinical inhibitors of FLT3 and WEE1 synergistically trigger apoptosis in leukemic cells that express FLT3-ITD. An accumulation of single and double strand DNA damage precedes this process. Mass spectrometry-based proteomic analyses show that FLT3-ITD and WEE1 sustain the expression of the ribonucleotide reductase subunit RRM2, which provides dNTPs for DNA replication. Unlike their strong pro-apoptotic effects on leukemia cells with FLT3-ITD, inhibitors of FLT3 and WEE1 do not damage healthy human blood cells and murine hematopoietic stem cells. Thus, pharmacological inhibition of FLT3-ITD and WEE1 might become an improved, rationally designed therapeutic option.
Assuntos
Apoptose , Proteínas de Ciclo Celular , Dano ao DNA , Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Proteínas Tirosina Quinases , Tirosina Quinase 3 Semelhante a fms , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Animais , Pirazóis , PirimidinonasRESUMO
Our previously reported HDAC6 inhibitor (HDAC6i) Marbostat-100 (4) has provided many arguments for further clinical evaluation. By the substitution of the acidic hydrogen of 4 for different carbon residues, we were able to generate an all-carbon stereocenter, which significantly improves the hydrolytic stability of the inhibitor. Further asymmetric synthesis has shown that the (S)-configured inhibitors preferentially bind to HDAC6. This led to the highly selective and potent methyl-substituted derivative S-29b, which elicited a long-lasting tubulin hyperacetylation in MV4-11 cells. Finally, a crystal structure of the HDAC6/S-29b complex provided mechanistic explanation for the high potency and stereoselectivity of synthesized compound series.
Assuntos
Carbolinas , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases , Humanos , Carbolinas/química , Carbolinas/farmacologia , Carbolinas/síntese química , Linhagem Celular Tumoral , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/síntese química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Morfolinas/síntese química , Morfolinas/química , Morfolinas/farmacologiaRESUMO
Overexpression of MYC is a genuine cancer driver in lymphomas and related to poor prognosis. However, therapeutic targeting of the transcription factor MYC remains challenging. Here, we show that inhibition of the histone deacetylase 6 (HDAC6) using the HDAC6 inhibitor Marbostat-100 (M-100) reduces oncogenic MYC levels and prevents lymphomagenesis in a mouse model of MYC-induced aggressive B-cell lymphoma. M-100 specifically alters protein-protein interactions by switching the acetylation state of HDAC6 substrates, such as tubulin. Tubulin facilitates nuclear import of MYC, and MYC-dependent B-cell lymphoma cells rely on continuous import of MYC due to its high turn-over. Acetylation of tubulin impairs this mechanism and enables proteasomal degradation of MYC. M-100 targets almost exclusively B-cell lymphoma cells with high levels of MYC whereas non-tumor cells are not affected. M-100 induces massive apoptosis in human and murine MYC-overexpressing B-cell lymphoma cells. We identified the heat-shock protein DNAJA3 as an interactor of tubulin in an acetylation-dependent manner and overexpression of DNAJA3 resulted in a pronounced degradation of MYC. We propose a mechanism by which DNAJA3 associates with hyperacetylated tubulin in the cytoplasm to control MYC turnover. Taken together, our data demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma.
Assuntos
Linfoma de Células B , Tubulina (Proteína) , Acetilação , Animais , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/genética , Camundongos , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Internal tandem duplications (ITDs) in the FMS-like tyrosine kinase-3 (FLT3) are causally linked to acute myeloid leukemia (AML) with poor prognosis. Available FLT3 inhibitors (FLT3i) preferentially target inactive or active conformations of FLT3. Moreover, they co-target kinases for normal hematopoiesis, are vulnerable to therapy-associated tyrosine kinase domain (TKD) FLT3 mutants, or lack low nanomolar activity. We show that the tyrosine kinase inhibitor marbotinib suppresses the phosphorylation of FLT3-ITD and the growth of permanent and primary AML cells with FLT3-ITD. This also applies to leukemic cells carrying FLT3-ITD/TKD mutants that confer resistance to clinically used FLT3i. Marbotinib shows high selectivity for FLT3 and alters signaling, reminiscent of genetic elimination of FLT3-ITD. Molecular docking shows that marbotinib fits in opposite orientations into inactive and active conformations of FLT3. The water-soluble marbotinib-carbamate significantly prolongs survival of mice with FLT3-driven leukemia. Marbotinib is a nanomolar next-generation FLT3i that represents a hybrid inhibitory principle.
