Downregulation of stromal syntenin sustains AML development.

The crosstalk between cancer and stromal cells plays a critical role in tumor progression. Syntenin is a small scaffold protein involved in the regulation of intercellular communication that is emerging as a target for cancer therapy. Here, we show that certain aggressive forms of acute myeloid leukemia (AML) reduce the expression of syntenin in bone marrow stromal cells (BMSC). Stromal syntenin deficiency, in turn, generates a pro-tumoral microenvironment. From serial transplantations in mice and co-culture experiments, we conclude that syntenin-deficient BMSC stimulate AML aggressiveness by promoting AML cell survival and protein synthesis. This pro-tumoral activity is supported by increased expression of endoglin, a classical marker of BMSC, which in trans stimulates AML translational activity. In short, our study reveals a vicious signaling loop potentially at the heart of AML-stroma crosstalk and unsuspected tumor-suppressive effects of syntenin that need to be considered during systemic targeting of syntenin in cancer therapy.

WHIM Syndrome-linked CXCR4 mutations drive osteoporosis.

WHIM Syndrome is a rare immunodeficiency caused by gain-of-function CXCR4 mutations. Here we report a decrease in bone mineral density in 25% of WHIM patients and bone defects leading to osteoporosis in a WHIM mouse model. Imbalanced bone tissue is observed in mutant mice combining reduced osteoprogenitor cells and increased osteoclast numbers. Mechanistically, impaired CXCR4 desensitization disrupts cell cycle progression and osteogenic commitment of skeletal stromal/stem cells, while increasing their pro-osteoclastogenic capacities. Impaired osteogenic differentiation is evidenced in primary bone marrow stromal cells from WHIM patients. In mice, chronic treatment with the CXCR4 antagonist AMD3100 normalizes in vitro osteogenic fate of mutant skeletal stromal/stem cells and reverses in vivo the loss of skeletal cells, demonstrating that proper CXCR4 desensitization is required for the osteogenic specification of skeletal stromal/stem cells. Our study provides mechanistic insights into how CXCR4 signaling regulates the osteogenic fate of skeletal cells and the balance between bone formation and resorption.

Niche-expressed Galectin-1 is involved in pre-B acute lymphoblastic leukemia relapse through pre-B cell receptor activation.

B-cell acute lymphoblastic leukemia (B-ALL) reflects the malignant counterpart of developing B cells in the bone marrow (BM). Despite tremendous progress in B-ALL treatment, the overall survival of adults at diagnosis and patients at all ages after relapse remains poor. Galectin-1 (GAL1) expressed by BM supportive niches delivers proliferation signals to normal pre-B cells through interaction with the pre-B cell receptor (pre-BCR). Here, we asked whether GAL1 gives non-cell autonomous signals to pre-BCR+ pre-B ALL, in addition to cell-autonomous signals linked to genetic alterations. In syngeneic and patient-derived xenograft (PDX) murine models, murine and human pre-B ALL development is influenced by GAL1 produced by BM niches through pre-BCR-dependent signals, similarly to normal pre-B cells. Furthermore, targeting pre-BCR signaling together with cell-autonomous oncogenic pathways in pre-B ALL PDX improved treatment response. Our results show that non-cell autonomous signals transmitted by BM niches represent promising targets to improve B-ALL patient survival.

Adhesion Molecules Involved in Stem Cell Niche Retention During Normal Haematopoiesis and in Acute Myeloid Leukaemia.

In the bone marrow (BM) of adult mammals, haematopoietic stem cells (HSCs) are retained in micro-anatomical structures by adhesion molecules that regulate HSC quiescence, proliferation and commitment. During decades, researchers have used engraftment to study the function of adhesion molecules in HSC's homeostasis regulation. Since the 90's, progress in genetically engineered mouse models has allowed a better understanding of adhesion molecules involved in HSCs regulation by BM niches and raised questions about the role of adhesion mechanisms in conferring drug resistance to cancer cells nested in the BM. This has been especially studied in acute myeloid leukaemia (AML) which was the first disease in which the concept of cancer stem cell (CSC) or leukemic stem cells (LSCs) was demonstrated. In AML, it has been proposed that LSCs propagate the disease and are able to replenish the leukemic bulk after complete remission suggesting that LSC may be endowed with drug resistance properties. However, whether such properties are due to extrinsic or intrinsic molecular mechanisms, fully or partially supported by molecular crosstalk between LSCs and surrounding BM micro-environment is still matter of debate. In this review, we focus on adhesion molecules that have been involved in HSCs or LSCs anchoring to BM niches and discuss if inhibition of such mechanism may represent new therapeutic avenues to eradicate LSCs.

The microenvironment of DLBCL is characterized by noncanonical macrophages recruited by tumor-derived CCL5.

