Efficacy and Biomarker Analysis of Adavosertib in Differentiated Thyroid Cancer

Simple Summary Adavosertib is a first-in-class Wee1 inhibitor that has demonstrated activity against certain cancers. We evaluated the effects of adavosertib in treating differentiated thyroid cancer (DTC) using four DTC cell lines (BHP7-13, K1, FTC-133, FTC-238). Adavosertib accumulated cells in the G2/M phase and induced apoptosis in four DTC cell lines. Baseline Wee1 levels correlated with adavosertib sensitivity. Single-agent adavosertib therapy was sufficient to inhibit the growth of K1 and FTC-133 tumor models. Adavosertib potentiated the anti-tumor effect of dabrafenib and trametinib in K1 xenografts harboring the BRAFV600E mutation, with promising safety profiles. Adavosertib also improved the anti-tumor efficacy of lenvatinib in FTC-133 xenografts. Our results suggest the clinical efficacy of adavosertib for DTC patient therapy. Abstract Differentiated thyroid cancer (DTC) patients are usually known for their excellent prognoses. However, some patients with DTC develop refractory disease and require novel therapies with different therapeutic mechanisms. Targeting Wee1 with adavosertib has emerged as a novel strategy for cancer therapy. We determined the effects of adavosertib in four DTC cell lines. Adavosertib induces cell growth inhibition in a dose-dependent fashion. Cell cycle analyses revealed that cells were accumulated in the G2/M phase and apoptosis was induced by adavosertib in the four DTC tumor cell lines. The sensitivity of adavosertib correlated with baseline Wee1 expression. In vivo studies showed that adavosertib significantly inhibited the xenograft growth of papillary and follicular thyroid cancer tumor models. Adavosertib therapy, combined with dabrafenib and trametinib, had strong synergism in vitro, and revealed robust tumor growth suppression in vivo in a xenograft model of papillary thyroid cancer harboring mutant BRAFV600E, without appreciable toxicity. Furthermore, combination of adavosertib with lenvatinib was more effective than either agent alone in a xenograft model of follicular thyroid cancer. These results show that adavosertib has the potential in treating DTC.


Interaction between Adavosertib and Targeted Therapies in DTC Cells
We analyzed the effects of adavosertib combined with sorafenib against DTC cells. The cytotoxic effects and IC 50 of sorafenib in four DTC cell lines were evaluated in this ( Figure S1) and prior studies [33], and the resulting data were employed for the combination therapy study of adavosertib and sorafenib. The combination therapy of adavosertib and sorafenib significantly improved cytotoxicity in the four DTC cell lines over single-agent therapy ( Figure 5A). Interactions between adavosertib and sorafenib were determined by calculating the combination index (CI) using the Chou-Talalay equation ( Figure 5B) [23,24]. The combination of adavosertib and sorafenib was synergistic in K1 (CI, 0.72-0.73), and ranged from synergistic to antagonist in BHP7-13, FTC-133, and FTC-238 (CI of 0.66-1.06, 0.97-1.02, and 0.53-1.07, respectively). The results demonstrate that the adavosertib and sorafenib combination was mostly synergistic in treating DTC cells.
We also assessed the therapeutic effects of the combination of adavosertib and lenvatinib in the four DTC cell lines by determining the dose-response curves and IC 50 of lenvatinib in the four DTC cell lines ( Figure S2A,B). The results were used to study the combination of adavosertib and lenvatinib, which had increased cytotoxicity over therapy with either agent alone ( Figure 5C). The Chou-Talalay Combination index was employed to evaluate the drug-drug interaction between adavosertib and lenvatinib and showed that the combination was synergistic in FTC-133 (CI, 0.43-0.67) and FTC-238 (CI, 0.53-0.96) and mostly synergistic in BHP7-13 and K1 (fraction affected ≥ 0.4) ( Figure 5D). The re-sults indicate that the adavosertib and lenvatinib combination was mainly synergistic in DTC cells.  BRAF V600E mutation is a common genetic alteration (45.7%) in PTC and appears less frequently (1.4%) in FTC [34]. It is associated with increased cancer-related mortality in PTC patients [35]. Dual BRAF and MEK inhibition therapy has emerged as a novel treatment alternative for patients with BRAF mutations; however, acquired resistance remains a concern [36]. The US FDA recently approved the combination of dabrafenib (a BRAF inhibitor) and trametinib (a MEK inhibitor) for treating BRAF V600E -mutated anaplastic thyroid cancer [37]. We assessed the therapeutic effects of adavosertib combined with dabrafenib and trametinib in K1 cells harboring the BRAF V600E mutation [38] by determining the dose-response curves and the IC 50 of dabrafenib and trametinib in K1 cells ( Figure S3A,B). The results were employed to study the triple combination of adavosertib and dabrafenib plus trametinib, which increased cytotoxicity over the single modality treatment ( Figure 5E). The triple combination treatment showed synergism in K1 cells (CI, 0.50-0.82) ( Figure 5F).

