Development of Mutant-Selective Allosteric EGFR Inhibitors for Drug-Resistant Lung Cancer

Abstract

<b>Abstract ID 16204</b> <b>Poster Board 517</b> Mutations in the epidermal growth factor receptor (EGFR) are common drivers of non-small cell lung cancer (NSCLC). Receptors harboring the most common EGFR point mutation, L858R, are effectively treated by tyrosine kinase inhibitors (TKIs), but resistance inevitably develops through additional T790M and/or C797S mutations. Acquisition of both resistance mutations renders tumors unresponsive to all clinically-approved EGFR TKIs and affects &gt;10,000 patients per year in the US. To address this, we utilized a combination of high-throughput screening and structure-based drug design to discover and develop mutant-selective, <i>allosteric</i> EGFR inhibitors that are unaffected by common ATP-site resistance mutations. Allosteric EGFR inhibitors work by stabilizing the inactive, “C-helix out” conformation of the kinase and are therefore antagonized by receptor dimerization, which induces the active conformation. To better understand the antagonistic effects of receptor dimerization on allosteric inhibition, we generated a constitutively dimerized pair of kinase domains suitable for biochemical assays. Using this synthetic dimer, we showed that allosteric inhibitor potency was decreased by ∼500-fold upon dimerization. We also assessed the effectiveness of allosteric inhibitors against a rare kinase domain duplication (KDD) variant of EGFR associated with lung cancers. This variant was resistant to allosteric inhibition as a result of constitutive kinase domain dimerization within a single receptor. We overcame these antagonistic effects through co-administration of the dimerization-blocking antibody cetuximab or the use of a highly potent allosteric inhibitor, such as JBJ-09-063, which disrupts EGFR dimers in cells. JBJ-09-063 displays a biochemical IC₅₀ of &lt;100 pM and single-agent efficacy <i>in&nbsp;vivo</i> in a number of NSCLC models, including those harboring both the T790M and C797S resistance mutations. We have shown that JBJ-09-063 and a series of ATP-competitive TKIs can simultaneously bind EGFR kinase, resulting in inhibition synergy. Dual-targeting benefits from positive binding cooperativity between inhibitors, which partially restores binding of the frontline therapy osimertinib to the resistant C797S variant. Cooperativity is facilitated by protein conformational changes and complementary electrostatic contacts between inhibitors. Leveraging “cooperative inhibition” with other allosteric inhibitor and TKI combinations represents a novel way to target difficult to treat variants. Thus, we are utilizing structural and molecular insights of simultaneous drug binding to facilitate the identification and development of novel drug combinations. T.S.B. is supported by a Ruth L. Kirschstein National Research Service Award (F32CA247198). This work was supported by the National Institutes of Health grant R01 CA201049 (M.J.E., N.S.G.), P01 CA154303 (M.J.E. and P.A.J.), R35 CA242461 (M.J.E.), and R35 CA220497 (P.A.J.).