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MIDH-associated DNA hypermethylation in acute myeloid leukemia reflects differentiation blockage rather than inhibition of TET-mediated demethylation.

ABSTRACT: Isocitrate dehydrogenases 1 and 2 (IDH1/2) are recurrently mutated in acute myeloid leukemia (AML), but their mechanistic role in leukemogenesis is poorly understood. The inhibition of TET enzymes by D-2-hydroxyglutarate (D-2-HG), which is produced by mutant IDH1/2 (mIDH1/2), has been suggested to promote epigenetic deregulation during tumorigenesis. In addition, mIDH also induces a differentiation block in various cell culture and mouse models. Here we analyze the genomic methylation patterns of AML patients with mIDH using Infinium 450K data from a large AML cohort and found that mIDH is associated with pronounced DNA hypermethylation at tens of thousands of CpGs. Interestingly, however, myeloid leukemia cells overexpressing mIDH, cells that were cultured in the presence of D-2-HG or TET2 mutant AML patients did not show similar methylation changes. In further analyses, we also characterized the methylation landscapes of myeloid progenitor cells and analyzed their relationship to mIDH-associated hypermethylation. Our findings identify the differentiation state of myeloid cells, rather than inhibition of TET-mediated DNA demethylation, as a major factor of mIDH-associated hypermethylation in AML. Furthermore, our results are also important for understanding the mode of action of currently developed mIDH inhibitors.

SUBMITTER: Wiehle L

PROVIDER: S-EPMC6551656 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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mIDH-associated DNA hypermethylation in acute myeloid leukemia reflects differentiation blockage rather than inhibition of TET-mediated demethylation.

Wiehle Laura L Raddatz Günter G Pusch Stefan S Gutekunst Julian J von Deimling Andreas A Rodríguez-Paredes Manuel M Lyko Frank F

Cell stress 20170920 1

Isocitrate dehydrogenases 1 and 2 (IDH1/2) are recurrently mutated in acute myeloid leukemia (AML), but their mechanistic role in leukemogenesis is poorly understood. The inhibition of TET enzymes by D-2-hydroxyglutarate (D-2-HG), which is produced by mutant IDH1/2 (mIDH1/2), has been suggested to promote epigenetic deregulation during tumorigenesis. In addition, mIDH also induces a differentiation block in various cell culture and mouse models. Here we analyze the ge ...[more]

PMID: 31225434

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Project description:BackgroundRecent massive sequencing studies have revealed that SWI/SNF complexes are among the most frequently altered functional entities in solid tumors. However, the role of SWI/SNF in acute myeloid leukemia is poorly understood. To date, SWI/SNF complexes are thought to be oncogenic in AML or, at least, necessary to support leukemogenesis. However, mutation patterns in SWI/SNF genes in AML are consistent with a tumor suppressor role. Here, we study the SWI/SNF subunit BCL7A, which has been found to be recurrently mutated in lymphomas, but whose role in acute myeloid malignancies is currently unknown.MethodsData mining and bioinformatic approaches were used to study the mutational status of BCL7A and the correlation between BCL7A expression and promoter hypermethylation. Methylation-specific PCR, bisulfite sequencing, and 5-aza-2'-deoxycytidine treatment assays were used to determine if BCL7A expression was silenced due to promoter hypermethylation. Cell competition assays after BCL7A expression restoration were used to assess the role of BCL7A in AML cell line models. Differential expression analysis was performed to determine pathways and genes altered after BCL7A expression restoration. To establish the role of BCL7A in tumor development in vivo, tumor growth was compared between BCL7A-expressing and non-expressing mouse xenografts using in vivo fluorescence imaging.ResultsBCL7A expression was inversely correlated with promoter methylation in three external cohorts: TCGA-LAML (N = 160), TARGET-AML (N = 188), and Glass et al. (2017) (N = 111). The AML-derived cell line NB4 silenced the BCL7A expression via promoter hypermethylation. Ectopic BCL7A expression in AML cells decreased their competitive ability compared to control cells. Additionally, restoration of BCL7A expression reduced tumor growth in an NB4 mouse xenograft model. Also, differential expression analysis found that BCL7A restoration altered cell cycle pathways and modified significantly the expression of genes like HMGCS1, H1-0, and IRF7 which can help to explain its tumor suppressor role in AML.ConclusionsBCL7A expression is silenced in AML by promoter methylation. In addition, restoration of BCL7A expression exerts tumor suppressor activity in AML cell lines and xenograft models.

Project description:A cute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase.

Dataset Information

MIDH-associated DNA hypermethylation in acute myeloid leukemia reflects differentiation blockage rather than inhibition of TET-mediated demethylation.

Publications

<i>mIDH</i>-associated DNA hypermethylation in acute myeloid leukemia reflects differentiation blockage rather than inhibition of TET-mediated demethylation.

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