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

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

Quantitative modelling of amino acid transport and homeostasis in mammalian cells

Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signaling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell’s endowment with amino acid transporters can be deconvoluted experimentally and used this data to computationally simulate amino acid translocation across the plasma membrane. Transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system are replicated in silico and can be applied to systems where only transcriptomic data are available. This work explains amino acid homeostasis at the systems-level, through a combination of secondary active transporters, functionally acting as loaders, harmonizers and controller transporters to generate a stable equilibrium of all amino acid concentrations

Do Amino Acid Antiporters Have Asymmetric Substrate Specificity?

Amino acid antiporters mediate the 1:1 exchange of groups of amino acids. Whether substrate specificity can be different for the inward and outward facing conformation has not been investigated systematically, although examples of asymmetric transport have been reported. Here we used LC–MS to detect the movement of 12C- and 13C-labelled amino acid mixtures across the plasma membrane of Xenopus laevis oocytes expressing a variety of amino acid antiporters. Differences of substrate specificity between transporter paralogs were readily observed using this method. Our results suggest that antiporters are largely symmetric, equalizing the pools of their substrate amino acids. Exceptions are the antiporters y+LAT1 and y+LAT2 where neutral amino acids are co-transported with Na+ ions, favouring their import. For the antiporters ASCT1 and ASCT2 glycine acted as a selective influx substrate, while proline was a selective influx substrate of ASCT1. These data show that antiporters can display non-canonical modes of transport

Impact of Inhibition of Glutamine and Alanine Transport on Cerebellar Glial and Neuronal Metabolism

The cerebellum, or “little brain”, is often overlooked in studies of brain metabolism in favour of the cortex. Despite this, anomalies in cerebellar amino acid homeostasis in a range of disorders have been reported. Amino acid homeostasis is central to metabolism, providing recycling of carbon backbones and ammonia between cell types. Here, we examined the role of cerebellar amino acid transporters in the cycling of glutamine and alanine in guinea pig cerebellar slices by inhibiting amino acid transporters and examining the resultant metabolism of [1-13C]d-glucose and [1,2-13C]acetate by NMR spectroscopy and LCMS. While the lack of specific inhibitors of each transporter makes interpretation difficult, by viewing results from experiments with multiple inhibitors we can draw inferences about the major cell types and transporters involved. In cerebellum, glutamine and alanine transfer is dominated by system A, blockade of which has maximum effect on metabolism, with contributions from System N. Inhibition of neural system A isoform SNAT1 by MeAIB resulted in greatly decreased metabolite pools and reduced net fluxes but showed little effect on fluxes from [1,2-13C]acetate unlike inhibition of SNAT3 and other glutamine transporters by histidine where net fluxes from [1,2-13C]acetate are reduced by ~50%. We interpret the data as further evidence of not one but several glutamate/glutamine exchange pools. The impact of amino acid transport inhibition demonstrates that the cerebellum has tightly coupled cells and that glutamate/glutamine, as well as alanine cycling, play a major role in that part of the brain

Ablation of the ASCT2 (SLC1A5) gene encoding a neutral amino acid transporter reveals transporter plasticity and redundancy in cancer cells

The neutral amino acid transporter solute carrier family 1 member 5 (SLC1A5 or ASCT2) is overexpressed in many cancers. To identify its roles in tumors, we employed 143B osteosarcoma cells and HCC1806 triple-negative breast cancer cells with or without ASCT2 deletion. ASCT2ko 143B cells grew well in standard culture media, but ASCT2 was required for optimal growth at < 0.5 mM glutamine, with tumor spheroid growth and monolayer migration of 143B ASCT2ko cells being strongly impaired at lower glutamine concentrations. However, the ASCT2 deletion did not affect matrix-dependent invasion. ASCT2ko 143B xenografts in nude mice exhibited a slower onset of growth and a higher number of small tumors than ASCT2wt 143B xenografts, but did not differ in average tumor size 25 days after xenotransplantation. ASCT2 deficiency was compensated by increased levels of sodium neutral amino acid transporter 1 (SNAT1 or SLC38A1) and SNAT2 (SLC38A2) in ASCT2ko 143B cells, mediated by a GCN2 EIF2alpha kinase (GCN2)-dependent pathway, but this compensation was not observed in ASCT2ko HCC1806 cells. Combined SNAT1 silencing and GCN2 inhibition significantly inhibited growth of ASCT2ko HCC1806 cells, but not of ASCT2ko 143B cells. Similarly, pharmacological inhibition of L-type amino acid transporter 1 (LAT1) and GCN2 significantly inhibited growth of ASCT2ko HCC1806 cells, but not of ASCT2ko 143B cells. We conclude that cancer cells with reduced transporter plasticity are more vulnerable to disruption of amino acid homeostasis than cells with a full capacity to upregulate redundant transporters by an integrated stress response.This work was supported in part by a Merck KGaA speed grant (to S. B.), Australian Research Council Discovery Project Grant DP180101702 (to S. B.), and Cancer Council New South Wales Grants RG17-04 and RG18-06 (to J. H.)

DataSheet2_Identification and characterization of a novel SNAT2 (SLC38A2) inhibitor reveals synergy with glucose transport inhibition in cancer cells.xlsx

SNAT2 (SLC38A2) is a sodium-dependent neutral amino acid transporter, which is important for the accumulation of amino acids as nutrients, the maintenance of cellular osmolarity, and the activation of mTORC1. It also provides net glutamine for glutaminolysis and consequently presents as a potential target to treat cancer. A high-throughput screening assay was developed to identify new inhibitors of SNAT2 making use of the inducible nature of SNAT2 and its electrogenic mechanism. Using an optimized FLIPR membrane potential (FMP) assay, a curated scaffold library of 33934 compounds was screened to identify 3-(N-methyl (4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide as a potent inhibitor of SNAT2. In two different assays an IC50 of 0.8–3 µM was determined. The compound discriminated against the close transporter homologue SNAT1. MDA-MB-231 breast cancer and HPAFII pancreatic cancer cell lines tolerated the SNAT2 inhibitor up to a concentration of 100 µM but in combination with tolerable doses of the glucose transport inhibitor Bay-876, proliferative growth of both cell lines was halted. This points to synergy between inhibition of glycolysis and glutaminolysis in cancer cells.</p