The Progression Of B-All Is Inhibited By The Therapy Of Methylation Inhibitor CPI-1205 And 6-Mercaptopurine And Possible Mechanism

Part I CPI-1205 COOPERATES WITH 6-MERCAPTOPURINE TO INHIBIT THE GROWTH OF B-ALL CELLSObjective: 6-Mercaptopurine(6-Mercaptopurine,6-MP)is metabolized to 6-TGN(6-Thioguanine Nucleotide)through HPRT(Hypoxanthine Guanine Phosphoribosyltransferase),or is metabolized to 6-MMP(6-Methyl-mercaptopurine)through TPMP(Thiopurine Methyltransferase),which interferes with purinesynthesis.And 6-MMP is related with serious toxic and side effects.Because methylation/demethylation regulates gene expression,methylation inhibitor 5-azacytidine can reactivate the HPRT gene whose expression is inhibited by promoter methylation.Additionally,methyltransferase shares methyl donor SAM(S-Adenosylmethionine)with TPMP.To reduce the dosage of 6-MP and its toxic and side effects,we screened methylation inhibitors,which can cooperate with 6-MP to inhibit the growth of acute B-lymphoblastic leukemia(B-ALL)cells,and uncovered the ratio and effective concentration for two drugs.Methods: Methylation inhibitors were screened through Drug Bank database combined with registration tests in the website of www.Clinical Trail.gov.cn.Four methylation inhibitors were selected.CCK-8 was used to detect and analyze the effects of different drug concentrations and the regimens of combination drug on the growth inhibition of B-ALL cells;The half inhibitory concentration(IC50)and drug combination index(CI)were calculated by Graph Prism and Calcusyn Software.Results: 0.01 n M-100μM 6-MP was used on B-ALL cell lines:Sup-B15,Reh,HAL-01 and BAL01.The IC50 concentrations were 2μM,1μM,1.5μM and 138μM,respectively.However,according to Drug Central 2021,6-MP has the higher incidence of hematotoxicity and recurrence,compared to other common chemotherapeutic drugs in pediatric B-ALL.On the other hand,through the results of Durgbank Database and Clinical Trail website,we screened and selected methylation inhibitors CPI-1205,Decitabine,Temozolomide and Zebularine.0.001 n M-100μM methylation inhibitors were acted on B-ALL cells: CPI-1205 inhibited the growth of Sup-B15,Reh and HAL-01 cells,the IC50 was about 50μM;Temozolomide inhibited the growth of Sup-B15 and HAL-01 cells,IC50 was about 70μM;and the rest ones were ineffective or IC50>100μM.Further,Sup-B15 and Reh cell lines were treated by 2μM,1μM respectively.Transcriptome sequencing analysis showed that the changes of RNA profile after 6-MP treatment were similar to those after methylation intervention.Hence,the therapy with CPI-1205 and 6-MP was analyzed.The growth inhibition of 10 n M-1μM 6-MP combined with 60μM CPI-1205 was>IC50in Sup-B15 and Reh cells.However,when the concentration ratio of6-MP:CPI-1205 was only 100:1,it had a good concentration linear correlation in Sup-B15;that was only 200:1 in Reh.And 300 n M 6-MP combined 30μM CPI-1205 can cause IC50 in Sup-B15 cells;150n M 6-MP combined 30μM CPI-1205 can cause IC50 in Reh cells.Additionally,30μM CPI-1205 combined with 300 n M 6-MP,administered twice in 48 hours,could inhibit the growth of some B-ALL tumor cells(3/8);and the inhibitory effect of 30μM CPI-1205 combined with 300 n M 6-MP on monocytes isolated from peripheral blood of non leukemia patients is weaker than that of 30μM CPI-1205 or 300 n M 6-MP.Conclusion: 30μM CPI-1205 combined with 300 n M/150 n M 6-MP inhibited the growth of Sup-B15/Reh cells;30μM CPI-1205 combined with300 n M 6-MP,administered twice in 48 hours,inhibited the growth of some B-ALL tumor cells,and its inhibitory effect on monocytes from peripheral blood of non leukemia patients was weaker than that of 30μM CPI-1205 or300 n M 6-MP.Part II CPI-1205 COMBINED WITH 6-MP PROMOTES APOPTOSIS OF B-ALL CELLSObjective: To investigate the change of EZH2 activity via CPI-1205 inhibition,the 6-MP