Evaluating melanoma drug response and therapeutic escape with quantitative proteomics

Abstract

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.

Fig. 1.

Scheme showing the signaling nodes covered by the LC-MRM assay.

Fig. 2.

LC-MRM detects increased HSP70 expression following treatment with the HSP90 inhibitor XL888. A, XL888 is associated with concentration-dependent decreases in cell growth in NRAS mutant melanoma cell lines. Four NRAS mutant melanoma cell lines were treated with increasing concentrations of XL888 for 72 h. Cell viability was measured using the Alamar Blue assay. B, LC-MRM quantification of HSP90α, HSP90β, and HSP70 isoform 1 (HSP71) induction following treatment of four NRAS mutant melanoma cell lines (M245, M318, WM1361A, and WM1366) with XL888 (300 nm, 0–48 h). Protein expression is shown in femtomoles/μg of total protein. C, in vivo efficacy of XL888. NRAS mutant M245 melanoma cells were grown as xenografts in SCID mice. Treatment with XL888 (100 mg/kg, daily) was initiated once tumors were palpable. Data show the tumor volume fold-change from baseline following treatment with either vehicle or XL888. D, LC-MRM analysis of HSP90α, HSP90β, and HSP71 expression following treatment with either vehicle or XL888. Protein expression is given as femtomoles/μg of total protein.

Fig. 3.

LC-MRM detection of HSP client proteins. A, LC-MRM analysis of NRAS mutant melanoma cell lines following treatment with XL888. NRAS mutant melanoma cell lines were treated with XL888 (300 nm, 0–48 h) before being analyzed by LC-MRM. B, Western blot validation confirming decreased levels in RTK expression following XL888 treatment. C, heat maps of relative quantification of RTK expression in fine needle aspirates taken from xenografts of human melanoma cells and from two clinical melanoma specimens.

Fig. 4.

LC-MRM as a platform for the detection of decreased HSP client expression in an in vivo model of acquired vemurafenib resistance following XL888 treatment. A, treatment of M229R vemurafenib-resistant xenografts with XL888 leads to significant levels of tumor shrinkage in vivo. M229R cells were allowed to form palpable xenografts, before treatment was initiated with either vehicle or XL888. Data show fold change from baseline after 14 days of treatment. B, LC-MRM analysis of xenograft specimens treated with either vehicle or XL888. C, LC-MRM quantification shows the decreased expression of proteins associated with PI3K/AKT/mTOR signaling in XL888-treated xenografts. D, immunofluorescence staining demonstrating the loss of PI3K/AKT/mTOR signaling in 1205LuR xenografts following XL888 treatment.

Fig. 5.

LC-MRM quantification of adaptive signaling in NRAS mutant melanoma following MEK inhibition. A, MEK inhibitor, AZD6244², leads to concentration-dependent decreases in the growth of NRAS mutant melanoma cell lines. Cells were treated with AZD6244 for 72 h, and levels of growth inhibition were quantified using the MTT assay. B, AZD6244 inhibits MAPK signaling in NRAS mutant melanoma cell lines. NRAS mutant melanoma cell lines were treated with increasing concentrations of AZD6244 (1 nm to 10 μm) for 1 h. Western blots show phospho-ERK levels and total ERK expression. C, LC-MRM peptide quantification indicates altered expression of multiple proteins in WM1366 and WM1361A cells following AZD6244 treatment. D, Western blot showing increased expression of WNT signaling proteins (β-catenin/APC), NFκB, and RTKs. E, siRNA knockdown of β-catenin cooperates with AZD6244 to limit phospho-ERK and cyclin D1 expression. Western blot showing the effects of β-catenin knockdown ± AZD6244 (1 μm) upon the expression of phospho-ERK, total ERK, cyclin B1, and cyclin D1. GADPH levels confirm even protein loading. F, siRNA knockdown of β-catenin enhances the level of AZD6244-mediated apoptosis in WM1366 cells. Levels of apoptosis were analyzed by annexin-V binding and flow cytometry. ²AZD is AZD-6244 (MEK inhibitor). NT is non-targeting siRNA used as a vehicle control. APC stands for Adenomatous Polyposis Coli.

Fig. 6.

Co-targeting of MEK and PDGFR limits therapeutic escape. A, LC-MRM analysis of absolute PDGFR expression (in mol normalized to GAPDH expression in fmol) following MEK inhibition. B, RTK array analysis demonstrates increased phosphorylation of PDGFR-β following MEK inhibition. WM131A and WM1366 cells were treated with AZD6244 (1 μm, 24 h) and analyzed on RTK arrays. Data show increased tyrosine phosphorylation of PDGFR-β following AZD6244 treatment. C, PDGFR inhibition overcomes escape from AZD6244 therapy. WM1361A and WM1366 cells were treated with vehicle, AZD6244 alone, crenolanib alone, or AZD6244 + crenolanib for 72 h. Levels of apoptosis were analyzed by annexin-V binding and flow cytometry. D, PDGFR inhibition prevents escape from AZD6244 treatment in long term colony formation assays. WM1366 and WM1361A cells were treated with vehicle, AZD6244 alone, crenolanib alone, or AZD6244 + crenolanib twice weekly for 4 weeks. Colonies were visualized by staining with crystal violet; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. *** indicates p values < 0.001.