ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway in breast cancer.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.

ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway.

ENPP1 expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer STING pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single cell RNA-seq (scRNA-seq), we show that ENPP1 overexpression drives primary breast tumor growth and metastasis by synergistically dampening extracellular cGAMP-STING mediated antitumoral immunity and activating immunosuppressive extracellular adenosine (eADO) signaling. In addition to cancer cells, stromal and immune cells in the tumor microenvironment (TME) also express ENPP1 that restrains their response to tumor-derived cGAMP. Enpp1 loss-of-function in both cancer cells and normal tissues slowed primary tumor initiation and growth and prevented metastasis in an extracellular cGAMP- and STING-dependent manner. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied total ENPP1 knockout, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Strikingly, we find that breast cancer patients with low ENPP1 expression have significantly higher immune infiltration and improved response to therapeutics impacting cancer immunity upstream or downstream of the cGAMP-STING pathway, like PARP inhibitors and anti-PD1. Altogether, selective inhibition of ENPP1's cGAMP hydrolase activity alleviates an innate immune checkpoint to boost cancer immunity and is therefore a promising therapeutic approach against breast cancer that may synergize with other cancer immunotherapies.

ENPP1's regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating STING signaling.

The metazoan innate immune second messenger 2'3'-cGAMP is present both inside and outside cells. However, only extracellular cGAMP can be negatively regulated by the extracellular hydrolase ENPP1. Here, we determine whether ENPP1's regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating stimulator of interferon genes (STING) signaling. We identified ENPP1H362A, a point mutation that cannot degrade the 2'-5' linkage in cGAMP while maintaining otherwise normal function. The selectivity of this histidine is conserved down to bacterial nucleotide pyrophosphatase/phosphodiesterase (NPP), allowing structural analysis and suggesting an unexplored ancient history of 2'-5' cyclic dinucleotides. Enpp1H362A mice demonstrated that extracellular cGAMP is not responsible for the devastating phenotype in ENPP1-null humans and mice but is responsible for antiviral immunity and systemic inflammation. Our data define extracellular cGAMP as a pivotal STING activator, identify an evolutionarily critical role for ENPP1 in regulating inflammation, and suggest a therapeutic strategy for viral and inflammatory conditions by manipulating ENPP1 activity.

Genetic deciphering of the antagonistic activities of the melanin-concentrating hormone and melanocortin pathways in skin pigmentation.

The genetic origin of human skin pigmentation remains an open question in biology. Several skin disorders and diseases originate from mutations in conserved pigmentation genes, including albinism, vitiligo, and melanoma. Teleosts possess the capacity to modify their pigmentation to adapt to their environmental background to avoid predators. This background adaptation occurs through melanosome aggregation (white background) or dispersion (black background) in melanocytes. These mechanisms are largely regulated by melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH), two hypothalamic neuropeptides also involved in mammalian skin pigmentation. Despite evidence that the exogenous application of MCH peptides induces melanosome aggregation, it is not known if the MCH system is physiologically responsible for background adaptation. In zebrafish, we identify that MCH neurons target the pituitary gland-blood vessel portal and that endogenous MCH peptide expression regulates melanin concentration for background adaptation. We demonstrate that this effect is mediated by MCH receptor 2 (Mchr2) but not Mchr1a/b. mchr2 knock-out fish cannot adapt to a white background, providing the first genetic demonstration that MCH signaling is physiologically required to control skin pigmentation. mchr2 phenotype can be rescued in adult fish by knocking-out pomc, the gene coding for the precursor of α-MSH, demonstrating the relevance of the antagonistic activity between MCH and α-MSH in the control of melanosome organization. Interestingly, MCH receptor is also expressed in human melanocytes, thus a similar antagonistic activity regulating skin pigmentation may be conserved during evolution, and the dysregulation of these pathways is significant to our understanding of human skin disorders and cancers.

Neutrophil-like HL-60 cells expressing only GFP-tagged ß-actin exhibit nearly normal motility.

Observations of actin dynamics in living cells using fluorescence microscopy have been foundational in the exploration of the mechanisms underlying cell migration. We used CRISPR/Cas9 gene editing to generate neutrophil-like HL-60 cell lines expressing GFP-ß-actin from the endogenous locus (ACTB). In light of many previous reports outlining functional deficiencies of labeled actin, we anticipated that HL-60 cells would only tolerate a monoallelic edit, as biallelic edited cells would produce no normal ß-actin. Surprisingly, we recovered viable monoallelic GFP-ß-actin cells as well as biallelic edited GFP-ß-actin cells, in which one copy of the ACTB gene is silenced and the other contains the GFP tag. Furthermore, the edited cells migrate with similar speeds and persistence as unmodified cells in a variety of motility assays, and have nearly normal cell shapes. These results might partially be explained by our observation that GFP-ß-actin incorporates into the F-actin network in biallelic edited cells at similar efficiencies as normal ß-actin in unedited cells. Additionally, the edited cells significantly upregulate γ-actin, perhaps helping to compensate for the loss of normal ß-actin. Interestingly, biallelic edited cells have only modest changes in global gene expression relative to the monoallelic line, as measured by RNA sequencing. While monoallelic edited cells downregulate expression of the tagged allele and are thus only weakly fluorescent, biallelic edited cells are quite bright and well-suited for live cell microscopy. The nondisruptive phenotype and direct interpretability of this fluorescent tagging approach make it a promising tool for studying actin dynamics in these rapidly migrating and highly phagocytic cells.

