The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma

Abstract

Most patients with BRAF(V600)-mutant metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options for salvage therapy are incompletely understood. We performed whole-exome sequencing on formalin-fixed, paraffin-embedded tumors from 45 patients with BRAF(V600)-mutant metastatic melanoma who received vemurafenib or dabrafenib monotherapy. Genetic alterations in known or putative RAF inhibitor resistance genes were observed in 23 of 45 patients (51%). Besides previously characterized alterations, we discovered a "long tail" of new mitogen-activated protein kinase (MAPK) pathway alterations (MAP2K2, MITF) that confer RAF inhibitor resistance. In three cases, multiple resistance gene alterations were observed within the same tumor biopsy. Overall, RAF inhibitor therapy leads to diverse clinical genetic resistance mechanisms, mostly involving MAPK pathway reactivation. Novel therapeutic combinations may be needed to achieve durable clinical control of BRAF(V600)-mutant melanoma. Integrating clinical genomics with preclinical screens may model subsequent resistance studies.

Figure 1. Genetic alterations in the context of RAF inhibitor therapy

(A) Schematic overview of tumor biopsy collection in the context of RAF inhibitor therapy, followed by whole exome sequencing and analysis. (B) Spectrum of putative resistance genes, including known genes (NRAS, BRAF, MEK1) and new genes (MEK2, MITF). Additional recurrently altered pathways (PI3K pathway) or genomic correlates of early resistance (HOXD8, RAC1) are also shown. Results are sorted by duration of therapy (weeks). CR = Complete response, PR = partial response, SD = stable disease, PD = progressive disease.

Figure 2. MEK2 mutations confer resistance to RAF/MEK but not ERK inhibition

(A) A stick plot of MAP2K2 (which encodes the MEK2 kinase); the location of putative resistance-associated mutations observed in the patient cohort are indicated. (B) The crystal structure for MEK2. The locations of somatically mutated bases are denoted in yellow; the first stretch of amino acids are missing from the MEK2 structure in PDB, so the V35M and L46F mutations cannot be shown on the structure. (C–E) Growth inhibition curves are shown for MEK2 mutants in the context of RAF (C), MEK (D), or ERK (E) inhibitors. (F) The effect of dabrafenib or trametinib on ERK1/2 phosphorylation (pERK 1/2) in wild-type A375 cells (BRAF^V600E) and those expressing wildtype MEK2 (WT) or mutant constructs for MEK2. The levels of pERK1/2, total ERK1/2, pMEK1/2, MEK1/2, and vinculin are shown for A375 cells expressing novel MEK2 mutations after a 16-hour incubation at various drug concentrations as indicated.

Figure 3. MEK1 mutations confer varying degrees of resistance to RAF/MEK but not ERK inhibition

(A) A stick plot of MAP2K1 (which encodes the MEK1 kinase); the location of putative resistance-associated mutations observed in the patient cohort are indicated. (B) The crystal structure for MEK1. The locations of somatically mutated bases are denoted in yellow. (C-E) Growth inhibition curves are shown for MEK1 mutants in the context of RAF (C), MEK (D), or ERK (E) inhibitors. (F) The effect of dabrafenib or trametinib on ERK1/2 phosphorylation (pERK 1/2) in wild-type A375 cells (BRAF^V600E) and those expressing wildtype MEK1 (WT) or mutant constructs for MEK1. The levels of pERK1/2, total ERK1/2, pMEK1/2, MEK1/2, and vinculin are shown for A375 cells expressing novel MEK2 mutations after a 16-hour incubation at various drug concentrations as indicated.

Figure 4. Acquired MITF amplification confers resistance to MAP kinase pathway inhibition

(A) Focal genomic amplification of MITF occurs in a post-relapse sample in the absence of additional, known resistance mechanisms. (B) Relative viability of BRAF^V600E-mutant WM266.4 cells following overexpression of wild type MITF, a DNA-binding impaired MITF mutant (MITF-m^R217Δ) or control (LacZ) in the presence of RAF, MEK, combined RAF/MEK, or ERK inhibitors. (C) Western blot analysis of WM266.4 expressing the constructs used in (B). Half-maximal drug response curves in BRAF^V600E-mutant melanoma cell lines SKMEL19 (D) and UACC62 (E) expressing either LacZ or the melanocyte-specific isoform of MITF (MITFm) in the presence of increasing concentrations of PLX4720.

Figure 5. Intra-tumor resistance heterogeneity

(A) Co-occuring NRAS (Integrated Genomics Viewer [IGV] compressed window) and MEK1^V60E (IGV regular window) missense mutations in a relapsed tumor sample (Patient 41). NRAS^Q61R occurs in mutually exclusive reads compared to a neighboring NRAS^T58I somatic missense mutation in this patient. (B) Acquired NRAS^Q61K missense mutation together with BRAF amplification in the same tumor (Patient 8). (C) A MEK2^L46F missense mutation coincident with BRAF amplification in a resistant tumor specimen (Patient 02 – no pre-treatment tumor sample was available in this case).

Figure 6. Combined inhibition of BRAF and PI3K can confer synergistic activity

(A) Copy number profile from a resistant tumor sample (Patient 36 – no pre-treatment tumor sample was available in this case) demonstrates a focal homozygous deletion in chromosome 10 that includes PTEN. (B) A375 (BRAF^V600E, PTEN^WT) and A2058 (BRAF^V600E, PTEN^null) cells were treated with multiples of their respective GI₅₀s for PLX4720 or GDC0941 alone and in combination for 4 d. Cell proliferation was assessed using the CellTiter-Glo reagent. Combined BRAF and PI3K inhibition demonstrated synergistic activity in A2058 cells but to a lesser extent in A375 cells. The combination index (CI) was determined at the GI₅₀ concentration using the Chou and Talalay method and Calcusyn (Biosoft). (C) A375 and A2058 cells were treated with 4xGI₅₀ concentrations of PLX4720, GDC0941 or a combination of both inhibitors for 20 h. Cell lysates were analyzed by Western blotting for the indicated proteins.