EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harboring activating mutations in the EGFR kinase1,2, but resistance arises rapidly, most frequently due to the secondary T790M mutation within the ATP-site of the receptor.3,4 Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant5,6, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond7. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternate mechanisms of action. Here we describe rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild type receptor. A crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays, but as a single agent is not effective in blocking EGFR-driven proliferation in cells due to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state8. We observe dramatic synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization9,10, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by L858R/T790M EGFR and by L858R/T790M/C797S EGFR, a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.

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 EGFR L858R/T790M/C797S signaling in vitro and in vivo . However, increased EGFR dimer formation limits treatment effi cacy and leads to drug resistance. Remarkably, osimertinib, an ATP-competitive covalent EGFR inhibitor, uniquely and signifi cantly 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 effi cacy in vitro and in vivo compared with either single agent alone. Collectively, our fi ndings 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 effi cacy 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.

Intermediary metabolism generates substrates for chromatin modification, enabling potential coupling of metabolic and epigenetic states. Here, we identify such a network as a major component of oncogenic transformation downstream of the LKB1/STK11 tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fueled by pronounced mTOR-dependent induction of the serine-glycine-one carbon pathway coupled to S-adenosylmethionine generation. In concert, DNA methyltransferases are upregulated, leading to elevation in DNA methylation, with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumourigenic growth of LKB1-mutant cells, while resulting in novel therapeutic vulnerabilities.