Abstract SY19-03: Rewiring of signaling associated protein complexes in cancer
Authors: Haura Eric B. 
Published: August 2015

Abstract
Attacking aberrantly activated kinases has now become a standard of care for certain cancers, such as subtypes of lung cancer driven by genetically altered receptor tyrosine kinases (RTK, i.e., EGFR, EML4-ALK). While genomic alterations in RTK identify patients likely to respond, the underlying molecular mechanisms that drive the magnitude of response as well as the duration of response remain unclear. In addition, acquired and intrinsic therapeutic resistance remains a problem precluding cure. While genomic strategies can be helpful in identifying tumors driven by RTK and susceptive to RTK inhibitors, increasing evidence suggest a role for multiple RTK activation on a single cancer cell. Importantly, RTK bypass can occur through stromal mechanisms, such as hepatocyte growth factor (HGF) driven MET activation, which cannot be assessed through genomic technology. A major challenge in the field is to accurately identify bypass RTK in individual tumors accounting for both tumor-cell autonomous features (genome) as well as tumor exogenous features (tumor stroma). Activation of RTK through diverse mechanisms, including gene mutation, gene amplification, or overproduction of activating growth factor ligands, leads to assembly of protein complexes on RTK that facilitate downstream signal transduction. Exploiting this fact, we have developed proximity ligation assays (PLA) to annotate protein complexes reflecting an activated EGFR, a key RTK in lung cancer and other cancers. We show that PLA can be developed starting with mass spectrometry based protein-protein interaction datasets to characterize in situ EGFR complexes reflecting active signaling in cancer tissues. We have developed one such assay reflecting EGFR in complex with its adaptor protein GRB2. We show these assays reflect EGFR kinase activity both in vitro using cancer cell lines and in vivo using mouse xenograft models of lung cancer. Importantly, these assays perform well in formalin fixed paraffin embedded (FFPE) tissues enabling wide spread utility in typical hospital tumor samples. We demonstrate feasibility and utility in characterizing EGFR:GRB2 complexes in nearly 300 patient derived xenograft models of cancer. Further, our data indicate good concordance of high PLA signals across three independent cohorts of lung cancer patients numbering 350 unique cases. Our human tumor data shows high PLA signal in tumors with activating EGFR mutation as expected but we can further identify tumors with high PLA signal that lack EGFR mutation or with KRAS mutation, thus representing tumor stromal activation of EGFR. In both the PDX and patient cohorts, high EGFR:GRB2 signal by PLA is associated with responsiveness to anti-EGFR therapy. Ongoing work examining MET, ALK, and FGFR protein complexes will be discussed. This work opens up the human protein interactome as a new class of molecular markers for disease in a more practical manner. Proteins, encoded by DNA, do not work in isolation but instead function as part of multi-protein complexes that drive both normal and disease physiology. Therefore, annotation of such complexes may allow a new view toward disease characterization. Importantly, while we demonstrate the utility of this approach in lung cancer, receptor tyrosine kinase signaling and tyrosine kinase inhibitor therapeutics, our approach described here could have utility across a wide spectrum of both signaling-associated complexes and different types of disease.
Citation Format: Eric B. Haura. Rewiring of signaling associated protein complexes in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY19-03. doi:10.1158/1538-7445.AM2015-SY19-03