Region-specific alteration of histone modification by LSD1 inhibitor conjugated with pyrrole-imidazole polyamide

// Kokiladevi Alagarswamy 1 , Ken-Ichi Shinohara 1 , Shihori Takayanagi 2 , Masaki Fukuyo 1 , Atsushi Okabe 1 , Bahityar Rahmutulla 1 , Natsumi Yoda 1 , Rui Qin 2 , Naoki Shiga 2 , Masahiro Sugiura 1 , Hiroaki Sato 1 , Kazuko Kita 1 , Takayoshi Suzuki 3 , Tetsuhiro Nemoto 2 and Atsushi Kaneda 1 1 Department of Molecular Oncology, School of Medicine, Chiba University, Chiba, Japan 2 Department of Pharmaceutical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan 3 Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan Correspondence to: Atsushi Kaneda, email: kaneda@chiba-u.jp Keywords: epigenome; histone modification; lysine-specific demethylase-1 inhibitor; pyrrole imidazole polyamide Received: December 23, 2017      Accepted: May 07, 2018      Published: June 29, 2018 ABSTRACT Epigenome regulates gene expression to determine cell fate, and accumulation of epigenomic aberrations leads to diseases, including cancer. NCD38 inhibits lysine-specific demethylase-1 (LSD1), a histone demethylase targeting H3K4me1 and H3K4me2, but not H3K4me3. In this study, we conjugated NCD38 with a potent small molecule called pyrrole (Py) imidazole (Im) polyamide, to analyze whether targets of the inhibitor could be regulated in a sequence-specific manner. We synthesized two conjugates using β-Ala (β) as a linker, i.e., NCD38-β-β-Py-Py-Py-Py (NCD38-β 2 P 4 ) recognizing WWWWWW sequence, and NCD38-β-β-Py-Im-Py-Py (NCD38-β 2 PIPP) recognizing WWCGWW sequence. When RKO cells were treated with NCD38, H3K4me2 levels increased in 103 regions with significant activation of nearby genes ( P = 0.03), whereas H3K4me3 levels were not obviously increased. H3K27ac levels were also increased in 458 regions with significant activation of nearby genes ( P = 3 × 10 −10 ), and these activated regions frequently included GC-rich sequences, but less frequently included AT-rich sequences ( P < 1 × 10 −15 ) or WWCGWW sequences ( P = 2 × 10 −13 ). When treated with NCD38-β 2 P 4 , 234 regions showed increased H3K27ac levels with significant activation of nearby genes ( P = 2 × 10 −11 ), including significantly fewer GC-rich sequences ( P < 1 × 10 −15 ) and significantly more AT-rich sequences ( P < 1 × 10 −15 ) compared with NCD38 treatment. When treated with NCD38-β 2 PIPP, 82 regions showed increased H3K27ac levels, including significantly fewer GC-rich sequences ( P = 1 × 10 −11 ) and fewer AT-rich sequences ( P = 0.005), but significantly more WWCGWW sequences ( P = 0.0001) compared with NCD38 treatment. These indicated that target regions of epigenomic inhibitors could be modified in a sequence-specific manner and that conjugation of Py-Im polyamides may be useful for this purpose.