A combinatorial strategy for targeting BRAF^V600E mutant cancers with BRAF^V600E inhibitor (PLX4720) and tyrosine kinase inhibitor (ponatinib)

Abstract

Purpose:

Most aggressive thyroid cancers are commonly associated with a BRAF^V600E mutation. Preclinical and clinical data in BRAF ^V600E cancers suggest that combined BRAF and MEK inhibitor treatment result in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAF^V600E thyroid cancer than single agent or BRAF and MEK inhibitors.

Experimental design:

The combined drug activity was analyzed to predict any synergistic effect using high throughput screening (HTS) of active drugs. We performed follow up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs.

Results:

The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion and migration in BRAF^V600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720 resistant BRAF^V600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (p < 0.05), lower number of metastases (p < 0.05) and longer survival (p < 0.05) compared to monotherapy and vehicle control.

Conclusions:

Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAF^V600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

Introduction

Approximately 10–15% of thyroid cancer have aggressive disease behavior and are associated with a high disease-specific mortality (1). Anaplastic thyroid cancer (ATC) is one of the most aggressive and fatal malignancies in humans (2,3). Treatment approaches including surgery, chemotherapy, radiotherapy and multimodal therapy, rarely result in a durable response or prolonged survival in patients with ATC (4–6). Multiple genetic aberrations are common in ATC, and a better understanding of the genetic alterations have led to important and effective treatment advances (6).

Among the different genetic mutations identified in different histologic subtypes of thyroid cancer, BRAF^V600E has been studied extensively because it is the most common driver and actionable mutation in thyroid cancer, with additional secondary mutations that promote progressive dedifferentiation into ATC (7). Monotherapy with a BRAF inhibitor may result in initial clinical benefits but this is for a short duration as acquired resistance is common and leads to a significant relapse in almost all cases (8). Several mechanisms of acquired resistance to BRAF inhibitor therapy have been reported, including tyrosine kinase (TK) upregulation, NRAS mutation, mutant BRAF amplification or alternative splicing, and MEK mutation (9,10).

Combination therapy using both a BRAF inhibitor and a MEK inhibitor was initially investigated in metastatic melanoma and was shown to be more effective compared to BRAF inhibition therapy alone (8). Subsequently, clinical trials combining inhibitors of both MEK and BRAF were conducted, as this combination was predicted to delay MAPK-driven acquired resistance, resulting in longer response duration and a higher tumor response rate (11). As a result of those studies, combination therapy with a BRAF and MEK inhibitors for BRAF ^V600E mutant ATC was recently approved by the U.S. Food and Drug Administration (FDA). However, both BRAF and MEK act on the same downstream target and act largely on the same pathway; hence, they may not target any alternatively activated pathways, such as upregulation of the upstream TK signaling pathways, which in turn, may result in acquired resistance for this combination therapy as well (12). Furthermore, studies have shown that other cancers treated with combined BRAF and MEK inhibitor treatment, such as melanoma, also develop resistance (13). Thus, approaches using a combination of BRAF and multitargeted TK inhibitors may prove even more effective in BRAF^V600E thyroid cancer.

In this study, we hypothesized that combination therapy with a BRAF inhibitor and a multitargeting TK inhibitor would be more effective in BRAF^V600E mutant thyroid cancer than BRAF inhibition alone. We performed high throughput screening (HTS) on BRAF^V600E mutant cells with BRAF^V600E inhibitors and TK inhibitors. The drug combination with synergistic activity (ponatinib and vemurafenib/PLX4720) was then studied in vitro and in vivo to determine its anticancer activity and the mechanism of action for synergism.

Materials and Method

The Animal Care and Use Committee of the National Cancer Institute, National Institutes of Health approved the animal study protocol. No human sample data was used in this study.

Cell lines

Human thyroid cancer cell lines 8505C and BCPAP cell harboring the BRAF^V600E mutation were purchased from the European Collection of Cell Culture (Salisbury, United Kingdom), BRAF^{Wild Type (WT)} cell lines, THJ-16T derived from a patient with ATC was a kind gift from Dr. John A. Copland (Mayo Clinic, Jacksonville, FL), and C643 was obtained from cell Line Service (GmbH, Eppelheim, Germany). All cell lines were maintained in Dulbecco’s Eagle medium (DMEM) containing 4,500 mg/L of D-glucose, 2 mmol/L of L-glutamine and 110 mg/L of sodium pyruvate, supplemented with 10% fetal bovine serum (FBS), thyroid stimulating hormone (TSH, 10 mU/M) (Sigma), penicillin (10,000 U/mL), streptomycin (10,000 U/mL), fungizone (250 ng/mL) and insulin (10 μg/mL) in a standard humidified incubator at 37ºC in 5% CO₂ and 95% O₂ atmosphere. All cell lines were authenticated by short-tandem repeat profiling. Cells lines were tested for Mycoplasma from Idexx BioAnalytics (Westbrook, ME) and were found negative for any contamination.

Cell proliferation assay

Cell proliferation assays were performed in 96-well plates in triplicate. Cells were plated in 96-well black bottom well plates (Greiner Bio-one, NC) at 1.5 × 10³ cells/well in 100 μL of culture medium. The next day, cells were supplemented with 100 μL of fresh medium containing drug(s) PLX4720 and/or ponatinib (Sellekchem, Houston, TX), or vehicle dimethyl sulphoxide (DMSO). Medium with drug/vehicle was replenished every 48 hours. Cell proliferation data was collected for 0, 24, 48, 72 and 96 hours after treatment. CyQUANT (ThermoFisher Scientific, Pittsburg, PA) cell proliferation assays were performed according to the manufacturer’s instructions. The cell numbers were determined using a fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) at 485 nm/538 nm.

