Changes in cell state are often accompanied by altered metabolic demands, and homeostasis depends on cells adapting to their changing needs. One major cell state change is senescence, which is associated with dramatic changes in cell metabolism, including increases in lipid metabolism, but how cells accommodate such alterations is poorly understood. Here we show that the transcription factor p53 increases recycling of the lipid headgroups required to meet the increased demand for membrane phospholipids during senescence. p53 activation increases the supply of phosphoethanolamine, an intermediate in the Kennedy pathway for de novo synthesis of phosphatidylethanolamine, in part by increasing lipid turnover and transactivating genes involved in autophagy and lysosomal catabolism that enable membrane turnover. Disruption of phosphoethanolamine conversion to phosphatidylethanolamine is well tolerated in the absence of p53 but results in dramatic organelle remodelling and perturbs growth and gene expression following p53 activation. Consistently, CRISPR–Cas9-based genetic screens reveal that p53-activated cells preferentially depend on genes involved in lipid metabolism and lysosomal function. Together, these results reveal lipid headgroup recycling to be a homeostatic function of p53 that confers a cell-state-specific metabolic vulnerability. Yashinskie, Zhu and colleagues show that p53 activation triggers increased synthesis and accumulation of phospholipids, with enhanced activation of autophagy and lysosomal catabolism programmes and increased reliance on lipid headgroup recycling.

1-Deoxysphingolipids are non-canonical sphingolipids linked to several diseases, yet their cellular effects are poorly understood. Here, we utilize lipid chemical biology approaches to investigate the role of 1-deoxysphingolipid metabolism on the properties and functions of secretory membranes. We applied organelle-specific bioorthogonal labeling to visualize the subcellular distribution of metabolically tagged sphingolipids. We observed that 1-deoxysphingolipids are retained in the endoplasmic reticulum (ER) and specifically in ER exit sites (ERESs), suggesting that they do not efficiently sort into vesicular carriers. Cell lines expressing disease-associated variants of serine palmitoyl-CoA transferase accumulated 1-deoxysphingolipids, which were accompanied by a reduction in ER membrane fluidity and enlargement of ERES. We found that the rates of membrane protein release from the ER were altered in response to 1-deoxysphingolipid metabolism in a manner dependent on the protein’s affinity for ordered or disordered membranes. The dysregulation of sphingolipid metabolism can thus alter secretory membrane properties and affect protein trafficking.

Lipids enable compartmentation and coordinate membrane-localized signaling events in cells, and dysregulation of lipid metabolism is linked to many disease states. However, limited tools are available for quantifying metabolic fluxes across the lipidome. To measure fluxes encompassing lipid homeostasis in cells and tissue slices, we apply stable isotope tracing, liquid chromatography-high-resolution mass spectrometry, and network-based isotopologue modeling to non-small cell lung cancer (NSCLC) models. Lipid metabolic flux analysis (Lipid-MFA) enables quantitation of fatty acid synthesis, elongation, headgroup assembly, and salvage reactions within virtually any biological system. Using Lipid-MFA, we observed decreased fatty acid synthase and very long-chain fatty acid (VLCFA) elongation fluxes, along with increased sphingolipid recycling, in p53-deficient versus liver kinase B1 (LKB1)-deficient NSCLC tumors using precision-cut lung slice culture. We also apply Lipid-MFA to demonstrate the unique trafficking of ceramides with distinct n-acyl chain lengths, highlighting the utility of this approach in elucidating molecular mechanisms in lipid homeostasis.

Lipids represent the most diverse pool of metabolites found in cells, facilitating compartmentation, signaling, and other functions. Dysregulation of lipid metabolism is linked to disease states such as cancer and neurodegeneration. However, limited tools are available for quantifying metabolic fluxes across the lipidome. To directly measure reaction fluxes encompassing compound lipid homeostasis, we applied stable isotope tracing, liquid chromatography-high-resolution mass spectrometry, and network-based isotopologue modeling to non-small cell lung cancer (NSCLC) models. Compound lipid metabolic flux analysis (CL-MFA) enables the concurrent quantitation of fatty acid synthesis, elongation, headgroup assembly, and salvage reactions within virtually any biological system. Here, we resolve liver kinase B1 (LKB1)-mediated regulation of sphingolipid recycling in NSCLC cells and precision-cut lung slice cultures. We also demonstrate that widely used tissue culture conditions drive cells to upregulate fatty acid synthase flux to supraphysiological levels. Finally, we identify previously uncharacterized isozyme specificity of ceramide synthase inhibitors. These results highlight the ability of CL-MFA to quantify lipid cycling in biological systems to discover biological function and elucidate molecular mechanisms in membrane lipid metabolism.

