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. 2018 Sep;103(9):1472-1483.
doi: 10.3324/haematol.2018.188185. Epub 2018 Jun 7.

Pharmacological Inhibition of Dihydroorotate Dehydrogenase Induces Apoptosis and Differentiation in Acute Myeloid Leukemia Cells

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Pharmacological Inhibition of Dihydroorotate Dehydrogenase Induces Apoptosis and Differentiation in Acute Myeloid Leukemia Cells

Dang Wu et al. Haematologica. .
Free PMC article

Abstract

Acute myeloid leukemia is a disorder characterized by abnormal differentiation of myeloid cells and a clonal proliferation derived from primitive hematopoietic stem cells. Interventions that overcome myeloid differentiation have been shown to be a promising therapeutic strategy for acute myeloid leukemia. In this study, we demonstrate that CRISPR/Cas9-mediated knockout of dihydroorotate dehydrogenase leads to apoptosis and normal differentiation of acute myeloid leukemia cells, indicating that dihydroorotate dehydrogenase is a potential differentiation regulator and a therapeutic target in acute myeloid leukemia. By screening a library of natural products, we identified a novel dihydroorotate dehydrogenase inhibitor, isobavachalcone, derived from the traditional Chinese medicine Psoralea corylifolia Using enzymatic analysis, thermal shift assay, pull down, nuclear magnetic resonance, and isothermal titration calorimetry experiments, we demonstrate that isobavachalcone inhibits human dihydroorotate dehydrogenase directly, and triggers apoptosis and differentiation of acute myeloid leukemia cells. Oral administration of isobavachalcone suppresses subcutaneous HL60 xenograft tumor growth without obvious toxicity. Importantly, our results suggest that a combination of isobavachalcone and adriamycin prolonged survival in an intravenous HL60 leukemia model. In summary, this study demonstrates that isobavachalcone triggers apoptosis and differentiation of acute myeloid leukemia cells via pharmacological inhibition of human dihydroorotate dehydrogenase, offering a potential therapeutic strategy for acute myeloid leukemia.

