Author Correction: Trans-differentiation of outer hair cells into inner hair cells in the absence of INSM1.

In Figs. 1e and 2g of this Letter, the labels 'actin' and 'VGLUT3', respectively, should have been in red instead of green font. This has been corrected online.

Nuclear-localized, iron-bound superoxide dismutase-2 antagonizes epithelial lineage programs to promote stemness of breast cancer cells via a histone demethylase activity.

The dichotomous behavior of superoxide dismutase-2 (SOD2) in cancer biology has long been acknowledged and more recently linked to different posttranslational forms of the enzyme. However, a distinctive activity underlying its tumor-promoting function is yet to be described. Here, we report that acetylation, one of such posttranslational modifications (PTMs), increases SOD2 affinity for iron, effectively changing the biochemical function of this enzyme from that of an antioxidant to a demethylase. Acetylated, iron-bound SOD2 localizes to the nucleus, promoting stem cell gene expression via removal of suppressive epigenetic marks such as H3K9me3 and H3K927me3. Particularly, H3K9me3 was specifically removed from regulatory regions upstream of Nanog and Oct-4, two pluripotency factors involved in cancer stem cell reprogramming. Phenotypically, cells expressing nucleus-targeted SOD2 (NLS-SOD2) have increased clonogenicity and metastatic potential. FeSOD2 operating as H3 demethylase requires H2O2 as substrate, which unlike cofactors of canonical demethylases (i.e., oxygen and 2-oxoglutarate), is more abundant in tumor cells than in normal tissue. Therefore, our results indicate that FeSOD2 is a demethylase with unique activities and functions in the promotion of cancer evolution toward metastatic phenotypes.

Trans-differentiation of outer hair cells into inner hair cells in the absence of INSM1.

The mammalian cochlea contains two types of mechanosensory hair cell that have different and critical functions in hearing. Inner hair cells (IHCs), which have an elaborate presynaptic apparatus, signal to cochlear neurons and communicate sound information to the brain. Outer hair cells (OHCs) mechanically amplify sound-induced vibrations, providing enhanced sensitivity to sound and sharp tuning. Cochlear hair cells are solely generated during development, and hair cell death-most often of OHCs-is the most common cause of deafness. OHCs and IHCs, together with supporting cells, originate in embryos from the prosensory region of the otocyst, but how hair cells differentiate into two different types is unknown1-3. Here we show that Insm1, which encodes a zinc finger protein that is transiently expressed in nascent OHCs, consolidates their fate by preventing trans-differentiation into IHCs. In the absence of INSM1, many hair cells that are born as OHCs switch fates to become mature IHCs. To identify the genetic mechanisms by which Insm1 operates, we compared the transcriptomes of immature IHCs and OHCs, and of OHCs with and without INSM1. In OHCs that lack INSM1, a set of genes is upregulated, most of which are normally preferentially expressed by IHCs. The homeotic cell transformation of OHCs without INSM1 into IHCs reveals a mechanism by which these neighbouring mechanosensory cells begin to differ: INSM1 represses a core set of early IHC-enriched genes in embryonic OHCs and makes them unresponsive to an IHC-inducing gradient, so that they proceed to mature as OHCs. Without INSM1, some of the OHCs in which these few IHC-enriched transcripts are upregulated trans-differentiate into IHCs, identifying candidate genes for IHC-specific differentiation.

Unique molecular signatures sustained in circulating monocytes and regulatory T cells in convalescent COVID-19 patients.

Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including convalescent COVID-19 and sero-negative controls. Flow cytometry analyses revealed reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells within the patients who have recovered from severe COVID-19. sc-RNA seq analysis identifies seven heterogeneous clusters of monocytes and nine Treg clusters featuring distinct molecular signatures in association with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocytes and Tregs expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters featuring S100 family genes: one monocyte cluster of S100A8 & A9 coupled with high HLA-I and another cluster of S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, as well as a unique TGF-ß high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-lived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (≥ 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.

Epigenetic reprogramming of host and viral genes by Human Cytomegalovirus infection in Kasumi-3 myeloid progenitor cells at early times post-infection.