Adavosertib Therapy Shows Anti-Tumor Efficacy in DTC Xenograft Models
With the in vitro promising results, we examined the in vivo effect of adavosertib monotherapy and combination with targeted therapies. Nude mice were inoculated with K1 cells in the right flank. Once the tumors reached a mean diameter of 7.0 mm, the mice were treated with vehicle, adavosertib (50 mg/kg), sorafenib (40 mg/kg), adavosertib (50 mg/kg) and sorafenib (40 mg/kg), dabrafenib (30 mg/kg) plus trametinib (0.6 mg/kg), and the triple combination of adavosertib (50 mg/kg), dabrafenib (30 mg/kg), and trametinib (0.6 mg/kg) (n = 4 per group) ( Figure 6A). Adavosertib, sorafenib, and dabrafenib plus trametinib significantly reduced tumor growth versus vehicle treatment (p < 0.001 for both comparisons). The combination of adavosertib and sorafenib and the triple combination of adavosertib, dabrafenib, and trametinib did not significantly improve the therapeutic efficacy over sorafenib and dabrafenib plus trametinib (p = 0.998 and p = 0.997, respectively). However, the triple combination of adavosertib, dabrafenib, and trametinib resulted in complete remission in 25% (1 of 4) of the K1 tumors between days 18 and 21, at which point the study was closed due to the control animals being close to the humane endpoints. This 25% complete response rate demonstrates significant promise, and the effect was not observed in the other five treatment groups. There was no obvious weight loss following multiple treatments in the treatment groups during the entire study ( Figure 6B). A group of mice photographed on day 15 revealed tumor dimension reduction following the drug treatment ( Figure 6C). We performed additional experiments to confirm the therapeutic efficacy of triple combination of adavosertib, dabrafenib, and trametinib in K1 xenografts ( Figure S4A). Triple combination therapy led to complete tumor regression in 60% (3/5) of the K1 tumors between days 14 and 21 after two cycles of treatment. There was no obvious weight change between the dual and triple combination therapy ( Figure S4B). These data strengthen our primary observation that the triple combination of adavosertib, dabrafenib, and trametinib had robust therapeutic effects against K1 tumors.
Nude mice bearing FTC-133 tumors with a mean diameter of 5.7 mm were treated with vehicle (n = 6), adavosertib (50 mg/kg, n = 6), sorafenib (40 mg/kg, n = 5), and combination therapy (n = 6) daily for three cycles of 5-days-on and 2-days-off treatment ( Figure 7A). Adavosertib, sorafenib, and adavosertib plus sorafenib significantly retarded FTC-133 tumor growth as compared with the vehicle treatment (p < 0.001 for both comparisons). The combination therapy of adavosertib and sorafenib did not significantly repress FTC-133 tumor growth compared with either single-agent treatment. When the study was closed, there was no significant reduction in body weight with the adavosertib, sorafenib, or combination therapy compared with placebo treatment ( Figure 7B). Representative nude mice with FTC-133 tumors were photographed on day 21 ( Figure 7C).  We also investigated the in vivo effect of adavosertib and lenvatinib in the FTC-133 xenograft model. Nude mice, inoculated with FTC-133 cells in the right flank, were treated with vehicle, adavosertib (50 mg/kg), lenvatinib (30 mg/kg), and the combination of adavosertib (50 mg/kg) and lenvatinib (30 mg/kg) (n = 5 per group), daily, for three cycles of 5-days-on and 2-days-off treatments ( Figure 7D) once the tumors reached a mean diameter of 5.6 mm. Adavosertib, lenvatinib, and adavosertib plus lenvatinib significantly reduced tumor growth versus the control treatment (p = 0.002, p = 0.003, and p < 0.001, respectively). The combination of adavosertib and lenvatinib significantly improved the therapeutic efficacy versus adavosertib and lenvatinib alone (p < 0.001 and p = 0.011, respectively). There was no obvious weight loss in the treatment groups compared with the control group during the study period ( Figure 7E). A group of mice photographed on day 21 demonstrated tumor size reduction after the drug treatment ( Figure 7F).
To evaluate the molecular effects of a single adavosertib (50 mg/kg) treatment in K1 and FTC-133 xenografts ( Figure S5), we performed immunoblotting, which showed that p-CDK1 (Tyr15) was reduced by 4 h in the K1 and FTC-133 tumors. The p-CHK1 (Ser345) expression was reduced in the K1 (24 h), but increased in the FTC-133 tumor (4 h). p-H2AX (Ser139) levels were increased in the K1 (4 and 8 h) and FTC-133 (8 h) tumors. Proliferating cell nuclear antigen (PCNA) levels, a marker of cell proliferation, were not significantly changed in the K1 and FTC-133 tumors during the study period. Cleaved caspase-3 levels were enhanced in the K1 and FTC-133 (by 8 h) tumors.

