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ASLAN003 for differentiation of AML
enasidenib and ivosidenib for relapsed/refractory AML with mutated IDH2 and mutated IDH1, respectively, by the USA Food and Drug Administration renewed enthu- siasm for differentiation therapy.
Pyrimidines and pyrimidine derivatives are the building blocks of both deoxyribonucleic acid (DNA) and ribonu- cleic acid (RNA), and protein glycosylation, which are the essential cellular components.11 Dihydroorotate dehydro- genase (DHODH) catalyzes the fourth enzymatic step in de novo pyrimidine biosynthesis, converting the ubiquinone-mediated oxidation of dihydroorotate to oro- tate.12,13 DHODH has been a therapeutic target for malar- ia, rheumatoid arthritis, and multiple sclerosis.14-16 Recently, an elegant study revealed an unexpected role of DHODH in the differentiation of AML blast cells.17 The DHODH inhibitor used in that study, brequinar, was originally discovered by Du Pont in 1985.17,18 However, clinical trials of brequinar in solid tumors demonstrated myelosuppression with predominant thrombocytopenia, which limit its potential use in AML.16,19
ASLAN003 (LAS186323) is a novel, bioavailable and potent small molecule DHODH inhibitor. The drug was discovered by Almirall, S.A. and global rights to the com- pound were granted to ASLAN Pharmaceuticals Singapore in 2012, which re-named it as ASLAN003. ASLAN003 is a potent inhibitor of human DHODH enzyme activity, with a half maximal inhibitory concentration (IC50) of 35 nM, and high plasma protein binding (>99%). In phase I single and multiple ascending dose clinical trials, ASLAN003 has been shown to be tolerated by healthy volunteers. In this study, we set out to investigate the effects of ASLAN003 on AML cell function in vitro and in vivo, as well as to eluci- date the molecular mechanism of DHODH inhibition of AML cell differentiation.
Methods
Cell lines and primary acute myeloid leukemia cells, drugs and chemicals
Details on the cell lines and primary bone marrow (BM) cell culture, drugs and chemicals are provided in the Online Supplementary Methods.
Cell viability assays, western blot analysis, polymerase chain reaction, and FACS analysis
Experiments were conducted as previously described.20,21 Details of the cell viability assays, western blot analysis, real- time quantitative reverse transcriptase polymerase chain reac- tion (qRT-PCR), and FACS analysis of myeloid cell surface anti- gens are available in the Online Supplementary Methods. The primer sequences are provided in Online Supplementary Table S1.
Wright-Giemsa staining and nitro blue tetrazolium assay
After exposure to ASLAN003 or dimethylsulfoxide (DMSO) for 96 h, AML cells (1 x 106) were harvested and distributed equally for Wright-Giemsa staining and nitro blue tetrazolium (NBT) reduction assay (details in the Online Supplementary Methods).
Assessment of mitochondrial membrane potential
The mitochondrial membrane potential was determined using a JC-10 Assay Kit (Sigma, MAK160). Details are provided in the Online Supplementary Methods.
RNA sequencing and data analysis
The experiments and subsequent analysis of KG-1 and MOLM- 14 cells treated with ASLAN003 or DMSO were performed as detailed in the Online Supplementary Methods.
Protein synthesis assays
Click-iT assays were performed using an O-propargyl- puromycin (OPP) Alexa Fluor® 488 Protein Synthesis Assay Kit from ThermoFisher (C10456) according to the manufacturer’s rec- ommendation. MOLM-14 and KG-1 cells were exposed to ASLAN0031μMor2μMfor1hbeforeOPP20mMwasadded for 1 h. DMSO was used as a control. Cells were washed in ice- cold phosphate-buffered saline and then fixed and permeabilized prior to FACS analysis using a LSRII flow cytometer (BD Biosciences).
In vivo efficacy of ASLAN003
The in vivo efficacy of ASLAN003 was tested in a human AML
cell line xenograft model and in human AML patient-derived xenograft (PDX) models.
For the human AML cell line xenograft model, we used female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, NGS mice (4-6 weeks old), pur- chased from The Jackson Laboratory (Bar Harbor, ME, USA) through InVivos (Singapore). The animals were maintained in spe- cific pathogen-free conditions. Exponentially growing THP-1 and MOLM-14 cells (3 × 106 each) were injected into mice via the tail vein. From the second day of inoculation of AML cells, the mice were administered either vehicle, as a control, or ASLAN003 50 mg/kg by oral gavage once daily in a 200 μL volume.
For the human AML PDX models, the AML-14 PDX line was established from a patient with AML-M4 with a normal kary- otype, while the AML-23 PDX line was established from a patient diagnosed with chronic myeloid leukemia in accelerated phase.
The protocols were reviewed and approved by the Institutional Animal Care and Use Committee in compliance with guidelines on the care and use of animals for scientific purpose. More details are provided in the Online Supplementary Methods.
Statistical analysis
A Student t-test (two-tailed paired) was used to examine the sta- tistical difference for in vitro cell line experiments, and P values <0.05 were considered to be statistically significant. Data are pre- sented as mean ± standard deviation (SD). Kaplan-Meier analyses were conducted using GraphPad Prism® version 7 (GraphPad Software; La Jolla, CA, USA) and statistical significance was calcu- lated by the log-rank test (P<0.05).
Data availability
RNA-sequencing data for MOLM-14 and KG-1 cells have been deposited in the Gene Expression Omnibus with accession num- ber GSE128950.
Results
ASLAN003 inhibits cell proliferation and induces cell differentiation of acute myeloid leukemia cell lines
ASLAN003 was found to inhibit leukemic cell prolifera- tion of THP-1, MOLM-14 and KG-1 with IC50 values of 152 nM, 582 nM, and 382 nM, respectively (Figure 1A). It is worth noting that cell viability was maintained at ~50% at ASLAN003 1 μM and higher (Figure 1A). This indicates that the mode of action of ASLAN003 differs from that of cytotoxic drugs, which cause increased cell death with higher doses. We also examined the selectivity of
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