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Discovery of a novel Nrf2 inhibitor that induces apoptosis of human acute myeloid leukemia cells.

ABSTRACT: Nuclear factor-erythroid 2-related factor 2 (Nrf2) is persistently activated in many human tumors including acute myeloid leukemia (AML). Therefore, inhibition of Nrf2 activity may be a promising target in leukemia therapy. Here, we used an antioxidant response element-luciferase reporter system to identify a novel pyrazolyl hydroxamic acid derivative, 1-(4-(tert-Butyl)benzyl)-3-(4-chlorophenyl)-N-hydroxy-1H pyrazole-5-carboxamide (4f), that inhibited Nrf2 activity. 4f had a profound growth-inhibitory effect on three AML cell lines, THP-1, HL-60 and U937, and a similar anti-growth effect in a chick embryo model. Moreover, flow cytometry of AML cells revealed increased apoptosis with 4f (10 ?M) treatment for 48 h. The protein levels of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase were enhanced in all three AML cell types. Furthermore, Nrf2 protein level was downregulated by 4f. Upregulation of Nrf2 by tert-butylhydroquinone (tBHQ) or Nrf2 overexpression could ameliorate 4f-induced growth inhibition and apoptosis. Treatment with 4f reduced both B-cell lymphoma-2 (Bcl-2) expression and Bcl-2/Bcl-2-associated X protein (Bax) ratio, which indicated that 4f induced apoptosis, at least in part, via mitochondrial-dependent signaling. Therefore, as an Nrf2 inhibitor, the pyrazolyl hydroxamic acid derivative 4f may be a promising agent in AML therapy.

SUBMITTER: Zhang J

PROVIDER: S-EPMC5352348 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Discovery of a novel Nrf2 inhibitor that induces apoptosis of human acute myeloid leukemia cells.

Zhang JinFeng J Su Le L Ye Qing Q Zhang ShangLi S Kung HsiangFu H Jiang Fan F Jiang GuoSheng G Miao JunYing J Zhao BaoXiang B

Oncotarget 20170101 5

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is persistently activated in many human tumors including acute myeloid leukemia (AML). Therefore, inhibition of Nrf2 activity may be a promising target in leukemia therapy. Here, we used an antioxidant response element-luciferase reporter system to identify a novel pyrazolyl hydroxamic acid derivative, 1-(4-(tert-Butyl)benzyl)-3-(4-chlorophenyl)-N-hydroxy-1H pyrazole-5-carboxamide (4f), that inhibited Nrf2 activity. 4f had a profound growth-inhi ...[more]

PMID: 28032588

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Project description:IntroductionThe emergence of resistance to the highly successful BCL2-directed therapy is a major unmet need in acute myeloid leukemia (AML), an aggressive malignancy with poor survival rates. Towards identifying therapeutic options for AML patients who progress on BCL2-directed therapy, we studied a clinical-stage CDK7 inhibitor XL102, which is being evaluated in solid tumors (NCT04726332).Materials and methodsTo determine the anti-proliferative effects of XL102, we performed experiments including time-resolved fluorescence resonance energy transfer, target occupancy, cell cycle and apoptosis-based assays. We also included genetically characterized primary myeloid blasts from de novo and relapsed/refractory AML patients. For mechanistic studies, CRISPR/Cas9 mediated knockout of CDK7 and c-Myc and immunoblotting were performed. NOD/SCID orthotropic and subcutaneous AML xenografts were used to determine anti-leukemic effects. To assess the synergistic effects of XL102 with Venetoclax, we performed RNA sequencing and gene set enrichment analysis using Venetoclax sensitive and resistant model systems.ResultsXL102, a highly specific, orally bioavailable covalent inhibitor of CDK7. Inhibitory effect on CDK7 by XL102 in primary myeloid blasts (n = 54) was in nanomolar range (mean = 300 nM; range = 4.0-952 nM). XL102 treated AML cells showed a reduction in phosphorylation levels of Serine 2/5/7 at carboxy-terminal domain of RNA polymerase II. T-loop phosphorylation of CDK1(Thr161) and CDK2(Thr160) was inhibited by XL102 in dose-dependent manner leading to cell-cycle arrest. c-Myc downregulation and enhanced levels of p53 and p21 in XL102 treated cells were observed. Increased levels of p21 and activation of p53 by XL102 were mimicked by genetic ablation of CDK7, which supports that the observed effects of XL102 are due to CDK7 inhibition. XL102 treated AML xenografts showed remarkable reduction in hCD45 + marrow cells (mean = 0.60%; range = 0.04%-3.53%) compared to vehicle control (mean = 38.2%; range = 10.1%-78%), with corresponding increase in p53, p21 and decrease in c-Myc levels. The data suggests XL102 induces apoptosis in AML cells via CDK7/c-Myc/p53 axis. RNA-sequencing from paired Venetoclax-sensitive and Venetoclax-resistant cells treated with XL102 showed downregulation of genes involved in proliferation and apoptosis.ConclusionTaken together, XL102 with Venetoclax led to synergistic effects in overcoming resistance and provided a strong rationale for clinical evaluation of XL102 as a single agent and in combination with Venetoclax.

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Discovery of a novel Nrf2 inhibitor that induces apoptosis of human acute myeloid leukemia cells.

Publications

Discovery of a novel Nrf2 inhibitor that induces apoptosis of human acute myeloid leukemia cells.

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