Title: Metabolism-mediated control of cellular differentiation
Description: Understanding the molecular mechanisms by which cells differentiate has important implications for cancer and stem cell therapies. Herein I examine whether genetic perturbations in metabolic enzymes are sufficient to cause cellular differentiation. Using mouse C2C12 muscle precursor cells, I systematically knocked down 50 enzymes involved in carbon catabolism using short hairpin RNAs, quantitated differentiation induced by the hairpin using high-content image analysis of myosin heavy chain intensity, and discovered three metabolic enzymes whose knockdown induced differentiation of C2C12 myoblasts: phosphoglycerate kinase (Pgk), hexose-6-phosphate dehydrogenase (H6pd) and ATP citrate lyase (Acly). I further confirmed gene knockdown by quantitative RT-PCR and protein expression, and was able to confirm the on-target effects of the shRNAs by rescue with the human ortholog, which abrogated the hairpin-induced differentiation response. To delineate a mechanism by which these enzymes can influence differentiation, I performed a mass spectrometry-based analysis of intracellular metabolites. During normal myogenic differentiation, changes consistent with reduced Pgk and H6pd activities were observed. Further investigation into a potential mechanism by which shH6pd mediates differentiation implicated an endoplasmic reticulum (ER)-stress response that is dependent on calcineurin signaling. Specifically, cyclosporine, a selective inhibitor of calcineurin, can rescue shH6pd-mediated differentiation.

Finally, I discovered a mechanistic link between Acly knockdown, chromatin acetylation, and cholesterol biosynthesis, which coordinates myoblast differentiation. Depletion of Acly lowers histone acetylation and induces differentiation, while treatment of myoblasts with an inhibitor of histone deacetylation prevents differentiation. Thus, through opposing chemical perturbations to shAcly, myoblast precursor cells can be maintained in culture despite signals for differentiation. I further show that small-molecule inhibition of cholesterol biosynthesis can phenocopy shAcly knockdown, presumably because Acly provides a key metabolic precursor, acetyl coA, for cholesterol biosynthesis. By extending these findings to cancer-cell differentiation, I show that treatment with statins, which inhibit cholesterol biosynthesis through inhibition of HMG CoA reductase, can induce differentiation of embryonal rhabdomyosarcoma. These studies identify three metabolic enzymes and their cellular pathways as potential regulators of myoblast differentiation, and show that targeting of these metabolic nodes could have implications for cancer differentiation therapy.