EZH2; Hippel-Lindau (VHL) or cereblon (CRBN)

YM181 and YM281 Degraded the EZH2 Protein through the VHL-Dependent Ubiquitin–Proteasome System [1]
As shown in Figure 3A, YM181 and YM281 abrogated both the EZH2 protein level and the H3K27me3 degree in a concentration-dependent manner in 24 h, and moreover had no significant effect on the protein level of EZH1 that is homologous to EZH2 (Figures 3B and S2). The maximum degradation efficacy reached 80% at a concentration of 2 μM. A time-course study demonstrated that the EZH2 degradation could be detected in 2 h, and this effect reached a maximum in 4 h (Figure 3C). It is reported that the PRC2 complex collapse in the absence of any core subunit. To test the influence of EZH2 degraders on the PRC2 complex, the protein levels of the other two subunits, EED and SUZ12, were examined in both SU-DHL-2 and 22Rv1 cells treated with YM181 at 2 μM (Figure 3D). Within 24 h, YM181 substantially reduced the levels of EED and SUZ12. The results demonstrated that YM181 and YM281 rapidly deleted the EZH2 protein and consequently destabilized the PRC2 complex due to the loss of integrity. (25,37,38) Alternatively, it is also possible that EED and/or SUZ12 in proximity to EZH2 may be ubiquitinated by the EZH2-PROTAC-mediated ternary complex formation In the following experiments, as shown in Figure 3E, the degradation effect of YM181 could be rescued by adding either a proteasome inhibitor MG132 or MLN-4924, an inhibitor of the neddylation that is essential to activate the VHL E3 ligase system. Meanwhile, the EZH2 inhibitor EPZ6438 competed with YM181 to occupy the catalytic pocket and subsequently prevented protein degradation. Furthermore, the addition of a synthetic VHL ligand VH032 also reversed the EZH2 degradation caused by YM181. To further confirm the requirement of VHL for YM181-induced EZH2 degradation, VHL was knocked down by siRNAs in 22Rv1 cells. The exposure of the VHL knockdown cells to YM181 did not reduce the EZH2 protein level that was largely degraded in the control cells (Figure 3F). Additionally, Co-IP experiments clearly displayed a significant increase of EZH2 ubiquitination mediated by both YM181 and YM281, while the whole protein level was decreased within 12 h (Figure 3G). These results confirmed that the degradation of EZH2 induced by the two compounds was indeed associated with the VHL-dependent ubiquitin–proteasome system.

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EZH2 Degraders Displayed Stronger Anticancer Effects than EPZ6438 in Lymphoma Cell Lines [1]
Given that EZH2 inhibitors are reportedly effective in the intervention of DLBCL, anticancer effects of EZH2 degraders were first tested in three DLBCL cell lines, SU-DHL-2, SU-DHL-4, and SU-DHL-6. The MTS assays confirmed that both YM181 and YM281 caused cell viability to decrease stronger than EPZ6438 in all three cell lines (Figure 4A). YM181 and YM281 could induce nearly complete cell viability inhibition compared to EPZ6438, although their initial effective concentrations were slightly higher. Meanwhile, EPZ6438 only achieved half inhibition in SU-DHL-2 and SU-DHL-4 cells and 70% inhibition in SU-DHL-6. Moreover, all of the other lymphoma cell lines that were resistant to the EZH2 inhibitor also showed a complete response to the EZH2 degraders (Figure S3A). Western blot analysis further confirmed that YM181 induced substantial EZH2 degradation (Figures 4B and S3B,C). EPZ6438 robustly reduced the H3K27me3 levels at low nanomolar concentrations, while YM181 required high doses. The installment of both the linker and the VHL ligand motif might cause an EZH2 inhibitory activity loss, explaining the reason why EZH2 degraders needed a higher effect-starting concentration. To study whether the linker and the VHL ligand warhead would bring off-target effects, we also evaluated the cytostatic effects of V6 and V7 that are structurally similar but do not induce EZH2 degradation. Neither V6 nor V7 induced significant cell viability inhibition (Figure S3D,E). Meanwhile, we synthesized YM620, an isomer of YM281, with alterations of two stereocenters in hydroxyproline, which diminish the binding affinity to VHL (Figure S3F). Indeed, YM620 did not have an apparent effect on the EZH2 protein level, while it substantially inhibited EZH2 enzymatic activity (Figure S3G). Compared to YM281, YM620 had a weaker antiproliferative capacity in two tested cancer cell lines (Figure S3H). All of these data excluded the off-target effects of our EZH2 degraders.

