RESUMO
Protein arginine methyltransferases (PRMT) are a widely expressed class of enzymes responsible for catalyzing arginine methylation on numerous protein substrates. Among them, type I PRMTs are responsible for generating asymmetric dimethylarginine. By controlling multiple basic cellular processes, such as DNA damage responses, transcriptional regulation, and mRNA splicing, type I PRMTs contribute to cancer initiation and progression. A type I PRMT inhibitor, GSK3368715, has been developed and has entered clinical trials for solid and hematologic malignancies. Although type I PRMTs have been reported to play roles in modulating immune cell function, the immunologic role of tumor-intrinsic pathways controlled by type I PRMTs remains uncharacterized. Here, our The Cancer Genome Atlas dataset analysis revealed that expression of type I PRMTs associated with poor clinical response and decreased immune infiltration in patients with melanoma. In cancer cell lines, inhibition of type I PRMTs induced an IFN gene signature, amplified responses to IFN and innate immune signaling, and decreased expression of the immunosuppressive cytokine VEGF. In immunocompetent mouse tumor models, including a model of T-cell exclusion that represents a common mechanism of anti-programmed cell death protein 1 (PD-1) resistance in humans, type I PRMT inhibition increased T-cell infiltration, produced durable responses dependent on CD8+ T cells, and enhanced efficacy of anti-PD-1 therapy. These data indicate that type I PRMT inhibition exhibits immunomodulatory properties and synergizes with immune checkpoint blockade (ICB) to induce durable antitumor responses in a T cell-dependent manner, suggesting that type I PRMT inhibition can potentiate an antitumor immunity in refractory settings.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteína-Arginina N-Metiltransferases , Animais , Arginina , Humanos , Imunidade , Camundongos , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.
Assuntos
Azacitidina , Leucemia Mieloide Aguda , Animais , Azacitidina/farmacologia , DNA/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/genética , Decitabina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , CamundongosRESUMO
Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.
Assuntos
Antineoplásicos/farmacocinética , Biomarcadores , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Arginina/metabolismo , Células Cultivadas , Cromatografia Líquida , Monitoramento de Medicamentos , Ativação Enzimática , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ribonucleoproteína Nuclear Heterogênea A1/sangue , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Espectrometria de Massas , Metilação , Camundongos , Terapia de Alvo Molecular , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Especificidade por SubstratoRESUMO
Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells. IMPLICATIONS: Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Histona Desmetilases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Técnicas de Inativação de Genes , Células HCT116 , Células HT29 , Histona Desmetilases/genética , Humanos , Mutação , Fosforilação , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , TransfecçãoRESUMO
Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/deficiência , Processamento Alternativo , Antineoplásicos/química , Biomarcadores , Linhagem Celular Tumoral , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Humanos , Metilação , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Proteína-Arginina N-Metiltransferases/química , Especificidade por SubstratoRESUMO
INTRODUCTION: This first-time-in-humans study assessed the safety, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity of GSK2879552 in patients with relapsed or refractory SCLC. METHODS: This phase I, multicenter, open-label study (NCT02034123) enrolled patients (≥18 years old) with relapsed or refractory SCLC (after ≥1 platinum-containing chemotherapy or refusal of standard therapy). Part 1 was a dose-escalation study; Part 2 was a dose-expansion study. Dose escalations were based on safety, PK, and PD. The primary end point (Part 1) was to determine the safety, tolerability, and recommended dose and regimen of GSK2879552. Secondary end points were to characterize PK and PD parameters and measure disease control rate at week 16. Part 2 was not conducted. RESULTS: Between February 4, 2014, and April 18, 2017, a total of 29 patients were allocated to one of nine dose cohorts (0.25 mg-3 mg once daily and 3-mg or 4-mg intermittent dosing). In all, 22 patients completed the study; 7 withdrew, primarily owing to adverse events (AEs). Most patients (24 of 29 [83%]) had at least one treatment-related AE, most commonly thrombocytopenia (12 of 29 [41%]). Twelve serious AEs (SAEs) were reported by nine patients; six were considered treatment related, the most common of which was encephalopathy (four SAEs). Three patients died; one death was related to SAEs. PK was characterized by rapid absorption, slow elimination, and a dose-proportional increase in exposure. CONCLUSIONS: GSK2879552 is a potent, selective inhibitor of lysine demethylase 1A and has demonstrated favorable PK properties but provided poor disease control and a high AE rate in patients with SCLC. The study was terminated, as the risk-benefit profile did not favor continuation.
