Uncontrolled cell proliferation is a hallmark of cancer and is often accompanied by reprogramming of energy metabolism. In addition to providing the building blocks required for uncontrolled proliferation, metabolic reprograming interferes with gene expression, enabling cancer cells to evade mechanisms that maintain tissue homeostasis. Imbalances in the metabolite pool, driven by metabolic rewiring, create an environment that inhibits activity of regulators of gene expression such as the alpha ketoglutarate (aKG)-dependent dioxygenase family. This family of enzymes includes the RNA demethylases FTO, ALKBH1 and ALKBH5 important regulators of RNA methylation. Recent studies have demonstrated that RNA methylation plays an important role in cell identity and is implicated in cancer establishment and progression. Fumarate, Succinate and (R)-2-hydroxyglutarate, known to accumulate in multiple types of cancer, are examples of metabolites known to inhibit 2-oxoglutarate (2OG)-dependent dioxygenase enzymes. To understand how energy reprograming remodels gene expression through modulation of RNA methylation dependent pathways we are performing comparative studies between established cancer cell lines (with chronic accumulation of these metabolites) with cells engineered to allow for acute inactivation of Fumarate Hydrogenase (FH) and Succinate Dehydrogenase (SDH) or inducible expression of mutated isocitrate dehydrogenase 1 (IDH1). This approach will allow us to understand how accumulation of fumarate (loss of FH) succinate (loss of SHD) or 2-HG (expression of mutated IDH1) contribute to the establishment, and progression, of cancer phenotype. Metabolite analysis shows similar metabolite accumulation in both groups of cells. Preliminary experiments suggest that changes in gene expression induced by chronic or acute accumulation of oncometabolites are distinct. For follow up studies, we have established protocols to measure, and map, multiple types of RNA methylation, including m6A, m6Am, 5mC, m1A and Am. Modifications in tRNAs are critical to maintain fidelity in translation. The modification 4SU, at position 8 of prokaryotic tRNA, has been shown to respond to change in growth rate. In order to understand how the 4SU modification is regulated and determine if a similar modification is present in eukaryotic RNAs, we develop a method that relies on orthogonal chemistry to capture 4SU modified tRNAs. With this method we are able to identify 4SU modified tRNAs at a genome level, and easily survey multiple growth conditions, greatly improving our ability to study this modification. Moving forward, we will use this method to identify 4SU modified tRNAs in bacteria with a negative impact on human health.
