Acute myeloid leukemia (AML) is a hematological malignancy featured by impaired differentiation and uncontrolled proliferation of myeloid blasts. FLT3 internal tandem duplication (ITD) presents in 30-40% patients with AML, which serves as an independent poor prognostic marker and an attractive therapeutic target. Up to date, several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved by FDA in the treatment of AML. However, FLT3 TKIs as single agents have limited effects to eliminate AML cells due to multiple drug-resistant mechanisms, including secondary FLT3 mutations, alternatively activated cell survival pathways, upregulation of FLT3 ligand, and downregulation of tumor suppressor genes. In the present study, we found that FLT3 TKIs decreased tumor suppressor p53 protein level by downregulation of YOD1 through miR-181 in FLT3-ITD mutant AML cells. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. By using transcriptome analysis with RNAseq, we identified multiple differentially expressed miRNAs in HCD-57 transformed by FLT3-ITD, compared with parental HCD-57 cells. miR-181a-5p and miR-181b-5p were among these highly differentially expressed miRNAs. These two miRNAs were predicted to bind in 3'UTR of deubiquitinase YOD1 by using TargetScanHuman 7.2, an online tool to predict biological targets of miRNAs. Previous studies have shown that the ubiquitination and protein level of p53 is dysregulated due to overexpressed E3 ligase MDM2/4 in AML cells, but few studies focused on deubiquitinase of p53. We found that deubiquitinase YOD1 interacted with p53 by immunoprecipitation. Overexpression of YOD1 prevented degradation of p53 led by cycloheximide, a protein synthesis inhibitor. We further found that overexpression of YOD1 resulted in decreased ubiquitination of p53, indicating that YOD1 stabilized p53 protein through deubiquitination. Subsequently, we detected the expression of miR-181, YOD1 and p53 in a FLT3-ITD positive AML cell line MV-4-11 treated by FLT3 TKIs sorafenib, sunitinib and quizartinib. Real-time quantitative PCR showed that the treatment of FLT3 TKIs upregulated the expression of miR-181a-5p/miR-181b-5p, and downregulated mRNA level of their predicted target YOD1. The mRNA level of p53 remained unchanged but its protein level decreased with enhanced ubiquitination in MV-4-11 cells treated by FLT3 TKIs in the ubiquitination assay. These data suggested that FLT3 TKIs could reduce the stability of p53 by regulating miRNA-targeted YOD1. In addition, we collected peripheral blood mononuclear cells (PBMCs) from patients with AML and age-matched healthy donors. We found increased expression of miR-181a-5p and miR-181b-5p and decreased expression of YOD1 in PBMCs from AML patients compared with healthy donors, suggesting a pathological role of miR-181 to regulate YOD1/53 pathway in AML. In conclusion, our data showed that FLT3 TKIs induced instability of p53 by miR-181 mediated downregulation of YOD1. YOD1 as a novel deubiquitinase of p53 could play important roles in drug-resistance and progression of AML.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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