8Background:
Venetoclax (VEN) is a novel selective BCL-2 inhibitor showing good tolerance in acute myeloid leukemia (AML), but the drug resistance increases with more clinical application. Newly combined VEN therapy strategies are needed to improve the efficacy of AML. Cladribine (CLA) is an adenosine-deaminase-resistant nucleoside used to treat AML. A previous study demonstrated that VEN and CLA synergistically affect chronic lymphocytic leukemia cells (CLL) in vitro by apoptotic pathways. However, it has not yet determined the anti-tumor effect of VEN plus CLA in AML
Aims: Here, we explored the synergistic effect of VEN plus CLA on cell proliferation arrest and apoptosis in AML and the underlying mechanisms.
Methods: The effect of VEN, CLA, and their combination on cell proliferation arrest, apoptosis, and cell cycle were examined using a Cell Counting Kit-8 (CCK-8) assay, PI staining, and Annexin-V/PI assay in AML cell lines and primary samples from AML patients. The mechanism underlying the synergism of the combination was investigated using RNA sequencing (RNA-seq), enrichment analysis, RT-qPCR, western blot, and co-immunoprecipitation (co-IP). A total of 72 AML patients’ BM samples and 72 normal controls from healthy volunteers were obtained from Zhongda Hospital Southeast University to analyze the association between DNA-PKcs expression level and clinical characteristics, another 2 patients’ samples were further treated with indicated drugs in vitro.
Results: CLA significantly sensitized the effect of VEN on cell proliferation arrest (Fig. 1A), inducing apoptosis (Fig. 1B), and G0/G1 phase arrest (Fig. 1C) in AML cells (U937, THP-1). Combination treatment upregulated PARP, cleaved caspase 9, and caspase 3, and downregulated BCL2 protein abundance (Fig. 1D), upraising p21Waf1/Cip1 expression and reducing CDK2/Cyclin E2 complex in the cells (Fig. 1E). CLA could overcome the resistance to VEN in AML cells by reducing the level of MCL-1 (Fig. 1D). To understand the mechanisms underlying the synergy, RNA-seq analysis was performed in U937 cells treated with VEN and CLA respectively, and 411 DEGs were identified (Fig. 1F). Cluster analysis indicated the accumulation of c-MYC and DNA-PKCS related pathways in DEGs (Fig. 1G). DNA-PKcs and c-MYC were downregulated upon combination therapy in AML (Fig 1H). Inhibition of DNA-PKcs by its inhibitor NU7441 preferentially killed cells with c-MYC (U937) overexpression, compared to THP-1 for 24h and 48h (Fig. 1I). Co-IP showed c-MYC interacted with DNA-PKcs (Fig. 1J), and inhibition of DNA-PKcs using NU7441 suppressed c-MYC mRNA and protein expression and cell proliferation in AML cells (Fig. 1K). DNA-PKcs was a positive correlation with c-MYC in the TCGA-AML dataset. Moreover, the DNA-PKcs expression level was increased in our AML patient cohort compared with healthy controls (Fig. 1L). Higher expression of DNA-PKcs was associated with poor outcomes in de novo and R/R AML cohorts (Fig 1L), and patients with DNA-PKcs overexpression had more cases of WT1 (n: 9 vs. 1, P=0.009) and TP53 (n: 10 vs. 4, P=0.098) mutations. The synergistic effect on cell proliferation arrest (Fig. 1M) and apoptosis (Fig. 1N) was also observed in primary cells from the AML patients with KARAS mutation and MLL-AF6 fusion (PT1), FLT3-ITD mutation, and NPM1 mutation (PT2).
Conclusion:
Venetoclax plus cladribine exert synergistic effects in AML cells particularly in the primary cells with the mutations in leukemia driver genes by suppressing DNA-PKcs/c-MYC signaling (Fig. 1O). Our results provide experimental evidence for further clinical application of the novel combination of venetoclax with cladribine in AML.
Keywords: Apoptosis, AML, Venetoclax, Cladribine