RESUMO
PURPOSE: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. EXPERIMENTAL DESIGN: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. RESULTS: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. CONCLUSIONS: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.
Assuntos
Histona Desmetilases com o Domínio Jumonji/genética , Leucemia de Células T/genética , Receptor Notch1/genética , Peptidase 7 Específica de Ubiquitina/genética , Animais , Carcinogênese/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Terapia Genética , Humanos , Células Jurkat , Leucemia de Células T/patologia , Leucemia de Células T/terapia , Camundongos , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ubiquitin specific protease 7 (USP7), the most widely studied among the nearly 100 deubiquitinating enzymes, supports cancer by positively affecting tumor growth and negatively affecting the patient's immune response to tumors. Great interest exists, therefore, in developing USP7 inhibitors for clinical evaluation. While the proteasome inhibitor field has enjoyed clinical success, very few clinically appropriate effectors of deubiquitinating (protease) or ubiquitinating (ligase) enzymes have appeared. The ubiquitin protease/ligase field is moving from the initial discovery of potent, selective modulators with cell proof of concept and in vivo activity to the optimization of these molecules to impart drug-like properties or the discovery of new inhibitor scaffolds by improved screening or rational design. This Perspective focuses on the current status of USP7 inhibitors from various organizations active in developing these compounds for the clinic and suggests undertakings that are both achievable and necessary to lead to successful clinical outcomes for USP7 inhibitors in cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Peptidase 7 Específica de Ubiquitina/metabolismo , Antineoplásicos/química , Ensaios Clínicos como Assunto , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Terapia de Alvo Molecular/métodos , Inibidores de Proteassoma/farmacologia , Conformação Proteica , Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/imunologiaRESUMO
Accumulation of Foxp3+ T-regulatory (Treg) cells in the tumor microenvironment is associated with tumor immune evasion and poor patient outcome in the case of many solid tumors. Current therapeutic strategies for blocking Treg functions are not Treg-specific, and display only modest and transient efficacy. Recent studies revealed that ubiquitin-specific protease 7 (USP7) is essential for Treg functions by stabilizing expression of Tip60 and Foxp3, which together are central to the development and maintenance of the Treg cell lineage. Pharmacological inhibition of USP7 is therefore a promising strategy for suppressing Treg functions and promoting anti-tumor immunity. Previously, we reported the P5091 series of small molecule USP7 inhibitors and demonstrated their direct anti-tumor activity in vivo using xenograft models. However, the precise mechanism of action of these compounds was not well defined. In this study, we report the development and characterization of P217564, a second-generation USP7 inhibitor with improved potency and selectivity. P217564 selectively targets the catalytic cleft of USP7 and modifies its active site cysteine (C223) by forming a covalent adduct. Irreversible inhibition of USP7 results in durable downstream biological responses in cells, including down-regulation of Tip60 and consequent impairment of Treg suppressive function. In addition, we demonstrate that both USP7 and various USP7 substrates are subjected to Lys48-mediated ubiquitin modification, consistent with increased proteasomal degradation of these proteins because of USP7 inhibition.
Assuntos
Proteínas de Drosophila/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Histona Acetiltransferases/metabolismo , Linfócitos T Reguladores/imunologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Domínio Catalítico , Linhagem Celular Tumoral , Humanos , Inibidores de Proteases/farmacologia , Peptidase 7 Específica de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
USP7 is a deubiquitinating enzyme that plays a pivotal role in multiple oncogenic pathways and therefore is a desirable target for new anti-cancer therapies. However, the lack of structural information about the USP7-inhibitor interactions has been a critical gap in the development of potent inhibitors. USP7 is unique among USPs in that its active site is catalytically incompetent, and is postulated to rearrange into a productive conformation only upon binding to ubiquitin. Surprisingly, we found that ubiquitin alone does not induce an active conformation in solution. Using a combination of nuclear magnetic resonance, mass spectrometry, computational modeling, and cell-based assays, we found that DUB inhibitors P22077 and P50429 covalently modify the catalytic cysteine of USP7 and induce a conformational switch in the enzyme associated with active site rearrangement. This work represents the first experimental insights into USP7 activation and inhibition and provides a structural basis for rational development of potent anti-cancer therapeutics.
