P407: SYNERGISTIC EFFECT OF A NOVEL COMBINATION OF HDACI CHIDAMIDE WITH CLADRIBINE ON CELL PROLIFERATION ARREST AND APOPTOSIS IN ACUTE MYELOID LEUKEMIA BY TARGETING MYC/HDAC2 SIGNALING

Abstract

Background: Chidamide (CHI), a novel Histone deacetylase inhibitor (HDACi), which is now involved in the salvage treatment of relapsed/refractory acute myeloid leukemia in a clinical trial. Cladribine (2-chloro-2’-deoxyadenosine (2-CdA)), is a purine nucleoside antimetabolite analog, which resists degradation by adenosine deaminase and therefore accumulates to cytotoxic levels. The anti-leukemia effects of either single drug have been reported by inducing cell apoptosis through suppressing multi-oncogenic pathways in human tumor cells. However, whether and how the combination of the two drugs exerts the synergistic anti-tumor effects are still undetermined in AML. Aims: This study is to investigate the synergistic therapeutic effects of 2-CdA in combination with CHI in AML. Methods: Cell Counting Kit-8 (CCK-8) cell proliferation assay, Annexin V apoptosis assay, Propidium Iodide cell cycle assay, and Western Blot (WB) were performed in U937 AML cells in presence of CHI only, 2-CdA only, CHI+ 2-CdA, and vehicle control for 48h. RNA-seq was performed in U937 cells treated with CHI and 2-CdA; the differentially expressed genes (DEGs) were identified by R studio, and pathway enrichment of the DEGs was analyzed with Metascape online tool. Co-Immunoprecipitation (co-IP) was used to examine the interaction of cMYC with HDAC1/2. Data were expressed as the mean ± standard deviation. T-tests statistical analysis was employed for determining the significant difference between groups. A synergistic effect was determined by the combination indices (CIs) with CompuSyn software. Results: The 50% inhibiting concentration (IC50) of CHI and 2-CdA on U937 cell proliferation is 9.358±1.448μmol/L and 0.024±3.071μmol/L, respectively. The combination of various doses of CHI with either IC50 or 1/2 IC50 of 2-CdA significantly decreases the survival rate of U937 cells compared to single drug control (Fig.1a). The CI analysis showed the high synergistic therapeutic effects of CHI with ether dose of 2-CdA (Fig. 1b). The combination of (8mM) CHI with (0.02mM) 2-CdA ignificantly elevated the apoptosis rate and induced the G0/G1 cell phase arrest of AML cells compared with either single drug alone (p<0.0001, Fig. 1c, d). Moreover, the expression of apoptotic markers, caspase-3, PARP-1, and caspase-9 is elevated, and the cell cycle marker p21 proteins were significantly upregulated, but Cyclin-dependent kinase CDK2 and Cyclin E2 down-regulated in the combination group compared to either single drug control (p<0.05, Fig. 1e). To further understand the underlying mechanism of the synergy, RNA-seq analysis was performed in U937 cells treated with either CHI or 2-CdA, respectively. The overlapped altered DEGs were identified, and cMYC is dramatically down-regulated. The pathway analysis of the altered DEGs showed the enrichment of MYC pathways further indicated the critical role of MYC (Fig. 1f). It is reported that cMYC is associated with the histone deacetylase complex. Indeed, the co-IP assay showed an interaction of MYC with HDAC2, not HDAC1 in U937 (Fig. 1g). These data suggested the role of cMYC/HDAC2 signaling in the synergistic effect of the combination of CHI with 2-CdA in the disease. Image:Summary/Conclusion: The combination of Chidamide and Cladribine has a synergistic effect on cell proliferation arrest, apoptosis, and cell cycle arrest in AML cells through MYC/HDAC2 pathway. Our data provide experimental evidence for the novel potential combination of AML therapy. More in vivo pre-clinical studies will be explored for future clinical trials.