The Benefit of Reactivating p53 under MAPK Inhibition on the Efficacy of Radiotherapy in Melanoma

Abstract

Radiotherapy (RT) in patients with melanoma historically showed suboptimal results, because the disease is often radioresistant due to various mechanisms such as scavenging free radicals by thiols, pigmentary machinery, or enhanced DNA repair. However, radiotherapy has been utilized as adjuvant therapy after the complete excision of primary melanoma and lymph nodes to reduce the rate of nodal recurrences in high-risk patients. The resistance of melanoma cells to radiotherapy may also be in relation with the constitutive activation of the MAPK pathway and/or with the inactivation of p53 observed in about 90% of melanomas. In this study, we aimed to assess the potential benefit of adding RT to BRAF-mutated melanoma cells under a combined p53 reactivation and MAPK inhibition in vitro and in a preclinical animal model. We found that the combination of BRAF inhibition (vemurafenib, which completely shuts down the MAPK pathway), together with p53 reactivation (PRIMA-1^Met) significantly enhanced the radiosensitivity of BRAF-mutant melanoma cells. This was accompanied by an increase in both p53 expression and activity. Of note, we found that radiation alone markedly promoted both ERK and AKT phosphorylation, thus contributing to radioresistance. The combination of vemurafenib and PRIMA-1^Met caused the inactivation of both MAPK kinase and PI3K/AKT pathways. Furthermore, when combined with radiotherapy, it was able to significantly enhance melanoma cell radiosensitivity. Interestingly, in nude mice bearing melanoma xenografts, the latter triple combination had not only a synergistic effect on tumor growth inhibition, but also a potent control on tumor regrowth in all animals after finishing the triple combination therapy. RT alone had only a weak effect. In conclusion, we provide a basis for a strategy that may overcome the radioresistance of BRAF-mutated melanoma cells to radiotherapy. Whether this will translate into a rational to use radiotherapy in the curative setting in BRAF-mutated melanoma patients deserves consideration.

Keywords: V600EBRAF inhibition; intrinsic and acquired resistance; melanoma; p53 activation; radiotherapy.

Figure 1

Combining p53 activation and B-Raf proto-oncogene serine/threonine kinase (BRAF) inhibition sensitizes melanoma BRAF mutant melanoma cells to irradiation. (A) In the workflow, cells were cultured on day 0, effectors were added on day 1, and radiotherapy (RT) was done on day 2. Western blot (WB) analysis followed two days after RT (day 4). Cell death and colony formation were evaluated on days 7 and 14, respectively. Effectors with fresh medium were changed every three days. (B) Clonogenic survival assay of human melanoma cell lines with intrinsic resistance (MM043) and acquired resistance (MM074-R) to vemurafenib with different irradiation doses (2 Gy, 5 Gy, and 10 Gy) alone or in combination with vemurafenib (Vemu, 0.1 µM) and/or PRIMA-1^Met (PRIMA-1^Met, 20 µM). Surviving fractions were calculated relative to plating efficiencies. Data were presented as the mean ± standard error of at least three independent experiments. Gy, Gray; CTR: untreated control; Vemu, vemurafenib; p53 Reactivation and Induction of Massive Apoptosis (PRIMA), PRIMA-1^Met; V + P: vemurafenib + PRIMA-1^Met. * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t-test) compared to RT alone. (C) The interaction between Vemu, PRIMA, and RT was examined using the combination index (CI) method of Chou and Talalay and CompuSyn software. CI = 1, additive effect, CI <1, synergism, CI >1, antagonism. (D) Cell death (apoptosis (annexin-V positive cells) + necrosis (7-AAD positive cells) analysis for irradiated and non-irradiated cells treated with 1 µM of vemurafenib and/or 40 µM of PRIMA-1^Met. Data are presented as means ± SD (n = 3) compared to non-irradiated cells, *** p < 0.001 (Student’s t-test). SD, standard deviation.

