The Effects And Underlying Mechanisms Of CDK7 Inhibitor THZ1 And The Combination With Azacitidine In Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia(AML)is the most common type of acute leukemia in adults.Elderly patients with AML tend to have a high frequency of adverse cytogenetics and molecular mutations with poor chemotherapy tolerance and dismal prognosis,with a 5-year overall survival(OS)rate of less than 30%.In recent years,the combination of the BCL2 inhibitor venetoclax and hypomethylating agents(HMAs)has become the first-line treatment for elderly AML patients.The introduction of small molecule inhibitors targeting IDH-1,IDH2,and FLT-3 has also expanded the treatment options for AML patients.However,most patients could inevitably experience primary and secondary drug resistance due to the high heterogeneity of AML.The treatment of AML remains an enormous challenge,and it is urgent to explore novel drugs with different mechanisms or multidrug combination regimens to improve the clinical outcomes of AML patients.Cyclin-dependent kinases(CDKs),which play an important role in regulating cell cycle and transcription,are significant targets in tumor therapy.THZ1,a covalent inhibitor of CDK7,has potent antitumor effects in both solid and hematologic malignancies.A previous study has shown that THZ1 induces apoptosis in AML cells with t(8;21)and induces dynamic changes in RNA polymerase Ⅱ.However,the role of THZ1 in AML cells without t(8;21)is unclear,furthermore,whether THZ1 can exert synergistic anti-leukemic effects in combination with HMA needs to be investigated.PurposeThe present study aimed to investigate the antileukemic effects and potential mechanisms of THZ1,alone and in combination with azacitidine in AML in vitro and in vivo.Methods1.The cell viability was detected by CCK-8 after AML cell lines treated with THZ1;the expression of apoptotic proteins,cyclin-dependent kinases,and proteins associated with transcription was detected by western blot;and flow cytometry was applied to detect the cell cycle and the percentage of apoptotic cells.2.AML cell lines and primary AML cells were treated with AZA,THZ1,and the combination of the two drugs,respectively,after which the cell viability was detected by CCK-8,and the combination index(CI)was calculated by CompuSyn software;the effects of the drugs on the expression of apoptotic proteins were examined by western blot;flow cytometry was applied to detect the percentage of apoptotic cells.3.AML cell lines were treated with AZA,THZ1,and the combination of the two drugs respectively,followed by the detection of mitochondrial membrane potential by the JC-1 method;the effects of drugs on the expression of BCL2 family were detected by western blot and qRT-PCR.RNA of drug-treated cells was extracted to perform RNA-sequencing,exploring the underlying antileukemic mechanisms of drugs.4.A luciferase-labeled MOLM-13 cell line(MOLM-13-Luc-GFP)was constructed and transplanted into NSG mice by tail vein injection to establish AML xenograft models,which were treated with placebo,AZA,THZ1,and the combination of the two drugs,respectively(n=9/group).The tumor burden in mice was measured by bioluminescence imaging every 7 days.Three mice in each group were randomly sacrificed after 10 days of treatment.Subsequently,the size and weight of spleen were measured and weighed;bone marrow cells were collected and stained by anti-human CD45 and CD33 antibodies for FACS analysis;spleen,liver,kidney,and lung tissues were processed for immunohistochemical and HE staining studies.The remaining mice were monitored for survival analysis.Results1.THZ1 inhibited AML cell proliferation in a dose-and time-dependent manner and increased the expression of apoptotic proteins.Flow cytometry results indicated that increasing drug concentration and duration of treatment raised the proportion of apoptotic cells.Furthermore,THZ1 decreased phosphorylated CDK1 and CDK2 expression,induced cell cycle arrest at G0/G1 phase,and inhibited phosphorylation of carboxy-terminal domain of RNA Pol II at multiple serine sites.2.The combination of AZA and THZ1 inhibited cell proliferation in AML cell lines and primary AML cells with CI<1 in most conditions;the combined treatment increased the expression of apoptotic proteins and elevated the proportion of Annexin V+cells.3.The combination of AZA and THZ1 decreased the mitochondrial membrane potential significantly and inhibited the expression of the MCL1 at protein level rather than mRNA level without significant changes in protein expression of BCL2 and BCL-XL.RNA-seq results showed that two Hallmark gene sets regulated by MYC were significantly enriched after the combination treatment.Western blot and qRT-PCR results confirmed that the combination of AZA and THZ1 could inhibit the expression of c-MYC at the protein and mRNA levels.4.MOLM-13 cell line stably expressing luciferase was transplanted into NSG mice,and BLI was used to confirm the establishment of the AML xenograft model.Mice were treated with the placebo,AZA monotherapy,THZ1 monotherapy,and the combination regimen(n=9/group).After 14 days of treatment,the mean BLI intensity in the combination group was significantly lower than AZA group(2.37×l07±1.48×107 vs.3.23×108±1.17×108,P<0.05);the mean BLI intensity in the THZ1 group was 1.91×108±1.07×108 without significant difference from the combination group(P=0.076).The median survival of the vehicle group,AZA group,THZ1 group,and combination group were 14,15,16,and 20 days,respectively.Log-rank test results revealed that AZA and THZ1 monotherapy had no significant effect on the survival of mice compared with the vehicle group,and the combination treatment of AZA and THZ1 prolonged the survival significantly(P<0.01).Mice in the combination treatment group had significantly reduced spleen weight(P<0.01)and the number of human CD45+/CD33+cells in the bone marrow(P<0.001)compared to the vehicle group.Immunohistochemical and HE staining results revealed that the combination treatment reduced the infiltration of leukemic cells in multiple organs of mice.ConclusionsThe study demonstrates that CDK7 inhibitor THZ1 inhibits proliferation and promotes apoptosis in AML cell lines in a time-dependent and dose-dependent manner.Our results verify for the first time that THZ1 exerts a synergistic antileukemia effect with AZA in AML cell lines and primary AML cells.Mechanistically,the combination of the two drugs inhibits the expression of the anti-apoptotic protein MCL1,decreases the mitochondrial membrane potential,and suppresses the expression of c-MYC at the mRNA and protein levels.In vivo experiments confirm that the combination of AZA and THZ1 reduces the tumor burden and significantly prolongs the survival of mice,showing potent anti-leukemic activity.