Extracellular cGAMP is a cancer cell-produced immunotransmitter involved in radiation-induced anti-cancer immunity.

2'3'-cyclic GMP-AMP (cGAMP) is an intracellular second messenger that is synthesized in response to cytosolic double-stranded DNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, we detected that cGAMP is continuously exported but then efficiently cleared by ENPP1, explaining why it has previously escaped detection. By developing potent, specific, and cell impermeable ENPP1 inhibitors, we found that cancer cells continuously export cGAMP in culture at steady state and at higher levels when treated with ionizing radiation (IR). In mouse tumors, depletion of extracellular cGAMP decreased tumor-associated immune cell infiltration and abolished the curative effect of IR. Boosting extracellular cGAMP with ENPP1 inhibitors synergized with IR to delay tumor growth. In conclusion, extracellular cGAMP is an anti-cancer immunotransmitter that could be harnessed to treat cancers with low immunogenicity.

Neural signatures of sleep in zebrafish.

Slow-wave sleep and rapid eye movement (or paradoxical) sleep have been found in mammals, birds and lizards, but it is unclear whether these neuronal signatures are found in non-amniotic vertebrates. Here we develop non-invasive fluorescence-based polysomnography for zebrafish, and show-using unbiased, brain-wide activity recording coupled with assessment of eye movement, muscle dynamics and heart rate-that there are at least two major sleep signatures in zebrafish. These signatures, which we term slow bursting sleep and propagating wave sleep, share commonalities with those of slow-wave sleep and paradoxical or rapid eye movement sleep, respectively. Further, we find that melanin-concentrating hormone signalling (which is involved in mammalian sleep) also regulates propagating wave sleep signatures and the overall amount of sleep in zebrafish, probably via activation of ependymal cells. These observations suggest that common neural signatures of sleep may have emerged in the vertebrate brain over 450 million years ago.

A screen for deeply conserved non-coding GWAS SNPs uncovers a MIR-9-2 functional mutation associated to retinal vasculature defects in human.

Thousands of human disease-associated single nucleotide polymorphisms (SNPs) lie in the non-coding genome, but only a handful have been demonstrated to affect gene expression and human biology. We computationally identified risk-associated SNPs in deeply conserved non-exonic elements (CNEs) potentially contributing to 45 human diseases. We further demonstrated that human CNE1/rs17421627 associated with retinal vasculature defects showed transcriptional activity in the zebrafish retina, while introducing the risk-associated allele completely abolished CNE1 enhancer activity. Furthermore, deletion of CNE1 led to retinal vasculature defects and to a specific downregulation of microRNA-9, rather than MEF2C as predicted by the original genome-wide association studies. Consistent with these results, miR-9 depletion affects retinal vasculature formation, demonstrating MIR-9-2 as a critical gene underpinning the associated trait. Importantly, we validated that other CNEs act as transcriptional enhancers that can be disrupted by conserved non-coding SNPs. This study uncovers disease-associated non-coding mutations that are deeply conserved, providing a path for in vivo testing to reveal their cis-regulated genes and biological roles.

MicroRNA-9 Couples Brain Neurogenesis and Angiogenesis.

In the developing brain, neurons expressing VEGF-A and blood vessels grow in close apposition, but many of the molecular pathways regulating neuronal VEGF-A and neurovascular system development remain to be deciphered. Here, we show that miR-9 links neurogenesis and angiogenesis through the formation of neurons expressing VEGF-A. We found that miR-9 directly targets the transcription factors TLX and ONECUTs to regulate VEGF-A expression. miR-9 inhibition leads to increased TLX and ONECUT expression, resulting in VEGF-A overexpression. This untimely increase of neuronal VEGF-A signal leads to the thickening of blood vessels at the expense of the normal formation of the neurovascular network in the brain and retina. Thus, this conserved transcriptional cascade is critical for proper brain development in vertebrates. Because of this dual role on neural stem cell proliferation and angiogenesis, miR-9 and its downstream targets are promising factors for cellular regenerative therapy following stroke and for brain tumor treatment.

The hypothalamic NPVF circuit modulates ventral raphe activity during nociception.

RFamide neuropeptide VF (NPVF) is expressed by neurons in the hypothalamus and has been implicated in nociception, but the circuit mechanisms remain unexplored. Here, we studied the structural and functional connections from NPVF neurons to downstream targets in the context of nociception, using novel transgenic lines, optogenetics, and calcium imaging in behaving larval zebrafish. We found a specific projection from NPVF neurons to serotonergic neurons in the ventral raphe nucleus (vRN). We showed NPVF neurons and vRN are suppressed and excited by noxious stimuli, respectively. We combined optogenetics with calcium imaging and pharmacology to demonstrate that stimulation of NPVF cells suppresses neuronal activity in vRN. During noxious stimuli, serotonergic neurons activation was due to a suppression of an inhibitory NPVF-ventral raphe peptidergic projection. This study reveals a novel NPVF-vRN functional circuit modulated by noxious stimuli in vertebrates.

Circadian and homeostatic regulation of structural synaptic plasticity in hypocretin neurons.

Neurons exhibit rhythmic activity that ultimately affects behavior such as sleep. In living zebrafish larvae, we used time-lapse two-photon imaging of the presynaptic marker synaptophysin in hypocretin/orexin (HCRT) neurons to determine the dynamics of synaptic modifications during the day and night. We observed circadian rhythmicity in synapse number in HCRT axons. This rhythm is regulated primarily by the circadian clock but is also affected by sleep deprivation. Furthermore, NPTX2, a protein implicated in AMPA receptor clustering, modulates circadian synaptic changes. In zebrafish, nptx2b is a rhythmic gene that is mostly expressed in hypothalamic and pineal gland cells. Arrhythmic transgenic nptx2b overexpression (hcrt:NPTX2b) increases synapse number and abolishes rhythmicity in HCRT axons. Finally, hcrt:NPTX2b fish are resistant to the sleep-promoting effects of melatonin. This behavioral effect is consistent with NPTX2b-mediated increased activity of HCRT circuitry. These data provide real-time in vivo evidence of circadian and homeostatic regulation of structural synaptic plasticity.

Characterization of two melanin-concentrating hormone genes in zebrafish reveals evolutionary and physiological links with the mammalian MCH system.

Melanin-concentrating hormone (MCH) regulates feeding and complex behaviors in mammals and pigmentation in fish. The relationship between fish and mammalian MCH systems is not well understood. Here, we identify and characterize two MCH genes in zebrafish, Pmch1 and Pmch2. Whereas Pmch1 and its corresponding MCH1 peptide resemble MCH found in other fish, the zebrafish Pmch2 gene and MCH2 peptide share genomic structure, synteny, and high peptide sequence homology with mammalian MCH. Zebrafish Pmch genes are expressed in closely associated but non-overlapping neurons within the hypothalamus, and MCH2 neurons send numerous projections to multiple MCH receptor-rich targets with presumed roles in sensory perception, learning and memory, arousal, and homeostatic regulation. Preliminary functional analysis showed that whereas changes in zebrafish Pmch1 expression correlate with pigmentation changes, the number of MCH2-expressing neurons increases in response to chronic food deprivation. These findings demonstrate that zebrafish MCH2 is the putative structural and functional ortholog of mammalian MCH and help elucidate the nature of MCH evolution among vertebrates.

Comparative expression of p2x receptors and ecto-nucleoside triphosphate diphosphohydrolase 3 in hypocretin and sensory neurons in zebrafish.

The hypocretin/orexin (HCRT/ORX) excitatory neuropeptides are expressed in a small population of lateral hypothalamic cells in mammals and fish. In humans, loss of these cells causes the sleep disorder narcolepsy. Identification of genes expressed in HCRT-producing cells may be revealing as to the regulation of sleep and the pathophysiology of narcolepsy. In this study, in situ hybridization analyses were performed to characterize the expression pattern of receptors and enzyme, which regulate ATP-mediated transmission in hypocretin cells of zebrafish larvae. The zebrafish cDNA encoding the ecto-nucleoside triphosphate diphosphohydrolase 3 (ENTPD3/NTPDase3) was isolated. This transcript was found to be expressed in zebrafish HCRT cells as previously reported in mammals. It was also expressed in the cranial nerves (gV, gVII, gIV and gX) and in primary sensory neurons (i.e., Rohon-Beard neurons) in the spinal cord. The expression of known zebrafish p2rx purinergic receptor family members was next studied and found to overlap with the entpd3 expression pattern. Specifically, p2rx2, p2rx3.1, p2rx3.2 and p2rx8 were expressed in the trigeminal ganglia and subsets of Rohon-Beard neurons. In contrast to mammals, p2rx2 was not expressed in HCRT cells; rather, p2rx8 was expressed with entpd3 in this hypothalamic region. The conservation of expression of these genes in HCRT cells and sensory neurons across vertebrates suggests an important role for ATP mediated transmission in the regulation of sleep and the processing of sensory inputs.