An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer

Abstract

Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.

Figure 1.. Characterization of JBJ-09-063 in enzymatic assays, in vitro BaF3 cellular studies and in vivo xenograft models.

(A) Chemical structures of JBJ-04-125-02 and JBJ-09-063 with orange circles depicting the key difference between the two molecules. The 1.8 Å co-crystal structure of JBJ-09-063 in complex with EGFR^T790M/V948R confirming an allosteric binding mode (PDB 7JXQ). JBJ-09-063 forms a critical hydrogen bond with D855 and a pi-stacking interaction with F856 in the DFG motif. (B) In vitro enzymatic inhibition assay of recombinant EGFR L858R/T790M kinase domain treated with increasing concentrations of allosteric inhibitors, JBJ-04-125-02, JBJ-09-063 and osimertinib. Results is graphed as percentage activity relative to DMSO control (mean ± SD). Cell growth and EGFR phosphorylation inhibition activity of JBJ-04-125-02 and JBJ-09-063 in (C) EGFR^L858R/T790M and (D) EGFR^{L858R/T790M/C797S} Ba/F3 cells as measured by Cell Titer Glo assay and Western Blot. Cell proliferation was graphed as a percentage relative to DMSO control. Data shown in A-C are representative experiments that were repeated at least three times. Efficacy studies examining the effect of allosteric inhibitors (JBJ-09-063, JBJ-04-125-02) and tyrosine kinase inhibitor (osimertinib) in (E) H1975 and (F) DFCI52 xenograft models harboring the EGFR^L858R/T790M mutation. Efficacy studies examining the effect of JBJ-09-063 or osimertinib as a single agent or in combination in (G) H3255GR-C797S and (H) DFCI52-C797S xenograft models harboring the EGFR^{L858R/T790M/C797S} mutation. Data is shown as a group mean of tumor volume in mm³ ± SEM relative to the start of treatment for all available data at the indicated timepoint (Study Days) with corresponding waterfall plots indicating maximum response in each group.

Figure 2.. JBJ-09-063 efficacy in human cancer cells is enhanced when combined with gefitinib.

(A) Cell viability and (B) Western Blot analyses of DFCI52 cells treated with indicated concentrations of osimertinib, JBJ-09-063 and JBJ-04-125-02. (C) Cell viability and (D) apoptosis measured as normalized Caspase3/7 activity and (E) Western Blot analyses of H3255GR cells treated with indicated concentrations of gefitinib, JBJ-09-063 and the combination of both agents. (F) Long-term cell growth assay measured as confluency (%) in H3255GR cells treated with indicated concentrations of gefitinib, JBJ-09-063 and the combination of both drugs for two weeks followed by drug withdrawal for an additional two weeks. Data shown in A-F a representative experiment that was repeated at least two times. All cell viability assays were graphed as a percentage of activity relative to DMSO control over indicated concentrations and all apoptosis experiments were graphed as normalized caspase 3/7 activity (in arbitrary units) over time. Statistical significance was determined using a one-way ANOVA on ranks (the Kruskal-Wallis Test) and Dunn’s multiple comparisons test. **P<0.005; ***P<0.0001. (G) Efficacy studies examining the effect of JBJ-09-063 as a single agent or in combination with gefitinib in DFCI52 xenograft model harboring the EGFR^L858R/T790M mutation. Data is shown as a group mean of tumor volume (mm³) ± SEM relative to the start of treatment for all available data at the indicated timepoint (Study Days).

Figure 3.. Forced dimerization of EGFR with other ERBB family members impart resistance to JBJ-09-063.

(A) Western Blot analyses of HEK293T/Cl.17 cells transiently transfected with EGFR^L858R/T790M or with different EGFR^KDD constructs and treated with DMSO, osimertinib or JBJ-09-063. Cell viability in EGFR^WT, EGFR^KDD-LT/WT, or EGFR^L858R/T790M Ba/F3 cells in the presence or absence of EGF treated with increasing concentrations of (B) JBJ-09-063 or (C) osimertinib. (D) Cell viability and (E) Western Blot analyses of H3255GR cells cultured in RPMI media and treated with indicated concentrations of compounds in the presence or absence of EGF or NRG1. Cell viability assays shown in B-D were graphed as a percentage of activity relative to DMSO control over indicated concentrations. (F) Long-term cell growth assay measured as confluency (%) in H3255GR cells cultured in ACL4 media (top panel) versus RPMI media (bottom panel) and treated with DMSO, JBJ-09-063 or osimertinib for two weeks followed by drug withdrawal for an additional two weeks. Cell proliferation was graphed as a percentage relative to DMSO control. (G) Homodimerization of EGFR visualized by proximation ligation assay (PLA) in H3255GR cells cultured in ACL4 media or RPMI media. Statistical significance was determined using the unpaired t-test **P<0.005. (H) EGFR homodimerization in H3255GR cells cultured in RPMI media visualized by PLA after pre-treatment with or without 10 ng/ml of EGF for 15 minutes followed by incubation with DMSO or JBJ-09-063 for 4 hours. Nuclei are stained in cyan and distinct punctate dots in red are PLA signal showing interaction of EGFR homodimers. Statistical significance was determined by ANOVA followed by Tukey’s multiple comparisons test. *P<0.0278; **P<0.0014. Data quantification in 3D-E was performed and graphed as EGFR:EGFR interactions (PLA dot/nucleus) over different culture media. Scale bar = 30 µm. All studies shown here are representative experiments that were repeated at least three times.

