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1 Supplementary Information Targeted Disruption of the EZH2/EED Complex Inhibits EZH2- dependent Cancer Woojin Kim 1,2,3, Gregory H. Bird 2,3,4, Tobias Neff 5, Guoji Guo 1,2,3, Marc A. Kerenyi 1,2,3, Loren D. Walensky 1,2,3,4 *, Stuart H. Orkin 1,2,3,6 * 1 Division of Pediatric Hematology/Oncology, Boston Children's Hospital, MA Harvard Medical School, Boston, MA 2115, 3 Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston MA Pediatric Hematology/Oncology/BMT, University of Colorado, Aurora, CO Howard Hughes Medical Institute, Boston, MA 2115, USA *Correspondence: stuart_orkin@dfci.harvard.edu (S.H.O.), loren_walensky@dfci.harvard.edu (L.D.W.) Nature Chemical Biology: doi:1.138/nchembio.1331

2 Supplementary Results Supplementary Figure 1 3, EZH2(42-68) A (42-68) 17% 46% [θ], Mean Residue Ellipticity 2, 1, -1-2 B (42-68) 27% Wavelength (nm) Supplementary Figure 1 Circular dichroism analysis of EZH2(42-68), A (42-68) and B (42-68) peptides. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S1

3 Supplementary Figure 2 EED Peptide input (nm) EZH2(42-68) A (42-68) MUT IP: FITC WB: HA FS Polarization units (mp) [EED],M Peptide K D 95% CI MUT 32 nm >1 μm Supplementary Figure 2 Comparative binding of glutamate mutants at the indicated concentrations to purified HA-EED (4 nm) as assessed by anti-fitc pull-down and western analysis with α-ha antibody (top). The results are representative of experiments performed in duplicate. FS, Fluorescence Scan. EED-binding affinity of and MUT peptides, as measured by fluorescence polarization (bottom). Data represent mean ± s. e. m. for experiments performed in triplicate. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S2

4 Supplementary Figure 3 Nucleus Peptide MUT Nucleus 1μm 1μm EED Peptide Merge EED Merge Supplementary Figure 3 Localization of EED (red) and and SAHEZH2MUT peptides (green) in COS-7 cells treated with 5 μm peptides for 8 hours. Nuclei are stained with DAPI (blue). Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S3

5 Supplementary Figure 4 EZH2 (42-68) MUT TAT-EZH2 (42-68) FSSNRQKILERTEILNQEWKQRRIQPV FSSNRXKILXRTQILNQEWKQRRIQPV FSSNRXKILXRTQILNQQWKQRRIQPV GRKKRRQRRRPQ-FSSNRQKILERTEILNQEWKQRRIQPV Fluorescence scan FITC peptides EZH2 (42-68) MUT TAT-EZH2 Peptide Markers TAT-EZH2 EZH2(42-68) WB:H3 loading Incubation (Hr) Supplementary Figure 4 Comparative cellular uptake and intracellular stability of EZH2 peptides. MLL-AF9 cells were treated with the indicated FITC-derivatized EZH2 petides (5 μm) and lysates prepared at 1, 2, 4, and 8 hours, followed by electrophoresis and fluorescence scan. We used unmodified FITC-EZH2 peptide as a negative control and the same sequence fused to the cell penetrating sequence TAT as a positive control Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S4

6 Supplementary Figure 5 Vehicle MUT IP: FITC WB: EED WB: β-actin FS Supplementary Figure 5 Binding of, but not MUT to native EED, as assessed by anti-fitc immunoprecipitation from peptide-treated (1 μm) MLL-AF9 cells and EED western analysis. FS, Fluorescence Scan Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S5

7 Supplementary Figure 6 Coomassie Blue Staining EZH1 EZH2 EZH1 EED EZH2 EED Flow through 2. 1st Wash 3. 2nd Wash 4. 1st Elute 5. 2nd Elute 6. Proteins left bound to resin after elution Supplementary Figure 6 Co-immunoprecipitation of HA-EED with FLAG-EZH1/FLAG-EZH2 by anti-flag agarose (coomassie blue staining) Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S6

8 Supplementary Figure 7 EED -/- Vehicle MUT Number of cells K27-Me3 Supplementary Figure 7 Flow cytometric analysis of -treated MLL-AF9 leukemia cells. decreased the number of cells manifesting H3Lys27 trimethylation. Cells were treated with peptides (1 μm) twice daily for 7 days. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S7

9 Supplementary Figure 8 Camptothecin Vehicle MUT AAD Annexin V Supplementary Figure 8 Apoptosis analysis of MLL-AF9 cells after extended incubation with peptides (1 μm twice daily dosing for 16 days). Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S8

10 Supplementary Figure 9 Vehicle 1μM MUT 1μM 1μM Supplementary Figure 9 -treated MLL-AF9 cells exhibited decreased colony-forming capacity compared to vehicle and MUT-treated cells, as evidenced by the low power images. Cells were treated as described in Methods. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S9

