BackgroundCurrent conventional chemotherapy for acute myeloid leukemia (AML) can achieve remission in over 70% of patients, but a majority of them will relapse within 5 years despite continued treatment. The relapse is postulated to be due to leukemia stem cells (LSCs), which are different from normal hematopoietic stem cells (HSCs). LIN28B is microRNA regulator and stem cell reprogramming factor. Overexpression of LIN28B has been associated with advance human malignancies and cancer stem cells (CSCs), including AML. However, the molecular mechanism by which LIN28B contributes to the development of AML remains largely elusive.MethodsWe modulated LIN28B expression in AML and non-leukemic cells and investigated functional consequences in cell proliferation, cell cycle, and colony-forming assays. We performed a microarray-based analysis for LIN28B-silencing cells and interrogated gene expression data with different bioinformatic tools. AML mouse xenograft model was used to examine the in vivo function of LIN28B.ResultsWe demonstrated that targeting LIN28B in AML cells resulted in cell cycle arrest, inhibition of cell proliferation and colony formation, which was induced by de-repression of let-7a miRNA. On the other hand, overexpression of LIN28B promoted cell proliferation. Data point to a mechanism where that inhibition of LIN28B induces metabolic changes in AML cells. IGF2BP1 was confirmed to be a novel downstream target of LIN28B via let-7 miRNA in AML. Notably, ectopic expression of LIN28B increased tumorigenicity, while silencing LIN28B led to slow tumor growth in vivo.ConclusionsIn sum, these results uncover a novel mechanism of an important regulatory signaling, LIN28B/let-7/IGF2BP1, in leukemogenesis and provide a rationale to target this pathway as effective therapeutic strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-017-0507-y) contains supplementary material, which is available to authorized users.

Differentiation therapies achieve remarkable success in acute promyelocytic leukemia, a subtype of acute myeloid leukemia. However, excluding acute promyelocytic leukemia, clinical benefits of differentiation therapies are negligible in acute myeloid leukemia except for mutant isocitrate dehydrogenase 1/2. Dihydroorotate dehydrogenase catalyses the fourth step of the de novo pyrimidine synthesis pathway. ASLAN003 is a highly potent dihydroorotate dehydrogenase inhibitor that induces differentiation, as well as reduces cell proliferation and viability, of acute myeloid leukemia cell lines and primary acute myeloid leukemia blasts including in chemo-resistant cells. Apoptotic pathways are triggered by ASLAN003, and it also significantly inhibits protein synthesis and activates AP-1 transcription, contributing to its differentiation promoting capacity. Finally, ASLAN003 substantially reduces leukemic burden and prolongs survival in acute myeloid leukemia xenograft mice and acute myeloid leukemia patient-derived xenograft models. Notably, the drug has no evident effect on normal hematopoietic cells and exhibits excellent safety profiles in mice, even after a prolonged period of administration. Our results, therefore, suggest that ASLAN003 is an agent targeting dihydroorotate dehydrogenase with potential in the treatment of acute myeloid leukemia. ASLAN003 is currently being evaluated in phase 2a clinical trial in acute myeloid leukemia patients. Article Summary 1. ASLAN003, a novel, potent dihydroorotate dehydrogenase inhibitor, induces differentiation of acute myeloid leukemia cells in vitro and in vivo. 2. ASLAN003 triggers apoptosis, inhibits protein synthesis and activates AP-1 transcription.

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.