Targeted disruption of the EZH2-EED complex inhibits EZH2-dependent cancer

Abstract

Enhancer of zeste homolog 2 (EZH2) is the histone lysine N-methyltransferase component of the Polycomb repressive complex 2 (PRC2), which, in conjunction with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog, regulates cell lineage determination and homeostasis. Enzymatic hyperactivity has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Here, we report the development of stabilized α-helix of EZH2 (SAH-EZH2) peptides that selectively inhibit H3 Lys27 trimethylation by dose-responsively disrupting the EZH2-EED complex and reducing EZH2 protein levels, a mechanism distinct from that reported for small-molecule EZH2 inhibitors targeting the enzyme catalytic domain. MLL-AF9 leukemia cells, which are dependent on PRC2, undergo growth arrest and monocyte-macrophage differentiation upon treatment with SAH-EZH2, consistent with observed changes in expression of PRC2-regulated, lineage-specific marker genes. Thus, by dissociating the EZH2-EED complex, we pharmacologically modulate an epigenetic 'writer' and suppress PRC2-dependent cancer cell growth.

Figure 1. Synthesis, EED-binding activity, and cellular penetration of SAH-EZH2 peptides

a) Design of hydrocarbon-stapled peptides to recapitulate the bioactive structure of the EED-binding domain of EZH2. The location of stapling position ‘A’ (47–51) is marked with red spheres and position ‘B’ (54–58) is marked with blue spheres. The alpha-helical EED-binding domain of EZH2 is colored gold and EED is colored gray. The native methionine of SAH-EZH2 was replaced with norleucine (‘N_L’) due to the incompatibility of sulfur with the metathesis reaction. b) Binding affinity of SAH-EZH2 peptides for EED as measured by fluorescence polarization assay. Data represents mean ± s.e.m. for experiments performed in triplicate. c) Sequences and binding affinities of SAH-EZH2 peptides. CI, confidence interval d) Anti-FITC immunoprecipitation of purified HA-EED (40 nM) upon incubation with the indicated amounts of FITC-labeled SAH-EZH2 peptides. The results are representative of experiments performed in duplicate. FS, Fluorescence Scan. Full gel image in Supplementary Fig. 21 e) Cellular penetration of SAH-EZH2 peptides. Lysates of treated MLL-AF9 leukemia cells were electrophoresed and analyzed by fluorescence scan. The results are representative of experiments performed in duplicate. FS, Fluorescence Scan. Full gel image in Supplementary Fig. 21

Figure 2. Sequence-dependent dissociation of EED-EZH1/EZH2 complexes and impairment of PRC2 activity by SAH-EZH2

a) Sequences of SAH-EZH2 and its mutants b) Enhanced cellular penetration of the E54Q (SAH-EZH2) and E54Q/E59Q (SAH-EZH2_MUT) mutants of SAH-EZH2_A(42-68), as measured by fluorescence scan of lysates from FITC-peptide treated MLL-AF9 leukemia cells at 8 hours. The results are representative of experiments performed in duplicate. FS, Fluorescence Scan. Full gel image in Supplementary Fig. 21 c) Comparative cellular uptake of EZH2, SAH-EZH2_A(42-68), SAH-EZH2, and SAH-EZH2_MUT peptides, as measured by confocal microscopy of FITC-peptide treated (5 μM) COS-7 cells at 8 hours. The results are representative of experiments performed in triplicate. d) Dissociation of HA-EED/EZH1 and HA-EED/EZH2 complexes (40 nM) by SAH-EZH2, but not SAH-EZH2_MUT, peptides at the indicated doses, as assessed by anti-HA pull-down and EZH1/EZH2 western analysis. FS, Fluorescence Scan. Full gel image in Supplementary Fig. 21 e) Dose-responsive suppression of H3K27 trimethylation by SAH-EZH2, administered to cultured MLL-AF9 cells twice daily for 7 days at 1, 3, and 10 μM dosing. Full gel image in Supplementary Fig. 21 f) Selective reduction of H3K27Me3 by SAH-EZH2, as assessed by trimethylation mark western analysis. MLL-AF9 leukemia cells were treated with SAH-EZH2 or SAH-EZH2_MUT (10 μM) twice daily for 7 days prior to analysis.