Figures

Figure 1.
Figure 1.
Dihydroorotate dehydrogenase is required for acute myeloid leukemia cells to maintain their malignant characteristics. (A) Kaplan-Meier survival curves for AML patients divided by level of DHODH expression. The P-value of the Kaplan-Meier survival analysis was determined using a log-rank test (see the Online Supplementary Methods). (B) Western blot analysis of the expression levels of DHODH in different cancer types. SiHa: cervical carcinoma; H1299, A549 and H446: lung carcinoma; HL60 and THP1: AML; Jurkat: acute T-cell leukemia; HepG2: hepatic carcinoma; U251: glioma. (C) Knockout of DHODH in HL60 cells was analyzed by western blot. (D) Knockout of DHODH impaired the growth of HL60 cells. Cell viability was evaluated by MTS assay at 24 h intervals up to 96 h in three independent experiments. The graph represents the means ± SD. The Student t-test was performed, **P<0.01. (E and F) DHODH knockout resulted in apoptosis of HL60 cells. Cell apoptosis was analyzed by flow cytometry and the expression levels of apoptosis-related proteins in HL60 cells was detected by western blot at 96 h after infection. (G) Flow cytometry demonstrated upregulation of cell surface markers CD14 and CD11b after knockout of DHODH in HL60 cells whereas there was no effect on CD33 and CD34 expression. The cells were measured at 96 h after infection. Data represent the mean ± SD of three independent experiments. (H) Knockout of DHODH resulted in reduced expression of MYC protein and upregulated expression of p21 in HL60 cells.
Figure 2.
Figure 2.
A natural product, isobavachalcone, is a newly identified direct dihydroorotate dehydrogenase inhibitor. (A) Graphical presentation of screening results of 337 compounds tested at a concentration of 10 μM in a DHODH enzymatic assay. Each dot represents one compound. (B) Chemical structure of isobavachalcone. (C) Dose-response curves of isobavachalcone and leflunomide in the DHODH enzymatic assay. (D) A thermofluor assay shows that isobavachalcone robustly stabilizes DHODH and produces a thermal shift over 14°C (ratio 1:10). (E) NMR measurement of direct binding between isobavachalcone and DHODH. Carr-Purcell-Meiboom-Gill NMR spectra for isobavachalcone (red), isobavachalcone in the presence of DHODH at 2.5 μM (green) and 5 μM (cyan). (F) Isothermal titration calorimetry of isobavachalcone binding to DHODH. Binding curves were fitted as a single binding event. (G) Computational docking analysis of the binding mode of isobavachalcone with DHODH. The structure is shown as a ribbon diagram and the isobavachalcone molecule (left) is presented as a sphere model based on PDB ID: 4YLW. The amino acid residues surrounding isobavachalcone (yellow sticks, right) are represented by slate sticks. Figure 1G was generated by PyMOL software (https://www.pymol.org/). IBC: isobavachalocone; LEF: leflunomide.
Figure 3.
Figure 3.
Isobavachalcone shows anti-proliferative activity against acute myeloid leukemia cells. (A) HL60 and THP1 cells were treated with increasing concentrations of isobavachalcone for 48 h, and cell viability was measured by MTS assay. (B) The time-response curve of 30 μM isobavachalcone on cell viability of HL60 and THP1 cells. (C) Recombinant DHODH protein or HL60 cell lysate was incubated with control or isobavachalcone-conjugated Sepharose 4B beads. Proteins bound to the beads were analyzed by western blot. (D) Cellular thermal shift assay shows that isobavachalcone stabilizes and targets DHODH in intact HL60 cells. Cells were incubated with isobavachalcone for 12 h and the assay was performed. (E) The IC50 value of isobavachalcone against DHODH-knockout HL60 cells. (F) HL60 cells were treated with isobavachalcone at the indicated concentrations for 72 h. Cell apoptosis was detected by flow cytometry using staining with annexin V, fluorescein isothiocyanate (FITC) and propidium iodine (PI). (G) The quantitative data of cell apoptosis in (F). (H) Changes in apoptosis-related proteins after treatment with isobavachalcone or uridine for 72 h. (I) Representative images of Hoechst 33258-stained cells were analyzed by fluorescence microscopy in HL60 cells treated with 30 μM isobavachalcone for 48 h. Red arrows indicate apoptotic cells. IBC: isobavachalocone.
Figure 4.
Figure 4.
Isobavachalcone induces HL60 cell differentiation. (A) HL60 cells were treated with isobavachalcone for 72 h and CD14 expression was detected by flow cytometry analysis. Right: quantification data of CD14 expression in HL60 cells. (B) HL60 cells were treated with isobavachalcone for 72 h and CD11b expression was detected by flow cytometry analysis. Right: quantification data of CD11b expression in HL60 cells. (C) Morphological changes associated with differentiation of HL60 cells were evidenced by Wright-Giemsa staining in the presence of 30 μM isobavachalcone. (D) HL60 cells were incubated with different concentrations of isobavachalcone for 24 h. After incubation, western blot assay was performed to examine the expression levels of MYC and p21. (E) The expression of MYC was analyzed by western blot 1.5, 3, 6 and 12 h after treatment with 30 μM isobavachalcone. (F) HL60 cells were treated with 30 μM isobavachalcone for 3 h with or without 10 μM MG132. The levels of MYC expression were subsequently examined by western blot analysis. (G) 293T cells were transfected with the MYC-luc reporter plasmid together with pRSVluc plasmid (as an internal control) and incubated with different concentrations of isobavachalcone for 24 h. (H) HL60 cells were treated with 30 μM isobavachalcone for 24 h, then MYC and p21 gene levels in HL60 cells were examined by reverse transcriptase polymerase chain reaction. Data represent mean ± SD of three independent experiments. A Student t-test was performed, *P<0.05, **P<0.01, ***P<0.001. IBC: isobavachalocone.
Figure 5.
Figure 5.
Isobavachalcone suppresses tumor growth in a subcutaneous HL60 xenograft mouse model. (A) Measurements of tumor volume in an HL60 xenograft mouse model treated with vehicle or the indicated dosages of compounds for 18 days. Changes in mean tumor sizes compared with the control group. Bars represent mean ± SD for eight animals in each group. (B) Effect of isobavachalcone on tumor weights. (C) The body weight of mice was measured every 3 days. (D) Histological morphology of tumor tissue, stained with hematoxylin and eosin, from the different groups. (E) Images of the major organ tissues, stained with hematoxylin and eosin, from the different groups of animals. (F) DHODH protein expression in tumors was examined at the time the animals were sacrificed. (G) The enzyme activity of DHODH in xenograft tumor tissues treated with vehicle, isobavachalcone or leflunomide was measured by fluorescence assay. (H) Western blot analysis of the changes of apoptosis-associated proteins at day 18 of HL60 xenograft tumors treated with vehicle, isobavachalcone or leflunomide. (I) Expression levels of MYC and p21 in tumors treated with vehicle, isobavachalcone, or leflunomide were estimated by western blot. Data represent means ± SD. A one-way ANOVA test was performed, **P<0.01 and ***P<0.001. IBC: isobavachalocone; LEF: leflunomide.
Figure 6.
Figure 6.
The combination of isobavachalcone and adriamycin shows synergistic antileukemic effects in vitro and in vivo. (A-D) Synergistic effects of the isobavachalcone and adriamycin combination on AML cells. AML cell lines (HL60, THP1, U937 and MOLM-13) were treated with several increasing concentrations of isobavachalcone and adriamycin alone or in combination for 48 h. The combination index (CI) calculation was performed using CalcuSyn software (Version 2.1; Biosoft). Drug combinations with a CI<1 are considered to be synergistic. (E) Synergistic effects of isobavachalcone and adriamycin combination therapy in an intravenous HL60 leukemia model. Mice with established tumors (4 per group) were divided into four groups and treated with vehicle, isobavachalcone, adriamycin or a combination of isobavachalcone and adriamycin. The P value was determined using the log-rank test, P=0.0003 for the survival analysis (E). (F) Leukemia cells isolated from the isobavachalcone-treated group exhibit morphological features of differentiation. ADR: adriamycin; IBC: isobavachalocone.

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