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Aberrant expression of the E26 transformation-specific (ETS) transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS:FLI1 and EWS:ERG fusions in Ewing sarcoma (EwS) and TMPRSS2:ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS:FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV, and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs), and are highly elevated in plasma of patients with EwS with metastatic disease. High human satellite 2 and 3 (HSAT2,3) levels in EWS:FLI1- or ERG- expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle, and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA, and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs that transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics., Introduction Ewing sarcoma (EwS) is a poorly differentiated and highly aggressive childhood and adolescent malignancy of bone and soft tissues, thought to originate from primitive bone marrow-derived mesenchymal stem cells [...]

Muscle fibers express particular isoforms of contractile proteins, depending on the fiber's function and the organism's developmental stage. In the adult, after a muscle injury, newly generated fibers transition through embryonic and neonatal myosins, prior to selecting their distinctive adult myosin isoform. In this issue of the JCI, Wang et al. discover a checkpoint that regulates the neonatal-to-adult myosin isoform transition. They found that HIF-1[alpha] regulated this checkpoint, with elevated HIF-1[alpha] levels blocking progression, while HIF-1[alpha] knockout accelerated the transition. They further related these findings to centronuclear myopathy, a disease in which HIF-1[alpha] is similarly elevated and neonatal myosin expression is maintained. These findings highlight a maturation checkpoint that impacts the skeletal muscle regeneration following ischemic injury, providing a pharmacologically accessible pathway in injury and diseases such as centronuclear myopathy., Regeneration parallels embryogenesis Skeletal muscle fibers come in many varieties with different properties of metabolism, contractile speed, and fatigability, depending on the physiological role of the muscle they belong to. [...]

A fundamental issue in regenerative medicine is whether there exist endogenous regulatory mechanisms that limit the speed and efficiency of the repair process. We report the existence of a maturation checkpoint during muscle regeneration that pauses myofibers at a neonatal stage. This checkpoint is regulated by the mitochondrial protein mitofusin 2 (Mfn2), the expression of which is activated in response to muscle injury. Mfn2 is required for growth and maturation of regenerating myofibers; in the absence of Mfn2, new myofibers arrested at a neonatal stage, characterized by centrally nucleated myofibers and loss of H3K27me3 repressive marks at the neonatal myosin heavy chain gene. A similar arrest at the neonatal stage was observed in infantile cases of human centronuclear myopathy. Mechanistically, Mfn2 upregulation suppressed expression of hypoxia-induced factor 1a (HIF1[alpha]), which is induced in the setting of muscle damage. Sustained HIF1[alpha] signaling blocked maturation of new myofibers at the neonatal-to-adult fate transition, revealing the existence of a checkpoint that delays muscle regeneration. Correspondingly, inhibition of HIF1[alpha] allowed myofibers to bypass the checkpoint, thereby accelerating the repair process. We conclude that skeletal muscle contains a regenerative checkpoint that regulates the speed of myofiber maturation in response to Mfn2 and HIF1[alpha] activity., Introduction In response to injury, skeletal muscle undergoes a synchronized sequence of events over several days, including clearance of muscle debris, revascularization of the damaged region, activation and proliferation of [...]

Hepatic inflammation is culpable for the evolution of asymptomatic steatosis to nonalcoholic steatohepatitis (NASH). Hepatic inflammation results from abnormal macrophage activation. We found that FoxO1 links overnutrition to hepatic inflammation by regulating macrophage polarization and activation. FoxO1 was upregulated in hepatic macrophages, correlating with hepatic inflammation, steatosis, and fibrosis in mice and patients with NASH. Myeloid cell conditional FoxO1 knockout skewed macrophage polarization from proinflammatory M1 to the antiinflammatory M2 phenotype, accompanied by a reduction in macrophage infiltration in liver. These effects mitigated overnutrition-induced hepatic inflammation and insulin resistance, contributing to improved hepatic metabolism and increased energy expenditure in myeloid cell FoxO1-knockout mice on a high-fat diet. When fed a NASH-inducing diet, myeloid cell FoxO1-knockout mice were protected from developing NASH, culminating in a reduction in hepatic inflammation, steatosis, and fibrosis. Mechanistically, FoxO1 counteracts Stat6 to skew macrophage polarization from M2 toward the M1 signature to perpetuate hepatic inflammation in NASH. FoxO1 appears to be a pivotal mediator of macrophage activation in response to overnutrition and a therapeutic target for ameliorating hepatic inflammation to stem the disease progression from benign steatosis to NASH., Introduction Nonalcoholic fatty liver (NAFL) affects approximately 30% of the population worldwide and its prevalence increases on par with the rising epidemic of obesity in both adults and children (1). [...]

