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ASLAN003 for differentiation of AML
apoptosis” and “positive regulation of myeloid cell differ- entiation”, while downregulated genes associated with ”co-translational protein targeting to membrane”, “rRNA metabolic process”, “ribosome biogenesis”, “translation” and “peptide biosynthetic process”, showed the most sig- nificant changes (Figure 3E). These data from transcrip- tome analysis suggest that ASLAN003 might impair pro- tein synthesis and induce the differentiation and apoptosis transcriptional program in AML cells.
ASLAN003 triggers apoptotic pathways
Fas (CD95, APO-1) belongs to the cell death receptor family. Upon binding to its ligand FasL, they form death- inducing signaling complex (DISC), initiating the extrinsic apoptosis cascade.22 Our RNA-sequencing data showed that the expression of FAS was upregulated after treat- ment (Figure 3B, C, Online Supplementary Table S2), sug- gesting that ASLAN003 could trigger the extrinsic apopto- sis pathway. Indeed, western blot analysis revealed a sig- nificant increase in cleaved caspase 8, the extrinsic path- way-specific caspase, in ASLAN003-treated KG-1 and MOLM-14 cells (Figure 4A).
Because DHODH is located on the inner mitochondrial membrane, we also assessed whether ASLAN003 could induce loss of mitochondrial membrane potential (ΔΨm), an indicator of the early phase of intrinsic (mitochondrial or BCL-2-regulated) apoptosis. In the DMSO-treated cells, the majority of cells had intact mitochondrial membranes. ASLAN003 was found to disrupt ΔΨm in a dose-depen-
dent manner in THP-1, MOLM-14 and KG-1 cells (Figure 4B). To investigate whether loss of ΔΨm is DHODH-spe- cific, we conducted mitochondrial membrane potential assays in MOLM-14 cells treated with brequinar, another DHODH inhibitor and cytarabine, a chemotherapeutic agent. Both brequinar and cytarabine could induce loss of ΔΨm (Online Supplementary Figure S3), suggesting that loss of ΔΨm is not DHODH-specific, most likely being a marker of cell health. However, given the localization of DHODH, the effect of its inhibitors on mitochondrial membrane potential might be direct, while the effect of cytarabine might be indirect. As a result of loss of ΔΨm, increased leakage of cytochrome c from mitochondria into the cytosol, a characteristic of activation of the intrinsic apoptosis pathway, was also observed in ASLAN003- treated cells (Figure 4A). ASLAN003 treatment also induced cleaved caspase-3 and -7 (Figure 4A). Altogether, these results suggest a role for both intrinsic and extrinsic pathways in ASLAN003-induced apoptosis.
ASLAN003 inhibits protein synthesis and induces differentiation of acute myeloid leukemia cells via activation of AP-1 transcription factors
Transcriptome and gene ontology analysis showed a greater enrichment of protein translation-related genes and ribosome proteins among the genes downregulated by ASLAN003. Furthermore, gene expression of four members of the family of eukaryotic translation initiation factors (eIF), namely EEF1B2, EIF4B, EIF3L, and EEF1B2P3,
Table 1. Clinical characteristics of patients with acute myeloid leukemia and myelodysplastic syndrome and their responses to ASLAN003 in an ex vivo assay.
Response group
Highly sensitive
AML (43%) MDS (50%)
Moderately sensitive
AML (43%) MDS (50%)
Resistant
AML (14%)
Diagnosis
AML-M1
AML-M5
AML-M2 AML with MDS AML-M5 AML-M4 MDS MDS MDS
AML-M2
AML-M1
AML-M4 Relapsed AML AML-M4 AML-M4 MDS MDS MDS
AML-M5a
AML-M1
Healthy donor
Karyotype
t(9;22)
Normal +8 t(8;21) Normal -7 Normal Normal Complex
Normal
-9 Normal +13 Inv(16) Inv(16) Normal -7 -13, +8
+8
+11
Normal
FLT3 NPM1
WT WT
WT WT ITD Mutant
NA NA NA NA WT WT NA NA NA NA NA NA
TKD NA
NA NA WT WT ITD NA TKD WT ITD WT NA NA WT WT NA NA
TKD Mutant
WT WT
WT WT
Patient ID
UPN1
UPN4 UPN6 UPN7 UPN9 UPN5 UPN16 UPN17 UPN18
UPN2
UPN8 UPN11 UPN12 UPN10 UPN13 UPN15 UPN19 UPN20
UPN3
UPN14
The response of primary bone marrow cells to ASLAN003 was classified into three categories: sensitive if any of myeloid markers CD11b, CD14, CD13 or CD33 increased ≥15%; moderately sensitive if the markers increased ≥5%, but <15%; and resistant if the markers increased <5%, by FACS analysis. FLT3: fms-like tyrosine kinase; NPM: nucleophosmin-1; ID:identify;AML:acute myeloid leukemia;MDS:myelodysplastic syndromes;WT:wildtype;UPD:unique patient number;ITD:internal tandem duplication;NA;not available;TKD: tyrosine kinase domain.
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