EZH2 Degraders Reduced Tumor Growth In Vivo and Decreased Cell Viability in Primary Lymphoma Patient Cells [1]
To investigate the antitumor activity of EZH2 degraders in vivo, a xenograft mouse model of DLBCL cell line SU-DHL-6 was first conducted. Consistent with in vitro results, YM281 (80 mg/kg) administered by intraperitoneal injection 6 times weekly for 3 weeks remarkably suppressed the tumor volume (Figure 5A). However, EPZ6438 at the equal molar dose to YM281 (42.5 mg/kg) failed. Neither EPZ6438 nor YM281 caused significant weight loss in mice (Figure S5A). Western blot analysis of tumor tissue showed that YM281 significantly reduced the EZH2 protein and H3K27me3 levels (Figure 5B). EPZ36438 did not prevent the tumor growth, although it substantially decreased the H3K27me3 level, indicating that the enzymatic inhibition of EZH2 might not be enough to attenuate lymphoma cells in vivo. The significant destabilization of EZH2 and inhibition of tumor proliferation (indicated by Ki67 staining) induced by YM281 was further confirmed by immunohistochemistry in the tumor slices (Figure 5C). The in vivo anticancer efficacy in DLBCL was validated in the mice xenograft model of Jeko-1, a mantle cell lymphoma cell line (Figures 5D–F and S5B). More importantly, the tumor weight was clearly associated with the EZH2 protein level in YM281 treated mice (Figure S5C,D). Finally, to evaluate the potential clinical implication of EZH2 degraders that showed promising efficacy in all different lymphoma cell lines, as shown in Figure S3A, primary lymphoma cells extracted from various lymphoma patient samples were used for further tests. First, EZH2 degrader YM281 induced dose-dependent EZH2 degradation in primary cells from one DLBCL patient (Figure 5G and Table S1). Furthermore, lymphoma primary cells from 11 cases of lymphoma patients, including two cases of BLBCL, were tested for the efficacy of YM281. Compared to EPZ6438, YM281 increased the activity of caspase-3 and -7 and meanwhile reduced the cell viability observed in adenosine triphosphate (ATP) assays (Figure 5H,I).

Cell Viability Assay [1]
Cell viability was determined by the MTS assay, as we reported previously.

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Cell Cycle and Apoptosis [1]
The cell cycle was measured by flow cytometry according to the commercial cell cycle analysis kit. Annexin-V and propidium iodide-based apoptosis analyses were measured by flow cytometry according to the commercial cell apoptosis analysis kit (BD, Franklin Lakes, NJ). The activity of Caspase-3/7 was evaluated by the Caspase-Glo 3/7 Assay through the manufacturer’s instructions.

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Measurement of Cellular ATP [1]
The cellular ATP concentration was detected using an ATP-based CellTiter-Glo Luminescent Cell Viability Kit according to the manufacturer’s instructions.

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Western Blot [1]
The cells were washed with phosphate-buffered saline (PBS) and then lysed in lysis buffer with a protease inhibitor cocktail. After the protein concentration normalization using a bicinchoninic acid (BCA) protein assay, the samples were separated by standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to poly(vinylidene fluoride) (PVDF) membranes. Subsequently, the membranes were blocked with 5% milk for 1 h at r.t. before blotting with the indicated first antibodies overnight at 4 °C. After being washed with TBST (Tris-buffered saline containing 0.1% Tween-20), the membranes were probed with the horseradish peroxidase-conjugated secondary antibodies for 1 h at r.t. Enhanced chemiluminescence was used for signal detection.

