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Science Spotlight

Methods for the masses: multiplexed growth signaling analyses by mass spec

From the Paulovich Lab, Clinical Research Division, and colleagues

In the age of personalized cancer therapy, genetic sequencing technologies allow clinicians to rapidly pinpoint mutations likely involved in driving patient-specific tumorigenesis and disease progression. These oncogenic mutations lead to the aberrant activation of signaling pathways involved in cellular growth and division. Targeted therapeutics that inhibit specific components of these pathways often yield dramatic responses in patients whose cancers rely on them for sustained growth and survival. Unfortunately, tumor relapse is a common eventuality in these settings. The mechanisms of therapeutic resistance can involve complex and sometimes paradoxical rewiring of signaling networks, highlighting an urgent need to deepen our understanding of the underlying biology for the development of new therapeutics and combination treatment strategies.

Dr. Amanda Paulovich in the Fred Hutch Clinical Research Division, who holds the Aven Foundation Endowed Chair, teamed up with colleagues at the Moffitt Cancer Center and Broad Institute to develop multiplexed assays for analyzing growth signaling by multiple reaction monitoring mass spectrometry (MRM), a targeted form of mass spectrometry that enables highly sensitive and specific measurements. Their work, led by Paulovich Lab staff scientist Dr. Jeffrey Whiteaker, and recently published in Cell Reports Methods, presents new community resources for simultaneously quantifying the abundance of proteins and phosphoproteins involved in three major networks of oncogenic growth signaling.

Multiple reaction monitoring mass spectrometry (MRM) allows for multiplexed measurements of tumor growth signaling pathway protein and phosphoprotein abundance.
Multiple reaction monitoring mass spectrometry (MRM) allows for multiplexed measurements of tumor growth signaling pathway protein and phosphoprotein abundance. Figure provided by Dr. Jeffrey Whiteaker.

Signal transduction pathways translate the input of external growth factors into the outputs of cellular growth and proliferation through phosphorylation cascades, a relay of chemical protein modifications mediated by a class of enzymes called kinases. Receptor tyrosine kinases (RTKs) are a family of surface proteins that bind to growth factor ligands, causing them to pair with other RTKs, which leads to the activation of the kinase catalytic domain and reciprocal phosphorylation of each of the receptors’ intracellular tails. The presence of these phosphate groups on the tails of the RTKs creates docking sites for downstream signaling proteins, triggering a complex choreography of molecular events through several signaling pathways, involving the successive phosphorylation and activation of downstream kinases. The mitogen-activated protein kinase (MAPK) pathway is one such network, activated upstream by rapidly accelerated fibrosarcoma (RAF) kinases and leading to downstream gene expression changes regulating cell proliferation and survival. A second signaling network induced by RTK stimulation is the Src homology 2-like serine/threonine-protein kinase B family (AKT) pathway, activated upstream by phosphatidylinositol 3-kinase (PI3K) and leading to downstream gene expression and protein function changes that promote cell division, survival and metabolic changes. 

Mutations that enhance RTK, MAPK and AKT network signaling are common oncogenic drivers of tumorigenesis, since they cause aberrant mitogenic signaling in the absence of external growth factors. Conversely, targeted inhibitors that bind and antagonize the protein components of these pathways serve as potent anti-tumor therapeutic agents, oftentimes taking the form of kinase inhibitors. Understanding how cancers exploit these interconnected signaling pathways, how they change in response to therapy, and how these networks become rewired as tumors acquire therapeutic resistance requires methods for comprehensively and simultaneously measuring flux through each of them.

Traditional methods for monitoring growth pathway signaling, namely western blot-based detection of phosphorylated proteins, are only semi-quantitative, difficult to multiplex, and limited by the specificity and quality of antibody-based reagents. Conversely, mass spectrometry-based methods are sensitive and exceedingly specific, readily multiplexed, and rendered highly quantitative through the use of spiked-in standards. The Paulovich lab and colleagues developed a set of MRM assays for quantifying growth pathway proteins and phosphoproteins that effectively replaces what would necessitate more than 60 western blots, with enhanced specificity and quantitative precision. “This work generates a tremendous resource for researchers interested in precisely quantifying protein expression and phosphorylation in signaling pathways,” said Dr. Whiteaker. “We were fortunate to be able to work with our colleagues at the Moffitt Cancer Center, Broad Institute, and Frederick National Laboratory in developing the assays and demonstrating their transferability.”

The recent Cell Reports Methods publication describes the development, validation, and proof-of-concept application of a suite of publicly available targeted proteomics assays for quantifying the abundance and phosphorylation of proteins involved in RTK, MAPK and AKT network signaling. The MRM workflow involves the digestion of proteins into peptides, which are subjected to liquid chromatography coupled to tandem mass spectrometry, tuned for measuring specific ion/fragment pairs that were hand-picked by the authors to reduce interferences. These methods are complemented by enrichment strategies to allow for the detection of low abundance analytes, including affinity chromatography enrichment of phosphorylated peptides and immunoaffinity enrichment of specific peptides of interest. For immunoaffinity enrichment, the assays incorporated a suite of custom monoclonal antibodies previously developed by the Paulovich Laboratory. Importantly, validation studies included a demonstration of assay portability, showing good concordance between measurements taken at three different laboratory sites.

In a proof of concept study, the authors utilized their new toolset for examining growth pathway signaling in the context of BRAF kinase inhibition, using the targeted small molecule PLX-4720. They treated PLX-4720-sensitive and -resistant melanoma and colon cancer cell lines, which carried activating mutations in either BRAF or the upstream rat sarcoma (RAS) proto-oncogene, and subjected the samples to MRM analyses. Encouragingly, BRAF inhibition selectively dampened MAPK signaling in treatment-sensitive BRAF-mutant cells. As has been previously established in the literature, the investigators observed that long-term BRAF inhibition led to paradoxical hyperactivation of MAPK signaling, through the release of negative feedback mechanisms, leading to resistance to therapy. Interestingly, in addition to enhanced MAPK signaling, RAS-mutant treatment-resistant cells also upregulated AKT pathway signaling in response to BRAF inhibition, indicating a potential second mechanism for resistance in this cell line. These results, both recapitulating well-known drug resistance mechanisms as well as uncovering previously uncharacterized signaling changes in response to therapy, support the utility of MRM-based assays for examining the rewiring of key signaling pathways in response to treatment.

“All the resources for applying the assays were made available to the research community via the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (​CPTAC) Assay Portal, and we expect they will impact preclinical and clinical biomarker, pharmacodynamic, and mechanism of action studies,” said Dr. Whiteaker. “In fact, the assays are already getting uptake! The assays were used in a recent study identifying a subset of pediatric brain tumors that may respond to MEK/MAPK inhibitors. It’s exciting to see our measurements contribute to such meaningful studies.”

This work was funded by the National Cancer Institute and the Moffitt Foundation.

UW/Fred Hutch Cancer Consortium member Amanda Paulovich contributed to this work.

Jeffrey R. Whiteaker, Kanika Sharma, Melissa A. Hoffman, Eric Kuhn, Lei Zhao, Alexandra R. Cocco, Regine M. Schoenherr, Jacob J. Kennedy, Ulianna Voytovich, Chenwei Lin, Bin Fang, Kiah Bowers, Gordon Whiteley, Simona Colantonio, William Bocik, Rhonda Roberts, Tara Hiltke, Emily Boja, Henry Rodriguez, Frank McCormick, Matthew Holderfield, Steven A. Carr, John M. Koomen, Amanda G. Paulovich. Targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP kinase, and AKT signaling. Cell Reports Methods. 2021, 100015, ISSN 2667-2375.