Figure 3. SAH-EZH2 induces cell cycle arrest and inhibits proliferation of MLL-AF9 leukemia cells

a) SAH-EZH2 (10 μM, twice daily), but not its mutant control, inhibits the proliferation of MLL-AF9 cells. Data represent mean ± s.e.m for independent experiments performed in triplicate. b) The anti-leukemic activity of SAH-EZH2 is not mediated by a pro-apoptotic effect, as evidenced by FACS analysis and Annexin V/7-AAD staining after 8 days of treatment with SAH-EZH2 peptides (10 μM, twice daily). c) Cell cycle analysis of SAH-EZH2-treated (10 μM, twice daily) cells. Cells were stained with BrdU-APC antibody and 7-AAD and analyzed by flow cytometry after 6 days of treatment. Data represent mean ± s.e.m for independent experiments performed in duplicate (see also Supplementary Table1). d) SAH-EZH2, but not its mutant control, upregulates the expression of p19ARF upon treatment of MLL-AF9 cells for 7 days (10 μM, twice daily), as assessed by western analysis. Full gel image in Supplementary Fig. 21

Figure 4. SAH-EZH2 induces monocyte/macrophage differentiation of MLL-AF9 leukemia cells

a) Suppression of colony-forming potential by SAH-EZH2 as compared to vehicle (water) and SAH-EZH2_MUT, p<0.02. MLL-AF9 cells were treated as described in Methods. Data represent mean and s.e.m for independent experiments performed in duplicate. b) Morphology of SAH-EZH2-treated MLL-AF9 leukemia cells. Whereas vehicle and SAH-EZH2_MUT -treated cells retain characteristic blastic features, SAH-EZH2-treated cells exhibit the morphology of monocyte/macrophage differentiation. c) Flow cytometric analysis of macrophage-specific cell surface marker expression (F4/80) of SAH-EZH2-treated MLL-AF9 cells at 8 days (10 μM, twice daily). d) Comparative changes in cell surface marker gene expression in MLL-AF9 cells treated with vehicle (water), SAH-EZH2, or SAH-EZH2_MUT for 8 days (10 μM, twice daily). CT-value was processed as described in Methods and the transcripts that increased/decreased by more than 6 fold were plotted. e) Real Time qPCR analysis of macrophage/monocyte-specific marker expression. SAH-EZH2-treated (10 μM, twice daily for 8 days) cells exhibit increased expression of macrophage/monocyte specific markers. Data represent mean and s.e.m for independent experiments performed in duplicate. p_Adam8<0.11, p_Fcer1a<0.14, p_ACE< 0.29. p values were calculated based on data comparison between SAH-EZH2 and SAH-EZH2_MUT treatments. f) Decreased CD133 expression in SAH-EZH2-treated (10 μM, twice daily for 8 days) MLL-AF9 leukemia cells, p_CD133<0.015. p values were calculated based on data comparison between SAH-EZH2 and SAH-EZH2_MUT treatments.

Figure 5. Cell Viability Effects of SAH-EZH2 and GSK126

a) Dose-responsive effects of SAH-EZH2 and GSK126 treatment on MLL-AF9 leukemia cell viability. Data represent mean ± s.e.m. for experiments performed in biological triplicates and technical duplicates. Cells were treated with SAH-EZH2 (twice daily) and GSK126 (single dose, as reported) in serum-containing media for 7 days. b) Effects of vehicle (water), SAH-EZH2, and GSK126 treatments on EZH2 protein level in MLL-AF9 cells. Cells were treated with SAH-EZH2 (twice daily) and GSK126 (single dose, and replenished at cell split due to confluency, as reported) for 7 days. Full gel image in Supplementary Fig. 21 c) Cell surface marker expression of MLL-AF9 cells treated with SAH-EZH2 and GSK126 as above for 8 days. Data represent mean ± s.e.m for independent experiments performed in biological and technical duplicates. d) Dose-responsive effects of SAH-EZH2 and GSK126 treatment on EZH2 wild-type (OCI-LY19) and EZH2 mutant (Karpas422) B-cell lymphoma cell lines. Cells were treated with SAH-EZH2 twice daily and GSK126 at the indicated doses for 12 days. Cells were split at days 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. e) Effect of combination treatment at the indicated doses with SAH-EZH2 and GSK126 in MLL-AF9 and Karpas422 cells. Calcusyn analysis revealed ED₅₀ combination indices (CI) of 0.11 and 0.74 for MLL-AF9 and Karpas422 cells, respectively (CI<1 indicates synergy). Cells were treated twice daily with SAH-EZH2 and single dose GSK126 (as reported) at the indicated concentrations for 7 days. Data represent mean ± s.e.m for experiments performed in biological triplicate.