Plasma membrane abundance dictates phagocytic capacity and functional cross-talk in myeloid cells.

Professional phagocytes like neutrophils and macrophages tightly control what they consume, how much they consume, and when they move after cargo uptake. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G protein subunit Gß4 exhibited profound plasma membrane expansion, accompanied by marked reduction in plasma membrane tension. These biophysical changes promoted the phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. We also found that Gß4-deficient neutrophils are defective in the normal inhibition of migration following cargo uptake. Sphingolipid synthesis played a central role in these phenotypes by driving plasma membrane accumulation in cells lacking Gß4. In Gß4 knockout mice, neutrophils not only exhibited enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. Together, these results reveal an unexpected, biophysical control mechanism central to myeloid functional decision-making.

Altered microbial bile acid metabolism exacerbates T cell-driven inflammation during graft-versus-host disease.

Microbial transformation of bile acids affects intestinal immune homoeostasis but its impact on inflammatory pathologies remains largely unknown. Using a mouse model of graft-versus-host disease (GVHD), we found that T cell-driven inflammation decreased the abundance of microbiome-encoded bile salt hydrolase (BSH) genes and reduced the levels of unconjugated and microbe-derived bile acids. Several microbe-derived bile acids attenuated farnesoid X receptor (FXR) activation, suggesting that loss of these metabolites during inflammation may increase FXR activity and exacerbate the course of disease. Indeed, mortality increased with pharmacological activation of FXR and decreased with its genetic ablation in donor T cells during mouse GVHD. Furthermore, patients with GVHD after allogeneic hematopoietic cell transplantation showed similar loss of BSH and the associated reduction in unconjugated and microbe-derived bile acids. In addition, the FXR antagonist ursodeoxycholic acid reduced the proliferation of human T cells and was associated with a lower risk of GVHD-related mortality in patients. We propose that dysbiosis and loss of microbe-derived bile acids during inflammation may be an important mechanism to amplify T cell-mediated diseases.

Autotaxin-lysolipid signaling suppresses a CCL11-eosinophil axis to promote pancreatic cancer progression.

Lipids and their modifying enzymes regulate diverse features of the tumor microenvironment and cancer progression. The secreted enzyme autotaxin (ATX) hydrolyzes extracellular lysophosphatidylcholine to generate the multifunctional lipid mediator lysophosphatidic acid (LPA) and supports the growth of several tumor types, including pancreatic ductal adenocarcinoma (PDAC). Here we show that ATX suppresses the accumulation of eosinophils in the PDAC microenvironment. Genetic or pharmacologic ATX inhibition increased the number of intratumor eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop, wherein ATX-LPA signaling negatively regulates the activity of the AP-1 transcription factor c-Jun, in turn suppressing the expression of the potent eosinophil chemoattractant CCL11 (eotaxin-1). Eosinophils were identified in human PDAC specimens, and rare individuals with high intratumor eosinophil abundance had the longest overall survival. Together with recent findings, this study reveals the context-dependent, immune-modulatory potential of ATX-LPA signaling in cancer.

A genetically encoded tool to increase cellular NADH/NAD+ ratio in living cells.

Impaired redox metabolism is a key contributor to the etiology of many diseases, including primary mitochondrial disorders, cancer, neurodegeneration and aging. However, mechanistic studies of redox imbalance remain challenging due to limited strategies that can perturb redox metabolism in various cellular or organismal backgrounds. Most studies involving impaired redox metabolism have focused on oxidative stress; consequently, less is known about the settings where there is an overabundance of NADH reducing equivalents, termed reductive stress. Here we introduce a soluble transhydrogenase from Escherichia coli (EcSTH) as a novel genetically encoded tool to promote reductive stress in living cells. When expressed in mammalian cells, EcSTH, and a mitochondrially targeted version (mitoEcSTH), robustly elevated the NADH/NAD+ ratio in a compartment-specific manner. Using this tool, we determined that metabolic and transcriptomic signatures of the NADH reductive stress are cellular background specific. Collectively, our novel genetically encoded tool represents an orthogonal strategy to promote reductive stress.

Plasma membrane abundance dictates phagocytic capacity and functional crosstalk in myeloid cells.