Discussion
In this study, we aimed at evaluating the therapeutic efficacy of adavosertib monotherapy and the combination of adavosertib and kinase inhibitors against DTC. Adavosertib efficiently reduced cell viability in four DTC cell lines, and adavosertib monotherapy significantly inhibited xenograft growth in PTC and FTC tumor models. Adavosertib potentiated the therapeutic effects of dabrafenib and trametinib in the K1 tumor model, without appreciable toxicity. The complete response rate of 25-60% in K1 xenografts in the triple combination therapy group compared with 0% in the other treatment groups is also notable. Adavosertib also improved the antitumor effect of lenvatinib in the FTC-133 model. These study findings suggest that adavosertib has the potential for treating patients with DTC.
Adavosertib accumulated the DTC cells in the G2/M phase, which might be one of the drug's treatment mechanisms for DTC. Adavosertib inhibited DTC cells in the G2 phase (instead of the M phase) in four DTC cell lines. Adavosertib induced a higher proportion of cells in the G2/M phase but did not increase the proportion of mitotic cells, demonstrating that adavosertib accumulates DTC cells in the G2 phase. CDK1 activation is essential for commitment to mitosis, and CDK1 activity is regulated by Wee1, Myt1, and CDC25C [39]. Wee1 inhibition by adavosertib might be insufficient to fully activate CDK1 for mitosis in DTC cells.
We employed two DTC xenograft models in this study, and adavosertib consistently inhibited tumor growth in both tumor models. These results demonstrate that singleagent adavosertib is effective for DTC therapy. The addition of adavosertib significantly improved the therapeutic efficacy of lenvatinib in a FTC tumor model. However, the therapeutic effect of adavosertib and sorafenib combination was not greater than that of single sorafenib therapy. An optimization of treatment regimen might improve the curative effects of the adavosertib and sorafenib combination therapy in DTC xenografts.
Adavosertib sensitivity was associated with lower Wee1 protein levels in thyroid cancer cell lines. Our data suggest that thyroid cancer cell lines with lower Wee1 expression rely on Wee1 activity, and interrupting Wee1 with adavosertib impairs cell growth more significantly than thyroid cancer cells with higher Wee1 expression. This potential predictive biomarker might help in designing the patient selection process for clinical trials.
There are numerous clinical studies using adavosertib as monotherapy and in combination with chemotherapy, with encouraging results revealing that these treatment regimens are generally tolerated [21,22,[44][45][46]. Our study provides preclinical data that warrant clinical trials to examine adavosertib for DTC therapy.

