A medium-throughput assay to profile the impact of Ras-disease mutants and -drugs on cellular differentiation

The Ras-MAPK pathway is overactivated in cancers and RASopathies, driving aberrant cell proliferation and differentiation. Currently no simple standard assay exists to quantify the extent to which differentiation is perturbed.
Here we implemented an automated, flow cytometry-based C2C12 cell differentiation assay to profile the impact of Ras-disease mutants and -drugs on cellular differentiation. We showed that in the heterogeneous C2C12 cell pool, differentiated cells arise from a major pool of transit amplifying cells that are replenished via asymmetric division and subsequent expansion from just a few Pax7+ progenitors.
Overexpression of wt K-Ras significantly expands the fraction of differentiated cells, while each oncogenic mutant shows a distinct ability to expand the Pax7+ progenitor pool at the expense of differentiation. This is consistent with a shift from asymmetric to pseudo-symmetric divisions induced by oncogenic K-Ras. Interestingly, RASopathy-associated mutants exhibit a phenotype that is intermediate between the former two.
We then profiled eight Ras pathway inhibitors on eight mutants of K-Ras, N-Ras and H-Ras. Our assay can quantitate how much inhibitors can restore differentiation and in parallel is able to distinguish this from simple anti-proliferative effects. Notably, all four tested K-RasG12C inhibitors can restore differentiation. However, they do have distinct profiles. We observed that AMG 510 shows essentially no toxicity and a unique promotion of differentiation.
Our differentiation assay has the potential to serve as a valuable tool for quantitating the effects of disease mutants and drugs on differentiation, thereby providing novel insights into cancer and developmental diseases