Mode of action of direct KRAS inhibition and development of KRAS inhibitor-based combination therapies

Abstract

As pancreatic ductal adenocarcinoma (PDAC) has still one of the least favourable survival rates among cancers, better treatment is urgently needed. This thesis aimed to make use of a new class of targeted therapies, the Kirsten Rat Sarcoma virus (KRAS)G12C inhibitors and to evaluate their selectivity and mechanisms in context of PDAC cell lines. Because these inhibitors are also known to be susceptible to resistance development, potential combination therapies were sought and evaluated. First of all, different KRASG12C inhibitors were tested on various PDAC cell lines with and without the KRASG12C mutation. Sotorasib, Adagrasib and MRTX-1257 revealed a selective reduction of cell viability in both Clonogenic and CellTiter-Glo® Assays specifically in KRASG12C mutated MiaPaCa2 cells. By the testing, Sotorasib was prioritized for the following experiments due to the specificity and the authorization for clinical use before this work has started. Further experiments were mainly done on human 2D cell lines which were either commercially available (MiaPaCa2) or generated from patient-derived organoids (PAN51T-2D). To comprehend the impact of KRASG12C inhibitors on signalling pathways, Western blot analysis was performed on cell lysates obtained from MiaPaCa2 and PAN51T-2D cells following different treatment durations of Sotorasib. On-target activity of Sotorasib was detected in both cell lines after all time points. In addition, a downregulation of downstream targets of KRAS was observed after a treatment time of 24 hours. However, after incubation with Sotorasib for 48 hours and 72 hours, a dose-dependent recovery of ERK phosphorylation was exhibited, suggesting that downstream pathways were reactivated, besides KRAS blockade. To overcome these resistance mechanisms, a Sotorasib-anchored unbiased drug screen with 126 different drugs, ranging from clinical approved drugs to preclinical compounds, was performed on MiaPaCa2 cells. Nine potential candidates were identified, of which three could be validated by Clonogenic, CellTiter-Glo® Assays, and the calculation of a Bliss-synergy score. These were the SHP2 inhibitor TNO155, the Son of Sevenless KRAS interaction inhibitor BI-3406, and the tyrosine kinase inhibitor Nintedanib. Ultimately, Western blot analysis was performed once more after treating both cell lines with Sotorasib and TNO155 in combination for 72 hours. This time, the pERK recovery could be prevented in the PAN51T-2D cell line, while the combined treatment had only little effect on the MiaPaCa2 cell line. Additionally, cell cycle analysis was performed via fluorescence-activated cell sorting, which found that a combination treatment led to an increased fraction of MiaPaCa2 cells entering G1 cell cycle arrest. Contrary, the treatment of PAN51T-2D cells with Sotorasib and TNO155 did not lead to a G1 cell cycle arrest. In summary, this work has identified the SHP2 inhibitor TNO155 as a potential synergistic partner for Sotorasib, with the ability to overcome potential resistance mechanisms. Nevertheless, more research is needed to fully understand the divergent cellular responses after the combination treatment.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed