Complementary Omics Strategies to Dissect p53 Signaling Networks Under Nutrient Stress

Signaling trough p53 is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

Effects of nitrogen and phosphorus on Microcystis aeruginosa growth and microcystin production

In the present study, the effects of nitrogen (N) and phosphorus (P) on the growth of Microcystis aeruginosa and the production of microcystins (MCs) were investigated. The results showed that the growth of M. aeruginosa was not merely determined by N or P, but both nutrients were limiting for the species. Moreover, an excess of N and constant P in the culture medium could stimulate the growth of M. aeruginosa, whereas the growth of the species was inhibited in the culture medium containing excess of P and constant N. The optimal growth of M. aeruginosa was at an N:P ratio of 0.1 with the maximal optical density of 1.197 at 680 nm (OD680), whereas the maximal microcystin-LR (MC-LR) content of 228.2 μg·L−1 observed in the culture medium with an N:P ratio of 5. Interestingly, MC-LR production occurred under conditions of N starvation, thereby suggesting that the growth rate of M. aeruginosa was not related to MC-LR production under conditions of nutrient stress.

Characterization of metabolic compartmentalization in the liver using spatially resolved metabolomics

Cells adapt their metabolism to physiological stimuli, and metabolic heterogeneity exists between cell types, within tissues, and subcellular compartments. The liver plays an essential role in maintaining whole-body metabolic homeostasis and is structurally defined by metabolic zones. These zones are well-understood on the transcriptomic level, but have not been comprehensively characterized on the metabolomic level. Mass spectrometry imaging (MSI) can be used to map hundreds of metabolites directly from a tissue section, offering an important advance to investigate metabolic heterogeneity in tissues compared to extraction-based metabolomics methods that analyze tissue metabolite profiles in bulk. We established a workflow for the preparation of tissue specimens for matrix-assisted laser desorption/ionization (MALDI) MSI and achieved broad coverage of central carbon, nucleotide, and lipid metabolism pathways. We used this approach to visualize the effect of nutrient stress and excess on liver metabolism. Our data revealed a highly organized metabolic compartmentalization in livers, which becomes disrupted under nutrient stress conditions. Fasting caused changes in glucose metabolism and increased the levels of fatty acids in the circulation. In contrast, a prolonged high-fat diet (HFD) caused lipid accumulation within liver tissues with clear zonal patterns. Fatty livers had higher levels of purine and pentose phosphate related metabolites, which generates reducing equivalents to counteract oxidative stress. This MALDI MSI approach allowed the visualization of liver metabolic compartmentalization at high resolution and can be applied more broadly to yield new insights into metabolic heterogeneity in vivo .

870 Targeting GCN2 kinase-driven stress response inactivation to restore tumor immunity in metastatic triple negative breast cancer

BackgroundPatients with PD-L1-positive metastatic triple-negative breast cancers (mTNBC) who have been treated with atezolizumab+nab-paclitaxel had a clinically meaningful overall survival extension of 9.5 months compared to nab-paclitaxel alone, although overall survival in overall population was not statistically significant. Unlike many other cancers, immunotherapy for breast cancer has had limited success, due to the fact that there are very few T cells in the tumor microenvironment of mTNBC patients. Identifying ways to boost immunotherapy responses could change the paradigm of mTNBC, a disease still difficult to treat. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the tumors, leads to depletion of nutrients such as functional/natural amino acids. This metabolically stressful milieu causes activation of nutrient stress pathways, autophagy, and repressed immune responses. A key meditator of this nutrient stress pathway is a cytoplasmic Ser/Thr protein kinase called General Control Nonderepressible 2 (GCN2), also called EIF2AK4. GCN2 switches on following reduction of amino acids, and its activity results in T cell inactivation, T cell death, regulatory T cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs).MethodsWe have developed and synthesized a series of novel small molecule immunotherapeutic agents that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit pharmacological responses in immune cells and in breast cancer cells.ResultsGCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF-4 phosphorylation inhibition assays were performed. We confirmed on-target efficacy and tested the potency of our lead GCN2 inhibitor HCI-1046. HCI-1046 demonstrated potent activity, with an IC50 of 36 nM in inhibiting GCN2 kinase and exhibited cellular efficacy with an IC50 of 0.1 to 1.0 μM range. Our preliminary results support the hypothesis that the inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in breast cancer models. In vivo PK studies of HCI-1046 in rodents showed excellent PK properties; 55% oral bioavailability, low clearance, and >5 hour half-life.ConclusionsThus, HCI-1046 is nominated as a pre-clinical agent. Additional data regarding evaluation of the effects of HCI-1046 on the MDSC-suppressive function on T cells using ELISpot assays with breast cancer patient samples, and mouse model efficacy studies will be discussed.ReferencesEkiz HA, Lai SA, Gundlapalli H, Haroun F, Williams MA, Welm AL. Inhibition of RON kinase potentiates anti-CTLA-4 immunotherapy to shrink breast tumors and prevent metastatic outgrowth. Oncoimmunology 2018;7(9):e1480286.Toogood PL. Small molecule immuno-oncology therapeutic agents. Bioorg Med Chem Lett 2018;28(3):319–329.Ravindran R, Loebbermann J, Nakaya HI, Khan N, Ma H, Gama L, Machiah DK, Lawson B, Hakimpour P, Wang YC, Li S, Sharma P, Kaufman RJ, Martinez J, Pulendran B. The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation. Nature 2016;531(7595):523–527.Brazeau JF, Rosse G. Triazolo[4,5-d]pyrimidine derivatives as inhibitors of GCN2. ACS Med Chem Lett 2014;5(4):282–3.

Effects of Nitrogen and Phosphorus Limitation on Fatty Acid Contents in Aspergillus oryzae

Aspergillus oryzae, commonly known as koji mold, has been widely used for the large-scale production of food products (sake, makgeolli, and soy sauce) and can accumulate a high level of lipids. In the present study, we showed the dynamic changes in A. oryzae mycelium growth and conidia formation under nitrogen and phosphorus nutrient stress. The fatty acid profile of A. oryzae was determined and the content of unsaturated fatty acid was found increased under nitrogen and phosphorus limitation. Oleic acid (C18:1), linoleic acid (C18:2), and γ-linolenic acid (C18:3) production were increased on five nitrogen and phosphorus limitation media, especially on nitrogen deep limitation and phosphorus limitation group, showing a 1. 2–, 1. 6–, and 2.4-fold increment, respectively, compared with the control. Transcriptomic analysis showed the expression profile of genes related to nitrogen metabolism, citrate cycle, and linoleic acid synthesis, resulting in the accumulation of unsaturated fatty acid. qRT-PCR results further confirmed the reliability and availability of the differentially expressed genes obtained from the transcriptome analysis. Our study provides a global transcriptome characterization of the nitrogen and phosphorus nutrient stress adaptation process in A. oryzae. It also revealed that the molecular mechanisms of A. oryzae respond to nitrogen and phosphorus stress. Our finding facilitates the construction of industrial strains with a nutrient-limited tolerance.