Assuntos
Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Tirosina Quinase 3 Semelhante a fms , Animais , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Simulação de Acoplamento Molecular , Mutação , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidoresRESUMO
Targeted protein degradation offers new opportunities to inactivate cancer drivers and has successfully entered the clinic. Ways to induce selective protein degradation include proteolysis targeting chimera (PROTAC) technology and immunomodulatory (IMiDs) / next-generation Cereblon (CRBN) E3 ligase modulating drugs (CELMoDs). Here, we aimed to develop a MYC PROTAC based on the MYC-MAX dimerization inhibitor 10058-F4 derivative 28RH and Thalidomide, called MDEG-541. We show that a subgroup of gastrointestinal cancer cell lines and primary patient-derived organoids are MDEG-541 sensitive. Although MYC expression was regulated in a CRBN-, proteasome- and ubiquitin-dependent manner, we provide evidence that MDEG-541 induced the degradation of CRBN neosubstrates, including G1 to S phase transition 1/2 (GSPT1/2) and the Polo-like kinase 1 (PLK1). In sum, we have established a CRBN-dependent degrader of relevant cancer targets with activity in gastrointestinal cancers.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Gastrointestinais/tratamento farmacológico , Talidomida/farmacologia , Tiazóis/farmacologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Talidomida/síntese química , Talidomida/química , Tiazóis/síntese química , Tiazóis/química , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3.
Assuntos
Leucemia Mieloide Aguda , Apoptose , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Histona Desacetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Mutants of the FLT3 receptor tyrosine kinase (RTK) with duplications in the juxtamembrane domain (FLT3-ITD) act as drivers of acute myeloid leukemia (AML). Potent tyrosine kinase inhibitors (TKi) of FLT3-ITD entered clinical trials and showed a promising, but transient success due to the occurrence of secondary drug-resistant AML clones. A further caveat of drugs targeting FLT3-ITD is the co-targeting of other RTKs which are required for normal hematopoiesis. This is observed quite frequently. Therefore, novel drugs are necessary to treat AML effectively and safely. Recently bis(1H-indol-2-yl)methanones were found to inhibit FLT3 and PDGFR kinases. In order to optimize these agents we synthesized novel derivatives of these methanones with various substituents. Methanone 16 and its carbamate derivative 17b inhibit FLT3-ITD at least as potently as the TKi AC220 (quizartinib). Models indicate corresponding interactions of 16 and quizartinib with FLT3. The activity of 16 is accompanied by a high selectivity for FLT3-ITD.
Assuntos
Antineoplásicos/farmacologia , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis/síntese química , Indóis/química , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Aberrant epigenetic changes in DNA methylation and histone modification by acetylation or deacetylation regulate the pathogenesis of many diseases. Especially selective inhibitors are getting more and more attention. We recently reported on a new class of potent and selective anti-inflammatory and antirheumatic histone deacetylase 6 (HDAC6) inhibitors (e.g., Marbostat-100). The attachment of a morpholinoethoxy part to the head group dramatically enhances the solubility, in particular the solubility in aqueous solutions, of the lead compound Marbostat-100. Here, we present the enantioselective synthesis of small-molecule compounds based on the tetrahydro-ß-carboline core system with improved solubility, and the influence of the stereochemistry on the biological activity. The enantiomers were synthesized in good enantiomeric excess (ee) purity and were potent and selective HDAC6 inhibitors, whereas the S-derivative S-21 is clearly the eutomer. The potency of our selective HDAC6 inhibitors is demonstrated by Ki values in the range of 0.5-2 nM toward HDAC6, and the selectivity was proved in cellular assays by Western blot analysis taking ac-tubulin as surrogate parameter.
Assuntos
Carbolinas/química , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/síntese química , Acetilação , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Humanos , Estrutura Molecular , Solubilidade , EstereoisomerismoRESUMO
Experiments with cultured mammalian cells represent an in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used media supplement worldwide. FCS contains a mixture of largely undefined growth factors and cytokines, which support cell proliferation. This undefined nature of FCS is a source of experimental variation, undesired immune responses, possible contaminations, and because of its way of production an ethical concern. Thus, alternative, defined, valid, and reliable media supplements should be characterized in a large number of experiments. Human platelet lysate (hPL) is increasingly appreciated as an alternative to FCS. Since it is unclear whether cells respond differentially to clinically relevant chemotherapeutics inducing replicative stress and DNA damage (Hydroxyurea, Irinotecan), induction of reactive oxygen species (ROS), the tyrosine kinase inhibitor (TKi) Imatinib, and novel epigenetic modifiers belonging to the group of histone deacetylase inhibitors (HDACi), we investigated these issues. Here we show that cancer cells derived from leukemia and colon cancer grow very similarly in culture media with FCS or outdated hPL. Notably, cells have practically identical proteomes under both culture conditions. Moreover, cells grown with FCS or hPL respond equally to all types of drugs and stress conditions that we have tested. In addition, the transfection of blood cells by electroporation can be achieved under both conditions. Furthermore, we reveal that class I HDACs, but not HDAC6, are required for the expression of the pan-leukemic marker WT1 under various culture conditions. Hence, hPL is a moderately priced substitute for FCS in various experimental settings.
