Anti-leukemia activity of histone deacetylase (HDAC) inhibitor LBH589 involves depletion of EZH2 and DNA methyltransferase (DNMT) 1 through disruption of their chaperone association with heat shock protein (hsp) 90

Abstract

10501 Background: LBH589 is a hydroxamate pan-HDAC inhibitor (HA-HDI) that also inhibits HDAC6, which induces acetylation of hsp90 and hsp70. This inhibits the ATP binding and chaperone function of hsp90. We recently reported that treatment with LBH589 depletes the levels of the polycomb repressive complex (PRC) 2 proteins EZH (enhancer of zeste homolog) 2, Suz12 and EED in AML cells. Since EZH2 has been shown to interact with and modulate the DNA methyltransferase activity of DNMT1, which affects its binding to the EZH2-targeted gene promoters, we determined the effects of LBH589 on the levels and interaction between EZH2 and DNMT1 in the CML blast crisis (CML-BC) K562 and LAMA-84 cells as well as in primary AML samples. Methods: Following exposure to 20 to 100 nM of LBH589 for 8 and 24 hours. immunoprecipitation and/or immunoblot analyses were performed to determine hsp90 binding and levels of DNMT1 and EZH2, as well as induction of JunB and loss of clonogenic survival of leukemia cells. Results: Treatment with LBH589 disrupted the chaperone association of DNMT1 and EZH2 with hsp90, resulting in polyubiquitylation and proteasomal degradation of DNMT1. Similar findings were also noted following treatment of leukemia cells with the hsp90 inhibitor, 17-DMAG. DNMT1 depletion by LBH589 was associated with induction of JunB in cultured and primary leukemia cells, which was further enhanced by co-treatment with decitabine. Consistent with this, methylation specific PCR analysis demonstrated increased demethylation of the JunB promoter. Co-treatment with decitabine and LBH589 or DMAG inhibited colony growth of the leukemia cells significantly more than treatment with either agent alone (p<0.05). Conclusions: These findings indicate that in addition to mediating histone acetylation and demethylation, LBH589 treatment induces epigenetic DNA demethylation by targeted depletion of EZH2 and DNMT1 in human leukemia cells. These results support the rationale for testing novel anti- leukemia combinations for epigenetic targeting that include LBH589, decitabine and/or histone methyltransferase inhibitors. No significant financial relationships to disclose.