11 Supplementary Figure CD Fold change 1..5 <Qdot 65-A> Lin + Lin - Kit Kit Kit - Sca Sca1 Kit + Sca1 - <PE-Cy7-A> FcgR + CD34 MLL AF9 L-GMP Lin + /Kit - 5K 1K 15K 2K 25K SSC-A Sca <Pacific Blue-A> Supplementary Figure 1 Decreased CD133 expression in differentiated leukemic Lin + /kit - blast cells p<.23. p values were calculated based on data comparison between undifferentiated MLL-AF9 L-GMP cells (Lin - Kit + Sca1 - FcgR + CD34 + ) and differentiated leukemic blasts (Lin + Kit - Sca1 - ), harvested from mice and FACS-sorted. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S1

12 Days MUT Days Vehicle C1498 M1 4 Number of cells(x16) 6 MUT HPC5 Number of cells(x16) 8 MLL-AF9 Number of cells(x16) Number of cells(x16) Supplementary Figure 11 MUT MUT Days MUT Days 15 2 HPC5 cells 5μm 5μm MLL-AF9 5μm HPC5 treated 2μm 2μm Supplementary Figure 11 Effect of peptides (1 μm, twice daily) on HPC5, C1498 and M1 cell proliferation (top). Data represent mean of cell counts and s.e.m. for experiments performed in triplicate. The non-leukemic HPC5 cell line showed no apparent morphologic differences in response to treatment. (middle) Comparative effect of on the morphology of MLL-AF9 (differentiated) and HPC5 (undifferentiated) cells observed under high-magnification. (bottom) Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S11

13 Supplementary Figure 12 Viability (Observed relative to vehicle) D GSK126 Water 1 nm 3 nm 1 µm 3 µm 1 µm Supplementary Figure 12 Effects of and GSK126 treatments on the viability of 32D, a non-leukemic transformed cell line. Cells were treated with (1 μm, twice daily) and GSK126 (1 μm, single dose, as reported 21 ) for 7 days. Data represent mean ± s.e.m for independent experiments performed in biological triplicate. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S12

14 Supplementary Figure 13 MLL-AF WB: H3K27Me WB: H3 loading GSK (μm) Supplementary Figure 13 Effects of vehicle (water),, and GSK126 treatment on EZH2 protein level in MLL-AF9 cells. Cells were treated with (twice daily) and GSK126 (single dose, and replenished at cell split due to confluency, as reported 21 ) for 7 days. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S13

15 Supplementary Figure MDA-MB231 GSK DU145 GSK126 Viability (Observed relative to vehicle) Viability (Observed relative to vehicle) Water 1 nm 3 nm 1 µm 3 µm 1 µm Water 1 nm 3 nm 1 µm 3 µm 1 µm Supplementary Figure 14 Effects of and GSK126 treatments on the viability of MDA-MB213 (breast cancer) and DU145 (prostate cancer) cells. Cells were treated twice daily with and single dose GSK126 (as reported 21 ) at the indicated concentrations for 7 days. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S14

16 Supplementary Figure 15 1 nm 3 nm 1 µm 3 µm 1 µm GSK126 Pfeiffer (EZH2 A677G) Viability (Observed relative to vehicle) Days Days Supplementary Figure 15 Dose-responsive effects of and GSK126 treatment on EZH2 mutant B-cell lymphoma cell line, Pfeiffer. Cells were treated with twice daily and GSK126 at the indicated doses for 12 days. Cells were split at day 4, 8 and 12 for viability measurement and compounds were replenished with fresh media to maintain concentration. Data represent mean ± s.e.m for experiments performed in biological triplicate and technical duplicate. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S15

17 Supplementary Figure 16 MLL-AF9 HPC5 C1498 M1 OCI-LY19 Karpas422 Pfeiffer Peptide uptake FS β-actin WB: β-actin Peptide treatment FS (μm) Supplementary Figure 16 Cellular uptake of. was added to cultured cells in serum-containing media at 1, 3 and 1 μm concentration and incubated for 4 hours, followed by fluorescence analysis of electrophoresed cellular lysates. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S16

18 Supplementary Figure 17 OCI-LY19 WB:EZH2 WB:β-actin Karpas422 WB:EZH2 WB:β-actin Pfeiffer WB:EZH2 WB:β-actin Vehicle GSK (μm) Supplementary Figure 17 Effect of treatment on EZH2 protein levels in B lymphoma cell lines. Cells were treated with 1, 3, and 1 μm dosing of SAH- EZH2 (twice daily) and GSK126 (single dose, followed by replenishment at cell split due to confluency, as reported 21 ) for 7 days, followed by anti-ezh2 western analysis Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S17

19 Supplementary Figure 18 Karpas WB: H3K27Me WB: H3 loading GSK (μm) Supplementary Figure 18 Effect of and GSK126 treatment on H3K27 trimethylation status in Karpas422 cells. Cells were treated with 1, 3, and 1 μm dosing of (twice daily) and GSK126 (single dose, followed by replenishment at cell split due to confluency 21 ) for 7 days, followed by anti-h3k27me3 western analysis. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S18

