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The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia.

Pappano WN, Guo J, He Y, Ferguson D, Jagadeeswaran S, Osterling DJ, Gao W, Spence JK, Pliushchev M, Sweis RF, Buchanan FG, Michaelides MR, Shoemaker AR, Tse C, Chiang GG - PLoS ONE (2015)

Bottom Line: A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2.Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed.In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.

View Article: PubMed Central - PubMed

Affiliation: Discovery Research, AbbVie Inc., 1 North Waukegan Road, North Chicago, IL 60064 United States of America.

ABSTRACT
Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2) is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1), but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines in vitro resulted in marked differentiation and morphological changes of these tumor cell lines. Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.

No MeSH data available.


Related in: MedlinePlus

A-366 has cellular activity comparable to known G9a/GLP inhibitors.(A) The chemical structure of A-366. (B) PC-3 prostate adenocarcinoma cells were incubated in triplicate with DMSO or the indicated concentrations of A-366 or UNC0638 for 72 hours. H3K9me2 levels were assessed by In-Cell Western assay. (C) In-Cell Western assay for total histone H3 levels from the same plate as shown in (B).
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pone.0131716.g001: A-366 has cellular activity comparable to known G9a/GLP inhibitors.(A) The chemical structure of A-366. (B) PC-3 prostate adenocarcinoma cells were incubated in triplicate with DMSO or the indicated concentrations of A-366 or UNC0638 for 72 hours. H3K9me2 levels were assessed by In-Cell Western assay. (C) In-Cell Western assay for total histone H3 levels from the same plate as shown in (B).

Mentions: In order to identify chemically distinct small molecule inhibitors of G9a, we performed a high-throughput screen of a chemical diversity subset of our internal compound collection. A singleton hit (spiro[cyclobutane-1,3’-indol]-2’amine) ultimately led to the generation of A-366 (Fig 1A, [20]). A-366 is a peptide-competitive inhibitor of G9a/GLP with an in vitro enzymatic IC50 of ~3 nM and >100-fold selectivity over other methyltransferases and other non-epigenetic targets [20]. The potency and selectivity of A-366 made it an ideal candidate to probe the cellular activities of G9a/GLP. To assess the relative cellular activity of A-366, we treated the human prostate adenocarcinoma cell line PC-3 with A-366 or the control compound UNC0638 before performing in-cell western blotting to measure histone H3 lysine 9 methylation. A-366 reduced the total levels of H3K9me2 in a time and concentration dependent manner with a cellular EC50 of ~300 nM (Fig 1B), similar to UNC0638. Full reduction of the H3K9me2 signal required at least 3 days of incubation, which is consistent with other inhibitors with an epigenetic mechanism of action (data not shown, [7, 19]). A-366 was also specific for dimethylation of H3 lysine 9 when compared to a panel of eight additional histone methyl modification antibodies (S1 Fig) as suggested by previous biochemical data [20]. In addition, treatment of cells with 10 μM UNC0638 showed reduced amounts of total H3 staining, which is suggestive of cytotoxicity, although this was not observed with A-366 treatment (Fig 1C). These results suggest that A-366 is a potent and cell-active inhibitor of G9a/GLP that could be used for further investigation of the biological functions of G9a/GLP.


The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia.

Pappano WN, Guo J, He Y, Ferguson D, Jagadeeswaran S, Osterling DJ, Gao W, Spence JK, Pliushchev M, Sweis RF, Buchanan FG, Michaelides MR, Shoemaker AR, Tse C, Chiang GG - PLoS ONE (2015)

A-366 has cellular activity comparable to known G9a/GLP inhibitors.(A) The chemical structure of A-366. (B) PC-3 prostate adenocarcinoma cells were incubated in triplicate with DMSO or the indicated concentrations of A-366 or UNC0638 for 72 hours. H3K9me2 levels were assessed by In-Cell Western assay. (C) In-Cell Western assay for total histone H3 levels from the same plate as shown in (B).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492996&req=5

pone.0131716.g001: A-366 has cellular activity comparable to known G9a/GLP inhibitors.(A) The chemical structure of A-366. (B) PC-3 prostate adenocarcinoma cells were incubated in triplicate with DMSO or the indicated concentrations of A-366 or UNC0638 for 72 hours. H3K9me2 levels were assessed by In-Cell Western assay. (C) In-Cell Western assay for total histone H3 levels from the same plate as shown in (B).
Mentions: In order to identify chemically distinct small molecule inhibitors of G9a, we performed a high-throughput screen of a chemical diversity subset of our internal compound collection. A singleton hit (spiro[cyclobutane-1,3’-indol]-2’amine) ultimately led to the generation of A-366 (Fig 1A, [20]). A-366 is a peptide-competitive inhibitor of G9a/GLP with an in vitro enzymatic IC50 of ~3 nM and >100-fold selectivity over other methyltransferases and other non-epigenetic targets [20]. The potency and selectivity of A-366 made it an ideal candidate to probe the cellular activities of G9a/GLP. To assess the relative cellular activity of A-366, we treated the human prostate adenocarcinoma cell line PC-3 with A-366 or the control compound UNC0638 before performing in-cell western blotting to measure histone H3 lysine 9 methylation. A-366 reduced the total levels of H3K9me2 in a time and concentration dependent manner with a cellular EC50 of ~300 nM (Fig 1B), similar to UNC0638. Full reduction of the H3K9me2 signal required at least 3 days of incubation, which is consistent with other inhibitors with an epigenetic mechanism of action (data not shown, [7, 19]). A-366 was also specific for dimethylation of H3 lysine 9 when compared to a panel of eight additional histone methyl modification antibodies (S1 Fig) as suggested by previous biochemical data [20]. In addition, treatment of cells with 10 μM UNC0638 showed reduced amounts of total H3 staining, which is suggestive of cytotoxicity, although this was not observed with A-366 treatment (Fig 1C). These results suggest that A-366 is a potent and cell-active inhibitor of G9a/GLP that could be used for further investigation of the biological functions of G9a/GLP.

Bottom Line: A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2.Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed.In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.

View Article: PubMed Central - PubMed

Affiliation: Discovery Research, AbbVie Inc., 1 North Waukegan Road, North Chicago, IL 60064 United States of America.

ABSTRACT
Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2) is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1), but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines in vitro resulted in marked differentiation and morphological changes of these tumor cell lines. Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.

No MeSH data available.


Related in: MedlinePlus