The ETO2 transcriptional cofactor maintains acute leukemia by driving a MYB/EP300-dependent stemness program.

Transcriptional cofactors of the ETO family are recurrent fusion partners in acute leukemia. We characterized the ETO2 regulome by integrating transcriptomic and chromatin binding analyses in human erythroleukemia xenografts and controlled ETO2 depletion models. We demonstrate that beyond its well-established repressive activity, ETO2 directly activates transcription of MYB, among other genes. The ETO2-activated signature is associated with a poorer prognosis in erythroleukemia but also in other acute myeloid and lymphoid leukemia subtypes. Mechanistically, ETO2 colocalizes with EP300 and MYB at enhancers supporting the existence of an ETO2/MYB feedforward transcription activation loop (e.g., on MYB itself). Both small-molecule and PROTAC-mediated inhibition of EP300 acetyltransferases strongly reduced ETO2 protein, chromatin binding, and ETO2-activated transcripts. Taken together, our data show that ETO2 positively enforces a leukemia maintenance program that is mediated in part by the MYB transcription factor and that relies on acetyltransferase cofactors to stabilize ETO2 scaffolding activity.

Pulmonary maternal immune activation does not cross the placenta but leads to fetal metabolic adaptation.

The fetal development of organs and functions is vulnerable to perturbation by maternal inflammation which may increase susceptibility to disorders after birth. Because it is not well understood how the placenta and fetus respond to acute lung- inflammation, we characterize the response to maternal pulmonary lipopolysaccharide exposure across 24 h in maternal and fetal organs using multi-omics, imaging and integrative analyses. Unlike maternal organs, which mount strong inflammatory immune responses, the placenta upregulates immuno-modulatory genes, in particular the IL-6 signaling suppressor Socs3. Similarly, we observe no immune response in the fetal liver, which instead displays metabolic changes, including increases in lipids containing docosahexaenoic acid, crucial for fetal brain development. The maternal liver and plasma display similar metabolic alterations, potentially increasing bioavailability of docosahexaenoic acid for the mother and fetus. Thus, our integrated temporal analysis shows that systemic inflammation in the mother leads to a metabolic perturbation in the fetus.

Malignant features of minipig melanomas prior to spontaneous regression.

In MeLiM minipigs, melanomas develop around birth, can metastasize, and have histopathologic characteristics similar to humans. Interestingly, MeLiM melanomas eventually regress. This favorable outcome raises the question of their malignancy, which we investigated. We clinically followed tens of tumors from onset to first signs of regression. Transcriptome analysis revealed an enrichment of all cancer hallmarks in melanomas, although no activating or suppressing somatic mutation were found in common driver genes. Analysis of tumor cell genomes revealed high mutation rates without UV signature. Canonical proliferative, survival and angiogenic pathways were detected in MeLiM tumor cells all along progression stages. Functionally, we show that MeLiM melanoma cells are capable to grow in immunocompromised mice, with serial passages and for a longer time than in MeLiM pigs. Pigs set in place an immune response during progression with dense infiltration by myeloid cells while melanoma cells are deficient in B2M expression. To conclude, our data on MeLiM melanomas reveal several malignancy characteristics. The combination of these features with the successful spontaneous regression of these tumors make it an outstanding model to study an efficient anti-tumor immune response.

Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues.

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues.

High caspase 3 and vulnerability to dual BCL2 family inhibition define ETO2::GLIS2 pediatric leukemia.

Pediatric acute myeloid leukemia expressing the ETO2::GLIS2 fusion oncogene is associated with dismal prognosis. Previous studies have shown that ETO2::GLIS2 can efficiently induce leukemia development associated with strong transcriptional changes but those amenable to pharmacological targeting remained to be identified. By studying an inducible ETO2::GLIS2 cellular model, we uncovered that de novo ETO2::GLIS2 expression in human cells led to increased CASP3 transcription, CASP3 activation, and cell death. Patient-derived ETO2::GLIS2+ leukemic cells expressed both high CASP3 and high BCL2. While BCL2 inhibition partly inhibited ETO2::GLIS2+ leukemic cell proliferation, BH3 profiling revealed that it also sensitized these cells to MCL1 inhibition indicating a functional redundancy between BCL2 and MCL1. We further show that combined inhibition of BCL2 and MCL1 is mandatory to abrogate disease progression using in vivo patient-derived xenograft models. These data reveal that a transcriptional consequence of ETO2::GLIS2 expression includes a positive regulation of the pro-apoptotic CASP3 and associates with a vulnerability to combined targeting of two BCL2 family members providing a novel therapeutic perspective for this aggressive pediatric AML subgroup.

