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failure, and may be fatal within weeks or months without treatment.1 AML is the most common acute leukemia, with an estimated incidence of about 19,000 cases in 2018 in the United States (USA) and a 5-year relative survival rate of approximately 60% in a population of children in the USA, based on data from 2007 to 2013.2 The major therapeutic paradigm for AML in the past several decades has been either chemotherapy with an anthracycline/cytarabine combination or allogeneic stem cell transplantation.3 Although traditional chemotherapy induces remission in de novo AML patients, only 20-30% of patients survive disease free in the long-term because of the high toxicity of chemotherapeutics, subsequent
4,5
patients.
The human dihydroorotate dehydrogenase (DHODH)
enzyme belongs to the class 2 DHODH family. It is anchored at the inner mitochondrial membrane.10 As an essential enzyme that catalyzes dihydroorotate to orotic acid, DHODH plays a critical role in the de novo pyrimi- dine biosynthesis of DNA and RNA.11 Rapidly proliferat- ing cells, such as cancer cells and lymphocytes, mainly depend on de novo pyrimidine biosynthesis to support their growth rate, indicating that this enzyme is a poten- tial target in the treatment of cancer and autoimmune dis- eases.10 A previous study suggested that DHODH is required for rapid proliferation of tumor cells, playing an important role in tumorigenesis and tumor develop- ment.12 Using a unique Homeobox A9-driven leukemia model, Sykes et al. recently found that DHODH is a novel metabolic target in differentiation therapy of AML.13,14 Their pioneer work offers a potential differentiation treat- ment strategy for patients with AML.13,14 Several DHODH inhibitors, such as brequinar, have already been evaluated in various clinical cancer trials, but severe adverse reac- tions limit their clinical application.15,16
In this study, we show that CRISPR-Cas9-mediated knockout of DHODH greatly impairs growth, increases apoptosis, and induces differentiation of two AML cell lines, HL60 and THP-1, indicating once again that DHODH is a potential therapeutic target. We identified a novel, direct inhibitor of DHODH, isobavachalcone, by screening a library of natural products. We demonstrate that isobavachalcone effectively triggers apoptosis and induces differentiation in AML cells via direct inhibition of DHODH. Furthermore, our results suggest that isobavachalcone, alone or in combination with adri- amycin, significantly prolongs survival in an intravenous HL60 leukemia model.
Methods
The Online Supplement contains detailed information on the experimental materials and methods.
All animal care and experimental procedures in this study
complied with the protocol approved by the Animal Care and Use Committee at East China University of Science and Technology.
Knockout of dihydroorotate dehydrogenase in HL60 and THP-1 cells
The guide RNA sequences targeting DHODH were designed and cloned into a LentiCRISPRv2 construct (Addgene, #52961).17 This construct along with psPAX2 (Addgene, #12260) and pMD2.G (Addgene, #12259) helper constructs were co-transfected into HEK-293T cells using Lipofectamine 2000 reagent (Invitrogen, NY, USA) to produce lentiviral supernatants. Viral production was subsequently concentrated 60X by ultracentrifugation. Cells were infected with lentiviral supplemented with polybrene (8 μg/mL) in 24-well plates and infected cells were selected in medium contain- ing puromycin (0.8 μg/mL). The knockout efficiency of single guide RNA (sgRNA) was determined by western blot analysis. The sgRNA targeting exon 2 of human DHODH were listed as follows: sgRNA1: 5’-TTCTTCGACATTGCCGTCGA-3’; sgRNA2: 5’- ACAAGGTCCCAAAGACAG-3’.
Cell apoptosis assay
Cells were seeded into six-well plates and incubated with the indicated concentrations of compounds. The apoptosis assays were performed using an AnnexinV-FITC Apoptosis Detection kit (eBioscience, MA, USA) according to the instructions.18 The apoptotic cells were analyzed using a BD FACS Calibur flow cytometer (BD Biosciences, NJ, USA).
Differentiation marker analysis
Cultured cells were harvested and washed with phosphate- buffered saline on ice, then resuspended in FACS buffer (phos- phate-buffered saline, pH 7.4, supplemented with 1 mM ethyl- enediaminetetraacetic acid and 2% fetal bovine serum).19 Antibodies of differentiation markers (CD11b, CD14, CD33 and CD34) were added and incubated for 1 h at 37°C in the dark. Flow cytometer data were collected and analyzed on a BD FACS Caliber using Cell Quest software (BD Biosciences, NJ, USA).
Wright-Giemsa staining
Cells were harvested, washed with phosphate-buffered saline and fixed with 95% ice-cold methanol for 30 min at 4°C.14 The cells were then seeded on the slide and allowed to dry in the air. Next, the cells were stained with Wright-Giemsa for 5 min and rinsed in deionized water. Finally, coverslips were fixed with Permount prior to microscopy (Nikon, Tokyo, Japan).
Statistical analysis
All values are expressed as the mean ± standard deviation of at least three independent experiments. GraphPad Prism 5.0 software (GraphPad software, CA, USA) was used for the statis- tical analysis. Comparisons between two groups were analyzed using the two-tailed Student t-test. For multiple comparisons, one-way ANOVA followed by Tukey multiple comparison tests were performed. P values < 0.05 are considered statistically sig- nificant.
Results
Dihydroorotate dehydrogenase overexpression is associated with poor prognosis in acute myeloid leukemia
We examined the relationship between DHODH expression and overall survival in AML patients. In the
On the other hand, although targeted therapies, such as the isoc- itrate dehydrogenase 2 (IDH2) inhibitor enasidenib and the FMS-like tyrosine kinase 3 (FLT3) inhibitor midostau- rin, were recently approved by the USA Food and Drug Administration for AML treatment,6 only relatively few AML patients with actionable mutations of IDH2 or FLT3 will benefit from these drugs.7-9 Development of innova- tive therapeutic agents is, therefore, a pressing need to improve the clinical efficacy and quality of life for AML
relapses, and development of drug resistance.
haematologica | 2018; 103(9)
Inhibition of DHODH induces differentiation in AML
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