Abstract 1610: MAPK inhibitor resistance leads to ligand-independent Ephrin A2 receptor signaling and the formation of new melanoma metastases
Authors: Paraiso Kim H. T. , Thakur Meghna Das , John Jobin K. , Fang Bin , Koomen John M. , Fedorenko Inna V. , Tsao Hensin , Flaherty Keith T. , Messina Jane L. , Pasquale Elena M. , Villagra Alejandro , Kirkwood John M. , Meier Friedegund , Sloot Sarah , Gibney Geoffrey T. , Stuart Darrin , Tawbi Hussein , Smalley Keiran S.M. 
Published: May 2015

Abstract
Melanoma is the deadliest form of skin cancer with the worst prognosis in patients with metastasis to distant sites such as brain, liver and bone. Studies have shown that MAPK reactivation is a key signaling event leading to BRAF inhibitor resistance. As such, clinical investigations are now underway to evaluate the efficacy of combining frontline BRAF plus MEK inhibitors. Though this approach appears to have meaningful clinical benefit, there are a number of patients who do not respond to therapy, or who through unknown mechanisms, succumb to refractory disease. In order to identify the dynamic changes that drive MAPK inhibitor resistance, we have developed a systems level approach combining mass spectrometry based phosphoproteomic and bioinformatics methodologies. Based on significant changes in tyrosine, threonine and serine phosphorylation events between naive and vemurafenib resistant melanoma cell lines, we have mapped a resistance interactome of ∼550 nodes. This resistance network was significantly enriched for pathways associated with metastatic disease where changes in network connectivity resulted in the appearance of new signaling hubs such as EGFR, EphA2, EphB4, STAT3, FAK1 and HDAC1. The clinical relevance of these findings was demonstrated in a retrospective study showing that 65% of BRAFV600E patients on vemurafenib therapy developed metastases at new sites, with 25% of these new metastases involving the brain. Consistent with our bioinformatics prediction, resistant lines had greater metastatic potential as seen by increased migration and invasion across matrigel and endothelial cell barriers. Extending our findings to a panel of BRAF and BRAF plus MEK inhibitor resistant cell lines, we found compelling evidence that EphA2 is essential for maintaining a resistance phenotype. This phenotype was associated with AKT activation and uncoupled Eph-ephrin signaling resulting in S897 phosphorylation and overexpression of EphA2. In vivo analysis of matched primary and metastatic tumors from vemurafenib resistant xenografts showed that EphA2 expression is increased in metastatic but not primary lesions. Importantly, analysis of clinical specimens from melanoma patients undergoing or failing vemurafenib therapy confirmed that EphA2 receptor expression is significantly upregulated in metastatic but not primary tumors. Functionally, EphA2 S897 phosphorylation was increased at the tumor leading edge of metastatic lesions while absent in primary lesions. Our results show that MAPK resistant melanomas can adaptively rewire their signaling leading to a more malignant phenotype and that through proteomics based approaches we can reliably identify resistance pathways that will translate into rational therapeutic strategies for disseminated disease.
Citation Format: Kim H. T. Paraiso, Meghna Das Thakur, Jobin K. John, Bin Fang, John M. Koomen, Inna V. Fedorenko, Hensin Tsao, Keith T. Flaherty, Jane L. Messina, Elena M. Pasquale, Alejandro Villagra, John M. Kirkwood, Friedegund Meier, Sarah Sloot, Geoffrey T. Gibney, Darrin Stuart, Hussein Tawbi, Keiran S.M. Smalley. MAPK inhibitor resistance leads to ligand-independent Ephrin A2 receptor signaling and the formation of new melanoma metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1610. doi:10.1158/1538-7445.AM2014-1610