1A: Identify mutant EGFR substrates using in vitro kinase assays on protein arrays. We sought to identify targets of mutant EGFR kinases using in vitro kinase assays on protein arrays. We have used human protein arrays developed by Invitrogen to perform kinase assays with purified proteins. These protoarrays had around 8000 human proteins spotted in duplicates in glass slides. We used WTEGFR, L858R EGFR, L858R/T790M EGFR in presence or absence of tyrosine kinase inhibitor, erlotinib. We identified several proteins which are phosphorylated in vitro by mutant kinases, but not WTEGFR. For example, we found STK3, PKCtheta, PAK3, MST4 (MASK), TBK1, LynA to be phosphorylated by L585R EGFR, but not WT EGFR. More recently we have collaborated with Dr. Heng Zhus laboratory at Johns Hopkins Medical Institute to use the custom protein arrays developed by Dr. Zhus group. These arrays have around 20,000 full-length human proteins spotted on glass slides. We are currently analyzing this data further to identify specific substrates of WT EGFR, L858R EGFR or L858R/T790M EGFR. We have also initiated validation of some of these targets of mutant EGFR by in vitro kinase assays using purified target proteins. We will also validate some of these targets in vivo by Western blots with phospho-specific antibodies (where available) and shRNA-mediated knockdown of respective target proteins in lung adenocarcinoma cells harboring the EGFR mutants. 1B: Compare the degree of interaction of proteins that associate with WT EGFR, L858R EGFR and Del E746-A750 EGFR. The two most common TKI-sensitizing lung cancer-specific EGFR mutations are constitutively active. However two small clinical trials of patients treated with erlotinib have shown that the prognosis of patients harboring the Del EGFR is better than those with L858R EGFR. Patients with Del EGFR respond better and have more prolonged progression free survival than those harboring the L858R EGFR. We undertook an approach of identifying EGFR-interacting proteins by mass spectrometry. We expressed WT EGFR, L858R EGFR and Del E746-A750 EGFR in NR6 cells, a variant of mouse 3T3 fibroblasts that do not express endogenous EGFR. We used stable isotope labeling with amino acids in cell culture (SILAC) to differentially label cells expressing each of the EGFR variants. Immunoprecipitation of EGFR with a monoclonal antibody specific to the extracellular domain of EGFR was performed to isolate EGFR-interacting proteins that were further identified by mass spectrometry. SILAC labeling of cells enabled relative quantitation of degree of interaction of proteins that interacted with both WT EGFR and mutant EGFRs. We identified several proteins that interacted specifically with mutant EGFRs. Of particular interest are receptor tyrosine kinases that interacted more with L858R and Del EGFR compared to WT EGFR (e.g. EPHA2, DDR2, AXL), suggesting cross talk of receptor tyrosine kinases in cells expressing mutant EGFRs. We are currently validating biochemically and functionally the significance of these interactions.
