RESUMO
Non-genotoxic reactivation of the p53 pathway by MDM2-p53 binding antagonists is an attractive treatment strategy for wild-type TP53 cancers. To determine how resistance to MDM2/p53 binding antagonists might develop, SJSA-1 and NGP cells were exposed to growth inhibitory concentrations of chemically distinct MDM2 inhibitors, Nutlin-3 and MI-63, and clonal resistant cell lines generated. The p53 mediated responses of parental and resistant cell lines were compared. In contrast to the parental cell lines, p53 activation by Nutlin-3, MI-63 or ionizing radiation was not observed in either the SJSA-1 or the NGP derived cell lines. An identical TP53 mutation was subsequently identified in both of the SJSA-1 resistant lines, whilst one out of three identified mutations was common to both NGP derived lines. Mutation specific PCR revealed these mutations were present in parental SJSA-1 and NGP cell populations at a low frequency. Despite cross-resistance to a broad panel of MDM2/p53 binding antagonists, these MDM2-amplified and TP53 mutant cell lines remained sensitive to ionizing radiation (IR). These results indicate that MDM2/p53 binding antagonists will select for p53 mutations present in tumours at a low frequency at diagnosis, leading to resistance, but such tumours may nevertheless remain responsive to alternative therapies, including IR.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Humanos , Mutação , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
Two libraries of substituted benzimidazoles were designed using a 'scaffold-hopping' approach based on reported MDM2-p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X-ray structure. Benzimidazole libraries were prepared using an efficient solution-phase approach and screened for inhibition of the MDM2-p53 and MDMX-p53 protein-protein interactions. Key examples showed inhibitory activity against both targets.
Assuntos
Benzimidazóis/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Benzimidazóis/química , Proteínas de Ciclo Celular , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: del(17p), del(11q), and associated p53 dysfunction predict for short survival and chemoresistance in B-cell chronic lymphocytic leukemia (CLL). DNA-dependent protein kinase (DNA-PK) is activated by DNA damage and mediates DNA double-strand break repair. We hypothesized that inhibiting DNA-PK would sensitize CLL cells to drug-induced DNA damage and that this approach could increase the therapeutic index of agents used to treat CLL. EXPERIMENTAL DESIGN: Fifty-four CLL cases were characterized for poor prognosis markers [del(17p), del(11q), CD38, and ZAP-70]. In selected cases, DNA-PK catalytic subunit (DNA-PKcs) expression and activity and p53 function were also measured. Ex vivo viability assays established sensitivity to fludarabine and chlorambucil and also tested the ability of a novel DNA-PK inhibitor (NU7441) to sensitize CLL cells to these drugs. The effects of NU7441 on fludarabine-induced DNA damage repair were also assessed (Comet assays and detection of gammaH2AX). RESULTS: DNA-PKcs levels correlated with DNA-PK activity and varied 50-fold between cases but were consistently higher in del(17p) (P = 0.01) and del(11q) cases. NU7441 sensitized CLL cells to chlorambucil and fludarabine, including cases with del(17p), del(11q), p53 dysfunction, or high levels of DNA-PKcs. NU7441 increased fludarabine-induced double-strand breaks and abrogated drug-induced autophosphorylation of DNA-PKcs at Ser2056. High DNA-PK levels predicted for reduced treatment-free interval. CONCLUSIONS: These data validate the concept of targeting DNA-PKcs in poor risk CLL, and demonstrate a mechanistic rationale for use of a DNA-PK inhibitor. The novel observation that DNA-PKcs is overexpressed in del(17p) and del(11q) cases indicates that DNA-PK may contribute to disease progression in CLL.