Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation

Abstract

Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ∼7% of human malignancies and ∼60% of melanomas. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.

Figure 1. In vitro models of PLX4032 acquired resistance display differential MAPK reactivation

a, Parental and PLX4032-resistant sub-lines were treated with increasing PLX4032 concentration (0, 0.01, 0.1, 1 and 10 μM), and the effects on MAPK signalling were determined by immunoblotting for p-MEK1/2 and p-ERK1/2 levels. Total MEK1/2, ERK1/2 and tubulin levels, loading controls. b, Heat map for B-RAF(V600E) signature genes in each of the cell lines treated with DMSO or PLX4032. Colour scale, log₂-transformed expression (red, high; green, low) for each gene (row) normalized by the mean of all samples. Blue box showing M249 R4 MAPK reactivation. Yellow box showing diminished, baseline expression of B-RAF(V600E) signature genes in M229 and M238 resistant sub-lines (FDR <0.05). The probeset number is shown after each gene.

Figure 2. PDGFRβ upregulation is strongly correlated with PLX4032 acquired resistance

a, Left, total levels of PDGFRβ and EGFR. A431, an EGFR-amplified cell line. Tubulin levels, loading control. Right, whole-cell extracts were incubated on the RTK antibody arrays, and phosphorylation status was determined by subsequent incubation with anti-phosphotyrosine horseradish peroxidase (each RTK spotted in duplicate, positive controls in corners, gene identity below). b, Anti-PDGFRβ immunohistochemistry of formalin-fixed, paraffin-embedded tissues. Prostate, negative control; placenta, positive control. Black bar, 50 μm. c, Relative RNA levels of PDGFRβ in M229 P/R5 and Pt48 R as determined by real-time, quantitative PCR (average of duplicates). d, Total PDGFRβ (left) and p-RTK (right) levels in Pt48 R versus M229 R5.

Figure 3. N-RAS upregulation correlates with a distinct subset of PLX4032 acquired resistance

a, Detection of a N-RAS(Q61K) allele in M249 R4 and Pt55 R. b, The levels of activated RAS (aRAS) and N-RAS (aN-RAS) eluted after pull-down using the RAS-binding domain (RBD) of RAF-1. The total levels of RAS, N-RAS, PDGFRβ and tubulin (loading control) from the whole-cell lysates are shown by immunoblotting. Effects of GDP and GTPγS pre-incubation on RBD pull-down and beads without RBD pull-down from Pt48 R lysates are shown as controls.

Figure 4. PDGFRβ- and N-RAS-mediated growth and survival pathways differentially predict MEK inhibitor sensitivity

a, Transduction of PDGFRβ shRNAs in M229 R5 and M238 R1 (1 μM PLX4032), RNA (relative to GAPDH) and protein knockdown, effects on p-ERK levels, cell cycle distribution, and apoptosis (when applicable). M229 R5 was also treated with 0.5 μM AZD6244. PI, propidium iodide. b, Transduction of N-RAS shRNAs in M249 R4 and Pt55 R (1 μM PLX4032), RNA and protein knockdown, effects on p-ERK levels and apoptosis. c, Survival curves for isogenic cell line pairs and melanoma cultures treated with the indicated AZD6244 concentration for 72 h (relative to DMSO-treated controls; mean ± s.e.m., n = 5). PLX4032-resistant cells were grown with PLX4032. Dashed line, 50% cell killing.