RAS isoform specific activities are disrupted by disease associated mutations during cell differentiation

Abstract

The Ras-MAPK pathway is aberrantly regulated in cancer and developmental diseases called RASopathies. While the impact of Ras on proliferation is typically assessed on various cancer cell lines, it is poorly established how Ras affects cellular differentiation.Here we implement the C2C12 myoblast cell line to systematically study the effect of Ras mutants and Ras-pathway drugs on differentiation. We first provide evidence that the Pax7+ progenitors divide asymmetrically to replenish the major pool of transit amplifying cells that are ready to differentiate. Intriguingly, Ras isoforms have distinct roles in the differentiating culture, where K-Ras is more important than N-Ras to maintain the progenitor pool, and H-Ras is significant for the expansion of differentiated cells. While overexpression of wild-type K-Ras increases the fraction of differentiated cells at the expense of transit amplifying cells, all oncogenic mutants block further differentiation of transit amplifying cells. Interestingly, RASopathy associated mutants display distinct phenotypes. Notably variants that are also NF1-GAP resistant block differentiation as their oncogenic counterparts, albeit less. Profiling of targeted Ras-pathway drugs on oncogenic Ras mutants reveals their distinct abilities to restore normal differentiation as compared to triggering cell death. Interestingly, the MEK-inhibitor trametinib could broadly restore differentiation, while the mTORC1 inhibitor rapamycin broadly suppressed differentiation.We expect that this quantitative assessment of the impact of Ras-pathway mutans and drug treatments on cellular differentiation will provide significant new insights into Ras biology and Ras-driven diseases.