Serial transplantation unmasks galectin-9 contribution to tumor immune escape in the MB49 murine model.

Mechanisms of tumor immune escape are quite diverse and require specific approaches for their exploration in syngeneic tumor models. In several human malignancies, galectin-9 (gal-9) is suspected to contribute to the immune escape. However, in contrast with what has been done for the infiltrating cells, the contribution of gal-9 produced by malignant cells has never been demonstrated in an animal model. Therefore, we derived isogenic clones-either positive or negative for gal-9-from the MB49 murine bladder carcinoma cell line. A progressive and consistent reduction of tumor growth was observed when gal-9-KO cells were subjected to serial transplantations into syngeneic mice. In contrast, tumor growth was unaffected during parallel serial transplantations into nude mice, thus linking tumor inhibition to the enhancement of the immune response against gal-9-KO tumors. This stronger immune response was at least in part explained by changing patterns of response to interferon-γ. One consistent change was a more abundant production of CXCL10, a major inflammatory factor whose production is often induced by interferon-γ. Overall, these observations demonstrate for the first time that serial transplantation into syngeneic mice can be a valuable experimental approach for the exploration of novel mechanisms of tumor immune escape.

Targeting the Respiratory Syncytial Virus N0-P Complex with Constrained α-Helical Peptides in Cells and Mice.

Respiratory syncytial virus (RSV) is the main cause of severe respiratory infection in young children worldwide, and no therapies have been approved for the treatment of RSV infection. Data from recent clinical trials of fusion or L polymerase inhibitors for the treatment of RSV-infected patients revealed the emergence of escape mutants, highlighting the need for the discovery of inhibitors with novel mechanisms of action. Here we describe stapled peptides derived from the N terminus of the phosphoprotein (P) that act as replication inhibitors. We demonstrate that these peptides inhibit RSV replication in vitro and in vivo by preventing the formation of the N0-P complex. The present strategy provides a novel means of targeting RSV replication with constrained macrocyclic peptides or small molecules and is broadly applicable to other viruses of the Mononegavirales order.

Chronic circadian disruption modulates breast cancer stemness and immune microenvironment to drive metastasis in mice.

Breast cancer is the most common type of cancer worldwide and one of the major causes of cancer death in women. Epidemiological studies have established a link between night-shift work and increased cancer risk, suggesting that circadian disruption may play a role in carcinogenesis. Here, we aim to shed light on the effect of chronic jetlag (JL) on mammary tumour development. To do this, we use a mouse model of spontaneous mammary tumourigenesis and subject it to chronic circadian disruption. We observe that circadian disruption significantly increases cancer-cell dissemination and lung metastasis. It also enhances the stemness and tumour-initiating potential of tumour cells and creates an immunosuppressive shift in the tumour microenvironment. Finally, our results suggest that the use of a CXCR2 inhibitor could correct the effect of JL on cancer-cell dissemination and metastasis. Altogether, our data provide a conceptual framework to better understand and manage the effects of chronic circadian disruption on breast cancer progression.

CLOUDED LEOPARD (NEOFELIS NEBULOSA) MORBIDITY AND MORTALITY IN CAPTIVE-BRED POPULATIONS: A COMPREHENSIVE RETROSPECTIVE STUDY OF MEDICAL DATA FROM 271 INDIVIDUALS IN EUROPEAN, ASIAN, AND AUSTRALIAN ZOOS.