Pharmacologic Agents
Adavosertib, sorafenib, lenvatinib, dabrafenib, and trametinib were purchased from Selleck Chemicals, and diluted in dimethyl sulfoxide (DMSO; Merck) to a concentration of 10 mmol/L and stored at −80 • C until in vitro use. For the in vivo experiments, adavosertib and lenvatinib were diluted in methyl cellulose (Merck) and distilled water (1:200 w/v) to a final concentration of 12 mg/mL. Sorafenib was dissolved in 50/50% Kolliphor EL (Merck) and ethanol (Merck) and further diluted with water to a final concentration of 14.4 mg/mL before use. Dabrafenib and trametinib were dissolved in 0.5% (hydroxypropyl) methylcellulose (Merck), 0.2% Tween 80 (Merck), and distilled water to concentrations of 8 mg/mL and 0.16 mg/mL, respectively. All drugs were stored at −80 • C until their in vivo use.

Cell Viability Assays and Drug Synergy Studies
DTC cells were cultured at a density of 2 × 10 3 cells per well in 24-well plates in 1 mL of media and incubated overnight. We added six serial two-fold dilutions of adavosertib, sorafenib, lenvatinib, dabrafenib, trametinib, or vehicle for 4-day treatments before cell viability was determined. After removing the culture media, the cells were washed with PBS and lysed with Triton X-100 (1.35%, Merck) to release intracellular lactate dehydrogenase (LDH). Cell viability was assessed using an LDH assay kit (Promega, Madison, WI, USA) and quantified LDH levels using spectrophotometry (Infinite M200 PRO, Tecan, Männedorf, Switzerland) according to the manufacturer's protocol. All experiments were performed in triplicate, and the results are shown as the percentage of cells normalized to the placebo samples, which were considered 100% viable. We determined IC 50 using CompuSyn software for each cell line on day 4 [23,24].
To study the drug combinations of adavosertib and targeted therapy, cells were cultured in 1 mL media for overnight at 2 × 10 3 cells per well in 24-well plates and cells were treated with vehicle, adavosertib and targeted therapy at a fixed-dose ratio, or combination therapy simultaneously for a 4-day course before evaluating cell viability. Six serial of twofold dilutions were investigated at the following starting doses for BHP7-13, K1, FTC-133, and FTC-238, respectively: adavosertib at 702.4, 356.4, 287.2, and 383.2 nmol/L, sorafenib at 2.8, 18.0, 23.6, and 29.6 µmol/L, and lenvatinib at 0. 2, 26.32, 11.84, and 29.96 µmol/L. To study the combination therapy of adavosertib and dabrafenib plus trametinib, K1 cells were incubated with vehicle, adavosertib, dabrafenib plus trametinib, or triple combination of adavosertib, dabrafenib, and trametinib simultaneously for 4-days. Six serial of two-fold dilutions were analyzed at the following starting doses for K1 cells: adavosertib at 356.4 nmol/L, dabrafenib at 1.0 nmol/L, and trametinib at 0.1 nmol/L for a 4-days course. These starting doses were determined using the IC 50 obtained in this and prior studies [33]. For the drug combinations, the Chou-Talalay method and CompuSyn software (Paramus, NJ, USA) were used to calculate the quantitative CI that determined an additive effect (CI = 1), synergy (CI < 1), and antagonism (CI > 1).

