PDK1 and SGK3 Contribute to the Growth of BRAF-Mutant Melanomas and Are Potential Therapeutic Targets

Abstract

Melanoma development involves members of the AGC kinase family, including AKT, PKC, and, most recently, PDK1, as elucidated recently in studies of Braf::Pten mutant melanomas. Here, we report that PDK1 contributes functionally to skin pigmentation and to the development of melanomas harboring a wild-type PTEN genotype, which occurs in about 70% of human melanomas. The PDK1 substrate SGK3 was determined to be an important mediator of PDK1 activities in melanoma cells. Genetic or pharmacologic inhibition of PDK1 and SGK3 attenuated melanoma growth by inducing G1 phase cell-cycle arrest. In a synthetic lethal screen, pan-PI3K inhibition synergized with PDK1 inhibition to suppress melanoma growth, suggesting that focused blockade of PDK1/PI3K signaling might offer a new therapeutic modality for wild-type PTEN tumors. We also noted that responsiveness to PDK1 inhibition associated with decreased expression of pigmentation genes and increased expression of cytokines and inflammatory genes, suggesting a method to stratify patients with melanoma for PDK1-based therapies. Overall, our work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment.

Figure 1. Loss of PDK1 inhibits the onset of melanoma development and delays metastasis

(A–B) Representative images of PDK1 WT (Braf^V600E::Cdkn2a^−/−::Pten^+/+::Pdk1^+/+) or PDK1 KO (Braf^V600E::Cdkn2a^−/−::Pten^+/+::Pdk1^−/−) mice 21 days after administration of 4-HT. (C–D) Representative images of the dorsal skin (C) and lymph nodes (D) from PDK1 WT and PDK1 KO mice 38 days after systemic administration of 4-HT. (E) Quantification of tumors in PDK1 WT and PDK1 KO mice (N = 12 and 8, respectively). (F) Kaplan-Meier survival curves of PDK1 WT and PDK1 KO mice (N = 16 and 18, respectively). P < 0.001 by log-rank (Mantel-Cox) test. (G–H) H&E stained (G) and S100 immunostained (H) skin sections from PDK1 WT and PDK1 KO mice 38 days after 4-HT administration. Bars = 100 μm. (I–J) H&E stained (I) and S100 (J) immunostained lymph nodes from PDK1 WT and PDK1 KO mice 38 days after administration of 4-HT. Bars = 100 μm. Graph shows mean ± SEM of S100+ cells from 3 animals per genotype. *P < 0.005. (K) Western blot analysis of the indicated proteins in primary melanoma cultures derived from PDK1 WT and PDK1 KO mice.

Figure 2. shRNA-mediated inhibition of PDK1 or SGK3 suppresses phosphorylation of target proteins, expression of cyclin D1, and growth of melanoma cells

(A) Western blot analysis of the indicated proteins in primary melanoma cultures derived from Braf^V600E::Cdkn2a^−/−::PTEN^−/− mice (YUMM1.5 and YUMM1.9) or Braf^V600E::Cdkn2a^−/−::PTEN^+/+ mice (SBM-A2 and SBM-A3). (B) Western blot analysis of the indicated proteins from YUMM1.5 and SBM-A2 melanoma cells expressing control, Sgk1-, Pdk1-, or Sgk3-targeted shRNA. (C–D) Colony formation assay of SBM-A2 (C) and YUMM1.5 (D) cultures expressing the indicated shRNAs. Individual wells shown are representative of three experiments with triplicate cultures. The graph represents the quantification of colony forming ability compared to control shRNA. Error bars represent SEM. (E) Representative images of YUMM1.5 spheroids expressing the indicated shRNAs. Relative spheroid sizes were quantified using ImageJ software (NIH) and are presented as the mean ± SEM of ≥6 spheroids per group.

