miRNA-1 promotes acute myeloid leukemia cell pathogenesis through metabolic regulation.

Acute myeloid leukemia (AML) is a heterogeneous and deadly disease characterized by uncontrolled expansion of malignant blasts. Altered metabolism and dysregulated microRNA (miRNA) expression profiles are both characteristic of AML. However, there is a paucity of studies exploring how changes in the metabolic state of the leukemic cells regulate miRNA expression leading to altered cellular behavior. Here, we blocked pyruvate entry into mitochondria by deleting the Mitochondria Pyruvate Carrier (MPC1) gene in human AML cell lines, which decreased Oxidative Phosphorylation (OXPHOS). This metabolic shift also led to increased expression of miR-1 in the human AML cell lines tested. AML patient sample datasets showed that higher miR-1 expression correlates with reduced survival. Transcriptional and metabolic profiling of miR-1 overexpressing AML cells revealed that miR-1 increased OXPHOS, along with key metabolites that fuel the TCA cycle such as glutamine and fumaric acid. Inhibition of glutaminolysis decreased OXPHOS in miR-1 overexpressing MV4-11 cells, highlighting that miR-1 promotes OXPHOS through glutaminolysis. Finally, overexpression of miR-1 in AML cells exacerbated disease in a mouse xenograft model. Together, our work expands current knowledge within the field by uncovering novel connections between AML cell metabolism and miRNA expression that facilitates disease progression. Further, our work points to miR-1 as a potential new therapeutic target that may be used to disrupt AML cell metabolism and thus pathogenesis in the clinic.

Interferon Gamma-Inducible NAMPT in Melanoma Cells Serves as a Mechanism of Resistance to Enhance Tumor Growth.

(1) Background: Immune cells infiltrate the tumor microenvironment and secrete inflammatory cytokines, including interferons (IFNs), to drive antitumor responses and promote tumor clearance. However, recent evidence suggests that sometimes, tumor cells can also harness IFNs to enhance growth and survival. The essential NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT) gene is constitutively expressed in cells during normal homeostasis. However, melanoma cells have higher energetic demands and elevated NAMPT expression. We hypothesized that interferon gamma (IFNγ) regulates NAMPT in tumor cells as a mechanism of resistance that impedes the normal anti-tumorigenic effects of IFNγ. (2) Methods: Utilizing a variety of melanoma cells, mouse models, Crispr-Cas9, and molecular biology techniques, we explored the importance of IFNγ-inducible NAMPT during melanoma growth. (3) Results: We demonstrated that IFNγ mediates the metabolic reprogramming of melanoma cells by inducing Nampt through a Stat1 binding site in the Nampt gene, increasing cell proliferation and survival. Further, IFN/STAT1-inducible Nampt promotes melanoma in vivo. (4) Conclusions: We provided evidence that melanoma cells directly respond to IFNγ by increasing NAMPT levels, improving their fitness and growth in vivo (control n = 36, SBS KO n = 46). This discovery unveils a possible therapeutic target that may improve the efficacy of immunotherapies involving IFN responses in the clinic.

A Stat1 bound enhancer promotes Nampt expression and function within tumor associated macrophages.

Tumor associated macrophage responses are regulated by distinct metabolic states that affect their function. However, the ability of specific signals in the local tumor microenvironment to program macrophage metabolism remains under investigation. Here, we identify NAMPT, the rate limiting enzyme in NAD salvage synthesis, as a target of STAT1 during cellular activation by interferon gamma, an important driver of macrophage polarization and antitumor responses. We demonstrate that STAT1 occupies a conserved element within the first intron of Nampt, termed Nampt-Regulatory Element-1 (NRE1). Through disruption of NRE1 or pharmacological inhibition, a subset of M1 genes is sensitive to NAMPT activity through its impact on glycolytic processes. scRNAseq is used to profile in vivo responses by NRE1-deficient, tumor-associated leukocytes in melanoma tumors through the creation of a unique mouse strain. Reduced Nampt and inflammatory gene expression are present in specific myeloid and APC populations; moreover, targeted ablation of NRE1 in macrophage lineages results in greater tumor burden. Finally, elevated NAMPT expression correlates with IFNγ responses and melanoma patient survival. This study identifies IFN and STAT1-inducible Nampt as an important factor that shapes the metabolic program and function of tumor associated macrophages.