Figure 4.. JBJ-09-063 is effective in TKI naïve EGFR L858R mutant models.

(A) Cell viability of EGFR^L858R Ba/F3 cells following treatment with gefitinib, osimertinib or JBJ-09-063. (B) Cell inhibitory activity and (C) Western Blot analyses of H3255 cells cultured in RPMI media and treated with indicated concentrations of specific inhibitors for 72 hours and 24 hours respectively. (D) Long-term cell growth activity measured as confluency (%) in H3255 parental cells cultured in RPMI media and treated with indicated concentrations of JBJ-09-063, gefitinib or osimertinib for two weeks followed by drug withdrawal for another two weeks. (E) Cell viability and (F) Western Blot analyses of A431 cells treated with increasing concentrations of JBJ-09-063 and afatinib. Data shown in A, B, D, E are representative experiments that were repeated at least three times. Cell viability was graphed as a percentage relative to DMSO control. (G) Efficacy studies examining the effect of JBJ-09-063 or afatinib as single agents in the A431 xenograft model. Mice in the in vivo studies were treated after tumor development for 30 days. Data is shown as a group mean of tumor volume ± SEM relative to the start of treatment (Tumor volume (mm³)) for all available data at the indicated timepoint (Study Days).

Figure 5.. JBJ-09-063 is effective in a broad range of osimertinib-resistant EGFR mutant contexts.

Crystal structure of EGFR in complex with (A) JBJ-09-063 (PDB 7JXQ) or (B) osimertinib (bottom, PDB 4ZAU). Sites of mutation are localized to the ATP binding site and should not affect allosteric inhibitor binding. Modeling of resistance mutations reveals steric clashes with osimertinib with the exception of C797S, which prevents osimertinib from forming a covalent adduct with the protein. (C) Cell proliferation and (D) Western Blot analyses of EGFR^L858R/T790M, EGFR^LT/C797S, EGFR^LT/L718Q, EGFR^LT/L792F, and EGFR^LT/G796S Ba/F3 cells treated with DMSO, osimertinib or JBJ-09-063. All data shown is a representative experiment that was repeated at least three times. Cell proliferation was graphed as a percentage relative to DMSO control. LT = L858R/T790M.

Figure 6.. L747S mutation is an on-target resistance mechanism to JBJ-09-063 but not to osimertinib.

(A) Mutagenesis studies showing the number of colonies that arose when EGFR^L858R/T790M Ba/F3 cells that were exposed to N-ethyl-N-nitrosourea (ENU) to induce sporadic mutation were treated with either DMSO, JBJ-09-063, osimertinib or combination treatment for 4 weeks. Sequence tracing and the percentage of L747S and C797S mutations frequencies present in JBJ-09-063- and osimertinib-resistant colonies were shown with purple arrows respectively. (B) Modeling of the L747S resistance mutation (PDB 7JXQ) using the most likely serine rotamer. (C) The side chain of L747 forms favorable hydrophobic contacts with the phenyl ring of the allosteric inhibitor that are lost in the L747S variant. (D) In vitro inhibition of EGFR L858R/T790M and EGFR L858R/T790M/L747S kinases with increasing concentrations of JBJ-09-063 and osimertinib were measured using an HTRF-based assay. Percentage of activity is relative to a 1% DMSO control. (E) Cell growth inhibition and (F) EGFR phosphorylation activity of EGFR^L858R/T790M or EGFR^LT/L747S Ba/F3 cells treated with JBJ-09-063 or osimertinib was measured by Cell Titer Glo assay and analyzed by Western Blot. Data shown is a representative experiment that was repeated at least three times. Cell viability was graphed as a percentage relative to DMSO control. LT=L858R/T790M.