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Inhibition of DHODH induces differentiation in AML
blot revealed that DHODH expression levels were not affected in isobavachalcone-treated tumors (Figure 5F). Interestingly, DHODH activity decreased in isobavachal- cone-treated tumors compared with vehicle-treated tumors, as determined by a cellular DHODH enzyme assay based on the use of a 4-trifluoromethyl-benzami- doxime fluorogenic reagent (Figure 5G). Hence, we hypothesized that isobavachalcone suppresses tumor growth by inhibiting DHODH enzymatic activity in vivo. We checked how isobavachalcone affects the expression levels of three apoptosis-related markers (cleaved cas- pase-9, cleaved caspase-3, and cleaved PARP) and two differentiation-related proteins (MYC and p21) in xenograft tumors. As expected, cleaved caspase-9, cleaved caspase-3 and cleaved PARP and p21 levels were noticeably elevated in the isobavachalcone-treated groups, while MYC protein level decreased (Figure 5H), consistent with the in vitro analysis (Figures 3H and 4D). Notably, isobavachalcone shows greater efficacy than leflunomide (Figure 5A-H).
The combination of isobavachalcone and adriamycin shows synergistic antileukemic effects in vitro
and in vivo
Adriamycin is a widely used chemotherapy drug for treatment of AML in the clinic. However, as monothera- py, its therapeutic efficacy is limited by problems such as acquired resistance.9 We investigated whether isobavachalcone sensitizes AML to adriamycin. We first determined the cytotoxicity of adriamycin in four human AML cell lines: HL60, THP-1, U937 and MOLM-13 (Online Supplementary Figure S9). We then examined the efficacy of a combination of isobavachalcone and adri- amycin across the four AML cell lines and found that the isobavachalcone and adriamycin combination led to a cooperative suppression of AML cell growth (Figure 6A- D). We further investigated the potency of combinational therapy using a disseminated HL60 model of AML. As shown in Figure 6E, mice treated with the isobavachal- cone and adriamycin combination had a significantly longer survival compared with that of animals in the other groups. Of note, treatment with isobavachalcone led to AML differentiation in vivo, as evidenced by Wright–Giemsa staining (Figure 6F).
We also investigated the effect of isobavachalcone on adriamycin-resistant HL60/adriamycin cells in vitro. As expected, the HL60/adriamycin cell line showed greater resistance to adriamycin (IC50 = 38.77±0.81 μM), (Online Supplementary Figure S10A) compared to HL60 cells (IC50 = 0.36±0.05 μM), (Online Supplementary Figure S9A). We found that isobavachalcone markedly suppressed HL60/adriamycin cell growth in a concentration-depen- dent manner (Online Supplementary Figure S10B). Online Supplementary Figure S10C shows that the combination of isobavachalcone and adriamycin had an enhanced antiproliferative effect in HL60/adriamycin cells com- pared with the effect of isobavachalcone or adriamycin alone in vitro. The values of the combination index, which were all <0.8, suggest a synergistic antitumor effect between isobavachalcone and adriamycin.33 Notably, the group co-administered isobavachalcone and adriamycin exhibited markedly coordinative anti-tumor activity com- pared with monotherapy and control groups (P<0.001, one-way ANOVA) (Online Supplementary Figure S10D,E). No obvious changes were observed in the animals’ body
weight, indicating that the combined therapy was well tolerated (Online Supplementary Figure S10F). In summary, isobavachalcone combined with adriamycin effectively suppresses the growth of adriamycin-resistant AML cells in vitro and in vivo, offering a potential drug combination strategy for AML therapy.
Discussion
Differentiation therapy is inspired by the observation that hormones and cytokines can induce differentiation ex vivo; it can, therefore, be a powerful way of irreversibly altering the phenotype of malignant cells.34 The high cure rates of acute promyelocytic leukemia by a combination of retinoic acid and arsenic underscore the success of dif- ferentiation therapy.34 However, approximately 90% of patients with AML other than acute promyelocytic leukemia do not benefit from the combination of retinoic and arsenic. New differentiation therapy strategies are urgently needed to improve the clinical outcome of these patients. In this study, we demonstrated that DHODH is a potent regulator of AML cell growth, apoptosis and dif- ferentiation. Specifically, genetic knockout or pharmaco- logical inhibition of DHODH overcomes a differentiation blockade of myeloid cells by promoting MYC degrada- tion. Via systematic screening of an in-house natural prod- uct library, we identified isobavachalcone as an effective, direct DHODH inhibitor. By targeting DHODH, isobavachalcone suppressed tumor growth, overcoming the differentiation blockade of AML in vitro and in vivo, without a significant toxic profile, thus making it a poten- tial therapeutic agent for AML differentiation.
MYC is a pro-oncogenic transcription factor that con- tributes to tumorigenesis and tumor progression of human cancers including leukemia.35,36 MYC levels are correlated with tumor cell progression and differentiation and myeloid cell differentiation is dependent on the sup- pression of this transcription factor.37 An earlier study revealed that inhibition of DHODH abrogates transcrip- tional elongation of the MYC gene in melanoma.38 The DHODH inhibitor leflunomide can reduce MYC expres- sion and consequently reduce proliferation of human melanoma cells.38 However, the ramifications of DHODH inhibition on MYC in AML are still unclear. In our exper- iments, the level of MYC expression was markedly low- ered after the silencing of DHODH, either by knockout of DHODH or by the introduction of the DHODH inhibitor. Isobavachalcone induced MYC degradation in a proteasome-dependent manner. In addition, extended isobavachalcone treatment inhibited MYC transcriptional activity in a luciferase reporter assay. Sykes and col- leagues reported that inhibition of DHODH overcame differentiation blockade in AML; however, the precise pathway affected by DHODH inhibition is still not understood.14 We suggest that inhibition of DHODH through down-regulation of MYC induces AML cell dif- ferentiation.
It was previously reported that isobavachalcone, a nat- urally occurring chalcone, exhibited anticancer activity in several types of malignancies. For instance, isobavachal- cone showed anti-cancer activities in a two-stage mouse skin cancer model.39 Yang et al. reported that isobavachal- cone impaired the growth and increased apoptosis of the ovarian carcinoma cell line OVCAR-8 and prostate cancer
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