Abstract 3209: Elucidating drug resistance mechanisms in melanoma with quantitative proteomics.
Authors: Remily-Wood Elizabeth , Paraiso Kim , Haarberg Eirik , Chen Y. Ann , Rebecca Vito , Smalley Keiran , Koomen John M. 
Published: August 2014

Introduction: Exciting breakthroughs have been made in targeted therapy for melanoma; however, the development of drug resistance remains a critical problem for patients due to the speed of onset and the numerous complex mechanisms. Parallel development of platform for quantitative protein expression enables biomarker monitoring to evaluate the multiple pathways through which tumor cells can acquire drug resistance. We have developed quantitative assays using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) to assess a diverse panel of &gt;70 proteins involved in signaling pathways, including apoptosis, receptor tyrosine kinases, PI3K/Akt/MTOR, and MAP kinases. Assays quantify changes in response to targeted therapeutic agents and examine subsequent resistance mechanisms in cell lines, xenografts, and biopsies from patients.

Experimental procedures: Naïve and BRAF V600E inhibitor-resistant melanoma cell lines were treated with vehicle control, BRAF V600E inhibitor (PLX4720), HSP90 inhibitor (XL-888, Exelixis), or MEK inhibitor (AZD6244). Immunoblotting and LC-MRM were used to evaluate protein expression levels. Heat shock proteins can be measured directly from whole cell lysates or tumor homogenates using a few micrograms of protein. For expression of cancer signaling proteins (many of which are HSP90 clients), 50 micrograms of total protein were fractionated by SDS-PAGE prior to digestion and LC-MRM quantification. Similar experiments were conducted with mouse xenograft tumors from treated and control mice; these measurements have also been applied to fine needle aspirates (FNAs) and core biopsies from patients in the context of a clinical trial.

Data summary: LC-MRM quantification is effective in quantifying these proteins in cell lines, xenografts, and biopsies or FNAs. The LC-MRM panel assay was able to screen for a diverse range of proteins revealing previously undescribed cellular responses to treatment and elucidate potential mechanisms of resistance. As an example, both LC-MRM data and Westerns showed consistent responses to HSP90 inhibition, including increased expression of HSP71 and reduction of HSP90 client levels. Proteins were also modulated in response and resistance to BRAF and MEK inhibitors.

Conclusion: LC-MRM quantifies biomarker panels and provides an assessment tool that can translated in a straightforward way from cell line models to patient specimens. This emerging technique holds great promise for biopsy-mandated clinical trials, particularly in diseases like melanoma that can be readily sampled. Measurements using this LC-MRM platform can be used to assess drug performance and patient response to evaluate treatment, detect drug resistance, and suggest rational combination therapies.

Citation Format: Elizabeth Remily-Wood, Kim Paraiso, Eirik Haarberg, Y. Ann Chen, Vito Rebecca, Keiran Smalley, John M. Koomen. Elucidating drug resistance mechanisms in melanoma with quantitative proteomics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3209. doi:10.1158/1538-7445.AM2013-3209