Plasmids and siRNA transfections

pCLXSN-c-JUN retroviral vector was purchased from Addgene (Cambridge, MA). c-JUN (2 μg) plasmid was transfected into Amp-phoenix cells, a retroviral packing cell line (a gift from Dr. George Xu, University of Pennsylvania), using Fugene6 transfection reagent (Promega, Madison, WI). At 24 hours post-transfection, fresh culture media was added, and the cells were incubated for an additional 48 hours before harvesting the retrovirus-containing media. The cell lines 8505C, BCPAP, C643 and THJ16T at approximately 50–70% confluence was then infected with c-JUN containing retrovirus in cell culture media containing 10% fetal bovine serum (FBS) for 48 hours at 37°C. The cells were treated with 1 μg/ml puromycin. The puromycin resistant cell clones were selected for further studies.

Combination drug activity calculation method

Combination drug effect on cell proliferation was determined using CompuSyn software (Version 1.0, http://www.compusyn.com), which is based on the median-effect principle (Chou) and the combination index-isobologram theorem (Chou-Talalay). The combination index (CI) values were calculated, where CI < 1.0 indicates a synergistic effect, CI = 1.0 indicates an additive effect and CI > 1 indicates an antagonistic effect (14). Drug combinations at non-constant ratios were used to calculate CI. For calculating the fold change in sensitivity to PLX4720, the IC50 was determined for a panel of BRAF^V600E mutant and BRAF^WT cell lines grown in the presence and absence of ponatinib. Experiments were performed independently three times.

Apoptosis assay

A Caspase-Glo 3/7 assay (Promega, Madison, WI) was used to measure caspase activity. Cells were plated in 96-well plates at a density of 2 × 10³ cells/well in 200 μl of media in triplicate. After 24 hours, fresh cell culture medium with the drug(s) or vehicle control was added to each well. After 48 hours, cells were analyzed for caspase 3/7 activity using the Caspase-Glo3/7 assay kit according to the manufacturer’s instruction. The relative luminescence was calculated and normalized to the total number of cells.

To detect the drug induced apoptosis, thyroid cancer cells were treated with drug(s) or vehicle (48 hrs), then the fraction of both floating and attached cells were pooled in 1X annexin V binding buffer and resuspended. Next, 400 μl of cell suspension containing 1 × 10⁵ cells were transferred to a 5 ml culture tubes. BioLegend (San Diego, CA) FITC annexin V Apoptosis Detection Kit was used for staining the cells. Further, 5 μl of FITC annexin V and 10 μl of FxCycle Violet (Invitrogen, Carlsbad, CA) were added to the cell suspension, vortexed gently and incubated for 15 min at room temperature in the dark before measurement by flow cytometry (FACS Canto II, BD Biosciences, San Jose, CA) with excitation blue laser (488 nm). FlowJo software (Ashland, OR) was used for analyzing the data.

Western blot and antibodies

Total cell lysates were prepared from the cells after treatment with the drug(s) or vehicle with 10 mM Tris buffer (pH 7.4) and 1% sodium dodecyl sulfate (SDS). The protein concentration was determined using the Pierce BCA assay kit solution (ThermoFisher Scientific, Pittsburg, PA). An equal amount of proteins was resolved by electrophoresis on 4–15% SDS-PAGE gradient gels and transferred to PVDF membrane, then immunostained overnight using specific antibodies (Supplementary Method). All primary (1:1000) and secondary antibodies (1:5000) were purchased from Cell Signaling Technologies (Danvers, MA). Band densitometry analysis was performed using ImageJ software (National Cancer Institute, Bethesda, MD).

Gene expression analysis

Total mRNA was extracted from the cells after treatment with the drug(s) or vehicle using a RNeasy Kit (Qiagen, Gaithersburg, MD) and was used for reverse transcription to make cDNA (Applied Biosystems, Forest City, CA). Quantitative real-time PCR was performed using reagents from Applied Biosystem and TaqMan probes (JUN, FOS, ThermoFisher Scientific, Pittsburg, PA).

Cell migration and invasion assay

The cell migration and invasion assays were performed according to the manufacture’s instruction (BD Biosciences, San Jose, CA). Briefly, the cells were seeded in triplicate into 6 well plates (5 × 10³ cells/well); after 24 hours, cells were treated with the drug(s) and vehicle for 24 hours. The cells were trypsinized, and then 5 × 10⁴ cells were seeded in 500 μL total volume of serum-free medium in the upper chamber of the transwell plate. In the bottom well, 750 μL of DMEM supplemented with 10% FBS was added to act as a chemoattractant. Cells were incubated at 37°C for 22 hours, at which point the cells that migrated and invaded were fixed and stained using the Diffi Quick Staining Kit (VWR, Bridgeport, NJ). Cells were then photographed and analyzed using ImageJ software (National Cancer Institute, Bethesda, MD).

Clonogenic assay

Cells were seeded in triplicate in collagen pre-coated 12 well plates (5 × 10² cells/well) and allowed to adhere overnight in culture media. The cells were then cultured with the drug(s) alone or in combination, or with the vehicle in complete media for 12–14 days. Growth media with vehicle or drug(s) was replaced every 48 hours. The cells were fixed with 0.4% buffered paraformaldehyde and then stained with 0.5% crystal violet in methanol for 10 min. The colonies were counted and photographed using a ChemiDoc system (Bio-Rad, Hercules, CA).

Screening of phosphoprotein kinase proteins

After treatment of the 8505C cell line, the relative phosphorylation level of 43 proteins was determined using the Proteome Profiler Human-Phospho-Kinase Array Kit (R&D System, Minneapolis, MN), according to the manufacturer’s instruction. The expression level of the phosphorylated proteins was quantified using the Biorad ChemiDoc system, and the spots were quantified by ImageJ software.

Virtual docking of drug binding

Docking analysis was performed using Autodock Vina and PyMol software (15).

