title: A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hypertrimethylation.
abstract: The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell-derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival.
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Heidi M
authors.initials: HM
authors.lastname: Ott
authors.affiliation: Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Alan P
authors.initials: AP
authors.lastname: Graves
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Melissa B
authors.initials: MB
authors.lastname: Pappalardi
authors.affiliation: Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Michael
authors.initials: M
authors.lastname: Huddleston
authors.affiliation: Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Wendy S
authors.initials: WS
authors.lastname: Halsey
authors.affiliation: Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Hughes
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Arthur
authors.initials: A
authors.lastname: Groy
authors.affiliation: Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Edward
authors.initials: E
authors.lastname: Dul
authors.affiliation: Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Yong
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Molecular Medicine Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Roland
authors.initials: R
authors.lastname: Annan
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Sharad K
authors.initials: SK
authors.lastname: Verma
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Knight
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Kruger
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Dashyant
authors.initials: D
authors.lastname: Dhanak
authors.affiliation: Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schwartz
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tummino
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania. Molecular Medicine Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.
authors.forename: Caretha L
authors.initials: CL
authors.lastname: Creasy
authors.affiliation: Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania. michael.t.mccabe@gsk.com.
authors.forename: Michael T
authors.initials: MT
authors.lastname: McCabe
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D000071221:Enhancer of Zeste Homolog 2 Protein; D015870:Gene Expression; D020869:Gene Expression Profiling; D020868:Gene Silencing; D006579:Heterozygote; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D063151:Polycomb Repressive Complex 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000378:metabolism / Q000401:mortality / Q000473:pathology; D011487:Protein Conformation; D016415:Sequence Alignment; D013379:Substrate Specificity; D015533:Transcriptional Activation
chemical_list: D006657:Histones; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2; D008239:Lysine