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Immunohistochemistry [1]
Tumors were harvested in formalin and dehydrated and embedded in paraffin. Tissue slides were then deparaffinized, hydrated, and rinsed in PBS. After being boiled in citrate buffer for 4–6 min for antigen retrieval, peroxide blocking was performed with 3% H2O2 at r.t. for 20 min. The sections were incubated with EZH2, H3K27me3, and Ki67 (Abcam), following the suggested concentration at 37 °C overnight. Then, the samples were probed with a secondary antibody at 37 °C for 1 h. All slides were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin.

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Immunoprecipitation [1]
The cells were harvested using NP40 containing a protease inhibitor cocktail. After 30 min, cell lysates were centrifuged at 4000g for 15 min at 4 °C, and the supernatants were incubated with EZH2 antibody or IgG overnight at 4 °C. Subsequent incubation with protein A/G-coated agarose beads continued for an additional 3 h at 4 °C. After the samples were washed six times with ice-cold NP40, the supernatants were removed by centrifugation at 800g for 2 min. The proteins were then separated from the beads using immunoblotting loading buffer for 5 min at 95 °C. The supernatants were collected for subsequent immunoblotting analysis after SDS gel separation.

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Caco-2 Cell Permeability Assay [1]
The Caco-2 cells were seeded onto polycarbonate 12-well Transwell filters at a density of 2 × 105 cells/well. The confluent monolayers obtained at 21 days were utilized to assess the in vitro permeability. Culture media in the apical and basolateral compartments were replaced every 2 days, and the integrity of the monolayer was detected by fluorescein. Before the experiments, the culture medium in both chambers was replaced with prewarmed Hank’s balanced salt solution (HBSS). The cultures were then stabilized at 37 °C for 30 min. For the permeation studies, 0.2 mL of drug formulation diluted with HBSS was added to the apical side, and the basolateral side was replaced with 1 mL fresh HBSS. The treated cells were incubated at 37 ± 0.5 °C. The amount of permeated drug was determined by collecting 50 μL of samples from the basolateral compartment, followed by replacement with 50 μL of fresh HBSS at 1, 2, and 3 h. The collected samples were evaporated and reconstituted with methanol, then the concentrations of drugs in the samples were determined by liquid chromatography–mass spectrometry (LC-MS). The apparent permeability (Papp) of ETP in various formulations was calculated as follows: where dQ/dt is the slope of the cumulative drug permeated versus time (μg/s), A is the surface area of the monolayer (1.12 cm2), V is the volume of basolateral side HBSS (1 mL), and C0 is the initial concentration of compounds on the apical side (μg/mL).

The animal experiment was conducted in compliance with a protocol approved by the Institutional Animals Care and Use Committee of Sun Yat-sen University Cancer Center and was carried out in the Center of Experiment Animal of Sun Yat-sen University (North Campus, approval no.: L102012019050K). Balb/c nude mice (female) were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. For SU-DHL-6 xenografts, and five million cells were injected subcutaneously. After 2 weeks, when tumors reached 100–200 mm3, mice were randomly divided into three groups (6 mice per group) and administrated with a vehicle control (80% PBS, 10% castor oil, and 10% DMSO) or indicated doses of compounds (YM281: 80 mg/kg; EPZ6438: 42.5 mg/kg) through intraperitoneal injection 6 times weekly. Tumor sizes and animal weights were measured 2–3 times per week. The mice were sacrificed after 3 weeks’ drug administration, and the tumor tissue was harvested for analyses. For Jeko-1 xenografts, two million cells were injected subcutaneously. Ten days later, most of the tumors grow about 100 mm3, mice were then randomly divided into three groups (5 mice per group), and intraperitoneally administrated daily with the control (70% PBS, 20% castor oil, and 10% DMSO) or indicated doses of compounds (YM281: 100 mg/kg; EPZ6438: 50 mg/kg). Tumor sizes and animal weights were measured every 3 days. The mice were sacrificed after 30 days’ drug treatment, and then the tumor tissue was harvested for analyses.[1]

[1]. Design, Synthesis, and Evaluation of VHL-Based EZH2 Degraders to Enhance Therapeutic Activity against Lymphoma. J Med Chem. 2021 Jul 22;64(14):10167-10184.

