Antitumor effects of CEP32496 on urothelial carcinoma with BRAF mutation (V595E) in dogs

Abstract

Canine urothelial carcinoma (UC) has a BRAF gene mutation (V595E) with a high prevalence rate (67 to 87%). CEP32496 is a novel and highly potent BRAF inhibitor. As described in the literature, the efficacy of BRAF inhibitors, including CEP32496, for UC with V595E is limited. The objective of this study was to evaluate the effects of CEP32496 alone and in combination with other BRAF inhibitors on the cell viability and tumor growth of 5 UC cell lines with V595E. For reference, 7 BRAF inhibitors, including GDC0879, PLX4720, encorafenib, vemurafenib, dabrafenib, AZ628, and RAF265, were also evaluated. The results revealed that CEP32496 had a sensitive half-maximal inhibitory concentration (IC₅₀) ranging from 16 nM to 150 nM. In addition, the combination of CEP32496 and vemurafenib resulted in lower IC₅₀s than those of individual treatment. The synergism of CEP32496 with vemurafenib may be due to the different mechanisms of cell proliferation between the 2 inhibitors. In the xenograft model, 10 mg/kg body weight of CEP32496 resulted in a significant decrease (P < 0.05) in tumor growth in mice, with severe and extensive necrosis on histopathological examination. These results suggested that CEP32496 exerts antitumor effects on both cell proliferation and tumor growth in UC with V595E.

Introduction

Urothelial carcinoma (UC), the classical term for transitional cell carcinoma, is the most common and aggressive tumor of the urinary tract and the bladder in dogs. Tumors originating from urothelial epithelial cells have a high tendency to infiltrate into adjacent tissues and develop distant metastasis (1–3). At diagnosis, metastatic lesions were detected in 20% of dogs with UC and in up to 50% of dogs at death (4–6). The metastatic dynamics of neoplastic cells are closely related to the prognosis of the disease (7–9), with the median survival time, after surgical resection and administration of carboplatin or piroxicam, being reported as 3.5 to 11.0 mo (4,9) and 2.5 to 8.1 mo (2,3,5), respectively.

In 2015, the v-raf murine sarcoma viral oncogene homolog B (BRAF) gene mutation in exon 15 (V595E), corresponding to human V600E, was identified and detected in canine UC with high prevalence rates of 67 to 87% (6,10). As BRAF is a serine/threonine kinase that activates the mitogen-activated protein kinase (MAPK) signaling pathway, V595E directly phosphorylates and activates its downstream MAPK kinase (MEK), followed by the activation of extracellular single-regulated kinase (ERK1/2), which induces activation of the MAPK signaling pathway (10–13).

Tumor cell lines are valuable tools for evaluating tumor characteristics and responses to antitumor drugs (14). In 5/18 UC cell lines with V595E that were used for cell proliferation assay of BRAF inhibitors (10,15–18), 3 with vemurafenib and 2 with sorafenib, the half maximal inhibitory concentration (IC₅₀) ranged from 7 to 45 μM (10,18). Meanwhile, the IC₅₀ of vemurafenib ranges from 13 to 50 nM in human melanoma with V600E (19,20).

CEP32496 is a novel and highly potent BRAF inhibitor. In this study, we investigated the antitumor effects of CEP32496, alone and in combination with other BRAF inhibitors, on cell viability and xenograft tumor growth in 5 UC cell lines with V595E.

Materials and methods

Ethics approval statement

All procedures involving animals in this study were carried out in accordance with the ethical standards of the University of Tokyo and approved by the Committee of Animal Experiments, Graduate School of Agricultural and Life Sciences, University of Tokyo (approval number P17-017).

Cell line validation statement

The 5 cell lines with V595E used in this study, TCCV-So, TCCV-Ka, TCCV-Ec, TCCV-Ni, and TCCV-Ic, were derived from the primary UC tissues of 5 dogs admitted to the Japan Animal Referral Medical Center. The cells and the breeds they were collected from are detailed in Table I. These cells were cloned at our Center by limiting the dilution culture.

Table I.

Five cell lines of dogs with urothelial carcinoma in this study.

Cell line	Breed	Age	Sex	Original tumor		Doubling time
				Tumor cell	Collecting site
TCCV-So	Miniature dachshund	11 y	NM	Urothelial epithelium	Urinary bladder	31 h
TCCV-Ka	Miniature dachshund	11 y, 10 mo	NF	Urothelial epithelium	Urinary bladder	22 h
TCCV-Ec	Siberian husky	9 y, 9 mo	NF	Epithelioid	Urethral tract	27 h
TCCV-Ni	Cairn terrier	13 y, 11 mo	NF	Urothelial epithelium	Urinary bladder	24 h
TCCV-Ic	Maltese	13 y, 7 mo	NF	Urothelial epithelium	Urinary bladder	21 h

y — Year; mo — Month; NM — Neutered male; NF — Neutered female.

