The oncogenic BRAF kinase inhibitor PLX4032/RG7204 does not affect the viability or function of human lymphocytes across a wide range of concentrations

Abstract

Purpose: PLX4032 (RG7204), an oncogenic BRAF kinase inhibitor undergoing clinical evaluation, has high response rates in early clinical trials in patients with advanced BRAF(V600E) mutant melanoma. Combining PLX4032 with immunotherapy may allow expanding the durability of responses. The effects of PLX4032 on immune cells were studied to explore the feasibility of future combinatorial approaches with immunotherapy for melanoma.

Experimental design: Peripheral blood mononuclear cells (PBMC) and BRAF(V600E) mutant melanoma cells were exposed to increasing concentrations of PLX4032 and the cell viability, proliferation, cell cycle, apoptosis, and phosphorylation of signaling proteins were analyzed. Effects of PLX4032 on antigen-specific T-cell function were analyzed by specific cytokine release and cytotoxicity activity.

Results: The 50% inhibition concentration (IC(50)) of PLX4032 for resting human PBMC was between 50 and 150 μmol/L compared with an IC(50) below 1 μmol/L for sensitive BRAF(V600E) mutant melanoma cell lines. Activated lymphocytes were even more resistant with no growth inhibition up to concentrations of 250 μmol/L. PLX4032 had a marginal effect on cell-cycle arrest, apoptotic cell changes or alteration of phosphorylated signaling molecules in lymphocytes. Functional analysis of specific antigen recognition showed preserved T-cell function up to 10-μmol/L concentration of PLX4032, whereas the cytotoxic activity of PLX4032 was maintained up to high concentrations of 50 μmol/L.

Conclusions: The preserved viability and function of lymphocytes exposed to high concentrations of PLX4032 suggest that this agent could be a potential candidate for combining with immunotherapy strategies for the treatment of patients with BRAF(V600)(E) mutant melanoma.

Fig. 1. Effects of PLX4032 on viability and proliferation of PBMC

A) Fifty percent inhibitory concentration (IC₅₀) values of PLX4032 against BRAF^V600E mutated melanoma cell lines with prior demonstration of sensitivity (M229) and resistance (M233) to PLX4032 (dark grey columns), compared to resting PBMC from three patients with metastatic melanoma (GA17, GA20, GA21, white columns), a healthy donor (H, grey column), and two samples of proliferating PBMC after activation with anti-CD3 and IL-2 (GA20-P and H-P, black columns). Data represents the mean of three experiments in triplictes. B) Quantification of Ki67-Ax700 staining by flow cytometry of PBMC cultured for 5 days in the presence of anti-CD3 and IL-2 and then exposed to PLX4032 or DMSO for 24 to 72 hours. Data represent the mean and s.e.m values from three independent replicate experiments.

Fig. 2. Analysis of cell cycle progression of melanoma cell lines and activated lymphocytes cultured in PLX4032

A) Two melanoma cell lines (M229, M233) and B) two PBMC cultures stimulated in anti-CD3 and IL-2 for 5 days as described in Figure 1 (GA20-P, H-P) were analyzed for cell cycle progression by DAPI staining using flow cytometry after exposure to PLX4032 for 72 hours. Ctrl= Control. Columns, means (n=3); bars, s.e.m; * p<0.05, **p<0.01.

Fig. 3. Apoptosis in response to PLX4032 exposure

A and B) Plasma membrane phospatidylserine exposure trough PI and Annexin V staining in PBMC (A) and melanoma cell lines (B) cultured in increasing concentrations of PLX4032 for 72 hours. C and D) Cleavage of PARP (PARP-Ax700) into fragment p85 in PBMC (C) and melanoma cell lines (D) exposed to PLX4032 as described above. SSP at 1μM was used as positive control. Columns, means (n=3); bars, s.e.m.

Fig. 4. Phosphorylation of cell signaling proteins in CD8+ T cells in the presence of increasing concentrations of PLX4032

Fold change compared to control in phosphoprotein levels for ERK1/2 (A), AKT (B), and p38 (C), as well as phosphorylated signal transducers and activators of transcription (STAT) proteins STAT1 (D), STAT3 (E), STAT5 (E) and STAT6 (F) in CD8+ T cells. P (filled triangles) represent five day proliferating PBMC. R (filled circles) represent resting PBMC. DMSO (0.0005%). Y-axis= percentage of fold change compared to controls. Points, means (n=3); bars, s.e.m.

Fig. 5. Specific IFN-γ secretion and lysis of HLA-A2.1 melanoma cells by the MART-1 TCR transgenic T cells

A to C) ELISA results showing IFN-γ secretion by MART-1 TCR transgenic T cells exposed to increasing concentrations of PLX4032 for 24 hours. A) M202 (MART-1+, HLA-A*0210+, black column), M238 (MART-1+, HLA-A*0210−, white column). B) M249 (MART-1+, HLA-A*0210−, white column), M249-A2.1 (MART-1+, HLA-A*0210+, black column). C) K562-A2.1 (MART-1-, HLA-A*0210+, white column), K562-A2.1 pulsed with MART-1_26–35 peptide (black column). Columns represent mean values from triplicate samples tested independently in three experiments; bars, s.d. D to F) Cr⁵¹ release assay of lytic activity against HLA-matched and -mismatched melanoma cells by MART-1 TCR transgenic cells exposed to 10μM PLX4032 for 24 hours. U = Untransduced; T = Transduced with MART-1 TCR retrovirus. Columns represent mean values from triplicate samples tested independently in four experiments; bars, s.d.