Mitochondrial Pyruvate Carrier 1 Promotes Peripheral T Cell Homeostasis through Metabolic Regulation of Thymic Development.

Metabolic pathways regulate T cell development and function, but many remain understudied. Recently, the mitochondrial pyruvate carrier (MPC) was identified as the transporter that mediates pyruvate entry into mitochondria, promoting pyruvate oxidation. Here we find that deleting Mpc1, an obligate MPC subunit, in the hematopoietic system results in a specific reduction in peripheral αß T cell numbers. MPC1-deficient T cells have defective thymic development at the ß-selection, intermediate single positive (ISP)-to-double-positive (DP), and positive selection steps. We find that early thymocytes deficient in MPC1 display alterations to multiple pathways involved in T cell development. This results in preferred escape of more activated T cells. Finally, mice with hematopoietic deletion of Mpc1 are more susceptible to experimental autoimmune encephalomyelitis. Altogether, our study demonstrates that pyruvate oxidation by T cell precursors is necessary for optimal αß T cell development and that its deficiency results in reduced but activated peripheral T cell populations.

A Low Iron Diet Protects from Steatohepatitis in a Mouse Model.

High tissue iron levels are a risk factor for multiple chronic diseases including type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). To investigate causal relationships and underlying mechanisms, we used an established NAFLD model-mice fed a high fat diet with supplemental fructose in the water ("fast food", FF). Iron did not affect excess hepatic triglyceride accumulation in the mice on FF, and FF did not affect iron accumulation compared to normal chow. Mice on low iron are protected from worsening of markers for non-alcoholic steatohepatitis (NASH), including serum transaminases and fibrotic gene transcript levels. These occurred prior to the onset of significant insulin resistance or changes in adipokines. Transcriptome sequencing revealed the major effects of iron to be on signaling by the transforming growth factor beta (TGF-ß) pathway, a known mechanistic factor in NASH. High iron increased fibrotic gene expression in vitro, demonstrating that the effect of dietary iron on NASH is direct. Conclusion: A lower tissue iron level prevents accelerated progression of NAFLD to NASH, suggesting a possible therapeutic strategy in humans with the disease.

Anti-inflammatory microRNA-146a protects mice from diet-induced metabolic disease.

Identifying regulatory mechanisms that influence inflammation in metabolic tissues is critical for developing novel metabolic disease treatments. Here, we investigated the role of microRNA-146a (miR-146a) during diet-induced obesity in mice. miR-146a is reduced in obese and type 2 diabetic patients and our results reveal that miR-146a-/- mice fed a high-fat diet (HFD) have exaggerated weight gain, increased adiposity, hepatosteatosis, and dysregulated blood glucose levels compared to wild-type controls. Pro-inflammatory genes and NF-κB activation increase in miR-146a-/- mice, indicating a role for this miRNA in regulating inflammatory pathways. RNA-sequencing of adipose tissue macrophages demonstrated a role for miR-146a in regulating both inflammation and cellular metabolism, including the mTOR pathway, during obesity. Further, we demonstrate that miR-146a regulates inflammation, cellular respiration and glycolysis in macrophages through a mechanism involving its direct target Traf6. Finally, we found that administration of rapamycin, an inhibitor of mTOR, was able to rescue the obesity phenotype in miR-146a-/- mice. Altogether, our study provides evidence that miR-146a represses inflammation and diet-induced obesity and regulates metabolic processes at the cellular and organismal levels, demonstrating how the combination of diet and miRNA genetics influences obesity and diabetic phenotypes.

Rab27-Dependent Exosome Production Inhibits Chronic Inflammation and Enables Acute Responses to Inflammatory Stimuli.

