MAP kinase pathway alterations in BRAF-mutant melanoma patients with acquired resistance to combined RAF/MEK inhibition

Abstract

Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.

FIGURE 1. Identification of a MEK2 mutation in a melanoma sample resistant to dabrafenib/trametinib

(A) Whole exome sequencing (WES) (left panel) and whole transcriptome sequencing (RNA-Seq) (right panel) of the tumor tissue from Patient 1 both before treatment and after the development of resistance to dabrafenib/trametinib revealed a Q60P mutation in MEK2 in the resistant tumor that was undetectable in the pre-treatment tumor. (B) The fraction of tumor cells (the cancer cell fraction, CCF) harboring each alteration was calculated for the pretreatment and resistant tumor samples. Direct comparison of the CCF for all alterations in the pretreatment and resistant tumor samples demonstrated alterations that occurred in the pretreatment sample only (lower right corner, blue), the resistant sample only (upper left corner, purple), or both samples (upper right corner, red). 15 missense mutations were identified occurring in the resistant sample only (upper left corner, purple), including the MEK2^Q60P mutation (see Supplementary Table 3).

FIGURE 2. Pharmacologic and biochemical characterization of the MEK2^Q60P mutation

(A-D) Growth inhibition curves are shown for dabrafenib/trametinib (A), as well as monotherapy with dabrafenib (B) or trametinib (C), and the ERK inhibitor VX-11e (D) for A375 (BRAF^V600E) melanoma cells (grey) and A375 cells expressing wild type MEK2 (MEK2 WT; blue) or MEK2^Q60P (red). (E) The effect of combined dabrafenib/trametinib treatment on ERK1/2 phosphorylation (pERK 1/2) in wild type A375 cells (BRAF^V600E) and those expressing wild type MEK2 (MEK2 WT) or MEK2^Q60P is shown. The levels of pERK1/2, total ERK1/2, pMEK1/2, total MEK1/2, and vinculin are shown for A375 cells expressing MEK2 mutations after a 16-hour incubation at various drug concentrations as indicated.

FIGURE 3. Additional secondary alterations in MAP kinase pathway identified in resistant tumors

(A) RNA-Seq of the tumor tissue from Patient 2 both before treatment and after the development of resistance to dabrafenib/trametinib revealed a BRAF splice isoform lacking exons 2-10 in the resistant tumor that was undetectable in the pre-treatment tumor. (B). Copy number analysis of whole exome data from Patient 3 demonstrates 2 highly amplified regions in the resistant tumor that are not amplified in the pre-treatment tumor. One of these regions contains the BRAF gene, while the second region contains multiple genes, including SAMD4B.