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ASLAN003 for differentiation of AML
ASLAN003 and brequinar on AML cells over normal CD34+CD38+ BM cells obtained from healthy donors. CD34+CD38+ cells are considered as dividing myeloid pro- genitor cells. The IC50 values of ASLAN003 and brequinar were 5.22 μM and 2.87 μM, respectively (Online Supplementary Figure S1A). ASLAN003 and brequinar were on average 11-fold more active in AML cells than in nor- mal CD34+CD38+ BM myeloid progenitor cells, suggesting a favorable therapeutic index for DHODH inhibitors.
Next we examined the differentiation effects of ASLAN003 on THP-1, MOLM-14 and KG-1 cells. Treatment of these leukemic cells with ASLAN003 consis- tently resulted in a substantial increase of CD11b in all three cell lines. The CD11b+ population was increased by 86.7%, 63.9% and 86.5% in THP-1, MOLM-14 and KG-1 cells treated with 100 nM ASLAN003 after normalization to values for DMSO-treated samples (P<0.001) (Figure 1B, Online Supplementary Figure S1B). CD14+ cells were also significantly increased in MOLM-14, THP-1 and KG-1 cells following treatment with ASLAN003 (P<0.01) (Figure 1B, Online Supplementary Figure S1B). Secondly, cells treat- ed with ASLAN003 displayed morphological changes with a lower nucleocytoplasmic ratio, condensed chro- matin, and increased nuclear lobulation, which are charac- teristics of myeloid maturation (Figure 1C). Thirdly, we employed a NBT reduction assay to evaluate functional evidence of myeloid maturation. After 96 h of treatment with ASLAN003 100 nM, 95.2% of the THP-1 cells, 62.4% of the MOLM-14 cells, and 93.6% of the KG-1 cells were positive for NBT reduction (P<0.001). At concentra- tions of ASLAN003 as low as 50 nM, more than 50% of the THP-1 cells showed increased NBT reduction com- pared with DMSO controls (P<0.001) (Figure 1D). Furthermore, in a time-dependent experiment, following 24 or 48 h exposure to 100 nM ASLAN003, almost 100% of THP-1 cells became CD11b+ (Figure 1E, Online Supplementary Figure S1C). Taken together, these results suggest that ASLAN003 can rapidly induce differentiation of AML cells.
Differentiation effect of ASLAN003 and brequinar as well as uridine rescue
Parallel experiments were carried out to compare the efficacy of ASLAN003 and brequinar. MOLM-14 cells were incubated with brequinar or ASLAN003 100 nM, a concentration similar to the half maximal effective con- centration (EC50) of ASLAN003 in MOLM-14 cells, for 96 h. After normalization to the respective controls, MOLM- 14 cells treated with brequinar had 33.1% of CD11b+ cells, while those treated with the same dose of ASLAN003 had 63.9% of CD11b+ cells (Figure 2A, Online Supplementary Figure S1D). These results showed a nearly two-fold high- er potency of ASLAN003 compared to brequinar (P<0.05).
Because DHODH coverts dihydroorotate into orotate, which is a precursor of uridine, inhibition of DHODH leads to a diminished uridine pool in cells.16 In both MOLM-14 and THP-1 cells, ASLAN003-mediated differ- entiation was completely rescued by addition of 50 μM uridine (Figure 2B), with no significant further rescue detected in the presence of higher concentrations of uri- dine (100 μM and 150 μM) (data not shown). Interestingly, uridine also rescued the cell viability of ASLAN003-treat- ed MOLM-14 and THP-1 cells (P<0.05) (Figure 2C). Overall, these data demonstrate that uridine could abro- gate the effects of ASLAN003 on cell differentiation and
cell viability, implying the on-target specificity of ASLAN003.
ASLAN003 decreases viability and induces differentiation in primary acute myeloid leukemia blasts and myelodysplastic syndrome samples
To confirm the clinical relevance of our observations in human AML cell lines, we examined the effect of ASLAN003 on cell viability and differentiation status in BM cells obtained from patients with de novo or relapsed AML and myelodysplastic syndrome (MDS). ASLAN003 displayed excellent potency in inducing differentiation and cell death in some primary AML blasts. For example, in patient UPN1 with AML-M1 with t(9;22) and a com- plex karyotype, exposure to ASLAN003 at the concentra- tions of 2 μM and 4 μM led, respectively, to 22% and 30% increases in CD11b+ cells, as well as 31% and 35% increases in CD14+ cells. Concomitantly, there were decreases of 18% and 27%, respectively, in cell viability. Furthermore, in patient UPN6 with AML-M2 with FLT3- ITD and NPM1 mutations, following incubation with ASLAN003 2 μM and 4 μM for 96 h, we observed 18% and 23% more CD13/CD33 double positive cells, accom- panying reduced cell viability. Among these tested sam- ples, UNP5 with deletion of chromosome 7 had the most sensitive response, with the CD11b+ population increasing by 62% in response to ASLAN003 1 μM. Importantly, ASLAN003 was still effective in promoting differentiation and cell death of myeloid cells in relapsed AML (UNP13). Morphological analysis and NBT assays demonstrated the features of neutrophil differentiation in ASLAN003-treat- ed AML blasts from selected cases (Online Supplementary Figure S2). In summary, the response of primary BM cells from AML patients to ASLAN003 was classified into three categories: sensitive if any of the myeloid markers CD11b, CD14, CD13 or CD33 increased ≥15%; moderately sensi- tive if the markers increased ≥5%, but <15%; and resistant if the markers did not increase or increased <5%. Among the AML samples, we observed six (43%) sensitive cases, six (43%) moderately sensitive cases and two (14%) resistant cases (Table 1).
For BM samples from MDS patients, three cases (50%) were sensitive to ASLAN003 and three cases (50%) were moderately sensitive (Table 1). Thus, on the bases of these data, MDS cells appear to be sensitive to ASLAN003 treat- ment. No resistant cases were seen, but the number of cases tested was limited.
Notably, ASLAN003, at the concentrations of 2 μM and 4 μM, was shown to have a negligible impact on cell via- bility and differentiation status of mononuclear cells from a healthy donor, suggesting that ASLAN003 is not toxic to normal hematopoietic cells (Table 1). Collectively, these experiments provide evidence that ASLAN003 treatment of primary cells obtained from either de novo or relapsed patients leads to myeloid differentiation and cell death.
Transcriptome analysis reveals the effects of ASLAN003 on apoptosis, differentiation, metabolism and translation initiation in acute myeloid leukemia cells
To understand the impact of ASLAN003 on transcrip- tional networks in AML, we performed RNA-sequencing on DMSO- and ASLAN003-treated KG-1 and MOLM-14 cells. These cells shared 320 upregulated genes and 225 downregulated genes (posterior probability of differential
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