Deguelin Induced Differentiation of NPM1-Mutant AML In Vivo and in Vitro

Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy due to abnormal proliferation of hematopoietic progenitor cells, with difficulty in treatment and high mortality. Nucleophosmin 1 (NPM1), a restricted nucleolar localization protein, shuttles between nucleus and cytoplasm. NPM1-mutant protein, aberrant cytoplasmic dislocation of nucleophosmin, occurs in about one third of AML. Deguelin exhibits significant inhibitory effects and induces apoptosis in a variety of cancer cell lines in vitro and in vivo. Our previous researches showed deguelin induced differentiation of NPMc+ AML cells potentially by targeting NPM1-mutant protein. Thus, we extend our investigations of deguelin-induced differentiation activity to an AML xenograft mouse model and clinical samples.

After sub-lethal whole body radiation, OCI/AML3 cells were injected intravenously in NOD/SCID mice. 7 days later, either deguelin or vehicle (2%DMSO) was injected intraperitoneally every day. Kaplan-Meier estimates showed 1mg/kg/day deguelin extended the longest survival of mice. The percentage of CD11b+ cells was 17.73%±3.03% in control group, 41.33%±13.22% and 76.15%±10.25% after deguelin-treated for 7 days and 14 days (P < 0.05). Thus, we used 14 days in the following experiments. The percent of CD114+ cells in the vehicle- and deguelin-treated group were 13.29%±10.79% and 47.36%±21.66%, respectively (P < 0.05). The percentage of CD14+ cells in the vehicle- and deguelin-treated group were 22.86%±15.54% and 83.49%±8.06%, respectively (P < 0.05). Wright's Giemsa staining of mouse bone marrow cells showed differentiated. The ratios of differentiated cells were 36.92%±4.99% and 77.01%±1.98% in control and deguelin group (P < 0.01). Median OS of vehicle-treated group was 23 days, while that of deguelin-treated group was 55 days (HR, 0.5818, 95% confidence interval [CI], 0.1623-1.001). Survival of deguelin-treated mice was significantly longer compared with mice injected with vehicle (P=0.0002). IHC and IF staining for mutated NPM1 protein showed bone marrow biopsy densities significantly reduced in deguelin-treated mice which was further confirmed by western blot assay (P < 0.05).

We analyzed the toxicity of deguelin (1mg/kg/day for 14 days) to healthy NOD/SCID mice. They had steady movement and no rigidity posture treated with deguelin. HE staining indicated no pathological changes in striatum, substantia nigra pars compacta (SNpc), liver, kidney and heart in deguelin-treated mice. Striatum and SNpc, subjected to immunostaining with anti-tyrosine hydroxylase (TH) antibody, showed deguelin did not alter TH immunoreactivity in striatum and SNpc in vivo (P >0.05). Plasmatic levels of liver enzymes (AST and ALT), renal function (Scr and BUN) and cardiac enzymes (CK-MB and cTnT) in deguelin-treated group had not statistical difference comparing with control group (P>0.05). Thus, deguelin did not cause neurotoxicity, hepatotoxicity, nephrotoxicity and cardiotoxicity to mice.

We collected 82 cases of patients with acute leukemia. 24 cases out of 68 AML patients carried mutated NPM1 gene (20 cases of type A; 1 case of type B; 2 cases of type D and 1 case of type I). Bone marrow mononuclear cells (BMMCs) of 6 AML patients were analyzed by FACS analysis. We analyzed patient (pt.) 1 to 4 with NPM1 mutation. FACS showed deguelin increased the percentage of CD11b+ and CD14+ cells of pt.1 in a dose-dependent manner. The percent of CD11b+ and CD14+ cells were 55% and 57.9% separately at 16 nM. Deguelin increased CD14 but CD11b expression of BMMCs of pt.2 in the concentration of 8 nM, 12 nM, 16 nM and 20 nM. The highest percentage of CD14+ cells was 85.5%. The expression of CD14 in cells from pt.3 increased after treating with 16 nM or 20 nM and the highest percentage of CD14+ cells was 49.4%. Deguelin induced differentiation of BMMCs from pt.4. The maximum percent of CD11b+ cells was 42.5% at 16 nM. Moreover, FACS analysis showed deguelin did not promote differentiation in BMMCs from pt.5 and 6 without NPM1 mutation. These results demonstrated deguelin induced differentiation of BMMCs cells from AML patients with NPM1 mutation in vitro. Taken together, mutated NPM1 protein may be one of targets of deguelin, which provides molecular basis for inducing differentiation in NPM1-mutant AML cells.