Host immunity contributes to the anti-melanoma activity of BRAF inhibitors

Abstract

The BRAF mutant, BRAF(V600E), is expressed in nearly half of melanomas, and oral BRAF inhibitors induce substantial tumor regression in patients with BRAF(V600E) metastatic melanoma. The inhibitors are believed to work primarily by inhibiting BRAF(V600E)-induced oncogenic MAPK signaling; however, some patients treated with BRAF inhibitors exhibit increased tumor immune infiltration, suggesting that a combination of BRAF inhibitors and immunotherapy may be beneficial. We used two relatively resistant variants of Braf(V600E)-driven mouse melanoma (SM1 and SM1WT1) and melanoma-prone mice to determine the role of host immunity in type I BRAF inhibitor PLX4720 antitumor activity. We found that PLX4720 treatment downregulated tumor Ccl2 gene expression and decreased tumor CCL2 expression in both Braf(V600E) mouse melanoma transplants and in de novo melanomas in a manner that was coincident with reduced tumor growth. While PLX4720 did not directly increase tumor immunogenicity, analysis of SM1 tumor-infiltrating leukocytes in PLX4720-treated mice demonstrated a robust increase in CD8(+) T/FoxP3(+)CD4(+) T cell ratio and NK cells. Combination therapy with PLX4720 and anti-CCL2 or agonistic anti-CD137 antibodies demonstrated significant antitumor activity in mouse transplant and de novo tumorigenesis models. These data elucidate a role for host CCR2 in the mechanism of action of type I BRAF inhibitors and support the therapeutic potential of combining BRAF inhibitors with immunotherapy.

Figure 1. PLX4720 activity against melanoma in vitro and in vivo.

(A) To determine proliferation IC₅₀ in vitro, BRAF^V600E mutant SM1 and SM1WT1 melanoma cells were seeded in 96-well plates and allowed to proliferate for 48 hours. Cells were then treated with a range of PLX4720 concentrations (n = 3). After 72 hours, cell number was determined using the sulforhodamine B method. IC₅₀ concentrations were determined using nonlinear regression (mean ± SEM). (B) PLX4720 and target pathways. SM1WT1 mouse melanoma cells were treated with 1 to 30 μM PLX4720 for 6 hours, after which cells were harvested and lysed (n = 3). Proteins were separated by SDS-PAGE, and ERK and pERK protein bands were visualized by immunoblotting (β-actin was used as a loading control). (C) PLX4720 is active in vivo against SM1 and SM1WT1 melanomas. Groups of 5 WT mice were inoculated with 2 × 10⁶ SM1 cells or 5 × 10⁵ SM1WT1 cells. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 3 to 6 or day 3 to 10 after tumor inoculation, respectively. Tumor sizes are represented as the mean ± SEM. Data are representative of 2 independent experiments. (D) Tumors were harvested from mice prior to drug treatment or after 4 days of PLX4720 treatment. Tumor cell lysates were prepared and proteins were separated by SDS-PAGE. ERK and pERK protein bands were visualized by immunoblotting (β-actin was used as a loading control). Each lane corresponds to an individual tumor.

Figure 2. PLX4720 suppresses tumor CCL2 release.

After 18 to 24 hours in vitro culture, supernatants from in vitro culture were collected for CCL2 analysis. Supernatant concentrations of CCL2 are presented. (A) 5 × 10⁴ SM1 and SM1WT1 cells were cultured in the presence of vehicle or 10 μM PLX4720. Experiments were performed in replicates of 5 wells. (B) Groups of B6 WT mice (n = 5–6) were inoculated with 1 × 10⁶ SM1WT1 cells. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 12 to 15 after tumor inoculation. At day 16, tumors were excised and tumor single cell suspensions were prepared. (C) Groups of Braf^V600E transgenic mice (n = 6–7) were induced for localized melanoma. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 28 to 49 after 4-HT application. At day 49, tumors were excised and tumor single cell suspensions were prepared. (A and B) 1 × 10⁵ tumor cells suspended in 100 μl volume were plated. (B and C) Experiments were performed in 1 well per tumor. (A) Statistical differences in CCL2 concentrations between vehicle- or PLX4720-treated SM1 and SM1WT1 cell lines were determined by an unpaired t test (***P < 0.001). (B) Statistical differences in CCL2 concentrations between vehicle- or PLX4720-treated SM1WT1 tumors were determined by an unpaired t test (**P < 0.01). (C) Statistical differences in CCL2 concentrations between vehicle- or PLX4720-treated Braf^V600E transgenic tumors were determined by an unpaired t test (*P < 0.05). (A–C) Data shown are representative of 2 independent experiments (mean ± SEM).

