Abstract
DNA (cytosine-5)-methyltransferase 3A (DNMT3A) mutations occur in ~20% of de novo acute myeloid leukemia (AML) patients, and >50% of these mutations in AML samples are heterozygous missense alterations within the methyltransferase domain at residue R882. DNMT3A R882 mutations in AML patients promote resistance to anthracycline chemotherapy and drive relapse. In this study, we performed high-throughput screening and identified that oridonin, an ent-kaurene diterpenoid extracted from the Chinese herb Rabdosia rubescens, inhibits DNMT3A R882 mutant leukemic cells at a low-micromolar concentration (IC50 = 2.1 µM) by activating both RIPK1-Caspase-8-Caspase-3-mediated apoptosis and RIPK1-RIPK3-MLKL-mediated necroptosis. The inhibitory effect of oridonin against DNMT3A R882 mutant leukemia cells can also be observed in vivo. Furthermore, oridonin inhibits clonal hematopoiesis of hematopoietic stem cells (HSCs) with Dnmt3a R878H mutation comparing to normal HSCs by inducing apoptosis and necroptosis. Overall, oridonin is a potential and promising drug candidate or lead compound targeting DNMT3A R882 mutation-driven clonal hematopoiesis and leukemia.
Highlights
DNA-methyltransferase 3A (DNMT3A) is one of the most frequently mutated genes in age-related clonal hematopoiesis (ARCH) which is related to the increases in the risk of hematological malignancies and the all-cause mortality [1,2,3]
The DNA (cytosine-5)-methyltransferase 3A (DNMT3A) R882H mutation in K562-R882H and K562-tdTomato cells was confirmed by Sanger sequencing, and the result showed that the 2645 site in the exon 23 of DNMT3A was successfully edited by CRISPR-Cas9 in these two cell lines (Fig. 1D)
To evaluate the response of DNMT3A R882H cells used in this study to chemotherapy drugs, K562-tdTomato, and K562-enhanced green fluorescent protein (EGFP) cells were mixed at the ratio of 1:1 and the mixed cells were treated with different commonly used chemotherapy drugs for the indicated time (Fig. 1E)
Summary
DNA (cytosine-5)-methyltransferase 3A (DNMT3A) is one of the most frequently mutated genes in age-related clonal hematopoiesis (ARCH) which is related to the increases in the risk of hematological malignancies and the all-cause mortality [1,2,3]. DNMT3A is one of the de novo DNA methyltransferases, and DNMT3A R882H AML cells exhibit decreased de novo methyltransferase activity and markedly hypomethylation throughout the genomes of these cells at specific CpGs [7]. It seems to be contradictory that hypomethylating agents, including azacytidine and decitabine, exhibit therapeutic effect on DNMT3A mutant AML patients [9, 10], which may be due to the changed flanking sequence preference of the aberrant DNMT3A enzyme that results in abnormal hypermethylation at some specific gene loci contributing to leukemogenesis [11, 12]. Highdose anthracycline therapies show certain efficacy for DNMT3A mutant leukemia in AML clinical trials, while the increased toxicity limits its application [13]
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