Professional phagocytes like neutrophils and macrophages tightly control what they eat, how much they eat, and when they move after eating. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G-protein subunit Gb4 exhibit profound plasma membrane expansion due to enhanced production of sphingolipids. This increased membrane allocation dramatically enhances phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. Gb4 deficient neutrophils are also defective in the normal inhibition of migration following cargo uptake. In Gb4 knockout mice, myeloid cells exhibit enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. These results reveal an unexpected, biophysical control mechanism lying at the heart of myeloid functional decision-making.

Sustained Minimal Residual Disease Negativity in Multiple Myeloma is Associated with Stool Butyrate and Healthier Plant-Based Diets.

PURPOSE: Sustained minimal residual disease (MRD) negativity is associated with long-term survival in multiple myeloma. The gut microbiome is affected by diet, and in turn can modulate host immunity, for example through production of short-chain fatty acids including butyrate. We hypothesized that dietary factors affect the microbiome (abundance of butyrate-producing bacteria or stool butyrate concentration) and may be associated with multiple myeloma outcomes. EXPERIMENTAL DESIGN: We examined the relationship of dietary factors (via a food frequency questionnaire), stool metabolites (via gas chromatography-mass spectrometry), and the stool microbiome (via 16S sequencing - α-diversity and relative abundance of butyrate-producing bacteria) with sustained MRD negativity (via flow cytometry at two timepoints 1 year apart) in myeloma patients on lenalidomide maintenance. The Healthy Eating Index 2015 score and flavonoid nutrient values were calculated from the food frequency questionnaire. The Wilcoxon rank sum test was used to evaluate associations with two-sided P < 0.05 considered significant. RESULTS: At 3 months, higher stool butyrate concentration (P = 0.037), butyrate producers (P = 0.025), and α-diversity (P = 0.0035) were associated with sustained MRD negativity. Healthier dietary proteins, (from seafood and plants), correlated with butyrate at 3 months (P = 0.009) and sustained MRD negativity (P = 0.05). Consumption of dietary flavonoids, plant nutrients with antioxidant effects, correlated with stool butyrate concentration (anthocyanidins P = 0.01, flavones P = 0.01, and flavanols P = 0.02). CONCLUSIONS: This is the first study to demonstrate an association between a plant-based dietary pattern, stool butyrate production, and sustained MRD negativity in multiple myeloma, providing rationale to evaluate a prospective dietary intervention.

Hyperpolarized [5-13C,4,4-2H2,5-15N]-L-glutamine provides a means of annotating in vivo metabolic utilization of glutamine.

Glutamine is consumed by rapidly proliferating cells and can provide the carbon and nitrogen required for growth through various metabolic pathways. However, delineating the metabolic fate of glutamine is challenging to interrogate in vivo. Hyperpolarized magnetic resonance, by providing high transient nuclear magnetic resonance signals, provides an approach to measure fast biochemical processes in vivo. Aminohydrolysis of glutamine at carbon-5 plays an important role in providing nitrogen and carbon for multiple pathways. Here, we provide a synthetic strategy for isotope-enriched forms of glutamine that prolongs glutamine-C5 relaxation times and thereby reveals in vivo reactions involving carbon-5. We investigate multiple enrichment states, finding [5-13C,4,4-2H2,5-15N]-L-glutamine to be optimal for hyperpolarized measurement of glutamine conversion to glutamate in vivo. Leveraging this compound, we explore pancreatic cancer glutamine metabolism in vivo. Taken together, this work provides a means for studying glutamine metabolic flux in vivo and demonstrates on-target effects of metabolic enzyme inhibitors.

MAIT and Vδ2 unconventional T cells are supported by a diverse intestinal microbiome and correlate with favorable patient outcome after allogeneic HCT.

