Cytoplasmatic compartmentalization by Bcr‐Abl promotes TET2 loss‐of‐function in chronic myeloid leukemia

Abstract

The loss‐of‐function of ten–eleven‐translocation (TET) 2, a Fe2+‐oxoglutarate‐dependent dioxygenase catalyzing 5 methyl cytosine (5mC) conversion into 5‐hydroxymethylcytosine (5hmC), contributes to the hematopoietic transformation in vivo. The aim of our study was to elucidate its role in the phenotype of chronic myeloid leukemia (CML), a myeloproliferative disease caused by the Bcr‐Abl rearranged gene. We first confirmed TET2 interaction with the Bcr‐Abl protein predicted by a Fourier‐based bioinformatic method. Such interaction led to TET2 cytoplasmatic compartmentalization in a complex tethered by the fusion protein tyrosine kinase (TK) and encompassing the Forkhead box O3a (FoxO3a) transcription factor. We then focused the impact of TET2 loss‐of‐function on epigenetic transcriptional regulation of Bcl2‐interacting mediator (BIM), a pro‐apoptotic protein transcriptionally regulated by FoxO3a. BIM downregulation is a critical component of CML progenitor extended survival and is also involved in the disease resistance to imatinib (IM). Here we reported that TET2 release from Bcr‐Abl protein following TK inhibition in response to IM triggers a chain of events including TET2 nuclear translocation, re‐activation of its enzymatic function at 5mC and recruitment at the BIM promoter followed by BIM transcriptional induction. 5hmC increment following TET2 re‐activation was associated with the reduction of histone H3 tri‐methylation at lysine 9 (H3K9me3), which may contribute with DNA de‐methylation reported elsewhere to recast a permissive epigenetic “landscape” for FoxO3a transcriptional activity. J. Cell. Biochem. 113: 2765–2774, 2012. © 2012 Wiley Periodicals, Inc.

DNA hypermethylation promotes the low expression of pro‐apoptotic BCL2L11 associated with BCR‐ABL1 fusion gene of chronic myeloid leukaemia

“…DNMT1 displacement was associated with a significant reduction of 5mC at the above-mentioned region of the BCL2L11 promoter paralleling the increment of 5hmC (P < 0Á05 or less) ( Fig 1A). The latter event was most likely due to the restored function of TenEleven-Translocation (TET) 2 dioxygenase (Mancini et al, 2012). As expected, DNA de-methylation resulted in a progressive increase of BCL2L11 transcription (P < 0Á05 or less) followed by a significant increment of all three BCL2L11 isoforms (P < 0Á001) and apoptosis induction (Fig 1B-D).…”

“…Low BCL2L11 expression in CML has been also ascribed to DNA hypermethylation at the gene promoter (Kuribara et al, 2004;Aichberger et al, 2005;San José-Eneriz et al, 2009). The putative role of epigenetic modifications in BCR-ABL1-associated BCL2L11 downmodulation is supported by our recent study proving the participation of 5 methylcytosine (5mC) hydroxylation in BCL2L11 transcriptional induction in response to IM (Mancini et al, 2012). Notably, the conversion of 5mC into 5 hydroxymethylcytosine (5hmC) impairs the chromatin recruitment of DNA methyltransferase (DNMT) 1, hence facilitating DNA de-methylation and gene re-expression Here we show that BCR-ABL1 TK is associated with DNMT1 enhanced recruitment at the BCL2L11 promoter and 5mC excess over 5hmC.…”

“…The putative role of epigenetic modifications in BCR-ABL1-associated BCL2L11 downmodulation is supported by our recent study proving the participation of 5 methylcytosine (5mC) hydroxylation in BCL2L11 transcriptional induction in response to IM (Mancini et al, 2012). Notably, the conversion of 5mC into 5 hydroxymethylcytosine (5hmC) impairs the chromatin recruitment of DNA methyltransferase (DNMT) 1, hence facilitating DNA de-methylation and gene re-expression (Boultwood & Wainscoat, 2007).…”

“…(D) BCL2L11 induction was followed by apoptotic cell death at 24 h of exposure to IM. ChIP, sodium dodecyl sulphate polyacrylamide gel electrophoresis and PCR for BCL2L11 were performed according to previously published procedures (Mancini et al, 2012). PCR amplification of BCL2L11 promoter was performed on equal amounts (50 ng) of DNA from ChIP products using the following primers: 5′ACT-CCACAAGCTGGGGAGCTGAT3′ (upper) and 5′AGGCCTCTCAGC-AGGCTGCAATT3′ (lower) and PCR conditions: 35 cycles of denaturation at 95°C for 30″, annealing at 58°C for 30″ and extension at 72°C for 30″.…”

CREB-binding protein (CBP) regulates β-adrenoceptor (β-AR)−mediated apoptosis

“…43 In chronic myeloid leukemia, changes in Bim promoter methylation status are associated with differences in Bim gene expression. 44 As CBP is a cofactor in c-Myc-regulated Bim expression, and CBP mutations in the HAT domain are associated with tumor development, 40 we investigated whether epigenetic changes in the Bim promoter occur during bAR signalling. Bisulphite sequence analysis of the Bim promoter from bAR responsive Ramos cells and the non-responsive LK-2 (CBP negative) cells showed a significant increase in CpG island methylation in the nonresponsive cells.…”