Xenograft ATC models

The Animal Care and Use Committee (ACUC) at the National Cancer Institute (NCI), National Institutes of Health approved the animal studies. For orthotopic ATC cell implantation, 5 × 10⁵ 8505C-Luc2 cells (Luc2 denotes cells with stable expression of a luciferase reporter) were implanted into the region of the thyroid gland of NOD Cg-Prkdcs^cid Il2rg^tm1WjI/SzJ mice. Tumor luminescence was measured using the Xenogen IVIS in vivo imaging system after intraperitoneal luciferin injection (10 μg/body weight of animals). After 1 week of orthotopic implantation, mice were randomized into four treatment groups. The mice were given a PLX4720 drug containing diet or vehicle control diet (Plexxikon, Berkley, CA). Group I mice were treated with the PLX4720 diet (417 mg drug per kd of chow, ppm), plus ponatinib (20 mg/day) by oral gavage. Group II mice were treated with the PLX4720 diet (417 ppm), plus 0.9% NaCl daily by oral gavage. Group III mice were treated with the vehicle control normal diet and ponatinib (20 mg/day) by oral gavage. Group IV control mice received the vehicle control normal diet and 0.9% NaCl daily by oral gavage. Mice were imaged and weighed weekly. They were treated for 21 days, after which all mice were euthanized using CO₂ inhalation. Tumors, regional lymph nodes, lungs and liver tissue were collected after euthanasia for histologic analysis.

We also used an ATC metastasis mouse model, in which 8505C-Luc2 cells (3 × 10⁴ cells/200 μl) were injected into the tail vein of 6-month-old NOD Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ mice. A week later, in vivo imaging was performed to confirm lung metastases and treatment was started. Mice were randomized into four groups. Mice were treated with oral gavage with 0.9% NaCl (control group), PLX4720 (30 mg/kg/day), ponatinib (20 mg/Kg/daily) or a combination PLX4720 (30 mg/kg/day) and ponatinib (20 mg/Kg/day). Mice were imaged and weighed weekly. Treatment was continued until the first mouse reached humane endpoint criteria, upon which all mice were euthanized using CO₂ inhalation. Tumors, lungs and liver tissue were collected after euthanasia for histologic examination. The survival time was calculated from the day that the 8505C-Luc2 cells were inoculated (day0) to the day the animal died. Kaplan Meier survival curves were drawn for each group.

Statistical Analyses

Data are presented as mean ± S.E.M or mean ± S.D values. The effect of treatment and differences among experimental group were assessed using analysis of variance (ANOVA) and appropriate post-hoc test. Kaplan-Meier survival analysis, using Mantel-Cox and Gehen-Breslow-Wilcoxon tests, was performed for the in vivo study. A two-tailed p value of ≤ 0.05 was considered statistically significant. GraphPad Prism software (version 8: GraphPad Software Inc.) was used to perform all statistical analyses. The following symbols are used to denote the statistical significance of data: Non significant = ns, * = p<0.05, ** = p<0.01, *** = p<0.001, and **** = p<0.0001.

Results

PLX4720 and ponatinib combination therapy have a synergistic activity in BRAF^V600E thyroid cancer cells

Based on our previous studies, thirty-two drugs and drug candidates were selected for pair-wise HTS (16–21). Combination TK inhibitor ponatinib and vemurafenib showed synergistic activity (Fig. 1A-D). The combination of vemurafenib and ponatinib had good synergistic effect, based on their effective HSA and Bliss score (22,23). We selected PLX4720, also a BRAF inhibitor, for further studies as it had a lower IC50 in BRAF^V600E mutant 8505C cells when used in combination with ponatinib (Supplemental Table S1). For follow up studies, we used two BRAF^V600E mutant cell lines (8505C, BCPAP) and two BRAF^WT cell lines (THJ-16T, C643).

Figure 1.

HTS matrix drug screen identifies synergy between BRAF inhibitor and tyrosine kinase inhibitor in ATC 8505c cell line. (A) a 10×10 combination response illustration plot representing drug synergy screen. Drug A is added to each column from left to right, concentration from high to low. Drug B is added to each row from top to bottom, concentration from high to low. (B) Complete dose-response curves for Vemurafenib and Ponatinib in 8505c cell line. Single agent Vemurafenib curve is shown in red, single agent Ponatinib curve is shown in blue, other curves represent dose-response from different combinations. (C) 10 × 10 matrix plot for the combination of Vemurafenib (0.0007 to 15 μM) and Ponatinib (0.0007 to 15 μM) in cell viability assay after data normalization in 0 – 100 scale. 100 means strong cell killing. (D) Data is shown in Excess HSA (highest single agent) format. Green means strong synergistic effect with Excess HAS >= 20, cyan means weak synergistic effect with Excess HSA < 20, yellow and red indicate possible antagonistic effect. The mean excess HSA and BLISS score for 8505C was 21.143 and 20.355, respectively. (E–H) Effect of PLX4720 and ponatinib on cellular proliferation. PLX4720 (5, 10, 15 μM), ponatinib (0.062, 0.185, 0.556 μM) or combinations of the two for up to 96 h were analyzed for cell viability. The X axis represents the elapsed time in hours and the Y axis represents the relative fluorescence unit. (I) The synergistic effect of combination PLX4720 and ponatinib treatment on BRAF^V600E thyroid cancer cell is depicted as an isobologram of drug combinations. The X axis represents the drug concentration and the Y axis represents their combination effect at FA of 0.5 (50% reduction of cell growth). The red and blue plots are the response of the single agents and the combination plots (green) indicates synergistic effect when cell growth inhibition was greater than ~50% (for fractional inhibition of Fa=0.50–0.90). Synergistic responses are between the two lines and above Fa=0.5 on Y axis. PLX = PLX4720, PTB = ponatinib in all the figures.

The proliferation assay showed the drugs had effect on all the cell lines tested, both single agents and in combination (Fig. 1E-H). Chou-Talaley algorithm was used to identify drug interaction. PLX4720 and ponatinib had a synergistic effect on BRAF^V600E cell lines (8505C, BCPAP), but the combination in BRAF^WT cell lines had mostly antagonistic effect (Fig. 1I, Table 1). These results suggested that the two drugs may have a complementary mechanism of action in BRAF^V600E mutant cell lines.

Table 1.

