Direct and Indirect Modulation of EGFR-Mediated Growth Signaling by Hsp90 Inhibition in NSCLCs Resistant to EGFR Tyrosine Kinase Inhibitors in vitro and in vivo

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Activating EGFR point mutations and deletions confer sensitivity to the reversible EGFR TKIs gefitinib and erlotinib. However, reversible EGFR TKIs are not effective against NSCLCs expressing EGFRs harboring exon 20 mutations, EGFRs carrying the T790M secondary mutation, or wild-type EGFR, in a large proportion of cases. Here, we propose Hsp90 inhibition as a novel strategy for NSCLCs lacking sensitivity to reversible EGFR TKIs. Hsp90 is a crucial chaperone for both conformational maturation and stability of many cellular proteins (clients), particularly oncogenic kinases. We previously reported that Hsp90 inhibition results in degradation of EGFRs with kinase domain point mutations and deletions. To assess exon 20 insertions, we used Ba/F3 cells transformed with exon 20 insertions identified in patient samples. These cells were resistant to erlotinib and the irreversible EGFR-ERBB2 inhibitor CL-387,785 (IC50s > 1 microM) but were sensitive to the Hsp90 inhibitor 17-DMAG (IC50s < 50 nM). Similarly, Ba/F3 cells expressing EGFR exon19 deletions+T790M identified in patient samples were also sensitive to 17-DMAG. In addition, a NSCLC cell line with EGFR E746_T751del_insI + T790M (H820) was resistant to both erlotinib and CL-387,785 (IC50 values > 10 and 0.9 microM, respectively) and sensitive to 17-DMAG (IC50 68 nM). Treatment of H820 cells with 17-DMAG caused marked depletion of several growth factor receptors, including EGFR, ERBB2, and IGF-1R, with a diminution of downstream signals and induction of apoptosis. In contrast, treatment of these cells with CL-387,785 resulted in incomplete depletion of downstream signals and persistent ERBB3 phosphorylation. 17-DMAG was also effective against H1975 cells and in a murine inducible bitransgenic model in which EGFR harboring L858R + T790M causes lung adenocarcinoma. Finally, 17-DMAG also was effective against a variety of NSCLC cell lines harboring wild-type EGFR. For example, H1781 cells, carrying wild-type EGFR and the ERBB2 G776insV_G/C mutation underwent apoptosis after depletion of mutant ERBB2 either by siRNA or 17-DMAG. 17-DMAG also compromised the proliferation and anchorage-independent growth of H1299, H460, and A549 NSCLC cell lines harboring wild-type EGFR, with acute depletion of HER2, IGF-1R, and other Hsp90 client proteins, including cdk4. In A549 cells, erlotinib and 17-DMAG synergistically compromised cell viability. Synergism was not readily demonstrated in EGFR-mutant cell lines already exquisitely sensitive to erlotinib. The result was recapitulated by co-treatment with erlotinib and the IGF-1R inhibitor AG1024, or siRNA targeting IGF-1R. In summary, Hsp90 inhibition is effective against NSCLCs not responsive to EGFR inhibitors. The mechanism underlying the synergism of EGFR and Hsp90 inhibition in EGFR wild-type cells is under investigation.