Chidamide Combined with Cytarabine Plays a Role in the Treatment of AML By Down-Regulating the MYC-RRP9 Pathway

Abstract

Purpose: To explore the therapeutic effect and mechanism of Chidamide combined with Cytarabine in acute myeloid leukemia (AML), and to provide a new treatment strategy for AML patients. Methods: The effects of Chidamide and Cytarabine alone or in combination on the phenotype of AML cell lines were detected by MTT, flow cytometry and Western blot. Transcriptome sequencing and GSEA software were used to explore the mechanism of Chidamide combined with Cytarabine in the treatment of AML. RRP9 overexpression and knockdown AML cell lines were constructed by lentivirus to explore the effects of RRP9 on the proliferation, drug sensitivity, precursor ribosomal RNA (rRNA) process and protein production capacity of AML cell lines. Mature rRNA was detected by qPCR, nascent RNA was detected by EU kit, nucleolar area was observed by electron microscope, and nascent protein was detected by protein synthesis kit. Lentivirus and chromatin immunoprecipitation (CHIP) were used to explore the upstream regulatory genes of RRP9. The effects of Chidamide combined with Cytarabine on the survival rate and molecular level of primary AML cells were detected by CCK-8 and qPCR, respectively. An AML mouse model was constructed to explore the effects of Chidamide and Cytarabine on tumor burden and survival time in AML mice. Results: Chidamide combined with Cytarabine was more effective in killing AML cell lines than single drug, and the combination index was less than 1. The apoptosis rate (P<0.01) and the apoptotic protein level (P<0.05) induced by double drug were both higher than those induced by single drug. Chidamide combined with Cytarabine significantly down-regulated MYC pathway RRP9. In this pathway, RRP9 was selected for further research. An AML cell line overexpressing RRP9 was constructed. RRP9 overexpression promoted the proliferation of AML cell line (p<0.01) and induced the AML cell line to be resistant to the dual-drug combination (P<0.01). Construction of RRP9 knockdown AML cell line. The mature rRNA (P<0.01), nascent RNA (P<0.05), nucleolar area (P<0.0001) and nascent protein level (P<0.05) of cells in the RRP9 knockdown group were significantly lower than those in the control group. In addition, Chidamide combined with Cytarabine reduced the levels of mature rRNA (P<0.05), nascent RNA, nucleolar area (P<0.05), and nascent protein (P<0.05) in AML cell lines. The expression levels of RRP9 in MYC-overexpressing cells and knockdown cells were higher and lower than those in the control group, respectively (P<0.05). CHIP showed that the enrichment abundance of MYC at the RRP9 promoter site was significantly higher than that of IgG (P<0.05). The downregulation of MYC by dual-drug combination was stronger than that of single-drug(P<0.05). Chidamide combined with Cytarabine had stronger killing effect on AML primary cells than single drug, and the combination index was less than 1. Chidamide combined with Cytarabine down-regulated MYC (P<0.05) and RRP9 (P<0.01) mRNA levels in AML primary cells. The results of in vivo imaging of small animals showed that Chidamide alone could significantly reduce the tumor burden in mice (P<0.01), and the combination with Cytarabine further reduced the tumor burden (P<0.01). The survival time of mice in the Chidamide combined with Cytarabine treatment group was significantly longer than that in the control group (median survival time 28 days (28-31 days) vs 23 days (19-26 days)) (P<0.001), and the difference was still statistically significant compared with the single drug (P<0.05). Conclusion: Chidamide combined with cytarabine has a synergistic therapeutic effect on AML cell lines, primary cells and AML mice, which inhibits mature rRNA, total RNA and protein synthesis in AML cells caused AML cell death by synergistically down-regulating the MYC-RRP9 pathway.