Assuntos
Benzoatos/uso terapêutico , Ciclopropanos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Terapia de Salvação , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Adolescente , Adulto , Idoso , Benzoatos/farmacocinética , Ciclopropanos/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Prognóstico , Carcinoma de Pequenas Células do Pulmão/patologia , Taxa de Sobrevida , Distribuição Tecidual , Adulto JovemRESUMO
Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Transativadores/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Medula Óssea/patologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Epigênese Genética/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Análise de Célula Única , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Disruption of epigenetic regulation is a hallmark of acute myeloid leukemia (AML), but epigenetic therapy is complicated by the complexity of the epigenome. Herein, we developed a long-term primary AML ex vivo platform to determine whether targeting different epigenetic layers with 5-azacytidine and LSD1 inhibitors would yield improved efficacy. This combination was most effective in TET2 mut AML, where it extinguished leukemia stem cells and particularly induced genes with both LSD1-bound enhancers and cytosine-methylated promoters. Functional studies indicated that derepression of genes such as GATA2 contributes to drug efficacy. Mechanistically, combination therapy increased enhancer-promoter looping and chromatin-activating marks at the GATA2 locus. CRISPRi of the LSD1-bound enhancer in patient-derived TET2 mut AML was associated with dampening of therapeutic GATA2 induction. TET2 knockdown in human hematopoietic stem/progenitor cells induced loss of enhancer 5-hydroxymethylation and facilitated LSD1-mediated enhancer inactivation. Our data provide a basis for rational targeting of cooperating aberrant promoter and enhancer epigenetic marks driven by mutant epigenetic modifiers. SIGNIFICANCE: Somatic mutations of genes encoding epigenetic modifiers are a hallmark of AML and potentially disrupt many components of the epigenome. Our study targets two different epigenetic layers at promoters and enhancers that cooperate to aberrant gene silencing, downstream of the actions of a mutant epigenetic regulator.This article is highlighted in the In This Issue feature, p. 813.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Animais , Azacitidina/farmacologia , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Dioxigenases , Elementos Facilitadores Genéticos , Epigenoma , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Genes Supressores de Tumor , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Distribuição Aleatória , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epigenetic dysregulation is a common feature of most cancers, often occurring directly through alteration of epigenetic machinery. Over the last several years, a new generation of drugs directed at epigenetic modulators have entered clinical development, and results from these trials are now being disclosed. Unlike first-generation epigenetic therapies, these new agents are selective, and many are targeted to proteins which are mutated or translocated in cancer. This review will provide a summary of the epigenetic modulatory agents currently in clinical development and discuss the opportunities and challenges in their development. As these drugs advance in the clinic, drug discovery has continued with a focus on both novel and existing epigenetic targets. We will provide an overview of these efforts and the strategies being employed.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética , Neoplasias/tratamento farmacológico , Proteína-Arginina N-Metiltransferases/uso terapêutico , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Descoberta de Drogas , Histona Acetiltransferases/antagonistas & inibidores , Inibidores de Histona Desacetilases/uso terapêutico , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Humanos , Terapia de Alvo Molecular , Neoplasias/genéticaRESUMO
Drugs that modify the epigenome are powerful tools for treating cancer, but these drugs often have pleiotropic effects, and identifying patients who will benefit from them remains a major clinical challenge. Here we show that medulloblastomas driven by the transcription factor Gfi1 are exquisitely dependent on the enzyme lysine demethylase 1 (Kdm1a/Lsd1). We demonstrate that Lsd1 physically associates with Gfi1, and that these proteins cooperate to inhibit genes involved in neuronal commitment and differentiation. We also show that Lsd1 is essential for Gfi1-mediated transformation: Gfi1 proteins that cannot recruit Lsd1 are unable to drive tumorigenesis, and genetic ablation of Lsd1 markedly impairs tumor growth in vivo. Finally, pharmacological inhibitors of Lsd1 potently inhibit growth of Gfi1-driven tumors. These studies provide important insight into the mechanisms by which Gfi1 contributes to tumorigenesis, and identify Lsd1 inhibitors as promising therapeutic agents for Gfi1-driven medulloblastoma.