PLX4720 and ponatinib combination show synergistic activity on BRAF^V600E thyroid cancer cells.^*

Cell Lines	PLX4720 5 μM+ Ponatinib 0.062 μM	PLX4720 5 μM+ Ponatinib 0.185 μM	PLX4720 5 μM+ Ponatinib 0.556 μM	PLX4720 10 μM+ Ponatinib 0.062 μM	PLX4720 10 μM+ Ponatinib 0.185 μM	PLX4720 10 μM+ Ponatinib 0.556 μM	PLX4720 15 μM+ Ponatinib 0.062 μM	PLX4720 15 μM+ Ponatinib 0.185 μM	PLX4720 15 μM+ Ponatinib 0.556 μM
8505C	4.22416	0.89812	0.45152	1.97866	9.36626	0.4395	1.11639	0.97061	0.6195
C6343	3.21843	2.86244	1.50454	3.39379	0.99065	1.66398	2.74089	2.61221	2.40038
BCPAP	0.56469	0.64187	0.97572	0.46722	0.9291	1.15374	0.45521	1.61142	1.01058
THJ-16T	1.37974	1.81091	1.56236	1.32762	2.22532	1.36109	1.74805	8.74873	1.72797

^*The combination index (CI) was calculated using the Chow-Talalay method with the PLX4720 and ponatinib combination treatment at 72-hours, which had a synergistic effect on BRAF^V600E cell lines (8505C, BCPAP). This was compared to BRAF^WT cell lines (THJ-16T, C643), which mostly had an antagonistic effect with the combination treatment. The CI is determined by the following range: CI < 1, synergist; CI =1 agonist; CI > 1, antagonist.

PLX4720 and ponatinib combination therapy inhibits colony formation, cellular invasion and migration in BRAF^V600E mutant thyroid cancer cells

We found that PLX4720 (15 μM) and ponatinib (0.556 μM) combination treatment reduced colony formation more than PLX4720 or ponatinib alone in 8505C and BCPAP cells compared to control (p<0.05, p< 0.01 and p<0.001, respectively) (Fig. 2A-D). The reduced colony formation with combination therapy was also seen in BRAF^WT cells but at higher doses (Supp. Fig S1A). Additionally, we found that PLX4720 and ponatinib combination most significantly inhibited cellular invasion and migration in BRAF^V600E cells compared to control (p<0.05, p<0.01 and p<0.001, respectively) and compared to PLX4720 (15 μM) (Fig. 2E).

Figure 2.

PLX4720 and ponatinib combination treatment inhibits colony formation, invasion and migration in BRAF^V600E mutant thyroid cancer cells. (A) PLX4720 and ponatinib combination inhibits colony formation in 8505C and BCPAP cells. BRAF^V600E and BRAF ^WT cells were incubated with increasing doses of PLX4720 (5, 10, 15 μM), ponatinib (0.062, 0.185, 0.556 μM) and their combinations for 12–14 day. Images are representative of three independent experiments. (B) Histogram representing the mean colony counts of 8505C. PLX4720 (15 μM) and ponatinib (0.556 μM) reduced colony formation more than PLX4720 and ponatinib alone in 8505C and BCPAP compared to control (**p< 0.01) as well as compared to PLX4720 (15 μM) alone (p<0.05). (C-D) Combination treatment with PLX4720 (15 μM) and ponatinib (0.556 μM) reduced colony formation more than PLX4720 and ponatinib alone in BCPAP cells compared to control (***p<0.001). (E) PLX4720 (15 μM) and ponatinib (0.556 μM) significantly inhibited cellular invasion and migration of BRAF^V600E thyroid cancer cells. The combination of PLX4720 (15 μM) and ponatinib (0.556 μM) showed the most significant inhibition of cellular invasion and migration compared to control (*p<0.05, **p<0.01, ***p<0.001) as well as compared to PLX4720 (15μM) alone (p<0.05, p<0.01). PLX = PLX4720 and PTB = ponatinib in all the figures.(@ in the figure denotes PLX vs other groups).

PLX4720 and ponatinib combination therapy induces apoptosis in BRAF^V600E mutant thyroid cancer cells

FACS analysis was performed after the cells were treated with PLX4720, ponatinib or their combination, and an increase in apoptosis was found with combination treatment in 8505C and BCPAP cell lines (Fig. 3A–D). A significant increase in caspase 3/7 activity was also observed in BRAF^V600E cells after 24 and 48-hour treatment with the drug combination (> 4.6-fold), PLX4720 (> 2.1-fold) and ponatinib (> 0.82-fold) as compared to vehicle control (p<0.001, p<0.0001).The combination treatment group also showed significantly higher caspase 3/7 activity when compared to single drug treatment groups (Fig. 3E, F). This suggests that the combination treatment results increased caspase3/7 activity. In the BRAF^WT cell lines the result was heterogenous with no consistent increase of caspase activity after treatment. In THJ-16T, the single agent treatment resulted in higher caspase activity when compared to control and compared to combination treatment (p<0.05, p<0.01), and combination treatment had significantly lower activity compared to PLX4720 alone (p<0.0001). Whereas in C643 cells, the highest caspase activity was observed after PLX4720 treatment and no difference between ponatinib and combination treatment was observed (p<0.05,p<0.01) (Supp. Fig. S1. B). Combination treatment showed significantly lower activity compared to PLX4720 alone (p<0.01).

Figure 3.

