Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo Christian Poscha,b,1, Homayoun Moslehia, Luzviminda Feeneya, Gary A. Greena, Anoosheh Ebaeea, Valentin Feichtenschlagerb, Kim Chonga, Lily Penga, Michelle T. Dimona, Thomas Phillipsa, Adil I. Daudc, Timothy H. McCalmontd, Philip E. LeBoitd, and Susana Ortiz-Urdaa aDepartment of Dermatology, Mount Zion Cancer Research Center, and dDepartment of Pathology, University of California, San Francisco, CA 94115; bDepartment of Dermatology, Rudolfstiftung Hospital, A-1030 Vienna, Austria; and cDepartment of Dermatology, University of California, San Francisco, CA 94143 Edited* by James E. Cleaver, University of California, San Francisco, CA, and approved January 23, 2013 (received for review September 14, 2012) Activating mutations in the neuroblastoma rat sarcoma viral onco- with metastatic melanoma. The lack of responses among the first gene homolog (NRAS) gene are common genetic events in malig- 14 patients led to the early closure of the trial. A paucity of ef- nant melanoma being found in 15–25% of cases. NRAS is thought to ficacy has also been observed for this approach in other RAS- activate both mitogen activated protein kinase (MAPK) and PI3K mutated malignancies. Recently, an oral mitogen activated protein signaling in melanoma cells. We studied the influence of different (MAP)/extracellular signal-regulated (ERK) kinase (MEK) in- components on the MAP/extracellular signal-regulated (ERK) kinase hibitor (MEK162) was tested in patients with metastatic mela- (MEK) and PI3K/mammalian target of rapamycin (mTOR)-signaling noma harboring murine sarcoma viral oncogene homolog B1 cascade in NRAS mutant melanoma cells. In general, these cells were (BRAF) or NRAS mutations with encouraging results (12). In this more sensitive to MEK inhibition compared with inhibition in the PI3K/ study, we evaluate in detail NRAS mutant primary melanomas, mTOR cascade. Combined targeting of MEK and PI3K was superior to melanoma metastases, and 10 human NRAS mutant melanoma MEK and mTOR inhibition in all NRAS mutant melanoma cell lines cell lines. The expression and role of MEK/ERK and PI3K/ 1,2 mammalian target of rapamycin (mTOR) phospho-proteins in via- tested, suggesting that PI3K signaling is more important for cell survival MEDICAL SCIENCES in NRAS mutant melanoma when MEK is inhibited. However, targeting bility, growth, and therapeutics of NRAS mutant melanoma tumors are assessed. Our data show that combined targeting of MEK and of PI3K/mTOR in combination with MEK inhibitors is necessary to 1,2 PI3K/mTOR is necessary to regress NRAS mutant melanoma, effectively abolish growth of NRAS mutant melanoma cells in vitro 1,2 thus opening the possibility of a beneficial treatment strategy. and regress xenografted NRAS mutant melanoma. Furthermore, we showed that MEK and PI3K/mTOR1,2 inhibition is synergistic. Expression Results analysis confirms that combined MEK and PI3K/mTOR inhibition pre- 1,2 NRAS Mutant Melanoma Activates the MEK/ERK, the PI3K/mTOR dominantly influences genes in the rat sarcoma (RAS) pathway and Pathway, or both. Levels of phospho-ERK, p-MEK phospho- growth factor receptor pathways, which signal through MEK/ERK murine thymoma viral oncogene homolog 1 (p-AKT), phospho- and PI3K/mTOR, respectively. Our results suggest that combined target- S6 ribosomal protein (p-S6), and phosphatase and tensin homolog ing of the MEK/ERK and PI3K/mTOR pathways has antitumor activity (PTEN) were measured in 14 primary melanomas and 18 me- and might serve as a therapeutic option in the treatment of NRAS tastases from 32 patient specimens of NRAS-mutated melanoma. mutant melanoma, for which there are currently no effective therapies. Sample information, including mutation status, is provided in Table S1. Protein levels were measured by immunohisto- ncogenic mutations in codons 12, 13, or 61 of the rat sarcoma chemistry (IHC) and evaluated as the average rating of staining O(RAS) family of small GTPases, Kirsten rat sarcoma viral intensity by four independent reviewers on a scale from 3 =+++ oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene positive to 0 = negative. Analysis of inter- and intrarater re- homolog (HRAS), and neuroblastoma RAS viral oncogene ho- liability can be found in Fig. S1. Across all patients, tumors dis- molog (NRAS) occur in approximately one-third of all human played the strongest staining for p-ERK (1.34 ± 0.14) and p-S6 cancers with NRAS mutations found in about 15–20% of mela- (1.16 ± 0.12). Comparing the staining patterns between nomas (1–7). Mutated RAS proteins activate signaling pathways patients, scoring results were divided into quartiles. Negative that promote the cell division cycle and cell growth and suppress staining was