Person:

Manning, Brendan

The mechanistic target of rapamycin complex 1 (mTORC1) supports proliferation through parallel induction of key anabolic processes, including protein, lipid, and nucleotide synthesis. We hypothesized that these processes are coupled to maintain anabolic balance in cells with mTORC1 activation, a common event in human cancers. Loss of the tuberous sclerosis complex (TSC) tumor suppressors results in activation of mTORC1 and development of the tumor syndrome TSC. We find that pharmacological inhibitors of guanylate nucleotide synthesis have selective deleterious effects on TSC-deficient cells, including in mouse tumor models. This effect stems from replication stress and DNA damage caused by mTORC1-driven ribosomal RNA synthesis, which renders nucleotide pools limiting. These findings reveal a metabolic vulnerability downstream of mTORC1 triggered by anabolic imbalance.

Targeted tandem mass spectrometry (LC–MS/MS) has been extremely useful for profiling small molecules extracted from biological sources, such as cells, bodily fluids and tissues. Here, we present a protocol for analysing incorporation of the non-radioactive stable isotopes carbon-13 (13C) and nitrogen-15 (15N) into polar metabolites in central carbon metabolism and related pathways. Our platform utilizes selected reaction monitoring (SRM) with polarity switching and amide hydrophilic interaction liquid chromatography (HILIC) to capture transitions for carbon and nitrogen incorporation into selected metabolites using a hybrid triple quadrupole (QQQ) mass spectrometer. This protocol represents an extension of a previously published protocol for targeted metabolomics of unlabeled species and has been used extensively in tracing the metabolism of nutrients such as 13C-labeled glucose, 13C-glutamine and 15N-glutamine in a variety of biological settings (e.g., cell culture experiments and in vivo mouse labelling via i.p. injection). SRM signals are integrated to produce an array of peak areas for each labelling form that serve as the output for further analysis. The processed data are then used to obtain the degree and distribution of labelling of the targeted molecules (termed fluxomics). Each method can be customized on the basis of known unlabeled Q1/Q3 SRM transitions and adjusted to account for the corresponding 13C or 15N incorporation. The entire procedure takes ~6–7 h for a single sample from experimental labelling and metabolite extraction to peak integration.