PLX4720 and ponatinib combination treatment induces apoptosis in BRAF^V600E mutant thyroid cancer cells. (A–B) FACS analysis using annexin V-FITC and PI was performed after the cells were incubated with PLX4720, ponatinib and their combination for 48 hours. There was increase (> 3.42-fold) in double-positive annexin/PI labeled cells (late apoptosis, right upper square) and annexin V single-positive cells (early apoptosis, left upper square) with combination treatment (PLX4720 15 μM and ponatinib 0.556 μM) than the single agents PLX4720 (>1.88-fold), ponatinib (0.690 fold) and vehicle control (DMSO) after 48 hours in 8505C cells. (C–D) Quantification of apoptosis of BRAF^V600E cells with treatment (percentage). The X axis represents the treatment groups and the Y axis represents the percentage of cells in each quadrant (% in quadrant from Figure 3A and B). (E–F) Effect on caspase 3/7 activity with treatment. There was a significant increase in caspase 3/7 activity in BRAF^V600E cells after 48 hours of treatments with the combination of drugs (> 4.6-fold) than the PLX4720 (> 2.1-fold), ponatinib (> 0.82-fold) compared to vehicle control (***p<0.001, ****p<0.0001). Also, the combination treatment showed significantly higher caspase 3/7 activity compared to individual drug treatments. The result is represented in the relative light unit (RLU, y axis). PLX = PLX4720, PTB = ponatinib. The results are mean ± SD.

PLX4720 and ponatinib combination therapy inhibits phosphorylation of ERK and MEK in BRAF^V600E mutant thyroid cancer cells

Combination treatment with PLX4720 and ponatinib significantly downregulated the phosphorylation of ERK and MEK in BRAF^V600E mutant cell compared to single agents and vehicle control (Fig. 4A, Supp. Fig. S1C) at 48 hrs of treatment. We also evaluated this effect over time (24 and 48 hours) and found greater reduction in phosphorylated-ERK (pERK) and phosphorylated-MEK (pMEK) at 48 hours as compared to 24 hours with combination treatment (Supp. Fig.S4 C,D ).

Figure 4.

PLX4720 and ponatinib combination reduces phosphorylation of ERK (P44/42) and MEK in BRAF^V600E mutant thyroid cancer cells. Both BRAF^V600E and BRAF^WT thyroid cancer cells were treated with either PLX4720 (10–20 μM), ponatinib (0.185–0.556 μM) and their combination, and phosphorylation of ERK and MEK were measured by Western blot. (A) No significant reduction of ERK and MEK phosphorylation was detected after up to 48 hours of incubation with single agents PLX4720 or ponatinib in BRAF^V600E cells; however, significant reduction in the phosphorylation of ERK and MEK was observed after treatment with the combination of PLX4720 and ponatinib in BRAF^V600E cells (8505C and BCPAP). (B) No synergistic effect and no changes in the phosphorylation of ERK and MEK were observed in BRAF^WT cells (C643 and THJ-16T) with single or combination drug treatments.

There was no synergistic effect or changes in the phosphorylation of ERK and MEK observed in BRAF^WT cell lines (Fig. 4B, Supp. Fig S1C ). Collectively, these data suggest the enhanced anticancer activity of combination drug treatment in BRAF^V600E mutant cell lines may be due to the more effective reduction in pERK and pMEK levels.

PLX4720 and ponatinib combination therapy reduces c-JUN levels in BRAF^V600E mutant thyroid cancer cells, and c-JUN mediates the antiproliferative activity of combination drug treatment

Because PLX4720 and ponatinib combination treatment had a synergistic effect on pMEK/pERK levels in BRAF^V600E mutant cells, we wanted to further explore the possible mechanism(s) that might cause this enhanced effect on pMEK/pERK in BRAF^V600E cells. The proteome profile human phospho-kinase array was used to explore the effect of combination PLX4720 and ponatinib treatment. Combination treatment decreased phosphorylation of p38a (T180/Y182), c-JUN (S63), JNK1/3 (T183/Y185) and HSP27 (S78/S82), and increased phosphorylation of STAT3 (S727), AKT1/2/3 (T308), EGF-R (Y1080), GDK3(α/β), β-catenin, p70S Kinase (S78/S82), CREB (S100) and p27 (T198) (Fig. 5A) (Supp. Table S2). c-JUN expression and phosphorylation was investigated as a possible mechanism of the synergistic drug action, since c-JUN and ERK crosstalk has been reported (24), and ponatinib has also been shown to act on c-JUN in breast cancer (25).

Figure 5.

PLX4720 and ponatinib combination inhibits c-JUN and other kinases in BRAF^V600E mutant thyroid cancer cells and c-JUN enhances the antiproliferative activity of combination treatment in vitro. (A) A proteome human phospho-kinase array was used to investigate the additional mechanism of action of PLX4720 and ponatinib combination. Combination drug treatments decreased phosphorylation of p38a (T180/Y182), c-Jun (S63), JNK1/3 (T183/Y185) and HSP27 (S78/S82) and upregulated phosphorylation of STAT3 (S727), AKT1/2/3 (T308), EGF-R (Y1080), GDK3(α/β), β-catenin, p70S Kinase (S78/S82), CREB (S100) and p27 (T198) in 8505C. The red box in the figure is around c-JUN (S63) and the band densitometry representation of this data is shown in the lower panel. (B) Validation of decreased c-JUN phosphorylation with drug combination treatment by Western blot. (C-F) Western blot showing c-JUN knockdown in 8505C, BCPAP,THJ16T and C643 cells. Bottom panel shows the significant relative knockdown by band densitometry in 8505C, BCPAP,THJ16T and C643 cells respectively (*p<0.05,**p<0.01,***p<0.001). (G) c-JUN knockdown affects cellular proliferation. c-JUN knockdown (siRNA1) reduced proliferation significantly compared to control siRNA (****p<0.0001) in 8505C. (H) Effect of drug treatment with c-JUN knockdown. Combination drug treatment and single agent reduced proliferation in c-JUN deficient 8505C cells significantly. Treatment with drug combination as compared to single agents decreased cellular proliferation 1.6-fold compared to controls (PLX4720 vs. combination, *p<0.01). (I) Cell number at 96 hrs of c-JUN knockdown cells and treatment groups compared to control (**p<0.002, **** p<0.0001) and PLX4720 (15μM) alone (p<0.0001). (@ in the figure denotes PLX vs other groups).

