Lactate Utilization Enables Metabolic Escape to Confer Resistance to BET Inhibition in Acute Myeloid Leukemia.

Impairing the BET family coactivator BRD4 with small-molecule inhibitors (BETi) showed encouraging preclinical activity in treating acute myeloid leukemia (AML). However, dose-limiting toxicities and limited clinical activity dampened the enthusiasm for BETi as a single agent. BETi resistance in AML myeloblasts was found to correlate with maintaining mitochondrial respiration, suggesting that identifying the metabolic pathway sustaining mitochondrial integrity could help develop approaches to improve BETi efficacy. Herein, we demonstrated that mitochondria-associated lactate dehydrogenase allows AML myeloblasts to utilize lactate as a metabolic bypass to fuel mitochondrial respiration and maintain cellular viability. Pharmacologically and genetically impairing lactate utilization rendered resistant myeloblasts susceptible to BET inhibition. Low-dose combinations of BETi and oxamate, a lactate dehydrogenase inhibitor, reduced in vivo expansion of BETi-resistant AML in cell line and patient-derived murine models. These results elucidate how AML myeloblasts metabolically adapt to BETi by consuming lactate and demonstrate that combining BETi with inhibitors of lactate utilization may be useful in AML treatment. SIGNIFICANCE: Lactate utilization allows AML myeloblasts to maintain metabolic integrity and circumvent antileukemic therapy, which supports testing of lactate utilization inhibitors in clinical settings to overcome BET inhibitor resistance in AML. See related commentary by Boët and Sarry, p. 950.

Plasma Cell Differentiation, Antibody Quality, and Initial Germinal Center B Cell Population Depend on Glucose Influx Rate.

Serum Ab concentrations, selection for higher affinity BCRs, and generation of higher Ab affinities are important elements of immune response optimization and functions of germinal center (GC) reactions. B cell proliferation requires nutrients to support the anabolism inherent in clonal expansion. Glucose usage by mouse GC B cells has been reported to contribute little to their energy needs, with questions raised as to whether glucose uptake or glycolysis increases in GC B cells compared with their naive precursors. Indeed, metabolism can be highly flexible, such that supply shortage along one pathway may be compensated by increased flux on others. We now show that reduction of the glucose transporter GLUT1 in mice after establishment of a preimmune B cell repertoire, even after initiation of the GC B cell gene expression program, decreased initial GC B cell population numbers, affinity maturation, and plasma cell outputs. Glucose oxidation was heightened in GC B cells, but this hexose flowed more into the pentose phosphate pathway, whose activity was important in controlling reactive oxygen species (ROS) and Ab-secreting cell production. In modeling how glucose usage by B cells promotes the Ab response, the control of ROS appeared insufficient. Surprisingly, the combination of galactose, which mitigated ROS, with provision of mannose, an efficient precursor to glycosylation, supported robust production of and normal Ab secretion by Ab-secreting cells under glucose-free conditions. Collectively, the findings indicate that GCs depend on normal glucose influx, especially in plasma cell production, but reveal an unexpected metabolic flexibility in hexose requirements.

Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rate.

Antibody secretion into sera, selection for higher affinity BCR, and the generation of higher Ab affinities are important elements of immune response optimization, and a core function of germinal center reactions. B cell proliferation requires nutrients to support the anabolism inherent in clonal expansion. Glucose usage by GC B cells has been reported to contribute little to their energy needs, with questions raised as to whether or not glucose uptake or glycolysis increases in GC B cells compared to their naïve precursors. Indeed, metabolism can be highly flexible, such that supply shortage along one pathway may be compensated by increased flux on others. We now show that elimination of the glucose transporter GLUT1 after establishment of a pre-immune B cell repertoire, even after initiation of the GC B cell gene expression program, decreased initial GC B cell population numbers, affinity maturation, and PC outputs. Glucose oxidation was heightened in GC B cells, but this hexose flowed more into the pentose phosphate pathway (PPP), whose activity was important in controlling reactive oxygen (ROS) and ASC production. In modeling how glucose usage by B cells promotes the Ab response, the control of ROS appeared insufficient. Surprisingly, the combination of galactose, which mitigated ROS, with provision of mannose - an efficient precursor to glycosylation - supported robust production of and normal Ab secretion by ASC under glucose-free conditions. Collectively, the findings indicate that GC depend on normal glucose influx, especially in PC production, but reveal an unexpected metabolic flexibility in hexose requirements. KEY POINTS: Glucose influx is critical for GC homeostasis, affinity maturation and the generation of Ab-secreting cells.Plasma cell development uses the Pentose Phosphate Pathway, and hexose sugars maintain redox homeostasis.PCs can develop and achieve robust Ab secretion in the absence of glucose using a combination of hexose alternatives.

