In vitro activity: I-CBP-112 is a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor that targets the bromodomain-containing transcription factors CREBBP (CBP) and EP300 with IC50 of 0.142 and 0.625 μM, respectively. It significantly enhances acetylation by p300 at the histone H3K18 and H3K23 sites. I-CBP112 stimulated H3K18ac by ~3-fold, I-CBP112 induced enhances acetylation of these same sites by CBP as well as at H4K5. The EC50’s of activation of I-CBP112 on p300- and CBP-mediated H3K18 acetylation are ~2 μM. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers.

Kinase Assay: I-CBP112 is a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor that targets the bromodomain-containing transcription factors CREBBP (CBP) and EP300 with IC50 of 0.142 and 0.625 μM, respectively. It significantly enhances acetylation by p300 at the histone H3K18 and H3K23 sites. I-CBP112 stimulated H3K18ac by ~3-fold, I-CBP112 induced enhances acetylation of these same sites by CBP as well as at H4K5. The EC50’s of activation of I-CBP112 on p300- and CBP-mediated H3K18 acetylation are ~2 μM.

Cell Assay: Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers. I-CBP112 is dissolved in DMSO and diluted with appropriate medium before use. Cells (6000 KG1a and 13000 LNCaP cells/well) are plated in 96-well flat-bottom plates approximately 24 h prior to drug treatment. After 24 h, 10–20% fetal bovine serum-containing medium is replaced with 2.5% serum medium, and cells are treated with I-CBP112 in 0.18% DMSO; 0.18% DMSO is shown to have negligible cell growth effects under the conditions used in our experiments. After being exposed to I-CBP112 for 66 h, cells are subjected to a final concentration of 0.476% [3H]thymidine per well and allowed to proliferate for an additional 6 h (exposure to I-CBP112 for a total of 72 h). Cells are harvested, and the counts of 3H in each well are taken relative to those treated with vehicle alone to quantify the effect of the ligand on proliferation