Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032

Abstract

Purpose: To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells.

Materials and methods: A large collection of melanoma cell lines (n=37) were treated with 0-8Gy IR and clonogenic survival assays used to generate survival curves to rank relative radiosensitivities among the cell lines. The ability of a B-RAF inhibitor, PLX-4032, to radiosensitize highly radioresistant B-Raf+ cells was also assessed by clonogenic cell survival and spheroid invasion assays and the effects of treatment on the cell cycle assessed by FACS.

Results: Melanoma cell lines displayed a very large, heterogeneous range of SF2 values (1.002-0.053) with a mean of 0.51. Cell lines with surviving fractions of 0.29 or less at SF2 and SF4 were observed at a high frequency of 18.9% and 70.2%, respectively. Treatment of B-Raf+ cells with the B-RAF inhibitor PLX-4032 in combination with radiation provided enhanced inhibition of both colony formation and invasion, and radiosensitized cells through an increase in G(1) arrest.

Conclusions: Our data suggest that melanomas are not uniformly radioresistant with a significant subset displaying inherent radiosensitivity. Pharmacologic inhibition of B-RAF with PLX-4032 effectively radiosensitized B-Raf+ melanoma cells suggesting that this combination approach could provide improved radiotherapeutic response in B-Raf+ melanoma patients.

FIG. 1. A significant subset of melanoma cell lines are radiosensitive

The percentage of cell lines with surviving fractions in the ranges indicated are shown for SF2 and SF4. A significant number of lines are radiosensitive with surviving fractions of 0.29 or less at SF2 and SF4 seen in 18.9 % and 70.2 %, respectively, of the cell lines.

FIG. 2. Melanoma survival curves

(A) Survival curves over 0–8 Gy were generated for 37 melanoma all cell lines and relative radioresistance subgrouped as low (), medium (), or high (). (B) Statistical comparisons of relative radioresistance among B-Raf+, N-Ras+, or BRaf/N-Ras WT melanoma cell lines. Fisher's exact test showed no significant differences between genotypes (P=0.49).

FIG. 3. PLX-4032 radiosensitizes B-Raf+ melanoma cells

(A) B-Raf+ cells (SKMel27, SKMel100, SK-Mel-181, SK-Mel-28) were fed fresh media containing DMSO or PLX-4032 for 48 h at the indicated concentrations to achieve partial inhibition with drug alone, irradiated at 6 Gy, trypsinized 2 hr post-irradiation and replated as single cells and colonies stained and counted after 2–3 weeks incubation. Surviving fractions were calculated by comparison to DMSO control cells. Control B-Raf/N-Ras WT (SKMel131) and N-Ras+ (SKMel119 and VMM39) cells were pretreated similarly with the highest dose of PLX-4032 (10.0 μM). (B) Enhancement ratios (ER) = SF radiation alone/PLX-4032 + radiation. (C) Protein lysates were collected from cells treated with PLX-4032 for 48 h at the concentrations described in (A) and western blot analyses performed with anti-pERK1/2 or anti-ERK1/2 as a loading control to show the levels of pERK1/2 at the time of radiation.

FIG. 3. PLX-4032 radiosensitizes B-Raf+ melanoma cells

(A) B-Raf+ cells (SKMel27, SKMel100, SK-Mel-181, SK-Mel-28) were fed fresh media containing DMSO or PLX-4032 for 48 h at the indicated concentrations to achieve partial inhibition with drug alone, irradiated at 6 Gy, trypsinized 2 hr post-irradiation and replated as single cells and colonies stained and counted after 2–3 weeks incubation. Surviving fractions were calculated by comparison to DMSO control cells. Control B-Raf/N-Ras WT (SKMel131) and N-Ras+ (SKMel119 and VMM39) cells were pretreated similarly with the highest dose of PLX-4032 (10.0 μM). (B) Enhancement ratios (ER) = SF radiation alone/PLX-4032 + radiation. (C) Protein lysates were collected from cells treated with PLX-4032 for 48 h at the concentrations described in (A) and western blot analyses performed with anti-pERK1/2 or anti-ERK1/2 as a loading control to show the levels of pERK1/2 at the time of radiation.

FIG. 4. Radiosensitization by PLX-4032 correlates with an increase of cells in G₁

Melanoma cells were pretreated with PLX-4032 (as in Fig. 3) or DMSO for 48 h, cells irradiated with 6 Gy or sham-irradiated, and DNA content assessed by FACS analyses 24 h post-irradiation. Shown are percentages of cells in each stage of the cell cycle.