A covalently bound inhibitor triggers EZH2 degradation through CHIP-mediated ubiquitination

Abstract

Enhancer of zeste homolog 2 (EZH2) has been characterized as a critical oncogene and a promising drug target in human malignant tumors. The current EZH2 inhibitors strongly suppress the enhanced enzymatic function of mutant EZH2 in some lymphomas. However, the recent identification of a PRC2- and methyltransferase-independent role of EZH2 indicates that a complete suppression of all oncogenic functions of EZH2 is needed. Here, we report a unique EZH2-targeting strategy by identifying a gambogenic acid (GNA) derivative as a novel agent that specifically and covalently bound to Cys668 within the EZH2-SET domain, triggering EZH2 degradation through COOH terminus of Hsp70-interacting protein (CHIP)-mediated ubiquitination. This class of inhibitors significantly suppressed H3K27Me3 and effectively reactivated polycomb repressor complex 2 (PRC2)-silenced tumor suppressor genes. Moreover, the novel inhibitors significantly suppressed tumor growth in an EZH2-dependent manner, and tumors bearing a non-GNA-interacting C668S-EZH2 mutation exhibited resistance to the inhibitors. Together, our results identify the inhibition of the signaling pathway that governs GNA-mediated destruction of EZH2 as a promising anti-cancer strategy.

Keywords: CHIP; EZH2; covalent inhibitor; oncoprotein; ubiquitination.

Figure 1. Screening and identification of gambogenic acid (GNA) to directly interact with EZH2

1. Immunofluorescence analysis demonstrated that a 12‐h treatment with 2 μM GNA decreased the abundance of endogenous EZH2 in the nuclei of HN‐6 head and neck cancer cells. Scale bar, 25 μm.

2. Immunofluorescence analysis demonstrated that biotinylated GNA (Bio‐GNA) co‐localized with EZH2 in the nuclei of mouse embryonic fibroblasts (MEFs). Scale bar, 25 μm.

3. In vitro binding assays coupled with immunoblot assays reveal that Bio‐GNA bound to EZH2 in the whole‐cell lysate derived from Cal‐27 head and neck cancer cells, whereas free, unconjugated GNA efficiently competed with Bio‐GNA to bind endogenous EZH2. After the cells were lysed to generate whole‐cell lysates, the indicated concentration of Bio‐GNA or free GNA was added to perform the in vitro binding assays.

4. Bio‐GNA (5 μM) binds to the recombinant C‐terminal portion of EZH2 in a time‐dependent manner.

Source data are available online for this figure.

Figure 2. EZH2 covalently binds to GNA and its derivatives

1. Recombinant wild‐type (WT) C‐terminal EZH2 and its indicated mutants (C688S, C699S, and C647S) were incubated with 5 μM Bio‐GNA in vitro for 1 h followed by immunoblotting with antibodies against biotin and EZH2.

2. Full‐length WT and the C668S mutant form of EZH2 (bottom panel) as well as full‐length and the S664C mutant form of EZH1 (upper panel) were incubated with 1 μM Bio‐GNA in vitro for 1 h followed by immunoblotting with antibodies against biotin and EZH2.

3. The MALDI‐TOF‐MS analysis illustrates the direct interaction between GNA and EZH2.

4. Immunoblotting assays revealed that Bio‐GNA binds to EZH2 in whole‐cell lysates derived from Cal‐27 and UMSCC12 head and neck cancer cells, whereas free GNA and GNA002 competed with Bio‐GNA to bind EZH2.

5. The octet assay indicated that GNA and GNA002 could compete with Bio‐GNA to bind the bacterially purified recombinant His‐EZH2‐SET domain. All experiments were performed in triplicate. The data are presented as the mean ± SD (n = 3).

Source data are available online for this figure.

Figure 3. The protein stability of PRC2 complex components and H3K27Me3 are decreased by GNA derivatives

1. Immunoblotting analysis demonstrated that long‐term (48 h) incubation of both GNA002 and GSK126 significantly reduced H3K27Me3 levels in Cal‐27 head and neck cancer cells. However, GNA002, but not GSK126, led to a significant reduction in EZH2 abundance.

2. Treatment with GNA002 for 24 h did not reduce EZH2 mRNA levels in either Cal‐27 or HN‐4 head and neck cancer cells. The data are presented as the mean fold change of expression ± SEM (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analysis was performed using one way ANOVA method.

3. Treatment with the 26S proteasome inhibitor MG132 (5 μM) reversed GNA002 (2 μM)‐induced reduction of EZH2 protein levels in Cal‐27 cells.

4. GNA002 increased the ubiquitination of ectopically expressed, Flag‐tagged wild‐type EZH2, but not the non‐GNA‐interacting C668S mutant form of EZH2, as detected by the anti‐ubiquitin antibody of the Flag immunoprecipitates recovered from HEK293 cells that were transfected with the indicated plasmids.

5. GNA002 treatment for 24 h decreased the association between EZH2 and EED within the PRC2 complex in Cal‐27 cells.

6. Immunoblotting assays demonstrated that GNA002 treatment (24 h) reduced the abundance of PRC complex components and H3K27Me3 in Cal‐27 cells in a dose‐dependent manner.

7. Cal‐27 cells were treated with 2 μM GNA002 for 24 h and subject to ChIP assays to determine the H3K27Me3 status within the promoter regions of characterized EZH2 gene targets, such as CB1, miR‐200C, and miR‐200C. The experiments were performed in triplicate. The data are presented as the mean ± SD (n = 3).

Source data are available online for this figure.