Assuntos
Antineoplásicos , Plaquetas/metabolismo , Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , Animais , Células Cultivadas , Humanos , SoroRESUMO
Drug resistance is a leading cause for treatment failure in many cancers, including neuroblastoma, the most common solid extracranial childhood malignancy. Previous studies from our lab indicate that histone deacetylase 10 (HDAC10) is important for the homeostasis of lysosomes, i.e. acidic vesicular organelles involved in the degradation of various biomolecules. Here, we show that depleting or inhibiting HDAC10 results in accumulation of lysosomes in chemotherapy-resistant neuroblastoma cell lines, as well as in the intracellular accumulation of the weakly basic chemotherapeutic doxorubicin within lysosomes. Interference with HDAC10 does not block doxorubicin efflux from cells via P-glycoprotein inhibition, but rather via inhibition of lysosomal exocytosis. In particular, intracellular doxorubicin does not remain trapped in lysosomes but also accumulates in the nucleus, where it promotes neuroblastoma cell death. Our data suggest that lysosomal exocytosis under doxorubicin treatment is important for cell survival and that inhibition of HDAC10 further induces DNA double-strand breaks (DSBs), providing additional mechanisms that sensitize neuroblastoma cells to doxorubicin. Taken together, we demonstrate that HDAC10 inhibition in combination with doxorubicin kills neuroblastoma, but not non-malignant cells, both by impeding drug efflux and enhancing DNA damage, providing a novel opportunity to target chemotherapy resistance.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Reparo do DNA , Doxorrubicina/farmacologia , Exocitose/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Neuroblastoma/tratamento farmacológico , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Exocitose/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Lisossomos/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologiaRESUMO
Various diseases are related to epigenetic modifications. Histone deacetylases (HDACs) and histone acetyl transferases (HATs) determine the pattern of histone acetylation, and thus are involved in the regulation of gene expression. First generation histone deacetylase inhibitors (HDACi) are unselective, hinder all different kinds of zinc dependent HDACs and additionally cause several side effects. Subsequently, selective HDACi are gaining more and more interest. Especially, selective histone deacetylase 6 inhibitors (HDAC6i) are supposed to be less toxic. Here we present a successful optimization study of tubastatin A, the synthesis and biological evaluation of new inhibitors based on hydroxamic acids linked to various tetrahydro-ß-carboline derivatives. The potency of our selective HDAC6 inhibitors, exhibiting IC50 values in a range of 1-10â¯nM towards HDAC6, was evaluated with the help of a recombinant human HDAC6 enzyme assay. Selectivity was proofed in cellular assays by the hyperacetylation of surrogate parameter α-tubulin in the absence of acetylated histone H3 analyzed by Western Blot. We show that all synthesized compounds, with varies modifications of the rigid cap group, were selective and potent HDAC6 inhibitors.