Assuntos
Carcinogênese/efeitos dos fármacos , Neoplasias Cerebelares/patologia , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases/metabolismo , Meduloblastoma/patologia , Fatores de Transcrição/metabolismo , Animais , Antibióticos Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/terapia , Proteínas de Ligação a DNA/genética , Doxorrubicina/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Histona Desmetilases/genética , Humanos , Meduloblastoma/genética , Meduloblastoma/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Células NIH 3T3 , Transplante de Neoplasias , Vírus Oncogênicos , Retroviridae , Fatores de Transcrição/genéticaRESUMO
Germinal center (GC) B cells feature repression of many gene enhancers to establish their characteristic transcriptome. Here we show that conditional deletion of Lsd1 in GCs significantly impaired GC formation, associated with failure to repress immune synapse genes linked to GC exit, which are also direct targets of the transcriptional repressor BCL6. We found that BCL6 directly binds LSD1 and recruits it primarily to intergenic and intronic enhancers. Conditional deletion of Lsd1 suppressed GC hyperplasia caused by constitutive expression of BCL6 and significantly delayed BCL6-driven lymphomagenesis. Administration of catalytic inhibitors of LSD1 had little effect on GC formation or GC-derived lymphoma cells. Using a CRISPR-Cas9 domain screen, we found instead that the LSD1 Tower domain was critical for dependence on LSD1 in GC-derived B cells. These results indicate an essential role for LSD1 in the humoral immune response, where it modulates enhancer function by forming repression complexes with BCL6.
Assuntos
Linfócitos B/fisiologia , Centro Germinativo/patologia , Histona Desmetilases/metabolismo , Linfoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Animais , Sistemas CRISPR-Cas , Carcinogênese , DNA Intergênico/genética , Centro Germinativo/imunologia , Histona Desmetilases/genética , Hiperplasia , Sinapses Imunológicas/genética , Íntrons/genética , Linfoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-6/genéticaRESUMO
Lysine specific demethylase 1 (LSD1) is a histone modifying enzyme that suppresses gene expression through demethylation of lysine 4 on histone H3. The anti-tumor activity of GSK2879552 and GSK-LSD1, potent, selective irreversible inactivators of LSD1, has previously been described. Inhibition of LSD1 results in a cytostatic growth inhibitory effect in a range of acute myeloid leukemia cell lines. To enhance the therapeutic potential of LSD1 inhibition in this disease setting, a combination of LSD1 inhibition and all-trans retinoic acid was explored. All-trans retinoic acid is currently approved for use in acute promyelocytic leukemia in which it promotes differentiation of abnormal blast cells into normal white blood cells. Combined treatment with all-trans retinoic acid and GSK2879552 results in synergistic effects on cell proliferation, markers of differentiation, and, most importantly, cytotoxicity. Ultimately the combination potential for LSD1 inhibition and ATRA will require validation in acute myeloid leukemia patients, and clinical studies to assess this are currently underway.
Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Tretinoína/farmacologia , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Benzoatos/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclopropanos/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Resultado do Tratamento , Tretinoína/administração & dosagemRESUMO
Epigenetic regulators are recurrently mutated and aberrantly expressed in acute myeloid leukemia (AML). Targeted therapies designed to inhibit these chromatin-modifying enzymes, such as the histone demethylase lysine-specific demethylase 1 (LSD1) and the histone methyltransferase DOT1L, have been developed as novel treatment modalities for these often refractory diseases. A common feature of many of these targeted agents is their ability to induce myeloid differentiation, suggesting that multiple paths toward a myeloid gene expression program can be engaged to relieve the differentiation blockade that is uniformly seen in AML. We performed a comparative assessment of chromatin dynamics during the treatment of mixed lineage leukemia (MLL)-AF9-driven murine leukemias and MLL-rearranged patient-derived xenografts using 2 distinct but effective differentiation-inducing targeted epigenetic therapies, the LSD1 inhibitor GSK-LSD1 and the DOT1L inhibitor EPZ4777. Intriguingly, GSK-LSD1 treatment caused global gains in chromatin accessibility, whereas treatment with EPZ4777 caused global losses in accessibility. We captured PU.1 and C/EBPα motif signatures at LSD1 inhibitor-induced dynamic sites and chromatin immunoprecipitation coupled with high-throughput sequencing revealed co-occupancy of these myeloid transcription factors at these sites. Functionally, we confirmed that diminished expression of PU.1 or genetic deletion of C/EBPα in MLL-AF9 cells generates resistance of these leukemias to LSD1 inhibition. These findings reveal that pharmacologic inhibition of LSD1 represents a unique path to overcome the differentiation block in AML for therapeutic benefit.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Leucemia Aguda Bifenotípica/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Leucemia Aguda Bifenotípica/genética , Leucemia Aguda Bifenotípica/metabolismo , Leucemia Aguda Bifenotípica/patologia , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas/genética , Elementos de Resposta , Transativadores/genéticaRESUMO
Most, if not all, human cancers exhibit altered epigenetic signatures that promote aberrant gene expression that contributes to cellular transformation. Historically, attempts to pharmacologically intervene in this process have focused on DNA methylation and histone acetylation. More recently, genome-wide studies have identified histone and chromatin regulators as one of the most frequently dysregulated functional classes in a wide range of cancer types. These findings have provided numerous potential therapeutic targets including many that affect histone methylation. These include histone lysine methyltransferases such as enhancer of zeste homolog 2 and DOT1L, protein arginine methyltransferases such as protein arginine methyltransferase 5, and histone lysine demethylases such as lysine-specific demethylase 1. This review presents the rationale for targeting histone methylation in oncology and provides an update on a few key targets that are being investigated in the clinic.
Assuntos
Epigênese Genética , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histonas/genética , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Metilação/efeitos dos fármacos , Neoplasias/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
Epigenetic machinery have become a major focus for new targeted cancer therapies. Our previous report described the discovery and biological activity of a potent, selective, orally bioavailable, irreversible inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) was sensitive to LSD1 inhibition. The SCLC lines that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes suggesting this may be used as a predictive biomarker of activity. This targeted mechanism coupled with a novel predictive biomarker make LSD1 inhibition an exciting potential therapy for SCLC.
RESUMO
SMYD3 is a lysine methyltransferase overexpressed in colorectal, breast, prostate, and hepatocellular tumors, and has been implicated as an oncogene in human malignancies. Methylation of MEKK2 by SMYD3 is important for regulation of the MEK/ERK pathway, suggesting the possibility of selectively targeting SMYD3 in RAS-driven cancers. Structural and kinetic characterization of SMYD3 was undertaken leading to a co-crystal structure of SMYD3 with a MEKK2-peptide substrate bound, and the observation that SMYD3 follows a partially processive mechanism. These insights allowed for the design of GSK2807, a potent and selective, SAM-competitive inhibitor of SMYD3 (Ki = 14 nM). A high-resolution crystal structure reveals that GSK2807 bridges the gap between the SAM-binding pocket and the substrate lysine tunnel of SMYD3. Taken together, our data demonstrate that small-molecule inhibitors of SMYD3 can be designed to prevent methylation of MEKK2 and these could have potential use as anticancer therapeutics.
