Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo

Abstract

Activating mutations in the neuroblastoma rat sarcoma viral oncogene homolog (NRAS) gene are common genetic events in malignant melanoma being found in 15-25% of cases. NRAS is thought to activate both mitogen activated protein kinase (MAPK) and PI3K signaling in melanoma cells. We studied the influence of different components on the MAP/extracellular signal-regulated (ERK) kinase (MEK) and PI3K/mammalian target of rapamycin (mTOR)-signaling cascade in NRAS mutant melanoma cells. In general, these cells were more sensitive to MEK inhibition compared with inhibition in the PI3K/mTOR cascade. Combined targeting of MEK and PI3K was superior to MEK and mTOR1,2 inhibition in all NRAS mutant melanoma cell lines tested, suggesting that PI3K signaling is more important for cell survival in NRAS mutant melanoma when MEK is inhibited. However, targeting of PI3K/mTOR1,2 in combination with MEK inhibitors is necessary to effectively abolish growth of NRAS mutant melanoma cells in vitro and regress xenografted NRAS mutant melanoma. Furthermore, we showed that MEK and PI3K/mTOR1,2 inhibition is synergistic. Expression analysis confirms that combined MEK and PI3K/mTOR1,2 inhibition predominantly influences genes in the rat sarcoma (RAS) pathway and growth factor receptor pathways, which signal through MEK/ERK and PI3K/mTOR, respectively. Our results suggest that combined targeting of the MEK/ERK and PI3K/mTOR pathways has antitumor activity and might serve as a therapeutic option in the treatment of NRAS mutant melanoma, for which there are currently no effective therapies.

Fig. 1.

(A) Representative +++ positive staining for p-MEK, p-ERK, p-AKT, and p-S6. (B) Representative negative staining for p-MEK, p-ERK, p-AKT, and p-S6. (C) Staining intensity of proteins in the MEK/ERK and PI3K/mTOR cascade across 32 different NRAS mutant melanomas. Error bars indicate the SD for the intensity scores from four independent raters. (D) Percentage of patients with positive and negative staining for p-MEK/p-ERK, p-AKT/p-S6, or both.

Fig. 2.

(A) Immunoblot analyses for effector proteins of the MEK/ERK and PI3K/mTOR pathway in six NRAS mutant melanoma cell lines. Dose-dependent reduction of p-ERK by the MEKi as well as p-AKT and p-S6 by the PI3K/mTORi_1,2. Induction of p-AKT by the MEKi in MM485, WM3629, and WM3670 cells. (MEKi: JTP-74057). Slight induction of p-ERK in MM485, Sk-Mel-2, and MaMel30I cells by PI3K/mTOR_1,2 inhibition (PI3K/mTORi_1,2: GSK2126458). (B) Growth response curves for six NRAS mutant melanoma cell lines treated with MEK inhibitors (Upper) or inhibitors in the PI3K/mTOR cascade (Lower). MEK inhibitors are more potent in reducing cell viability than inhibitors in the PI3K/mTOR cascade with PI3K/mTORi_1,2 being more potent than selective PI3Ki, mTORi_1,2, mTOR₁, or AKTi. (Assay results are the mean of three replicates ± SD.)

Fig. 3.

(A) Immunoblot analyses for six NRAS mutant cell lines incubated with different combinations of inhibitors. The combination of the MEKi+PI3K/mTORi_1,2 completely suppressed p-ERK, p-AKT, and p-S6 levels in D04, MaMel30I, WM3670, and MM485 cells. MEKi+PI3Ki was more potent in reducing p-AKT, but had lower impact on p-S6 levels than MEKi+mTORi_1,2. [MEKi = 10 nM (JTP-74057, PD325901) inhibitors in the PI3K/mTOR pathway = 160 nM (PI3K/mTORi_1,2: GSK2126458, BEZ235; PI3Ki: GDC-0941; mTORi_1,2: PP242; mTORi₁: rapamycin; AKTi: GSK690693]. (B) Growth response curves for six NRAS mutant melanoma cell lines with the inhibitor combinations indicated. MEKi+PI3K/mTORi_1,2 was most effective in reducing viability in all cell lines followed by the combination of MEKi+PI3Ki in 9 of 10 cell lines. (Assay results are the mean of three replicates ± SD.)

Fig. 4.

(A) Induction of cleaved caspase 3 by the combination MEKi+PI3K/mTORi_1,2 (arrow) in D04, MaMel30I, MM485, and WM3060 cells. (B) Flow cytometric analysis to determine the number of apoptotic cells. Six NRAS mutant cell lines were treated with DMSO (control), a MEKi, a PI3K/mTORi_1,2, or the combination of both for 24 h. Column charts display the average of Annexin V+/PI+ (apoptotic) cells and Annexin V+/PI− (preapoptotic) cells for all cell lines treated with the indicated conditions. (MEKi: JTP-74057 = 10 nM; PI3K/mTORi_1,2: GSK2126458 = 160 nM; *P < 0.05; n = 2.)

Fig. 5.

Treatment of established tumors in a nude mouse xenograft model of NRAS mutant melanoma with tumor size reduction after initiation of treatment (arrow) with the combination of a MEKi (1.5 mg⋅kg⁻¹⋅d⁻¹) and PI3K/mTORi_1,2 (1.5 mg⋅kg⁻¹⋅d⁻¹), but not with either MEKi or PI3K/mTORi_1,2 alone. (cell lines: Sk-Mel-2, MaMel30I, WM3629, MM485, D04; MEKi: JTP-74057; PI3K/mTORi_1,2: GSK2126458) Results are the mean tumor volume at indicated time points ± SD; n = 4 mice per group.

Fig. 6.

Immunoblot analyses of tumors from mice treated with the conditions indicated. (A) The combination MEKi+PI3K/mTORi_1,2 markedly reduced phospho-protein levels in WM3629 tumors and (B) completely abolished expression of p-ERK, p-AKT, and p-S6 in D04 tumors. (A) Dramatic decrease of cyclin D1 levels in WM3629 and (B) a strong induction of cleaved caspase 3 was seen in D04 tumors.

Fig. 7.

(A and C) Number of genes equal to or more than twofold up- and down-regulated in the three treatment groups compared with the vehicle control for cell lines D04 and MaMel30I. (B) The combination MEKi+PI3K/mTORi_1,2 completely abolished cyclin D1 levels in Sk-Mel-2, WM3629, WM3670, and MM415 cells. (D) Biological processes regulated by MEKi+PI3K/mTORi_1,2. Bars represent the number of genes regulated by the combination treatment. gen., generation; metab., metabolism; prec., precursor.