The Effect And Possible Mechanism Of Dihydroorotate Dehydrogenase Inhibitors On T-ALL Cells

PART Ⅰ DIHYDROOROTIC DEHYDROGENASE（DHODH） INHIBITOR SUPPRESSES T-ALL CELL GROWTHObjective:T-cell acute lymphoblastic leukemia（T-ALL）is one of the subtypes of leukemia that is difficult to treat,prone to relapse,and has a poor prognosis.When the relapse occurs,T-ALL patients are prone to develop resistance to traditional chemotherapeutic drugs.The search for novel target drugs is a topical concern for researchers in countries around the world.Pyrimidine nucleotides are essential to sustain life.Studies have demonstrated that an increased level of de novo pyrimidine synthesis promotes the rapid proliferation of tumor cells and induces the development of drug resistance in acute myeloid leukemia,glioma,breast cancer,lung cancer,etc.Targeting Dihydroorotate Dehydrogenase（DHODH）,a key enzyme in the de novo synthesis pathway,inhibits the growth of tumor cells.Nevertheless,the role of DHODH in T-ALL is still unclear.Methods:The public databases,such as GEO and TARGET,were used to analyze DHODH gene expression.WGCNA was used to recognize co-expression genes of DHODH in T-ALL.Bone marrow samples from T-ALL patients were collected,and DHODH expression in T-ALL was detected and verified by RNA-Seq.CCK8,flow cytometry,and EDU were utilized to explore the effects of Teriflunomide（TRF）,a DHODH inhibitor,on T-ALL cell lines,patient tumor cells,and T-ALL mice.RNA-Seq and data analysis,reactive oxygen species（ROS）,RT-PCR,and WB were employed to preliminarily explore the possible mechanism of TRF in suppressing T-ALL cell growth.Results:R2 online platform and GSE26713 data analysis revealed that DHODH mRNA expression was elevated in T-ALL patients compared with normal ones.WGCNA analysis indicated that DHODH co-expressed genes were enriched in OXPHOS,ROS,TCA cycle,pyrimidine metabolism,glycolysis pathways,etc.Compared with T-ALL patients in the DHODH^lowgroup,those in the DHODH^high group had a higher incidence of WBC>100×10⁹/L,MLL gene rearrangement,and positive MRD at 29 days after induction chemotherapy.RNA-Seq data of bone marrow tumor cells of 13T-ALL patients and normal cells of 4 individuals showed that DHODH was highly expressed in T-ALL.DHODH inhibitors TRF were applied to MOLT4 and JURKAT cells for48h with half maximal inhibitory concentration（IC50）of 21.42μM and19.00μM,respectively.TRF caused enhanced apoptosis,increased S-phase cells,and elevated ROS levels in MOLT4 and JURKAT cells.TRF was applied to the tumor cells of 5 T-ALL patients for 48h and inhibited in 4 cases.NSG leukemia mouse models were established with MOLT4 and JURKAT cells treated by 10μM TRF for 48h.Compared to the control group,the drug-treated mice had fewer tumor cells in the bone marrow,smaller spleen size,reduced tumor cell infiltration in the tissues,and prolonged survival time.RNA-Seq was performed on MOLT4 cells treated or untreated by TRF.GSEA analysis showed that genes of the drug-treated group were enriched in the P53 signaling pathway.B-cell translocation gene 2（BTG2）,the possible key gene,was identified by Venn and Protein-Protein Interaction Networks（PPI）.Compared with the control group,RT-PCR and WB validation showed that the mRNA and protein levels of P53 and BTG2 were elevated in drug-treated MOLT4 and JURKAT cells.The growth suppression and cycle-blocking effect of TRF were found to be weakened in BTG2knockdown MOLT4 and JURKAT cells.Conclusion:DHODH gene is high-expressed in T-ALL.DHODH inhibitor TRF suppressed the growth,induced apoptosis,and blocked the cell cycle of T-ALL cells,which may function through BTG2 and P53 signaling.PART Ⅱ CONSTRUCTION AND IDENTIFICATION OF DNR-RESISTANT T-ALL CELLSObjective: Daunorubicin（DNR）is one of the main anthracyclines in the current first-line induction chemotherapy regimen VDLP（Vincristine + Daunorubicin + Mentholase + Glucocorticoids）for T-ALL,as well as a widely used drug for re-induction therapy when relapse.DNR has a strong effect on dose-dependent efficacy.If DNR becomes resistant,it will weaken its effectiveness and increase the toxic side effects.Currently,there