The clouded leopard (Neofelis nebulosa) is classified as vulnerable on the International Union for the Conservation of Nature Red List of Threatened Species. However, diseases affecting this species across zoo populations are not well documented. The primary objective of this retrospective study was to identify common and significant causes of morbidity and mortality in captive-bred clouded leopards from European, Asian, and Australian institutions. Medical records from 44 zoological parks that held 271 clouded leopards from 1934 to 2017 were reviewed. Major causes of mortality in the dead leopards (n = 141) were respiratory disease (17%), maternal neglect and starvation (12%), generalized infectious disease (10%), digestive disease (10%), and trauma (10%). Six animals lived more than 20 yr and two were older than 22 yr. Diseases were recorded 344 times (average of two per leopard) in 166 living leopards. The body systems most frequently affected by disease in these 166 individuals were, in order of frequency, integumentary (prevalence = 21%), digestive (21%), respiratory (16%), musculoskeletal (12%), and urinary (10%) systems. Neoplasia (7%) was less frequent, followed by cardiovascular (5%), genital (3%), and viral (3%) disorders. Extensive, self-induced alopecia on the tail and dorsum was the most frequently reported dermatological disease, which is proposed to be called the "clouded leopard alopecia syndrome." The most common neoplasm was pheochromocytoma (1%), followed by squamous cell carcinoma of the paw pads, pleural mesothelioma and multicentric lymphomas (<1% each). Dilated cardiomyopathy (2%) was the most common cardiovascular disease. Bronchopneumonia (7%), enteritis (4%), and nephritis (4%) were the most frequently reported respiratory, digestive, and renal diseases, respectively. Diagnosed disease incidence was significantly higher in Europe. This paper reports the results of a comprehensive study of the causes of morbidity and mortality in European, Asian, and Australian clouded leopard zoo populations.

Multilayer intraclonal heterogeneity in chronic myelomonocytic leukemia.

The functional diversity of cells that compose myeloid malignancies, i.e., the respective roles of genetic and epigenetic heterogeneity in this diversity, remains poorly understood. This question is addressed in chronic myelomonocytic leukemia, a myeloid neoplasm in which clinical diversity contrasts with limited genetic heterogeneity. To generate induced pluripotent stem cell clones, we reprogrammed CD34+ cells collected from a patient with a chronic myelomonocytic leukemia in which whole exome sequencing of peripheral blood monocyte DNA had identified 12 gene mutations, including a mutation in KDM6A and two heterozygous mutations in TET2 in the founding clone and a secondary KRAS(G12D) mutation. CD34+ cells from an age-matched healthy donor were also reprogrammed. We captured a part of the genetic heterogeneity observed in the patient, i.e. we analyzed five clones with two genetic backgrounds, without and with the KRAS(G12D) mutation. Hematopoietic differentiation of these clones recapitulated the main features of the patient's disease, including overproduction of granulomonocytes and dysmegakaryopoiesis. These analyses also disclosed significant discrepancies in the behavior of hematopoietic cells derived from induced pluripotent stem cell clones with similar genetic background, correlating with limited epigenetic changes. These analyses suggest that, beyond the coding mutations, several levels of intraclonal heterogeneity may participate in the yet unexplained clinical heterogeneity of the disease.

Α de novo 3.8-Mb inversion affecting the EDA and XIST genes in a heterozygous female calf with generalized hypohidrotic ectodermal dysplasia.

BACKGROUND: In mammals, hypohidrotic ectodermal dysplasia (HED) is a genetic disorder that is characterized by sparse hair, tooth abnormalities, and defects in cutaneous glands. Only four genes, EDA, EDAR, EDARADD and WNT10A account for more than 90% of HED cases, and EDA, on chromosome X, is involved in 50% of the cases. In this study, we explored an isolated case of a female Holstein calf with symptoms similar to HED. RESULTS: Clinical examination confirmed the diagnosis. The affected female showed homogeneous hypotrichosis and oligodontia as previously observed in bovine EDAR homozygous and EDA hemizygous mutants. Under light microscopy, the hair follicles were thinner and located higher in the dermis of the frontal skin in the affected animal than in the control. Moreover, the affected animal showed a five-fold increase in the number of hair follicles and a four-fold decrease in the diameter of the pilary canals. Pedigree analysis revealed that the coefficient of inbreeding of the affected calf (4.58%) was not higher than the average population inbreeding coefficient (4.59%). This animal had ten ancestors in its paternal and maternal lineages. By estimating the number of affected cases that would be expected if any of these common ancestors carried a recessive mutation, we concluded that, if they existed, other cases of HED should have been reported in France, which is not the case. Therefore, we assumed that the causal mutation was dominant and de novo. By analyzing whole-genome sequencing data, we identified a large chromosomal inversion with breakpoints located in the first introns of the EDA and XIST genes. Genotyping by PCR-electrophoresis the case and its parents allowed us to demonstrate the de novo origin of this inversion. Finally, using various sources of information we present a body of evidence that supports the hypothesis that this mutation is responsible for a skewed inactivation of X, and that only the normal X can be inactivated. CONCLUSIONS: In this article, we report a unique case of X-linked HED affected Holstein female calf with an assumed full inactivation of the normal X-chromosome, thus leading to a severe phenotype similar to that of hemizygous males.