intracellular concentration and cell functions by CPI-1205 combined with 6-MP,based on effectively inhibiting the growth of B-ALL cells.Methods: Edu and Ki67 were used to detect cell proliferation;The cell cycle was detected by Flow Cytometry;Flow Cytometry and DAPI staining were used to detect apoptosis at different time points;The total activity of H3K27 methyltransferase were detected by enzyme labeling instrument and the level of H3K27me3 were detected by Colorimetric.The intracellular concentration of 6-MP was detected by HPLC-MSResults: Sup-B15 was treated by 30μM CPI-1205 combined with0.3μM 6-MP,and Reh was treated by 30μM CPI-1205 combined with0.15μM 6-MP.After 48 hours of treatment,the cell functions was tested.Compared with 6-MP treatment,it was found that CPI-1205 combined with 6-MP could effectively inhibit the proliferation of B-ALL cells;in Sup-B15 cells,the proportion of cells in G0/G1 phase was decreased and the proportion in G2/M phase was increased,but in Reh cells,the proportion of cells in G0/G1 and G2/M phase was increased,and the proportion in S phase was decreased;And the apoptosis cells were increased obviously,after 24 hours(Sup-B15 3.88±0.21 vs 6.47±0.56,Reh 8.51±4.08 vs11.2±4.89)and 48 hours(Sup-B15 3.05±1.27 vs 3.27±1.56,Reh 6.49±2.27 vs 8.79±3.62).Further,compared with CPI-1205 treatment,CPI-1205 combined with 6-MP treatment had no effect on the activity of H3K27 methyltransferase and the level of H3K27me3 in Sup-B15 cells;Compared with 6-MP treatment,CPI-1205 combined with 6-MP treatment promoted the intracellular concentration of 6-MP to increase about triple.Conclusion: Compared to treatment with 6-MP,CPI-1205 combined with 6-MP inhibited the cell proliferation and promoted apoptosis of B-ALL cells.The combination of CPI-1205 with 6-MP had no effect on the total activity of H3K27 methyltransferase and the level of H3K27me3,but increased the intracellular concentration of 6-MP in B-ALL cells.Part III THE MECHANISM OF CPI-1205 COOPERATING WITH6-MP TO PROMOTE APOPTOSIS IN B-ALL CELLSObjective: To explore the possible molecular mechanism of CPI-1205 cooperating with 6-MP to promote apoptosis and increase of 6-MP intracellular concentration in B-ALL cellsMethods: RNA-Seq was used to detect and analyze the changes of gene expression and signaling pathway after drug treatment in Sup-B15 and Reh cells.Q-PCR,WB,confocal and GSE data were used to analyze and identify gene expression changes.The stable knockdown cell lines of TRIB3 and SLC43A3 were constructed and identified(Si-TRIB3-Sup,Si-SLC43A3-Sup);CCK-8 and apoptosis assay were used to detect the effect of impaired TRIB3 and SLC43A3 on cell functions;30μM CPI-1205,0.3μM 6-MP,30μM CPI-1205 combined 0.3μM 6-MP treated Si-TRIB3-Sup,Si-SLC43A3-Sup cells,and the role of TRIB3 and SLC43A3 gene on effectiveness of drugs was analyzed.The possible relationship between TRIB3 and SLC43A3 was simulated by String and Cytoscape software,and was identified by Q-PCR.Resluts: After filtering and Gene Function Enrichment,apoptosis related pathways were enriched in Top20,and 15 genes related with apoptosis were highly expressed in Reh and Sup-B15 cells after CPI-1205 combined with 6-MP treatment.The ATF4,TRIB3 and CASP4 m RNA in Sup-B15 cells were detected at different time points and different drug concentrations.Compared to the treatment with 0.3μM 6-MP,the expression level of TRIB3 m RNA was increased and the protein was about twice in Sup-B15 cells with CPI-1205 combined with 6-MP treatment.GSE61905 database analysis showed that the expression of TRIB3 was higher in the thiopurine sensitive strain than that in the thiopurine resistant strain.Impaired TRIB3 gene promoted the growth of Sup-B15 cells and inhibited the level of apoptosis.CPI-1205 combined with 6-MP treatment could inhibited the growth of Si-TRIB3-Sup cells,but the ratio of inhibition