The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. Here, we find that EZH2 deletion or pharmacologic ...inhibition suppresses GC formation and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.
•EZH2 is required for germinal center formation and immunoglobulin affinity maturation•Conditional expression of an EZH2 mutant lymphoma allele drives GC hyperplasia in vivo•Mutant EZH2 functions in part through aberrant repression of GC-specific bivalent promoters•EZH2 cooperates with BCL2 to initiate and maintain diffuse large B cell lymphomas
Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive ...complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation unmethylated H3K27 (H3K27me0):me1:me2 kcat/Km ratio = 9:6:1 and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 kcat/Km ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 kcat/Km ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.
In eukaryotes, post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repressive complex 2 ...(PRC2) and is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 overexpression is implicated in tumorigenesis and correlates with poor prognosis in several tumour types. Additionally, somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 occur in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. The Y641 residue is the most frequently mutated residue, with up to 22% of germinal centre B-cell DLBCL and follicular lymphoma harbouring mutations at this site. These lymphomas have increased H3K27 tri-methylation (H3K27me3) owing to altered substrate preferences of the mutant enzymes. However, it is unknown whether specific, direct inhibition of EZH2 methyltransferase activity will be effective in treating EZH2 mutant lymphomas. Here we demonstrate that GSK126, a potent, highly selective, S-adenosyl-methionine-competitive, small-molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and markedly inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma.
The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a ...drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2.
CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. ...CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC
= 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC
values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases.
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.
EZH2/PRC2 catalyzes transcriptionally repressive methylation at lysine 27 of histone H3 and has been associated with numerous cancer types. Point mutations in EZH2 at Tyr641 and Ala677 identified in ...non-Hodgkin lymphomas alter substrate specificity and result in increased trimethylation at histone H3K27. Interestingly, EZH2/PRC2 is activated by binding H3K27me3 marks on histones, and this activation is proposed as a mechanism for self-propagation of gene silencing. Recent work has identified GSK126 as a potent, selective, SAM-competitive inhibitor of EZH2 capable of globally decreasing H3K27 trimethylation in cells. Here we show that activation of PRC2 by an H3 peptide trimethylated at K27 is primarily an effect on the rate-limiting step (kcat) with no effect on substrate binding (Km). Additionally, GSK126 is shown to have a significantly longer residence time of inhibition on the activated form of EZH2/PRC2 as compared to unactivated EZH2/PRC2. Overall inhibition constant (Ki*) values for GSK126 were determined to be as low as 93 pM and appear to be driven by slow dissociation of inhibitor from the activated enzyme. The data suggest that activation of EZH2 allows the enzyme to adopt a conformation that possesses greater affinity for GSK126. The long residence time of GSK126 may be beneficial in vivo and may result in durable target inhibition after drug systemic clearance.
There is little primate risk factor data in the literature evaluating the relationship between proposed mechanisms of PPAR agonist-induced hepatocarcinogenesis at clinically relevant therapeutic ...exposures. These studies were conducted to characterize the hepatic effects of fenofibrate and ciprofibrate in the cynomolgus monkey. Male cynomolgus monkeys were given fenofibrate (250, 1250 or 2500 mg/kg/day) or ciprofibrate (3, 30, 150 or 400 mg/kg/day) for up to 15 days. The highest doses used were ∼4 times (fenofibrate) and 9.4 times (ciprofibrate) the human therapeutic exposure for these agents based on AUC (area under the curve). For both compounds, there was a treatment-related increase in liver weight and periportal hepatocellular hypertrophy, which was related to increases in peroxisomes (up to 2.8 times controls) and mitochondria (up to 2.5 times controls). An increase in smooth endoplasmic reticulum probably contributed to the hypertrophy. There was no indication of cell proliferation as determined by the number of mitotic figures and this was confirmed by evaluating cell proliferation by immunohistochemical staining for the Ki-67 antigen. Consistent with the findings by light microscopy, there was no treatment-related effect on the level of mRNA for proteins known to be involved in the control of hepatocyte cell division or apoptosis (e.g. P21, Cyclin D1, PCNA, CDKN1A). Furthermore, there was minimal indication of oxidative stress. Thus, there was no evidence of lipofuscin accumulation, and there was no remarkable increase in the mRNA levels for most proteins known to respond to oxidative stress (e.g. catalase, glutathione peroxidase). A mild induction in the mRNA levels of cellular β-oxidation and detoxification enzymes (e.g. acyl CoA oxidase, thioredoxin reductase) was observed. Collectively, the data from these studies suggest that the primate responds to PPARα agonists in a manner that is different from the rodent suggesting that the primate may be refractory to PPAR-induced hepatocarcinogenesis.
Psychotic disorders most commonly appear during the late teenage years and early adulthood. A focused and rapid clinical response by an integrated health team can help to improve the quality of life ...of the patient, leading to a better long-term prognosis. The Vancouver Coastal Health early psychosis intervention program covers a catchment area of approximately 800,000 people in the cities of Vancouver and Richmond, Canada. The program provides a multidisciplinary approach to supporting patients under the age of 30 who have recently experienced first-break psychosis. The program addresses the needs of the treatment environment, medication, and psychological therapies. A critical part of this support includes a program to specifically improve patients' physical health. Physical health needs are addressed through a two-pronged, parallel approach. Patients receive routine metabolic health assessments during their first year in the program, where standard metabolic parameters are recorded. Based on the results of clinical interviews and laboratory tests, specific actionable interventions are recommended. The second key strategy is a program that promotes healthy lifestyle goal development. Patients work closely with occupational therapists to develop goals to improve cardiometabolic health. These programs are supported by an active research environment, where patients are able to engage in studies with a focus on improving their physical health. These studies include a longitudinal evaluation of the effects of integrated health coaching on maintaining cardiometabolic health in patients recently admitted to the program, as well as a clinical study that evaluates the effects of low versus higher metabolic risk antipsychotic drugs on central adiposity. An additional pharmacogenomic study is helping to identify genetic variants that may predict cardiometabolic changes following treatment with antipsychotic drugs.
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