HCMV establishes latency in myeloid cells. Using the Kasumi-3 latency model, we previously showed that lytic gene expression is activated prior to establishment of latency in these cells. The early events in infection may have a critical role in shaping establishment of latency. Here, we have used an integrative multi-omics approach to investigate dynamic changes in host and HCMV gene expression and epigenomes at early times post infection. Our results show dynamic changes in viral gene expression and viral chromatin. Analyses of Pol II, H3K27Ac and H3K27me3 occupancy of the viral genome showed that 1) Pol II occupancy was highest at the MIEP at 4 hours post infection. However, it was observed throughout the genome; 2) At 24 hours, H3K27Ac was localized to the major immediate early promoter/enhancer and to a possible second enhancer in the origin of replication OriLyt; 3) viral chromatin was broadly accessible at 24 hpi. In addition, although HCMV infection activated expression of some host genes, we observed an overall loss of de novo transcription. This was associated with loss of promoter-proximal Pol II and H3K27Ac, but not with changes in chromatin accessibility or a switch in modification of H3K27.Importance.HCMV is an important human pathogen in immunocompromised hosts and developing fetuses. Current anti-viral therapies are limited by toxicity and emergence of resistant strains. Our studies highlight emerging concepts that challenge current paradigms of regulation of HCMV gene expression in myeloid cells. In addition, our studies show that HCMV has a profound effect on de novo transcription and the cellular epigenome. These results may have implications for mechanisms of viral pathogenesis.

Differential DNA methylation and transcriptional signatures characterize impairment of muscle stem cells in pediatric human muscle contractures after brain injury.

Limb contractures are a debilitating and progressive consequence of a wide range of upper motor neuron injuries that affect skeletal muscle function. One type of perinatal brain injury causes cerebral palsy (CP), which affects a child's ability to move and is often painful. While several rehabilitation therapies are used to treat contractures, their long-term effectiveness is marginal since such therapies do not change muscle biological properties. Therefore, new therapies based on a biological understanding of contracture development are needed. Here, we show that myoblast progenitors from contractured muscle in children with CP are hyperproliferative. This phenotype is associated with DNA hypermethylation and specific gene expression patterns that favor cell proliferation over quiescence. Treatment of CP myoblasts with 5-azacytidine, a DNA hypomethylating agent, reduced this epigenetic imprint to TD levels, promoting exit from mitosis and molecular mechanisms of cellular quiescence. Together with previous studies demonstrating reduction in myoblast differentiation, this suggests a mechanism of contracture formation that is due to epigenetic modifications that alter the myogenic program of muscle-generating stem cells. We suggest that normalization of DNA methylation levels could rescue myogenesis and promote regulated muscle growth in muscle contracture and thus may represent a new nonsurgical approach to treating this devastating neuromuscular condition.

Cadmium-mediated pancreatic islet transcriptome changes in mice and cultured mouse islets.

Type II diabetes mellitus (T2DM) is a multifactorial disease process that is characterized by insulin resistance and impairment of insulin-producing pancreatic islets. There is evidence that environmental exposure to cadmium contributes to the development of T2DM. The presence of cadmium in human islets from the general population and the uptake of cadmium in ß-cells have been reported. To identify cadmium-mediated changes in gene expression and molecular regulatory networks in pancreatic islets, we performed next-generation RNA-Sequencing (RNA-Seq) in islets following either in vivo (1 mM CdCl2 in drinking water) or ex-vivo (0.5 µM CdCl2) exposure. Both exposure regiments resulted in islet cadmium concentrations that are comparable to those found in human islets from the general population. 6-week in vivo cadmium exposure upregulates the expression of five genes: Synj2, Gjb1, Rbpjl, Try5 and 5430419D17Rik. Rbpjl is a known regulator of ctrb, a gene associated with diabetes susceptibility. With 18-week in vivo cadmium exposure, we found more comprehensive changes in gene expression profile. Pathway enrichment analysis showed that these secondary changes were clustered to molecular mechanisms related to intracellular protein trafficking to the plasma membrane. In islet culture, cadmium ex vivo significantly induces the expression of Mt1, Sphk1, Nrcam, L3mbtl2, Rnf216 and Itpr1. Mt1 and Itpr1 are known to be involved in glucose homeostasis. Collectively, findings reported here revealed a complex cadmium-mediated effect on pancreatic islet gene expression at environmentally relevant cadmium exposure conditions, providing the basis for further studies into the pathophysiological processes arising from cadmium accumulation in pancreatic islets.

