Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance

Abstract

Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥50% of tumours expressing the BRAF(V600E) oncoprotein. Moreover, the marked tumour regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance. However, as most patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is critical to providing improved therapy. Here we investigate the cause and consequences of vemurafenib resistance using two independently derived primary human melanoma xenograft models in which drug resistance is selected by continuous vemurafenib administration. In one of these models, resistant tumours show continued dependency on BRAF(V600E)→MEK→ERK signalling owing to elevated BRAF(V600E) expression. Most importantly, we demonstrate that vemurafenib-resistant melanomas become drug dependent for their continued proliferation, such that cessation of drug administration leads to regression of established drug-resistant tumours. We further demonstrate that a discontinuous dosing strategy, which exploits the fitness disadvantage displayed by drug-resistant cells in the absence of the drug, forestalls the onset of lethal drug-resistant disease. These data highlight the concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance. Such observations may contribute to sustaining the durability of the vemurafenib response with the ultimate goal of curative therapy for the subset of melanoma patients with BRAF mutations.

Figure 1. Resistance to vemurafenib in a primary human melanoma xenograft model

a, Mice bearing subcutaneous HMEX1906 tumours were dosed with vehicle (n = 10), 5 mg kg⁻¹ (n = 8), 15 mg kg⁻¹ (n = 8) or 45 mg kg⁻¹ (n = 10) vemurafenib twice daily (mean tumour volume ± s.e.m.). b, Continuous dosing of tumour-bearing mice over an extended time leads to the emergence of resistant tumours. The tumour circled in red was excised, subdivided and re-implanted to be used for further analysis. c, Parental tumours (n = 3 untreated and treated, mean pERK levels ± s.e.m. for the three different tumours) and resistant tumours were treated with 45 mg kg⁻¹ vemurafenib, and lysates were collected 3 h after the drug dose to measure pathway inhibition using pERK levels. d, The pharmacodynamics of pERK1/2 were evaluated over multiple time points for eight resistant tumours (red) and three parental tumours (blue).

Figure 2. Resistant tumours show increased BRAF(V600E) expression

a, BRAF protein level was determined by western blot (with actin as a loading control) in parental and resistant tumours (all lysates were collected 3 h after the drug dose). b, BRAF mRNA was measured by quantitative polymerase chain reaction with reverse transcription (RT–qPCR), (n = 3 untreated and treated independent parental tumours, BRAF mRNA levels ± s.e.m.). c, BRAF copy number was determined by qPCR of genomic DNA (n = 3 untreated and treated independent parental tumours, BRAF copy number levels ± s.e.m.).

Figure 3. Vemurafenib-resistant tumour cells require continuous exposure to vemurafenib

a, Parental (top) and vemurafenib-resistant (bottom) tumour-derived cells were imaged after 1 day (left), after 10 days (middle) in culture (0.05% dimethylsulphoxide (DMSO)), and after 10 days of culture in 0.05 μM vemurafenib (Vem; right). Original magnification, ×40. b, Parental and vemurafenib-resistant cells were treated with the indicated concentrations of vemurafenib and AZD6244 for 72 h, and viability was determined using the Cell Titer-Glo ATP-based luminescence assay, with DMSO-treated parental cells set as the control. c, A parallel plate similar to b was set up and corresponding pERK1/2 levels were measured from samples. d, BRAF protein level was determined in parental and resistant tumour cells by western blot. e, Resistant and parental tumour cells were subjected to BRAF siRNA, treated with vemurafenib or control (DMSO), and cell viability was determined by Cell Titer-Glo assay after 3 days of culture. NTC, non-targeting control. f, BRAF knockdown efficiency was determined by western blot with actin as loading control. g, Model correlating BRAF→MEK→ERK pathway activity and tumour-cell proliferation. b, c and e show mean percentage ± s.e.m., n = 6.

Figure 4. Intermittent dosing of vemurafenib can be exploited to forestall the development of drug resistance in vivo

a, Vemurafenib-resistant tumours were implanted and then mice were dosed with either vehicle or 45 mg kg⁻¹ vemurafenib twice daily (mean percentage ± s.e.m., n = 30) and monitored for tumour establishment over a period of 100 days. b, Vemurafenib-resistant tumours were implanted into nude mice and dosed with 45 mg kg⁻¹ vemurafenib twice daily immediately after implant. Once tumours reached a volume of ~1,500 mm³, mice were switched from vemurafenib to vehicle control (blue line), while one mouse remained on vemurafenib (red line). FNAs (purple arrows) were taken from the tumours before and after drug withdrawal to evaluate pERK. c, Lysates collected from the FNA were used to measure pERK, bars represent the pERK1/2 levels from seven different tumours (separated by dotted grey lines), while mice were dosed with vemurafenib (dark blue bars) or vehicle (light blue bars). The growth kinetics for each tumour is represented by the line graph above the pERK1/2 bars and FNA sampling is depicted by arrows (dark blue, on drug; light blue, off drug). d, Tumour growth kinetics of naive parental HMEX1906 tumours with seven tumours dosed continuously (top) and nine tumours dosed intermittently (bottom). Intermittent dosing of vemurafenib was carried out on a 4-week on drug (green arrow) and 2-week off drug (red arrow) schedule with 15 mg kg⁻¹ vemurafenib twice daily. e, Kaplan–Meier curve of data in d (n = 7, continuous dosing and n = 9, intermittent dosing) and Supplementary Fig. 6a (n = 7, continuous dosing and n = 8, intermittent dosing), shows that there is a significant survival advantage with an intermittent dosing (solid lines) compared to a continuous dosing schedule (dashed lines). The end point for euthanasia was predetermined as a tumour size of 1,200 mm³.