A molecular docking technique was used to evaluate the potential binding of ponatinib and PLX4720 in the protein cavity of JNK (PDB id: 2ELJ). The JNK protein is crystallized and the bis-anilino-pyrolopyrimidine inhibitor binds with amino acids (GLU109, MET111, and ASN114), providing a binding site for virtual screening. Ponatinib binds to the ligand binding site of JNK with a binding energy of −9.5 Kcal/mol and stabilized itself by forming hydrogen bonds with MET111 (3.1 Å) and ASP112 (3.4 Å). PLX4720 binds with a binding energy of −7.8 Kcal/mol and stabilized itself by forming hydrogen bonds with ILE32 (2.6 Å). This suggests the potential binding of ponatinib with the ligand binding site of JNK and the amino acid residue MET111 is more stable than the binding with PLX4720 and might be involved in inhibition of JNK activity leading to lowering in phosphorylation of c-JUN. Also, the different binding sites of the two drugs might indicate that the drugs when used in combination have the potential to bind at different sites reducing the chance of competitive inhibition and possibly increasing the activity of their combined use (Supp. Fig. S1D).

Combination treatment had an effect on the phosphorylation of JNK/c-JUN in BRAF^V600E cells as seen in the phospho-kinase array; hence, we performed further experiments to validate the inhibition of JNK/c-JUN activity by combination treatment. The combination treatment downregulated c-JUN phosphorylation in BRAF^V600E cells as shown by Western blot (Fig. 5B). As c-JUN, in combination with c-Fos, forms the AP-1 early response transcription factor, the mRNA expression levels of both c-JUN and c-FOS were analyzed and a significant downregulation by the single agents and combination treatment was seen (p< 0.01, p< 0.001) (Supp. Fig. S2A).

Next, we wanted to determine whether the activity of the combination of PLX4720 and ponatinib was dependent on c-JUN. Therefore, c-JUN was ectopically overexpressed in BRAF^V600E mutant cells (Supp. Fig. S2B). No significant change was observed in cellular proliferation with c-JUN overexpression with single agent treatment or combination treatment (Supp. Fig. S2C,D). Knockdown of c-JUN was done in 8505C, BCPAP, C643 and THJ16T (Fig 5C-F ).Knockdown of c-JUN in 8505C (Fig. 5C) cells resulted in reduced cellular proliferation. c-JUN siRNA1 showed significant reduction in proliferation compared to control siRNA which was consistent with the level of knockdown as siRNA1 had a greater knockdown of c-JUN than siRNA2 (p < 0.0001) (Fig. 5G). More importantly, c-JUN knockdown in BRAF^V600E mutant cells was associated with greater sensitivity to combination treatment compared to control (p<0.0001) and PLX4720 (15 μM) alone (p<0.0001) (Fig. 5H, I). Combination treatment significantly reduced colony formation compared to single agent and vehicle control (Supp. Fig. S2E,F). Cellular proliferation after c-JUN knockdown was studied on BRAF^WT cells as well (Supp. Fig.S5 A-D).

PLX4720 and ponatinib combination treatment reduces tumor growth and metastasis in vivo

Combination PLX4720 and ponatinib treatment significantly reduced tumor growth (70.3%, p< 0.05) compared to vehicle control group and PLX4720 alone (p<0.05), whereas in the single agent groups (PLX4720– 49.4%; ponatinib- 19.95 %) there was no significant difference compared to vehicle control (Fig. 6 A, B). Mice in the control group (25 ± 1.7 gm), PLX4720 group (25 ± 1.9 gm) and ponatinib group (20.4 ± 4.0 gm) had significant lower weights as compared to the combination treatment group (29.4 ± 3.2 gm) (p< 0.05), who maintained their body weight and showed no signs of illness or cachexia (Fig 6C). Hematoxylin and eosin staining demonstrated that pulmonary metastases were lower in mice treated with PLX4720 (single) and combination treatment (PLX4720+ponatinib) when compared to control group (p < 0.01; p< 0.001). No significant difference was seen in the ponatinib group (Fig. 6D).

Figure 6.

PLX4720 and ponatinib combination treatment reduces tumor growth in an orthotopic model and decreases metastasis in a tail-vein metastasis model. (A) Ex-vivo orthotopic tumor luciferase activity in control and treatment groups showing tumor burden. Representative image of three mice from each group. Mice in the combination group (PLX4720 and ponatinib) had lower tumor burden and showed no signs of sickness. (B) There was a significant reduction of whole-body luciferase signal in the mice treated with the drug combination (70.3%, p < 0.05) than the single agent groups (PLX-49.4% and Ponatinib-19.95%, respectively) compared to the control and PLX4720 alone (p<0.05). (C) The mice in control (25 ± 1.67), PLX4720 (25 ± 1.92) and ponatinib (20.40 ± 4.03) group had lower body weight and cachexia compared to combination treatment. The mice in the combination group maintained their body weight and showed no sign of cachexia (*p<0.05). All the values are in mean ± SD. (D) Metastasis analysis in lungs from H & E staining. Metastasis is significantly low in PLX420 treated mice and combination treated mice compared to control (**p<0.01,***p<0.001). The scoring was performed at 4X magnification. (E) Tail vein metastasis model study. Survival curve analysis showed the mice in the combination group (PLX4720 and ponatinib, 100%) survived longer compared to the control group (50%). The X axis represents the elapsed time in weeks and the Y axis the percentage surviving. Kaplan-Meier survival analysis using Mantel-Cox and Gehen-Breslow-Wilcoxon test was performed to determine statistical significance in survival. There was a significant longer survival in the combination group (*p<0.05). (F) Tumor burden measured by body luciferase signal indicated the lowest tumor burden in the combination treatment [non-significant (n.s.)]. (G) Representative image of H & E staining. There was lower pulmonary metastasis in mice treated with the combination of PLX4720 and ponatinib, but it was not statistically significant based on randomly selected three fields (4x magnification) and counting the number of tumor foci. In the orthotopic model, 20 skid mice (NOD Cg-Prkdcs^cid Il2rg^tm1WjI/SzJ), 8505C-Luc2 cells tagged with a luciferase reporter gene were implanted orthotopically into the right thyroid lobe, and a week after tumor implantation, luciferase activity was detected by in vivo bioluminescence. In the metastasis model, 8505C-Luc2 cells were injected in the tail vein of NOD Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ mice (n = 40) to see the effect of treatment. (@ in the figure denotes PLX vs other groups).