Hepatic deletion of X-box binding protein 1 impairs bile acid metabolism in mice.

The unfolded protein response (UPR) is an adaptive response to endoplasmic reticulum stress and the inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1) pathway of the UPR is important in lipid metabolism. However, its role in bile acid metabolism remains unknown. We demonstrate that liver-specific Xbp1 knockout (LS-Xbp1-/-) mice had a 45% reduction in total bile acid pool. LS-Xbp1-/- mice had lower serum 7α-hydroxy-4-cholesten-3-one (C4) levels compared with Xbp1fl/fl mice, indicating reduced cholesterol 7α-hydroxylase (CYP7A1) synthetic activity. This occurred without reductions of hepatic CYP7A1 protein expression. Feeding LS-Xbp1-/- mice cholestyramine increased hepatic CYP7A1 protein expression to levels 2-fold and 8-fold greater than cholestyramine-fed and chow-fed Xbp1fl/fl mice, respectively. However, serum C4 levels remained unchanged and were lower than both groups of Xbp1fl/fl mice. In contrast, although feeding LS-Xbp1-/- mice cholesterol did not increase CYP7A1 expression, serum C4 levels increased significantly up to levels similar to chow-fed Xbp1fl/fl mice and the total bile acid pool normalized. In conclusion, loss of hepatic XBP1 decreased the bile acid pool and CYP7A1 synthetic activity. Cholesterol feeding, but not induction of CYP7A1 with cholestyramine, increased CYP7A1 synthetic activity and corrected the genotype-specific total bile acid pools. These data demonstrate a novel role of IRE1α/XBP1 regulating bile acid metabolism.

Divergent target recognition by coexpressed 5'-isomiRs of miR-142-3p and selective viral mimicry.

Sequence heterogeneity at the ends of mature microRNAs (miRNAs) is well documented, but its effects on miRNA function are largely unexplored. Here we studied the impact of miRNA 5'-heterogeneity, which affects the seed region critical for target recognition. Using the example of miR-142-3p, an emerging regulator of the hematopoietic lineage in vertebrates, we show that naturally coexpressed 5'-variants (5'-isomiRs) can recognize largely distinct sets of binding sites. Despite this, both miR-142-3p isomiRs regulate exclusive and shared targets involved in actin dynamics. Thus, 5'-heterogeneity can substantially broaden and enhance regulation of one pathway. Other 5'-isomiRs, in contrast, recognize largely overlapping sets of binding sites. This is exemplified by two herpesviral 5'-isomiRs that selectively mimic one of the miR-142-3p 5'-isomiRs. We hypothesize that other cellular and viral 5'-isomiRs can similarly be grouped into those with divergent or convergent target repertoires, based on 5'-sequence features. Taken together, our results provide a detailed characterization of target recognition by miR-142-3p and its 5'-isomiR-specific viral mimic. We furthermore demonstrate that miRNA 5'-end variation leads to differential targeting and can thus broaden the target range of miRNAs.

MicroRNA-mediated transformation by the Kaposi's sarcoma-associated herpesvirus Kaposin locus.

