P488: THZ1, A COVALENT CDK7 INHIBITOR, INDUCES APOPTOSIS IN ACUTE MYELOID LEUKEMIA CELLS AND EXERTS SYNERGISTIC ANTILEUKEMIA ACTIVITY WITH AZACITIDINE

Abstract

Background: Acute myeloid leukemia (AML) is a malignant blood cancer that develops mainly in elderly adults and has dismal clinical outcomes. Azacitidine (AZA) is still the preferred treatment for elderly patients unfit for intensive chemotherapy. However, the remission rate of azacitidine monotherapy is dismal, which necessitates the multidrug combination therapy. THZ1, a CDK7 inhibitor, has been proven to be effective in various cancers by regulating transcription and the cell cycle and inducing cell death. Here, we investigated the antitumor effect of THZ1 monotherapy and its combination treatment with AZA. Aims: To investigate the antitumor activity of the CDK7 inhibitor - THZ1 in acute myeloid leukemia (AML). Methods: We first examined the cell viability of THZ1 against THP-1, MOLM-13, OCI-AML3 cell lines using CCK-8 assays. We used flow cytometry to detect apoptosis after cells were stained with Annexin V-FITC/Propidium Iodide (PI), and western blot to detect the expression levels of proteins. We detect cell cycle by flow cytometry after cells were stained with 50 µg/ml PI and then analyzed with ModFit LT 5.0. RNA-sequencing analysis was performed to explore the potential mechanism of THZ1 in AML cells. In the double-drug combination experiment, we used CCK-8 assays to detect the effect of drugs on the viability of the three cell lines, used CompuSyn software to calculate the combination index (CI), which indicates additive effects (CI = 1.0), synergism (CI < 1.0), and antagonism (CI > 1.0). Results: THZ1 decreased viability and induced apoptosis in THP1, MOLM-13, and OCI-AML3 cells in a dose- and time-dependent manner. Besides, THZ1 inhibited phosphorylation of Ser2, Ser5, and Ser7 residues of RNA Pol II CTD and induced cell cycle arrest at G0/G1 phase in AML cells. RNA-sequencing analysis revealed that THZ1 induced changes in the expression of genes involved in apoptosis, the cell cycle, and DNA repair. Combined treatment with AZA and THZ1 showed a synergetic effect (CI < 1.0). Western blot analysis of apoptosis markers, including cleaved caspase3 and PARP1, demonstrated that THZ1 increased the expression of these markers and potentiated AZA-related apoptosis. Compared with monotherapy, combined treatment with AZA and THZ1 resulted in more Annexin V positive cells detected by flow cytometry, which further verified the synergistic effect of the two drugs. Western blot results indicate that combined treatment with AZA and THZ1 downregulates MCL1 at the protein level without an apparent decrease in BCL2 protein expression. Image:Summary/Conclusion: Our data demonstrate that the CDK7 inhibitor THZ1 induces the apoptosis of AML cells and exerts synergistic antileukemia activity with azacytidine, which provide the rationale for combination treatment with AZA and THZ1 for AML.