Microbial diversity is associated with improved outcomes in recipients of allogeneic hematopoietic cell transplantation (allo-HCT), but the mechanism underlying this observation is unclear. In a cohort of 174 patients who underwent allo-HCT, we demonstrate that a diverse intestinal microbiome early after allo-HCT is associated with an increased number of innate-like mucosal-associated invariant T (MAIT) cells, which are in turn associated with improved overall survival and less acute graft-versus-host disease (aGVHD). Immune profiling of conventional and unconventional immune cell subsets revealed that the prevalence of Vδ2 cells, the major circulating subpopulation of γδ T cells, closely correlated with the frequency of MAIT cells and was associated with less aGVHD. Analysis of these populations using both single-cell transcriptomics and flow cytometry suggested a shift toward activated phenotypes and a gain of cytotoxic and effector functions after transplantation. A diverse intestinal microbiome with the capacity to produce activating ligands for MAIT and Vδ2 cells appeared to be necessary for the maintenance of these populations after allo-HCT. These data suggest an immunological link between intestinal microbial diversity, microbe-derived ligands, and maintenance of unconventional T cells.

Gut microbiome correlates of response and toxicity following anti-CD19 CAR T cell therapy.

Anti-CD19 chimeric antigen receptor (CAR) T cell therapy has led to unprecedented responses in patients with high-risk hematologic malignancies. However, up to 60% of patients still experience disease relapse and up to 80% of patients experience CAR-mediated toxicities, such as cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. We investigated the role of the intestinal microbiome on these outcomes in a multicenter study of patients with B cell lymphoma and leukemia. We found in a retrospective cohort (n = 228) that exposure to antibiotics, in particular piperacillin/tazobactam, meropenem and imipenem/cilastatin (P-I-M), in the 4 weeks before therapy was associated with worse survival and increased neurotoxicity. In stool samples from a prospective cohort of CAR T cell recipients (n = 48), the fecal microbiome was altered at baseline compared to healthy controls. Stool sample profiling by 16S ribosomal RNA and metagenomic shotgun sequencing revealed that clinical outcomes were associated with differences in specific bacterial taxa and metabolic pathways. Through both untargeted and hypothesis-driven analysis of 16S sequencing data, we identified species within the class Clostridia that were associated with day 100 complete response. We concluded that changes in the intestinal microbiome are associated with clinical outcomes after anti-CD19 CAR T cell therapy in patients with B cell malignancies.

M24B aminopeptidase inhibitors selectively activate the CARD8 inflammasome.

Inflammasomes are multiprotein complexes that sense intracellular danger signals and induce pyroptosis. CARD8 and NLRP1 are related inflammasomes that are repressed by the enzymatic activities and protein structures of the dipeptidyl peptidases 8 and 9 (DPP8/9). Potent DPP8/9 inhibitors such as Val-boroPro (VbP) activate both NLRP1 and CARD8, but chemical probes that selectively activate only one have not been identified. Here we report a small molecule called CQ31 that selectively activates CARD8. CQ31 inhibits the M24B aminopeptidases prolidase (PEPD) and Xaa-Pro aminopeptidase 1 (XPNPEP1), leading to the accumulation of proline-containing peptides that inhibit DPP8/9 and thereby activate CARD8. NLRP1 is distinct from CARD8 in that it directly contacts DPP8/9's active site; these proline-containing peptides, unlike VbP, do not disrupt this repressive interaction and thus do not activate NLRP1. We expect that CQ31 will now become a valuable tool to study CARD8 biology.

Hodgkin Lymphoma Presenting With Spinal Cord Compression: Challenges for Diagnosis and Initial Management.

Hodgkin lymphoma (HL) can present with extra-nodal disease, but spinal cord compression is exceptionally rare. We describe a 15-year-old presenting with hip/back pain with normal initial examination. Persistent pain and raised inflammatory markers prompted further investigation with MRI, which revealed an epidural mass causing spinal cord compression. On examination, there was no palpable lymphadenopathy or cauda equina syndrome, but absent lower limb reflexes were noted. Following multidisciplinary discussion, it was determined that cauda equina syndrome was imminent and therefore surgical debulking was undertaken, both to prevent this complication and establish a diagnosis. At surgery, the tumor was highly vascular. Frozen section confirmed lesional material. Following surgery, and given the frozen section findings, a short course of steroids was commenced to reduce any peri-surgical edema. Unfortunately, histopathology was ultimately non-diagnostic, due to failure of immunohistochemistry on technically challenging material. Consequently, ultrasound-guided excision biopsy of a (non-palpable) cervical lymph node was performed five days later; histopathology showed typical effacement of the normal architecture and a conspicuous population of CD15/CD30-positive larger pale cells present, confirming nodular sclerosis classic HL, despite recent steroids. We review the available literature for HL presenting with spinal cord compression and describe the challenges for diagnosis and initial management in such cases.

