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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

In vivo efficacy study of A-366 in MV4;11 flank xenografts.(A) MV4;11 cells were implanted subcutaneously in SCID/bg mice and allowed to establish tumors of ~200 mm3. A-366 was administered to tumor-bearing mice at 30 mg/kg/day by osmotic mini-pump for 14 days. Tumors were measured at the indicated time points and tumor growth was plotted as a function of time. (B) In a parallel PK/PD arm carried out in MV4;11 tumor-bearing animals, plasma and tissues were collected at the indicated time points and analyzed for A-366 levels. (C) A-366-treated tumors from the indicated time points were harvested and analyzed by AlphaLISA for reductions in H3K9me2 relative to vehicle.
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pone.0131716.g005: In vivo efficacy study of A-366 in MV4;11 flank xenografts.(A) MV4;11 cells were implanted subcutaneously in SCID/bg mice and allowed to establish tumors of ~200 mm3. A-366 was administered to tumor-bearing mice at 30 mg/kg/day by osmotic mini-pump for 14 days. Tumors were measured at the indicated time points and tumor growth was plotted as a function of time. (B) In a parallel PK/PD arm carried out in MV4;11 tumor-bearing animals, plasma and tissues were collected at the indicated time points and analyzed for A-366 levels. (C) A-366-treated tumors from the indicated time points were harvested and analyzed by AlphaLISA for reductions in H3K9me2 relative to vehicle.

Mentions: To assess the potential role that G9a/GLP inhibition may have in an in vivo setting we tested A-366 in a AML flank xenograft model using MV4;11 cells. However, initial MTD studies with A-366 dosed intraperitoneally (IP) showed toxicity in the animals at 10 mg/kg, which would limit exposures to a level not predicted to be efficacious for inhibiting tumor growth. To achieve a projected efficacious exposure while mitigating the presumed Cmax-driven toxicity, osmotic mini-pump (OMP) dosing was utilized. Mice treated with either vehicle control or A-366 administered via osmotic mini-pump at 30 mg/kg/day for two weeks showed no overt toxicity. We observed a modest 45% tumor growth inhibition resulting from A-366 treatment in this model (Fig 5A). During this in vivo efficacy trial, a separate arm of tumor-bearing size matched cohorts was sacrificed after 2, 7 and 14 days of A-366 treatment. Plasma and tissue specimens were collected from these treated tumor-bearing animals to assess the PK/PD relationship to the efficacy arms. We found that A-366 had significant accumulation in the tumors and other tissues tested (Fig 5B). Finally, the levels of global H3K9me2 were assessed in the MV4;11 tumors from mice in the PK/PD arm of the study and we found a slow but steady decrease in total H3K9me2 levels (Fig 5C). The combination of modest in vivo efficacy in MV4;11 xenografts aligns well with the slow kinetics of H3K9me2 inhibition despite the significant accumulation of A-366 observed in these tumors.


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)

In vivo efficacy study of A-366 in MV4;11 flank xenografts.(A) MV4;11 cells were implanted subcutaneously in SCID/bg mice and allowed to establish tumors of ~200 mm3. A-366 was administered to tumor-bearing mice at 30 mg/kg/day by osmotic mini-pump for 14 days. Tumors were measured at the indicated time points and tumor growth was plotted as a function of time. (B) In a parallel PK/PD arm carried out in MV4;11 tumor-bearing animals, plasma and tissues were collected at the indicated time points and analyzed for A-366 levels. (C) A-366-treated tumors from the indicated time points were harvested and analyzed by AlphaLISA for reductions in H3K9me2 relative to vehicle.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131716.g005: In vivo efficacy study of A-366 in MV4;11 flank xenografts.(A) MV4;11 cells were implanted subcutaneously in SCID/bg mice and allowed to establish tumors of ~200 mm3. A-366 was administered to tumor-bearing mice at 30 mg/kg/day by osmotic mini-pump for 14 days. Tumors were measured at the indicated time points and tumor growth was plotted as a function of time. (B) In a parallel PK/PD arm carried out in MV4;11 tumor-bearing animals, plasma and tissues were collected at the indicated time points and analyzed for A-366 levels. (C) A-366-treated tumors from the indicated time points were harvested and analyzed by AlphaLISA for reductions in H3K9me2 relative to vehicle.
Mentions: To assess the potential role that G9a/GLP inhibition may have in an in vivo setting we tested A-366 in a AML flank xenograft model using MV4;11 cells. However, initial MTD studies with A-366 dosed intraperitoneally (IP) showed toxicity in the animals at 10 mg/kg, which would limit exposures to a level not predicted to be efficacious for inhibiting tumor growth. To achieve a projected efficacious exposure while mitigating the presumed Cmax-driven toxicity, osmotic mini-pump (OMP) dosing was utilized. Mice treated with either vehicle control or A-366 administered via osmotic mini-pump at 30 mg/kg/day for two weeks showed no overt toxicity. We observed a modest 45% tumor growth inhibition resulting from A-366 treatment in this model (Fig 5A). During this in vivo efficacy trial, a separate arm of tumor-bearing size matched cohorts was sacrificed after 2, 7 and 14 days of A-366 treatment. Plasma and tissue specimens were collected from these treated tumor-bearing animals to assess the PK/PD relationship to the efficacy arms. We found that A-366 had significant accumulation in the tumors and other tissues tested (Fig 5B). Finally, the levels of global H3K9me2 were assessed in the MV4;11 tumors from mice in the PK/PD arm of the study and we found a slow but steady decrease in total H3K9me2 levels (Fig 5C). The combination of modest in vivo efficacy in MV4;11 xenografts aligns well with the slow kinetics of H3K9me2 inhibition despite the significant accumulation of A-366 observed in these tumors.

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