Abstract 1043: Combined targeting of chromatin modifying enzymes LSD1, histone deacetylases (HDACs) and EZH2 has superior efficacy against human acute myeloid leukemia cells.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL LSD1 or KDM1A is a FAD-dependent demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K)4 on histone H3, reducing the permissive H3K4Me3 chromatin mark. LSD1 inhibition attenuates growth of pluripotent cancer cells with OCT4 and SOX2 expression. LSD1 complexes with HDAC1/2 and Co-REST, and high LSD1 expression confers poor prognosis in cancers. Previous studies have shown that HDAC inhibitors downregulate LSD1 thru Sp1 inhibition. Inhibition of LSD1 leads to increase in H3K4Me3-a permissive mark for gene expression. HCI2509 is an FAD-binding pocket, non-MAOA and MAOB LSD1 inhibitor. In the present studies, we determined the chromatin-modifying and cytotoxic effects of HCI2509 alone and in combination with the pan-histone deacetylase inhibitor, panobinostat (PS) in cultured (HL-60 and OCI-AML3) and primary human acute myeloid leukemia (AML) cells. Treatment with HCI2509 (100 to 500 nM), dose-dependently increased the levels of H3K4Me2 & Me3, p16 and p27, which was associated with inhibition of cell proliferation as measured by a decrease in Ki-67 expression. Chromatin immunoprecipitation followed by PCR demonstrated that treatment with HCI2509 increased the H3K4Me3 mark on the promoters of KLF4, HMOX1, and CDH1 in AML cells. Treatment with HCI2509 also induced C/EBPα expression and features of morphologic differentiation of cultured and primary AML cells. Treatment with HCI2509 (25 mg/kg B.I.W. via IP injection) significantly improved the survival of NOD/SCID mice bearing OCI-AML3- AML xenografts. We have previously reported that treatment with PS (Novartis Pharma) depleted PRC2 complex proteins EZH2, and SUZ12 but also modestly depleted LSD1 expression in AML cells. Co-treatment with PS enhanced the chromatin modifying effects of HCI2509 on K4 of Histone H3 in AML cells. Co-treatment with HCI2509 and PS synergistically induced apoptosis of the cultured AML cells (combination indices, CI <1.0). This was associated with greater induction of p16, and p27. Co-treatment with HCI2509 and PS also induced significantly greater loss of viability of primary AML cells but not of normal CD34+ cells. We have previously reported that treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, dose-dependently depleted EZH2, and SUZ12 levels in cultured and primary AML cells. Combined treatment with HCI2509 enhanced the apoptosis of AML cells induced by DZNep. Taken together, these findings indicate that combined targeted depletion of the level and activity of LSD1 and HDACs by PS and HCI2509, along with PS-mediated depletion of PRC2 proteins, exerts superior and selective cytotoxic activity against AML cells. These findings also support the in vivo testing of combined epigenetic therapies in the treatment of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1043. doi:1538-7445.AM2012-1043