Figure 3. Pharmacological inhibition of PDK1 or SGK3 inhibits target protein phosphorylation and melanoma cell growth

(A) Western blot analysis of the indicated proteins in YUMM1.5, YUMM1.9, SBM-A2, and SBM-A3 cells treated for 24 h with DMSO (vehicle), 10 μM GSK2334470 (PDK1i), or 10 μM GSK650394 (SGKi). (B–C) Spheroid formation by YUMM1.5 (B) and YUMM1.9 (C) cells incubated with 5 or 20 μM inhibitors. Spheroid volumes were quantified on the indicated days as described for Figure 2. Results are the mean ± SEM of ≥6 spheroids per group.

Figure 4. Inhibition of PDK1 or SGK3 induces cell cycle arrest in G1

Cell cycle distribution of SBM-A3 and YUMM1.5 cells stably expressing control shRNA (shSCR) or Pdk1- or Sgk3-targeted shRNAs. Left: Percentage of cells in the indicated cell cycle phases. Values are the mean ± SEM of biological triplicates. Right: Representative FACS profiles of the DNA content (propidium iodide staining) of cells expressing the indicated shRNAs. targeted*P < 0.05, **P < 0.001, ***P < 0.0001.

Figure 5. Combination screen of PDK1i against 45 candidate test agents

(A) Heat map of model-free area under the curve (AUC) of GSK2334470 at each of the two concentrations tested with unsupervised clustering of test agents (rows) and cell lines (columns). Red dots mark Bortezomib, Carfilzomib, and BKM-120 combinations with 10 μM GSK2334470. Dilutions of test agents were combined with either 0.1% DMSO vehicle, 2 μM GSK2334470, or 10 μM GSK2334470 and incubated with cell lines and assayed after 3 days with CellTiterGlo as described in Methods. Cell lines tested were YUROB, YUHEF (WT BRAF,NRAS), YUMAC, YUSIK (BRAF), YUGASP (NRAS). (B) Western blot analysis of the indicated proteins in mouse melanoma cells SBM-A2 and YUMM1.9, and human melanoma cell lines UACC903 and Mel501 treated for 24 h with 0.1, 1, or 5 μM of GSK2334470 (PDK1i) in the presence or absence of the proteasome inhibitor bortezomib (BTZ) at 1 nM (SBM-A2) or 4 nM (YUMM1.9, UACC903, Mel501). (C) Growth of YUMM1.5 spheroids treated with the indicated concentrations of GSK2334470 (PDK1i) and BTZ alone or in combination. Relative areas were calculated as described for Figure 2. Values are the mean ± SEM of ≥6 spheroids per group.

Figure 6. PDK1 and PI3K/mTOR inhibitors synergize to suppress melanoma cell growth

(A) Western blot analysis of the indicated proteins in YUMM1.9 and SBM-A2 cells treated for 24 h with 0.1, 1, or 5 μM of GSK2334470 (PDK1i) in the presence or absence of 1 or 3 μM of the dual PI3K/mTOR inhibitor BKM120. (B) Western blot analysis of the indicated proteins in the human melanoma cell lines UACC903 and Mel501 treated with 0.1, 1, or 5 μM of GSK2334470 (PDK1i) in the presence or absence of 1 or 3 μM of BKM120. (C) Growth of YUMM1.5 spheroids treated with the indicated concentrations of GSK2334470 (PDK1i) and BKM120 alone or in combination. Relative areas were calculated as described for Figure 2. Values are the mean ± SEM of ≥6 spheroids per group.

Figure 7. Stratification of human melanomas by sensitivity to PDK1 inhibition

(A) The mean IC50 for GSK2334470 inhibition of growth of 19 human melanoma cell lines was used to segregate cell lines into sensitivity or resistance to the inhibitor. (B) Heat map of the results of the IPA analysis showing the genes/pathways most significantly altered (P < 0.05; fold change >1.5) by GSK2334470 (1178 differentially expressed genes). (C) qPCR analysis of Dct, Tyr, and Il6 mRNA in YUMM1.5 cells treated with DMSO or GSK2334470 at 5 μM for 24 h (Dct and Tyr) or at 10 μM for 6 h (Il6). Values are the mean ± SEM of biological triplicates. *P < 0.05, **P < 0.01.