Inhibition of the proline metabolism rate-limiting enzyme P5CS allows proliferation of glutamine-restricted cancer cells.

Glutamine is a critical metabolite for rapidly proliferating cells as it is used for the synthesis of key metabolites necessary for cell growth and proliferation. Glutamine metabolism has been proposed as a therapeutic target in cancer and several chemical inhibitors are in development or in clinical trials. How cells subsist when glutamine is limiting is poorly understood. Here, using an unbiased screen, we identify ALDH18A1, which encodes P5CS, the rate-limiting enzyme in the proline biosynthetic pathway, as a gene that cells can downregulate in response to glutamine starvation. Notably, P5CS downregulation promotes de novo glutamine synthesis, highlighting a previously unrecognized metabolic plasticity of cancer cells. The glutamate conserved from reducing proline synthesis allows cells to produce the key metabolites necessary for cell survival and proliferation under glutamine-restricted conditions. Our findings reveal an adaptive pathway that cancer cells acquire under nutrient stress, identifying proline biosynthesis as a previously unrecognized major consumer of glutamate, a pathway that could be exploited for developing effective metabolism-driven anticancer therapies.

Establishment and Application of a Novel In Vitro Model of Microglial Activation in Traumatic Brain Injury.

Mechanical impact-induced primary injury after traumatic brain injury (TBI) leads to acute microglial pro-inflammatory activation and consequently mediates neurodegeneration, which is a major secondary brain injury mechanism. However, the detailed pathologic cascades have not been fully elucidated, partially because of the pathologic complexity in animal TBI models. Although there are several in vitro TBI models, none of them closely mimic post-TBI microglial activation. In the present study, we aimed to establish an in vitro TBI model, specifically reconstituting the pro-inflammatory activation and associated neurodegeneration following TBI. We proposed three sets of experiments. First, we established a needle scratch injured neuron-induced microglial activation and neurodegeneration in vitro model of TBI. Second, we compared microglial pro-inflammatory cytokines profiles between the in vitro TBI model and TBI in male mice. Additionally, we validated the role of injured neurons-derived damage-associated molecular patterns in amplifying microglial pro-inflammatory pathways using the in vitro TBI model. Third, we applied the in vitro model for the first time to characterize the cellular metabolic profile of needle scratch injured-neuron-activated microglia, and define the role of metabolic reprogramming in mediating pro-inflammatory microglial activation and mediated neurodegeneration. Our results showed that we successfully established a novel in vitro TBI model, which closely mimics primary neuronal injury-triggered microglial pro-inflammatory activation and mediated neurodegeneration after TBI. This in vitro model provides an advanced and highly translational platform for dissecting interactions in the pathologic processes of neuronal injury-microglial activation-neuronal degeneration cascade, and elucidating the detailed underlying cellular and molecular insights after TBI.SIGNIFICANCE STATEMENT Microglial activation is a key component of acute neuroinflammation that leads to neurodegeneration and long-term neurologic outcome deficits after TBI. However, it is not feasible to truly dissect primary neuronal injury-induced microglia activation, and consequently mediated neurodegeneration in vivo Furthermore, there is currently lacking of in vitro TBI models closely mimicking the TBI primary injury-mediated microglial activation. In this study, we successfully established and validated a novel in vitro TBI model of microglial activation, and for the first time, characterized the cellular metabolic profile of microglia in this model. This novel microglial activation in vitro TBI model will help in elucidating microglial inflammatory activation and consequently associated neurodegeneration after TBI.

Bone marrow adipocytes fuel emergency hematopoiesis after myocardial infarction.

After myocardial infarction (MI), emergency hematopoiesis produces inflammatory myeloid cells that accelerate atherosclerosis and promote heart failure. Since the balance between glycolysis and mitochondrial metabolism regulates hematopoietic stem cell homeostasis, metabolic cues may influence emergency myelopoiesis. Here, we show in humans and female mice that hematopoietic progenitor cells increase fatty acid metabolism after MI. Blockade of fatty acid oxidation by deleting carnitine palmitoyltransferase (Cpt1A) in hematopoietic cells of Vav1Cre/+Cpt1Afl/fl mice limited hematopoietic progenitor proliferation and myeloid cell expansion after MI. We also observed reduced bone marrow adiposity in humans, pigs and mice following MI. Inhibiting lipolysis in adipocytes using AdipoqCreERT2Atglfl/fl mice or local depletion of bone marrow adipocytes in AdipoqCreERT2iDTR mice also curbed emergency hematopoiesis. Furthermore, systemic and regional sympathectomy prevented bone marrow adipocyte shrinkage after MI. These data establish a critical role for fatty acid metabolism in post-MI emergency hematopoiesis.

Induction of a Timed Metabolic Collapse to Overcome Cancer Chemoresistance.

Cancer relapse begins when malignant cells pass through the extreme metabolic bottleneck of stress from chemotherapy and the byproducts of the massive cell death in the surrounding region. In acute myeloid leukemia, complete remissions are common, but few are cured. We tracked leukemia cells in vivo, defined the moment of maximal response following chemotherapy, captured persisting cells, and conducted unbiased metabolomics, revealing a metabolite profile distinct from the pre-chemo growth or post-chemo relapse phase. Persisting cells used glutamine in a distinctive manner, preferentially fueling pyrimidine and glutathione generation, but not the mitochondrial tricarboxylic acid cycle. Notably, malignant cell pyrimidine synthesis also required aspartate provided by specific bone marrow stromal cells. Blunting glutamine metabolism or pyrimidine synthesis selected against residual leukemia-initiating cells and improved survival in leukemia mouse models and patient-derived xenografts. We propose that timed cell-intrinsic or niche-focused metabolic disruption can exploit a transient vulnerability and induce metabolic collapse in cancer cells to overcome chemoresistance.

Synergistic substrate cofeeding stimulates reductive metabolism.

Advanced bioproduct synthesis via reductive metabolism requires coordinating carbons, ATP and reducing agents, which are generated with varying efficiencies depending on metabolic pathways. Substrate mixtures with direct access to multiple pathways may optimally satisfy these biosynthetic requirements. However, native regulation favouring preferential use precludes cells from co-metabolizing multiple substrates. Here we explore mixed substrate metabolism and tailor pathway usage to synergistically stimulate carbon reduction. By controlled cofeeding of superior ATP and NADPH generators as 'dopant' substrates to cells primarily using inferior substrates, we circumvent catabolite repression and drive synergy in two divergent organisms. Glucose doping in Moorella thermoacetica stimulates CO2 reduction (2.3 g gCDW-1 h-1) into acetate by augmenting ATP synthesis via pyruvate kinase. Gluconate doping in Yarrowia lipolytica accelerates acetate-driven lipogenesis (0.046 g gCDW-1 h-1) by obligatory NADPH synthesis through the pentose cycle. Together, synergistic cofeeding produces CO2-derived lipids with 38% energy yield and demonstrates the potential to convert CO2 into advanced bioproducts. This work advances the systems-level control of metabolic networks and CO2 use, the most pressing and difficult reduction challenge.