Abstract 4448: Novel tyrosine kinase/MEK inhibitor combinations impair the viability and invasive potential of TNBC cell lines

Abstract This study demonstrates that the tyrosine kinase inhibitors ALW-II-41-27 and ponatinib enhance the efficacy of MEK inhibition in multiple TNBC cell lines, suggesting potential for combination therapy. This project aimed to identify compensatory mechanisms triggered by MAPK inhibitors (MAPKi) in TNBC and to develop novel treatment strategies to effectively block these mechanisms. To this end, we performed a conditioned medium (CM) experiment in which we identified secreted cytokines as potential mediators of drug resistance. We then performed a cytokine array and receptor tyrosine kinase (RTK) array to identify the cytokines and RTKs implicated in this process. Finally, we targeted some of these previously identified RTKs in combination with trametinib and tested the efficacy of the combination on the viability of 11 TNBC cell lines. Two combinations were tested for further experiments to characterize their efficacy and mechanism of action, including colony forming assay, transcriptomic analysis and wound healing assays. We identified several cytokines secreted in response to the pan-Raf inhibitor CCT196969, the MEK inhibitor (MEKi) trametinib and the ERK inhibitor (ERKi) ulixertinib such as FGF-basic, FGF-19, MIF and angiogenin. CM from drug treated cells induced the activation of several RTKs such as FGFR1, FGFR3, Insulin-R, ALK and DTK in untreated TNBC cells. To validate the significance of these upregulated RTKs, we used a panel of kinase inhibitors. This led us to identify two synergistic combinations: trametinib with the EPHA2 inhibitor ALW-II-41-27 and trametinib with ponatinib, which targets multiple RTKs, including EPHA2. These combinations demonstrated efficacy in other TNBC cell lines over a longer period and, notably, prevented cell re-growth even after an additional eight days without treatment. This was in contrast to trametinib single treatment, where cells resumed growth under similar conditions. Our transcriptomic analyses of the combination treatment with trametinib and ALW-II-41-27, either singly or in combination, across a panel of cell lines revealed significant pathway alterations. Notably, we observed increased HER2 and HER3 transcription following trametinib treatment, which we could confirm at the protein level by Western blotting. Lastly, the trametinib/ALW-II-41-27 combination revealed a significant reduction in cell migration and invasion compared to trametinib treatment alone. These findings indicate that ponatinib and ALW-II-41-27 combined with MAPKi could be a promising novel therapeutic approach for TNBC. Citation Format: Célia Asdih, Ariane Lehmann, Jasmin Traichel, Eva Oswald, Julia Schueler, Rhena Klar, Patrick Metzger, Melanie Boerries, Tilman Brummer, Ricarda Griffin. Novel tyrosine kinase/MEK inhibitor combinations impair the viability and invasive potential of TNBC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4448.