Extracellular vesicles, including exosomes, have recently been implicated as novel mediators of immune cell communication in mammals. However, roles for endogenously produced exosomes in regulating immune cell functions in vivo are just beginning to be identified. In this article, we demonstrate that Rab27a and Rab27b double-knockout (Rab27DKO) mice that are deficient in exosome secretion have a chronic, low-grade inflammatory phenotype characterized by elevated inflammatory cytokines and myeloproliferation. Upon further investigation, we found that some of these phenotypes could be complemented by wild-type (WT) hematopoietic cells or administration of exosomes produced by GM-CSF-expanded bone marrow cells. In addition, chronically inflamed Rab27DKO mice had a blunted response to bacterial LPS, resembling endotoxin tolerance. This defect was rescued by bone marrow exosomes from WT, but not miR-155-/-, cells, suggesting that uptake of miR-155-containing exosomes is important for a proper LPS response. Further, we found that SHIP1 and IRAK-M, direct targets of miR-155 that are known negative regulators of the LPS response, were elevated in Rab27DKO mice and decreased after treatment with WT, but not miR-155-/-, exosomes. Together, our study finds that Rab27-dependent exosome production contributes to homeostasis within the hematopoietic system and appropriate responsiveness to inflammatory stimuli.

Antitumor immunity is defective in T cell-specific microRNA-155-deficient mice and is rescued by immune checkpoint blockade.

MicroRNA-155 (miR-155) regulates antitumor immune responses. However, its specific functions within distinct immune cell types have not been delineated in conditional KO mouse models. In this study, we investigated the role of miR-155 specifically within T cells during the immune response to syngeneic tumors. We found that miR-155 expression within T cells is required to limit syngeneic tumor growth and promote IFNγ production by T cells within the tumor microenvironment. Consequently, we found that miR-155 expression by T cells is necessary for proper tumor-associated macrophage expression of IFNγ-inducible genes. We also found that immune checkpoint-blocking (ICB) antibodies against programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) restored antitumor immunity in miR-155 T cell-conditional KO mice. We noted that these ICB antibodies rescued the levels of IFNγ-expressing T cells, expression of multiple activation and effector genes expressed by tumor-infiltrating CD8+ and CD4+ T cells, and tumor-associated macrophage activation. Moreover, the ICB approach partially restored expression of several derepressed miR-155 targets in tumor-infiltrating, miR-155-deficient CD8+ T cells, suggesting that miR-155 and ICB regulate overlapping pathways to promote antitumor immunity. Taken together, our findings highlight the multifaceted role of miR-155 in T cells, in which it promotes antitumor immunity. These results suggest that the augmentation of miR-155 expression could be used to improve anticancer immunotherapies.

miR-155 promotes FLT3-ITD-induced myeloproliferative disease through inhibition of the interferon response.

FLT3-ITD+ acute myeloid leukemia (AML) accounts for ∼25% of all AML cases and is a subtype that carries a poor prognosis. microRNA-155 (miR-155) is specifically overexpressed in FLT3-ITD+ AML compared with FLT3 wild-type (FLT3-WT) AML and is critical for the growth of FLT3-ITD+ AML cells in vitro. However, miR-155's role in regulating FLT3-ITD-mediated disease in vivo remains unclear. In this study, we used a genetic mouse model to determine whether miR-155 influences the development of FLT3-ITD-induced myeloproliferative disease. Results indicate that miR-155 promotes FLT3-ITD-induced myeloid expansion in the bone marrow, spleen, and peripheral blood. Mechanistically, miR-155 increases proliferation of the hematopoietic stem and progenitor cell compartments by reducing the growth-inhibitory effects of the interferon (IFN) response, and this involves targeting of Cebpb. Consistent with our observations in mice, primary FLT3-ITD+ AML clinical samples have significantly higher miR-155 levels and a lower IFN response compared with FLT3-WT AML samples. Further, inhibition of miR-155 in FLT3-ITD+ AML cell lines using CRISPR/Cas9, or primary FLT3-ITD+ AML samples using locked nucleic acid antisense inhibitors, results in an elevated IFN response and reduces colony formation. Altogether, our data reveal that miR-155 collaborates with FLT3-ITD to promote myeloid cell expansion in vivo and that this involves a multitarget mechanism that includes repression of IFN signaling.