Cell morphology (microscopically), doubling time (cell proliferation assay), xenograft tumor growth in severe combined immunodeficiency (SCID) mice, and xenograft tumor histopathology (carried out by 2 Board-certified pathologists) were evaluated. V595E mutations were confirmed by polymerase chain reaction (PCR)-restriction fragment length polymorphism using primer pairs (reverse primer: 5′-TGG CCT CAA TTC TTA CCA TCC AC-3′ and forward primer: 5′-GTA ATG CTT GCT TTG CTA GGA-3′) and restriction enzyme with BtsIMutl (New England Biolabs Japan, Tokyo, Japan) (13). All cell lines used in this study were submitted to the Biotherapy Institute of Japan (Koto-ku, Tokyo, Japan) to detect mycoplasma infection. All lines were free of mycoplasma based on real-time PCR (RT-PCR).

Cell culture

All cell lines were maintained in a humidified atmosphere 5% CO₂ at 37°C in Dulbecco’s Modified Eagle’s Medium (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA), supplemented with 20% fetal bovine serum (Gibco, Thermo Fisher Scientific) and 1% penicillin-streptomycin (Sigma-Aldrich, Burlington, Massachusetts, USA).

Reagents

CEP32496 (Selleck Co. Ltd., Tokyo, Japan) was used for this study. For references, 7 BRAF inhibitors [1 first generation (GDC0879), 4 second generation (PLX4720, encorafenib, vemurafenib, and dabrafenib), and 2 third generation (AZ628 and RAF265)] were also evaluated. Notably, 1 first- and 4 second-generation BRAF inhibitors were adenosine triphosphate (ATP)-competitive inhibitors (19,21–24).

Cell viability assay

The inhibitory effect of CEP32496 on the viability of the cell lines was assessed using the Premix WST-1 Cell Proliferation Assay System (Takara Bio, Kusatsu, Shiga, Japan) according to the manufacturer’s protocol with slight modification. Briefly, each cell line was seeded at an optimal cell density diluted in 100 μL of culture medium in 96-well flat-bottom plate (Falcon #353072: Corning, New York, USA) as follows: 800 cells/well for TCCV-Ec, 1000 cells/well for TCCV-So, 2000 cells/well for TCCV-Ka and TCCV-Ni, and 3000 cells/well for TCCV-Ic. All cell lines were then cultured in similar conditions.

After 24 h, cells were gently washed with culture medium twice and treated with 100 μL of a different concentration of BRAF inhibitors as follows; 0, 3, 10, 30, 100, 300, and 1000 nM for CEP32496 and encorafenib; 0, 1, 10, 100, 1000, 5000, and 10 000 nM for PLX4720; and 0, 50, 100, 500, 1000, 5000, and 10 000 nM for GDC0879, AZ628, RAF265, vemurafenib, and dabrafenib. After incubation for 24, 48, and 72 h, 10 μL/well of WST-1 was added and the cultures were incubated for an additional 1, 2, and 3 h, respectively.

The absorbance of 450 nm (reference: 690 nm) was measured using a multiplate reader (POWERSCAN HT; Sumitomo Pharma, Tokyo, Japan) and the IC₅₀ was calculated from the percentage (%) of the cell viability curve. All experiments were repeated 3 times and each value of the cell viability percentage was represented as mean ± standard deviation (SD) of triplicate wells.

Combination treatment with CEP32496 and vemurafenib

The efficacy of the combination treatment of BRAF inhibitors, especially with third- and second-generation inhibitors, such as the MEK inhibitor sorafenib and the MAPK inhibitor vemurafenib, has been reported for human melanoma with V600E (25,26). As CEP32496 is a third-generation and highly potent BRAF inhibitor, the effects of its combination with vemurafenib were examined in all cell lines.

Briefly, CEP32496 (0 to 1000 nM) was treated with the half IC₅₀ of vemurafenib (1.5 μM: reading from cell viability curve); conversely, vemurafenib (0 to 10 000 nM) was treated with the half IC₅₀ of CEP32496 (15 nM: ditto). All experiments were repeated 3 times and each value of the cell viability percentage was represented as the mean ± SD of triplicate wells. In addition, the combination index (CI) for CEP32496 and vemurafenib was calculated for each cell line using the Chou-Talalay’s CI method (27).

Antitumor effects of CEP32496 on xenograft tumor with TCCV-Ka

To create xenograft tumors, 1.0 × 10⁷ TCCV-Ka cells with V595E in 200 μL of physiological saline (TERUMO, Tokyo, Japan) were subcutaneously inoculated into the lower flanks of 5-week-old female mice with severe combined immunodeficiency (SCID) (total: 20 mice) (FOX CHASE SCID CB-17/lcr-scid/scidJcl; CLEA Japan, Tokyo, Japan) according to our previous study (13). The inoculated mice were housed in cages (5 mice/cage) with free access to water and fed a standard purified rodent growth diet (AIN-93G; Oriental Yeast, Tokyo, Japan). The day when a mass was palpable at the inoculation site was designated as Day 3.

