Differential activity of MEK and ERK inhibitors in BRAF inhibitor resistant melanoma

Abstract

Acquired resistance to BRAF inhibitors often involves MAPK re-activation, yet the MEK inhibitor trametinib showed minimal clinical activity in melanoma patients that had progressed on BRAF-inhibitor therapy. Selective ERK inhibitors have been proposed as alternative salvage therapies. We show that ERK inhibition is more potent than MEK inhibition at suppressing MAPK activity and inhibiting the proliferation of multiple BRAF inhibitor resistant melanoma cell models. Nevertheless, melanoma cells often failed to undergo apoptosis in response to ERK inhibition, because the relief of ERK-dependent negative feedback activated RAS and PI3K signalling. Consequently, the combination of ERK and PI3K/mTOR inhibition was effective at promoting cell death in all resistant melanoma cell models, and was substantially more potent than the MEK/PI3K/mTOR inhibitor combination. Our data indicate that a broader targeting strategy concurrently inhibiting ERK, rather than MEK, and PI3K/mTOR may circumvent BRAF inhibitor resistance, and should be considered during the clinical development of ERK inhibitors.

Keywords: Acquired resistance; BRAF inhibitors; ERK inhibitors; MEK inhibitors; Melanoma.

Figure 1

ERK and MEK inhibitors display variable activity towards BRAF inhibitor resistant sublines expressing a BRAF splice variant or mutant N‐RAS A. Viability curves of the parental SKMel28 and isogenic sublines, BR2 and BR4, treated with indicated concentrations of the MEK inhibitor trametinib and ERK inhibitor VX‐11e for 72 h (relative to DMSO‐treated controls; mean ± SD; n = 2). B. SKMel28 parental and dabrafenib‐resistant sublines were treated with DMSO (Control, C), 10 nM MEK inhibitor trametinib (M) or 10 μM ERK inhibitor VX‐11e (E) for 24 h. Western blots of lysates showing protein markers of MAPK activity and cell cycle progression. C. PARP cleavage was determined 72 h after treating melanoma cells with inhibitors as described above. Both full length and major cleaved PARP proteins shown. D. Cell cycle distribution of indicated cell lines treated with DMSO (control), 10 nM trametinib (MEK inhibitor) or 10 μM VX‐11e (ERK inhibitor) for 72 h (mean ± SD; n = 4). *Significant differences for subG1 and S phase when MEK compared to ERK inhibitor treatment (p < 0.05).

Figure 2

Resistant melanomas with RTK activation retain anti‐proliferative sensitivity to ERK but not MEK inhibition with no apoptotic sensitivity to either inhibitor A. Viability curves of the parental and isogenic melanoma sublines treated with indicated concentrations of the MEK inhibitor trametinib and ERK inhibitor VX‐11e for 72 h (relative to DMSO‐treated controls; mean ± SD; n = 2). B. Parental and BRAF inhibitor‐resistant sublines were treated with DMSO (C), 10 nM MEK inhibitor trametinib (M) or 10 μM ERK inhibitor VX‐11e (E) for 24 h. Western blots of lysates showing protein markers of MAPK activity and cell cycle progression. C. PARP cleavage was determined 72 h after treating sublines with inhibitors as described above. Both full length and major cleaved PARP proteins shown. D. Cell cycle distribution of the indicated cell lines treated with either DMSO (control), 10 nM trametinib (MEK inhibitor) or 10 μM VX‐11e (ERK inhibitor) for 72 h (mean ± SD; n = 4). *Significant differences for subG1 and S phase when MEK compared to ERK inhibitor treatment (p < 0.05).

Figure 3

Patient‐derived short‐term melanoma cell lines with acquired resistance to BRAF inhibitors arrest in response to ERK inhibition A. Patient derived MAPK resistant cell lines were treated with DMSO (C), 10 nM MEK inhibitor trametinib (M) or 10 μM ERK inhibitor VX‐11e (E) for 24 h. Western blots of lysates showing protein markers of MAPK activity and cell cycle progression. B. PARP cleavage was determined 72 h after treating cells with inhibitors as described above. Both full length and major cleaved PARP proteins shown. C. Cell cycle distribution of the indicated cell lines treated with either DMSO (control), 10 nM trametinib (MEK inhibitor) or 10 μM VX‐11e (ERK inhibitor) for 72 h (mean ± SD; n = 4). *Significant differences for subG1 and S phase when MEK compared to ERK inhibitor treatment (p < 0.05).

Figure 4

Combination ERK inhibitor with BEZ235 is more active than the MEK inhibitor/BEZ235 combination at promoting cell death A. The indicated cell lines were treated DMSO (C), 10 nM trametinib (M) or 10 μM VX‐11e (E) for 24 h. Western blots of lysates showing protein markers of MAPK and AKT activity. WMD013 and Patient 3 cell lines were directly compared to SKMel28 and BR2 cell lines. B. Whole cell lysates from the indicated cell lines were subjected to pull‐down (PD) assays with GST‐bound CRAF RAS‐binding domain after 24 h treatment with DMSO (C) or 10 μM VX‐11e (E). Whole cell lysates (total RAS) and pull down products (activated RAS) were immunoblotted with a pan‐RAS antibody. C. Histogram showing the percentage sub‐G1 population of cell lines treated with either DMSO (control), 10 nM trametinib (MEK inhibitor), 10 μM VX‐11e (ERK inhibitor), 2 uM BEZ235 or combinations as indicated for 72 h (mean ± SD; n = 4). *Significant differences between the ERK/BEZ235 inhibitor combination compared with the MEK/BEZ235 inhibitor combination (p < 0.05).