Therapy Of Neuroblastoma With The BET And CBP/P300 Dual Inhibitor NEO2734 And Approved Anticancer Drugs

Background:Neuroblastoma is the most common extracranial solid tumor in children,and one of the main causes of cancer-related death in children.The most common site of neuroblastoma is the abdomen,and the most common organ is adrenal medulla.Patients with neuroblastoma are divided into low,medium and high risk groups according to age,tumor histopathology,cell differentiation,DNA index(ploidy),11 q deletion and MYCN amplification status.High-risk neuroblastoma accounts for nearly 50% of all cases,mainly including children with metastatic disease(M phase),more than 18 months old and MYCN amplification.Children with low/medium risk neuroblastoma have a very good prognosis,but patients with neuroblastoma caused by amplification of MYCN oncogene and overexpression of N-Myc oncoprotein have a poor prognosis.Although multimodal therapy including surgery,radiotherapy,high-dose chemotherapy,differentiation therapy and GD2 targeted monoclonal antibody immunotherapy is performed,half of the patients relapse and the 5-year survival rate is less than 10%.Therefore,there is an urgent need for new drugs to improve survival and reduce long-term side effects to treat patients with high-risk neuroblastoma.Transcriptional super-enhancers play a critical role in tumorigenesis by inducing the transcription and dramatic over-expression of oncogenes,such as MYCN,and super-enhancer activity is controlled by the BET bromodomain protein BRD4(super-enhancer “reader”)and the histone acetyltransferases CBP and p300(super-enhancer “writers”).The dual BET and CBP/p300 co-inhibitor NEO2734 blocks the activity of BRD4,CBP/p300 and super-enhancers,has shown promising anticancer effects against myeloma,lymphoma and leukemia but does not induce cancer remission in vivo.Aims:1.To examine the anti-cancer effect of NEO2734 on MYCN gene amplified neuroblastoma,and to identify the approved oncology drug which exerts the best synergistic anticancer effects with NEO2734.2.To determine the mechanism through which NEO2734 and the approved oncology drug exert considerable synergistic anticancer effects against MYCN gene amplified neuroblastoma.Methods:1.MYCN-amplified SK-N-BE(2)neuroblastoma cells were treated with vehicle control,NEO2734 at1μM,each of the 166 Approved Oncology Drugs(AODs)from the US National Cancer Institute at 1μM,or combination of NEO2734 and each of the 166 AODs for 72 hours,followed by Alama blue cell viability assays and synergy analysis with the fractional product method.AODs which exerted synergy with NEO2734 and AODs which reduced cell viability by >90% on their own were subjected to secondary screening..2.For the secondary screening,MYCN-amplified SK-N-BE(2)and CHP134 neuroblastoma cells were treated with vehicle control,multiple doses of NEO2734 or AOD,or combination,followed by Alamar blue assays and synergy analysis to determine which AOD exerted the best synergistic anticancer effects with NEO2734.3.MRC-5 normal embryonic fibroblasts were treated with vehicle control,NEO2734,Ceritinib,or combination,followed by Alamar blue assays to exclude cytotoxicity of the combination on normal cells.4.SK-N-BE(2)and CHP134 cells were treated with vehicle control,NEO2734,Ceritinib,or combination,followed by flow cytometry analysis of cell death.5.Western blotting and real-time fluorescence quantitative RT-PCR were used to determine the expression of N-Myc protein and m RNA in MYCN-amplified SK-N-BE(2)and CHP134 neuroblastoma cells after treatment with vehicle control,NEO2734,Ceritinib or combination,and to find out the mechanism of synergistic anticancer effect of the drugs.6.CBP,p300 and CBP/p300 were knocked down in SK-N-BE(2)neuroblastoma cells.Western blotting and real-time fluorescence quantitative RT-PCR were used to detect knockdown efficiency and N-Myc protein and m RNA expression.The effects of CBP and/or p300 knockdown on cell proliferation was detected by colony formation assay,and further confirmed by tumor cell xenotransplantation in BALB/C nude mice.Results:1.Treatment with NEO2734 dose-dependently reduced MYCN gene-amplified neuroblastoma cell proliferation.2.After primary and secondary screening,the tyrosine kinase inhibitor Ceritinib were identified as the AOD exerting the best synergistic anticancer effect with NEO2734 without toxicity to normal non-malignant cells.3.In the flow cytometry experiment,NEO2734 and Ceritinib synergistically induced apoptosis in MYCN gene-amplified neuroblastoma cell lines.4.RT-PCR and immunoblot analysis showed that treatment with NEO2734 decreased the expression of N-Myc m RNA and protein,but combination of NEO2734 and Ceritinib did not synergistically reduce N-Myc m RNA and protein expression in MYCN gene-amplified neuroblastoma cells.Conclusions:NEO2734 alone can reduce the expression of N-Myc m RNA and protein in MYCN oncogene amplified neuroblastoma cells and induce growth inhibition of neuroblastoma cells.Screening of 166 AODs identified Ceritinib as the AOD exerting the best synergistic anticancer effects with NEO2734 in MYCN gene-amplified neuroblastoma cells without toxicity to normal non-malignant cells.NEO2734 and Ceritinib synergistically induces MYCN gene-amplified neuroblastoma cell apoptosis,but does not synergistically reduce N-Myc m RNA and protein expression.The synergistic anticancer mechanism of NEO2734 and Certinib needs to be further explored and verified in vitro and in vivo in order before carrying out potential clinical trials.