Inhibition of mTOR Reverses the Ability of c-MYC To Block the Differentiation of Murine Granulocytes.

The proto-oncogene c-MYC encodes a bHLH leucine-zipper transcription factor that acts as a potent differentiation inhibitor and as a promoter of cell cycle progression. Additionally, c-MYC and mTOR are widely acknowledged as major regulators of cell growth, the process by which cells accumulate biomass. Both c-MYC overexpression and constitutive activation of the PI 3-kinase/mTOR pathway are frequent events in acute myeloid leukaemia; a haemopoietic malignancy characterised by failure to undergo terminal differentiation. In this study we used the MPRO system, an in vitro murine myeloid terminal differentiation model, to study the relationship between cell growth effectors and differentiation in more detail. Enforced expression of c-MYC by the addition of 4-hydoxytamoxifen (4-OHT) to MPRO cells carrying the inducible mycER fusion protein prevents downregulation of growth and impairs retinoid-induced terminal myeloid differentiation. Because downregulation of mTOR limits growth we hypothesised that inhibition of mTOR may reverse the phenotypic effects of c-MYC. Strikingly, pharmacological inhibition of mTOR by rapamycin, but not growth limitation by other means such as amino acid deprivation or cycloheximide treatment, restores differentiation in granulocytes with enforced c-MYC expression (74.0±2.0% mature granulocytes in the rapamycin+4-OHT-treated mycER MPROs vs 4.5±1.0% in the cells treated with 4-OHT alone and 92.9±1.1% in vehicle-treated controls). Rapamycin restores cell growth parameters to levels equivalent to those of vehicle-treated control cells undergoing physiological granulocytic differentiation (2–3 fold reduction in ³⁵S methionine incorporation in rapamycin+4-OHT-treated and vehicle-treated controls compared to levels in 4-OHT-treated cells) and facilitates cell cycle exit (26.0±7.14% of rapamycin+4-OHT-treated cells and 10.7±2.48% of vehicle-treated control cells in S phase vs 51.9±1.72% of tamoxifen-treated cells in S phase as determined by BrdU incorporation). Interestingly, rapamycin attenuates c-MYC’s ability to activate transcription of its target genes without impairing activation of RARα target genes. c-mycER protein levels are reduced while levels of retrovirus-driven c-mycER mRNA do not fall in rapamycin-treated MPRO mycER cells. Furthermore, c-mycER protein levels decrease in rapamycin-treated MPRO cells expressing the ER fusion of the mycT58A point mutation known to increase protein stability. Overall, this data suggests that rapamycin restores the differentiation potential of granulocytes in the MPRO mycER system by regulating c-MYC at the level of protein translation, rather than at the level of gene transcription or protein degradation. These findings suggest that mTOR could be targeted to influence terminal differentiation regulated by MYC in haemopoietic malignancies.