Tazemetostat

The DFCI Experimental Therapeutics Core performed the xenograft study. This study was approved by the DFCI Institutional Animal Care and Use Committee and is compliant with the NIH Guide for the Care and Use of Laboratory Animals. 13-week-old female NOD scid gamma mice (Jackson Labs; Bar Harbor, ME) were implanted subcutaneously with 5×10⁶ MKL-1 cells in 100 μL PBS with 50% Matrigel. Tumors grew to a range of 91.9 mm³-188.5 mm³ prior to treatment randomization. 9 mice were treated with 400 mg/kg tazemetostat (MedChemExpress; Monmouth Junction, NJ) and 8 with vehicle (0.5% methylcellulose with 0.1% Tween 80 in sterile water; pH adjusted to 4.0 with 1N HCl) BID by oral gavage. Tumor volume was measured twice weekly until tumors reached endpoint volume (2,000 mm³) or became ulcerated, when mice were euthanized. Tumors were flash frozen in liquid nitrogen. For immunoblotting, frozen tumors were ground with mortar and pestle and disrupted in RIPA with the Qiagen TissueRuptor II (Hilden, Germany).

For the cell line soft agar experiments, tazemetostat (MedChemExpress) was dissolved in DMSO for a stock concentration of 2 mM and placed into the cell-containing top layer of agarose and overlaying medium at the noted concentrations. For PDX soft agars, PDXs were pre-treated with the noted concentrations of tazemetostat for 48-hours prior to seeding in addition to tazemetostat being placed into the cell-containing top layer of agarose and overlaying medium. For cell cycle analysis, cells were treated with 2 μM tazemetostat for 96-hours prior to sample preparation.