RESUMEN
The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.
Asunto(s)
Antineoplásicos , Leucemia Mieloide Aguda , Adenosina Trifosfatasas/metabolismo , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Reparación del ADN , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Proteína que Contiene ValosinaRESUMEN
RUVBL1 and RUVBL2 are ATPases associated with diverse cellular activities (AAAs) that form a complex involved in a variety of cellular processes, including chromatin remodeling and regulation of gene expression. RUVBLs have a strong link to oncogenesis, where overexpression is correlated with tumor growth and poor prognosis in several cancer types. CB-6644, an allosteric small-molecule inhibitor of the ATPase activity of the RUVBL1/2 complex, interacts specifically with RUVBL1/2 in cancer cells, leading to cell death. Importantly, drug-acquired-resistant cell clones have amino acid mutations in either RUVBL1 or RUVBL2, suggesting that cell killing is an on-target consequence of RUVBL1/2 engagement. In xenograft models of acute myeloid leukemia and multiple myeloma, CB-6644 significantly reduced tumor growth without obvious toxicity. This work demonstrates the therapeutic potential of targeting RUVBLs in the treatment of cancer and establishes a chemical entity for probing the many facets of RUVBL biology.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , Antineoplásicos/farmacología , Azepinas/farmacología , Benzamidas/farmacología , Proteínas Portadoras/antagonistas & inhibidores , ADN Helicasas/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Células HCT116 , Humanos , Mutación , Unión ProteicaRESUMEN
Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375-86. ©2017 AACR.
Asunto(s)
Adenosina Trifosfatasas/genética , Indoles/administración & dosificación , Mieloma Múltiple/tratamiento farmacológico , Proteínas Nucleares/genética , Factor Nuclear 1 de Respiración/genética , Inhibidores de Proteasoma/administración & dosificación , Pirimidinas/administración & dosificación , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Proteínas Nucleares/antagonistas & inhibidores , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Ubiquitina/genética , Respuesta de Proteína Desplegada/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
p97 is a AAA-ATPase with multiple cellular functions, one of which is critical regulation of protein homeostasis pathways. We describe the characterization of CB-5083, a potent, selective, and orally bioavailable inhibitor of p97. Treatment of tumor cells with CB-5083 leads to accumulation of poly-ubiquitinated proteins, retention of endoplasmic reticulum-associated degradation (ERAD) substrates, and generation of irresolvable proteotoxic stress, leading to activation of the apoptotic arm of the unfolded protein response. In xenograft models, CB-5083 causes modulation of key p97-related pathways, induces apoptosis, and has antitumor activity in a broad range of both hematological and solid tumor models. Molecular determinants of CB-5083 activity include expression of genes in the ERAD pathway, providing a potential strategy for patient selection.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Homeostasis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Sistemas CRISPR-Cas , Línea Celular Tumoral , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HEK293 , Humanos , Indoles/química , Indoles/farmacología , Células K562 , Ratones Desnudos , Ratones SCID , Estructura Molecular , Terapia Molecular Dirigida/métodos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pirimidinas/química , Pirimidinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ubiquitinadas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The AAA-ATPase p97 plays vital roles in mechanisms of protein homeostasis, including ubiquitin-proteasome system (UPS) mediated protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Herein we describe our lead optimization efforts focused on in vitro potency, ADME, and pharmaceutical properties that led to the discovery of a potent, ATP-competitive, D2-selective, and orally bioavailable p97 inhibitor 71, CB-5083. Treatment of tumor cells with 71 leads to significant accumulation of markers associated with inhibition of UPS and ERAD functions, which induces irresolvable proteotoxic stress and cell death. In tumor bearing mice, oral administration of 71 causes rapid accumulation of markers of the unfolded protein response (UPR) and subsequently induces apoptosis leading to sustained antitumor activity in in vivo xenograft models of both solid and hematological tumors. 71 has been taken into phase 1 clinical trials in patients with multiple myeloma and solid tumors.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Antineoplásicos/química , Indoles/química , Proteínas Nucleares/antagonistas & inhibidores , Pirimidinas/química , Administración Oral , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Apoptosis , Disponibilidad Biológica , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Xenoinjertos , Humanos , Indoles/farmacocinética , Indoles/farmacología , Ratones Desnudos , Simulación del Acoplamiento Molecular , Trasplante de Neoplasias , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Relación Estructura-Actividad , Ubiquitina/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
The ATP-dependent degradation of polyubiquitylated proteins by the 26S proteasome is essential for the maintenance of proteome stability and the regulation of a plethora of cellular processes. Degradation of substrates is preceded by the removal of polyubiquitin moieties through the isopeptidase activity of the subunit Rpn11. Here we describe three crystal structures of the heterodimer of the Mpr1-Pad1-N-terminal domains of Rpn8 and Rpn11, crystallized as a fusion protein in complex with a nanobody. This fusion protein exhibits modest deubiquitylation activity toward a model substrate. Full activation requires incorporation of Rpn11 into the 26S proteasome and is dependent on ATP hydrolysis, suggesting that substrate processing and polyubiquitin removal are coupled. Based on our structures, we propose that premature activation is prevented by the combined effects of low intrinsic ubiquitin affinity, an insertion segment acting as a physical barrier across the substrate access channel, and a conformationally unstable catalytic loop in Rpn11. The docking of the structure into the proteasome EM density revealed contacts of Rpn11 with ATPase subunits, which likely stabilize the active conformation and boost the affinity for the proximal ubiquitin moiety. The narrow space around the Rpn11 active site at the entrance to the ATPase ring pore is likely to prevent erroneous deubiquitylation of folded proteins.
Asunto(s)
Endopeptidasas/química , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/química , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas de Saccharomyces cerevisiae/química , Cristalografía , Dimerización , Endopeptidasas/metabolismo , Modelos Biológicos , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismoRESUMEN
Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.
RESUMEN
Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma and non-Hodgkin's lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for improved dosing flexibility and patient convenience over intravenously administered agents. The lead compound, 2-Me-5-thiazole-Ser(OMe)-Ser(OMe)-Phe-ketoepoxide (58) (PR-047), selectively inhibited CT-L activity of both the constitutive proteasome (beta5) and immunoproteasome (LMP7) and demonstrated an absolute bioavailability of up to 39% in rodents and dogs. It was well tolerated with repeated oral administration at doses resulting in >80% proteasome inhibition in most tissues and elicited an antitumor response equivalent to intravenously administered carfilzomib in multiple human tumor xenograft and mouse syngeneic models. The favorable pharmacologic profile supports its further development for the treatment of malignant diseases.