Abstract 2071: Protein aggregate formation predicts clinical responses to EGFR tyrosine kinase inhibitors
Authors: French Pim , Gao Ya , Wit Maurice de , Mercieca Darlene , Heer Iris de , Valkenburg Bart , Royen Martin van , Aerts Joachim , Smitt Peter Sillevis 
Published: August 2019

Abstract
Although many tumours depend on EGFR signalling for growth, benefit from EGFR tyrosine kinase inhibitors (TKIs) is restricted to patients harboring specific activating mutations and even then restricted to specific TKIs only. We here performed functional analysis on EGFR to understand why only specific mutation-TKI combinations respond. We find that the addition of TKIs (n=5 inhibitors tested) to mutated-EGFR expressing cells resulted in a rapid and massive formation of protein aggregates, but only with EGFR-mutations where clinical responses have been documented (L858R, ΔE746-A750) and only with TKIs with proven clinical benefit (n=4). No aggregates are formed with inhibitors that are clinically inactive (lapatinib) or with clinically unresponsive mutations (EGFRvIII, EGFRwt). Constructs harboring secondary resistance mutations only formed aggregates in the presence of osimertinib. Western blot, quantitative imaging analysis, revers phase phosphoprotein arrays (RPPA) and RT-qPCR was performed on five different lung cancer cell lines to demonstrate that all TKIs effectively inhibit EGFR phosphorylation and downstream pathway activation. Aggregate formation therefore occurs in addition to EGFR dephosphorylation. We next tested whether our assay is able to predict clinical responses to EGFR TKIs. For this, we first generated eleven different mutation constructs and show that our in-vitro assay directly correlates with cell survival in eleven different EGFR-mutated cell-lines, with striking concordance of IC50 values. We then generated more mutation constructs (total n=31) to show that our in-vitro assay predicts clinical benefit to EGFR TKIs in pulmonary adenocarcinoma patients (median survival 7.0 vs 13 months, HR 0.21, P=0.0004), including prediction of mutations where the response to inhibitors has thus-far not been documented (n=7). Our in-vitro assay therefore provides a clinically important asset to predict whether a tumor harboring an unknown mutation will respond to EGFR-TKIs, and if so, which TKI is most effective. We propose a model whereby only clinically effective inhibitors induce a conformational change, which occurs only in the context of specific activating mutations and in addition to EGFR dephosphorylation. Since this model only depends on the mutation present and the inhibitor used, response to EGFR TKIs is largely independent of the genetic background of the tumor. All patients with sensitive EGFR mutations should, regardless of the type of tumor, be considered for treatment with EGFR-TKIs.
Citation Format: Pim French, Ya Gao, Maurice de Wit, Darlene Mercieca, Iris de Heer, Bart Valkenburg, Martin van Royen, Joachim Aerts, Peter Sillevis Smitt. Protein aggregate formation predicts clinical responses to 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 2071.