Discussion

HTS amenable 3D technologies such as Alvetex® scaffolds have the potential to improve the physiological relevance of cell culture and thus influence the active chemistry identified in screening campaigns. In this study a multicellular phenotypic screen was employed to determine if 3D cell culture can influence the KRAS targeting potential of compounds.
The RAS gene family (HRAS, KRAS and NRAS) comprise the most frequently mutated oncogene family in human cancer (~30%). The KRAS locus is most often mutated in human cancer and mutant RAS proteins are endowed with a diverse set of biological capabilities that effect homeostatic mechanisms that control cell growth and survival. The term ‘undruggable’ is widely used to describe KRAS, this is due to the picomolar affinity of GTP & GDP binding to KRAS and the lack of any deep hydrophobic pockets on its surface. Inhibition of Downstream effectors has also proven difficult due to the vast complexity of the signalling network.
Published data has suggested that cells grown in a 3D platform rather than a 2D monolayer reveal a dependency on mutant KRAS by showing differential sensitivity to multiple MEK inhibitors.
A multicellular phenotypic cell growth/viability screen was developed in both 2D microtitre plates and 3D Alvetex® scaffold plates employing KRAS G12C and KRAS G12S mutants and KRAS WT cell lines. KRAS knockdown using siRNA inhibited cell growth in only KRAS mutant cell lines demonstrating the potential of the multicellular screen to differentiate between activated and WT KRAS, hence enabling the identification of small molecule inhibitors of the KRAS mutant pathway.

A 4K subset of compounds from the Kinase targeting collection was used to validate the assay. Both assay formats were shown to meet HTS QC criteria. The comparative screen resulted in identification of compounds uniquely active in 2D and in 3D as well as compounds specific for KRAS mutant cell lines vs. KRAS WT. The 3D assay format was found to have a lower hit rate than the 2D. IC50 follow up studies also showed that the compounds indentified in 3D were generally shown to be more potent than those identified in 2D. Assessment of actives from the single concentration screen in MEK and ERK inhibition assays suggests potential of 3D to enrich for Kinase inhibitors in KRAS specific pathways.