ASLAN003, a Novel and Potent Dihydroorotate Dehydrogenase (DHODH) Inhibitor, Induces Differentiation of Acute Myeloid Leukemia

Abstract

Background: Differentiation therapies achieve remarkable success in acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML). However, clinical benefits of differentiation therapies are negligible in non-APL AML, which accounts for the majority of AML cases. Dihydroorotate dehydrogenase (DHODH) regulates the fourth step of the de novo pyrimidine synthesis pathway. DHODH is a key therapeutic target for auto-immune diseases and cancer, particularly differentiation of AML. ASLAN003 is a novel, potent small molecule DHODH inhibitor being developed in AML by ASLAN Pharmaceuticals. Methods: We investigated activity of ASLAN003 in AML cell lines and primary bone marrow (BM) cells (NUS Leukemia Tissue Bank) from patients with AML (N = 14) or myelodysplastic syndromes (MDS) (N = 6) and healthy control (N = 1). We performed CTG assay, FACS analysis of cell viability and myeloid markers, wright-giemsa staining, NBT reduction assay, and qRT-PCR analysis of key lineage transcription factors to evaluate the effects of ASLAN003 on cell growth, differentiation, apoptosis, and gene expression changes in vitro. Two AML cell lines and 1 leukemic patient derived xenograft (PDX) line (NUS Leukemia Tissue Bank) were studied in NSG xenograft mice. Mice were administrated with vehicle control or ASLAN003 50 mg/kg by oral gavage once daily. Results: ASLAN003 inhibited leukemic cell growth of THP-1, MOLM-14 and KG-1 with IC50 of 152, 582 and 382 nM, respectively, at 48 h. Treatment of these leukemia cells with ASLAN003 for 96 h consistently resulted in remarkable increase of CD11b (p < 0.001) and displayed morphologic changes of terminal differentiation and positivity for NBT reduction. ASLAN003 was active in differentiation with an EC50 of 28, 85, and 56 nM, in these 3 lines, respectively. ASLAN003 induced approximately 2-fold higher CD11b+ cells than Brequinar (BRQ), another DHODH inhibitor. Addition of uridine rescued differentiation and improved cell viability in ASLAN003 treated-cells, implying on-target specificity of ASLAN003. Mechanistically, ASLAN003 induced differentiation through induction of myeloid lineage transcription factor Runx1, Pu.1, Gif1 and repression of HoxA9, Gata1. The response of primary BM cells to ASLAN003 was classified into 3 categories: sensitive if any of myeloid markers CD11b, CD14, CD13 or CD33 increased ≥ 15%; moderate: ≥ 5%, but < 15%; resistant: < 5%. Among AML samples, we observed 6 (43%) sensitive cases, 6 (43%) moderate cases and 2 (14%) resistant cases. Three (50%) MDS samples displayed sensitive response and 3 cases (50%) showed moderate response. The healthy control sample was resistant to ASLAN003. Importantly, ASLAN003 promoted differentiation and cell death of myeloid cells in one relapsed AML case. Morphologic analysis and NBT assay demonstrated the features of neutrophil differentiation in selected ASLAN003-treated primary AML blasts. For in vivo experiments, significantly prolonged survival was seen in ASLAN003-treated groups when compared to vehicle control group in both MOLM-14 (p = 0.031) and THP-1 (p < 0.001) xenograft models. ASLAN003 substantially reduced disseminated tumors and leukemic infiltration into liver in xenografted mice. The human CD45+ cells were significantly reduced in BM, peripheral blood, spleen and liver, with significantly increased differentiation of AML cells (CD11b and CD14 positive cells) in BM of treated mice in both models (p < 0.01). We also evaluated the therapeutic efficacy of ASLAN003 in one PDX line, AML-14. At the end of experiments (day 77 post treatment), all PDX mice were alive in both control and ASLAN003 group. The leukemic burden was significantly lower in ASLAN003-treated PDXs than in vehicle-treated PDXs (p = 0.04). Overall, these data demonstrate potent in vivo efficacy of ASLAN003 in inducing myeloid differentiation of blast cells and the drug appears highly tolerable even after prolonged administration. Conclusion: ASLAN003 is a novel, highly potent DHODH inhibitor that induces terminal differentiation, inhibits cell growth and promotes cell death of AML blasts, including relapsed AML blasts. ASLAN003 prolongs survival and shows therapeutic effects in mice bearing different AML cell lines and reduces leukemic burden in an AML PDX model. Currently, ASLAN003 efficacy is being evaluated in a Phase IIa clinical trial in patients with AML (NCT03451084; Ting, ASH abstract 2018). Disclosures Seet: ASLAN Pharmaceuticals: Employment, Equity Ownership. Ooi:ASLAN Pharmaceuticals: Employment, Equity Ownership. Lindmark:ASLAN Pharmaceuticals: Employment, Equity Ownership. McHale:ASLAN Pharmaceuticals: Employment, Equity Ownership. Chng:Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Aslan: Research Funding; Merck: Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

Differentiation therapy of myeloid leukemia: four decades of development

“…Earlier attempts to target DHODH with inhibitors (e.g., teriflunomide, brequinar, and leflunomide) in several cancers were unsuccessful because of either low potency or off-target effects. 118 Subsequently, more potent and selective DHODH inhibitors (ASLAN003, BAY2402234 and PTC299) (Table 4) have been developed, which show anti-leukemic activity in various preclinical models; 117,[119][120][121] and are in early phase clinical trials.…”

DHODH and cancer: promising prospects to be explored

“…As for the importance of DHODH in tumorigenesis and metastasis, DHODH becomes an attractive target for anti-malignance drug development. Until now, many DHODH inhibitors with different structures have been reported [ 7 ], such as canonical DHODH inhibitors BRQ [ 125 ], leflunomide [ 2 ], teriflunomide [ 126 ], ALASN003 [ 127 ], BAY2202234 [ 43 ], and other novel inhibitors which show DHODH inhibition effects including viral growth inhibitory factor [ 128 ], PARP inhibitor [ 129 ], myeloid differentiation inducing agent [ 60 ], p53 activating factor [ 61 ], a natural product isobavachalcone [ 70 ], or vascular endothelial growth factor A (VEGF-A) mRNA translation inhibitor [ 40 ]. Over 90 patent applications involving DHODH inhibitors have been filed in the last decade [ 107 , 130 ].…”

Emerging Therapies for Acute Myelogenus Leukemia Patients Targeting Apoptosis and Mitochondrial Metabolism

“…ASLAN003 is a novel, potent small molecule DHDOH inhibitor, developed in AML by Aslan Pharmaceuticals. This inhibitor was more active than brequinar, considered the reference standard of DHODH inhibitors, in inducing differentiation of AML blasts [214]. The large majority of primary AML cells is highly or moderately sensitive to ASLAN003 [214].…”

“…This inhibitor was more active than brequinar, considered the reference standard of DHODH inhibitors, in inducing differentiation of AML blasts [214]. The large majority of primary AML cells is highly or moderately sensitive to ASLAN003 [214]. This inhibitor displayed clear anti-leukemic activity in various in vivo AML models [214] and is under evaluation in a phase IIa study in patients with AML.…”

“…The large majority of primary AML cells is highly or moderately sensitive to ASLAN003 [214]. This inhibitor displayed clear anti-leukemic activity in various in vivo AML models [214] and is under evaluation in a phase IIa study in patients with AML. A preliminary evaluation of this ongoing clinical study showed early signs of clinical activity and a favorable safety profile [215].…”