YAP/TEAD interaction

In vitro, CA3 significantly reduces the growth of esophageal adenocarcinoma cells. Inducing apoptosis, decreasing tumor sphere formation, and reducing the number of ALDH1+ cells are all possible effects of CA3. After cotransfection of each transcriptional factor's individual promoter luciferases in 293T cells, CA3 specifically inhibits Tead/YAP1 transcriptional activity but has no effect on Super-TOP/Wnt, CBF1/Notch, or AP-1. The CSC properties that are enriched in radiation-resistant esophageal adenocarcinoma cells are preferentially inhibited by CA3[1].

In a xenograft model, CA3 exhibits potent antitumor activity with no discernible toxicity[1].

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CA3 exerts strong antitumor effects in inducible high YAP xenograft mouse model in vivo[1]
To further confirm the antitumor effects of CA3 result from targeting YAP1 in vivo, we utilized inducible YAP1 high SKGT-4 (Dox+) cells xenograft model. Nude mice were implanted with Dox− and Dox+ SKGT-4 cells at 1 × 106 cells per mouse. At this concentration, we observed that only Dox+ SKGT-4 cells were able to form tumors after 10–14 days of injection (Fig. 5B), indicating that YAP1 is necessary to drive tumor growth in vivo. To determine the effects of treatment with CA3 in inhibition of YAP-inducible tumor growth in vivo, we randomly placed mice bearing Dox+ SKGT-4 EAC xenografts in two groups and then gave them treatment with control phosphate-buffered saline or CA3 at 1 mg/kg. At the end of our 3-week dosing schedule, SKGT-4 xenograft tumor weights and volumes and the mice’s body weights were measured. Results from in vivo SKGT-4 Dox+ xenograft model demonstrated that mice with Dox+ SKGT-4 xenografts treated with CA3 greatly reduced tumor sizes and weights in vivo (Fig.5B&5C), whereas tumors did not form in mice implanted with Dox− SKGT-4 cells over the whole experimental period (Figure 5B). Mice’s body weights did not differ significantly between CA3 treatment and control group (Fig 5D). In addition, immunohistochemistry further confirmed in mice tumor tissues that expression of YAP1, SOX9 and KI67 was greatly reduced in mice with SKGT-4 Dox+ treated with CA3 (Fig. 5E). Thus, CA3 effectively suppress EAC tumor growth in vivo and these effects owe, at least in part, to inhibition of the stemness genes YAP1 and SOX9.

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CA3 synergizes with 5-FU in inhibiting growth of EAC cells in vitro and in vivo[1]
To determine the effects of treatment with CA3 alone or in combination with 5-FU on inhibition of the growth of EAC cell lines, we first seeded four EC cell lines with constitutive high YAP1 expression (SKGT-4, JHESO, OACP, and YES-6) in 96-well plates and treated them with CA3 alone, 5-FU alone, or the combination of CA3 and 5-FU at indicated concentrations. The results shown in Fig. 6A demonstrated that although CA3 produced dose-dependent decreases in the growth of these four cell lines, the combination of CA3 and 5-FU significantly synergized to inhibit their growth especially in the combination of CA3 and 5-FU at the maximum extent. To further examine the inhibition of CA3 on EAC cells are YAP1 dependent, we treated Dox+ (YAP1 induced) and Dox− SKGT4 (PIN20YAP1) cells with CA3 alone, 5-FU alone, or the combination, we found that CA3 alone preferentially inhibited the growth of YAP1 high SKGT-4 cells (Dox+) compared to YAP1 low SKGT-4 cells (Dox−) and in a dose dependent manner (Fig. 6B). Moreover, treatment with the combination of CA3 and 5-FU produced the greatest inhibition of growth of both Dox+ and Dox− SKGT-4 cells (Fig.6B). These findings indicated that treatment with CA3 synergize with 5-FU in GAC cell growth inhibition.

The SOX9 luciferase reporter was described previously. The 5 × -UAS-luciferase reporter and Gal4-TEAD4 constructs, also described previously, were obtained from Dr. Johnson of MD Anderson Cancer Center. Transient cotransfection esophageal adenocarcinoma cells with the SOX9 luciferase reporter and a Renilla vector or 5 × -UAS-luciferase reporter and Gal4-TEAD4 with a CMV-β-gal construct was performed as described previously.

SKGT-4 and JHESO cells are seeded onto 6-well plates (1 × 105/well) in DMEM and cultured for 24 hours to allow for cell attachment. The cells are then exposed for 48 hours to 0.1% DMSO (control) or CA3 at various doses as recommended. The cells are then collected, fixed with methanol, washed, treated with RNase A, stained with propidium iodide for DNA, and subjected to flow cytometry analysis of DNA histograms and cell-cycle phase distributions.

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Cell proliferation assay[1]
The EAC cells and their resistant counterparts were treated with 0.1% dimethyl sulfoxide (control),CA3 at different doses For combination treatment experiments, treatment of the cells with CA3, 5-FU, or a combination at different concentrations was administered for 6 days as indicated, and the cell viability was assessed using an MTS assay as described previously. All assays were performed in triplicate and repeated at least three times.