Figure 1

Combining p53 activation and B-Raf proto-oncogene serine/threonine kinase (BRAF) inhibition sensitizes melanoma BRAF mutant melanoma cells to irradiation. (A) In the workflow, cells were cultured on day 0, effectors were added on day 1, and radiotherapy (RT) was done on day 2. Western blot (WB) analysis followed two days after RT (day 4). Cell death and colony formation were evaluated on days 7 and 14, respectively. Effectors with fresh medium were changed every three days. (B) Clonogenic survival assay of human melanoma cell lines with intrinsic resistance (MM043) and acquired resistance (MM074-R) to vemurafenib with different irradiation doses (2 Gy, 5 Gy, and 10 Gy) alone or in combination with vemurafenib (Vemu, 0.1 µM) and/or PRIMA-1^Met (PRIMA-1^Met, 20 µM). Surviving fractions were calculated relative to plating efficiencies. Data were presented as the mean ± standard error of at least three independent experiments. Gy, Gray; CTR: untreated control; Vemu, vemurafenib; p53 Reactivation and Induction of Massive Apoptosis (PRIMA), PRIMA-1^Met; V + P: vemurafenib + PRIMA-1^Met. * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t-test) compared to RT alone. (C) The interaction between Vemu, PRIMA, and RT was examined using the combination index (CI) method of Chou and Talalay and CompuSyn software. CI = 1, additive effect, CI <1, synergism, CI >1, antagonism. (D) Cell death (apoptosis (annexin-V positive cells) + necrosis (7-AAD positive cells) analysis for irradiated and non-irradiated cells treated with 1 µM of vemurafenib and/or 40 µM of PRIMA-1^Met. Data are presented as means ± SD (n = 3) compared to non-irradiated cells, *** p < 0.001 (Student’s t-test). SD, standard deviation.

Figure 2

Mitogen-activated protein kinase (MAPK) kinase and PI3K pathways stimulation confers resistance to radiotherapy (RT) in BRAF mutant melanoma cells in vitro. Effect of RT with 2 Gy, 5 Gy, and 10 Gy in combination with 1 µM of vemurafenib and/or 20 µM of PRIMA-1^Met on the PI3K/AKT, MAPK, and p53/p21 pathways. (A) Cells with intrinsic resistance to vemurafenib (MM043) and (B) MM074-R with acquired resistance to vemurafenib. β-actin was used as loading control.

Figure 3

p53 activator (PRIMA-1^Met) and BRAF inhibitor (vemurafenib) radiosensitise ^V600EBRAF mutant melanoma in vivo. (A) Swiss nude mice (nu/nu) were injected by either MM043 and treated with CTR (DMSO), vemurafenib (45 mg/kg), PRIMA-1^Met (50 mg/kg), or vemurafenib and PRIMA-1^Met. One day later, the mice were irradiated on the right leg. Data are presented as means ± SEM (n = 9) compared to untreated tumors (B) Tumor volume of mice, non-irradiated (left panel), irradiated (right panel), untreated or treated with vemurafenib and/or PRIMA-1^Met in the period between D0 and D36. ** p < 0.01, *** p < 0.001 (two-way ANOVA) compared to non-irradiated.

Figure 3

p53 activator (PRIMA-1^Met) and BRAF inhibitor (vemurafenib) radiosensitise ^V600EBRAF mutant melanoma in vivo. (A) Swiss nude mice (nu/nu) were injected by either MM043 and treated with CTR (DMSO), vemurafenib (45 mg/kg), PRIMA-1^Met (50 mg/kg), or vemurafenib and PRIMA-1^Met. One day later, the mice were irradiated on the right leg. Data are presented as means ± SEM (n = 9) compared to untreated tumors (B) Tumor volume of mice, non-irradiated (left panel), irradiated (right panel), untreated or treated with vemurafenib and/or PRIMA-1^Met in the period between D0 and D36. ** p < 0.01, *** p < 0.001 (two-way ANOVA) compared to non-irradiated.

Figure 4

MAPK kinase and PI3K pathways reactivation confers resistance of BRAF mutant melanoma cells to radiation in a nude mice model. (A) Immunohistochemistry staining for Ki67, p53, p21, pERK, and pAKT in non-irradiated cells and irradiated cells, untreated or treated with vemurafenib (45 mg/kg) and/or PRIMA-1^Met (50 mg/kg). (B) Relative expression of immunohistochemistry data reporting Ki67, p53, p21, pERK, and pAKT compared to control. Data are presented as means ± SEM from three mice compared to untreated tumors (CTR). The level of significance is indicated by * p < 0.05 (Student’s t-test).