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1.
Blood ; 137(5): 661-677, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33197925

RESUMO

A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).


Assuntos
Acetamidas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Isoindóis/farmacologia , Leucemia Mieloide Aguda/patologia , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Piperidonas/farmacologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Acetamidas/uso terapêutico , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Humanos , Isoindóis/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Células-Tronco Neoplásicas/enzimologia , Proteína do Fator Nuclear 45/fisiologia , Proteínas do Fator Nuclear 90/fisiologia , Fatores de Terminação de Peptídeos/metabolismo , Piperidonas/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise , Bibliotecas de Moléculas Pequenas , Estresse Fisiológico , Serina-Treonina Quinases TOR/fisiologia , Células U937 , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Br J Haematol ; 172(6): 889-901, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26914976

RESUMO

Pomalidomide is an IMiD(®) immunomodulatory agent, which has shown clinically significant benefits in relapsed and/or refractory multiple myeloma (rrMM) patients when combined with dexamethasone, regardless of refractory status to lenalidomide or bortezomib. (Schey et al, ; San Miguel et al, 2013; Richardson et al, 2014; Scott, ) In this work, we present preclinical data showing that the combination of pomalidomide with dexamethasone (PomDex) demonstrates potent anti-proliferative and pro-apoptotic activity in both lenalidomide-sensitive and lenalidomide-resistant MM cell lines. PomDex also synergistically inhibited tumour growth compared with single-agent treatment in xenografts of lenalidomide-resistant H929 R10-1 cells. Typical hallmarks of IMiD compound activity, including IKZF3 (Aiolos) degradation, and the downregulation of interferon regulatory factor (IRF) 4 and MYC, seen in lenalidomide-sensitive H929 MM cell lines, were also observed in PomDex-treated lenalidomide-resistant H929 MM cells. Remarkably, this resulted in strong, synergistic effects on the induction of apoptosis in both lenalidomide-sensitive and resistant MM cells. Furthermore, gene expression profiling revealed a unique differential gene expression pattern in PomDex-treated samples, highlighted by the modulation of pro-apoptotic pathways in lenalidomide-resistant cells. These results provide key insights into molecular mechanisms of PomDex in the lenalidomide-resistant setting.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dexametasona/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunomodulação/efeitos dos fármacos , Lenalidomida , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Talidomida/administração & dosagem , Talidomida/análogos & derivados , Talidomida/uso terapêutico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
3.
Br J Haematol ; 164(2): 233-44, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24206017

RESUMO

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Talidomida/análogos & derivados , Talidomida/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Mieloma Múltiplo/tratamento farmacológico , Peptídeo Hidrolases/imunologia , Isoformas de RNA , Talidomida/uso terapêutico , Ubiquitina-Proteína Ligases
4.
Br J Haematol ; 154(3): 325-36, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21707574

RESUMO

Overexpression of the transcription factor interferon regulatory factor-4 (IRF4), which is common in multiple myeloma (MM), is associated with poor prognosis. Patients with higher IRF4 expression have significantly poorer overall survival than those with low IRF4 expression. Lenalidomide is an IMiD immunomodulatory compound that has both tumouricidal and immunomodulatory activity in MM. This study showed that lenalidomide downregulated IRF4 levels in MM cell lines and bone marrow samples within 8 h of drug exposure. This was associated with a decrease in MYC levels, as well as an initial G1 cell cycle arrest, decreased cell proliferation, and cell death by day 5 of treatment. In eight MM cell lines, high IRF4 levels correlated with increased lenalidomide sensitivity. The clinical significance of this observation was investigated in 154 patients with MM. Among MM patients with high levels of IRF4 expression, treatment with lenalidomide led to a significantly longer overall survival than other therapies in a retrospective analysis. These data confirm the central role of IRF4 in MM pathogenesis; indicate that this is an important mechanism by which lenalidomide exerts its antitumour effects; and may provide a mechanistic biomarker to predict response to lenalidomide.


Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/biossíntese , Fatores Reguladores de Interferon/biossíntese , Mieloma Múltiplo/metabolismo , Talidomida/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Proliferação de Células , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Genes myc , Humanos , Fatores Reguladores de Interferon/antagonistas & inibidores , Fatores Reguladores de Interferon/genética , Lenalidomida , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Prognóstico , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Talidomida/farmacologia , Talidomida/uso terapêutico , Resultado do Tratamento , Células Tumorais Cultivadas
5.
Blood ; 111(9): 4690-9, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18305219

RESUMO

Decreased p27(Kip1) levels are a poor prognostic factor in many malignancies, and can occur through up-regulation of SCF(Skp2) E3 ligase function, resulting in enhanced p27 ubiquitination and proteasome-mediated degradation. While proteasome inhibitors stabilize p27(Kip1), agents inhibiting SCF(Skp2) may represent more directly targeted drugs with the promise of enhanced efficacy and reduced toxicity. Using high-throughput screening, we identified Compound A (CpdA), which interfered with SCF(Skp2) ligase function in vitro, and induced specific accumulation of p21 and other SCF(Skp2) substrates in cells without activating a heat-shock protein response. CpdA prevented incorporation of Skp2 into the SCF(Skp2) ligase, and induced G(1)/S cell-cycle arrest as well as SCF(Skp2)- and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCF(Skp2) inhibitors as a novel class of antitumor agents.


Assuntos
Antineoplásicos/farmacologia , Autofagia , Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos , Humanos , Mieloma Múltiplo/tratamento farmacológico
6.
J Med Chem ; 63(13): 6648-6676, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32130004

RESUMO

Many patients with multiple myeloma (MM) initially respond to treatment with modern combination regimens including immunomodulatory agents (lenalidomide and pomalidomide) and proteasome inhibitors. However, some patients lack an initial response to therapy (i.e., are refractory), and although the mean survival of MM patients has more than doubled in recent years, most patients will eventually relapse. To address this need, we explored the potential of novel cereblon E3 ligase modulators (CELMoDs) for the treatment of patients with relapsed or refractory multiple myeloma (RRMM). We found that optimization beyond potency of degradation, including degradation efficiency and kinetics, could provide efficacy in a lenalidomide-resistant setting. Guided by both phenotypic and protein degradation data, we describe a series of CELMoDs for the treatment of RRMM, culminating in the discovery of CC-92480, a novel protein degrader and the first CELMoD to enter clinical development that was specifically designed for efficient and rapid protein degradation kinetics.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Mieloma Múltiplo/patologia , Recidiva , Estereoisomerismo , Falha de Tratamento , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Elife ; 72018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30234487

RESUMO

The cereblon modulating agents (CMs) including lenalidomide, pomalidomide and CC-220 repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the K48-linked polyubiquitination of CRL4CRBN neomorphic substrates via a sequential ubiquitination mechanism. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, it will be of fundamental interest to explore if loss of UBE2G1 activity is linked to clinical resistance to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins.


Assuntos
Peptídeo Hidrolases/metabolismo , Proteólise , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Fator de Transcrição Ikaros/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Talidomida/análogos & derivados , Talidomida/farmacologia , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
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