Although immobility is a common cause of muscle atrophy, the mechanism underlying this causality is unclear. We here show that Kruppel-like factor 15 (KLF15) and IL-6 are upregulated in skeletal muscle of limb-immobilized mice and that mice with KLF15 deficiency in skeletal muscle or with systemic IL-6 deficiency are protected from immobility-induced muscle atrophy. A newly developed [Ca.sup.2+] bioimaging revealed that the cytosolic [Ca.sup.2+] concentration ([[Ca.sup.2+]].) of skeletal muscle is reduced to below the basal level by immobilization, which is associated with the downregulation of Piezo1. Acute disruption of Piezol in skeletal muscle induced Klf15 and Il6 expression as well as muscle atrophy, which was prevented by antibodies against IL-6. A role for the Piezo1/KLF15/IL-6 axis in immobility-induced muscle atrophy was validated in human samples. Our results thus uncover a paradigm for [Ca.sup.2+] signaling in that a decrease in [[Ca.sup.2+]] from the basal level triggers a defined biological event., Introduction A decline in skeletal muscle mass can lead to a variety of detrimental conditions and a consequent shortening of life (1). Although many pathological conditions--including neuromuscular, cardiovascular, metabolic, inflammatory, [...]

A combination of anti-CTLA-4 plus anti-PD-1/PD-L1 is the most effective cancer immunotherapy but causes high incidence of immune-related adverse events (irAEs). Here we report that targeting of HIF-1[alpha] suppressed PD-L1 expression on tumor cells and tumor-infiltrating myeloid cells, but unexpectedly induced PD-L1 in normal tissues by an IFN-[gamma]-dependent mechanism. Targetingthe HIF-1[alpha]/PD-L1 axis in tumor cells reactivated tumor-infiltrating lymphocytes and caused tumor rejection. The HIF-1[alpha] inhibitor echinomycin potentiated the cancer immunotherapeutic effects of anti-CTLA-4 therapy, with efficacy comparable to that of anti-CTLA-4 plus anti-PD-1 antibodies. However, while anti-PD-1 exacerbated irAEs triggered by ipilimumab, echinomycin protected mice against irAEs by increasing PD-L1 levels in normal tissues. Our data suggest that targeting HIF-1[alpha] fortifies the immune tolerance function of the PD-1/PD-L1 checkpoint in normal tissues but abrogates its immune evasion function in the tumor microenvironment to achieve safer and more effective immunotherapy., Introduction Current immunotherapeutic strategies, articulated as immune checkpoint blockade, aim to release physiological immune tolerance checkpoints for the benefit of immunotherapeutic effect. As such, immune-related adverse events (irAEs) are considered [...]

Multiple beneficial cardiovascular effects of HDL depend on sphingosine-1-phosphate (S1P). S1P associates with HDL by binding to apolipoprotein M (ApoM). Insulin resistance is a major driver of dyslipidemia and cardiovascular risk. However, the mechanisms linking alterations in insulin signaling with plasma lipoprotein metabolism are incompletely understood. The insulin-repressible FoxO transcription factors mediate key effects of hepatic insulin action on glucose and lipoprotein metabolism. This work tested whether hepatic insulin signaling regulates HDL-S1P and aimed to identify the underlying molecular mechanisms. We report that insulin-resistant, nondiabetic individuals had decreased HDL-S1P levels, but no change in total plasma S1P. This also occurred in insulin-resistant db/db mice, which had low ApoM and a specific reduction of S1P in the HDL fraction, with no change in total plasma S1P levels. Using mice lacking hepatic FoxOs (L-FoxO1,3,4), we found that hepatic FoxOs were required for ApoM expression. Total plasma S1P levels were similar to those in controls, but S1P was nearly absent from HDL and was instead increased in the lipoprotein-depleted plasma fraction. This phenotype was restored to normal by rescuing ApoM in L-FoxO1,3,4 mice. Our findings show that insulin resistance in humans and mice is associated with decreased HDL-associated S1P. Our study shows that hepatic FoxO transcription factors are regulators of the ApoM/S1P pathway., Introduction Individuals with insulin resistance and type 2 diabetes have low levels of HDL cholesterol, and low HDL-cholesterol levels are inversely correlated with cardiovascular disease (1). However, clinical trials have [...]

The heart utilizes multiple adaptive mechanisms to maintain pump function. Compensatory cardiac hypertrophy reduces wall stress and oxygen consumption, thereby protecting the heart against acute blood pressure elevation. The nuclear effector of the Hippo pathway, Yes-associated protein 1 (YAP), is activated and mediates compensatory cardiac hypertrophy in response to acute pressure overload (PO). In this study, YAP promoted glycolysis by upregulating glucose transporter 1 (GLUT1), which in turn caused accumulation of intermediates and metabolites of the glycolytic, auxiliary, and anaplerotic pathways during acute PO. Cardiac hypertrophy was inhibited and heart failure was exacerbated in mice with YAP haploinsufficiency in the presence of acute PO. However, normalization of GLUT1 rescued the detrimental phenotype. PO induced the accumulation of glycolytic metabolites, including L-serine, L-aspartate, and malate, in a YAP-dependent manner, thereby promoting cardiac hypertrophy. YAP upregulated the GLUT1 gene through interaction with TEA domain family member 1 (TEAD1) and HIF-1[alpha] in cardiomyocytes. Thus, YAP induces compensatory cardiac hypertrophy through activation of the Warburg effect., Introduction Cardiac hypertrophy is an initial response of the heart to hemodynamic overload, including high blood pressure, termed pressure overload (PO). Long-term cardiac PO also often elicits other pathological effects [...]