Figure 3. PLX4720 antitumor activity is host CCR2 dependent.

(A) Groups of 5 WT, Ccl2^–/–, or Ccr2^–/– mice were inoculated with 5 × 10⁵ SM1WT1 cells. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 3 to 10 after tumor inoculation. (B and C) Groups of 5 WT or Ccl2^–/– mice were inoculated with 5 × 10⁵ SM1WT1 cells. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 3 to 10 after tumor inoculation. Some groups of mice additionally received cIg or anti-CCL2 mAb (20 μg i.p.) on days 2, 3, 10, 17, and 24. Tumor sizes are represented as the mean ± SEM. Statistical differences in tumor sizes between mice treated with vehicle or PLX4720 therapy were determined by a Mann-Whitney test (*P < 0.05). (A–C) Data are representative of 2 independent experiments.

Figure 4. CCR2⁺ TILs.

A group of B6 WT mice (n = 10) was inoculated with 1 × 10⁶ SM1WT1 cells. At day 21, tumors were excised and FACS analyses were performed on TILs. Frequencies of CCR2⁺ cells in (A) CD11b⁺ cells, (B) NK cells, (C) CD8⁺ T cells, and (D) CD4⁺Foxp3⁺ Tregs (top 2 right-most images) and CD4⁺Foxp3^– T cells (bottom 2 images) from TILs are shown. The data shown for CCR2⁺ cells are FACS plots concatenated from 10 individual mice.

Figure 5. PLX4720 enhances the intratumor CD8⁺ T cell/Treg ratio and proportion of NK cells.

Groups of B6 WT mice (n = 5–6) were inoculated with 1 × 10⁶ SM1WT1 cells. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 12 to 20 after tumor inoculation. At day 21, tumors were excised, and FACS analyses were performed on TILs. Frequencies of (A) NK cells, (B) total T cells, (C) CD8⁺ T cells, (D) CD4⁺ T cells, (E) CD4⁺FoxP3⁺ Tregs, and (F) CD11b⁺ Gr-1⁺ cells, gated on (A–E) CD45.2⁺ or (F) CD45.2⁺ CD3^– CD19^– TILs from vehicle- or PLX4720-treated mice, are shown. (G) The CD8⁺ T/Treg ratio calculated from C and E as shown. Statistical differences in (A–F) frequencies of respective cell subsets or (G) cell ratios between mice treated with vehicle or PLX4720 therapy were determined by an unpaired t test (*P < 0.05; **P < 0.01; ***P <0.001). Data shown are pooled from (F) 2 or (A–E and G) 3 independent analyses. Individual symbols represent individual mice; horizontal bars indicate the mean. Live CD45.2⁺ TIL numbers ranged from 0.32 × 10³ cells/mm² to 2.86 × 10³ cells/mm², with no statistical significance observed between vehicle- or PLX4720-treated mice.

Figure 6. PLX4720 antitumor activity is CD8⁺ T cell dependent.