Assuntos
Inibidores de Proteases/farmacologia , Tiofenos/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Domínio Catalítico , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/química , Relação Estrutura-Atividade , Especificidade por Substrato , Tiofenos/química , Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismoRESUMO
Foxp3+ T-regulatory (Treg) cells are known to suppress protective host immune responses to a wide variety of solid tumors, but their therapeutic targeting is largely restricted to their transient depletion or "secondary" modulation, e.g. using anti-CTLA-4 monoclonal antibody. Our ongoing studies of the post-translational modifications that regulate Foxp3 demonstrated that the histone/protein acetyltransferase, Tip60, plays a dominant role in promoting acetylation, dimerization and function in Treg cells. We now show that the ubiquitin-specific protease, Usp7, controls Treg function largely by stabilizing the expression and promoting the multimerization of Tip60 and Foxp3. Genetic or pharmacologic targeting of Usp7 impairs Foxp3+ Treg suppressive functions, while conventional T cell responses remain intact. As a result, pharmacologic inhibitors of Usp7 can limit tumor growth in immunocompetent mice, and promote the efficacy of antitumor vaccines and immune checkpoint therapy with anti-PD1 monoclonal antibody in murine models. Hence, pharmacologic therapy with Usp7 inhibitors may have an important role in future cancer immunotherapy.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Histona Acetiltransferases/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transativadores/metabolismo , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Animais , Autoimunidade/genética , Autoimunidade/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Expressão Gênica , Imunidade , Ativação Linfocitária/imunologia , Lisina Acetiltransferase 5 , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neoplasias/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Carga Tumoral , Peptidase 7 Específica de Ubiquitina , Proteases Específicas de Ubiquitina/genéticaRESUMO
Screening of our internal compound collection for inhibitors of the transforming growth factor beta1 (TGF-beta1) type I receptor (ALK5) identified several hits. Optimization of the dihydropyrroloimidazole hit 2 by introduction of a 2-pyridine and 3,4-methylenedioxyphenyl group gave 7, a selective ALK5 inhibitor. With this information, optimization of the triarylimidazole hit 8 gave the selective inhibitor 14, which inhibits TGF-beta1-induced fibronectin mRNA formation while displaying no measurable cytotoxicity in the 48 h XTT assay.
Assuntos
Receptores de Ativinas Tipo I/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Imidazóis/síntese química , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fibronectinas/biossíntese , Fibronectinas/genética , Humanos , Imidazóis/química , Imidazóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Serina-Treonina Quinases , RNA Mensageiro/biossíntese , Receptor do Fator de Crescimento Transformador beta Tipo I , Proteína Smad3 , Relação Estrutura-Atividade , Transativadores/metabolismo , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Inhibitors of the cancer-related cysteine isopeptidase human ubiquitin-specific proteases 7 (USP7) and 47 (USP47) are considered to have potential as cancer therapeutics, owing to their ability to stabilize the tumor suppressor p53 and to decrease DNA polymerase ß (Polß), both of which are potential anticancer effects. A new class of dual small molecule inhibitors of these enzymes has been discovered. Compound 1, a selective inhibitor of USP7 and USP47 with moderate potency, demonstrates inhibition of USP7 in cells and induces elevated p53 and apoptosis in cancer cell lines. Compound 1 has been shown to demonstrate modest activity in human xenograft multiple myeloma and B-cell leukemia in vivo models. This activity may be the result of dual inhibition of USP7 and USP47. To address issues regarding potency and developability, analogues of compound 1 have been synthesized and tested, leading to improvements in potency, solubility, and metabolic reactivity profile. Further optimization is expected to yield preclinical candidates and, ultimately, clinical candidates for the treatment of multiple myeloma, prostate cancer, and other cancers.
RESUMO
Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.