As most cancer deaths are due to metastatic disease, an ATC metastasis mouse model was used to test the efficacy of combination treatment. Survival analysis showed a significantly longer survival time in mice treated with combination PLX4720 and ponatinib, with 100% of the mice surviving, as compared to control and single agent treated groups (p<0.05) (Fig. 6E). Mice in the control and ponatinib groups had significantly lower body weight and signs of cachexia (≤0.8gm), while mice treated with PLX4720 showed no weight loss or signs of cachexia (≥ 1.5gm) when compared to baseline body weight (p<0.01). Interestingly, mice in the combination treatment group had higher body weight from baseline (≥3.4 gm) and no signs of cachexia (Fig. 6 F). The mice in the combination group also showed reduced tumor progression (not statistically significant, but this may be due to the different route of PLX4720 administration) compared to the single agent and control groups based on whole body luminescence measurements (Supp. Fig. S3A). Tumor burden as measured by body luciferase signal indicated the lowest tumor burden in the combination treatment group (Supp. Fig. S3B). Hematoxylin and eosin staining demonstrated that lung metastasis was not significantly different with treatment (Fig 6G, Supp. Fig. S3D), but liver metastasis was significantly lower in mice treated with combination ponatinib and PLX4720 (p < 0.01); (Supp. Fig. S3C, Supp. Fig S3E).

PLX4720 and ponatinib combination treatment overcomes BRAF inhibitor (PLX4720 > 60 μM) resistance in 8505C cells

Resistance to BRAF inhibition occurs in BRAF^V600E mutant cancers. Therefore, 8505C cells that were resistant to PLX4720 treatment (not sensitive to 30 μM with an IC50 of 60 μM) were used to test if the combination treatment could be effective (Supp. Fig. 2G). Combination PLX4720 and ponatinib treatment significantly inhibited cellular proliferation (PLX4720 15 μM and ponatinib 0.185 μM, PLX 4720 30 μM and ponatinib 0.185 μM, PLX 4720 30 μM and ponatinib 0.556 μM, PLX4720 60 μM and ponatinib 0.185, PLX4720 60 μM and ponatinib 0.556 μM) as compared to control (p<0.0001) and PLX4720 alone (p<0.001)(Supp. Fig. S2H, Supp. Fig. S3F). We also observed reduced number and size of colonies when treated with combination PLX4720 and ponatinib (Supp. Fig S3G). Chou-Talalay analysis of PLX4720 and ponatinib in 8505C-resistant cells demonstrated that the synergism of the drugs at the respective doses that make the combination effective, although the single drugs at those doses were not effective (Supp. Fig. S4A). Western blot analysis demonstrated lower pERK levels with combination treatment in 8505C-resistant cells (Supp. Fig. S4B).

Discussion

In this study, we report the combination of PLX4720 and ponatinib has synergistic anticancer activity in BRAF^V600E mutant cells based on in vitro and in vivo studies. The synergistic mechanism of action results in more effective inhibition of the MAPK pathway with lower pERK and c-JUN levels, and induces caspase-dependent apoptosis. Moreover, combination PLX4720 and ponatinib is able to overcome resistance in PLX4720 resistant BRAF^V600E mutant cell.

Approximately 20% of patients harboring activating mutations in BRAF develop intrinsic resistance and do not respond to BRAF inhibitors (26), which limits the effectiveness of the treatment. Thus, combination therapy with BRAF and MEK inhibitors have been studied and may achieve a synergistic therapeutic effect, which could reduce treatment dose and toxicity. Dabrafenib and trametinib (BRAF^V600E and MEK inhibitors) was approved by the U.S. FDA to treat BRAF^V600E mutant ATC, and it is the only combination treatment being used for this type of cancer. The combination has also been approved for BRAF-mutated melanoma and squamous cell carcinoma of the lungs. Although recent reports suggest that BRAF and MEK inhibitors used in combination in BRAF^V600E mutant melanoma have initial good response rates, most patients develop resistance to the combination therapy (27–30). Our preclinical data suggests a novel combination of PLX4720 and ponatinib might be effective as there is clearly room for improvements in the current therapies available for ATC.

In this study, we report for the first time that a novel combination of drugs, the BRAF inhibitor PLX4720 and the FDA-approved multi-target TK inhibitor ponatinib, synergistically reduces p-MEK and p-ERK compared to single drug treatments in BRAF^V600E cells. The chemoresistance of ponatinib has been frequently raised as critical issues in the preclinical and clinical settings, this might result in lower effect of ponatinib in vivo compared to in vitro study (31).

Pharmacologic inhibitors of BRAF^V600E are usually the drug of choice for first-line therapy in BRAF^V600E tumors as this is a targetable mutation that drives cancer initiation/progression (32). The effect of vemurafenib in combination with ponatinib showed a synergistic effect in BRAF^V600E mutant cells. The BRAF^V600E inhibitor, vemurafenib, has provided a major advance for the treatment of patients with BRAF^V600E mutant metastatic melanoma. However, in BRAF^V600E mutant thyroid cancer it has been found to be relatively resistant to vemurafenib treatment (33,34) and cell lines have a higher IC50 than melanoma cells and the reason for this difference in response remains unclear (35). Furthermore, PLX4720 treatment alone had a similar effect on cellular proliferation and colony formation in BRAF^WT and BRAF^V600E cell lines, results similar to previous studies (36). The presence of additional mutations (excluding BRAF) and altered signaling pathways present in the cell lines tested may account for this. Furthermore, although PLX4720 showed a similar effect on cellular proliferation in BRAF^V600E negative cell lines, the effect on pERK/ERK and pMEK/MEK was variable. For our follow up studies, PLX4720, a second-generation BRAF^V600E inhibitor, was used because of its much stronger affinity and favorable pharmacokinetic properties. Nucera and colleagues (36), reported that PLX4720 (10 μM) reduced the MEK/ERK phosphorylation in BRAF^V600E mutant 8505C cells. PLX4720 (5, 10, 15 μM) in combination with ponatinib (0.185 μM, 0.556 μM) inhibited cell migration, invasion and colony formation in BRAF^V600E mutant 8505C and BCPAP cell lines. Therefore, the combination of BRAF inhibition and the multi-target TK inhibitor ponatinib can be useful in targeting thyroid cancer and other cancers harboring a BRAF^V600E mutation.

