1377P Differential impact of EGFR exon 20 insertion location on tyrosine kinase inhibitor sensitivity

EGFR exon 20 insertion mutations (EGFRex20ins) can be classified as near-loop and far-loop based on their location. The underlying structural difference between near- vs. far-loop EGFRex20ins and its impact on various other novel EGFR TKIs response are not well understood. We assessed in vitro activity of multiple EGFR TKIs against a panel of BaF3 cells expressing EGFRex20ins mutations. Molecular Dynamics (MD) modeling of near- vs. far-loop EGFRex20ins, with and without binding of EGFR TKIs was performed. Clinical responses (tumor size reduction and progression free survival) were analyzed by near- vs. far-loop insertion location in ZENITH20 and EXCLAIM trials. In vitro testing with different EGFRex20ins indicated a favorable IC50 for afatinib, CLN-081, and poziotinib in near-loop (A767-P772) versus far-loop (H773-R776) insertions, whereas mobocertinib had similar IC50 values in near- and far-loop insertions. MD modeling using free-energy calculations indicated that wildtype EGFR displayed distinct inactive and active states with a relatively high transitional energy barrier. In comparison, the near-loop (S768insSVD) exhibited multiple states separated by shallow barriers whereas the far-loop (H773insNPH) was conformationally more rigid. The differential p-loop orientation change in near- vs. far-loop insertions played an active role in TKI binding, stabilization, and selectivity. Clinical data supported the in vitro differential impact of insertion location. In the ZENITH20 trial (n=82), in pre-treated EGFRex20ins lung cancer patients, poziotinib had superior activity in near-loop than far-loop tumors both on tumor size reduction (-25.9% vs. -9.8%, p=0.0014) and PFS (11.1 vs. 3.5 months, Log Rank p=0.016). In the EXCLAIM trial platinum-pretreated patients (n=76), mobocertinib demonstrated robust activity in both groups as assessed by tumor size reduction (near -38.5% vs. far -34.1%, p=0.59) and PFS (12.0 vs. 13.0, Log Rank p=0.99). Preclinical and clinical data indicated that near- vs far-loop EGFRex20 insertions differentially impact sensitivity to individual TKIs. Thus, knowledge of insertion location may allow for more effectively tailored TKI therapy.