UNLABELLED: The human oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) expresses a set of ∼20 viral microRNAs (miRNAs). miR-K10a stands out among these miRNAs because its entire stem-loop precursor overlaps the coding sequence for the Kaposin (Kap) A/C proteins. The ectopic expression of KapA has been reported to lead to transformation of rodent fibroblasts. However, these experiments inadvertently also introduced miR-K10a, which raises the question whether the transforming activity of the locus could in fact be due to miR-K10a expression. To answer this question, we have uncoupled miR-K10a and KapA expression. Our experiments revealed that miR-K10a alone transformed cells with an efficiency similar to that when it was coexpressed with KapA. Maintenance of the transformed phenotype was conditional upon continued miR-K10a but not KapA protein expression, consistent with its dependence on miRNA-mediated changes in gene expression. Importantly, miR-K10a taps into an evolutionarily conserved network of miR-142-3p targets, several of which are expressed in 3T3 cells and are also known inhibitors of cellular transformation. In summary, our studies of miR-K10a serve as an example of an unsuspected function of an mRNA whose precursor is embedded within a coding transcript. In addition, our identification of conserved miR-K10a targets that limit transformation will point the way to a better understanding of the role of this miRNA in KSHV-associated tumors. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus. The viral Kaposin locus has known oncogenic potential, which has previously been attributed to the encoded KapA protein. Here we show that the virally encoded miR-K10a miRNA, whose precursor overlaps the KapA-coding region, may account for the oncogenic properties of this locus. Our data suggest that miR-K10a mimics the cellular miRNA miR-142-3p and thereby represses several known inhibitors of oncogenic transformation. Our work demonstrates that functional properties attributed to a coding region may in fact be carried out by an embedded noncoding element and sheds light on the functions of viral miR-K10a.

Aberrant overexpression of CD14 on granulocytes sensitizes the innate immune response in mDia1 heterozygous del(5q) MDS.

The myelodysplastic syndromes (MDSs) include a spectrum of stem cell malignancies characterized by an increased risk of developing acute myeloid leukemia. Heterozygous loss of chromosome 5q (del[5q]) is the most common cytogenetic abnormality in MDS. DIAPH1 is localized to 5q31 and encodes one of the formin proteins, mDia1, which is involved in linear actin polymerization. Mice with mDia1 deficiency develop hematologic features with age mimicking human myeloid neoplasm, but its role in the pathogenesis of MDS is unclear. Here we report that mDia1 heterozygous and knockout mice develop MDS phenotypes with age. In these mice, CD14 was aberrantly overexpressed on granulocytes in a cell-autonomous manner, leading to a hypersensitive innate immune response to lipopolysaccharide (LPS) stimuli through CD14/Toll-like receptor 4 signaling. Chronic stimulation with LPS accelerated the development of MDS in mDia1 heterozygous and knockout mice that can be rescued by lenalidomide. Similar findings of CD14 overexpression were observed on the bone marrow granulocytes of del(5q) MDS patients. Mechanistically, mDia1 deficiency led to a downregulation of membrane-associated genes and a specific upregulation of CD14 messenger RNA in granulocytes, but not in other lineages. These results underscore the significance of mDia1 heterozygosity in deregulated innate immune responses in del(5q) MDS.

Hepatocyte X-box binding protein 1 deficiency increases liver injury in mice fed a high-fat/sugar diet.

Fatty liver is associated with endoplasmic reticulum stress and activation of the hepatic unfolded protein response (UPR). Reduced hepatic expression of the UPR regulator X-box binding protein 1 spliced (XBP1s) is associated with human nonalcoholic steatohepatitis (NASH), and feeding mice a high-fat diet with fructose/sucrose causes progressive, fibrosing steatohepatitis. This study examines the role of XBP1 in nonalcoholic fatty liver injury and fatty acid-induced cell injury. Hepatocyte-specific Xbp1-deficient (Xbp1(-/-)) mice were fed a high-fat/sugar (HFS) diet for up to 16 wk. HFS-fed Xbp1(-/-) mice exhibited higher serum alanine aminotransferase levels compared with Xbp1(fl/fl) controls. RNA sequencing and Gene Ontogeny pathway analysis of hepatic mRNA revealed that apoptotic process, inflammatory response, and extracellular matrix structural constituent pathways had enhanced activation in HFS-fed Xbp1(-/-) mice. Liver histology demonstrated enhanced injury and fibrosis but less steatosis in the HFS-fed Xbp1(-/-) mice. Hepatic Col1a1 and Tgfß1 gene expression, as well as Chop and phosphorylated JNK (p-JNK), were increased in Xbp1(-/-) compared with Xbp1(fl/fl) mice after HFS feeding. In vitro, stable XBP1-knockdown Huh7 cells (Huh7-KD) and scramble control cells (Huh7-SCR) were generated and treated with palmitic acid (PA) for 24 h. PA-treated Huh7-KD cells had increased cytotoxicity measured by lactate dehydrogenase release, apoptotic nuclei, and caspase3/7 activity assays compared with Huh7-SCR cells. CHOP and p-JNK expression was also increased in Huh7-KD cells following PA treatment. In conclusion, loss of XBP1 enhances injury in both in vivo and in vitro models of fatty liver injury. We speculate that hepatic XBP1 plays an important protective role in pathogenesis of NASH.

Genome-wide analysis of small RNAs expressed by Yersinia pestis identifies a regulator of the Yop-Ysc type III secretion system.

Small noncoding RNA (sRNA) molecules are integral components of the regulatory machinery for many bacterial species and are known to posttranscriptionally regulate metabolic and stress-response pathways, quorum sensing, virulence factors, and more. The Yop-Ysc type III secretion system (T3SS) is a critical virulence component for the pathogenic Yersinia species, and the regulation of this system is tightly controlled at each step from transcription to translocation of effectors into host cells. The contribution of sRNAs to the regulation of the T3SS in Yersinia has been largely unstudied, however. Previously, our lab identified a role for the sRNA chaperone protein Hfq in the regulation of components of the T3SS in the gastrointestinal pathogen Yersinia pseudotuberculosis. Here we present data demonstrating a similar requirement for Hfq in the closely related species Yersinia pestis. Through deep sequencing analysis of the Y. pestis sRNA-ome, we found 63 previously unidentified putative sRNAs in this species. We identified a Yersinia-specific sRNA, Ysr141, carried by the T3SS plasmid pCD1 that is required for the production of multiple T3SS proteins. In addition, we show that Ysr141 targets an untranslated region upstream of yopJ to posttranscriptionally activate the synthesis of the YopJ protein. Furthermore, Ysr141 may be an unstable and/or processed sRNA, which could contribute to its function in the regulation of the T3SS. The discovery of an sRNA that influences the synthesis of the T3SS adds an additional layer of regulation to this tightly controlled virulence determinant of Y. pestis.

Tissue-specific reprogramming leads to angiogenic neutrophil specialization and tumor vascularization in colorectal cancer.

Neutrophil (PMN) tissue accumulation is an established feature of ulcerative colitis (UC) lesions and colorectal cancer (CRC). To assess the PMN phenotypic and functional diversification during the transition from inflammatory ulceration to CRC we analyzed the transcriptomic landscape of blood and tissue PMNs. Transcriptional programs effectively separated PMNs based on their proximity to peripheral blood, inflamed colon, and tumors. In silico pathway overrepresentation analysis, protein-network mapping, gene signature identification, and gene-ontology scoring revealed unique enrichment of angiogenic and vasculature development pathways in tumor-associated neutrophils (TANs). Functional studies utilizing ex vivo cultures, colitis-induced murine CRC, and patient-derived xenograft models demonstrated a critical role for TANs in promoting tumor vascularization. Spp1 (OPN) and Mmp14 (MT1-MMP) were identified by unbiased -omics and mechanistic studies to be highly induced in TANs, acting to critically regulate endothelial cell chemotaxis and branching. TCGA data set and clinical specimens confirmed enrichment of SPP1 and MMP14 in high-grade CRC but not in patients with UC. Pharmacological inhibition of TAN trafficking or MMP14 activity effectively reduced tumor vascular density, leading to CRC regression. Our findings demonstrate a niche-directed PMN functional specialization and identify TAN contributions to tumor vascularization, delineating what we believe to be a new therapeutic framework for CRC treatment focused on TAN angiogenic properties.

Physics-driven structural docking and protein language models accelerate antibody screening and design for broad-spectrum antiviral therapy.