Changes in cell state are often accompanied by altered metabolic demands, and homeostasis depends on cells adapting to their changing needs. One major cell state change is senescence, which is associated with dramatic changes in cell metabolism, including increases in lipid metabolism, but how cells accommodate such alterations is poorly understood. Here, we show that the transcription factor p53 enables recycling of the lipid headgroups required to meet the increased demand for membrane phospholipids during senescence. p53 activation increases supply of phosphoethanolamine (PEtn), an intermediate in the Kennedy pathway for de novo synthesis of phosphatidylethanolamine (PE), by transactivating genes involved in autophagy and lysosomal catabolism that enable membrane turnover. Disruption of PEtn conversion to PE is well-tolerated in the absence of p53 but results in dramatic organelle remodeling and perturbs growth and gene expression following p53 activation. Consistently, CRISPR-Cas9-based genetic screens reveal that p53-activated cells preferentially depend on genes involved in lipid metabolism. Together, these results reveal lipid headgroup recycling to be a homeostatic function of p53 that confers a cell-state specific metabolic vulnerability.

Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family are increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe the resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach adds to the toolbox for laboratories to quantify and characterize acyl chain diversity across the lipidome.

Proliferating cells rely on acetyl-CoA to support membrane biogenesis and acetylation. Several organelle-specific pathways are available for provision of acetyl-CoA as nutrient availability fluctuates, so understanding how cells maintain acetyl-CoA homeostasis under such stresses is critically important. To this end, we applied 13 C isotope tracing cell lines deficient in these mitochondrial [ATP-citrate lyase (ACLY)]–, cytosolic [acetyl-CoA synthetase (ACSS2)]–, and peroxisomal [peroxisomal biogenesis factor 5 (PEX5)]–dependent pathways. ACLY knockout in multiple cell lines reduced fatty acid synthesis and increased reliance on extracellular lipids or acetate. Knockout of both ACLY and ACSS2 (DKO) severely stunted but did not entirely block proliferation, suggesting that alternate pathways can support acetyl-CoA homeostasis. Metabolic tracing and PEX5 knockout studies link peroxisomal oxidation of exogenous lipids as a major source of acetyl-CoA for lipogenesis and histone acetylation in cells lacking ACLY, highlighting a role for inter-organelle cross-talk in supporting cell survival in response to nutrient fluctuations.

Diabetes represents a spectrum of disease in which metabolic dysfunction damages multiple organ systems including liver, kidneys and peripheral nerves 1 , 2 . Although the onset and progression of these co-morbidities are linked with insulin resistance, hyperglycaemia and dyslipidaemia 3 – 7 , aberrant non-essential amino acid (NEAA) metabolism also contributes to the pathogenesis of diabetes 8 – 10 . Serine and glycine are closely related NEAAs whose levels are consistently reduced in patients with metabolic syndrome 10 – 14 , but the mechanistic drivers and downstream consequences of this metabotype remain unclear. Low systemic serine and glycine are also emerging as a hallmark of macular and peripheral nerve disorders, correlating with impaired visual acuity and peripheral neuropathy 15 , 16 . Here we demonstrate that aberrant serine homeostasis drives serine and glycine deficiencies in diabetic mice, which can be diagnosed with a serine tolerance test that quantifies serine uptake and disposal. Mimicking these metabolic alterations in young mice by dietary serine or glycine restriction together with high fat intake markedly accelerates the onset of small fibre neuropathy while reducing adiposity. Normalization of serine by dietary supplementation and mitigation of dyslipidaemia with myriocin both alleviate neuropathy in diabetic mice, linking serine-associated peripheral neuropathy to sphingolipid metabolism. These findings identify systemic serine deficiency and dyslipidaemia as novel risk factors for peripheral neuropathy that may be exploited therapeutically.

