Novel EGFR mutations that cause drug resistance to irreversible pyrimidine but not quinazoline based EGFR inhibitors
http://cancerres.aacrjournals.or ... etingAbstracts/4832
Background: Oncogenic EGFR T790M causes drug resistance to quinazoline based EGFR kinase inhibitors by increasing ATP affinity (Yun PNAS 2008). Mutant selective irreversible pyrimidine EGFR kinase inhibitor, WZ4002, is effective in non-small cell lung cancer (NSCLC) models harboring EGFR T790M (Zhou Nature 2009). We aimed to determine potential mechanisms of resistance to WZ4002 and explore alternative strategies to overcome acquired resistance to pyrimidine based EGFR inhibitors. Methods and Results: We performed an ENU mutagenesis screen in Ba/F3 cells expressing EGFR L858R, L858R/T790M, Del E746_A750 and Del E746_A750/T790M followed by culture in the presence of WZ4002 (100 nM or 1 μM). Using RT-PCR, we sequenced resistant clones for secondary EGFR mutations. No EGFR T790M mutations were identified. We detected novel secondary EGFR L718Q (9/27; 33%) or L844V (1/27; 3%) mutations in the drug resistant cells. We also recovered the EGFR C797S (1/27; 3%) mutation previously known to prevent covalent binding and decrease potency of WZ4002. Unlike EGFR T790M, EGFR L718Q and EGFR L844V did not lead to constitutive EGFR phosphorylation, were not transforming in Ba/F3 cells and required EGF for proliferation and survival. The EGFR L858R/L844V Ba/F3 cells were resistant to WZ4002 (IC50 0.7 μM) but sensitive to irreversible quinazoline EGFR inhibitors CL-387,785, HKI-272 (neratinib) and BIBW2992 (afatinib) (IC50 values all < 10 nM). Similar findings were observed with the EGFR L858R/L718Q cells although the IC50 values were slightly higher (100 nM or less) and with the Del E746_A750/L718Q and Del E746_A750/L844V cells. All triple mutants harboring EGFR T790M (e.g. L858R/T790M/L844V) were resistant to WZ4002 and irreversible quinazoline EGFR inhibitors. EGFR L858R/L844V and L858R/L718Q Ba/F3 cells were growth inhibited by clinical concentrations (1 μM) of gefitinib and the combination of 1 μM gefitinib and 100 nM WZ4002 completely prevented the emergence of resistant clones in our ENU assay. Using structural modeling, both the L718Q and L844V mutations likely lead to steric hindrance and could impact WZ4002 binding. To verify this hypothesis, we developed a biotinylated-WZ4002 compound and used it to assay binding to different EGFR mutant proteins. This agent effectively bound to EGFR L858R and DelE746_A750 (with or without T790M). However, in the presence of a concurrent L718Q or L844V mutation, protein binding was significantly reduced, consistent with the reduced in vitro efficacy in the Ba/F3 cells. Conclusions: We identify novel EGFR mutations that confer drug resistance to irreversible pyrimidine but not quinazoline EGFR kinase inhibitors. Our findings have implications for understanding drug resistance mechanisms and for the development of combinations of EGFR kinase inhibitors as therapies for cancer patients. |
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