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D. Wu et al.
Kaplan-Meier survival analyses (see Online Supplementary Methods), we found that high expression of DHODH was significantly correlated with poor prognosis in patients with cytogenetically normal AML based on data from a previous microarray study20 (P=2.5 × 10-3) (Figure 1A), suggesting a potential clinical role of human DHODH in AML.
Dihydroorotate dehydrogenase is required for maintenance of acute myeloid leukemia cancer cell malignancy
We next examined the levels of DHODH expression in a panel of human cancer cell lines including AML. We found that DHODH expression was higher in AML than in other cancer cell lines (Figure 1B), consistent with the results of bioinformatics analysis (Online Supplementary Figure S1) using large-scale cancer cell lines from the Cancer Cell Line Encyclopedia database.21 We next silenced DHODH completely by a CRISPR/Cas9 knock- out system in both HL60 and THP-1 cells. A substantial knockout of the DHODH protein was observed in the knockout groups compared with the control group (Figure 1C and Online Supplementary Figure S2A). Notably, DHODH knockout impaired the growth of HL60 and THP-1 cells (Figure 1D and Online Supplementary Figure S2B). Knockout of DHODH caused an increase of HL60 cell apoptosis from 1.34±0.21% to 23.47±1.23% (sgRNA1) or 26.18±0.84% (sgRNA2), compared to the control (Figure 1E). Similar cell apoptosis induction was observed in THP-1 (from 4.72±0.41% to 19.93±1.74% (sgRNA1) or 21.79±1.32% (sgRNA2) (Online Supplementary Figure S2C).
Western blot analysis further showed significant upreg- ulation of three apoptosis-related markers (cleaved PARP, cleaved caspase-3 and cleaved caspase-9) in HL60 and THP-1 cells, thus revealing an apoptotic mechanism (Figure 1F and Online Supplementary Figure S2D).
We observed that DHODH-knockout significantly increased the expression of CD11b and CD14 (differenti- ation markers of myeloid cells), whereas it had no effect on CD33 and CD34, in either HL60 (Figure 1G) or THP-1 cells (Online Supplementary Figure S2E), suggesting that DHODH-knockout induces myeloid differentiation of AML cells. The best-known MYC protein family mem- ber, MYC, a crucial myeloid cell differentiation modula- tor, is frequently overexpressed in AML.22 Bioinformatics analysis revealed that DHODH is highly co-expressed with MYC in AML patients (Online Supplementary Figure S3) according to RNA-sequencing data from The Cancer Genome Atlas.23 We found that DHODH knockout signif- icantly reduced the expression of MYC protein in HL60 and THP-1 cells (Figure 1H and Online Supplementary Figure S2F). p21 is transcriptionally suppressed by MYC in cancer cells.24 Notably, MYC loss induced by DHODH inhibition accompanied an elevation of protein expres- sion of p21 in HL60 and THP-1 (Figure 1H and Online Supplementary Figure S2F). Taken together, abrogation of DHODH activity in AML markedly alleviated malignant characteristics, indicating that DHODH is a potential therapeutic target in AML.
Isobavachalcone is a novel dihydroorotate dehydrogenase inhibitor
Through screening an in-house natural product library using the 2,6-dichloroindophenol assay (see Online
Supplementary Methods), we found that, at the concentra- tion of 10 μM, isobavachalcone, a chalcone derived from traditional Chinese medicine Psoralea corylifolia, showed the greatest inhibitory activity on recombinant human DHODH protein among 337 natural products (Figure 2A). The chemical structure of isobavachalcone is presented in Figure 2B. Specifically, isobavachalcone showed a half maximal inhibitory concentration (IC50) value of 0.13 μM on DHODH, which is approximately 2-fold stronger than that of leflunomide, a Food and Drug Administration- approved DHODH inhibitor for the treatment of rheuma- toid arthritis (Figure 2C).25 To further examine the direct interaction between isobavachalcone and DHODH, we first performed a thermal shift assay, a commonly used assay to evaluate ligand-protein interaction.26 Figure 2D reveals that isobavachalcone significantly stabilized DHODH protein with an over 14°C melting temperature (DTm) increase in the presence of a 10-fold molar excess of DHODH (Figure 2D), suggesting a direct interaction between isobavachalcone and DHODH. Furthermore, we observed a dose-dependent attenuation of signal in the Carr-Purcell-Meiboom-Gill nuclear magnetic resonance (NMR) spectra, confirming that DHODH influences the state of isobavachalcone (Figure 2E).27 In addition, isobavachalcone bound to DHODH with a KD value of 1.33 μM (Figure 2F), according to an isothermal titration calorimetry experiment, which is consistent with results of the thermal shift assay and NMR experiments.
We next performed kinetic analysis of isobavachalcone against human DHODH using a Lineweaver-Burk plot. We found that isobavachalcone is a competitive inhibitor against coenzyme Q0 and uncompetitive for the substrate dihydroorotate (Online Supplementary Figure S4). Figure 2G reveals that isobavachalcone occupies the “ubiquinone channel”, a well-known ligand binding pocket of DHODH (PDB ID: 4YLW),28 as determined from molecular docking simulation (see Online Supplementary Methods). Specifically, several hydrophobic contacts are involved in the binding between isobavachalcone and amino acid residues of DHODH, including Tyr38, Gln47, Ala55, Ala59, Leu67, Phe98, Val143, Thr360 and Pro364. In brief, we identified a direct, high potential DHODH inhibitor, isobavachalcone, which could be a therapeutic agent for AML.
Isobavachalcone inhibits the proliferation of acute myeloid leukemia cells via inhibition of dihydroorotate dehydrogenase
We next investigated DHODH protein expression in four human AML cell lines: HL60, THP-1, U937 and MOLM-13. Among these four AML cell lines, HL60 has a high level of DHODH protein and is sensitive to isobavachalcone (Online Supplementary Figure S5A,B). Figure 3A,B shows that isobavachalcone significantly sup- presses HL60 and THP-1 cell proliferation in a concentra- tion-dependent and time-dependent manner. The cell- counting assay further revealed isobavachalcone concen- tration- and time-dependent suppression of HL60 cell growth (Online Supplementary Figure S5C). Importantly, Figure 3C illustrates that both recombinant DHODH and endogenous DHODH in HL60 cells bind directly bind to isobavachalcone-conjugated-Sepharose 4B beads, but not to Sepharose 4B beads alone. Isobavachalcone stabilized DHODH in HL60 cells in a cellular thermal shift assay (Figure 3D), suggesting that isobavachalcone inhibits DHODH directly in AML cells. Notably, knockout of
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haematologica | 2018; 103(9)