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KINOMEscan selectivity profiling of JBJ-04-125-02 and IC50 of EAI045, JBJ-02-112-05 and JBJ-04-125-02 in BaF3 cells.

JBJ-04-125-02 is more potent than EAI045 and JBJ-02-112-05 in the presence of cetuximab and is effective in inhibiting EGFR-dependent proliferation and phosphorylation in L858R/ C797S Ba/F3 cells.

The combination treatment of JBJ-04-125-02 and osimertinib do not result in any toxicity issues associated with weight loss but osimertinib at clinically relevant dose (25 mg/kg) is too potent to exhibit additive effect when combined with JBJ-04-125-02.

JBJ-04-125-02 as a single agent does not result in any toxicity issues associated with weight loss.

Allosteric kinase inhibitors offer a potentially complementary therapeutic strategy to ATP-competitive kinase inhibitors due to their distinct sites of target binding. In this study, we identify and study a mutant-selective EGFR allosteric inhibitor, JBJ-04-125-02, which as a single agent can inhibit cell proliferation and EGFRL858R/T790M/C797S signaling in vitro and in vivo. However, increased EGFR dimer formation limits treatment efficacy and leads to drug resistance. Remarkably, osimertinib, an ATP-competitive covalent EGFR inhibitor, uniquely and significantly enhances the binding of JBJ-04-125-02 for mutant EGFR. The combination of osimertinib and JBJ-04-125-02 results in an increase in apoptosis, a more effective inhibition of cellular growth, and an increased efficacy in vitro and in vivo compared with either single agent alone. Collectively, our findings suggest that the combination of a covalent mutant–selective ATP-competitive inhibitor and an allosteric EGFR inhibitor may be an effective therapeutic approach for patients with EGFR-mutant lung cancer.Significance:The clinical efficacy of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutant lung cancer is limited by acquired drug resistance, thus highlighting the need for alternative strategies to inhibit EGFR. Here, we identify a mutant EGFR allosteric inhibitor that is effective as a single agent and in combination with the EGFR TKI osimertinib.This article is highlighted in the In This Issue feature, p. 813

Supplemental methods for structural modeling of inhibitor binding, pharmacokinetic assays, in vivo studies, crosslinking and biotinylated drug pull-down assays.

Crystallographic data collection and refinement statistics.

Not all tyrosine kinase inhibitors (TKIs) can enhance or bind at the same time as an allosteric inhibitor (AI) and co-binding of TKIs and AIs do not necessarily translate into increased drug potency.

The relative allelic fraction of T790M does not change the potency of JBJ-04-125-02 in H3255 cells but the presence of EGF blunts the efficacy of JBJ-04-125-02 in Ba/F3 and H1975 cells.