Dissecting the Role of Mutant RAS Signaling to Drive High-Yield Cellular Reprogramming

The oncogene HRAS regulates cell proliferation, differentiation, growth, and cell death. However, its role in direct conversion remains poorly understood. Direct conversion converts common, easily accessible cell types into rare, difficult to isolate cell types. While proliferation increases conversion rates, the mechanistic contribution of HRAS and signaling networks in these cell fate transitions remains poorly characterized. Addition of two oncogenic mutants, HRAS G12V and p53DD, to a transcription-factor based reprogramming ‘cocktail’ results in a 500-fold increase in the direct conversion of mouse embryonic fibroblasts (MEFs) to induced motor neurons (iMNs). Here, we identify the biphasic effect of MAPK signaling on reprogramming and identify an optimal RAS expression for conversion. Our findings indicate that genetic encoding through multicistronic cassette design impacts reprogramming via differential HRAS G12V expression levels. We hypothesize that differences in gene order, linkers (IRES vs P2A), and cloning ‘seams’ lead to different levels of expression of HRAS G12V and p53DD, which in turn drive different reprogramming efficiencies. Our results revealed that cassettes with either too-low or too-high HRAS G12V levels resulted in lower iMN yields, while cassettes with moderate HRAS G12V expression produced higher yields. Since excessive RAS levels reduced yield, we tested to see if inhibiting MAPK signaling downstream of RAS with a small molecule MEK inhibitor could rescue reprogramming. We found that slight inhibition of the MAPK pathway increased reprogramming yield for cassettes with high HRAS G12V expression levels. Unlike MAPK inhibition, inhibition of the AKT pathway did not rescue reprogramming. This result indicates that it is excessive MAPK signaling, but not AKT signaling, that reduces reprogramming at high HRAS G12V expression levels. These findings suggest a ‘sweet spot’ of MAPK activation for optimal proliferation and reprogramming. Small molecule inhibition of downstream RAS targets offers an additional level of control over RAS signaling levels. In the future, high-yield reprogramming has tremendous potential for use in disease modeling, therapeutic screening, and cellular therapies. Understanding and engineering signaling pathways during reprogramming are important steps towards this goal.