ERK mutations confer resistance to mitogen-activated protein kinase pathway inhibitors

Abstract

The use of targeted therapeutics directed against BRAF(V600)-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAF(V600)-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor-resistant alleles were sensitive to RAF/MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents.

Figure 1

The landscape of ERK1 and ERK2 resistance alleles following ERK or RAF/MEK inhibitor mutagenesis screens. A and B, the frequency of mutations from the ERK1 (A) and ERK2 (B) random mutagenesis screens is graphed at cDNA base pair resolution for VX-11e (red), trametinib (GSK′212; blue), and trametinib + dabrafenib (′212 + ′436; green). Amino acid substitutions for the most recurrently mutated nucleotides are highlighted in red (ERK inhibitor), blue (MEK inhibitor), and green (RAF + MEK inhibitor). Some amino acids were identified by more than one drug screen (black). Analogous alterations between ERK1 and ERK2 are underlined. C and D, Venn diagram depicting the overlap of all significantly mutated nucleotides in ERK1 or ERK2 from the ERK inhibitor screen (C) or the single-agent MEK and combined RAF/MEK inhibitor screens (D).

Figure 2

Pharmacologic and biochemical validation of ERK resistance alleles from ERK inhibitor screens. A, viability of A375 cells expressing tet-inducible ERK inhibitor resistance mutations exposed to VX-11e with or without DOX is shown as a heat map. GFP, wild-type ERK1/2, and kinase-dead ERK1^K71R/ERK2^K54R served as controls. Alleles are sorted by sensitivity to VX-11e + DOX. ERK2 alleles identified by sequencing drug-resistant colonies (black asterisks), or in both drug-resistant colonies and the ERK inhibitor random mutagenesis screen (red asterisks), are indicated. B, A375 cells expressing ERK inhibitor resistance mutations were treated as above except with SCH722984 (SCH'984). Viability was depicted as in A. Gray squares indicate mutations that were not tested. C, expression of phospho-ERK (pERK), phospho-RSK (pRSK), DUSP6, and cyclin D1 was examined in A375 cells expressing ERK resistance mutations or controls. Cells were treated with DOX in the presence or absence of VX-11e. D, structural localization of validated ERK1 and ERK2 mutations (spheres) mapped onto available ERK2 crystal structure data (cartoon) cocrystallized with VX-9a, a parent compound of VX-11e [PDB:3I60]. The glycine-rich loop (teal), the αC-helix (magenta), and the activation loop (blue) are indicated.

Figure 3

Kinase activity of ERK mutants from ERK inhibitor resistance screens. A and B, the kinase activity of ERK1 (A) and ERK2 (B) was examined in lysates from A375 cells expressing validated ERK inhibitor resistance alleles or controls (wild-type and kinase-dead ERK1^K71R/ERK2^K54R) in the absence or presence of VX-11e using IP kinase assays. pERK and pRSK expression in the input was analyzed by immunoblotting.

Figure 4

Pharmacologic and biochemical validation of ERK mutations that confer resistance to RAF/MEK inhibitors. A, A375 cells expressing tet-inducible RAF/MEK inhibitor resistance mutations were analyzed for viability in the presence of trametinib (GSK′212), dabrafenib (GSK′436), or trametinib + dabrafenib (GSK′212 + GSK′436), with or without DOX. Cell viability was normalized to DMSO, and values are depicted in the heat map. Alleles are sorted by sensitivity to trametinib + DOX. GFP, wild-type ERK1/2, and kinase-dead ERK1^K71R and ERK2^K54R served as controls. B, structural localization of validated RAF/MEK inhibitor–resistant alleles (spheres) mapped onto the ERK2 crystal structure (cartoon; PDB: 2ERK). The αC-helix (magenta) and the activation loop (blue) are labeled. The nonvalidating/ untested analogous ERK1/2 alleles are labeled in gray. C and D, cells expressing validated ERK1 (C) or ERK2 (D) RAF/MEK inhibitor resistance alleles were exposed to DOX with or without trametinib and pERK, pRSK, DUSP6, and cyclin D1 expression was analyzed by immunoblotting.

Figure 5

Kinase activity of ERK mutants from RAF/MEK inhibitor resistance screens. A and B, the kinase activity of ERK1 (A) and ERK2 (B) was examined in lysates from A375 cells expressing validated RAF/MEK inhibitor resistance alleles or controls (wild-type and kinase-dead ERK1^K71R/ERK2^K54R) treated with trametinib (GSK′212). pERK and pRSK were monitored in the input cell lysates by immunoblotting.

Figure 6

Drug-sensitivity studies of ERK resistance mutants using RAF, MEK, and ERK inhibitors. A, viability of A375 cells expressing tet-inducible ERK1/2 resistance alleles from Figs. 2 and 4 following exposure to VX-11e, trametinib (GSK′212), dabrafenib (GSK′436), or trametinib + dabrafenib (GSK′212+GSK ′436) in the presence or absence of DOX. Normalized viability is depicted by heat map. Alleles are sorted by their sensitivity to VX-11e in the presence of DOX. B and C, viability of SKMEL-19 cells expressing ERK1 (B) or ERK2 (C) resistance alleles after exposure to VX-11e, SCH772984 (SCH′984), trametinib (GSK′212), and trametinib + dabrafenib (GSK′212 + GSK′436) is graphed (left). Normalized viability is depicted by heat map (right). VX-11e–resistant alleles (red) and RAF/MEK inhibitor– resistant alleles (blue) are indicated.

Figure 7

Lethality of ERK resistance mutants in BRAF-mutant melanoma cells. A, viability of A375 cells expressing tetinducible ERK1/2 resistance alleles after exposure to DOX. Controls (GFP, wild-type ERK1/2, and kinase-dead ERK1^K71R/ERK2^K54R; green), ERK inhibitor resistance mutations (red), and RAF/MEK inhibitor resistance mutations (blue) are shown. ERK1^C82Y is resistant to RAF, MEK, and ERK inhibitors (purple). B, phase contrast micrographs of A375 cells expressing tet-inducible RAF/MEK inhibitor–resistant alleles (ERK1^A206V and ERK1^S219P) or controls treated with DOX (top), or DOX + trametinib (GSK′212; bottom) for 24 hours (magnification, ×10).