Exploring Novel Targets And Drugs And Drug Synergistic Effects For Osteosarcoma Treatment

ObjectiveThis study aims to explore new therapeutic targets and drugs for osteosarcoma,and to study mechanisms underlying the drug synergistic effects.MethodsFirstly,we utilized CRISPR screening to identify drug targets that could synergize doxorubicin,a first-line chemotherapy drug for osteosarcoma.The significant negative enrichment genes were validated throgh the single gene knockout cell line and gene inhibior.Further,to address the limitation of CRISPR screening,we adopted high-throughput drug library screening to figure out effective drugs for osteosarcoma.Synergistic effects between several HDACs inhibitors and the clinical first-line drugs were also explored.Otherwise,we have focused on the synergistic effect of the targets obtained through the two screening methods.Synergistic effects were analyzed by Chou-Talalay method.Cell titer reagent was used to detect cell viability,and the CI value was calculated based on cell inhibition rate.The CI<1 indicated synergistic effect between two drugs.Further,143B cell subcutaneous xenograft was useed to validate the in vivo efficacy and toxicity of drugs and drug combinations.Flow cytometry,q PCR,Western blot,immunofluorescence,etc.were applied to elucidate the mechanisms underlying the synergistic effect.ResultsCRISPR library screening confirmed that protein serine/threonine kinase-related genes(PRKDC,CSNK2A1,PIK3R4,etc.)were negatively enriched after doxorubicin treatment,and PRKDC gene(encoding DNA-PKcs protein)knockout increased the proportion of apoptotic cells induced by doxorubicin while decreased the Ki-67~+cells.Chou-Talalay analysis confirmed that the CI value for doxorubicin and AZD7648(a DNA-PKcs inhibitor)combination was less than 1 at various combinatorial concentrations,and such combination significantly increased the proportion of apoptotic cells and decreased the Ki-67~+cells.In vivo experiment showed that the combination of two drugs could further reduce the tumor volume,but had no significant effect on body weight of mice.High-throughput drug library screening has identified 31 drugs with IC50 values of less than 10μM in 4 osteosarcoma cell lines,5 of which were HDACs inhibitors.Further,Chou-Talalay analysis showed that the CI values of HDACs inhibitors(including PXD101)and doxorubicin combinatorial treatment were less than 1 at multiple concentration gradients,and the combinatorial treatment significantly increased the proportion of apoptotic cells and decreased the Ki-67~+cells.Interestingly,we also demonstrated that CI values of the combination between PXD101and AZD7648 were less than 1 at multiple concentrations.Combinatorial treatment could significantly increase the proportion of apoptotic cells,while reducing the Ki-67~+cells.Combinatorial therapy could further reduce the tumor volume compared with single drug,but has no significant effect on body weight in vivo.Mechanistically,one of the functional modes of doxorubicin is to bind to the DNA-TOP2A complex and inhibit the function of TOP2A,thereby causing DNA damage.Our results showed that the combination of AZD7648 and doxorubicin could further increase the expression ofγH2AX,a marker of cellular DNA damage and fragmentation.And further experiments confirmed that such combination could down-regulate the expression of TOPs protein,but has no effect on its m RNA expression.Meanwhile,knockdown of PRKDC could lead to down-regulation of TOP2A protein.Moreover,doxorubicin could lead to the increase of DNA-PKcs Ser2056 phosphorylation,the combinatorial use of DNA-PKcs inhibitors could inhibit NHEJ repair in cells and aggravate DNA damage.Similarly,PXD101 combining with doxorubicin also resulted in the increase ofγH2AX.And PXD101 could affect the transcriptional process of TOP2A and TOP2B genes,downregulating the expression of both their m RNA and protein.In addition,the combination of PXD101 and AZD7648 could inhibit TOP1 protein expression.And PXD101 could further reduce TOP1 protein expression after PRKDC gene knockout.ConclusionsCollectively,this study showed that doxorubicin,HDACs inhibitor,and DNA-PKcs inhibitor synergistically suppressed the growth of osteosarcoma reciprocally via interfering the expresison of their targets,and that such combinations could be potential alternative for osteosarcoma treatment in future clinical applications.