Assuntos
Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/química , Simulação de Acoplamento Molecular , Sítios de Ligação , Inibidores Enzimáticos/química , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , MAP Quinase Quinase Quinase 2/metabolismo , Mutação , Ligação Proteica , S-Adenosilmetionina/farmacologiaRESUMO
Epigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inactivator of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes, suggesting this may be used as a predictive biomarker of activity.
Assuntos
Antineoplásicos/administração & dosagem , Benzoatos/administração & dosagem , Ciclopropanos/administração & dosagem , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Histona Desmetilases/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Administração Oral , Animais , Antineoplásicos/farmacologia , Benzoatos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclopropanos/farmacologia , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Dados de Sequência Molecular , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Innovative therapies are needed for advanced Non-Small Cell Lung Cancer (NSCLC). We have undertaken a genomics based, hypothesis driving, approach to query an emerging potential that epigenetic therapy may sensitize to immune checkpoint therapy targeting PD-L1/PD-1 interaction. NSCLC cell lines were treated with the DNA hypomethylating agent azacytidine (AZA - Vidaza) and genes and pathways altered were mapped by genome-wide expression and DNA methylation analyses. AZA-induced pathways were analyzed in The Cancer Genome Atlas (TCGA) project by mapping the derived gene signatures in hundreds of lung adeno (LUAD) and squamous cell carcinoma (LUSC) samples. AZA up-regulates genes and pathways related to both innate and adaptive immunity and genes related to immune evasion in a several NSCLC lines. DNA hypermethylation and low expression of IRF7, an interferon transcription factor, tracks with this signature particularly in LUSC. In concert with these events, AZA up-regulates PD-L1 transcripts and protein, a key ligand-mediator of immune tolerance. Analysis of TCGA samples demonstrates that a significant proportion of primary NSCLC have low expression of AZA-induced immune genes, including PD-L1. We hypothesize that epigenetic therapy combined with blockade of immune checkpoints - in particular the PD-1/PD-L1 pathway - may augment response of NSCLC by shifting the balance between immune activation and immune inhibition, particularly in a subset of NSCLC with low expression of these pathways. Our studies define a biomarker strategy for response in a recently initiated trial to examine the potential of epigenetic therapy to sensitize patients with NSCLC to PD-1 immune checkpoint blockade.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Imunoterapia/métodos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Antígeno B7-H1/biossíntese , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Epigenômica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais , Regulação para Cima/efeitos dos fármacosRESUMO
While DNA methyltransferase1 (DNMT1) is classically known for its functions as a maintenance methyltransferase enzyme, additional roles for DNMT1 in gene expression are not as clearly understood. Several groups have shown that deletion of the catalytic domain from DNMT1 does not abolish repressive activity of the protein against a reporter gene. In our studies, we examine the repressor function of catalytically inactive DNMT1 at endogenous genes. First, potential DNMT1 target genes were identified by searching for genes up-regulated in HCT116 colon cancer cells genetically disrupted for DNMT1 (DNMT1(-/-) hypomorph cells). Next, the requirement for DNMT1 activity for repression of these genes was assessed by stably restoring expression of wild-type or catalytically inactive DNMT1. Both wild-type and mutant proteins are able to occupy the promoters and repress the expression of a set of target genes, and induce, at these promoters, both the depletion of active histone marks and the recruitment of a H3K4 demethylase, KDM1A/LSD1. Together, our findings show that there are genes for which DNMT1 acts as a transcriptional repressor independent from its methyltransferase function and that this repressive function may invoke a role for a scaffolding function of the protein at target genes.