Therapeutic Response and Molecular Mechanisms of Novel Combination Regimen Azacitidine Plus Homoharringtonine in Acute Myeloid Leukemia with Poor Outcome

Background：Homoharringtonine (HHT) is a natural alkaloid derivate from Cephalotaxus. HHT-based regimens have an advantage over the standard induction therapy in acute myeloid leukemia (AML). Azacitidine(AZA), a DNA hypomethylation agent, has therapeutic efficacy on AML. This study is to examine the therapeutic response and underlying mechanisms of AZA plus HHT-based regimens for AML with poor outcome in a registered clinical study (ClinicalTrials.gov ID: NCT04248595). Methods：Patients were enrolled into two treatment arms: arm A(AZA+HIA/HDA) or arm B( AZA+HAG). In arm A, AZA(75mg/m²/d on days1-5) was administered in combination with HIA/HDA regimen [HHT 2mg/m²/d on days4-8, idarubicin 6mg/m²/d or daunorubicin 40 mg/m²/d on days4-6, and cytarabine 100mg/m²/d on days4-10] for patients age<60. In arm B, AZA(75mg/m²/d on days1-7) was given in combination with HAG regimen [HHT 1mg/m²/d on days4-17(days4-10 for unfit patients), cytarabine 10mg/m² SC every 12 hours on days4-17(days4-10 for unfit patients), and G-CSF 200ug/m²/d from day4 until WBC>10*10^9/L] for patients age≥60 or ineligible for intensive chemotherapy. Therapeutic response was evaluated by [complete remission(CR) + CR with incomplete blood count recovery(CRi) + partial remission(PR)]. Mutation analysis was conducted using next-generation sequence panel targeting 58 frequently mutated genes. CCK-8 cell proliferation and Annexin V apoptosis assay were performed in cells treated with AZA, HHT, AZA+HHT, and control for 48h. A synergistic effect was observed in the cells treated with HHT and AZA, and analyzed by CalcuSyn software. The whole-genome transcriptome was identified by RNA-seq in U937 cells treated with HHT，AZA or control for 48h. The gene expressions in the paired patients' samples and the cell lines were accessed by qPCR. Results：A total of 32 patients, including relapsed/refractory(9/32), MDS/MPN transformed(7/32), de novo AML(16/32) were enrolled. The therapeutic response rate is 71.9% (21/32 CR/CRi, 2/32 PR) in the whole cohort, 90.0%(9/10 CR/CRi) in arm A, and 63.6%(12/22 CR/CRi, 2/22 PR) in arm B. CR/CRi was achieved after the first cycle in 77.8%(7/9) relapsed/refractory, 42.9%(3/7) MDS/MPN transformed, and 68.8%(11/16) de novo with unfavorable prognostic markers. All patients were tolerated through the treatment process with myelosuppression as the most common adverse event. The AZA+HHT-based regimens were sensitive in the patients with multiple gene variants involving DNA methylation(TET2, DNMT3A), metabolism(IGH1, IGH2), progenitor development and cell proliferation (FLT3, IKZF1, GATA2, KRAS, RUNX1, BCOR，and WT1). Notably, about 2/3 of the gene variants were undetectable and 1/3 significantly reduced in CR/CRi patients (Table 1). While in 9 non-remission(NR) patients, multiple variants in TP53, NRAS, PTPN11, CEBPA, ASXL1, FLT3，and PHF6 were detected without changes or even increased after induction. Furthermore, a synergistic effect of AZA+HHT on cell proliferation arrest and apoptosis was observed in AML cells compared to the single drug (Fig 1A). A similar result was observed for apoptosis in the pre-induction cells from CR/CRi patients, but not in the cells from NR patients (Fig 1B). In addition, RNA-seq analysis identified 2886 common differentially expressed genes (DEGs) upon HHT or AZA treatment. Pathways and GO term analysis indicated the DEGs are mainly involved in transcriptional and epigenomic regulation, metabolism, apoptosis, cell cycle regulation, etc. Notably, RNA-seq and qPCR data showed that the mRNA levels of oncogene c-MYC and glucose metabolism regulation gene IDH1 are significantly down-regulated; however, the tumor suppressor TET2 is up-regulated in the cells treated by HHT or AZA. Importantly, the expression of c-MYC/IDH1 was significantly decreased, but TET2 increased in the paired samples from CR/CRi patients, but no differences from NR patients(Fig 1C). Together, our data indicated that AZA+HHT exerts the anti-leukemia effect by down-regulation of c-MYC/IDH1 and up-regulation of TET2. Conclusions：The presented clinical data for the first time showed the novel combination-based regimens are effective and well-tolerated in AML with poor outcomes. Novel AZA+HHT combination-based regimens apply synergistic anti-leukemia efficacy by targeting c-MYC/IDH1/TET2 axis.