in Si-TRIB3-Sup cells was lower than that in Sup-B15 cells.On the other hand,GSE61905 data analysis showed that SLC43A3 was highly expressed in thiopurine sensitive cell lines compared with thiopurine resistant cell lines;In Sup-B15 cell line,after CPI-1205 combined with 6-MP treatment,SLC43A3 m RNA and membrane protein SLC43A3 were higher than those of 6-MP treatment.After knockdown of SLC43A3 gene,Sup-B15 cells had higher growth rate and lower apoptosis level.CPI-1205 combined with6-MP treatment could inhibited the growth of Si-SLC43A3-Sup cells,but the ratio of inhibition in Si-SLC43A3-Sup cells was lower than that in Sup-B15 cells.String and Cytoscape software simulated the signaling pathway including the genes related to transporter and apoptosis.We found that SLC43A3 regulated multiple pathways related apoptosis including TRIB3.And the TRIB3 m RNA level was decreased by impaired SLC43A3.Conclusion: Compared with 6-MP treatment,CPI-1205 combined with 6-MP treatment increased the expression levels of TRIB3 and SLC43A3,thus promoting apoptosis at 48 hour;and impaired SLC43A3down-regulated the expression of TRIB3 m RNA.Part IV CPI-1205 COMBINATED WTIH 6-MP TO DELAYTHE PROGRESSION OF B-ALL LEUKEMIC MICEObjective: To investigate the efficacy of CPI-1205 combined with6-MP treatment in B-ALL mice model.Methods: Sup-B15 cells was treated by 30μM CPI-1205,0.3μM6-MP,30μM CPI-1205 combined with 0.3μM 6-MP and control DMSO for48 hours,Sup-B15 cells were injected into NOD/SCID mice via vein to construct leukemic mice model;According to the concentration used in B-ALL cells,intraperitoneal injection of different concentrations of CPI-1205(25.92mg/kg,12.96mg/kg,6.48mg/kg and 3.24mg/kg,the corresponding injection volume of diluted drugs is VμL,1/2VμL,1/4VμL and 1/8VμL),6-MP(8.509mg/kg,4.254mg/kg and 2.127mg/kg,the corresponding injection volume of diluted drugs is VμL,1/2VμL and1/4VμL),once one day or once/2 days,total of 5 times.Flow cytometry and bone marrow smears were used to dynamically monitor the tumorigenesis;recording the survival time,collecting tissue samples and evaluating the tissue infiltration by H&E staining to analyse the effect of treatment.Resluts: The leukemic mice derived from Sup-B15 cells treated by CPI-1205,6-MP and CPI-1205 combined with 6-MP,the h CD45+ cells at33 rd day and blast cells at the end stage were least in CPI-1205 combined with 6-MP treatment group;compared with OS among different groups,OS in DMSO group was shorter than that in untreated cell group(28.33 ±3.66 days vs 38.00 ± 1.57days),compared with DMSO group,CPI-1205(33.33±5.12 days),6-MP(39.33±7.11 days)and CPI-1205 combined with6-MP(41.83±5.67 days)treatment groups were longer than that of DMSO group;the degree of liver and spleen infiltration was lowest in CPI-1205 combined with 6-MP treatment group.Drugs were injected into leukemic mice derived from Sup-B15 cells via intraperitoneal,at the 33 rd day,partly dead was present in all groups,especially in the 1/2V CPI-1205 treatment group and 1/4V 6-MP treatment group and the mortality was 66.67%(2/3);there was no significant difference on the number of blast cells in bone marrow smears.There was no significant difference on OS between the groups by intraperitoneal injection V,1/2V,1/4V and1/8V CPI-1205,the OS of 1/4V(6.48mg/kg)and 1/8V(3.24mg/kg)CPI-1205 group was longer slightly,but the less infiltration is observed in 1/8V group.After intraperitoneal injection 6-MP,OS in 1/2V(4.254mg/kg)6-MP group was significantly better than that in V or1/4V 6-MP group,and infiltration was less.Conclusion: Compared with 6-MP treatment,the progress of leukemic mice derived from Sup-B15 and treated by CPI-1205 combined with 6-MP,is delayed;the effective concentrations of CPI-1205 and 6-MP in the treatment of Sup-B15-derived leukemic mice were 3.24mg/kg and4.254mg/kg respectively.