Alloreactive T cells deficient of the short-chain fatty acid receptor GPR109A induce less graft-versus-host disease.

The intestinal microbiota is essential for the fermentation of dietary fiber into short-chain fatty acids (SCFA) such as butyrate, acetate, and propionate. SCFAs can bind to the G-protein-coupled receptors GPR43 and GPR109A (HCAR2), with varying affinities to promote cellular effects in metabolism or changes in immune function. We explored the role of GPR109A as the main receptor for butyrate in mouse models of allogeneic hematopoietic cell transplantation (allo-HCT) and graft-versus-host disease (GVHD). Deletion of GPR109A in allo-HCT recipients did not affect GVHD, but transplantation of T cells from GPR109A knockout (KO) (Gpr109a-/-) mice into allo-HCT recipient mice significantly reduced GVHD morbidity and mortality compared with recipients of wild-type (WT) T cells. Recipients of Gpr109a-/- T cells exhibited less GVHD-associated target organ pathology and decreased proliferation and homing of alloreactive T cells to target tissues. Although Gpr109a-/- T cells did not exhibit immune deficits at a steady state, following allo-activation, Gpr109a-/- T cells underwent increased apoptosis and were impaired mitochondrial oxidative phosphorylation, which was reversible through antioxidant treatment with N-acetylcysteine (NAC). In conclusion, we found that GPR109A expression by allo-activated T cells is essential for metabolic homeostasis and expansion, which are necessary features to induce GVHD after allo-HCT.

Vinblastine monotherapy induction prior to radiotherapy for patients with intracranial germinoma during the COVID-19 pandemic.

BACKGROUND: Patients with localized intracranial germinoma have excellent survival. Reducing treatment burden and long-term sequelae is a priority. Intensive inpatient chemotherapy (e.g., carboPEI = carboplatin/etoposide/ifosfamide) has been effectively employed to reduce radiotherapy treatment volume/dose. Outpatient-based carboplatin monotherapy is associated with excellent outcomes in metastatic testicular seminoma (an identical pathology), and successful vinblastine monotherapy induction (with 77% tumor volume reduction after just two weekly vinblastine doses) has recently been reported in an intracranial germinoma patient. METHODS: Adapted UK guidelines for germ cell tumor management were distributed during the COVID-19 pandemic, including nonstandard treatment options to reduce hospital visits and/or admissions. This included vinblastine monotherapy for intracranial germinoma (6 mg/m2 intravenously, or 4 mg/m2 for moderate count suppression, delivered weekly). We describe two such patients treated using this approach. RESULTS: A 30-year-old male with a localized pineal tumor received 12-week vinblastine induction, with >60% volume reduction, prior to definitive radiotherapy. A 12-year-old female with a metastatic suprasellar tumor and progression at all sites of disease whilst awaiting proton radiotherapy received two vinblastine doses with good early response, including 36% primary tumor volume reduction. The patients tolerated vinblastine well. CONCLUSION: Patients with intracranial germinoma have excellent outcomes, and reduction of late effects remains a priority. The description of vinblastine monotherapy in these intracranial germinoma patients warrants further exploration.

Connecting copper and cancer: from transition metal signalling to metalloplasia.

Copper is an essential nutrient whose redox properties make it both beneficial and toxic to the cell. Recent progress in studying transition metal signalling has forged new links between researchers of different disciplines that can help translate basic research in the chemistry and biology of copper into clinical therapies and diagnostics to exploit copper-dependent disease vulnerabilities. This concept is particularly relevant in cancer, as tumour growth and metastasis have a heightened requirement for this metal nutrient. Indeed, the traditional view of copper as solely an active site metabolic cofactor has been challenged by emerging evidence that copper is also a dynamic signalling metal and metalloallosteric regulator, such as for copper-dependent phosphodiesterase 3B (PDE3B) in lipolysis, mitogen-activated protein kinase kinase 1 (MEK1) and MEK2 in cell growth and proliferation and the kinases ULK1 and ULK2 in autophagy. In this Perspective, we summarize our current understanding of the connection between copper and cancer and explore how challenges in the field could be addressed by using the framework of cuproplasia, which is defined as regulated copper-dependent cell proliferation and is a representative example of a broad range of metalloplasias. Cuproplasia is linked to a diverse array of cellular processes, including mitochondrial respiration, antioxidant defence, redox signalling, kinase signalling, autophagy and protein quality control. Identifying and characterizing new modes of copper-dependent signalling offers translational opportunities that leverage disease vulnerabilities to this metal nutrient.

