Abstract 2532: Disrupting resistance to EGFR-specific tyrosine kinase inhibitors in TP53-mutated lung carcinoma cell lines by proteasome inhibition
Authors: Orth Franziska , Schaefer Benjamin , Harsch Annika , Weller Sandra , Beigl Tobias , Barber Lynn , Kopp Hans-Georg , Essmann Frank 
Published: April 2023

Abstract
Lung cancer is the leading cause of cancer related death. The most common form is non-small cell lung cancer (NSCLC). In a large proportion of NSCLC, driver mutations in protein kinases are found. One of the most frequently affected gene is the epidermal growth factor receptor (EGFR). Anti-cancer drugs have been developed that specifically inhibit mutated EGFR and are effective in anti-cancer therapy. However, resistance mediating mutations occur regularly during therapy limiting both treatment outcome and therapeutic options for follow up therapy. In sarcoma cell lines, resistance has already been overcome by a combination of proteasome inhibitors (PIs) with BH3 mimetics (Muenchow et al., 2020). In this case, drugs were combined that have an independent mechanism of action. In ALK-rearranged/TP53-mutated NSCLC cells, ALK-TKI resistance is also overcome by proteasome inhibition that mediates induction of the pro-apoptotic protein NOXA (Tanimoto et al., 2020). Since NOXA inhibits anti-apoptotic BCL-2 proteins, we hypothesize that combined treatment with receptor tyrosine kinase inhibitors (RTKIs) and PI or BCL-2 inhibitors (BCL2I) is an effective strategy for the treatment of EGFR mutated NSCLC tumors. To explore this hypothesis, we investigated cell death induction by combined administration of RTKI and PI in NSCLC cells differing in their mutational status, i.e., RTKI resistance. In addition, we analyzed the expression of effector BCL-2 family proteins (BAX, BAK, BOK) to determine their relevance to cell death induction and their informative value as predictive markers.
By flow cytometric analysis of apoptosis markers (exposure of phosphatidyl serine, loss of mitochondrial membrane potential) we showed that the RTKI Osimertinib and PI Bortezomib synergistically induce cell death in H1650 and HCC4006 cells, independent of RTKI resistance. Importantly, Western blot analysis showed RTKI-induced accumulation of the pro-apoptotic BH3-only protein NOXA in the HCC4006 and H1650 cancer cell lines. Additionally, the H1975 showed a similar effect combining Osimertinib and ABT-199, a BCL2I, in flow cytometric analysis and Western blot.
In summary, the presented experiments support the hypothesized synergistic cell death induction by RTK-inhibitors and proteasome inhibition as well as BCL-2-inhibition.
Studying the molecular mechanism of synergistic efficacy of RTKI/PI or RTKI/BCL2I combination therapies may form the basis for a general therapeutic concept which relies on simultaneous blocking of driver mutations and survival-promoting BCL-2 proteins.
Citation Format: Franziska Orth, Benjamin Schaefer, Annika Harsch, Sandra Weller, Tobias Beigl, Lynn Barber, Hans-Georg Kopp, Frank Essmann. Disrupting resistance to EGFR-specific tyrosine kinase inhibitors in TP53-mutated lung carcinoma cell lines by proteasome inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2532.