Chuvash polycythemia is an inherited disease caused by a homozygous germline [VHL.sup.R200W] mutation, which leads to impaired degradation of HIF2[alpha], elevated levels of serum erythropoietin, and erythrocytosis/polycythemia. This phenotype is recapitulated by a mouse model bearing a homozygous [Vhl.sup.R200W] mutation. We previously showed that iron- regulatory protein 1-knockout (lrp1-knockout) mice developed erythrocytosis/polycythemia through translational derepression of Hif2[alpha], suggesting that IRP1 could be a therapeutic target to treat Chuvash polycythemia. Here, we fed [Vhl.sup.R200W] mice supplemented with Tempol, a small, stable nitroxide molecule and observed that Tempol decreased erythropoietin production, corrected splenomegaly, normalized hematocrit levels, and increased the lifespans of these mice. We attribute the reversal of erythrocytosis/polycythemia to translational repression of Hif2[alpha] expression by Tempol-mediated increases in the IRE-binding activity of Irp1, as reversal of polycythemia was abrogated in [Vhl.sup.R200W] mice in which /rp1 was genetically ablated. Thus, a new approach to the treatment of patients with Chuvash polycythemia may include dietary supplementation of Tempol, which decreased Hif2[alpha] expression and markedly reduced life-threatening erythrocytosis/polycythemia in the [Vhl.sup.R200W] mice., Introduction Chuvash polycythemia is an autosomal recessive disorder that is endemic to the Chuvash population of the Russian Federation (1) and the Italian island of Ischia, but has also been [...]

Myc activation is a primary oncogenic event in many human cancers; however, these transcription factors are difficult to inhibit pharmacologically, suggesting that Myc-dependent downstream effectors may be more tractable therapeutic targets. Here, we show that Myc overexpression induces endoplasmic reticulum (ER) stress and engages the inositol-requiring enzyme 1[alpha] (IRE1[alpha])/X-box binding protein 1 (XBP1) pathway through multiple molecular mechanisms in a variety of c- Myc- and N-Myc-dependent cancers. In particular, Myc-overexpressing cells require IRE1[alpha]/XBP1 signaling for sustained growth and survival in vitro and in vivo, dependent on elevated stearoyl-CoA-desaturase 1 (SCD1) activity. Pharmacological and genetic XBP1 inhibition induces Myc-dependent apoptosis, which is alleviated by exogenous unsaturated fatty acids. Of note, SCD1 inhibition phenocopies IRE1[alpha] RNase activity suppression in vivo. Furthermore, IRE1[alpha] inhibition enhances the cytotoxic effects of standard chemotherapy drugs used to treat c-Myc-overexpressing Burkitt's lymphoma, suggesting that inhibiting the IRE1[alpha]/XBP1 pathway is a useful general strategy for treatment of Myc- driven cancers., Introduction The Myc family of proto-oncogenes (MYC, MYCN, MYCL) encodes bHLHZ (basic helix-loop-helix leucine zipper) transcription factors, which regulate thousands of genes coordinating numerous cellular processes, including proliferation, differentiation, self-renewal, [...]

Patients with congenital lymphedema suffer from tissue swelling in part due to mutations in genes regulating lymphatic valve development. Lymphatic valve leaflets grow and are maintained throughout life in response to oscillatory shear stress (OSS), which regulates gene transcription in lymphatic endothelial cells (LECs). Here, we identified the first transcription factor, Foxo1, that repressed lymphatic valve formation by inhibiting the expression of valve- forming genes. We showed that both embryonic and postnatal ablation of Foxo1 in LECs induced additional valve formation in postnatal and adult mice in multiple tissues. Our quantitative analyses revealed that after deletion, the total number of valves in the mesentery was significantly (P < 0.01) increased in the [Foxo1.sup.LEC-KO] mice compared with [Foxo1.sup.fl/fl] controls. In addition, our quantitative real-time PCR (RT-PCR) data from cultured LECs showed that many valve-forming genes were significantly (P < 0.01) upregulated upon knockdown of FOXO1. To confirm our findings in vivo, rescue experiments showed that [Foxc2.sup.+/-] mice, a model of lymphedema-distichiasis, had 50% fewer lymphatic valves and that the remaining valves exhibited backleak. Both valve number and function were completely restored to control levels upon Foxo1 deletion. These findings established FOXO1 as a clinically relevant target to stimulate de novo lymphatic valve formation and rescue defective valves in congenital lymphedema., Introduction The lymphatic vasculature plays an essential role in maintaining fluid homeostasis in the body. The lymphatic capillaries absorb interstitial fluid to form lymph and the collecting lymphatic vessels return [...]