Western Blot Analysis
DTC cells were treated with adavosertib (500 nmol/L) or vehicle for 24 and 48 h after plating overnight the cells at 1 × 10 6 cells in 100-mm petri dishes in 10 mL of media. Cell pellets were dissolved using immunoprecipitation lysis buffer containing protein phosphatase inhibitor mixture (Bionovas, Toronto, ON, Canada), sonicated, and clarified by centrifugation. Equal amounts of protein lysate were separated by 12% Tris-HCl gels, transferred to polyvinylidene difluoride membranes, blocked with 5% fat-free milk, and exposed to the primary antibody followed by a secondary antibody conjugated to horseradish peroxidase. Proteins were detected by an enhanced chemiluminescence kit (PerkinElmer, Waltham, MA, USA) using UVP ChemStudio PLUS touch (Analytik Jena, Jena, Germany).
Band densitometry was performed using Molecular Imager VersaDoc MP 4000 system software (Bio-Rad, Hercules, CA, USA). The ratios of Wee1, PLK1, p-CDK1 (Tyr15), p-CHK1 (Ser345), AXL, cyclin E1, and Myt1 to β-actin were calculated in each cell line to determine the relative expression using untreated FTC-133 cell values as reference. Original western blot images could be viewed in File S1.

Flow Cytometry Analysis for Cell Cycle
To elucidate the effect of adavosertib on cell cycle distribution, we seeded cells overnight at 4 ×

In Vivo Flank Xenograft Tumor Therapy
The animals were handled following the protocol approved by the Animal Care and Use Committee at Chang Gung Memorial Hospital, Linkou, Taiwan (approved on  22 March 2019, permission no. 2019010202). K1 and FTC-133 flank tumors were established by injecting 1 million cells in 100 µL of extracellular matrix gel (Merck): culture medium (1:1) into the subcutaneous flanks of 8-9-week-old female athymic nude mice (National Laboratory Animal Center, Taiwan). These DTC cell lines were chosen because they are more tumorigenic in vivo.

Statistical Analysis
The statistical analysis was conducted using SPSS statistical software (version 22.0, SPSS Inc., Chicago, IL, USA), employing Student's t-test to compare two groups of data. Dif-ferences between more than two treatment groups were determined by two-way ANOVA followed by a post hoc Scheffe test. Results are expressed as mean ± standard error and all p-value of < 0.05 were considered statistically significant.

Conclusions
Our results reveal that adavosertib treatment induces cytotoxicity in DTC cells. Two DTC xenograft models showed the therapeutic efficacy and safety of adavosertib. Adavosertib potentiates the therapeutic efficacy of dabrafenib and trametinib in the K1 xenograft model and the therapeutic efficacy of lenvatinib in FTC-133 tumors. Our findings have important clinical implications for using adavosertib in treating DTC, as a single drug and in combination therapies. Further clinical investigations of adavosertib in treating this disease are indicated.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cancers13143487/s1, Figure S1: Sorafenib treatment reduced cell viability in the FTC-238 cell line, Figure S2: Lenvatinib treatment reduced cell viability in 4 DTC cell lines, Figure S3: Dabrafenib and trametinib treatment reduce cell viability in K1 cells, Figure S4: Triple combination therapy of adavosertib, dabrafenib, and trametinib robustly retards subcutaneous xenograft growth in a papillary thyroid cancer model, Figure S5: Immunoblot analysis of p-CDK1, p-CHK1, p-H2AX, PCNA, and cleaved caspase-3 expression in K1 and FTC-133 tumors treated with adavosertib, File S1: The whole original western blot figures.  Institutional Review Board Statement: Not applicable as this study did not involve humans.
Informed Consent Statement: Not applicable as this study did not involve humans.
Data Availability Statement: All data were contained within the article and supplementary material.