EPZ004777 hydrochloride 

For assessment of cell proliferation and viability in human cell lines, exponentially growing cells are plated, in triplicate, in 96-well plates at a density of 3×10⁴ cells/well in a final volume of 150 μL. Cells are incubated in the presence of 3 μM (proliferation curve), or increasing concentrations (IC₅₀ determination) of EPZ004777 up to 50 μM. Viable cell number is determined every 3-4 days for up to 18 days using the Guava Viacount assay and analyzed on a Guava EasyCyte Plus instrument. On days of cell counts, growth media and EPZ004777 are replaced and cells split back to a density of 5×10⁴ cells/well. Total cell number is expressed as split-adjusted viable cells per well. For each cell line, IC₅₀ values are determined from concentration-dependence curves at each time point using Graphpad Prism software. Experiments to determine IC₅₀ values continued until IC₅₀ values stabilized (day 18 for THP-1 cells, day 14 for all other cell lines)^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Nine-week-old female nude mice (nu/nu) are injected subcutaneously with MV4-11 cells in the right flank (200 μL of a 5×10⁷ cells/mL suspension in a 1:1 mixture of PBS and Matrigel). Mice are randomized to treatment groups when tumor sizes reached 300-400 mm³. Six mice received subcutaneous implant of osmotic pumps, containing 50 mg/mL EPZ004777 in 10% ethanol, 90% water, and five control mice received no pump implant. Six days after pump implant, animals are sacrificed and tumor samples from treated and control animals are collected for immunoblot analysis. For the disseminated leukemia model, MV4-11 cells are transduced with the pMMP-LucNeo retrovirus. Eight-week-old female NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice are purchased from Jackson Laboratories. A total of 1×10⁷ MV4-11-LucNeo cells are injected intravenously via the lateral tail vein. Engraftment of disseminate leukemia is determined by bioluminescence imaging after injection of 75 mg/kg of D-luciferin. Animals with documented leukemia are divided into treatment groups consisting of vehicle (15% ethanol, 50% PEG300, 35% water) loaded osmotic pumps, or EPZ004777 at 50, 100, or 150 mg/mL. Osmotic pumps are replaced after one week. Irritation caused by compound precipitation is observed in the 100 and 150 mg/mL dose groups, precluding additional pump replacements. Animals are monitored daily for clinical symptoms, and are euthanized when they displayed signs of distress consistent with terminal leukemic disease. Log-rank analysis is used to determine statistical significance of the survival curves.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Daigle SR, et al. Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell. 2011 Jul 12;20(1):53-65.  [Content Brief]

  [2]. Chen L, et al. Abrogation of MLL-AF10 and CALM-AF10-mediated transformation through genetic inactivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l. Leukemia. 2013 Apr;27(4):813-22.  [Content Brief]

  [3]. Deshpande AJ, et al. Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l.Blood. 2013 Mar 28;121(13):2533-41.  [Content Brief]