Abstract 2200: Potent in vitro activity of Tarloxotinib for EGFR C797S and other mutations refractory to current EGFR tyrosine kinase inhibitors
Authors: Suda Kenichi , Nishino Masaya , Koga Takamasa , Fujino Toshio , Kobayashi Yoshihisa , Mitsudomi Tetsuya , Vellanki Avanish , Tirunagaru Vijaya G. 
Published: August 2019

Abstract
Introduction: EGFR tyrosine kinase inhibitors (TKIs) are key drugs in the treatment of lung cancers with activating EGFR mutations. However, inherent resistance such as exon 20 insertions and acquired resistance such as T790M / C797S hamper further improvement of treatment outcomes in lung cancer patients with EGFR mutations. In this study, we evaluated efficacy of a novel hypoxia-activated EGFR-TKI, tarloxotinib, for these resistant EGFR mutations using Ba/F3 models.
Methods: Ba/F3 cells with either of exon 20 insertions (A763insFQEA, V769insASV, D770insSVD, or H773insNPH) or Del 19/T790M/C797S in cis (triple mutations) were generated as previously described (Nishino, et al. Lung Cancer, in press). Ba/F3 cells with Del 19 alone was used as sensitive control. Growth inhibitory assays against these Ba/F3 cells were performed for tarloxotinib-E (activated form), tarloxotinib (pro-drug before activation), afatinib, poziotinib, and osimertinib. In vitro kinase assays for various EGFR and HER2 mutations at ATP Km were used to test the potency of Tarloxotinib-E.
Results: Tarloxotinib-E was highly potent across various EGFR and HER2 mutations, including C797S double mutants in the in vitro kinase assays with an IC50 of &lt;0.38 nM. Low nM potency was observed for EGFR C797S triple mutants (d746-750/T790M/C797S, T790M/C797S/L858R). Tarloxotinib showed about 100 times higher IC50 values compared with tarloxotinib-E in all tested Ba/F3 cells, indicating that tarloxotinib-E can selectively inhibit cell growth in lung cancers with EGFR mutations. Poziotinib and tarloxotinib-E were effective against all Ba/F3 cells with exon 20 insertions (IC50 &lt; 10 nM), while afatinib and osimertinib effectively killed only the Ba/F3 cells with A763insFQEA mutation. For Ba/F3 cells with Del19/C797S mutation, afatinib, poziotinib and tarloxtinib-E showed potent activity with an IC50 of 2.8, 1.6 and 5.1 nM respectively. For Ba/F3 cells with Del 19/T790M/C797S, tarloxotinib-E also exhibited activity (IC50 198 nM), while the triple mutations conferred resistance to the other TKIs.
Conclusion: As the hypoxia-activated fragmentation of tarloxotinib into tarloxotinib-E broadens the therapeutic window versus competing small molecule EGFR TKIs, direct IC50 comparisons across agents may not be relevant as tarloxotinib may be dosed higher. These data highlight potential opportunity for tarloxotinib in the treatment of lung cancers with inherent or acquired resistance to current available EGFR-TKIs.
Citation Format: Kenichi Suda, Masaya Nishino, Takamasa Koga, Toshio Fujino, Yoshihisa Kobayashi, Tetsuya Mitsudomi, Avanish Vellanki, Vijaya G. Tirunagaru. Potent in vitro activity of Tarloxotinib for EGFR C797S and other mutations refractory to current EGFR tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2200.