Figure 4. EZH2 protein levels are decreased largely through the E3 ubiquitin ligase CHIP

1. Immunofluorescence analysis indicated that EZH2 and CHIP proteins co‐localized in the nuclei of HN‐6 head and neck cancer cells. Scale bar, 75 μm.

2. Detection of the endogenous interaction between EZH2 and CHIP by co‐immunoprecipitation in Cal‐27 head and neck cancer cells.

3. Immunoblotting analysis demonstrated that the depletion of endogenous CHIP using two independent lentiviral shRNA constructs led to elevated basal EZH2 levels and resistance to GNA002‐induced EZH2 degradation in UMSCC‐12 head and neck cancer cells.

4. Ectopic expression of CHIP promoted the ubiquitination of WT‐EZH2, but not the non‐GNA‐interacting C668S mutant EZH2, only when challenged with GNA002 in HEK293 cells for 24 h.

5. Immunoblotting analysis to monitor changes in endogenous EZH2 abundance following the lentiviral shRNA‐mediated depletion of endogenous CHIP, Smurf1, or Smurf2 in UMSCC‐12 cells.

6. Depletion of endogenous CHIP, but not endogenous Smurf2, conferred resistance to EZH2 degradation induced by a 48‐h GNA002 treatment in UMSCC‐12 cells.

Source data are available online for this figure.

Figure 5. EZH2 degradation induced by GNA derivatives suppresses in vivo tumor growth

1. Nude mice bearing Cal‐27 xenograft tumors were orally administered GNA002 (p.o., 100 mg/kg, once per day, n = 10), GSK126 (i.p., 50 mg/kg, once per day, n = 10), cisplatin (i.p., 5 mg/kg, once per week, n = 10), or vehicle control (p.o., once per day, n = 10). Tumor sizes were measured daily, converted to tumor volume and plotted against days of treatment. The data are presented as the mean percentage change of tumor volume ± standard error of the mean (SEM). *P < 0.05, **P < 0.01.

2. Representative images of the xenografted tumors after vehicle or GNA002 treatments.

3. Body weights of the treated mice from (A) were recorded daily after the indicated treatments. The data are presented as the mean ± standard error of the mean (SEM). *P < 0.05.

4. Oral GNA002 treatment (100 mg/kg) for 4 weeks led to a decrease in H3K27Me3 levels in the collected Cal‐27 xenografted tumors.

5. Immunoblotting assays indicated that GNA002 incubation for 48 h not only reduced H3K27Me3 levels as GSK126 did, but also significantly reduced the EZH2 protein abundance in the wild‐type EZH2‐expressing lymphoma Daudi cells.

6. Nude mice bearing Daudi xenograft tumors were orally administered GNA002 (p.o., 100 mg/kg, once per day, n = 10) or vehicle control (p.o., once per day, n = 10). Tumor sizes were measured every 3 days. The data are presented as the mean percentage change of tumor volume ± SEM. *P < 0.05.

7. Immunoblotting assays indicated that GNA002 incubation for 48 h not only reduced H3K27Me3 levels as GSK126 did, but also significantly reduced mutant EZH2 protein expression in Pfeiffer cells that harbor gain‐of‐function EZH2 mutation.

8. Nude mice bearing Pfeiffer xenograft tumors were orally administered GNA002 (p.o., 100 mg/kg, once daily, n = 10) or vehicle control (p.o., once daily, n = 10). Tumor sizes were measured every 3 days. The data are presented as the mean percentage change of tumor volume ± SEM. *P < 0.05, ***P < 0.001.

Source data are available online for this figure.

Figure 6. The non‐GNA‐interacting EZH2‐C668S mutant rescues GNA002‐induced reduction of tumor growth

1. The anchorage‐independent growth of WT‐EZH2‐expressing but not C668S‐expressing UMSCC‐12 head and neck cancer cells was retarded by 1 μM GNA002 treatment for up to 6 weeks.

2. Quantification of the results that were obtained in (A). The experiments were performed in triplicate. Mean ± SEM.

3. Depletion of endogenous CHIP in UMSCC‐12 cells partially conferred resistance to GNA002‐mediated suppression of anchorage‐independent growth at the end of 6 weeks.

4. Quantification of the results that were obtained in (C). The experiments were performed in triplicate. Mean ± SEM.

5. Nude mice bearing WT‐EZH2‐ or C668S‐EZH2‐expressing UMSCC‐12 xenografted tumors were orally administered GNA002 (p.o., 100 mg/kg, once daily, n = 10) or vehicle control (p.o., once daily, n = 10). The data are presented as the mean percentage change of tumor volume ± SEM. One way ANOVA analysis was used to assess the statistical significance. ***P < 0.001.

6. Nude mice bearing shGFP‐ or shCHIP‐expressing UMSCC‐12 xenograft tumors were orally administered GNA002 (p.o., 100 mg/kg, once daily, n = 10) or vehicle control (p.o., once daily, n = 10). The data are presented as the mean tumor volume ± SEM, One way ANOVA analysis was used to assess the statistical significance. ***P < 0.001.

7. A proposed model to illustrate the molecular mechanisms underlying GNA derivatives as a novel class of EZH2 inhibitors. GNA and GNA derivatives specifically interact with EZH2 but not other methyltransferases by forming a covalent bond between the C8 equivalent atom of the GNA derivatives and the S atom of the Cys668 residue in the SET domain of EZH2. Subsequently, GNA binding to EZH2 induces CHIP‐dependent polyubiquitination and the subsequent degradation of EZH2, thereby leading to a total termination of the EZH2 oncogenic functions in human cancer cells.