RESUMO
The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.
Assuntos
Neoplasias/tratamento farmacológico , Nucleosídeos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Imidas/farmacologia , Camundongos , Neoplasias/genética , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Pirazóis , Pirimidinas , Sulfetos , Ubiquitina/antagonistas & inibidores , Ubiquitina/química , Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Guanylyl cyclase C (GCC) is a cell-surface protein that is expressed by normal intestinal epithelial cells, more than 95% of metastatic colorectal cancers (mCRC), and the majority of gastric and pancreatic cancers. Due to strict apical localization, systemically delivered GCC-targeting agents should not reach GCC in normal intestinal tissue, while accessing antigen in tumor. We generated an investigational antibody-drug conjugate (TAK-264, formerly MLN0264) comprising a fully human anti-GCC monoclonal antibody conjugated to monomethyl auristatin E via a protease-cleavable peptide linker. TAK-264 specifically bound, was internalized by, and killed GCC-expressing cells in vitro in an antigen-density-dependent manner. In GCC-expressing xenograft models with similar GCC expression levels/patterns observed in human mCRC samples, TAK-264 induced cell death, leading to tumor regressions and long-term tumor growth inhibition. TAK-264 antitumor activity was generally antigen-density-dependent, although some GCC-expressing tumors were refractory to TAK-264-targeted high local concentrations of payload. These data support further evaluation of TAK-264 in the treatment of GCC-expressing tumors.
Assuntos
Anticorpos Monoclonais/imunologia , Imunoconjugados/farmacologia , Oligopeptídeos/metabolismo , Receptores de Enterotoxina/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Western Blotting , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Feminino , Células HEK293 , Humanos , Mucosa Intestinal/enzimologia , Camundongos , Camundongos SCID , Receptores de Enterotoxina/genética , Receptores de Enterotoxina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Small ubiquitin-like modifier (SUMO) family proteins regulate target-protein functions by post-translational modification. However, a potent and selective inhibitor targeting the SUMO pathway has been lacking. Here we describe ML-792, a mechanism-based SUMO-activating enzyme (SAE) inhibitor with nanomolar potency in cellular assays. ML-792 selectively blocks SAE enzyme activity and total SUMOylation, thus decreasing cancer cell proliferation. Moreover, we found that induction of the MYC oncogene increased the ML-792-mediated viability effect in cancer cells, thus indicating a potential application of SAE inhibitors in treating MYC-amplified tumors. Using ML-792, we further explored the critical roles of SUMOylation in mitotic progression and chromosome segregation. Furthermore, expression of an SAE catalytic-subunit (UBA2) S95N M97T mutant rescued SUMOylation loss and the mitotic defect induced by ML-792, thus confirming the selectivity of ML-792. As a potent and selective SAE inhibitor, ML-792 provides rapid loss of endogenously SUMOylated proteins, thereby facilitating novel insights into SUMO biology.
Assuntos
Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Sumoilação , Proliferação de Células/efeitos dos fármacos , Segregação de Cromossomos/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes myc , Humanos , Mitose/efeitos dos fármacos , Neoplasias/genética , Neoplasias/patologia , Processamento de Proteína Pós-Traducional , Células Tumorais CultivadasRESUMO
Multiple pathways have been proposed to explain how proteasome inhibition induces cell death, but mechanisms remain unclear. To approach this issue, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity to bortezomib (Velcade (R); PS-341). This screen identified 100 genes whose knockdown affected lethality to bortezomib and to a structurally diverse set of other proteasome inhibitors. A comparison of three cell lines revealed that 39 of 100 genes were commonly linked to cell death. We causally linked bortezomib-induced cell death to the accumulation of ASF1B, Myc, ODC1, Noxa, BNIP3, Gadd45alpha, p-SMC1A, SREBF1, and p53. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death.
Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Morte Celular/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Pirazinas/farmacologia , RNA Interferente Pequeno/genética , Bortezomib , Morte Celular/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dano ao DNA , Técnicas de Silenciamento de Genes , Células HCT116 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Ribossomos/efeitos dos fármacos , Serina-Treonina Quinases TOR , TransfecçãoRESUMO
Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle, as its perturbation causes defects in centrosome separation, spindle pole organization, and chromosome congression. Moreover, Aurora A disruption leads to cell death via a mechanism that involves aneuploidy generation. However, the link between the immediate functional consequences of Aurora A inhibition and the development of aneuploidy is not clearly defined. In this study, we delineate the sequence of events that lead to aneuploidy following Aurora A inhibition using MLN8054, a selective Aurora A small-molecule inhibitor. Human tumor cells treated with MLN8054 show a high incidence of abnormal mitotic spindles, often with unseparated centrosomes. Although these spindle defects result in mitotic delays, cells ultimately divide at a frequency near that of untreated cells. We show that many of the spindles in the dividing cells are bipolar, although they lack centrosomes at one or more spindle poles. MLN8054-treated cells frequently show alignment defects during metaphase, lagging chromosomes in anaphase, and chromatin bridges during telophase. Consistent with the chromosome segregation defects, cells treated with MLN8054 develop aneuploidy over time. Taken together, these results suggest that Aurora A inhibition kills tumor cells through the development of deleterious aneuploidy.