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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 does not impact the proliferation of MCF-7 cells despite inhibition of H3K9me2.(A) MCF-7 and MDA-MB-231 breast cancer cell lines were treated with the indicated concentrations of A-366 or UNC0638 for 14 days before assessing colony formation. (B) MCF-7 and MDA-MB-231 cells were treated with the indicated concentrations of A-366 or UNC0638 for 3 days and subjected to western blot analysis of global H3K9me2 and total histone H3.
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pone.0131716.g002: A-366 does not impact the proliferation of MCF-7 cells despite inhibition of H3K9me2.(A) MCF-7 and MDA-MB-231 breast cancer cell lines were treated with the indicated concentrations of A-366 or UNC0638 for 14 days before assessing colony formation. (B) MCF-7 and MDA-MB-231 cells were treated with the indicated concentrations of A-366 or UNC0638 for 3 days and subjected to western blot analysis of global H3K9me2 and total histone H3.

Mentions: A previous report suggested that inhibition of G9a/GLP by RNA interference or continuous dosing with UNC0638 reduced the clonogenicity of MCF-7 but not MDA-MB-231 breast cancer cells [19]. We attempted to recapitulate this result with our novel G9a/GLP inhibitor, A-366. While the treatment of MCF-7 cells with UNC0638 reproduced the published results, A-366 had no impact on cellular clonogenicity (Fig 2A). Neither compound affected MDA-MBA-231 cells in this assay format, with the exception of the highest concentration tested for UNC0638 (10 μM). Notably, even 14 days of treatment with 10 μM A-366 had no impact on the growth of either of these cell lines despite being well above the cellular EC50 shown in Fig 1B. To test whether inadequate target inhibition might account for these observations, we assessed these compounds’ impact on global H3K9me2 levels in both breast cancer cell lines. However, A-366 and UNC0638 exhibited nearly identical cellular potency in reducing global H3K9me2 signal (Fig 2B). The treatment of MCF-7 cells consistently resulted in more H3K9me2 reduction than in the MDA-MB-231 cells, suggesting that the MDA-MB-231 cell line may have more contribution from additional H3K9 methyltransferases or that the turnover of H3K9me2 by histone demethylases may be more rapid in MCF-7 cells.


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 does not impact the proliferation of MCF-7 cells despite inhibition of H3K9me2.(A) MCF-7 and MDA-MB-231 breast cancer cell lines were treated with the indicated concentrations of A-366 or UNC0638 for 14 days before assessing colony formation. (B) MCF-7 and MDA-MB-231 cells were treated with the indicated concentrations of A-366 or UNC0638 for 3 days and subjected to western blot analysis of global H3K9me2 and total histone H3.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131716.g002: A-366 does not impact the proliferation of MCF-7 cells despite inhibition of H3K9me2.(A) MCF-7 and MDA-MB-231 breast cancer cell lines were treated with the indicated concentrations of A-366 or UNC0638 for 14 days before assessing colony formation. (B) MCF-7 and MDA-MB-231 cells were treated with the indicated concentrations of A-366 or UNC0638 for 3 days and subjected to western blot analysis of global H3K9me2 and total histone H3.
Mentions: A previous report suggested that inhibition of G9a/GLP by RNA interference or continuous dosing with UNC0638 reduced the clonogenicity of MCF-7 but not MDA-MB-231 breast cancer cells [19]. We attempted to recapitulate this result with our novel G9a/GLP inhibitor, A-366. While the treatment of MCF-7 cells with UNC0638 reproduced the published results, A-366 had no impact on cellular clonogenicity (Fig 2A). Neither compound affected MDA-MBA-231 cells in this assay format, with the exception of the highest concentration tested for UNC0638 (10 μM). Notably, even 14 days of treatment with 10 μM A-366 had no impact on the growth of either of these cell lines despite being well above the cellular EC50 shown in Fig 1B. To test whether inadequate target inhibition might account for these observations, we assessed these compounds’ impact on global H3K9me2 levels in both breast cancer cell lines. However, A-366 and UNC0638 exhibited nearly identical cellular potency in reducing global H3K9me2 signal (Fig 2B). The treatment of MCF-7 cells consistently resulted in more H3K9me2 reduction than in the MDA-MB-231 cells, suggesting that the MDA-MB-231 cell line may have more contribution from additional H3K9 methyltransferases or that the turnover of H3K9me2 by histone demethylases may be more rapid in MCF-7 cells.

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