20 Supplementary Figure 19 Viability (Observed relative to vehicle) MLL-AF9 GSK126 µm.1 µm.3 µm 1 µm 3 µm Viability (Observed relative to vehicle) Karpas422 GSK126 µm.1 µm.3 µm 1 µm 3 µm µm. 3 µm 1 µm 3 µm µm.3 µm 1 µm 3 µm Supplementary Figure 19 Effect of and GSK126, administered singly and in combination, on the cell viability of MLL-AF9 and Karpas422 cells. Cells were treated twice daily with SAH- EZH2 and single dose GSK126 (as reported 21 ) at the indicated concentrations for 7 days. Data represent mean ± s.e.m for independent experiments performed in biological triplicate. Nature Chemical Biology: doi:1.138/nchembio.1331 Kim, et al. Figure S19

21 Supplementary Figure 2 Mass spectrum of peptide FITC- A (4-68) Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

22 Supplementary Figure 2 Mass spectrum of peptide FITC- A (42-68) Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

23 Supplementary Figure 2 Mass spectrum of peptide FITC- A (42-64) Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

24 Supplementary Figure 2 Mass spectrum of peptide FITC- B (4-68) Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

25 Supplementary Figure 2 Mass spectrum of peptide FITC- B (42-68) Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

26 Supplementary Figure 2 Mass spectrum of peptide FITC- B (42-64) Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

27 Supplementary Figure 2 Mass spectrum of peptide FITC-EZH2(42-68) Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

28 Supplementary Figure 2 Mass spectrum of peptide FITC- A (42-68)E54Q Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

29 Supplementary Figure 2 Mass spectrum of peptide FITC- A (42-68)E54Q/E59Q Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

30 Supplementary Figure 2 Mass spectrum of peptide Ac- A (42-68)E54Q Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

31 Supplementary Figure 2 Mass spectrum of peptide Ac- A (42-68)E54Q/E59Q Max: m/z Nature Chemical Biology: doi:1.138/nchembio.1331

32 Supplementary Figure 21 Full gel image of Western blot Figure 1d HA-EED western blot Figure 1e Peptide uptake fluorescence scan Figure 2b Peptide uptake fluorescence scan Figure 1d Peptide input fluorescence scan Figure 1e β-actin western blot Figure 2b β-actin western blot Figure 1e Peptide scan Fluorescence scan Figure 2b Peptide treatment fluorescence scan Figure 2d Flag-EZH1 western blot Figure 2d Flag-EZH2 western blot Figure 2e H3K27Me3 western blot Figure 2d peptide treatment fluorescence scan Figure 2d peptide treatment fluorescence scan Figure 2e H3 Western blot Figure 2f H3K4Me3 western blot Figure 2f H3K9Me3 western blot Figure 2f H3K36Me3 western blot Figure 3d p19 ARF western blot Figure 5b EZH2 western blot Figure 2f H3K27Me3 western blot Figure 2f Loading Figure 3d β-actin western blot β-actin western blot Kim, et al. Figure S21 Nature Chemical Biology: doi:1.138/nchembio.1331

33 Supplementary Table 1 Cell cycle analysis of -treated cells. Vehicle MUT G /G 1 4. ± ± ± 3.3 S 53. ± ± ± 3.6 G 2 /M 6.9 ± ± ±.4 Data represent mean ± s.e.m for independent experiments performed in duplicate Nature Chemical Biology: doi:1.138/nchembio.1331

34 Supplementary Table 2 Sequences, modification and molecular weight of stapled peptides Name Peptide sequence N-terminus Expected Observed Appears in Modification Mass Mass A(4-68) SN LFSSNRXKILXRTEILNQEWKQRRIQPV FITC Fig. 1 A(42-68) FSSNRXKILXRTEILNQEWKQRRIQPV FITC Fig. 1, 2a, 2b, and 2c A(42-64) FSSNRXKILXRTEILNQEWKQRR FITC Fig. 1 Sup. Fig. 1 and 2 B(4-68) SN LFSSNRQKILERTXILNXEWKQRRIQPV FITC Fig. 1 B(42-68) FSSNRQKILERTXILNXEWKQRRIQPV FITC Fig. 1 and Sup. Fig. 1 B(42-64) FSSNRQKILERTXILNXEWKQRR FITC Fig. 1 EZH2(42-68) FSSNRQKILERTEILNQEWKQRRIQPV FITC Fig. 2, Sup. Fig. 1, 2 and 4 A-E54Q () FSSNRXKILXRTQILNQEWKQRRIQPV FITC Fig. 2, 3a, 3d, 4 and 5 Sup. Fig, 2-5, 7-9 and A-E54,59Q ( MUT) FSSNRXKILXRTQILNQQWKQRRIQPV FITC Fig. 2, 3a, 3d and 4 Sup. Fig. 2-5, 7-9 and 11 A-E54Q FSSNRXKILXRTQILNQEWKQRRIQPV Acetylation Fig. 3b and 3c Sup. Fig. 7 and 8 A-E54,59Q FSSNRXKILXRTQILNQQWKQRRIQPV Acetylation Fig. 3b and 3c Sup. Fig. 7 and 8 N L =Norleucine, X=Non-natural olefinic amino acid, Sup. Fig.= Supplementary Figure Nature Chemical Biology: doi:1.138/nchembio.1331

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