Assuntos
Carbolinas/farmacologia , Desenho de Fármacos , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carbolinas/síntese química , Carbolinas/química , Ciclo Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desacetilase 6 de Histona/deficiência , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Epigenetic modifiers of the histone deacetylase (HDAC) family contribute to autoimmunity, cancer, HIV infection, inflammation, and neurodegeneration. Hence, histone deacetylase inhibitors (HDACi), which alter protein acetylation, gene expression patterns, and cell fate decisions, represent promising new drugs for the therapy of these diseases. Whereas pan-HDACi inhibit all 11 Zn2+-dependent histone deacetylases (HDACs) and cause a broad spectrum of side effects, specific inhibitors of histone deacetylase 6 (HDAC6i) are supposed to have less side effects. We present the synthesis and biological evaluation of Marbostats, novel HDAC6i that contain the hydroxamic acid moiety linked to tetrahydro-ß-carboline derivatives. Our lead compound Marbostat-100 is a more potent and more selective HDAC6i than previously established well-characterized compounds in vitro as well as in cells. Moreover, Marbostat-100 is well tolerated by mice and effective against collagen type II induced arthritis. Thus, Marbostat-100 represents a most selective known HDAC6i and the possibility for clinical evaluation of a HDAC isoform-specific drug.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Benzamidas/uso terapêutico , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/toxicidade , Antirreumáticos/síntese química , Antirreumáticos/farmacologia , Antirreumáticos/toxicidade , Artrite Experimental/induzido quimicamente , Artrite Reumatoide/induzido quimicamente , Benzamidas/líquido cefalorraquidiano , Benzamidas/farmacologia , Benzamidas/toxicidade , Sítios de Ligação , Carbolinas/síntese química , Carbolinas/farmacologia , Carbolinas/uso terapêutico , Carbolinas/toxicidade , Linhagem Celular Tumoral , Colágeno Tipo II , Células HEK293 , Desacetilase 6 de Histona/química , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/toxicidade , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Ácidos Hidroxâmicos/toxicidade , Masculino , Camundongos Endogâmicos DBA , Simulação de Acoplamento Molecular , Peixe-ZebraRESUMO
Signal transducers and activators of transcription (STATs) are latent, cytoplasmic transcription factors. Janus kinases (JAKs) and activated CDC42-associated kinase-1 (ACK1/TNK2) catalyse the phosphorylation of STAT1 and the expression of its target genes. Here we demonstrate that catalytically active ACK1 promotes the phosphorylation and nuclear accumulation of STAT1 in transformed kidney cells. These processes are associated with STAT1-dependent gene expression and an interaction between endogenous STAT1 and ACK1. Moreover, the E3 ubiquitin ligase seven-in-absentia homolog-2 (SIAH2), which targets ACK1 through valine-909 for proteasomal degradation, attenuates the ACK1-STAT1 signalling node. We further show that ACK1 promotes the phosphorylation and nuclear accumulation of STAT3 in cultured cells and that the levels of ACK1 correlate positively with the levels of tyrosine phosphorylated STAT3 in primary lung adenocarcinoma (ADC) cells. Global analysis of ACK1 interaction partners validated the interaction of ACK1 with heat shock protein 90 (HSP90α/ß). Inhibition of this chaperone with the novel drug Onalespib (AT13387) demonstrates that HSP90 is an upstream regulator of the ACK1-dependent phosphorylation of STAT1 and STAT3. In addition to these molecular insights, our data offer a pharmacological strategy to control the ACK1-STAT signalling axis.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Benzamidas/farmacologia , Células HEK293 , Humanos , Isoindóis/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Tirosina Quinases/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Células Tumorais Cultivadas , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Fms-like tyrosine kinase 3 (FLT3) with internal tandem duplications (ITD) is a major oncoprotein in acute myeloid leukemia (AML), and confers an unfavorable prognosis. Interference with FLT3ITD signaling is therefore pursued as a promising therapeutic strategy. In this study we show that abrogation of FLT3ITD glycoprotein maturation using low doses of the N-glycosylation inhibitor tunicamycin has anti-proliferative and pro-apoptotic effects on FLT3ITD-expressing human and murine cell lines. This effect is mediated in part by arresting FLT3ITD in an underglycosylated state and thereby attenuating FLT3ITD-driven AKT and ERK signaling. In addition, tunicamycin caused pronounced endoplasmatic reticulum stress and apoptosis through activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activation of the gene encoding CCAAT-enhancer-binding protein homologous protein (CHOP). PERK inhibition with a small molecule attenuated CHOP induction and partially rescued cells from apoptosis. Combination of tunicamycin with potent FLT3ITD kinase inhibitors caused synergistic cell killing, which was highly selective for cell lines and primary AML cells expressing FLT3ITD. Although tunicamycin is currently not a clinically applicable drug, we propose that mild inhibition of N-glycosylation may have therapeutic potential in combination with FLT3 kinase inhibitors for FLT3ITD-positive AML.