Copper depletion modulates mitochondrial oxidative phosphorylation to impair triple negative breast cancer metastasis.

Copper serves as a co-factor for a host of metalloenzymes that contribute to malignant progression. The orally bioavailable copper chelating agent tetrathiomolybdate (TM) has been associated with a significant survival benefit in high-risk triple negative breast cancer (TNBC) patients. Despite these promising data, the mechanisms by which copper depletion impacts metastasis are poorly understood and this remains a major barrier to advancing TM to a randomized phase II trial. Here, using two independent TNBC models, we report a discrete subpopulation of highly metastatic SOX2/OCT4+ cells within primary tumors that exhibit elevated intracellular copper levels and a marked sensitivity to TM. Global proteomic and metabolomic profiling identifies TM-mediated inactivation of Complex IV as the primary metabolic defect in the SOX2/OCT4+ cell population. We also identify AMPK/mTORC1 energy sensor as an important downstream pathway and show that AMPK inhibition rescues TM-mediated loss of invasion. Furthermore, loss of the mitochondria-specific copper chaperone, COX17, restricts copper deficiency to mitochondria and phenocopies TM-mediated alterations. These findings identify a copper-metabolism-metastasis axis with potential to enrich patient populations in next-generation therapeutic trials.

Lactate dehydrogenase A-dependent aerobic glycolysis promotes natural killer cell anti-viral and anti-tumor function.

Natural killer (NK) cells are cytotoxic lymphocytes capable of rapid cytotoxicity, cytokine secretion, and clonal expansion. To sustain such energetically demanding processes, NK cells must increase their metabolic capacity upon activation. However, little is known about the metabolic requirements specific to NK cells in vivo. To gain greater insight, we investigated the role of aerobic glycolysis in NK cell function and demonstrate that their glycolytic rate increases rapidly following viral infection and inflammation, prior to that of CD8+ T cells. NK cell-specific deletion of lactate dehydrogenase A (LDHA) reveals that activated NK cells rely on this enzyme for both effector function and clonal proliferation, with the latter being shared with T cells. As a result, LDHA-deficient NK cells are defective in their anti-viral and anti-tumor protection. These findings suggest that aerobic glycolysis is a hallmark of NK cell activation that is key to their function.

MYC-mediated early glycolysis negatively regulates proinflammatory responses by controlling IRF4 in inflammatory macrophages.

MYC activates different metabolic programs in a cell-type- and cell-status-dependent manner. However, the role of MYC in inflammatory macrophages has not yet been determined. Metabolic and molecular analyses reveal that MYC, but not hypoxia inducible factor 1 (HIF1), is involved in enhancing early glycolytic flux during inflammatory macrophage polarization. Ablation of MYC decreases lactate production by regulating lactate dehydrogenase (LDH) activity and causes increased inflammatory cytokines by regulating interferon regulatory factor 4 (IRF4) in response to lipopolysaccharide. Moreover, myeloid-specific deletion of MYC and pharmacological inhibition of the MYC/LDH axis enhance inflammation and the bacterial clearance in vivo. These results elucidate the potential role of the MYC/LDH/IRF4 axis in inflammatory macrophages by connecting early glycolysis with inflammatory responses and suggest that modulating early glycolytic flux mediated by the MYC/LDH axis can be used to open avenues for the therapeutic modulation of macrophage polarization to fight against bacterial infection.

Mitochondrial NADP(H) generation is essential for proline biosynthesis.

The coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but rather led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.

Translation in amino-acid-poor environments is limited by tRNAGln charging.

An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNAGln in amino-acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core-binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino-acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNAGln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as key sensors of glutamine availability in mammalian cells.