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J. Zhou et al.
were alive and no obvious signs of disease were observed in either the control or ASLAN003 group. However, we found that the leukemic burden was significantly less in ASLAN003-treated PDX than in vehicle-treated PDX (P=0.04) (Figure 6C). The weight of the mice increased gradually and similarly in both groups during the treat- ment (P=0.42) (Online Supplementary Figure S5C). For AML-23, an aggressive PDX line, all animals with vehicle- treated xenografts (n=9) succumbed to the disease within a month (median survival 20 days). In contrast, 50% of the animals with ASLAN003-treated PDX (n=8) were still active and alive at the end of experiments on day 37 (P=0.0002) (Figure 6D). Importantly, the percentages of human CD45+ leukemia cells were significantly reduced in BM, peripheral blood, spleen, and liver of the ASLAN003 group as compared to the control group. The in vivo differ- entiation effect of ASLAN003 was also confirmed by the observation of increased human CD11b+ and CD14+ cells in BM (Figure 6E). As for the AML-14 line, there was no statistical difference between the body weight of the AML-23 control group and the ASLAN003-treated group (P=0.73) (Online Supplementary Figure S5C). Overall, these data demonstrate that ASLAN003 treatment mediates therapeutic efficacy in AML-PDX by extending survival, reducing leukemic burden and inducing differentiation. Notably, ASLAN003 appears safe and well tolerated even after prolonged in vivo administration.
Discussion
Recently, DHODH has been demonstrated to be a novel target for differentiation therapy in AML.24 Brequinar is the first DHODH inhibitor that has shown potency in inducing differentiation, but its clinical use is impeded by its hematologic toxicity and ineffectiveness in early trials in patients with solid tumors.19,25,26 Several other DHODH inhibitors have been described. PTC299, an inhibitor of VEGFA mRNA translation, has also been shown to target DHODH and to have broad activity against hematologic cancer cells in a preclinical setting.27 Isobavachalcone, a natural product, has been reported to target DHODH, resulting in apoptosis and differentiation of AML cell lines at a high concentration (10 μM) in vitro.28 BAY 2402234, a novel DHODH inhibitor, induces differ- entiation and inhibits proliferation in multiple AML sub- types and is currently being evaluated in a phase I trial in myeloid malignancies.29
In this study, we comprehensively characterize ASLAN003, a novel, potent DHODH inhibitor. ASLAN003 induces massive differentiation of AML cell lines, as well as primary AML and MDS cells. ASLAN003 triggers apoptotic pathways in AML cell lines. Multiple mechanisms may account for these effects of ASLAN003 on leukemia cells. In general, myeloid leukemia cells have a higher proliferation rate compared to normal myeloid cells, thus requiring more energy (ATP) and abundant amounts of precursors for many biosynthetic pathways. The de novo biosynthesis of pyrimidine provides multiple essential precursors for such pathways. By targeting DHODH, a key enzyme in pyrimidine biosynthesis, ASLAN003 significantly depletes pyrimidine nucleotides, leaving insufficient precursors for leukemia cells to biosynthesize DNA, RNA, and proteins. Consistently, our RNA-sequencing data revealed that a large family of genes
associated with protein translation initiation was the top and the largest class downregulated by ASLAN003 treat- ment. We further experimentally validated that ASLAN003 inhibits protein synthesis in AML cells. A growing body of evidence supports a critical onco-addic- tion on active protein translation in AML cells. Aberrant protein translation contributes to arrested differentiation of myeloid cells and leukemogenesis.30,31 EIF4B, one mem- ber of the eIF family, is downregulated by ASLAN003. EIF4B has been found to stimulate translation of a partic- ular set of genes with long, structured 5’-untranslated regions, such as MYC, BCL-2, and XIAP, which promote cell survival and proliferation.32 Ribavirin, which blocks the binding of eIF4E to mRNA, has been shown to induce complete or partial remission in some relapsed AML patients.33
Notably, our study has determined that ASLAN003- meditated AP-1 activation is important for the reversal of the blocked differentiation of AML cells. Transcription fac- tors for AP-1 comprise several families of protein dimers, mainly JUN (c-Jun, JunB and JunD), FOS (c-Fos, FosB, Fra1, and Fra2) and ATF (ATFa, ATF-2, and ATF-3).34 It is known that AP-1 transcription factors are implicated in the differ- entiation of leukemia cells.35 Deletion of JunB in transgenic mice causes leukemogenic stem cell expansion, resulting in a myeloproliferative disorder which resembles early human chronic myelogenous leukemia.36 Early studies demonstrated that cytarabine treatment induced differen- tiation of AML cells and enhanced JUN/AP-1 activity was observed.37 Overexpression of c-Fos overrides the blockage of differentiation mediated by c-Myc and potentiates inter- leukin-6-induced differentiation in AML cells.38 In agree- ment with these findings, our data indicate a vital role for AP-1 in ASLAN003-induced differentiation of AML. The differentiation effect of ASLAN003 is almost completely negated in KG-1 cells and partially abrogated in MOLM-14 cells by co-treatment with an AP-1 inhibitor, T-5224.
In further support of its clinical relevance, ASLAN003 induced primary AML blast myelocytic differentiation and decreased viability. It is worth noting that these therapeu- tic effects were achieved not only in samples from patients with de novo AML, but also in samples from patients with relapsed disease. In our study, a once daily dose of ASLAN003 50 mg/kg for 2 to 11 weeks in two AML cell line xenografts and two PDX models of NSG mice did not affect the animals’ body weight, indicating that the drug is safe and well-tolerated.
In summary, our study demonstrates that ASLAN003 is a novel, potent DHODH inhibitor characterized by anti- AML efficacy in vitro and in vivo and remarkable tolerability. We also provide molecular mechanisms through which ASLAN003 exerts multiple actions, including induction of apoptotic pathways, inhibition of protein translation and activation of AP-1 transcription factors. Taken together, our findings support the further development of ASLAN003 for clinical use in AML, a disease for which novel therapies are much needed. ASLAN003 has been granted orphan drug designation for the treatment of AML by the Food and Drug Administration and is currently being evaluated in a phase IIa trial in AML (ClinicalTrials.gov: NCT03451084).
Acknowledgments
This work was supported by a research fund from ASLAN Pharmaceuticals and the Singapore National Research
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haematologica | 2020; 105(9)