Renneville Aline, Van Galen Peter, Canver Matthew C, McConkey Marie, Krill-Burger John M, Dorfman David M, Holson Edward B, Bernstein Bradley E, Orkin Stuart H, Bauer Daniel E, Ebert Benjamin L
Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA; Harvard Medical School, Boston, MA; Laboratory of Hematology, Biology and Pathology Center, Centre Hospitalier Régional Universitaire de Lille, Lille, France;
Harvard Medical School, Boston, MA; Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA; Department of Pathology and Center for Cancer Research, Massachusetts General Hospital, Boston, MA;
Blood. 2015 Oct 15;126(16):1930-9. doi: 10.1182/blood-2015-06-649087. Epub 2015 Aug 28.
Fetal hemoglobin (HbF, α2γ2) induction is a well-validated strategy for sickle cell disease (SCD) treatment. Using a small-molecule screen, we found that UNC0638, a selective inhibitor of EHMT1 and EHMT2 histone methyltransferases, induces γ-globin expression. EHMT1/2 catalyze mono- and dimethylation of lysine 9 on histone 3 (H3K9), raising the possibility that H3K9Me2, a repressive chromatin mark, plays a role in silencing γ-globin expression. In primary human adult erythroid cells, UNC0638 and EHMT1 or EHMT2 short hairpin RNA-mediated knockdown significantly increased γ-globin expression, HbF synthesis, and the percentage of cells expressing HbF. At effective concentrations, UNC0638 did not alter cell morphology, proliferation, or erythroid differentiation of primary human CD34(+) hematopoietic stem and progenitor cells in culture ex vivo. In murine erythroleukemia cells, UNC0638 and Ehmt2 CRISPR/Cas9-mediated knockout both led to a marked increase in expression of embryonic β-globin genes Hbb-εy and Hbb-βh1. In primary human adult erythroblasts, chromatin immunoprecipitation followed by sequencing analysis revealed that UNC0638 treatment leads to genome-wide depletion in H3K9Me2 and a concomitant increase in the activating mark H3K9Ac, which was especially pronounced at the γ-globin gene region. In RNA-sequencing analysis of erythroblasts, γ-globin genes were among the most significantly upregulated genes by UNC0638. Further increase in γ-globin expression in primary human adult erythroid cells was achieved by combining EHMT1/2 inhibition with the histone deacetylase inhibitor entinostat or hypomethylating agent decitabine. Our data provide genetic and pharmacologic evidence that EHMT1 and EHMT2 are epigenetic regulators involved in γ-globin repression and represent a novel therapeutic target for SCD.
胎儿血红蛋白（HbF，α2γ2）诱导是镰状细胞病（SCD）治疗中一项经过充分验证的策略。通过小分子筛选，我们发现UNC0638，一种EHMT1和EHMT2组蛋白甲基转移酶的选择性抑制剂，可诱导γ珠蛋白表达。EHMT1/2催化组蛋白3上赖氨酸9（H3K9）的单甲基化和二甲基化，这增加了H3K9Me2（一种抑制性染色质标记）在沉默γ珠蛋白表达中起作用的可能性。在原代成人人类红细胞中，UNC0638以及EHMT1或EHMT2短发夹RNA介导的敲低显著增加了γ珠蛋白表达、HbF合成以及表达HbF的细胞百分比。在有效浓度下，UNC0638不会改变原代人类CD34(+)造血干细胞和祖细胞在体外培养中的细胞形态、增殖或红细胞分化。在鼠红细胞白血病细胞中，UNC0638和Ehmt2 CRISPR/Cas9介导的敲除均导致胚胎β珠蛋白基因Hbb-εy和Hbb-βh1的表达显著增加。在原代成人人类成红细胞中，染色质免疫沉淀测序分析表明，UNC0638处理导致全基因组范围内H3K9Me2减少，同时激活标记H3K9Ac增加，这在γ珠蛋白基因区域尤为明显。在成红细胞的RNA测序分析中，γ珠蛋白基因是UNC0638上调最显著的基因之一。通过将EHMT1/2抑制与组蛋白去乙酰化酶抑制剂恩替诺特或低甲基化剂地西他滨联合使用，可进一步增加原代成人人类红细胞中γ珠蛋白的表达。我们的数据提供了遗传学和药理学证据，表明EHMT1和EHMT2是参与γ珠蛋白抑制的表观遗传调节因子，代表了SCD的一个新的治疗靶点。
