The PRC (polycomb repressive complex) 2 is a multi-protein complex, which includes EZH2, SUZ12 and EED proteins. The PRC2 complex possesses histone methyltransferase (HMTase) activity mediated by the SET domain of EZH2, which methylates histone H3 on lysine (K)-27. We have previously reported that the treatment with the hydroxamate, pan-HDAC inhibitor LBH589 acetylates and inhibits the ATP binding and chaperone function of hsp90, as well as depletes the levels of EZH2, Suz12 and EED in AML cells (

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). Recently, within the PRC2, EZH2 was shown to interact with and modulate the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, which affects their binding to the EZH2-targeted gene promoters. Thus EZH2 may serve as a recruitment platform for DNA methyltransferases, thereby mechanistically linking the two epigenetic silencing systems, maintaining the chromatin in the silenced state and inhibiting gene expression. In the present studies we determined the effects of hydroxamate pan-HDAC inhibitor LBH589 on the interaction between the PRC2 proteins EZH2 and EED and DNMT1 in CML blast crisis (CML-BC) K562 and LAMA-84 cells. Treatment with ≥50 nM LBH589 for 24 hours disrupted the interaction between DNMT1 and Ezh2, as well as depleted the levels of DNMT1 in a dose and time dependent manner. LBH589 (≥50 nM) also disrupted the binding of DNMT1 to hsp90. This was associated with increased polyubiquitylation and accumulation of DNMT1 in the detergent insoluble fraction of the leukemia cells. Notably, similar findings were obtained following treatment with the hsp90 inhibitor 17-DMAG (250 nM). Taken together, these findings suggest that DNMT1 has chaperone association with hsp90, which is disrupted by LBH589. Additionally, RT-PCR analysis demonstrated that treatment with 100 nM LBH589 for 8 hours depleted the mRNA expression of DNMT1 in K562 cells, suggesting that both transcriptional and post-transcriptional mechanisms are involved in LBH589 mediated down regulation of DNMT1. Depletion of DNMT1 due to LBH589 treatment was associated with induction of JunB mRNA expression. This may be because JunB is repressed by DNA hypermethylation in CML-BC cells. A similar upregulation of JunB mRNA and protein in K562 cells also followed shRNA mediated depletion of DNMT1 (down to 40%). These findings indicate that treatment with pan-HDAC inhibitor LBH589 not only induces histone acetylation and depletes the levels of histone methyl transferase EZH2, but also down regulates DNMT1 through transcriptional mechanism and proteasome degradation. The latter effect may be due to LBH589 mediated acetylation and inhibition of chaperone function of hsp90, an effect also seen with treatment with 17-DMAG.

Topics:
dna (cytosine-5-)-methyltransferase 1, ezh2 gene, hdac protocol, histone deacetylase, hsp90 heat-shock proteins, molecular chaperones, mechlorethamine, rna, messenger, dna modification methylases, blast phase

Disclosures: Co-author PA is an employee of Novartis Pharmaceutical Inc.; Co-author KB has research funding from Novartis Pharmaceutical Inc.; Co-author KB has received honoraria from Novartis Pharmaceutical Inc.

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2006, The American Society of Hematology
2006
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