Acquired resistance to BRAFi reverses senescence-like phenotype in mutant BRAF melanoma

Abstract

Targeting MAPK pathway in mutant BRAF melanoma with the specific BRAF inhibitor vemurafenib showed robust initial responses in the majority of patients followed by relapses due to acquired resistance to the drug. In ^V600EBRAF melanoma cell lines, senescence-associated β-galactosidase activity is often encountered in a constitutive manner or induced after MAPK inhibition. However, the link between the senescence-like phenotype and the resistance to BRAF inhibition is not fully understood yet. Our data validate a senescence-like phenotype (low cell proliferation, high cell volume, and high β-Gal activity) in mutant BRAF cells. Vemurafenib increased β-Gal activity in 4 out of 5 sensitive lines and in 2 out of 5 lines with intrinsic resistance to the drug. Interestingly, the 3 lines with acquired resistance to vemurafenib became depending on the drug for proliferation. In absence of drug, these lines showed a lower cell proliferation rate together with a substantial increase of β-Gal activity both in vitro and in vivo. In all settings, the senescence-like phenotype was significantly associated with an inhibition of pRB and cyclin D1, explaining the inhibition of cell proliferation. In conclusion, β-Gal activity is increased by ^V600EBRAF inhibition in the majority of sensitive and intrinsically resistant melanoma cells. Acquired resistance to vemurafenib is associated with a dependence to the drug for cell proliferation and tumor growth, and, in this case, drug removal stimulate β-Gal activity suggesting that the senescence-like phenotype could contribute to the acquired resistance to BRAF inhibition.

Keywords: V600EBRAF; acquired resistance; melanoma; senescence; vemurafenib.

Figure 1. Effect of ^V600EBRAF mutation on ERK and AKT signaling, cell proliferation, β-Gal activity and cell morphology

(A) proliferation rate assessed by crystal violet in mutant BRAF melanoma cell lines (MM032, MM043, MM050, MM074, MM164 and SKMEL-28) in comparison with wild BRAF cell lines (HBL and LND1). (B) Constitutive phosphorylation and expression levels of ERK and AKT assessed by Western blotting. β-actin is used as loading control. (C) β-Gal activity by in situ staining of indicated cell lines. (D) Detection of fluorescein-di-beta-D-galactopyranoside (FDG), a substrate for β-galactosidase, by flow cytometry in mutant BRAF melanoma lines in comparison with wild type lines. (E) Phase contrast images of mutant BRAF melanoma cell lines of indicated cell lines. (F) Box plot representing the cell volume of indicated cell lines.

Figure 2. Effect of BRAF inhibition by vemurafenib on cell growth and death in sensitive and intrinsically resistant mutant BRAF melanoma cell lines

(A) Growth inhibition IC50 values after 3 days of vemurafenib exposure in the 10 mutant BRAF melanoma lines. (B) Cytotoxic effect of vemurafenib (0.1 µM) as determined by cell counting at day 15 relative to day 0. Data shown are derived from three independent experiments. Means ± SEM are indicated. ^*p < 0.05, ^***p < 0.001 (Student’s t-test). (C) Apoptosis induced by cell exposure to 0.1 µM of vemurafenib (vemu) for 15 days as evaluated by the percentage of annexin V-positive cells. Data are presented as means ± SEM (n = 3) compared to untreated cells (CTR). ^***p < 0.001 (Student’s t-test).

Figure 3. Effect of BRAF inhibition by vemurafenib on β-Gal activity and signaling pathways in sensitive and intrinsically resistant mutant BRAF melanoma cell lines

(A) Evaluation of β-Gal activity by in situ staining of indicated cell lines exposed or not to vemurafenib (0.1 µM) for 14 days. (B) The activity of β-gal was quantified by the rate of conversion of ortho-nitrophenyl-β-D-galactopyrannoside (ONPG) in mutant BRAF melanoma lines after 14 days of treatment with vemurafenib (0.1 µM). Data are presented as means ± SEM (n = 3) compared to untreated cells (CTR). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (Student’s t-test). (C) Constitutive phosphorylation and expression levels of ERK, AKT, pRB and cyclin D1 assessed by Western blot analysis of the indicated melanoma cell lines after 24 hours of treatment with 1 µM vemurafenib. β-actin is used as loading control.

Figure 4. Effect of acquired resistance to BRAF inhibition by vemurafenib on cell proliferation, apoptosis and β-Gal activity

(A) Scheme presenting the establishment of MM050-R, MM074-R and SKMEL-28-R lines with acquired resistance to vemurafenib by exposing parental MM050, MM074 and SKMEL-28 lines to increasing concentrations (0.1-2 µM) of the drug over 12 weeks. (B) Growth inhibition IC50 values after 3 days of vemurafenib exposure in parental and resistant cell lines. (C) Time kinetics of cell proliferation in parental and acquired resistance cell lines exposed to 2 µM vemurafenib (+vemu) or after 14 days of washing out of vemurafenib (w/o vemu) for 1, 2, 3 and 4 days. (D) Apoptosis as evaluated by the percentage of annexin V-positive cells in acquired resistance cell (R) under 2 µM vemurafenib and in cells after washing out the drug (w/o) in comparison with parental sensitive cells (C). (E) Evaluation of β-Gal activity by in situ staining in parental and acquired resistance cell lines under 2 µM vemurafenib (+vemu) or after 14 days of washing out vemurafenib (w/o vemu). (F) The activity of β-gal was quantified by the rate of conversion of ortho-nitrophenyl-β-D-galactopyrannoside (ONPG) in both parental and acquired resistance cell lines exposed to 2 µM vemurafenib (+vemu) or after 14 days of washing out vemurafenib (w/o vemu).

Figure 5. Drug addiction/dependency phenomenon in acquired resistance cells to vemurafenib

(A) Western blots illustrating the evaluation of pERK, ERK, pAKT, AKT, PTEN, cyclin D1, pRB, p53, p21 and ^V600EBRAF (VE1) in parental sensitive line (MM074) and in line with acquired resistance (MM074-R) line treated with 2 µM vemurafenib (vemu) or after 14 days of washing out vemurafenib (w/o). β-actin is used as loading control. (B) Growth curves for tumors grafted in mice with parental and resistant cells untreated as control and (C) treated with vemurafenib (45 mg/kg). Data are presented as means tumor volumes (mm³) ± SEM. (D) Senescence and proliferation markers in xenograft tumors. Tumors were analyzed for their histological appearance of from hematoxylin and eosin staining, the activity of β-Gal in frozen tissues, and the expression of the proliferation marker Ki67 in paraffin-embedded tissues. Positive cells were counted for each group and means ± SEM.