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. 2022 Nov 15;3(11):100815. doi: 10.1016/j.xcrm.2022.100815

Figure 1.

Figure 1

Evaluation of the combined effects of RMC-4550 (SHP2i) and LY3214996 (ERKi) administration in murine and human KRAS-mutant pancreatic cancer cell lines

(A) Western blot analysis with murine cancer cell line KCP_K2101 derived from KCP mouse model (KRASG12D) of spontaneous tumor formation and in human cancer cell lines: MiaPaCa-2 (KRASG12C) and Panc10.05 (KRASG12D). Cells were treated as depicted and collected for lysis at the indicated time points. Protein extracts were probed with specific antibodies against total RSK-1, phosphorylated RSK-1 (pRSK-1), and alpha-tubulin (as loading control). Numerical values indicate the pRSK-1/RSK-1 ratio quantified by densitometry. The blots are representative of at least three independent experiments. RSK-1, ribosomal S6 kinase 1.

(B) Synergistic effects of SHP2i and ERKi administration were evaluated by colony-formation assay in the KRAS-mutant cell lines used in (A). SHP2i and ERKi were combined at the indicated concentrations. Representative crystal violet staining of cells is shown (top panel). Box matrices below the plate scans depict quantification of growth inhibition in relation to control wells (middle panel). Bottom panel: calculation of the combination index (CI) scores from the growth inhibition values (shown above) via CompuSyn software demonstrating strong synergism between SHP2i and ERKi across a wide range of combinatorial concentrations. CI <0.75 (shades of green) indicates synergism, CI = 0.75–1.25 (shades of blue) indicates additive effects, and CI >1.25 (shades of red) indicates antagonism. Experiments were repeated independently at least three times each, with similar results.

(C) Apoptosis was analyzed in cell lines treated with either DMSO, SHP2i alone, ERKi alone, or a combination of SHP2i and ERKi at the indicated concentration in real time (top panel). GFP signal coupled to cleaved caspase 3 was quantified as readout. Bar plots for selected time points (48 h for KCP_K2101 and 76 h for MiaPaCa-2 and Panc10.05) show the fraction of GFP-positive cells (AU) (top panel) and the fold change GFP signal (bottom panel). AU, arbitrary units; GFP, green fluorescent protein. Experiments were repeated independently at least three times each. Results represent mean ± SD. ∗p < 0.05, ∗∗∗∗p < 0.0001, as determined by ordinary one-way ANOVA test.

See also Figure S1.