A Study of Cellular Amino Acid Homeostasis Identifies Novel Targets, Inhibitors and Combination Therapies in Cancer

Abstract

Amino acid homeostasis is maintained by a combination of transmembrane transport processes, metabolic pathways, protein synthesis and turnover, and signalling systems to furnish cells, tissues, organs, and organisms with optimal levels of these nutrients. Understanding how these elements support amino acid homeostasis in cancer cells is critical to identifying novel vulnerabilities in cancer metabolism and is the subject of this thesis. To this end, a computational model of amino acid homeostasis was constructed and validated by comparing simulated amino acid equilibria in various media against experimental data from A549 lung adenocarcinoma cells and U87-MG glioma cells. A promising degree of correlation between the computational and empirical datasets was found and the model has been used to generate new research questions and hypotheses. Among these, it outlined the sodium-coupled neutral amino acid transporter SNAT2 as a viable target for pharmacological blockade to curtail cancer cell proliferation. As a result, a high-throughput screen for the identification of novel small molecule inhibitors of SNAT2 was conducted and yielded a shortlist of hit compounds. One such compound was found to inhibit SNAT2 in mammalian cells but showed preferential selectivity for a paralog of SNAT2, called SNAT4, when assayed in Xenopus laevis oocytes. Nevertheless, its cytotoxicity was tested in cancer cells, and it was found to be well tolerated at low micromolar concentrations. However, when combined with the glucose transport inhibitor Bay876 synergistic effects amounting to the suppression of HPAFII pancreatic cancer cell proliferation were observed. SNAT2 expression is partially controlled by the integrated stress response. This complex pathway senses various cellular stressors and initiates a translational program aimed at recovering the cell from these stressors. Among these is amino acid insufficiency, sensed by GCN2. Rather than simply inhibiting SNAT2 or other downstream effectors of GCN2, a study was conceived to explore the sensitisation of cancer cells to a slew of kinase, transporter and metabolic enzyme inhibitors by a GCN2 inhibitor called TAP20, to identify synergistic combination therapies. CDK, MEK1/2 and ERK1/2 inhibitors among others were found to synergise well and warrant further investigations into the mechanistic relationships underpinning these synergies. Lastly, these and other findings stress the importance of integrating metabolic fluxes and cellular signalling systems to improve upon the model of cellular amino acid homeostasis. Such a task is necessary to better identify novel drug targets and polytherapies in the treatment of cancer and other diseases. Show more