Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling

Abstract

A detailed understanding of the mechanisms by which tumors acquire resistance to targeted anticancer agents should speed the development of treatment strategies with lasting clinical efficacy. RAF inhibition in BRAF-mutant melanoma exemplifies the promise and challenge of many targeted drugs; although response rates are high, resistance invariably develops. Here, we articulate overarching principles of resistance to kinase inhibitors, as well as a translational approach to characterize resistance in the clinical setting through tumor mutation profiling. As a proof of principle, we performed targeted, massively parallel sequencing of 138 cancer genes in a tumor obtained from a patient with melanoma who developed resistance to PLX4032 after an initial dramatic response. The resulting profile identified an activating mutation at codon 121 in the downstream kinase MEK1 that was absent in the corresponding pretreatment tumor. The MEK1(C121S) mutation was shown to increase kinase activity and confer robust resistance to both RAF and MEK inhibition in vitro. Thus, MEK1(C121S) or functionally similar mutations are predicted to confer resistance to combined MEK/RAF inhibition. These results provide an instructive framework for assessing mechanisms of acquired resistance to kinase inhibition and illustrate the use of emerging technologies in a manner that may accelerate personalized cancer medicine.

Fig 1.

Kinase oncogene dependence and principles of drug resistance. Tumor genetic alterations (denoted by the red star) may activate protein kinase oncogenes, which in turn dysregulate a cell signaling pathway, resulting in oncogene dependence. Such tumors often respond to treatment using pharmacologic inhibitors of the mutated kinase oncoprotein; however, resistance to such agents is common. Categories of resistance to kinase inhibitors include secondary mutation (denoted by the green star), amplification, or activation of the target kinase, or bypass of the oncogenic pathway, both leading to downstream reactivation and disease progression. Bypass mechanisms activating alternative pathways have also been described (alternate pathway, see Emerging Mechanisms of Resistance to Kinase Oncogene Inhibition). In principle, reactivation of the oncogenic pathway through additional, as yet uncharacterized mechanisms (denoted by question mark) should also confer acquired resistance to targeted therapies.

Fig 2.

A 38-year-old man with BRAF-mutant melanoma and miliary, subcutaneous metastatic deposits. Photographs were taken (A) before initiation of PLX4032, (B) after 15 weeks of therapy with PLX4032, and (C) after relapse, after 23 weeks of therapy.

Fig 3.

Pharmacologic and biochemical characterization of the MEK1^C121S mutation. Growth inhibition curves for (A) the RAF inhibitor PLX4720 and (B) the MEK inhibitor AZD6244 are shown for wild-type A375 (BRAF^V600E) melanoma cells (solid black) and A375 cells expressing MEK1^C121S (red), wild-type MEK1 (MEK-WT; blue), or a constitutively active MEK1 variant (MEK-DD; gold). Effect of (C) PLX4720 and (D) AZD6244 on ERK1/2 phosphorylation (pERK 1/2) in wild-type A375 cells and those expressing MEK-WT, MEK1^C121S, or MEK-DD. The levels of pERK1/2, total ERK1/2, MEK1/2, and α-tubulin are shown for A375 cells expressing MEK1 mutations after a 16-hour incubation at 0, 0.08, 0.4, 2, 5, and 10 μmol/L drug concentrations. (E) In vitro kinase assay measuring pERK in A375 cells expressing MEK1 mutations. Relative levels of pERK compared with total ERK1 and total MEK1 are shown.

Fig 4.

MEK1 mutations arising from an in vitro mutagenesis screen for resistance to RAF inhibition. (A) Recurrent mutations across the MEK1 coding sequence from a PLX4720 mutagenesis screen (based on approximately 1,000 sequenced clones) are shown. The corresponding amino acid substitutions from high-scoring mutations (> 0.4%) are indicated. (B and C) Locations of selected resistance alleles are indicated within the crystal structure of MEK1. Adenosine triphosphate (orange) and an allosteric, arylamine MEK inhibitor (PD318088; purple) are shown. Helix C (green) and helix A (red) are indicated. Mutations found to confer clinical resistance to RAF inhibition (C121S) and MEK inhibition (P124L) are indicated (blue spheres). Candidate mutations found in the mutagenesis screen are shown in blue. B and C show alternative views of the same crystal structure, with a 45-degree and slight inferior oblique rotation. bp, base pair.