Neutrophil trafficking to the site of infection requires Cpt1a-dependent fatty acid ß-oxidation.

Cellular metabolism influences immune cell function, with mitochondrial fatty acid ß-oxidation and oxidative phosphorylation required for multiple immune cell phenotypes. Carnitine palmitoyltransferase 1a (Cpt1a) is considered the rate-limiting enzyme for mitochondrial metabolism of long-chain fatty acids, and Cpt1a deficiency is associated with infant mortality and infection risk. This study was undertaken to test the hypothesis that impairment in Cpt1a-dependent fatty acid oxidation results in increased susceptibility to infection. Screening the Cpt1a gene for common variants predicted to affect protein function revealed allele rs2229738_T, which was associated with pneumonia risk in a targeted human phenome association study. Pharmacologic inhibition of Cpt1a increases mortality and impairs control of the infection in a murine model of bacterial pneumonia. Susceptibility to pneumonia is associated with blunted neutrophilic responses in mice and humans that result from impaired neutrophil trafficking to the site of infection. Chemotaxis responsible for neutrophil trafficking requires Cpt1a-dependent mitochondrial fatty acid oxidation for amplification of chemoattractant signals. These findings identify Cpt1a as a potential host determinant of infection susceptibility and demonstrate a requirement for mitochondrial fatty acid oxidation in neutrophil biology.

Acly Deficiency Enhances Myelopoiesis through Acetyl Coenzyme A and Metabolic-Epigenetic Cross-Talk.

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation.

MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function.

Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

BET Inhibition Enhances the Antileukemic Activity of Low-dose Venetoclax in Acute Myeloid Leukemia.

PURPOSE: The BCL2 inhibitor, venetoclax, has transformed clinical care in acute myeloid leukemia (AML). However, subsets of patients do not respond or eventually acquire resistance. Venetoclax-based regimens can lead to considerable marrow suppression in some patients. Bromodomain and extraterminal inhibitors (BETi) are potential treatments for AML, as regulators of critical AML oncogenes. We tested the efficacy of novel BET inhibitor INCB054329, and its synergy with venetoclax to reduce AML without induction of hematopoietic toxicity. EXPERIMENTAL DESIGN: INCB054329 efficacy was assessed by changes in cell cycle and apoptosis in treated AML cell lines. In vivo efficacy was assessed by tumor reduction in MV-4-11 cell line-derived xenografts. Precision run-on and sequencing (PRO-seq) evaluated effects of INCB054329. Synergy between low-dose BETi and venetoclax was assessed in cell lines and patient samples in vitro and in vivo while efficacy and toxicity was assessed in patient-derived xenograft (PDX) models. RESULTS: INCB054329 induced dose-dependent apoptosis and quiescence in AML cell lines. PRO-seq analysis evaluated the effects of INCB054329 on transcription and confirmed reduced transcriptional elongation of key oncogenes, MYC and BCL2, and genes involved in the cell cycle and metabolism. Combinations of BETi and venetoclax led to reduced cell viability in cell lines and patient samples. Low-dose combinations of INCB054329 and venetoclax in cell line and PDX models reduced AML burden, regardless of the sensitivity to monotherapy without development of toxicity. CONCLUSIONS: Our findings suggest low dose combinations of venetoclax and BETi may be more efficacious for patients with AML than either monotherapy, potentially providing a longer, more tolerable dosing regimen.