Newly identified LMO3-BORCS5 fusion oncogene in Ewing sarcoma at relapse is a driver of tumor progression.

Recently, we detected a new fusion transcript LMO3-BORCS5 in a patient with Ewing sarcoma within a cohort of relapsed pediatric cancers. LMO3-BORCS5 was as highly expressed as the characteristic fusion oncogene EWS/FLI1. However, the expression level of LMO3-BORCS5 at diagnosis was very low. Sanger sequencing depicted two LMO3-BORCS5 variants leading to loss of the functional domain LIM2 in LMO3 gene, and disruption of BORCS5. In vitro studies showed that LMO3-BORCS5 (i) increases proliferation, (ii) decreases expression of apoptosis-related genes and treatment sensitivity, and (iii) downregulates genes involved in differentiation and upregulates proliferative and extracellular matrix-related pathways. Remarkably, in vivo LMO3-BORCS5 demonstrated its high oncogenic potential by inducing tumors in mouse fibroblastic NIH-3T3 cell line. Moreover, BORCS5 probably acts, in vivo, as a tumor-suppressor gene. In conclusion, functional studies of fusion oncogenes at relapse are of great importance to define mechanisms involved in tumor progression and resistance to conventional treatments.

Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.

A miR-150/TET3 pathway regulates the generation of mouse and human non-classical monocyte subset.

Non-classical monocyte subsets may derive from classical monocyte differentiation and the proportion of each subset is tightly controlled. Deregulation of this repartition is observed in diverse human diseases, including chronic myelomonocytic leukemia (CMML) in which non-classical monocyte numbers are significantly decreased relative to healthy controls. Here, we identify a down-regulation of hsa-miR-150 through methylation of a lineage-specific promoter in CMML monocytes. Mir150 knock-out mice demonstrate a cell-autonomous defect in non-classical monocytes. Our pulldown experiments point to Ten-Eleven-Translocation-3 (TET3) mRNA as a hsa-miR-150 target in classical human monocytes. We show that Tet3 knockout mice generate an increased number of non-classical monocytes. Our results identify the miR-150/TET3 axis as being involved in the generation of non-classical monocytes.

Gallein, a Gßγ subunit signalling inhibitor, inhibits metastatic spread of tumour cells expressing OR51E2 and exposed to its odorant ligand.

OBJECTIVE: We previously reported that the olfactory receptor OR51E2, overexpressed in LNCaP prostate cancer cells, promotes cell invasiveness upon stimulation of its agonist ß-ionone, and this phenomenon increases metastatic spread. Furthermore, we showed that the induced cell invasiveness involves a PI3 kinase dependent signalling pathway. We report here the results of a new investigation to address whether gallein, a small inhibitor of G protein ßγ subunit interaction with PI3 kinase, can inhibit ß-ionone effects both in vitro and in vivo. RESULTS: We demonstrate that gallein can inhibit the ß-ionone-induced cell invasiveness in vitro, as well as the spread of metastases in vivo. LNCaP cell invasiveness, assessed using spheroid cultures in collagen gels in vitro, was increased by ß-ionone and the effect was reversed by co-administration of gallein. LNCaP tumour cells, subcutaneously inoculated to immunodeficient mice, generated more metastases in vivo when ß-ionone was applied through the skin. Furthermore, the intraperitoneal injection of gallein inhibited this increased metastasis spread. Our results thus support the role of OR51E2 in the ß-ionone observed effects, and suggest that gallein could be a potential new agent in personalized medicine of the tumours expressing OR51E2.

ETO2-GLIS2 Hijacks Transcriptional Complexes to Drive Cellular Identity and Self-Renewal in Pediatric Acute Megakaryoblastic Leukemia.

Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.

Gene transfer into hematopoietic stem cells reduces HLH manifestations in a murine model of Munc13-4 deficiency.

