EPZ-6438 (Tazemetostat)

Tazemetostat (EPZ-6438) concentration-dependently reduces global H3K27Me3 levels in wild-type or SMARCB1 mutant cells, and induces strong antiproliferative effects with IC50 ranging from 32 nM to 1000 nM in SMARCB1-deleted MRT cell lines. It induces gene expression of neuronal differentiation and cell cycle inhibition, while inhibtis expression of Hedgehog pathway genes, MYC and EZH2. [1] The antiproliferative effect of this compound is enhanced by either NSC-9900 or Hexadecadrol in several EZH2 mutant lymphoma cell lines. [2]

In SCID mice bearing s.c. G401 xenografts, Tazemetostat (EPZ-6438) induces tumor stasis during the administration period and produces a significant tumor growth delay with minimal effect on body weight. [1]

• Méthodes biochimiques

  Le Tazemetostat (EPZ-6438) est incubé pendant 30 min avec 40 μL par puits de 5 nM PRC2 (concentration finale d'essai dans 50 μL est de 4 nM) dans 1X tampon d'essai (20 mM Bicine [pH 7,6], 0,002% Tween-20, 0,005% gélatine de peau bovine et 0,5 mM DTT). 10 μL par puits de mélange de substrat comprenant du tampon d'essai, 3 H-SAM, SAM non marqué et un peptide représentant les résidus 21-44 de l'histone H3 contenant une biotine C-terminale (ajoutée à une lysine à terminaison amide C-terminale) sont ajoutés pour initier la réaction (les deux substrats sont présents dans le mélange réactionnel final à leurs valeurs Km respectives, un format d'essai appelé «conditions équilibrées»). Les concentrations finales des substrats et l'état de méthylation du peptide substrat sont indiqués pour chaque enzyme. Les réactions sont incubées pendant 90 min à température ambiante et stoppées avec 10 μL par puits de 600 μM de SAM non marqué, puis transférées dans une plaque flashplate 384 puits et lavées après 30 min.

• Lignées cellulaires

  Mutant cell lines (G401, A204, G402, KYM-1), Wild type cell line (RD, 293, SJCRH30)

• Concentrations

  ~10 μM

• Temps d'incubation

  7 days

• Méthode

  For the adherent cell line proliferation assays, plating densities for each cell line are determined based on growth curves (measured by ATP content) and density over a 7-d time course. On the day before compound treatment, cells are plated in either 96-well plates in triplicate (for the day 0–7 time course) or 6-well plates (for replating on day 7 for the remainder of the time course). On day 0, cells are either untreated, DMSO-treated, or treated with Tazemetostat (EPZ-6438) starting at 10 µM and decreasing in either threefold or fourfold dilutions. Plates are read on day 0, day 4, and day 7 using Cell Titer Glo, with compound/media being replenished on day 4. On day 7, the six-well plates are trypsinized, centrifuged, and resuspended in fresh media for counting by Vi-Cell. Cells from each treatment are replated at the original density in 96-well plates in triplicate. Cells are allowed to adhere to the plate overnight, and cells are treated as on day 0. On days 7, 11, and 14, plates are read using Cell Titer Glo, with compound/media being replenished on day 11. Averages of triplicates are used to plot proliferation over the time course, and calculate IC50 values. For cell cycle and apoptosis, G401 and RD cells are plated in 15-cm dishes in duplicate at a density of 1 × 10⁶ cells per plate. Cells are incubated with this compound at 1 µM, in a total of 25 mL, over a course of 14 d, with cells being split back to original plating density on day 4, 7, and 11. Cell cycle analysis and TUNEL assay are performed using a Guava flow cytometer, following the manufacturer’s protocol.

• Modèles animaux

  SCID mice bearing s.c. G401 xenografts.

• Posologies

  ~500 mg/kg

• Administration

  Oral administration