Establishment 6-MP And 6-TG Resistant Acute Lymphoblastic Leukemia Cell Lines And Its Resistance Mechanism Research

Objective:To investigate the thiopurine resistance mechanism of acute lymphoblastic leukemia,we set out to estabolish acute lymphoblastic leukemia resistant cell lines by 6-MP or 6-TG inducing.We analysis the mechanism of drug resistance in the resistant cells with the methods of whole exome sequencing,molecular cytology and metabolomics.Methods:The resistant cell lines Reh-6MPR and Reh-6TGR were established by human acute lymphoblastic leukemia cell Reh treating with increasing dosing of 6-MP and 6-TG respectively;The resistance of resistant cells was measured by drug sensitivity assay,apoptosis and DDR biomarkers by western blot;The characteristics of resistant cell growth were analysed by growth curve and cell cycle;The mutation of genes in resistant cells was detected by whole exome sequencing;The drug metabolism and purine metabolism in resistant cells wastested by the method of LC/MS;The protein expression in resistant cells was detected by Western blot;The overexprssion of target protein in resistant cells were accomplished by lentivirus infection.Results:We have successfully established stable resistant cell line Reh-6MPR induced by 6-MP and Reh-6TGR induced by 6-TG,Reh-6MPR is 959 and 1161-fold more resistant to6-MP and 6-TG compared with parental Reh respectively,Reh-6TGR is 699 and 956-fold more resistant to 6-MP and 6-TG compared with parental Reh respectively;The appotosis of6-MP and 6-TG to resistant cells were decreased significantly;The expression of the DDR biomarkers?-H2AX,the phosphor-CHK2?T68?and the apoptosis biomarker cleaved PARP were lower change in the resistant cell line Reh-6MPR than those in parental Reh cells;The resistant cells grow slower than Reh cells;6-MP and 6-TG were accumulated in Reh-6MPR and Reh-6TGR compared with parental Reh cells,and its metabolites TIMP and TGMP were reduced significantly.There was a same hypoxanthine phosphoribosyl transferase 1?HPRT1?mutation in Reh-6MPR and Reh-6TGR cell which associated with thiopurine metabolism,an adenine?A?was inserted between the mRNA base sequence 495 and 496 sites,the amino acid sequence was frameshift from 165 amino acids?V165fs?;We marked the purine salvage synthesis with ¹³C₅,¹⁵N₄-Hypoxanthine,the results revealed the production of[¹³C₅,¹⁵N₄]-IMP from[¹³C₅,¹⁵N₄]-hypoxanthine?HX?were decreased,showed a loss funciton of HPRT1;The purine metabolites Hypoxanthine?IMP?AICAR and Inosine were increased;The overexpression of HPRT1 wildtype gene can improve the drug sensitivity to 6-MP and 6-TG in resistant cell line Reh-66MPR.Conclusion:The mutation of HPRT1 V165fs caused the loss function of HPRT1 and induced drug resistance to 6-MP and 6-TG in the resistant cell line Reh-6MPR and Reh-6TGR,the resistance can be reversed by re-expression of HPRT1 wildtype genes.