Assuntos
Antineoplásicos/farmacologia , Duplicação Gênica , Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/farmacologia , Sequências de Repetição em Tandem , Tirosina Quinase 3 Semelhante a fms/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Glicosilação/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Tumorais Cultivadas , Tunicamicina/farmacologia , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
The transcription factors NF-κB and p53 as well as their crosstalk determine the fate of tumor cells upon therapeutic interventions. Replicative stress and cytokines promote signaling cascades that lead to the co-regulation of p53 and NF-κB. Consequently, nuclear p53/NF-κB signaling complexes activate NF-κB-dependent survival genes. The 18 histone deacetylases (HDACs) are epigenetic modulators that fall into four classes (I-IV). Inhibitors of histone deacetylases (HDACi) become increasingly appreciated as anti-cancer agents. Based on their effects on p53 and NF-κB, we addressed whether clinically relevant HDACi affect the NF-κB/p53 crosstalk. The chemotherapeutics hydroxyurea, etoposide, and fludarabine halt cell cycle progression, induce DNA damage, and lead to DNA fragmentation. These agents co-induce p53 and NF-κB-dependent gene expression in cell lines from breast and colon cancer and in primary chronic lymphatic leukemia (CLL) cells. Using specific HDACi, we find that the class I subgroup of HDACs, but not the class IIb deacetylase HDAC6, are required for the hydroxyurea-induced crosstalk between p53 and NF-κB. HDACi decrease the basal and stress-induced expression of p53 and block NF-κB-regulated gene expression. We further show that class I HDACi induce senescence in pancreatic cancer cells with mutant p53.
Assuntos
Histona Desacetilases/metabolismo , NF-kappa B/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Dano ao DNA , DNA de Neoplasias/metabolismo , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Hidroxiureia/farmacologia , Modelos Biológicos , Mutação/genética , Neoplasias/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Histone deacetylases (HDACs) catalyze the deacetylation of lysine residues in their target proteins. This biochemical modification can have profound effects on the functions of these proteins and a dysregulation of HDAC activity contributes to severe diseases, including neoplastic transformation. In the following chapter, we present a strategy that allows to distinguish between the inhibition of the class I HDACs HDAC1, 2, and 3 and of the class IIb HDAC HDAC6. This method is based on Western blot and relies on the detection of hyperacetylated substrates of class I or class IIb HDACs in lysates from cells that were treated with histone deacetylase inhibitors (HDACi).
Assuntos
Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Animais , Benzamidas/farmacologia , Western Blotting/métodos , Linhagem Celular Tumoral , Epigênese Genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/genética , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/genética , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Histona Desacetilases/genética , Histonas/genética , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Camundongos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Panobinostat , Piridinas/farmacologia , Ácido Valproico/farmacologiaRESUMO
Chimeric compounds combine the structural features of inhibitors of histone deacetylases (HDACi) and tyrosine kinase inhibitors (TKi), and therefore unite the effects of a dual-targeting strategy in one compound. Here, we describe the generation of such hybrid molecules. Small molecules, known as TKi, are combined with a Zn2+ chelating motive, preferentially a hydroxamic acid, in addition. The resulting small molecules also can inhibit histone deacetylases, which are dependent on the catalytically active Zn2+. Moreover, we summarize how the growth-inhibitory effects of these combined compounds can be determined with a simple proliferation assay with a leukemic cell line.
Assuntos
Antineoplásicos/síntese química , Quelantes/química , Técnicas de Química Sintética , Inibidores de Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Inibidores de Proteínas Quinases/química , Antineoplásicos/farmacologia , Cátions Bivalentes , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quelantes/farmacologia , Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/patologia , Inibidores de Proteínas Quinases/farmacologia , Zinco/química , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
The treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) induces granulocytic differentiation. This process renders APL cells resistant to cytotoxic chemotherapies. Epigenetic regulators of the histone deacetylases (HDACs) family, which comprise four classes (I-IV), critically control the development and progression of APL. We set out to clarify the parameters that determine the interaction between ATRA and histone deacetylase inhibitors (HDACi). Our assays included drugs against class I HDACs (MS-275, VPA, and FK228), pan-HDACi (LBH589, SAHA), and the novel HDAC6-selective compound Marbostat-100. We demonstrate that ATRA protects APL cells from cytotoxic effects of SAHA, MS-275, and Marbostat-100. However, LBH589 and FK228, which have a superior substrate-inhibitor dissociation constant (Ki) for the class I deacetylases HDAC1, 2, 3, are resistant against ATRA-dependent cytoprotective effects. We further show that HDACi evoke DNA damage, measured as induction of phosphorylated histone H2AX and by the comet assay. The ability of ATRA to protect APL cells from the induction of p-H2AX by HDACi is a readout for the cytoprotective effects of ATRA. Moreover, ATRA increases the fraction of cells in the G1 phase, together with an accumulation of the cyclin-dependent kinase inhibitor p21 and a reduced expression of thymidylate synthase (TdS). In contrast, the ATRA-dependent activation of the transcription factors STAT1, NF-κB, and C/EBP hardly influences the responses of APL cells to HDACi. We conclude that the affinity of HDACi for class I HDACs determines whether such drugs can kill naïve and maturated APL cells.