The tumor volume (mm³) [length (mm) × width (mm²)/2] was measured 5 d per wk using a caliper. CEP32496 was peritoneally administered 5 d a wk at doses of 0 (vehicle: 10% dimethyl-sulfoxide, DMSO), 2.5 mg/kg body weight (BW), 5 mg/kg BW, and 10 mg/kg BW in 200 μL of 10% DMSO according to previous studies (22,23). On Day 18, the mice were euthanized with isoflurane and the tumors were resected for histopathological examination. Tumor volume data were represented as mean ± SD of 3 mice, and Student’s t-test was used to compare the results to those with vehicle treatment. Statistical significance was set at P < 0.05.

Histopathological analysis of xenograft tumors treated with CEP32496

For histopathological examination, the resected xenograft tumor tissues were fixed in 10% neutral buffered formalin, routinely paraffin-embedded, sectioned at 4 μm, and stained with hematoxylin and eosin. Histopathological examination was carried out by 2 veterinary pathologists certified by the Japanese College of Veterinary Pathologists.

Results

Inhibitory effects of CEP32496 on cell viability in cell lines with V595E

The inhibitory effects of CEP32496 and 7 BRAF inhibitors on the viability of 5 cell lines with V595E are shown in Figure 1 (a to e). Although the IC₅₀s were based on the percentage of cell viability curves, the IC₅₀ of CEP32496 in the 5 cell lines ranged from 16 to 150 nM, indicating a sensitive IC₅₀. Meanwhile, the IC₅₀s of the 7 referential BRAF inhibitors varied from 130 nM (encorafenib in TCCV-Ec) to ≥ 8000 nM (Dabrafenib in TCCV-Ka, and GDC0879 and RAF265 in TCCV-Ic Table II).

Figure 1.

(a to e) Inhibitory effects of CEP32496 and 7 referential BRAF inhibitors on the viability of 5 cell lines with V595E. Although IC₅₀s were read from the percentage of the cell viability curves, the IC₅₀ of CEP32496 in all cell lines ranged from 16 to 150 nM, indicating a sensitive IC₅₀. All experiments were carried out at least 3 times, and each value of cell viability (%) was represented as mean ± standard deviation.

Table II.

Half maximal inhibitory concentration (IC₅₀; nM) of CEP32496 and additional referential BRAF inhibitors for urothelial cell carcinoma cell lines with V595E.

	TCCV-So	TCCV-Ka	TCCV-Ec	TCCV-Ni	TCCV-Ic
GDC0879	—*	—	—	1000	8000
PLX4720	4000	—	3000	400	—
Encorafenib	800	800	130	—	—
Vemurafenib	—	—	3000	1800	2500
Dabrafenib	700	8000	700	1100	—
AZ628	—	1800	3000	1000	650
RAF265	—	—	140	1100	8000
CEP32496	16	150	20	33	25

^*Cannot read.

Combination treatment with CEP32496 and vemurafenib

Cell viability following combination treatment with CEP32496 and vemurafenib, which was approved as the most effective and predominant inhibitor by the Food and Drug Administration, was evaluated in 5 cell lines with V595E. The results (Figure 2) showed that the viability percentage at each time point was lower than that of the individual treatment. The IC₅₀ reading following combination treatment with CEP32496 and vemurafenib in each cell line is shown in Table III. Most combination indexes indicated synergism (CI < 1) between the 2 drugs.

Figure 2.

Inhibitory effects of combination treatment with CEP32496 and vemurafenib on the cell viability of the 5 cell lines. The viability of cells treated with CEP32496 and vemurafenib was lower than that of individual treatment. All experiments were carried out at least 3 times, and each value of cell viability (%) was represented as mean ± standard deviation.

Table III.

Half maximal inhibitory concentration (IC₅₀; nM) of combination treatment with CEP32496 and vemurafenib for 5 UC cell lines with V595E.

Combination	Cell line
	TCCV-So	TCCV-Ka	TCCV-Ec	TCCV-Ni	TCCV-Ic
CEP32496
Solo	200	180	180	100	80
+ Vemurafenib (1.5 μM)	70	60	8	60	40
Vemurafenib
Solo	10 000	3000	180	1100	4000
+ CEP32496 (15 nM)	3000	300	7	1000	180
Combination index	0.34	0.13	0.09	1.51	0.55

Antitumor effects of CEP32496 on xenograft tumor growth

The effects of CEP32496 on xenograft tumor growth at 0 (vehicle), 2.5 mg/kg BW, 5 mg/kg BW, and 10 mg/kg BW are shown in Figure 3 a, whereas the histopathological features and changes in body weight are shown in Figures 3 b and 3 c, respectively. The xenograft tumor growth in the vehicle treatment group increased gradually until Day 7 and was variable thereafter as the tumor volume increased from 400 to 600 mm³.

