RESUMO
Among acute leukemias, mixed-lineage leukemia-rearranged (MLL-r) leukemia is associated with poor prognosis. Bromodomain and extra-terminal inhibitors (BETi) are promising agents for treatment of hematological malignancies; however, the mechanisms underlying sensitivity to BETi and biomarkers to predict sensitivity are yet to be clarified. Here, we established OTX015-resistant MLL-r cell lines (OTX015-R cells) and used them to explore therapeutic targets in BETi-resistant MLL-r leukemia. OTX015-R cells exhibited resistance to various BETi, and levels of bromodomain-containing protein 4 (BRD4) and BRD4-regulated molecules, such as c-MYC and B-cell/CLL lymphoma-2 (BCL-2), were remarkably increased in OTX015-R cells relative to those in the parental cells; however, BRD4 mRNA transcript levels were not elevated. These results suggest that overexpression of BRD4 protein, through suppression of BRD4 degradation, may contribute to BETi-resistance. Notably, expression of ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5) was increased in OTX015-R cells. Further, a UCHL5 inhibitor, b-AP15, and UCHL5 knockdown had antitumor effects by degrading BRD4. In addition, sensitivity to OTX015 was partially recovered in OTX015-R cells pretreated with b-AP15. Furthermore, cyclin-dependent kinase 4/6 (CDK4/6) inhibition decreased UCHL5 expression, suppressed OTX015-R cell proliferation, and induced apoptosis. These results indicate that the CDK4/6-UCHL5-BRD4 axis confers resistance to BETi by suppressing BRD4 degradation. We propose that this pathway is a potential novel therapeutic target in BETi-resistant MLL-r leukemia with BRD4 overexpression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Leucemia/patologia , Proteína de Leucina Linfoide-Mieloide/genética , Proteólise , Fatores de Transcrição/metabolismo , Ubiquitina Tiolesterase/metabolismo , Acetanilidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Compostos Heterocíclicos com 3 Anéis/farmacologia , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidoresRESUMO
Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEMLuc/GFP cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL.
Assuntos
Antineoplásicos/farmacologia , Proteína p300 Associada a E1A/antagonistas & inibidores , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proteína p300 Associada a E1A/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Rearranjo Gênico/efeitos dos fármacos , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multiple myeloma (MM) is characterized by the clonal proliferation of neoplastic plasma cells. Despite a stream of new molecular targets based on better understanding of the disease, MM remains incurable. Epigenomic abnormalities contribute to the pathogenesis of MM. bromodomain 4 (BRD4), a member of the bromodomain and extraterminal (BET) family, binds to acetylated histones during M/G1 transition in the cell cycle promoting progression to S phase. In this study, we investigated the effects of a novel BET inhibitor CG13250 on MM cells. CG13250 inhibited ligand binding to BRD4 in a dose-dependent manner and with an IC50 value of 1.1 µM. It inhibited MM proliferation in a dose-dependent manner and arrested cells in G1, resulting in the induction of apoptosis through caspase activation. CG13250 inhibited the binding of BRD4 to c-MYC promoter regions suppressing the transcription of the c-MYC gene. Administered in vivo, CG13250 significantly prolonged survival of an orthotopic MM-bearing mice. In conclusion, CG13250 is a novel bromodomain inhibitor that is a promising molecular targeting agent against MM.