title: EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression.
abstract: 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.
authors: 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;
authors: Aline
authors: A
authors: Renneville
authors: 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;
authors: Peter
authors: P
authors: Van Galen
authors: Harvard Medical School, Boston, MA;
authors: Matthew C
authors: MC
authors: Canver
authors: Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA;
authors: Marie
authors: M
authors: McConkey
authors: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA;
authors: John M
authors: JM
authors: Krill-Burger
authors: Department of Pathology, Brigham and Women's Hospital, Boston, MA;
authors: David M
authors: DM
authors: Dorfman
authors: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA;
authors: Edward B
authors: EB
authors: Holson
authors: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA; Department of Pathology and Center for Cancer Research, Massachusetts General Hospital, Boston, MA; Howard Hughes Medical Institute, Chevy Chase, MD;
authors: Bradley E
authors: BE
authors: Bernstein
authors: Harvard Medical School, Boston, MA; Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA; and Howard Hughes Medical Institute, Boston, MA.
authors: Stuart H
authors: SH
authors: Orkin
authors: Harvard Medical School, Boston, MA; Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA; and.
authors: Daniel E
authors: DE
authors: Bauer
authors: Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA; Harvard Medical School, Boston, MA; Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA;
authors: Benjamin L
authors: BL
authors: Ebert
mesh_terms: D000755:Anemia, Sickle Cell / Q000188:drug therapy / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D044127:Epigenesis, Genetic / Q000187:drug effects*; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism*; D041905:Erythroid Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D005319:Fetal Hemoglobin / Q000096:biosynthesis*; D006649:Histocompatibility Antigens / Q000378:metabolism; D011495:Histone-Lysine N-Methyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D011799:Quinazolines / Q000494:pharmacology*
chemical_list: D006649:Histocompatibility Antigens; D011799:Quinazolines; C561310:UNC 0638; D005319:Fetal Hemoglobin; C455847:EHMT1 protein, human; C445405:EHMT2 protein, human; C533711:G9a protein, mouse; C513479:GLP protein, mouse; D011495:Histone-Lysine N-Methyltransferase