Unbiased Proteomic and Phosphoproteomic Analysis Identifies Response Signatures and Novel Susceptibilities After Combined MEK and mTOR Inhibition in BRAFV600E Mutant Glioma

Abstract

The mitogen-activated protein kinase pathway is one of the most frequently altered pathways in cancer. It is involved in the control of cell proliferation, invasion, and metabolism, and can cause resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer, and glioma, are driven by a constitutively activating missense mutation (V600E) in the v-Raf murine sarcoma viral oncogene homolog B (BRAF) component of the pathway. Mitogen-activated protein kinase kinase (MEK) inhibition is initially effective in targeting these cancers, but reflexive activation of mammalian target of rapamycin (mTOR) signaling contributes to frequent therapy resistance. We have previously demonstrated that combination treatment with the MEK inhibitor trametinib and the dual mammalian target of rapamycin complex 1/2 inhibitor TAK228 improves survival and decreases vascularization in a BRAF^V600E mutant glioma model. To elucidate the mechanism of action of this combination therapy and understand the ensuing tumor response, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAF^V600E mutant glioma xenografts after short-course treatment with trametinib and TAK228. We identified 13,313 proteins and 30,928 localized phosphosites, of which 12,526 proteins and 17,444 phosphosites were quantified across all samples (data available via ProteomeXchange; identifier PXD022329). We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the mitogen-activated protein kinase and mTOR pathways. Combination therapy also increased apoptotic signaling, suppressed angiogenesis signaling, and broadly suppressed the activity of the cyclin-dependent kinases. In response to combination therapy, both epidermal growth factor receptor and class 1 histone deacetylase proteins were activated. This study reports a detailed (phospho)proteomic analysis of the response of BRAF^V600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies new targets for the development of rational combination therapies for BRAF-driven tumors.

Keywords: TAK228; angiogenesis; apoptosis; drug targets; glioma; mouse models; phosphoproteome; sapanisertib; signal transduction; tandem MS; trametinib.

Graphical abstract

Fig. 1

Global proteomics and phosphoproteomics separated tumors into groups based on treatment with trametinib monotherapy, TAK228 monotherapy, or combination therapy.A, proteomic and phosphoproteomic metrics. Thirteen thousand three hundred thirteen proteins were identified, of which 12,526 were quantified across all 20 samples. Thirty thousand nine hundred twenty-eight localized phosphosites were identified, of which 17,444 were quantified across all 20 samples. If all contributing PSMs were mouse, then the protein or phosphosite was considered mouse (stroma). If contributing PSMs were either distinctly human or human and mouse, then the protein or phosphosite was considered human (tumor). For phosphosites, in parentheses are listed the total number of phosphosites (10,767 human and 3912 mouse) that could be normalized to their respective protein. Only normalized phosphosites were used for all subsequent analysis. Principal component analysis of human proteome (B) and phosphoproteome (C) shows distinct grouping of the four treatment groups (vehicle, trametinib, TAK228, and combination), with the exception that one TAK228 sample more closely grouped with the vehicle samples. This TAK228 sample was included in all analyses given that the mTOR signaling pathway was appropriately inhibited in this sample (Fig. 2A). Volcano plots of human proteome (D) and human phosphoproteome (E) of mean difference (combination—vehicle) versus −log(p). For these analyses, FDR cutoff was <0.01. FDR, false discovery rate; mTOR, mammalian target of rapamycin; PSM, peptide-to-spectrum match.

Fig. 2

Trametinib–TAK228 combination therapy inhibited both the MAPK and mTOR pathways.A, heat map of log2(abundance) of MAPK and mTOR pathway phosphorylation sites. All sites included were statistically significantly decreased in combination as compared with vehicle based on prior volcano plot (Fig. 1E and supplemental Table S9). Diagram of MAPK and mTOR signaling pathways (B) with proteins whose phosphorylations are statistically significantly reduced in combination as compared with vehicle shaded blue. MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin.

Fig. 3

Global proteomic–phosphoproteomic analysis identified distinct response signatures associated with trametinib, TAK228, or combination therapy. Venn diagrams showing statistically significant unique and shared protein (A) or phosphosite (B) changes in each treatment condition (compared with vehicle). C, kinase-substrate enrichment analysis (KSEA) kinase z-scores are represented in a bar graph showing inhibition of mTOR activity after TAK228 and combination treatment, inhibition of MAPK signaling after trametinib and combination treatment, and inhibition of cyclin-dependent kinases after combination treatment. Only kinases for which the FDR was <0.05 were included. FDR, false discovery rate; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin.

Fig. 4

Trametinib–TAK228 combination therapy broadly inhibited progrowth signaling pathways, while activating pathways involved in RNA processing. Reactome protein pathways significantly altered (FDR <0.05) in an over-representation analysis (ORA) of statistically significantly altered proteins and phosphosites after trametinib–TAK228 combination therapy are represented in a scatter plot of fold enrichment versus −log10(FDR). FDR, false discovery rate.

Fig. 5

Trametinib–TAK228 combination therapy activated apoptotic signaling and inhibited angiogenesis in BRAF-mutant glioma. Stacked bar graphs of log2(abundance) of proteins and phosphorylation sites demonstrating statistically significant (A) increases in human proteins and phosphosites involved in apoptosis in combination compared with vehicle in BRAF-mutant glioma, (B) decreases in human proteins and phosphosites involved in angiogenesis/VEGF signaling in BRAF-mutant glioma. FDR <0.01 for all data points included. BRAF, v-Raf murine sarcoma viral oncogene homolog B; VEGF, vascular endothelial growth factor.

Fig. 6

Druggable protein analysis after combination trametinib–TAK228 therapy identified a number of potentially targetable upregulated proteins, including epidermal growth factor receptor (EGFR) and class 1 HDACs. Aligned dot plots of log2(abundance) of listed phosphosites in each treatment condition, vehicle, trametinib monotherapy, TAK228 monotherapy, and trametinib–TAK228 combination therapy: (A) two EGFR phosphosites, one activating (EGFR-Y1172) and one inhibitory (EGFR-T693), showing that the level of EGFR-Y1172 was statistically significantly increased after combination therapy, and the level of the EGFR-T693 was statistically significantly decreased after trametinib monotherapy and combination therapy; (B) two class 1 HDAC-activating phosphosites, HDAC1-S421 and HDAC2-S424, showing that the levels of both of these activating phosphorylations were statistically significantly increased after combination therapy, but the level of HDAC-S424 was decreased after trametinib monotherapy. For A and B, ∗∗∗p < 0.001, ∗∗p between 0.001 and 0.01, ∗p between 0.01 and 0.05. C, the table listing FDA-approved inhibitors for EGFR and HDAC1/2. EGFR, epidermal growth factor receptor; FDA, Food and Drug Administration; HDAC, histone deacetylase.