Therapeutic antibodies have become one of the most influential therapeutics in modern medicine to fight against infectious pathogens, cancer, and many other diseases. However, experimental screening for highly efficacious targeting antibodies is labor-intensive and of high cost, which is exacerbated by evolving antigen targets under selective pressure such as fast-mutating viral variants. As a proof-of-concept, we developed a machine learning-assisted antibody generation pipeline that greatly accelerates the screening and re-design of immunoglobulins G (IgGs) against a broad spectrum of SARS-CoV-2 coronavirus variant strains. These viruses infect human host cells via the viral spike protein binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2). Using over 1300 IgG sequences derived from convalescent patient B cells that bind with spike's receptor binding domain (RBD), we first established protein structural docking models in assessing the RBD-IgG-ACE2 interaction interfaces and predicting the virus-neutralizing activity of each IgG with a confidence score. Additionally, employing Gaussian process regression (also known as Kriging) in a latent space of an antibody language model, we predicted the landscape of IgGs' activity profiles against individual coronaviral variants of concern. With functional analyses and experimental validations, we efficiently prioritized IgG candidates for neutralizing a broad spectrum of viral variants (wildtype, Delta, and Omicron) to prevent the infection of host cells in vitro and hACE2 transgenic mice in vivo. Furthermore, the computational analyses enabled rational redesigns of selective IgG clones with single amino acid substitutions at the RBD-binding interface to improve the IgG blockade efficacy for one of the severe, therapy-resistant strains - Delta (B.1.617). Our work expedites applications of artificial intelligence in antibody screening and re-design even in low-data regimes combining protein language models and Kriging for antibody sequence analysis, activity prediction, and efficacy improvement, in synergy with physics-driven protein docking models for antibody-antigen interface structure analyses and functional optimization.

Critical Role for the Human Cytomegalovirus Major Immediate Early Proteins in Recruitment of RNA Polymerase II and H3K27Ac To an Enhancer-Like Element in OriLyt.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects most of the population. The complex 236 kbp genome encodes more than 170 open reading frames, whose expression is temporally regulated by both viral transcriptional regulators and cellular factors that control chromatin and transcription. Here, we have used state of the art genomic technologies to investigate the viral transcriptome in conjunction with 2 key transcriptional regulators: Pol II and H3K27Ac. Although it is well known that the major immediate early (IE) proteins activate early gene expression through both direct and indirect interactions, and that histone modifications play an important role in regulating viral gene expression, the role of the IE proteins in modulating viral chromatin is not fully understood. To address this question, we have used a virus engineered for conditional expression of the IE proteins combined with RNA and Chromatin immunoprecipitation (ChIP) analyses to assess the role of these proteins in modulating both viral chromatin and gene expression. Our results show that (i) there is an enhancer-like element in OriLyt that is extraordinarily enriched in H3K27Ac; (ii) in addition to activation of viral gene expression, the IE proteins play a critical role in recruitment of Pol II and H3K27Ac to this element. IMPORTANCE HCMV is an important human pathogen associated with complications in transplant patients and birth defects. The complex program of viral gene expression is regulated by both viral proteins and host factors. Here, we have investigated the role of the immediate early proteins in regulating the viral epigenome. Our results show that the viral immediate early proteins bring about an enormous enrichment of H3K27Ac marks at the OriLyt RNA4.9 promoter, concomitant with an increase in RNA4.9 expression. This epigenetic characteristic adds importantly to the view that OriLyt has structural and functional characteristics of a strong enhancer that, we now discover, is regulated by IE proteins.

Post-ischemic inactivation of HIF prolyl hydroxylases in endothelium promotes maladaptive kidney repair by inducing glycolysis.

Ischemic acute kidney injury (AKI) is common in hospitalized patients and increases the risk for chronic kidney disease (CKD). Impaired endothelial cell (EC) functions are thought to contribute in AKI to CKD transition, but the underlying mechanisms remain unclear. Here, we identify a critical role for endothelial oxygen sensing prolyl hydroxylase domain (PHD) enzymes 1-3 in regulating post-ischemic kidney repair. In renal endothelium, we observed compartment-specific differences in the expression of the three PHD isoforms in both mice and humans. We found that post-ischemic concurrent inactivation of endothelial PHD1, PHD2, and PHD3 but not PHD2 alone promoted maladaptive kidney repair characterized by exacerbated tissue injury, fibrosis, and inflammation. Single-cell RNA-seq analysis of the post-ischemic endothelial PHD1, PHD2 and PHD3 deficient (PHDTiEC) kidney revealed an endothelial glycolytic transcriptional signature, also observed in human kidneys with severe AKI. This metabolic program was coupled to upregulation of the SLC16A3 gene encoding the lactate exporter monocarboxylate transporter 4 (MCT4). Strikingly, treatment with the MCT4 inhibitor syrosingopine restored adaptive kidney repair in PHDTiEC mice. Mechanistically, MCT4 inhibition suppressed pro-inflammatory EC activation reducing monocyte-endothelial cell interaction. Our findings suggest avenues for halting AKI to CKD transition based on selectively targeting the endothelial hypoxia-driven glycolysis/MCT4 axis.

