1. Volume 20, Issue 11, Pages 1329-1339 (November 2013)
Identification of EZH2 and EZH1 Small Molecule Inhibitors with Selective Impact on Diffuse Large B Cell Lymphoma Cell Growth 
Shivani Garapaty-Rao, Christopher Nasveschuk, Alexandre Gagnon, Eric Y. Chan, Peter Sandy, Jennifer Busby, Srividya Balasubramanian, Robert Campbell, Feng Zhao, Louise Bergeron, James E. Audia, Brian K. Albrecht, Jean-Christophe Harmange, Richard Cummings, Patrick Trojer 
Chemistry & Biology 
Volume 20, Issue 11, Pages (November 2013)
DOI: /j.chembiol
Copyright © 2013 Elsevier Ltd Terms and Conditions

2. Chemistry & Biology 2013 20, 1329-1339DOI: (10. 1016/j. chembiol. 2013
Copyright © 2013 Elsevier Ltd Terms and Conditions

3. Figure 1
Characterization of a Highly Selective EZH2 and EZH1 Small Molecule Inhibitor
(A) Determination of the inhibitory potential of compound 3 on PRC2 reconstituted with WT EZH2 (left), Y641N mutant EZH2 (middle), and WT EZH1 (right). PRC2 IC50 values are determined from ten point dose response curves run in duplicate ± SEM.
(B) Determination of the mechanism of action of compound 3. Activity assays were carried out under balanced conditions with 2.5 or 25 nM PRC2 (1× enzyme and 10× enzyme, respectively), with oligonucleosomes (10× NUC) or SAM (10× SAM) in excess. The IC50 value in presence of 10× SAM shifts 8.3-fold. Data are derived from ten point dose response curves run in duplicate ± SEM.
(C) The inhibitory potential of compound 3 was tested against a panel of HKMTs, including WHSC1, SETD7, DOT1L, EHMT2, and SETD8. No inhibition was detected to up to 80 μM. All enzyme assays were carried out under balanced conditions as ten point dose response curves run in duplicate ± SEM.
(D) HeLa cells were treated with 3.75, 7.5, 15, or 30 μM of compound 3 for 6 days and whole cell extracts analyzed by western blotting for levels of EZH2, EZH1, SUZ12, EED, H3K27me3, H3K27me2, H3K27me1, H3K4me3, H3K9me3, and H3K36me3. GAPDH and H4 levels served as loading controls.
See also Figure S1.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2013 Elsevier Ltd Terms and Conditions

4. Figure 2
Inhibition of EZH2 HMT Activity Results in Global Reduction of H3K27me3
(A) HeLa cells were treated with DMSO or compound 3 (30 μM) for 3 days, cells were fixed, permeabilized, and analyzed for H3K27me3 levels by immunofluorescence microscopy (IFM). Counterstaining with Hoechst dye was used to illustrate similar cell numbers in both treatment groups.
(B) Distribution analysis of HeLa cells treated with DMSO or 15 and 30 μM of compound 3 for 3 days and analyzed for H3K27me3 levels by IFM. Each peak represents the distribution of H3K27me3 intensity across the entire cell population. This analysis is used to calculate the average H3K27me3 intensity for each treatment group. For further information, see also Figure S2.
(C and D) Determination of EC50 (C) and GI50 (D) values with respect to changes in global H3K27me3 levels (using a Mesoscale ELISA assay) and effects on viability (using a luminescent viability cell assay). Data are represented as the mean of three independent experiments ± SD.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2013 Elsevier Ltd Terms and Conditions

5. Figure 3
Inhibition of EZH2 HKMT Activity Affects H3K27me3 Species to Various Degrees
LC-MS/MS was used to analyze histone modification levels in HT (A) and SUDHL6 (B) cells upon treatment with 5, 15, and 25 μM of compound 1 or compound 3 for 4 days. Each bar represents a distinct histone modification or combination thereof. The experiment was carried out in triplicate ± SD.
See also Figures S2F and S2G and Table S1.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2013 Elsevier Ltd Terms and Conditions

6. Figure 4
Inhibition of EZH2 HKMT Activity Does Not Affect Growth of PC3 Prostate Cancer Cells
(A) PC3 cells were incubated with compound 1 and 3 (30 μM) for 4 and 10 days and H3K27me3 levels were assessed by western blot. Total H4 levels and GAPDH served as loading controls.
(B) PC3 cell viability was assessed upon treatment with DMSO or compound 1 and 3 (30 μM) for 4, 7, and 10 days using a luminescent cell viability assay. Shown is one representative of three experiments.
(C) Expression level of the known EZH2 target genes, DAB2IP and SLIT2, were assessed upon treatment with DMSO or compound 1 and 3 (30 μM) for 1, 2, 4, 7, and 10 days. Relative expression levels were graphed as a change compared to the DMSO-treated sample at day 1. Data are represented as the mean of triplicate experiments ± SD.
(D) EZH2 transcript (upper) and protein levels (lower) were determined upon siRNA-mediated knockdown for 1, 2, 4, 7, and 10 days (for transcript) and 4, 7, and 10 days for protein. H3K27me3 levels were determined as well (lower). EZH2 transcript level data are represented as the mean of triplicate experiments ± SD. GAPDH served as a loading control.
(E) PC3 cell viability was assessed upon siRNA-mediated knockdown of EZH2 for 4, 7, and 10 days using a luminescent cell viability assay. Shown is one representative of three experiments.
(F) DAB2IP transcript levels were assessed upon siRNA-mediated knockdown of EZH2 for 1, 2, 4, 7, and 10 days. Relative expression levels were graphed as a relative change in comparison to nontargeting siRNA control (siNTC) at day 1. Data are represented as the mean of triplicate experiments ± SD.
See also Figure S3 and Table S3.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2013 Elsevier Ltd Terms and Conditions

7. Figure 5
GCB-DLBCL Cells Are Selectively Growth Inhibited and Gene Expression Is Altered upon EZH2 Inhibition
(A) GCB-DLBCL cell viability was assessed in response to compound treatment. Pfeiffer (left) and OCI-LY19 (right) cells were treated with compound 1 (top) or compound 3 (bottom) for 4, 7, 10, and 14 days and viability was determined at each dose by luminescent cell viability assays. Experiments were carried out in triplicate ± SD.
(B) H3K27me3 levels were assessed in Pfeiffer (left) and OCI-LY19 (right) cells upon treatment with compounds 1 (top) or 3 (bottom). H3K27me3 levels were normalized to total H3. Experiments were carried out in triplicate ± SD. For more information, see also Figure S4.
(C) Transcript levels of ABAT, EPB41L1, APOL1, CEACAM1, PIGZ, SOX9, SESN3, and CDKN2A were determined in Pfeiffer cells upon treatment with various concentrations of compound 3. Experiments were carried out in triplicate ± SD. For further information, see also Figure S5 and Table S3.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2013 Elsevier Ltd Terms and Conditions

8. Chemistry & Biology 2013 20, 1329-1339DOI: (10. 1016/j. chembiol. 2013
Copyright © 2013 Elsevier Ltd Terms and Conditions