and C. in 41 therapy resistant tumors from four xenograft NSCLC versions. We identified unique and tumor-specific tyrosine phosphorylation rewiring in tumors resistant to treatment with the irreversible third generation EGFR-inhibitor, osimertinib, or the novel dual-targeting EGFR/Met antibody, JNJ-61186372. Tumor-specific increases in tyrosine-phosphorylated peptides from EGFR family members, Shc1 and Gab1 or Src family kinase substrates were observed, underscoring a differential ability of tumors to uniquely escape EGFR Rabbit Polyclonal to TUBGCP3 inhibition. Although most resistant tumors within each treatment group displayed a marked inhibition of EGFR as well as Src family kinase (SFK) signaling, the combination of EGFR inhibition (osimertinib) and SFK inhibition (saracatinib or dasatinib) led to further decrease in cell growth signaling rewiring that would have been masked by analysis of cell population averages. Keywords: Epidermal growth factor receptor (EGFR), drug resistance, non-small cell lung cancer (NSCLC), phosphoproteomics, mass spectrometry Introduction Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality, and its prevalence continues to increase worldwide (1). Activating mutations in the kinase domain name of the epidermal growth factor receptor (EGFRoccur at a high frequency in NSCLC patients, and are especially frequent in never-smokers. Despite initial survival benefit from therapy with EGFR tyrosine kinase Leflunomide inhibitors (TKIs), overall patient survival remains suboptimal and recurrence rates are high (2). Most patients experience acquired resistance to EGFR-targeted therapy in less than a year after starting treatment (3C5). Even in the context of improved EGFR TKIs, overcoming therapeutic resistance remains a significant clinical challenge (6,7). A better understanding of the molecular mechanisms underlying resistant cancer cell growth and survival is required to direct future therapies for advanced state NSCLC. To date, knowledge of therapy resistance mechanisms has facilitated the development of three generations of EGFR TKIs. First-generation EGFR TKIs such as erlotinib and gefitinib bind competitively and reversibly to the ATP-binding site of Leflunomide the EGFR tyrosine kinase domain name. Clinical trials confirmed superior response rates and improved progression-free survival in NSCLC patients with activating EGFR mutations, also known as sensitizing mutations, such as L858R and the in-frame exon 19 deletion (ex19del) (4,5,8). However, about 50C60% of patients acquire resistance to TKI therapy through restored EGFR signaling conferred by the secondary T790M EGFR gatekeeper mutation (8C11). Consequently, second-generation irreversible EGFR TKIs such as afatinib, dacomitinib and neratinib were introduced as a strategy to overcome first-generation EGFR TKI resistance. However, despite promising activity against T790M, these TKIs displayed limited clinical efficacy due to dose-limiting toxicity caused by simultaneous inhibition of wild type EGFR (12). Ultimately, third-generation EGFR-TKIs were designed to selectively target T790M and EGFR TKI-sensitizing mutations over the wild-type receptor. Recently, one of these, the irreversible TKI osimertinib, received US Food and Drug Administration (FDA) approval for advanced Leflunomide NSCLC Leflunomide by showing promising clinical efficacy (6,13). However, despite significant increase in progression-free survival with osimertinib as compared to platinum-pemetrexed in T790M-expressing NSCLC patients, development of resistance still limited the efficacy of this treatment (7,14). Given the challenge of therapy resistance, there have been considerable efforts over the past decade to define resistance mechanisms at a molecular level. In this respect, it has become clear that resistance does not merely evolve around the targetable driver of disease as exemplified by EGFR T790M. A recurrent theme involves the additional engagement of bypass signaling pathways driven by other receptor tyrosine kinases (RTKs) to support tumor cell growth and survival (15). For instance, multiple studies have highlighted activation of hepatocyte growth factor receptor (Met), by increased expression of the receptor due to gene amplification or by increased expression of the ligand hepatocyte growth factor (HGF), as an important resistance mechanism to both first and third generation EGFR TKIs (9,11,16C18). Other RTK-mediated resistance mechanisms include activation of human epidermal growth factor receptor 2 (HER2), insulin-like growth factor 1 receptor (IGF1R) and Axl (19C21). The frequent occurrence of bypass signaling resistance suggests the need for an approach whereby co-targeting multiple pathways could serve as a strategy to delay or overcome resistance. Bi-specific antibodies present one such approach, and have recently been approved in leukemia, with several other bi-specific antibodies in advanced clinical development (22,23). For NSCLC, the novel bi-specific antibody JNJ-61186372 targeting EGFR and Met was recently reported to be effective in EGFR TKI resistant preclinical models (24,25). However, even in.