This file includes extended experimental procedures including 1. Analysis of RNA-sequencing and Cancer Genome Atlas data 2. Generation of shRNA Cell Lines, 3. Automated cell counting, 4. Liquid chromatography and mass spectrometry, 5. BrdU staining of murine tissue; supplementary references and supplementary figure legends

OBJECTIVES:The non-essential amino acids serine, glycine, and alanine, as well as diverse sphingolipid species, are implicated in inherited neuro-retinal disorders and are metabolically linked by serine palmitoyltransferase (SPT), a key enzyme in membrane lipid biogenesis. To gain insight into the pathophysiological mechanisms linking these pathways to neuro-retinal diseases we compared patients diagnosed with two metabolically intertwined diseases: macular telangiectasia type II (MacTel), hereditary sensory autonomic neuropathy type 1 (HSAN1), or both. METHODS:We performed targeted metabolomic analyses of amino acids and broad sphingolipids in sera from a cohort of MacTel (205), HSAN1 (25) and Control (151) participants. RESULTS:MacTel patients exhibited broad alterations of amino acids, including changes in serine, glycine, alanine, glutamate, and branched-chain amino acids reminiscent of diabetes. MacTel patients had elevated 1-deoxysphingolipids but reduced levels of complex sphingolipids in circulation. A mouse model of retinopathy indicates dietary serine and glycine restriction can drive this depletion in complex sphingolipids. HSAN1 patients exhibited elevated serine, lower alanine, and a reduction in canonical ceramides and sphingomyelins compared to controls. Those patients diagnosed with both HSAN1 and MacTel showed the most significant decrease in circulating sphingomyelins. CONCLUSIONS:These results highlight metabolic distinctions between MacTel and HSAN1, emphasize the importance of membrane lipids in the progression of MacTel, and suggest distinct therapeutic approaches for these two neurodegenerative diseases.

(1) Figure S1 shows Metabolic flux analysis of p53+/+ and p53-/- cells in monolayer and spheroid culture. (2) Figure S2 shows Mechanism of induction of SREBP2 target genes in p53-deficient spheroid cultures. (3) Figure S3 shows Induction of cell cycle arrest and apoptosis by simvastatin in spheroid cultures. (4) Figure S4 shows Blockade of cholesterol and ubiquinone synthesis by statins. (5) Figure S5 shows Effect of simvastatin on TCA cycle flux and failure of ubiquinone to restore viability in monolayer cells. (6) Figure S6 shows Inhibition of apoptosis in p53-deficient spheroid cultures by uridine. (7) Figure S7 shows Genotyping of cells used for organoid cultures and lack of effect of leflunomide on intestinal hyperproliferation.

Figure S1 shows the effect of statins on cholesterol and coenzyme Q levels and synthesis. Figure S2 shows statins reduce oxidative phosphorylation and hence increase glucose uptake and lactate secretion. Figure S3 shows the metabolic rewiring that occurs as a consequence of statin-mediated ROS production. Figure S4 shows simvastatin treatment in vivo does not alter cholesterol or dolichol synthesis or systemic coenzyme Q synthesis. Figure S5 shows simvastatin and AZD6244 are synergistic.

TElevated production of collagen-rich extracellular matrix (ECM) is a hallmark of cancer associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. How to target ECM production to oppose cancer is yet unclear, since targeting CAFs has been shown to restrain but also promote cancer progression. Metabolic rewiring is a hallmark of CAFs. Here we find that proline, which is a highly abundant amino acid in collagen proteins, is newly synthesised from glutamine to make tumour collagen in breast cancer xenografts, and that its production is elevated in breast cancer CAFs. PYCR1 is the rate-limiting enzyme for proline synthesis and is highly expressed in the tumour stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. PYCR1 and COL1A1 are overexpressed in patients with invasive ductal carcinoma with poor prognosis. Both collagen and glutamine-derived proline synthesis in CAFs are enhanced by increased pyruvate dehydrogenase-derived acetyl-CoA levels, via gene expression regulation through the epigenetic regulator histone acetyl transferase EP300. Altogether, our work unveils unprecedented roles of CAF metabolism to support pro-tumorigenic collagen production. PYCR1 is a recognised cancer cell vulnerability and potential target for therapy, hence, our work provides evidence that targeting PYCR1 in tumours may have the additional benefit of halting the production of pro-tumorigenic ECM.

Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids (SLs) that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I, resulting in the synthesis and accumulation of 1-deoxysphingolipids (deoxySLs). These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine levels can promote 1-deoxySL synthesis, they impact numerous other metabolic pathways important for cancer cells. Here, we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxySL toxicity in cancer cells. We determined that both alanine treatment and SPTLC1C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, we found that spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxySL synthesis was induced via SPTLC1C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxySL synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.

Despite the clinical benefit of androgen‐deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration‐resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, we show that the ER membrane‐associated protein THEM6 regulates intracellular levels of ether lipids and is essential to trigger the induction of the ER stress response (UPR). Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid‐mediated activation of ATF4. Finally, we demonstrate that high THEM6 expression is associated with poor survival and correlates with high levels of UPR activation in PCa patients. Altogether, our results highlight THEM6 as a novel driver of therapy resistance in PCa as well as a promising target for the treatment of CRPC. Resistance to androgen‐deprivation therapy (ADT) leads to the development of lethal castration‐resistant prostate cancer. This study highlights THEM6 as a clinically relevant protein that supports ADT‐induced rewiring of lipid metabolism and sustains UPR activation, thus promoting ADT resistance. Resistance to androgen‐deprivation therapy (ADT) leads to the development of lethal castration‐resistant prostate cancer. This study highlights THEM6 as a clinically relevant protein that supports ADT‐induced rewiring of lipid metabolism and sustains UPR activation, thus promoting ADT resistance.

Type 2 diabetes represents a disease spectrum in which chronic metabolic dysfunction damages multiple organ systems including liver, kidneys, and peripheral nerves1,2. While onset and progression of these co-morbidities are linked with insulin resistance, hyperglycaemia and dyslipidemia3-7, aberrant amino acid metabolism also contributes to pathogenesis of diabetes and potentially its complications8-10. Serine and glycine are closely related non-essential amino acids11,12 that are consistently reduced in patients with metabolic syndrome10,13-16, but the mechanistic drivers of serine deficiency and the downstream metabolic and phenotypic consequences remain unclear. Low systemic serine, a serine-opathy, is also emerging as a hallmark of macular and peripheral nerve disorders. Specifically, serine deficiency correlates positively with impaired visual acuity and peripheral neuropathy (PN)17-19. Here we demonstrate that aberrant serine homeostasis in the liver drives serine and glycine deficiencies in genetically obese and hyperglycaemic mice. This serine-opathy can be diagnosed with a serine tolerance test that quantifies systemic serine disposal. Mimicking these metabolic alterations via dietary serine/glycine restriction together with high fat intake dramatically accelerates thermal hypoalgesia in mice and reduces epidermal sensory nerve density, which are accompanied by extensive sciatic nerve lipid remodeling. These phenotypes were subsequently normalized by myriocin, linking serine-associated PN with sphingolipid biosynthesis. These findings identify systemic serine deficiency and dyslipidemia as novel risk factors for PN that may be exploited therapeutically.

ABSTRACT Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. In patients, THEM6 expression correlates with progressive disease and is associated with poor survival. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, THEM6 is located at the endoplasmic reticulum (ER) membrane and controls lipid homeostasis by regulating intracellular levels of ether lipids. Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated induction of ATF4. Finally, we show that THEM6 is required for the establishment of the MYC-induced stress response. Thus, similar to PCa, THEM6 loss significantly impairs tumorigenesis in the MYC-dependent subtype of triple negative breast cancer. Altogether, our results highlight THEM6 as a novel component of the treatment-induced stress response and a promising target for the treatment of CRPC and MYC-driven cancer.