Abstract 692: A novel resistance mechanism to PLX4032 in the human melanoma cell line MEL-XY3 involves acquisition of stem cell-like characteristics
Authors: Madorsky Florencia , Barón Antonela , Mordoh José 
Published: May 2015

Abstract
Activating BRAF mutations, in particular V600E, occurs in approximately 50% of cutaneous melanomas. PLX4032 (vemurafenib), an ATP-competitive BRAF inhibitor, displays remarkable activity, leading to an increase in progression-free and overall survival in patients with BRAF V600E mutated melanoma, pointing to a strong dependence of this tumor on the MAP kinase pathway. However, patients treated with this drug almost invariably recur, and several resistance mechanisms have already been described. In this work, we have investigated the effects of long-term treatment (5 weeks - 5 months) with PLX4032 in the human melanoma cell line MEL-XY3. The sensitivity of the heterozygous, BRAF mutated (V600E) parental MEL-XY3 cells (XY3 wt) to PLX4032 was investigated, and the IC50 was &lt;0.25 μM. When XY3 wt cells were grown in the presence of 10 μM PLX4032 for periods up to five months, most of the cells died. However, some clusters of flattened, quiescent cells remained viable; we named such cells MEL-XY3 SUR. After removing PLX4032 from the culture medium, MEL-XY3 SUR were able to resume growth and their sensitivity to the drug was equal to XY3 wt. We sought to investigate if such quiescent cells presented stem cell-like characteristics. We found that the expression of the stem cell marker CD271 (NGFR) increased 100 - 1.000 fold as determined by RT-qPCR, and the increase in CD271 expression was confirmed by flow cytometry, immunofluorescence and western blot. Expression of the putative stem cell marker ABCB5 also increased around 25-fold as measured by RT-qPCR, but expression of CD133 was reduced 50-300 fold. Coincidently, in some plates maintained with 10 μM PLX4032, sparsed colonies with outgrowth of cellular masses appeared. When such colonies were isolated, they grew rapidly and were 200-fold more resistant to PLX4032 than XY3 wt, with an IC50 around 50 μM. Consequently, such cells, that we named MEL-XY3 RES, display more conventional resistance mechanisms. Therefore, at least two resistance mechanisms may appear after long term exposure to PLX4032: one which probably involves a shift to non-growing, surviving cells with stem cell-like characteristics and which retain the parental cell line sensitivity to the drug, and another which may involve acquisition of resistance mechanisms that bypass the PLX4032 inhibition of BRAF. We hypothesize that the stem cell-like surviving mechanism would be relevant to allow regrowth after discontinuation of PLX4032 treatment, whereas the conventional resistance mechanism would explain tumor escape during treatment. It does not escape notice that long-term culture in the presence of PLX4032 could be a method to select stem-like cells from BRAF-mutated tumor cells.
Citation Format: Florencia Madorsky, Antonela Barón, José Mordoh. A novel resistance mechanism to PLX4032 in the human melanoma cell line MEL-XY3 involves acquisition of stem cell-like characteristics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 692. doi:10.1158/1538-7445.AM2014-692