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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 induces differentiation and affects viability in MV4;11 cells.(A) MV4;11 cells were incubated with A-366 for 14 days. Cells were fixed, stained with an anti-CD11b antibody and analyzed by flow cytometry. (B) MV4;11 cells from (A) additionally assayed for cellular proliferation (Cell Titer-Glo) and viability (trypan blue exclusion) following A-366 treatment for 14 days. (C) Cell cycle DNA content analysis was performed by propidium iodide staining in MV4;11 cells treated for 14 days with A-366. (D) Wright-Giemsa staining of MV4;11 cells treated with DMSO or 5 μM A-366 for 10 days.
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pone.0131716.g004: A-366 induces differentiation and affects viability in MV4;11 cells.(A) MV4;11 cells were incubated with A-366 for 14 days. Cells were fixed, stained with an anti-CD11b antibody and analyzed by flow cytometry. (B) MV4;11 cells from (A) additionally assayed for cellular proliferation (Cell Titer-Glo) and viability (trypan blue exclusion) following A-366 treatment for 14 days. (C) Cell cycle DNA content analysis was performed by propidium iodide staining in MV4;11 cells treated for 14 days with A-366. (D) Wright-Giemsa staining of MV4;11 cells treated with DMSO or 5 μM A-366 for 10 days.

Mentions: One of the characteristic features of leukemia cells is a blockade of differentiation at a distinct stage of blood cell maturation [22]. As a result, differentiation therapy has been tested and validated clinically in several oncology indications. For example, two established clinical regimens for differentiation therapy, the treatment of acute promyelocytic leukemia with all-trans retinoic acid (ATRA) [23] and azacitidine treatment of myelodysplastic syndrome [24], provide proof-of-principle for this approach. Recently, G9a has been implicated in mediating T helper cell differentiation and function by studies of tissue-specific, G9a conditional knockout mice [25]. We hypothesized that inhibiting G9a function may likewise translate to the inhibition of leukemia cells by differentiation. As a first test of this hypothesis, we treated the acute myelocytic leukemia (AML) cell line MV4;11 with A-366. Long-term treatments resulted in dose-dependent differentiation of this tumor cell line, as observed by an increase in the differentiation marker CD11b (Fig 4A). A-366 treatment also resulted in inhibited proliferation and a decrease in viability corresponding to the dose response observed for CD11b staining (Fig 4B). DNA content analysis was also performed to assess a potential mechanism of action for the reduced viability observed in MV4;11 cells treated long-term with either A-366. We found that MV4;11 cells underwent a dose-dependent transition towards sub G1/G0 content consistent with the dose response observed for CD11b differentiation of this cell line (Fig 4C). Of note, we observed that shorter treatments (3 and 7 days) with A-366 produced similar but less dramatic effects, consistent with the delayed kinetics of an epigenetic mechanism of action (data not shown). Finally, we observed morphological changes following A-366 treatment, such as increased cytoplasm:nucleus ratio and nuclear lobulation, indicative of differentiation (Fig 4D).


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 induces differentiation and affects viability in MV4;11 cells.(A) MV4;11 cells were incubated with A-366 for 14 days. Cells were fixed, stained with an anti-CD11b antibody and analyzed by flow cytometry. (B) MV4;11 cells from (A) additionally assayed for cellular proliferation (Cell Titer-Glo) and viability (trypan blue exclusion) following A-366 treatment for 14 days. (C) Cell cycle DNA content analysis was performed by propidium iodide staining in MV4;11 cells treated for 14 days with A-366. (D) Wright-Giemsa staining of MV4;11 cells treated with DMSO or 5 μM A-366 for 10 days.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131716.g004: A-366 induces differentiation and affects viability in MV4;11 cells.(A) MV4;11 cells were incubated with A-366 for 14 days. Cells were fixed, stained with an anti-CD11b antibody and analyzed by flow cytometry. (B) MV4;11 cells from (A) additionally assayed for cellular proliferation (Cell Titer-Glo) and viability (trypan blue exclusion) following A-366 treatment for 14 days. (C) Cell cycle DNA content analysis was performed by propidium iodide staining in MV4;11 cells treated for 14 days with A-366. (D) Wright-Giemsa staining of MV4;11 cells treated with DMSO or 5 μM A-366 for 10 days.
Mentions: One of the characteristic features of leukemia cells is a blockade of differentiation at a distinct stage of blood cell maturation [22]. As a result, differentiation therapy has been tested and validated clinically in several oncology indications. For example, two established clinical regimens for differentiation therapy, the treatment of acute promyelocytic leukemia with all-trans retinoic acid (ATRA) [23] and azacitidine treatment of myelodysplastic syndrome [24], provide proof-of-principle for this approach. Recently, G9a has been implicated in mediating T helper cell differentiation and function by studies of tissue-specific, G9a conditional knockout mice [25]. We hypothesized that inhibiting G9a function may likewise translate to the inhibition of leukemia cells by differentiation. As a first test of this hypothesis, we treated the acute myelocytic leukemia (AML) cell line MV4;11 with A-366. Long-term treatments resulted in dose-dependent differentiation of this tumor cell line, as observed by an increase in the differentiation marker CD11b (Fig 4A). A-366 treatment also resulted in inhibited proliferation and a decrease in viability corresponding to the dose response observed for CD11b staining (Fig 4B). DNA content analysis was also performed to assess a potential mechanism of action for the reduced viability observed in MV4;11 cells treated long-term with either A-366. We found that MV4;11 cells underwent a dose-dependent transition towards sub G1/G0 content consistent with the dose response observed for CD11b differentiation of this cell line (Fig 4C). Of note, we observed that shorter treatments (3 and 7 days) with A-366 produced similar but less dramatic effects, consistent with the delayed kinetics of an epigenetic mechanism of action (data not shown). Finally, we observed morphological changes following A-366 treatment, such as increased cytoplasm:nucleus ratio and nuclear lobulation, indicative of differentiation (Fig 4D).

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