is little research on the mechanism of DNR resistance in T-ALL,and the construction of its cell resistance model is helpful for the in-depth exploration of the resistance mechanism of T-ALL.Methods: DNR-sensitive T-ALL cells were screened as target cells for resistance induction by CCK8.DNR-resistant T-ALL cell model was constructed by stepwise dose-escalation sequential exposure.DNR-resistant T-ALL cells were identified and functionally validated by short tandem repeat sequence（STR）and flow cytometry,respectively.Gene transcriptome alterations within T-ALL cells due to DNR resistance were analyzed by RNA-Seq.Results: DNR-sensitive MOLT4,JURKAT,and CCRF-CEM cells（named MOLT4-S,JURKAT-S,and CCRF-CEM-S,respectively）were screened as resistance-inducing target cells by CCK8.DNR-resistant T-ALL cells were successfully induced by stepwise dose-escalation sequential exposure（named MOLT4-R,JURKAT-R,and CCRF CEM-R）,whose resistance index [RI=IC50（R）/（S）] were MOLT4-R RI=566.9,JURKAT-R RI=152,and CCRF-CEM-R RI=116.4,respectively.STR results showed that the matching degree between DNR-sensitive cells and DNR-resistant cells was ≥80%.Compared with DNR-sensitive T-ALL cells,DNR-resistant TALL cells showed enhanced proliferation ability,no significant changes in cell cycle,and only a decrease of JURKAT-R CD1a+ cells and MOLT4-R CD5+ cells in immunophenotype.Resistance stability assay showed MOLT4-R RI=36.6,JURKAT-R RI=22.5,and CCRF-CEM-R RI=10.6.RNA-Seq and GSEA suggested that there were large differences in biological processes,cellular localization,molecular functions,and signaling pathways between DNR-sensitive and DNR-resistant T-ALL cells,especially in hypoxia,P53,apoptosis,and KRAS signaling pathways.Conclusion: DNR persistently resistant T-ALL cells were constructed.Compared with DNR-sensitive T-ALL cells,DNR-resistant T-ALL cells exhibited greater proliferative capacity and inconsistent gene transcriptome profiles.PART Ⅲ DHODH INHIBITOR IMPROVES CHEMOSENSITIVITY OF DNR-RESISTANT T-ALL CELLSObjective: Research had demonstrated that drug combinations can overcome chemoresistance in tumors,and this section investigated the effect of the DHODH inhibitor TRF on DNR-resistant T-ALL cells.Methods: The correlation of DHODH mRNA expression and drug sensitivity was predicted based on the Genomics of Drug Sensitivity in Cancer（GDSC）database.The optimal drug concentration of TRF in combination with DNR was calculated by Calcusyn software and the Synergyfinder website.The effects of different TRF concentrations and TRF combined with DNR on cell survival,apoptosis,and cell cycle of drugresistant T-ALL cells（MOLT4-R and JURKAT-R）were detected by CCK8 and flow cytometry.Results: As predicted by GDSC database,DHODH expression in T-ALL patient cells was found to correlate with drug sensitivity to doxorubicin,etoposide,methotrexate,vincristine,lenalidomide,and sorafenib.CCK8 results showed that TRF could inhibit the growth of the two drug-resistant T-ALL cells（MOLT4-R and JURKAT-R）with IC50 of 72.89μM and 93.98μM,respectively.Flow cytometry showed that TRF promoted apoptosis in DNR-resistant T-ALL cells and blocked the cell cycle in the S phase.CCK8 assay revealed that DNR combined with TRF applied to MOLT4-R and JURKAT-R cells at a concentration of 1:100 ratio,and the combination index（CI）was calculated by Calcusyn to be <1,suggesting a synergistic effect of the two drugs.Heatmap analysis and synergy model simulation on Synergyfinder online website suggested that the synergy scores（SC）of MOLT4-R cells were 32.71 and 34.03 for DNR 0.1μM combined with TRF 10μM and DNR 0.2μM combined with TRF 20μM,respectively;The SC of JURKAT-R cells were 28.18 and 34.48 when DNR 0.2μM combined with TRF 20μM and DNR 0.4μM combined with TRF 40μM,respectively.MOLT4-R and JURKAT-R cells showed stronger growth inhibitory and apoptosis-promoting effects when DNR 0.2μM combined with TRF 20μM than single drug use.Conclusion: TRF inhibited the growth of DNR-resistant T-ALL cells.A proportional concentration of DNR and TRF within a certain concentration range had a synergistic effect on DNR-resistant T-ALL cells.