ABSTRACT
BACKGROUND: Certain paediatric nervous system malignancies have dismal prognoses. Retinoic acid (RA) is used in neuroblastoma treatment, and preclinical data indicate potential benefit in selected paediatric brain tumour entities. However, limited single-agent efficacy necessitates combination treatment approaches. METHODS: We performed drug sensitivity profiling of 76 clinically relevant drugs in combination with RA in 16 models (including patient-derived tumouroids) of the most common paediatric nervous system tumours. Drug responses were assessed by viability assays, high-content imaging, and apoptosis assays and RA relevant pathways by RNAseq from treated models and patient samples obtained through the precision oncology programme INFORM (n = 2288). Immunoprecipitation detected BCL-2 family interactions, and zebrafish embryo xenografts were used for in vivo efficacy testing. RESULTS: Group 3 medulloblastoma (MBG3) and neuroblastoma models were highly sensitive to RA treatment. RA induced differentiation and regulated apoptotic genes. RNAseq analysis revealed high expression of BCL2L1 in MBG3 and BCL2 in neuroblastomas. Co-treatments with RA and BCL-2/XL inhibitor navitoclax synergistically decreased viability at clinically achievable concentrations. The combination of RA with navitoclax disrupted the binding of BIM to BCL-XL in MBG3 and to BCL-2 in neuroblastoma, inducing apoptosis in vitro and in vivo. CONCLUSIONS: RA treatment primes MBG3 and NB cells for apoptosis, triggered by navitoclax cotreatment.
Subject(s)
Apoptosis , Drug Synergism , Medulloblastoma , Neuroblastoma , Tretinoin , Zebrafish , Humans , Animals , Tretinoin/pharmacology , Medulloblastoma/drug therapy , Medulloblastoma/pathology , Medulloblastoma/metabolism , Medulloblastoma/genetics , Apoptosis/drug effects , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Neuroblastoma/metabolism , Neuroblastoma/genetics , Cell Line, Tumor , Aniline Compounds/pharmacology , Xenograft Model Antitumor Assays , Sulfonamides/pharmacology , bcl-X Protein/genetics , bcl-X Protein/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Mice , Cerebellar Neoplasms/drug therapy , Cerebellar Neoplasms/pathology , Cerebellar Neoplasms/metabolism , Cerebellar Neoplasms/genetics , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , N-Myc Proto-Oncogene ProteinABSTRACT
In addition to involvement in epigenetic gene regulation, histone deacetylases (HDACs) regulate multiple cellular processes through mediating the activity of non-histone protein substrates. The knockdown of HDAC8 isozyme is associated with the inhibition of cell proliferation and apoptosis enhancement in several cancer cell lines. As shown in several studies, HDAC8 can be considered a potential target in the treatment of cancer forms such as childhood neuroblastoma. The present work describes the development of proteolysis targeting chimeras (PROTACs) of HDAC8 based on substituted benzhydroxamic acids previously reported as potent and selective HDAC8 inhibitors. Within this study, we investigated the HDAC8-degrading profiles of the synthesized PROTACs and their effect on the proliferation of neuroblastoma cells. The combination of in vitro screening and cellular testing demonstrated selective HDAC8 PROTACs that show anti-neuroblastoma activity in cells.
Subject(s)
Histone Deacetylase Inhibitors , Histone Deacetylases , Neuroblastoma , Humans , Cell Line, Tumor/metabolism , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/chemistry , Histone Deacetylases/metabolism , Neuroblastoma/metabolism , Proteolysis , Repressor Proteins/metabolismABSTRACT
Histone deacetylases (HDACs) are an attractive therapeutic target for a variety of human diseases. Currently, all four FDA-approved HDAC-targeting drugs are nonselective, pan-HDAC inhibitors, exhibiting adverse side effects at therapeutic doses. Although selective HDAC inhibition has been proposed to mitigate toxicity, the targeted catalytic domains are highly conserved. Herein, we describe a series of rationally designed, conformationally constrained, benzanilide foldamers which selectively bind the catalytic tunnel of HDAC8. The series includes benzanilides, MMH371, MMH409, and MMH410, which exhibit potent in vitro HDAC8 activity (IC50 = 66, 23, and 66 nM, respectively) and up to 410-fold selectivity for HDAC8 over the next targeted HDAC. Experimental and computational analyses of the benzanilide structure docked with human HDAC8 enzyme showed the adoption of a low-energy L-shaped conformer that favors HDAC8 selectivity. The conformationally constrained HDAC8 inhibitors present an alternative biological probe for further determining the clinical utility and safety of pharmacological knockdown of HDAC8 in diseased cells.
Subject(s)
Anilides/chemistry , Anilides/pharmacology , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/pharmacology , Repressor Proteins/antagonists & inhibitors , Catalytic Domain/drug effects , Drug Design , Histone Deacetylases/metabolism , Humans , Molecular Docking Simulation , Repressor Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Human histone deacetylase 8 (HDAC8) is a highly promising target for neuroblastoma and other types of cancer. Several HDAC inhibitors are approved for the treatment of special cancer subtypes or are evaluated in clinical trials. By far the most drugs or drug candidates contain a hydroxamate group that chelates the catalytic zinc ion within HDACs. Most hydroxamate inhibitors are more or less unselective, although there are considerable exceptions demonstrating the general feasibility to develop at least HDAC isoenzyme selective inhibitors. In addition, hydroxamates have recently come under discussion regarding their potential for mutagenicity. Recently, PD-404,182 was discovered as a selective and potent non-hydroxamate inhibitor of HDAC8. However, this active compound turned out to be decomposed in the presence of glutathion (GSH). Here, we describe the synthesis of significantly improved analogs of PD-404,182 that demonstrate both, great selectivity for HDAC8 and also chemical stability in the presence of GSH. The compounds are characterized with respect to structure-activity relationship, binding mode and target engagement in neuroblastoma cells by combining biochemical and biophysical methods with chemoinformatics.