The HSP90 inhibitor ganetespib potentiates the antitumor activity of EGFR tyrosine kinase inhibition in mutant and wild-type non-small cell lung cancer

Abstract

Small molecule inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase activity, such as erlotinib and gefitinib, revolutionized therapy for non-small cell lung cancer (NSCLC) patients whose tumors harbor activating EGFR mutations. However, mechanisms to overcome the invariable development of acquired resistance to such agents, as well as realizing their full clinical potential within the context of wild-type EGFR (WT-EGFR) disease, remain to be established. Here, the antitumor efficacy of targeted EGFR tyrosine kinase inhibitors (TKIs) and the HSP90 inhibitor ganetespib, alone and in combination, were evaluated in NSCLC. Ganetespib potentiated the efficacy of erlotinib in TKI-sensitive, mutant EGFR-driven NCI-HCC827 xenograft tumors, with combination treatment causing significant tumor regressions. In erlotinib-resistant NCI-H1975 xenografts, concurrent administration of ganetespib overcame erlotinib resistance to significantly improve tumor growth inhibition. Ganetespib co-treatment also significantly enhanced antitumor responses to afatinib in the same model. In WT-EGFR cell lines, ganetespib potently reduced cell viability. In NCI-H1666 cells, ganetespib-induced loss of client protein expression, perturbation of oncogenic signaling pathways, and induction of apoptosis translated to robust single-agent activity in vivo. Dual ganetespib/erlotinib therapy induced regressions in NCI-H322 xenograft tumors, indicating that the sensitizing properties of ganetespib for erlotinib were conserved within the WT-EGFR setting. Mechanistically, combined ganetespib/erlotinib exposure stabilized EGFR protein levels in an inactive state and completely abrogated extracellular-signal-regulated kinase (ERK) and AKT signaling activity. Thus, selective HSP90 blockade by ganetespib represents a potentially important complementary strategy to targeted TKI inhibition alone for inducing substantial antitumor responses and overcoming resistance, in both the mutant and WT-EGFR settings.

Fig. 1

Ganetespib confers superior antitumor efficacy in combination with erlotinib in NCI-HCC827 NSCLC xenografts. a Mice bearing NCI-HCC827 tumors (n = 8 mice/group) were i.v. administered ganetespib or p.o. dosed with erlotinib (each at 50 mg/kg) on a weekly dosing regimen, either alone or in combination. Data are expressed as mean and SEM for each time point. Numerical T/C values are indicated to the right of each growth curve. Combinatorial ganetespib plus erlotinib therapy was significantly more efficacious than either agent alone (combination vs. erlotinib, *p = 0.0003). b Body weights were measured five times per week for the first 2 weeks of dosing and twice per week thereafter. Mean values are plotted against vehicle controls

Fig. 2

Combinations of ganetespib with EGFR TKIs overcome erlotinib resistance and confer superior antitumor efficacy in NCI-H1975 NSCLC xenografts. a NCI-H1975 cells were incubated with the indicated concentrations of ganetespib for 24 h. Cell lysates were analyzed by Western blotting. b Mice bearing NCI-H1975 tumors (n = 8 mice/group) were i.v. administered ganetespib (50 mg/kg) or p.o. dosed with erlotinib (200 mg/kg) on a weekly dosing regimen, either alone or in combination. Data are expressed as mean and SEM for each time point. Numerical T/C values are indicated to the right of each growth curve. Erlotinib monotherapy had no activity in this resistant xenograft model; however, the combination of ganetespib and erlotinib was significantly more efficacious than ganetespib treatment alone (*p < 0.0001). c NCI-H1975 cells were incubated with the indicated concentrations of ganetespib or afatinib, both alone and in combination, for 24 h. Cell lysates were analyzed by Western blotting. d Mice bearing NCI-H1975 tumors (n = 8 mice/group) were i.v. dosed with ganetespib (50 mg/kg) once weekly and afatinib (5 mg/kg) administered p.o. five times/week, either alone or in combination. Combinatorial ganetespib plus afatinib therapy was significantly more efficacious than either agent alone (*p < 0.001). e Mice bearing NCI-H1975 tumors (n = 8 mice/group) were i.v. dosed with ganetespib (50 mg/kg) once weekly and afatinib (20 mg/kg) administered p.o. five times/week, either alone or in combination. The combination of ganetespib and erlotinib was significantly more efficacious than single-agent therapy, resulting in tumor regressions (*p < 0.0001)

Fig. 3

Ganetespib inhibits oncogenic signaling and suppresses tumor growth in WT-EGFR NSCLC models. a NCI-H1666 cells were incubated with the indicated concentrations of ganetespib for 24 h. Cell lysates were analyzed by Western blotting. b NCI-H1666 cells were treated with 100 nM ganetespib for the indicated time periods. Cell lysates were analyzed by Western blotting. c Mice bearing NCI-H1666 tumors (n = 4 mice/group) were i.v. administered ganetespib (150 mg/kg) on a weekly dosing regimen for 3 weeks. Data are expressed as mean and SEM for each time point. Numerical T/C values are indicated to the right of each growth curve

Fig. 4

Ganetespib potentiates the activity of erlotinib to induce tumor regression in WT-EGFR NSCLC. a NCI-H322 cells were incubated with the graded concentrations of erlotinib for 24 h, with or without 500 nM ganetespib. Cell lysates were analyzed by Western blotting. b Mice bearing NCI-H322 tumors (n = 8 mice/group) were i.v. dosed with ganetespib (50 mg/kg) once weekly and erlotinib (25 mg/kg) administered p.o. five times/week, either alone or in combination. Numerical T/C values are indicated to the right of each growth curve, and the errors bars are the SEM. The combination of ganetespib and erlotinib was significantly more efficacious than either agent alone, resulting in tumor regressions (combination vs. erlotinib, *p < 0.0001)