Pan-HDAC inhibitor LBH589 depletes EZH2 and DNMT1 by inhibiting chaperone association of hsp90 with EZH2 and DNMT1.

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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 recently reported that 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 (Mol Cancer Ther 5 (12), 2006). Recently, within the PRC2 complex, 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. In the present studies we determined the effects of LBH589 on the interaction between the PRC2 proteins EZH2 and EED and DNMT1 in the 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, which were restored by co-treatment with the proteasome inhibitor bortezomib. 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 and DMAG. Additionally, RT-PCR analysis demonstrated that treatment with LBH589 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 levels. Depletion of DNMT1 due to LBH589 treatment was also associated with induction of JunB mRNA expression. This may be because JunB is known to be repressed by DNA hypermethylation in CML-BC cells. A similar upregulation of JunB mRNA and protein in K562 cells was also observed following shRNA mediated depletion of DNMT1 (down to 40%). These findings indicate that treatment with pan-HDAC inhibitor LBH589 not only depletes EZH2, but also attenuates DNMT1 levels, which results from LBH589 mediated acetylation and inhibition of chaperone function of hsp90. Thus LBH589 and DMAG disrupt EZH2 based recruitment platform for DNA methyltransferases, thereby targeting and downregulating two epigenetic silencing systems with ensuing derepression of methylated genes.