The PTPN2/PTPN1 inhibitor ABBV-CLS-484 unleashes potent anti-tumour immunity.

Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance1,2. The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity3-6. However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8+ T cell function by enhancing JAK-STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.

In vivo CRISPR screens reveal the landscape of immune evasion pathways across cancer.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.

Hepatic Fis1 regulates mitochondrial integrated stress response and improves metabolic homeostasis.

Mitophagy and mitochondrial integrated stress response (ISR) are 2 primary protective mechanisms to maintain functional mitochondria. Whether these 2 processes are coordinately regulated remains unclear. Here we show that mitochondrial fission 1 protein (Fis1), which is required for completion of mitophagy, serves as a signaling hub linking mitophagy and ISR. In mouse hepatocytes, high fat diet (HFD) feeding induces unresolved oxidative stress, defective mitophagy and enhanced type I interferon (IFN-I) response implicated in promoting metabolic inflammation. Adenoviral-mediated acute hepatic Fis1 overexpression is sufficient to reduce oxidative damage and improve glucose homeostasis in HFD-fed mice. RNA-Seq analysis reveals that Fis1 triggers a retrograde mitochondria-to-nucleus communication upregulating ISR genes encoding anti-oxidant defense, redox homeostasis, and proteostasis pathways. Fis1-mediated ISR also suppresses expression of IFN-I-stimulated genes through activating transcription factor 5 (Atf5), which inhibits the transactivation activity of interferon regulatory factor 3 (Irf3) known to control IFN-I production. Metabolite analysis demonstrates that Fis1 activation leads to accumulation of fumarate, a TCA cycle intermediate capable of increasing Atf5 activity. Consequently, hepatic Atf5 overexpression or monomethyl fumarate (MMF) treatment improves glucose homeostasis in HFD-fed mice. Collectively, these results support the potential use of small molecules targeting the Fis1-Atf5 axis, such as MMF, to treat metabolic diseases.

Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity.

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape1,2. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape3-5. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution6. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.

Mitochondrial TNAP controls thermogenesis by hydrolysis of phosphocreatine.

Adaptive thermogenesis has attracted much attention because of its ability to increase systemic energy expenditure and to counter obesity and diabetes1-3. Recent data have indicated that thermogenic fat cells use creatine to stimulate futile substrate cycling, dissipating chemical energy as heat4,5. This model was based on the super-stoichiometric relationship between the amount of creatine added to mitochondria and the quantity of oxygen consumed. Here we provide direct evidence for the molecular basis of this futile creatine cycling activity in mice. Thermogenic fat cells have robust phosphocreatine phosphatase activity, which is attributed to tissue-nonspecific alkaline phosphatase (TNAP). TNAP hydrolyses phosphocreatine to initiate a futile cycle of creatine dephosphorylation and phosphorylation. Unlike in other cells, TNAP in thermogenic fat cells is localized to the mitochondria, where futile creatine cycling occurs. TNAP expression is powerfully induced when mice are exposed to cold conditions, and its inhibition in isolated mitochondria leads to a loss of futile creatine cycling. In addition, genetic ablation of TNAP in adipocytes reduces whole-body energy expenditure and leads to rapid-onset obesity in mice, with no change in movement or feeding behaviour. These data illustrate the critical role of TNAP as a phosphocreatine phosphatase in the futile creatine cycle.

Tumor-Derived PGE2 Gives NK Cells a Headache.

Tumors use active immunosuppressive mechanisms to evade immune recognition and shape the local inflammatory environment. In this issue of Immunity, Bonavita et al. report that tumor-derived PGE2 blocks early activation of natural killer cells and interferes with subsequent adaptive immune cell recruitment to the tumor.

Bmal1 integrates mitochondrial metabolism and macrophage activation.

Metabolic pathways and inflammatory processes are under circadian regulation. Rhythmic immune cell recruitment is known to impact infection outcomes, but whether the circadian clock modulates immunometabolism remains unclear. We find that the molecular clock Bmal1 is induced by inflammatory stimulants, including Ifn-γ/lipopolysaccharide (M1) and tumor-conditioned medium, to maintain mitochondrial metabolism under metabolically stressed conditions in mouse macrophages. Upon M1 stimulation, myeloid-specific Bmal1 knockout (M-BKO) renders macrophages unable to sustain mitochondrial function, enhancing succinate dehydrogenase (SDH)-mediated mitochondrial production of reactive oxygen species as well as Hif-1α-dependent metabolic reprogramming and inflammatory damage. In tumor-associated macrophages, aberrant Hif-1α activation and metabolic dysregulation by M-BKO contribute to an immunosuppressive tumor microenvironment. Consequently, M-BKO increases melanoma tumor burden, whereas administering the SDH inhibitor dimethyl malonate suppresses tumor growth. Therefore, Bmal1 functions as a metabolic checkpoint that integrates macrophage mitochondrial metabolism, redox homeostasis and effector functions. This Bmal1-Hif-1α regulatory loop may provide therapeutic opportunities for inflammatory diseases and immunotherapy.

Interleukin-13 drives metabolic conditioning of muscle to endurance exercise.

Repeated bouts of exercise condition muscle mitochondria to meet increased energy demand-an adaptive response associated with improved metabolic fitness. We found that the type 2 cytokine interleukin-13 (IL-13) is induced in exercising muscle, where it orchestrates metabolic reprogramming that preserves glycogen in favor of fatty acid oxidation and mitochondrial respiration. Exercise training-mediated mitochondrial biogenesis, running endurance, and beneficial glycemic effects were lost in Il13-/- mice. By contrast, enhanced muscle IL-13 signaling was sufficient to increase running distance, glucose tolerance, and mitochondrial activity similar to the effects of exercise training. In muscle, IL-13 acts through both its receptor IL-13Rα1 and the transcription factor Stat3. The genetic ablation of either of these downstream effectors reduced running capacity in mice. Thus, coordinated immunological and physiological responses mediate exercise-elicited metabolic adaptations that maximize muscle fuel economy.

Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

Macrophage alternative activation confers protection against lipotoxicity-induced cell death.

OBJECTIVE: Alternative activation (M2) of adipose tissue resident macrophage (ATM) inhibits obesity-induced metabolic inflammation. The underlying mechanisms remain unclear. Recent studies have shown that dysregulated lipid homeostasis caused by increased lipolysis in white adipose tissue (WAT) in the obese state is a trigger of inflammatory responses. We investigated the role of M2 macrophages in lipotoxicity-induced inflammation. METHODS: We used microarray experiments to profile macrophage gene expression regulated by two M2 inducers, interleukin-4 (Il-4), and peroxisome proliferator-activated receptor delta/gamma (Pparδ/Pparγ) agonists. Functional validation studies were performed in bone marrow-derived macrophages and mice deprived of the signal transducer and activator of transcription 6 gene (Stat6; downstream effector of Il-4) or Pparδ/Pparγ genes (downstream effectors of Stat6). Palmitic acid (PA) and ß-adrenergic agonist were employed to induce macrophage lipid loading in vitro and in vivo, respectively. RESULTS: Profiling of genes regulated by Il-4 or Pparδ/Pparγ agonists reveals that alternative activation promotes the cell survival program, while inhibiting that of inflammation-related cell death. Deletion of Stat6 or Pparδ/Pparγ increases the susceptibility of macrophages to PA-induced cell death. NLR family pyrin domain containing 3 (Nlrp3) inflammasome activation by PA in the presence of lipopolysaccharide is also increased in Stat6-/- macrophages and to a lesser extent, in Pparδ/γ-/- macrophages. In concert, ß-adrenergic agonist-induced lipolysis results in higher levels of cell death and inflammatory markers in ATMs derived from myeloid-specific Pparδ/γ-/- or Stat6-/- mice. CONCLUSIONS: Our data suggest that ATM cell death is closely linked to metabolic inflammation. Within WAT where concentrations of free fatty acids fluctuate, M2 polarization regulated by the Stat6-Ppar axis enhances ATM's tolerance to lipid-mediated stress, thereby maintaining the homeostatic state.

Genetic Control of Fatty Acid ß-Oxidation in Chronic Obstructive Pulmonary Disease.

Bioenergetics homeostasis is important for cells to sustain normal functions and defend against injury. The genetic controls of bioenergetics homeostasis, especially lipid metabolism, remain poorly understood in chronic obstructive pulmonary disease (COPD), the third leading cause of death in the world. Additionally, the biological function of most of the susceptibility genes identified from genome-wide association studies (GWASs) in COPD remains unclear. Here, we aimed to address (1) how fatty acid oxidation (FAO), specifically ß-oxidation, a key lipid metabolism pathway that provides energy to cells, contributes to cigarette smoke (CS)-induced COPD; and (2) whether-and if so, how-FAM13A (family with sequence similarity 13 member A), a well-replicated COPD GWAS gene, modulates the FAO pathway. We demonstrated that CS induced expression of carnitine palmitoyltransferase 1A (CPT1A), a key mitochondrial enzyme for the FAO pathway, thereby enhancing FAO. Pharmacological inhibition of FAO by etomoxir blunted CS-induced reactive oxygen species accumulation and cell death in lung epithelial cells. FAM13A promoted FAO, possibly by interacting with and activating sirutin 1, and increasing expression of CPT1A. Furthermore, CS-induced cell death was reduced in lungs from Fam13a-/- mice. Our results suggest that FAM13A, the COPD GWAS gene, shapes the cellular metabolic response to CS exposure by promoting the FAO pathway, which may contribute to COPD development.

Identity Crisis: CD301b(+) Mononuclear Phagocytes Blur the M1-M2 Macrophage Line.

Obesity shifts the immune phenotype from M2 macrophage polarization to M1, which causes metabolic dysfunction. In this issue of Immunity, Kumamoto et al. (2016) identify a tissue-resident mononuclear phagocyte population that promotes weight gain and glucose intolerance but are defined by the M2 marker CD301b.

Hepatic Bmal1 Regulates Rhythmic Mitochondrial Dynamics and Promotes Metabolic Fitness.

Mitochondria undergo architectural/functional changes in response to metabolic inputs. How this process is regulated in physiological feeding/fasting states remains unclear. Here we show that mitochondrial dynamics (notably fission and mitophagy) and biogenesis are transcriptional targets of the circadian regulator Bmal1 in mouse liver and exhibit a metabolic rhythm in sync with diurnal bioenergetic demands. Bmal1 loss-of-function causes swollen mitochondria incapable of adapting to different nutrient conditions accompanied by diminished respiration and elevated oxidative stress. Consequently, liver-specific Bmal1 knockout (LBmal1KO) mice accumulate oxidative damage and develop hepatic insulin resistance. Restoration of hepatic Bmal1 activities in high-fat-fed mice improves metabolic outcomes, whereas expression of Fis1, a fission protein that promotes quality control, rescues morphological/metabolic defects of LBmal1KO mitochondria. Interestingly, Bmal1 homolog AHA-1 in C. elegans retains the ability to modulate oxidative metabolism and lifespan despite lacking circadian regulation. These results suggest clock genes are evolutionarily conserved energetics regulators.