The Antioxidant Protein Peroxiredoxin 4 Is Epigenetically Down Regulated in Acute Promyelocytic Leukemia

The antioxidant peroxiredoxin (PRDX) protein family comprises 6 members, which are implicated in a variety of cellular responses, including growth factor signal transduction. PRDX4 resides in the endoplasmic reticulum (ER), where it locally controls oxidative stress by reducing H2O2levels. We recently provided evidence for a regulatory function of PRDX4 in signal transduction from a myeloid growth factor receptor, the granulocyte colony-stimulating factor receptor (G-CSFR). Upon activation, the ligand-induced G-CSFR undergoes endocytosis and routes via the early endosomes where it physically interacts with ER-resident PRDX4. PRDX4 negatively regulates G-CSFR mediated signaling. Here, we investigated whether PRDX4 is affected in acute myeloid leukemia (AML); genomic alterations and expression levels of PRDX4 were investigated. We show that genomic abnormalities involving PRDX4 are rare in AML. However, we find a strong reduction in PRDX4 expression levels in acute promyelocytic leukemia (APL) compared to normal promyelocytes and different molecular subtypes of AML. Subsequently, the possible role of DNA methylation and histone modifications in silencing of PRDX4 in APLs was investigated. We show that the reduced expression is not due to methylation of the CpG island in the promoter region of PRDX4 but correlates with increased trimethylation of histone 3 lysine residue 27 (H3K27me3) and lysine residue 4 (H3K4me3) at the transcriptional start site (TSS) of PRDX4, indicative of a bivalent histone code involved in transcriptional silencing. These findings suggest that the control of G-CSF responses by the antioxidant protein PRDX4 may be perturbed in APL

The gene encoding thioredoxin-interacting protein (TXNIP) is a frequent virus integration site in virus-induced mouse leukemia and is overexpressed in a subset of AML patients

Thioredoxin-interacting protein (TXNIP) is involved in reactive oxygen species-induced stress responses. In a screen for novel disease genes in murine leukemia virus (MLV)-induced mouse leukemias, we identified Txnip as a frequent target for proviral integration. Ectopic TXNIP expression inhibited the proliferation of myeloid progenitor cells. TXNIP transcript and protein levels were significantly elevated in human AML blasts of certain patients, particularly those harboring translocation t(8;21). Nucleotide sequencing revealed no abnormalities in the TXNIP coding region in AML. These findings suggest that deregulated TXNIP expression contributes to MLV-induced murine leukemia as well as human AML.</p

The gene encoding thioredoxin-interacting protein (TXNIP) is a frequent virus integration site in virus-induced mouse leukemia and is overexpressed in a subset of AML patients

Thioredoxin-interacting protein (TXNIP) is involved in reactive oxygen species-induced stress responses. In a screen for novel disease genes in murine leukemia virus (MLV)-induced mouse leukemias, we identified Txnip as a frequent target for proviral integration. Ectopic TXNIP expression inhibited the proliferation of myeloid progenitor cells. TXNIP transcript and protein levels were significantly elevated in human AML blasts of certain patients, particularly those harboring translocation t(8;21). Nucleotide sequencing revealed no abnormalities in the TXNIP coding region in AML. These findings suggest that deregulated TXNIP expression contributes to MLV-induced murine leukemia as well as human AML.</p

Reduced PRDX4 protein and transcript levels in APL.

<p><b>A</b>. Lysates of APL and AML samples were analyzed for PRDX4 protein levels. Because human <i>PRDX4</i> gene is located on the X-chromosome, male samples and female samples were compared with male and female AML control samples respectively to rule out the possibility of X-inactivation involvement. Patient number followed by F indicates female sample while patient number followed by M indicates male sample. <b>B. Upper panel</b>: Western blot analysis of PRDX4 expression in different normal bone marrow fractions (myeloblasts, promyelocytes, neutrophils). <b>Lower panel</b>: Micrographs of May Grunwald Giemsa stained fractions of cells used for Western blotting.</p

Bivalent H3K27me3 and H3K4me3 marks are present at the TSS of <i>PRDX4.</i>

<p>ChIP using αH3K27me3 and an IgG control (<b>A</b>) and αH3K4me3 and an IgG control (<b>B</b>) showing an enrichment of both H3K27me3 and H3K4me3 at the TSS of <i>PRDX4</i> in APL cells. To rule out a possible involvement of X-inactivation, and thereby H3K27me3, in silencing of <i>PRDX4</i> expression, male APL samples are compared to a male AML control and female APL samples are compared to a female AML control.</p

Expression of PRDX transcripts in AML and APL.

<p><b>A</b>. Graphical representation of expression of PRDX family members in APL patients clustered based on expression of ∼2000 genes as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016340#pone.0016340-Valk1" target="_blank">[24]</a>. Cluster 12 is exclusively formed by APL patients, as indicated by the red bars indicating the presence of t(15;17). Cluster 11 comprises AML patients with normal karyotype and an underlying NPM1 mutation. Cluster 13 is formed by AML patients with t(8;21). Histograms represent MAS5-normalized expression values. <b>B</b>. Expression levels of the peroxiredoxin gene family in APL (n = 22) were compared to transcript levels in AML (n = 439). Significant differences were calculated using a Wilcoxon test. *  =  p-value <0.001, **  =  p-value <0.0001.</p