Figure 3.

Antitumor effects of CEP32496 on xenograft tumors with TCCV-Ka. a — Tumor growth of the xenograft TCCV-Ka treated with CEP32496 showed similar growth patterns at 0 (vehicle), 2.5 mg/kg BW, and 5 mg/kg BW. However, tumor growth was significantly lower (P < 0.05) at 10 mg/kg BW. b — On histopathology, typical features of xenograft UC tumor were observed in the vehicle and 2.5 mg/kg BW treatment arms, whereas necrosis was observed in the 5 mg/kg BW treatment arm. Notably, more severe and extensive necrosis was observed at 10 mg/kg BW. Tumor volume data are represented as mean ± standard deviation and Student’s t-test was used to compare the results to those for the vehicle. Bar = 100 μm. c — No significant differences in body weight were observed among the 4 treatment groups.

Tumor growth in the 2.5 mg/kg BW and 5 mg/kg BW treatment groups showed similar growth patterns to that of the vehicle, whereas tumor growth in the 10 mg/kg BW treatment group increased gradually until Day 7, then decreased thereafter, finally achieving a significantly lower volume (200 to 400 mm³; P < 0.05) than that of the vehicle. In addition, typical histopathological features of xenograft tumors, such as the formation of glandular structure, solid proliferation of pleomorphic tumor cells, and vascular formation, were observed in the vehicle and 2.5 mg/kg BW treatment groups. With tumor cell invasion and vascular formation, mild necrosis in the neoplastic foci was observed in mice treated with 5 mg/kg BW of CEP32496, whereas more severe and widespread necrosis was observed in mice treated with 10 mg/kg BW of CEP32496.

Discussion

Canine urothelial carcinoma (UC) is characterized by a highly invasive nature, rapid growth, frequent distant metastasis, weak response to chemotherapy, and poor prognosis (1–3,5,8). Despite the high prevalence of V595E as a driver mutation (6,10,11), there are few reports on BRAF inhibitors as treatment for canine UC with V595E (10,17). In human malignant tumors with V600E, most BRAF inhibitors exert profound inhibitory effects below 200 nM of IC₅₀ (28,29).

In this study, the IC₅₀ of CEP32496 in the 5 UC cell lines ranged from 16 to 150 nM; however, those of 7 reference BRAF inhibitors ranged from 130 to ≥ 8000 nM, indicating different selectivity and sensitivity for individual cell lines that may be associated with the affinity and/or structure of the BRAF-activating conformation (10,30,31).

In each cell line, the IC₅₀ results of combined CEP32496 and vemurafenib were lower than those of the individual treatment. In addition, most CIs indicated synergism (CI < 1) between the 2 drugs (20,27). CEP32496 binds to the site adjacent to the ATP-binding pocket to maintain the inactive conformation of the BRAF kinase, whereas vemurafenib binds to the ATP-binding pocket in the active conformation of BRAF (32,33). The different binding sites of the 2 BRAF inhibitors may explain the synergism between the 2 drugs. Intraperitoneal treatment of xenograft tumors with CEP32496 resulted in dose-dependent effects. For example, the growth of xeno-grafted TCCV-Ka cells was significantly inhibited by 10 mg/kg BW of CEP32496. Histopathologically, necrosis was observed after the treatment with 5 mg/kg BW of CEP32496, whereas more severe and extensive findings were observed after treatment with 10 mg/kg BW.

Urothelial carcinoma (UC) cells expressing V595E, particularly TCCV-Ka, reportedly coincided with phosphorylated (activated) BRAF, similar to the acquired resistance to BRAF inhibitors (12,34). Apart from BRAF inhibitors, CEP32496 is a multi-kinase inhibitor (35) that is involved in the inhibition of receptor tyrosine kinase and/or MEK, both of which might have different inhibitory mechanisms from those of BRAF inhibitors in the MAPK signaling pathway. As CEP32496 has both activities of anti-BRAF and escape from acquired resistance of BRAF, combination treatment with CEP32496 and BRAF inhibitors might have a high potential to treat UC with V595E in dogs.

In conclusion, CEP32496 exhibited a sensitive IC₅₀ in all UC cell lines with V595E and synergism with vemurafenib. In addition, CEP32496 significantly inhibited the tumor growth of UC with V595E in a dose-dependent manner. These results indicated that CEP32496 has inhibitory effects on both cell proliferation and tumor growth that can potentially be effective for UC with V595E in dogs.