defined as a score ≤0.75 (quartile 1), and positive apoptosis. Small interfering RNA (siRNA)-mediated depletion of staining was defined as a score >0.75 (quartile 2, 3, 4). Positive NRAS in melanoma cell lines inhibits proliferation and renders staining for p-ERK or p-S6 or both was found in 95.24% of cells sensitive to chemotherapy, making mutant NRAS and its samples stained, with 42.86% of patients positive for both pro- signaling effectors relevant targets for melanoma therapy (8, 9). teins, 28.57% positive only for p-MEK, and 23.81% positive only Efforts at developing therapeutics that inhibit mutant RAS di- for p-S6 (Fig. 1). In 28.6% of patient samples, p-ERK/p-MEK rectly have so far not been successful. The high affinity of RAS for staining was negative. This was predominantly the case in mel- GTP and the high concentrations of GTP intracellularly has anoma metastasis although it was not statistically significant; meant that the identification of small molecules, which selectively 57.9% of slides expressed PTEN, with an average staining prevent accumulation of RAS-GTP, has not been possible (10). Targeting mutant NRAS with siRNA is still limited to preclinical fi models because of the signi cant challenge in delivering antisense Author contributions: C.P., A.I.D., T.H.M., P.E.L., and S.O.-U. designed research; C.P., H.M., oligonucleotides in vivo. The response of NRAS mutant mela- L.F., G.A.G., A.E., V.F., K.C., L.P., and T.P. performed research; T.H.M. and P.E.L. contrib- noma and other melanomas to various chemotherapeutic regi- uted new reagents/analytic tools; C.P., G.A.G., A.E., V.F., K.C., L.P., M.T.D., A.I.D., and ments has been very scarce with only 6% of patients responding S.O.-U. analyzed data; and C.P. and S.O.-U. wrote the paper. (11). Alternatively, farnesyltransferase inhibitors (FTIs) were The authors declare no conflict of interest. thought to inhibit RAS activation by blocking farnesylation, a key *This Direct Submission article had a prearranged editor. posttranslational modification step of RAS that is essential for 1To whom correspondence should be addressed. E-mail: [email protected]. RAS function. One FTI, R115777 (also known as tipifarnib), was This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. evaluated in a single-agent, single-arm phase II trial in patients 1073/pnas.1216013110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1216013110 PNAS | March 5, 2013 | vol. 110 | no. 10 | 4015–4020 Downloaded by guest on September 28, 2021 Combination of MEK and PI3K/mTOR1,2 Inhibitors Is Most Effective in Reducing Cell Viability in NRAS Mutant Melanoma. We further ex- amined the growth inhibitory effect of different combinations of selective inhibitors. Combining a MEKi with a PI3Ki, mTORi1, mTORi1,2, or AKTi resulted in greater growth inhibition than with a MEKi alone in all cell lines. The combination of MEKi+PI3Ki was superior to combinations of MEKi with mTORi1,mTORi1,2, or AKTi. However, the strongest decrease in cell viability was seen with MEKi+PI3K/mTORi1,2 (Fig. 3 and Fig. S3). As expected, immunoblot analyses revealed “paradoxical” AKT hyperphosphorylation when cells were treated with the AKTi. This previously described effect is not due to enhance- ment of upstream signals to compensate for AKT signal loss, but rather is related to occupation of the AKT nucleotide-binding pocket by the AKT inhibitor (14,15). We further found that MEKi+mTORi1,2 reduced levels of p-S6 more potently than MEKi+PI3Ki. Interestingly, the latter has a stronger effect in the reduction of viability in NRAS cell lines. Only the combination of MEKi and PI3K/mTORi1,2 showed complete suppression of p-ERK, p-AKT, and p-S6 in D04, MaMel30I, MM485, WM3670, WM3060, and WM1366 and almost complete suppression in Sk-Mel-2 and WM3629 cells (Fig. 3 and Fig. S3). Combined Inhibition of MEK/ERK and PI3K/mTOR Pathways Induces Apoptosis in NRAS Mutant Melanoma Cells. +++ To evaluate if growth Fig. 1. (A) Representative positive staining for p-MEK, p-ERK, p-AKT, and inhibition induced with single or combination treatment is due to p-S6. (B) Representative negative staining for p-MEK, p-ERK, p-AKT, and p-S6. apoptosis, we determined the number of cells with phosphatidyl (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 serine exposed outside the cell membrane by labeling with an Annexin V-FITC antibody. Cells with loss of membrane integrity, intensity scores from four independent raters. (D) Percentage of patients with fl fi positive and negative staining for p-MEK/p-ERK, p-AKT/p-S6, or both. re ecting the latest stage of cell death, were identi ed by propi- dium iodide (PI) staining. Results confirmed that the reduction of cell count in viability assays is a result of apoptosis (16).