Traditional EZH2 inhibitors are developed to suppress the enzymatic methylation activity, and they may have therapeutic limitations due to the nonenzymatic functions of EZH2 in cancer development. Here, we report proteolysis-target chimera (PROTAC)-based EZH2 degraders to target the whole EZH2 in lymphoma. Two series of EZH2 degraders were designed and synthesized to hijack E3 ligase systems containing either von Hippel-Lindau (VHL) or cereblon (CRBN), and some VHL-based compounds were able to mediate EZH2 degradation. Two best degraders, YM181 and YM281, induced robust cell viability inhibition in diffuse large B-cell lymphoma (DLBCL) and other subtypes of lymphomas, outperforming a clinically used EZH2 inhibitor EPZ6438 (tazemetostat) that was only effective against DLBCL. The EZH2 degraders displayed promising antitumor activities in lymphoma xenografts and patient-derived primary lymphoma cells. Our study demonstrates that EZH2 degraders have better therapeutic activity than EZH2 inhibitors, which may provide a potential anticancer strategy to treat lymphoma.[1]

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In this work, the therapeutic efficacy of EZH2 inhibitors was limited to DLBCL cell lines among the tested lymphoma cell lines (Figure 1A). We wonder whether the direct EZH2 degradation via PROTAC technology could be developed to improve their targeting capacity to the other types of lymphoma cells. In our current study, we developed PROTAC-based EZH2 degraders and investigated their efficiency of EZH2 degradation and therapeutic efficacy in various types of lymphoma in vitro and in vivo. Our study revealed that only VHL-targeting compounds enabled the EZH2 degradation with an appropriate linker at 7 or 9 atoms length. Compared to the parental EZH2 inhibitor EPZ6438, our two best EZH2 degraders YM181 and YM281 selectively degraded EZH2 over EZH1, and they exhibited effective antiproliferative activity both in DLBCL and other types of lymphoma cell lines. Furthermore, the EZH2 degrader showed an apparent advantage to prevent in vivo tumor growth in lymphoma xenografts without obvious toxicity at the efficacious doses.
However, the incomplete EZH2 degradation and the modest cellular potencies for YM281 and YM181 in the inhibition of cell viability at low concentrations suggest that there is still room for further optimization. In the future, a structure and activity relationship study on different linker scaffoldings with the same linking length as YM181 and YM281 may be warranted to obtain more potent EZH2 degraders. Meanwhile, cancer cells that may not depend on EZH2 for their tumorigenesis, for example, pancreatic cancer cell AsPC1 and lung cancer cell NCI-H460, are not sensitive to YM181 and YM181, although the compounds were able to decrease their EZH2 levels (Figure S6). By measuring cell permeability with Caco-2 cells, both YM181 and YM281 showed their apparent permeability ability largely compromised compared to the parental EHZ2 inhibitor EPZ6438 (Table S2), indicating the importance to improve their oral bioavailability through further structural optimization. Overall, our results demonstrate that EZH2 degraders may have better therapeutic potential than EZH2 inhibitors against lymphomas. The exact mechanism of action and the application of our EZH2 degraders in other cancers are under investigation.[1]

C34H43N3O7

605.721129655838

605.31

2750350-39-9

166176920

Yellow to brown solid powder

4.5

3

8

14

44

1050

0

C(O)(=O)COCCCOC1=CC=C(C2=CC(N(CC)C3CCOCC3)=C(C)C(C(NCC3=C(C)C=C(C)NC3=O)=O)=C2)C=C1

CLDOGTJZRUKKBD-UHFFFAOYSA-N

InChI=1S/C34H43N3O7/c1-5-37(27-11-15-42-16-12-27)31-19-26(25-7-9-28(10-8-25)44-14-6-13-43-21-32(38)39)18-29(24(31)4)33(40)35-20-30-22(2)17-23(3)36-34(30)41/h7-10,17-19,27H,5-6,11-16,20-21H2,1-4H3,(H,35,40)(H,36,41)(H,38,39)

2-[3-[4-[3-[(4,6-dimethyl-2-oxo-1H-pyridin-3-yl)methylcarbamoyl]-5-[ethyl(oxan-4-yl)amino]-4-methylphenyl]phenoxy]propoxy]acetic acid

Tazemetostat de(methylene morpholine)-O-C3-O-C-COOH; 2750350-39-9;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 160 mg/mL (264.15 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)