RESUMO
Inhibitor of DNA binding 1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types, including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin-specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a ubiquitin-rhodamine-based high-throughput screening, we identified small-molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose-dependent manner. A known USP1 inhibitor, pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary acute myelogenous leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small-molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy.
Assuntos
Proteínas de Arabidopsis/antagonistas & inibidores , Proteína 1 Inibidora de Diferenciação/metabolismo , Leucemia/metabolismo , Inibidores de Proteases/farmacologia , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Proteínas de Arabidopsis/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática/efeitos dos fármacos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Ensaios de Triagem em Larga Escala , Recombinação Homóloga/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Leucemia/genética , Camundongos , Pimozida/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ligação Proteica , Proteólise/efeitos dos fármacos , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , DNA/biossíntese , Endodesoxirribonucleases/metabolismo , Animais , Pesquisa Biomédica , Proteínas Estimuladoras de Ligação a CCAAT/genética , Endodesoxirribonucleases/genética , Humanos , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: DNA double-strand breaks (DSBs) caused by ionizing radiation or by the stalling of DNA replication forks are among the most deleterious forms of DNA damage. The ability of cells to recognize and repair DSBs requires post-translational modifications to histones and other proteins that facilitate access to lesions in compacted chromatin, however our understanding of these processes remains incomplete. UHRF1 is an E3 ubiquitin ligase that has previously been linked to events that regulate chromatin remodeling and epigenetic maintenance. Previous studies have demonstrated that loss of UHRF1 increases the sensitivity of cells to DNA damage however the role of UHRF1 in this response is unclear. RESULTS: We demonstrate that UHRF1 plays a critical role for facilitating the response to DSB damage caused by gamma-irradiation. UHRF1-depleted cells exhibit increased sensitivity to gamma-irradiation, suggesting a compromised cellular response to DSBs. UHRF1-depleted cells show impaired cell cycle arrest and an impaired accumulation of histone H2AX phosphorylation (gammaH2AX) in response to gamma-irradiation compared to control cells. We also demonstrate that UHRF1 is required for genome integrity, in that UHRF1-depleted cells displayed an increased frequency of chromosomal aberrations compared to control cells. CONCLUSIONS: Our findings indicate a critical role for UHRF1 in maintenance of chromosome integrity and an optimal response to DSB damage.
RESUMO
Mus81 (methyl methansulfonate UV sensitive clone 81) and Eme1 (essential meiotic endonuclease 1, also known as MMS4) form a heterodimeric endonuclease that is critical for genomic stability and the response to DNA crosslink damage and replication blockade. However, relatively little is known as to how this endonuclease is regulated following DNA damage. Here, we report mammalian Eme1 interacts with Np95, an E3 ubiquitin ligase that participates in chromatin modification, replication-linked epigenetic maintenance and the DNA damage response. Np95 and Eme1 co-localize on nuclear chromatin following exposure of cells to camptothecin, an agent that promotes the collapse of replication forks. The observed co localization following DNA damage was found to be dependent on an intact RING finger, the structural motif that encodes the E3 ubiquitin ligase activity of Np95. Taken together, these findings link Mus81-Eme1 with the replication-associated chromatin modifier functions of Np95 in the cellular response to DNA damage.
