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.
Highlights
From the ‡Departments of Molecular Oncology, ¶Cutaneous Oncology, ʈChemical Biology and Molecular Medicine, Moffitt Cancer Center and Research Institute, Tampa, Florida 33612
HSP90 Inhibition Is Associated with Induction of HSP70 in Vitro and in Vivo—Inhibition of HSP90 is known to be associated with compensatory increases in the expression of the highly abundant chaperone protein HSP70
We began using the Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) platform to analyze the altered expression of chaperones following treatment with HSP90 inhibitor
Summary
From the ‡Departments of Molecular Oncology, ¶Cutaneous Oncology, ʈChemical Biology and Molecular Medicine, Moffitt Cancer Center and Research Institute, Tampa, Florida 33612. We have developed a novel multiplexed LC-MRM assay to quantify the expression of Ͼ80 key signaling proteins in cell line models and fine needle aspirates from accessible melanoma lesions [22]. We present the proof-of-principle for monitoring multiple signaling proteins in melanomas treated with either HSP90 or MEK inhibitors.
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