Patients with mutations in the UNC13D gene (coding for Munc13-4 protein) suffer from familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a life-threatening immune and hyperinflammatory disorder. The only curative treatment is allogeneic hematopoietic stem cell (HSC) transplantation, although the posttreatment survival rate is not satisfactory. Here, we demonstrate the curative potential of UNC13D gene correction of HSCs in a murine model of FHL3. We generated a self-inactivating lentiviral vector, used it to complement HSCs from Unc13d-deficient (Jinx) mice, and transplanted the cells back into the irradiated Jinx recipients. This procedure led to complete reconstitution of the immune system (ie, to wild-type levels). The recipients were then challenged with lymphocytic choriomeningitis virus to induce hemophagocytic lymphohistiocytosis (HLH)-like manifestations. All the clinical and biological signs of HLH were significantly reduced in mice having undergone HSC UNC13D gene correction than in nontreated animals. This beneficial effect was evidenced by the correction of blood cytopenia, body weight gain, normalization of the body temperature, decreased serum interferon-γ level, recovery of liver damage, and decreased viral load. These improvements can be explained by the restoration of the CD8+ T lymphocytes' cytotoxic function (as demonstrated here in an in vitro degranulation assay). Overall, our results demonstrate the efficacy of HSC gene therapy in an FHL-like setting of immune dysregulation.

Chondrocytes play a major role in the stimulation of bone growth by thyroid hormone.

Thyroid hormone (T3) is required for postnatal skeletal growth. It exerts its effect by binding to nuclear receptors, TRs including TRα1 and TRß1, which are present in most cell types. These cell types include chondrocytes and osteoblasts, the interactions of which are known to regulate endochondral bone formation. In order to analyze the respective functions of T3 stimulation in chondrocytes and osteoblasts during postnatal growth, we use Cre/loxP recombination to express a dominant-negative TRα1(L400R) mutant receptor in a cell-specific manner. Phenotype analysis revealed that inhibiting T3 response in chondrocytes is sufficient to reproduce the defects observed in hypothyroid mice, not only for cartilage maturation, but also for ossification and mineralization. TRα1(L400R) in chondrocytes also results in skull deformation. In the meantime, TRα1(L400R) expression in mature osteoblasts has no visible effect. Transcriptome analysis identifies a number of changes in gene expression induced by TRα1(L400R) in cartilage. These changes suggest that T3 normally cross talks with several other signaling pathways to promote chondrocytes proliferation, differentiation, and skeletal growth.

SDHA is a tumor suppressor gene causing paraganglioma.

Mitochondrial succinate-coenzyme Q reductase (complex II) consists of four subunits, SDHA, SDHB, SDHC and SDHD. Heterozygous germline mutations in SDHB, SDHC, SDHD and SDHAF2 [encoding for succinate dehydrogenase (SDH) complex assembly factor 2] cause hereditary paragangliomas and pheochromocytomas. Surprisingly, no genetic link between SDHA and paraganglioma/pheochromocytoma syndrome has ever been established. We identified a heterozygous germline SDHA mutation, p.Arg589Trp, in a woman suffering from catecholamine-secreting abdominal paraganglioma. The functionality of the SDHA mutant was assessed by studying SDHA, SDHB, HIF-1alpha and CD34 protein expression using immunohistochemistry and by examining the effect of the mutation in a yeast model. Microarray analyses were performed to study gene expression involved in energy metabolism and hypoxic pathways. We also investigated 202 paragangliomas or pheochromocytomas for loss of heterozygosity (LOH) at the SDHA, SDHB, SDHC and SDHD loci by BAC array comparative genomic hybridization. In vivo and in vitro functional studies demonstrated that the SDHA mutation causes a loss of SDH enzymatic activity in tumor tissue and in the yeast model. Immunohistochemistry and transcriptome analyses established that the SDHA mutation causes pseudo-hypoxia, which leads to a subsequent increase in angiogenesis, as other SDHx gene mutations. LOH was detected at the SDHA locus in the patient's tumor but was present in only 4.5% of a large series of paragangliomas and pheochromocytomas. The SDHA gene should be added to the list of genes encoding tricarboxylic acid cycle proteins that act as tumor suppressor genes and can now be considered as a new paraganglioma/pheochromocytoma susceptibility gene.