Among cancer entities lung cancer ranks first in incidence and mortality, with ~85% of cases classified as non-small cell lung cancer (NSCLC). In lung cancer, driver mutations are commonly found in receptor tyrosine kinases with Epidermal Growth Factor Receptor (EGFR) constituting the major driver of oncogenesis (29%, Chevallier et al. 2021). Mutant EGFR is targeted by multiple specific tyrosine kinase inhibitors (RTKis) for anti-cancer therapy. Therapy resistance against such RTKis and subsequent disease progression occurs frequently and remains a severe challenge. A promising approach to overcome resistance is the combination of RTKi with BH3-mimetics or proteasome inhibition (Muenchow et al. 2021, Tanimoto et al. 2021, Weller et al. 2022). The proposed mechanism is the induction of NOXA, which inhibits the anti-apoptotic Bcl-2 protein MCL-1. In this study, we assess the role of BCL-2 proteins in RTKi resistance in NSCLC cell lines and the effect of direct BCL-2 inhibition.

NSCLC cell lines of differing EGFR mutation status (HCC4006 [ex19del] and H1975 [L858R/T790M]) were cultured in the presence of EGFR-TKI osimertinib (OSI) to generate EGFR-TKI resistant cell lines. Successful generation of resistance was verified via flow cytometric analysis of apoptotic cell death induction and via live-cell imaging-based proliferation assessment. Expression of Bcl-2 proteins was analyzed by RNAseq and Western Blot. Cells were incubated with OSI and/or Bcl-2 inhibitors and apoptotic cell death induction as well as cell viability were measured by flow cytometry and luminescence-based assays.

RNAseq revealed an upregulation of BCL-2 in the RTKi resistant cell lines and derived clonal cell lines suggesting a resistance mechanism. We found resistant cell lines to be more sensitive to apoptosis induction via BCL-2 inhibition than parental cells, highlighting their dependency on anti-apoptotic proteins. Especially BCL-xL- and MCL-1-specific inhibitors demonstrated strong efficacy in combination with RTKi in NSCLC cells and RTKi-resistant NSCLC cells. Furthermore, combination of osimertinib with BCL-2i was sufficient to overcome RTKi resistance likely mediated by BCL-2 overexpression

We find that RTKi resistant NSCLC cell lines show increased sensitivity towards inhibition of Bcl-2 proteins and that RTKi resistance of NSCLC cells is overcome through inhibition of these proteins by specific Bcl-2 inhibitors. These findings represent a general therapeutic approach that relies on the simultaneous blockade of driver mutation mediated EGFR activity and survival-promoting Bcl-2 proteins.