Previous studies have shown that cancer cells are able to adapt signaling pathway circuits in response to drug treatment by establishing alternate signaling routes. Hence one critical aspect to improve cancer treatment is not only to inhibit the primary oncogenic pathway that reduces cell proliferation, but simultaneously to prevent functional redundancies and pathway crosstalk that facilitates survival of cancer cell populations, rendering tumors resistant to therapy (37). As such, studies based on network pharmacology principles have tried to identify synergistic multitarget intervention strategies to improve clinical efficacies. Our study was designed to target the most commonly activated pathway BRAF/MEK/ERK, with a BRAF^V600E inhibitor (PLX4720) and a multitarget TK inhibitor ponatinib.

A link between ERK and JNK signaling in melanoma, a cancer with high rates of BRAF^V600E mutations, has been reported (38). Constitutively active ERK affects the c-JUN oncogene, its upstream kinase JNK and its downstream targets RAC1 and cyclin D1. Understanding how these signaling pathways are re-wired offers new targets for therapy. Crosstalk between these two pathways can contribute to a robust and integrated signaling transduction network involved in cell proliferation (24). In breast cancer metastasis, ponatinib represses the expression of BCLM-associated genes, mainly through the ERK/c-JUN signaling pathway by inhibiting the transcription of JUN and accelerating the degradation of the c-JUN protein (25). These investigators identified ponatinib as a new drug to inhibit BCLM, and c-JUN was found to be a crucial factor and a potential drug target. We investigated the effects of PLX4720 and ponatinib on c-JUN as a possible mechanism for their synergistic action. Our data showed a lower level of c-JUN and JNK kinase activity after combination treatment in BRAF^V600E mutant cells, suggesting a functional interaction of ERK and JNK pathway, and the inhibitory effect of PLX4720 and ponatinib on this crosstalk might cause the synergistic effect. We also discovered that c-JUN knockdown along with the combination PLX4720 and ponatinib significantly inhibited cell proliferation and colony formation in BRAF^V600E mutant cells, demonstrating the important role c-JUN has in BRAF^V600E thyroid cancer cells. Interestingly, JNK is a serine/threonine kinase and not a TK, but inhibition of its effector c-JUN by combination treatment suggests that it might not be the direct target but gets regulated from upstream inhibition. These results suggest that c-JUN mediates some of the synergistic effect of combination PLX4720 and ponatinib treatment but is not the sole mediator as illustrated by our knockdown studies showing c-JUN is not the sole regulator of cellular proliferation.

Developing resistance to BRAF^V600E inhibition alone is a significant problem, thus we performed experiments on PLX4720-resistant 8505C cells generated by established protocol (39,40). Thyroid cancer cell lines are known to have complex mutational profiles and harbor concurrent alteration in many genes (41). The 8505C cell line has mutations in TP53, EGFR, PIK3R1, PIK3R2, NF2, SMARCA4, SMARCD1 and TERT in addition to BRAF^V600E (19,40,41). The presence of an activating BRAF^V600E mutation generally predict response to BRAF inhibitors but resistance to this treatment develops (39). Interestingly, we found, in the resistant cells, reduced phosphorylation of the downstream MEK1/2 (on Ser217/221) and ERK1/2 (on T202/Y204) signaling pathway targeted by PLX4720 at its IC50 dose, unlike the parental cells. Ponatinib as a single agent was effective in the PLX4720-resistant 8505C cells. Although the concentration we used might not be achievable in vivo, this observation in the in vitro system may be due to the additional mutations present in this cell line. Resistant cell lines have also been reported to acquire a more invasive phenotype characterized by increased cell mobility and metastatic capacity (40).

Upregulation of a distinct receptor TK has been shown to sustain signaling through signaling pathway, despite continued inhibition of the primary oncoprotein with the targeted drug (42). Thus, drug combinations to block proliferation pathways are in development, but the fundamental combinatorial principle is still elusive. Here, we propose the combination of a BRAF mutant inhibitor and a multitargeted TK inhibitor might prove to be effective in overcoming BRAF inhibitor resistance, as it can target multiple pathways.

In summary, the current study demonstrates that the combination of PLX4720 and ponatinib has significant and synergistic anticancer activity in BRAF^V600E mutant thyroid cancer cells in vitro and in vivo. Combination treatment with PLX4720 and ponatinib is a highly promising new combination targeted therapy for BRAF^V600E mutant thyroid cancer, as well as for BRAF inhibitor-resistant BRAF ^V600E mutant cell. Our preclinical studies suggest that combination PLX4720 and ponatinib treatment should be tested in clinical trials.

Translational Relevance: -.

Most aggressive thyroid cancers are commonly associated with a BRAF^V600E mutation. Recent preclinical and clinical findings on BRAF mutant cancers suggest that combination therapy with BRAF and MEK inhibitors may result in a response, but resistance is common, leading to disease progression. We used quantitative high-throughput screening (qHTS) on BRAF^V600E mutant cells with BRAF^V600E inhibitors and tyrosine kinae (TK) inhibitors for identifying new therapeutic indications and found PLX4720 showed good synergism with ponatinib. The synergistic activity of this combination was validated on cellular proliferation, colony formation, invasion, migration and was seen to induce apoptosis. In vivo study also showed the effectiveness of this drug combination in regressing tumor and lung metastasis in orthotopic model. Our preclinical evaluation of PLX4720 and ponatinib shows that this novel combination is a potential candidate for clinical trial in BRAF^V600E thyroid cancer.