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Flow cytometry and apoptotic analysis[1]
Analysis of EAC cell apoptosis using flow cytometry was performed as described previously. In brief, SKGT-4 and JHESO cells were seeded onto six-well plates (1 × 105 per well) in Dulbecco’s modified Eagle’s medium and cultured for 24 hours to allow for cell attachment. The cells were then treated with 0.1% dimethyl sulfoxide(control) or CA3 at different doses as indicated for 48 hours. Next, the cells were harvested, fixed with methanol, washed, treated with RNase A, and stained for DNA with propidium iodide, and their DNA histograms and cell-cycle phase distributions were analyzed using flow cytometry with a FACS Calibur instrument.

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Protein extraction and Western blot analysis[1]
Proteins were isolated from EAC cells treated with CA3 as indicated and analyzed using Western blotting as described previously.

In vivo xenograft mouse model[1]
In vivo experiments have been conducted in accordance with an Institutional Animal Care and Use Committee (IACUC). SKGT-4 (PIN20YAP1) cells (1 × 106) without (Dox−) or with (Dox+) YAP1 induction by Doxycycline were inoculated into nude mice (n = 5/group). The mice in the Dox+ group were fed drinking water containing 2.5% sucrose and 2.5% Doxycycline, whereas those in the Dox− group were fed water containing only 2.5% sucrose. After 10 days, CA3 was introperitoneally injected into the animals in the Dox+ group at 1 mg/kg/mouse three times a week for total 3 weeks.

In a JHESO xenograft model of EAC, 2 × 106 JHESO cells were subcutaneously injected into nude mice (n = 5/group). After about 10 days, the mice underwent intraperitoneal injection of CA3 at 1 mg/kg/mouse, 5-FU at 30 mg/kg/mouse, or a combination of them three times a week for total 3 weeks. A control group was given phosphate-buffered saline at 100 µl/mouse. The mice’s tumor volumes, tumor weights, and body weights were measured as described previously (4). All measurements were compared using an unpaired Student t-test.

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PBS; 1 mg/kg; i.p.

JHESO xenograft mice model of esophageal adenocarcinoma

[1]. Mol Cancer Ther . 2018 Feb;17(2):443-454.

Mounting evidence suggests that the Hippo coactivator Yes-associated protein 1 (YAP1) is a major mediator of cancer stem cell (CSC) properties, tumor progression, and therapy resistance as well as often a terminal node of many oncogenic pathways. Thus, targeting YAP1 may be a novel therapeutic strategy for many types of tumors with high YAP1 expression, including esophageal adenocarcinoma. However, effective YAP1 inhibitors are currently lacking. Here, we identify a small molecule (CA3) that not only has remarkable inhibitory activity on YAP1/Tead transcriptional activity but also demonstrates strong inhibitory effects on esophageal adenocarcinoma cell growth especially on YAP1 high-expressing esophageal adenocarcinoma cells both in vitro and in vivo Remarkably, radiation-resistant cells acquire strong cancer stem cell (CSC) properties and aggressive phenotype, while CA3 can effectively suppress these phenotypes by inhibiting proliferation, inducing apoptosis, reducing tumor sphere formation, and reducing the fraction of ALDH1+ cells. Furthermore, CA3, combined with 5-FU, synergistically inhibits esophageal adenocarcinoma cell growth especially in YAP1 high esophageal adenocarcinoma cells. Taken together, these findings demonstrated that CA3 represents a new inhibitor of YAP1 and primarily targets YAP1 high and therapy-resistant esophageal adenocarcinoma cells endowed with CSC properties. Mol Cancer Ther; 17(2); 443-54. ©2017 AACR.

C23H27N3O5S2

489.61

489.139

C, 56.42; H, 5.56; N, 8.58; O, 16.34; S, 13.10

300802-28-2

654092

White to off-white solid powder

1.5±0.1 g/cm3

741.7±70.0 °C at 760 mmHg

402.4±35.7 °C

0.0±2.6 mmHg at 25°C

1.714

3.66

1

8

4

33

870

0

S(C1C=CC2=C(/C(/C3C=C(C=CC2=3)S(N2CCCCC2)(=O)=O)=N/O)C=1)(N1CCCCC1)(=O)=O

XYZXEEIUKQGUHB-UHFFFAOYSA-N

InChI=1S/C23H27N3O5S2/c27-24-23-21-15-17(32(28,29)25-11-3-1-4-12-25)7-9-19(21)20-10-8-18(16-22(20)23)33(30,31)26-13-5-2-6-14-26/h7-10,15-16,27H,1-6,11-14H2

N-[2,7-bis(piperidin-1-ylsulfonyl)fluoren-9-ylidene]hydroxylamine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: 2.5 mg/mL (5.11 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.25 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.25 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5% DMSO+40% PEG 300+5% Tween 80+50% ddH2O: 0.5mg/ml

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