Lactate-dependent transcriptional regulation controls mammalian eye morphogenesis.

Mammalian retinal metabolism favors aerobic glycolysis. However, the role of glycolytic metabolism in retinal morphogenesis remains unknown. We report that aerobic glycolysis is necessary for the early stages of retinal development. Taking advantage of an unbiased approach that combines the use of eye organoids and single-cell RNA sequencing, we identify specific glucose transporters and glycolytic genes in retinal progenitors. Next, we determine that the optic vesicle territory of mouse embryos displays elevated levels of glycolytic activity. At the functional level, we show that removal of Glucose transporter 1 and Lactate dehydrogenase A gene activity from developing retinal progenitors arrests eye morphogenesis. Surprisingly, we uncover that lactate-mediated upregulation of key eye-field transcription factors is controlled by the epigenetic modification of histone H3 acetylation through histone deacetylase activity. Our results identify an unexpected bioenergetic independent role of lactate as a signaling molecule necessary for mammalian eye morphogenesis.

Checkpoint kinase 1/2 inhibition potentiates anti-tumoral immune response and sensitizes gliomas to immune checkpoint blockade.

Whereas the contribution of tumor microenvironment to the profound immune suppression of glioblastoma (GBM) is clear, tumor-cell intrinsic mechanisms that regulate resistance to CD8 T cell mediated killing are less understood. Kinases are potentially druggable targets that drive tumor progression and might influence immune response. Here, we perform an in vivo CRISPR screen to identify glioma intrinsic kinases that contribute to evasion of tumor cells from CD8 T cell recognition. The screen reveals checkpoint kinase 2 (Chek2) to be the most important kinase contributing to escape from CD8 T-cell recognition. Genetic depletion or pharmacological inhibition of Chek2 with blood-brain-barrier permeable drugs that are currently being evaluated in clinical trials, in combination with PD-1 or PD-L1 blockade, lead to survival benefit in multiple preclinical glioma models. Mechanistically, loss of Chek2 enhances antigen presentation, STING pathway activation and PD-L1 expression in mouse gliomas. Analysis of human GBMs demonstrates that Chek2 expression is inversely associated with antigen presentation and T-cell activation. Collectively, these results support Chek2 as a promising target for enhancement of response to immune checkpoint blockade therapy in GBM.

Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming.

Endothelial cell (EC) metabolism has emerged as a driver of angiogenesis. While hypoxia inactivates the oxygen sensors prolyl-4 hydroxylase domain-containing proteins 1-3 (PHD1-3) and stimulates angiogenesis, the effects of PHDs on EC functions remain poorly defined. Here, we investigated the impact of chemical PHD inhibition by dimethyloxalylglycine (DMOG) on angiogenic competence and metabolism of human vascular ECs. DMOG reduced EC proliferation, migration, and tube formation capacities, responses that were associated with an unfavorable metabolic reprogramming. While glycolytic genes were induced, multiple genes encoding sub-units of mitochondrial complex I were suppressed with concurrent decline in nicotinamide adenine dinucleotide (NAD+) levels. Importantly, the DMOG-induced defects in EC migration could be partially rescued by augmenting NAD+ levels through nicotinamide riboside or citrate supplementation. In summary, by integrating functional assays, transcriptomics, and metabolomics, we provide insights into the effects of PHD inhibition on angiogenic competence and metabolism of human vascular ECs.