Adipocyte iron regulates leptin and food intake.

Dietary iron supplementation is associated with increased appetite. Here, we investigated the effect of iron on the hormone leptin, which regulates food intake and energy homeostasis. Serum ferritin was negatively associated with serum leptin in a cohort of patients with metabolic syndrome. Moreover, the same inverse correlation was observed in mice fed a high-iron diet. Adipocyte-specific loss of the iron exporter ferroportin resulted in iron loading and decreased leptin, while decreased levels of hepcidin in a murine hereditary hemochromatosis (HH) model increased adipocyte ferroportin expression, decreased adipocyte iron, and increased leptin. Treatment of 3T3-L1 adipocytes with iron decreased leptin mRNA in a dose-dependent manner. We found that iron negatively regulates leptin transcription via cAMP-responsive element binding protein activation (CREB activation) and identified 2 potential CREB-binding sites in the mouse leptin promoter region. Mutation of both sites completely blocked the effect of iron on promoter activity. ChIP analysis revealed that binding of phosphorylated CREB is enriched at these two sites in iron-treated 3T3-L1 adipocytes compared with untreated cells. Consistent with the changes in leptin, dietary iron content was also directly related to food intake, independently of weight. These findings indicate that levels of dietary iron play an important role in regulation of appetite and metabolism through CREB-dependent modulation of leptin expression.

Dietary iron controls circadian hepatic glucose metabolism through heme synthesis.

The circadian rhythm of the liver maintains glucose homeostasis, and disruption of this rhythm is associated with type 2 diabetes. Feeding is one factor that sets the circadian clock in peripheral tissues, but relatively little is known about the role of specific dietary components in that regard. We assessed the effects of dietary iron on circadian gluconeogenesis. Dietary iron affects circadian glucose metabolism through heme-mediated regulation of the interaction of nuclear receptor subfamily 1 group d member 1 (Rev-Erbα) with its cosuppressor nuclear receptor corepressor 1 (NCOR). Loss of regulated heme synthesis was achieved by aminolevulinic acid (ALA) treatment of mice or cultured cells to bypass the rate-limiting enzyme in hepatic heme synthesis, ALA synthase 1 (ALAS1). ALA treatment abolishes differences in hepatic glucose production and in the expression of gluconeogenic enzymes seen with variation of dietary iron. The differences among diets are also lost with inhibition of heme synthesis with isonicotinylhydrazine. Dietary iron modulates levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a transcriptional activator of ALAS1, to affect hepatic heme. Treatment of mice with the antioxidant N-acetylcysteine diminishes PGC-1α variation observed among the iron diets, suggesting that iron is acting through reactive oxygen species signaling.

Upregulation of cytosolic NADP+-dependent isocitrate dehydrogenase by hyperglycemia protects renal cells against oxidative stress.

Hyperglycemia-induced oxidative stress is widely recognized as a key mediator in the pathogenesis of diabetic nephropathy, a complication of diabetes. We found that both expression and enzymatic activity of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) were upregulated in the renal cortexes of diabetic rats and mice. Similarly, IDPc was induced in murine renal proximal tubular OK cells by high hyperglycemia, while it was abrogated by co-treatment with the antioxidant N-Acetyl-Cysteine (NAC). In OK cells, increased expression of IDPc by stable transfection prevented hyperglycemia-mediated reactive oxygen species (ROS) production, subsequent cellular oxidative stress and extracellular matrix accumulation, whereas these processes were all stimulated by decreased IDPc expression. In addition, production of NADPH and GSH in the cytosol was positively correlated with the expression level of IDPc in OK cells. These results together indicate that upregulation of IDPc in response to hyperglycemia might play an essential role in preventing the progression of diabetic nephropathy, which is accompanied by ROS-induced cellular damage and fibrosis, by providing NADPH, the reducing equivalent needed for recycling reduced glutathione and low molecular weight antioxidant thiol proteins.