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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 inhibits H3K9me2 similar to UNC0638, but does not inhibit cell growth of MOLT-16 and HT-1080 cells.(A) The T-cell ALL cell line MOLT-16 was treated for 5 days with A-366 or UNC0638 before assessing their proliferation. (B) MOLT-16 cells were treated for 3 days with compounds and lysed. Lysates were normalized and western blot analysis was performed for H3K9me2, total H3 and G9a. (C) HT-1080 fibrosarcoma cells were treated for 2 or 5 days with A-366 or UNC0638 before assessing their proliferation. (D) H3K9me2 AlphaLISA was performed on HT-1080 cells treated for 2 days with compounds and % inhibition of H3K9me2 was calculated compared to untreated control cells.
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pone.0131716.g003: A-366 inhibits H3K9me2 similar to UNC0638, but does not inhibit cell growth of MOLT-16 and HT-1080 cells.(A) The T-cell ALL cell line MOLT-16 was treated for 5 days with A-366 or UNC0638 before assessing their proliferation. (B) MOLT-16 cells were treated for 3 days with compounds and lysed. Lysates were normalized and western blot analysis was performed for H3K9me2, total H3 and G9a. (C) HT-1080 fibrosarcoma cells were treated for 2 or 5 days with A-366 or UNC0638 before assessing their proliferation. (D) H3K9me2 AlphaLISA was performed on HT-1080 cells treated for 2 days with compounds and % inhibition of H3K9me2 was calculated compared to untreated control cells.

Mentions: The discrepancy in cell proliferation EC50s for A-366 and UNC0638 despite equivalent EC50s for H3K9me2 inhibition suggested that UNC0638 may have off-target effects. To test this hypothesis, we treated additional cell lines with A-366 compared to UNC0638. In the T-cell acute lymphoblastic leukemia (ALL) cell line MOLT-16, UNC0638 inhibited cell proliferation at concentrations consistent with its H3K9me2 EC50; however, A-366 had no impact on the proliferation of this cell line even though it inhibited H3K9me2 as potently as UNC0638 (Fig 3A and 3B). We further assessed the effects of these compounds in the human fibrosarcoma cell line HT-1080. UNC0638 exhibited anti-proliferative effects after only 2 days of treatment (1.4 μM EC50, Fig 3C). Treatment for an additional 3 days resulted in a reduction of the proliferation EC50 to 0.5 μM (Fig 3C), in line with the observed H3K9me2 cellular EC50 of 0.3 μM. However, A-366 had no impact on HT-1080 proliferation up to 10 μM. The cellular inhibition of G9a/GLP is also not consistent with these results as these two compounds have nearly identical impact on global H3K9me2 levels in this cell line at the relevant concentrations of interest (Fig 3D). We also performed an unbiased screen of an additional 38 cell lines from numerous cancer types to assess the impact of A-366 (S1 Table). We found that A-366 had no overt anti-proliferative effects in any cell line tested up to 10 μM, often in contradiction to the quinazoline-based inhibitor UNC0638. Finally, we tested A-366 in human PBMCs and found that A-366 also had no impact on viability on these non-transformed cells (S2 Fig).


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 inhibits H3K9me2 similar to UNC0638, but does not inhibit cell growth of MOLT-16 and HT-1080 cells.(A) The T-cell ALL cell line MOLT-16 was treated for 5 days with A-366 or UNC0638 before assessing their proliferation. (B) MOLT-16 cells were treated for 3 days with compounds and lysed. Lysates were normalized and western blot analysis was performed for H3K9me2, total H3 and G9a. (C) HT-1080 fibrosarcoma cells were treated for 2 or 5 days with A-366 or UNC0638 before assessing their proliferation. (D) H3K9me2 AlphaLISA was performed on HT-1080 cells treated for 2 days with compounds and % inhibition of H3K9me2 was calculated compared to untreated control cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131716.g003: A-366 inhibits H3K9me2 similar to UNC0638, but does not inhibit cell growth of MOLT-16 and HT-1080 cells.(A) The T-cell ALL cell line MOLT-16 was treated for 5 days with A-366 or UNC0638 before assessing their proliferation. (B) MOLT-16 cells were treated for 3 days with compounds and lysed. Lysates were normalized and western blot analysis was performed for H3K9me2, total H3 and G9a. (C) HT-1080 fibrosarcoma cells were treated for 2 or 5 days with A-366 or UNC0638 before assessing their proliferation. (D) H3K9me2 AlphaLISA was performed on HT-1080 cells treated for 2 days with compounds and % inhibition of H3K9me2 was calculated compared to untreated control cells.
Mentions: The discrepancy in cell proliferation EC50s for A-366 and UNC0638 despite equivalent EC50s for H3K9me2 inhibition suggested that UNC0638 may have off-target effects. To test this hypothesis, we treated additional cell lines with A-366 compared to UNC0638. In the T-cell acute lymphoblastic leukemia (ALL) cell line MOLT-16, UNC0638 inhibited cell proliferation at concentrations consistent with its H3K9me2 EC50; however, A-366 had no impact on the proliferation of this cell line even though it inhibited H3K9me2 as potently as UNC0638 (Fig 3A and 3B). We further assessed the effects of these compounds in the human fibrosarcoma cell line HT-1080. UNC0638 exhibited anti-proliferative effects after only 2 days of treatment (1.4 μM EC50, Fig 3C). Treatment for an additional 3 days resulted in a reduction of the proliferation EC50 to 0.5 μM (Fig 3C), in line with the observed H3K9me2 cellular EC50 of 0.3 μM. However, A-366 had no impact on HT-1080 proliferation up to 10 μM. The cellular inhibition of G9a/GLP is also not consistent with these results as these two compounds have nearly identical impact on global H3K9me2 levels in this cell line at the relevant concentrations of interest (Fig 3D). We also performed an unbiased screen of an additional 38 cell lines from numerous cancer types to assess the impact of A-366 (S1 Table). We found that A-366 had no overt anti-proliferative effects in any cell line tested up to 10 μM, often in contradiction to the quinazoline-based inhibitor UNC0638. Finally, we tested A-366 in human PBMCs and found that A-366 also had no impact on viability on these non-transformed cells (S2 Fig).

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