(A) Groups of 5 WT mice were inoculated with 5 × 10⁵ SM1WT1 cells. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 3 to 10 after tumor inoculation. Some groups of mice were additionally treated with cIg, anti-CD4, anti-CD8β, or anti-asialoGM1 (100 μg i.p. each) on days 2, 3, 10, 17, and 24 after tumor inoculation to deplete T cell subsets or NK cells. (B) Groups of 5 WT, Ifng^–/–, or Pfp^–/– mice were inoculated with 5 × 10⁵ SM1WT1 cells. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 3 to 10 after tumor inoculation. Some groups of Pfp^–/– mice were additionally treated with anti–IFN-γ (250 μg i.p.) on days 2, 3, 10, 17, and 24 after tumor inoculation to neutralize IFN-γ. (C) Groups of 5 WT, gld, or Trail^–/– mice were inoculated with 5 × 10⁵ SM1WT1 cells. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 3 to 10 after tumor inoculation. Tumor sizes are represented as the mean ± SEM. Statistical differences in tumor sizes between mice treated with vehicle and those treated with PLX4720 therapy for each group were determined by a Mann-Whitney test (*P < 0.05; **P < 0.01). Data are representative of 2 independent experiments.

Figure 7. Synergistic antitumor activity of PLX4720 and anti-CD137.

Groups of 5 WT mice were inoculated with 5 × 10⁵ SM1WT1 cells. Mice received (A) vehicle or (B) PLX4720 (20 mg/kg i.p.) daily from day 7 to 11 after tumor inoculation. Some groups of mice were additionally treated with cIg, anti–CTLA-4, anti–PD-1, anti-Tim3, or anti-CD137 (250 μg i.p. each) on days 12, 14, 16, and 18 after tumor inoculation. (C and D) Groups of 5 WT mice were inoculated with 5 × 10⁵ SM1WT1 cells. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 12 to 16 after tumor inoculation and cIg or anti-CD137 (100 μg i.p.) on days 17, 19, 21, and 23 after tumor inoculation. Some groups of WT mice were additionally treated with anti–IFN-γ (250 μg i.p.) or anti-CD8β (100 μg i.p.) on days 11, 12, 19, and 26 after tumor inoculation. Tumor sizes are represented as the mean ± SEM. Statistical differences in tumor sizes between mice treated with control versus combination therapy for each group were determined by a Mann-Whitney test (*P < 0.05).

Figure 8. Combination antitumor activity of PLX4720/anti-CD137 against de novo BRAF^V600E-driven mouse melanomas.

Groups of Braf^V600E transgenic mice (n = 6–7) were induced for localized melanoma by 4-HT application on day 0. Mice received vehicle or PLX4720 (20 mg/kg i.p.) daily from day 28 to 49. Some groups of mice additionally received cIg or anti-CD137 (100 μg i.p.) on days 28, 32, 36, 40, 44, and 48. (A) At the indicated time, tumor sizes (height; mm) were recorded and represented as the mean ± SEM. Statistical differences in tumor sizes between different groups of mice were determined by a Mann-Whitney test (**P < 0.01; ^#P < 0.0001). (B) At day 49, tumors were excised and weighed. Statistical differences in tumor weights between different groups of mice were determined by a Mann-Whitney test (*P < 0.05, PLX4720 and clg compared with vehicle and anti-CD137; ^#P < 0.001, vehicle and clg compared with PLX4720 and clg [P = 0.0004] or PLX4720 and anti-CD137 [P = 0.0001] and vehicle and anti-CD137 compared with PLX4720 and anti-CD137 [P = 0.0008]). Data shown are pooled from 2 independent experiments. Individual symbols represent individual tumors; horizontal bars indicate the mean.

Figure 9. Anti-CCL2 and anti-CD137 also suppress SM1WT1 tumor growth.

Groups of 5 WT mice were inoculated with 5 × 10⁵ SM1WT1 cells. Mice received a first treatment course of vehicle or PLX4720 (20 mg/kg i.p.) daily from day 12 to 16 or cIg or anti-CCL2 (20 μg i.p.) on days 12, 14, 16, and 18 after tumor inoculation. This was sequentially followed by cIg or anti-CD137 (100 μg i.p.) on days 17, 19, 21, and 23 after tumor inoculation. Tumor sizes are represented as the mean ± SEM. Statistical differences in tumor sizes between mice treated with control versus combination therapy for each group were determined by a Mann-Whitney test (*P < 0.05).