Abstract 549: Characterization of the selective inhibitory effect of KRas inhibitors in different cellular assay formats

• Published in: Cancer research (Chicago, Ill.) Ill.), 2023-04, Vol.83 (7_Supplement), p.549-549
• Main Authors: Fimm-Todt, Franziska, Dikici, Ezgi, Schaich, Katharina, Siedentopf, Oliver, Lauterwasser, Joachim, Feger, Daniel, Ehlert, Jan Erik
• Format: Article
• Language: English
• ISSN: 1538-7445; 1538-7445
• DOI: 10.1158/1538-7445.AM2023-549

The proto-oncogene KRas is a well-described small GTPase that functions as a molecular switch for major physiological signaling pathways involved in cell proliferation, differentiation and survival. It has been shown that activating mutations in KRas are among the most common oncogenic drivers of tumorigenesis. Missense mutations of KRas result in constitutive activation due to impaired hydrolysis of GTP which enhances tumor-promoting downstream signaling pathways. Most KRas mutations are located in exon 2 or 3 including the most frequently changed glycine 12 which is present in most pancreatic cancers as well as in colorectal cancers and lung adenocarcinomas. Although direct inhibition of KRas is challenging due to its small size, smooth surface and limited druggable pockets on its surface, first promising drug candidates for selected KRas mutants have been developed in recent years (e.g. Sotorasib, MRTX1133, BI-2852). It was the aim of this study to characterize the selective inhibitory effects of KRas inhibitors in a panel of cancer cell lines harboring different KRas mutations like G12C, G12V or G12D. So, we applied different cellular techniques including phosphorylation assays as well as 2D proliferation and 3D spheroid assays to screen KRas inhibitors for their effect on distinct KRas mutants. Firstly, inhibitors were tested in a cellular assay monitoring phosphorylation of the downstream target ERK at residues Thr202/Tyr204. Analysis applying the cellular pERK AlphaLisa assay showed a high selectivity of several inhibitors for specific KRas mutants, while RAF and MEK inhibitors showed no significant selectivity in the panel of cancer cell lines tested here. Next, KRas inhibitors were tested in a cellular 2D proliferation and a 3D spheroid assay to determine the inhibitory effect on cell growth in both settings. In line with published data, for some inhibitors we could observe a high selectivity for a specific KRas mutant which was even more pronounced in the 3D format as compared to the 2D setup. Most likely due to the fact that 3D growth assays mimic physiological conditions more closely than 2D settings. Interestingly, in the 2D and 3D assays we could not find such a clear inhibitory effect of the MEK and RAF inhibitors as compared to the cellular pERK assay. These results indicate that the described cellular pERK AlphaLisa assay established for selected cancer cell lines could be a useful tool to screen newly developed KRas inhibitors for their s