Tissue invasion by tumor cells induces a host inflammatory response that variably impacts tumorigenesis. This has been well documented for tumor-associated macrophages (TAMs) that could play a pro/M2- or an anti/M1-tumoral function. TAMs frequently infiltrate diffuse large B-cell lymphoma (DLBCL), an aggressive neoplasm arising from germinal center-experienced B cells. However, the pathway leading to the presence of TAMs in DLBCL remains unknown, and their impact is unclear. Here, we show that some DLBCL tumor cells expressed the chemokine CCL5, enabling the differential recruitment of blood monocytes through their expression of CCR1 and CCR5. CCL5 expression by DLBCL was not related to molecular subtypes, and healthy tonsillar B cells did not produce this chemokine, implying a posttransformation event. A single-cell analysis revealed that most DLBCL TAMs had a noncanonical gene signature with the concomitant expression of M1 and M2 genes. The presence of noncanonical TAMs may explain the lack of impact of macrophages on DLBCL development reported in some survival studies.

Flow Cytometry Analysis of Mouse Hematopoietic Stem and Multipotent Progenitor Cells.

Flow cytometry has been widely used to detect a single event by means of multiparametric fluorescence measurements. Here we describe a method to analyze subsets of hematopoietic stem and progenitor cells isolated from long bones of mice. We further show that this method allows for comparing JAM-C protein expression between subsets of hematopoietic stem and progenitor cells.

Chronic IL-15 Stimulation and Impaired mTOR Signaling and Metabolism in Natural Killer Cells During Acute Myeloid Leukemia.

Natural Killer (NK) cells are potent anti-leukemic immune effectors. However, they display multiple defects in acute myeloid leukemia (AML) patients leading to reduced anti-tumor potential. Our limited understanding of the mechanisms underlying these defects hampers the development of strategies to restore NK cell potential. Here, we have used a mouse model of AML to gain insight into these mechanisms. We found that leukemia progression resulted in NK cell maturation defects and functional alterations. Next, we assessed NK cell cytokine signaling governing their behavior. We showed that NK cells from leukemic mice exhibit constitutive IL-15/mTOR signaling and type I IFN signaling. However, these cells failed to respond to IL-15 stimulation in vitro as illustrated by reduced activation of the mTOR pathway. Moreover, our data suggest that mTOR-mediated metabolic responses were reduced in NK cells from AML-bearing mice. Noteworthy, the reduction of mTOR-mediated activation of NK cells during AML development partially rescued NK cell metabolic and functional defects. Altogether, our data strongly suggest that NK cells from leukemic mice are metabolically and functionally exhausted as a result of a chronic cytokine activation, at least partially IL-15/mTOR signaling. NK cells from AML patients also displayed reduced IL-2/15Rß expression and showed cues of reduced metabolic response to IL-15 stimulation in vitro, suggesting that a similar mechanism might occur in AML patients. Our study pinpoints the dysregulation of cytokine stimulation pathways as a new mechanism leading to NK cell defects in AML.

GRASP55 Is Dispensable for Normal Hematopoiesis but Necessary for Myc-Dependent Leukemic Growth.

Grasp55 is a ubiquitous Golgi stacking protein involved in autophagy, protein trafficking, and glucose deprivation sensing. The function of Grasp55 in protein trafficking has been attributed to its PDZ-mediated interaction with the C-terminal PDZ-binding motifs of protein cargos. We have recently shown that such an interaction occurs between Grasp55 and the adhesion molecule Jam-C, which plays a central role in stemness maintenance of hematopoietic and spermatogenic cells. Accordingly, we have found that Grasp55-deficient mice suffer from spermatogenesis defects similar to Jam-C knockout mice. However, whether Grasp55 is involved in the maintenance of immunohematopoietic homeostasis through regulation of protein transport and Jam-C expression remains unknown. In this study, we show that Grasp55 deficiency does not affect hematopoietic stem cell differentiation, engraftment, or mobilization, which are known to depend on expression of Grasp55-dependent protein cargos. In contrast, using an Myc-dependent leukemic model addicted to autophagy, we show that knockdown of Grasp55 in leukemic cells reduces spleen and bone marrow tumor burden upon i.v. leukemic engraftment. This is not due to reduced homing of Grasp55-deficient cells to these organs but to increased spontaneous apoptosis of Grasp55-deficient leukemic cells correlated with increased sensitivity of the cells to glucose deprivation. These results show that Grasp55 plays a role in Myc-transformed hematopoietic cells but not in normal hematopoietic cells in vivo.

Bi-allelic JAM2 Variants Lead to Early-Onset Recessive Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder characterized by a combination of neurological, psychiatric, and cognitive decline associated with calcium deposition on brain imaging. To date, mutations in five genes have been linked to PFBC. However, more than 50% of individuals affected by PFBC have no molecular diagnosis. We report four unrelated families presenting with initial learning difficulties and seizures and later psychiatric symptoms, cerebellar ataxia, extrapyramidal signs, and extensive calcifications on brain imaging. Through a combination of homozygosity mapping and exome sequencing, we mapped this phenotype to chromosome 21q21.3 and identified bi-allelic variants in JAM2. JAM2 encodes for the junctional-adhesion-molecule-2, a key tight-junction protein in blood-brain-barrier permeability. We show that JAM2 variants lead to reduction of JAM2 mRNA expression and absence of JAM2 protein in patient's fibroblasts, consistent with a loss-of-function mechanism. We show that the human phenotype is replicated in the jam2 complete knockout mouse (jam2 KO). Furthermore, neuropathology of jam2 KO mouse showed prominent vacuolation in the cerebral cortex, thalamus, and cerebellum and particularly widespread vacuolation in the midbrain with reactive astrogliosis and neuronal density reduction. The regions of the human brain affected on neuroimaging are similar to the affected brain areas in the myorg PFBC null mouse. Along with JAM3 and OCLN, JAM2 is the third tight-junction gene in which bi-allelic variants are associated with brain calcification, suggesting that defective cell-to-cell adhesion and dysfunction of the movement of solutes through the paracellular spaces in the neurovascular unit is a key mechanism in CNS calcification.

Toward Therapeutic Targeting of Bone Marrow Leukemic Niche Protective Signals in B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) represents the malignant counterpart of bone marrow (BM) differentiating B cells and occurs most frequently in children. While new combinations of chemotherapeutic agents have dramatically improved the prognosis for young patients, disease outcome remains poor after relapse or in adult patients. This is likely due to heterogeneity of B-ALL response to treatment which relies not only on intrinsic properties of leukemic cells, but also on extrinsic protective cues transmitted by the tumor cell microenvironment. Alternatively, leukemic cells have the capacity to shape their microenvironment towards their needs. Most knowledge on the role of protective niches has emerged from the identification of mesenchymal and endothelial cells controlling hematopoietic stem cell self-renewal or B cell differentiation. In this review, we discuss the current knowledge about B-ALL protective niches and the development of therapies targeting the crosstalk between leukemic cells and their microenvironment.

Dynamic trafficking and turnover of JAM-C is essential for endothelial cell migration.

Junctional complexes between endothelial cells form a dynamic barrier that hinders passive diffusion of blood constituents into interstitial tissues. Remodelling of junctions is an essential process during leukocyte trafficking, vascular permeability, and angiogenesis. However, for many junctional proteins, the mechanisms of junctional remodelling have yet to be determined. Here, we used receptor mutagenesis, horseradish peroxidase (HRP), and ascorbate peroxidase 2 (APEX-2) proximity labelling, alongside light and electron microscopy (EM), to map the intracellular trafficking routes of junctional adhesion molecule-C (JAM-C). We found that JAM-C cotraffics with receptors associated with changes in permeability such as vascular endothelial cadherin (VE-Cadherin) and neuropilin (NRP)-1 and 2, but not with junctional proteins associated with the transmigration of leukocytes. Dynamic JAM-C trafficking and degradation are necessary for junctional remodelling during cell migration and angiogenesis. By identifying new potential trafficking machinery, we show that a key point of regulation is the ubiquitylation of JAM-C by the E3 ligase Casitas B-lineage lymphoma (CBL), which controls the rate of trafficking versus lysosomal degradation.

CD146 deficiency promotes plaque formation in a mouse model of atherosclerosis by enhancing RANTES secretion and leukocyte recruitment.

AIMS: The progression of atherosclerosis is based on the continued recruitment of leukocytes in the vessel wall. The previously described role of CD146 in leukocyte infiltration suggests an involvement for this adhesion molecule in the inflammatory response. In this study, we investigated the role of CD146 in leukocyte recruitment by using an experimental model of atherogenesis. METHODS AND RESULTS: The role of CD146 was explored in atherosclerosis by crossing CD146-/- mice with ApoE-/- mice. CD146 -/-/ApoE -/- and ApoE -/- mice were fed a Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. The arterial wall was significantly thicker in CD146-deficient mice. After 24 weeks of Western diet, a significant increase of atheroma in both total aortic lesion and aortic sinus of CD146-null mice was observed. In addition, atherosclerotic lesions were more inflammatory since plaques from CD146-deficient mice contained more neutrophils and macrophages. This was due to up-regulation of RANTES secretion by macrophages in CD146-deficient atherosclerotic arteries. This prompted us to further address the function of CD146 in leukocyte recruitment during acute inflammation by using a second experimental model of peritonitis induced by thioglycollate. Neutrophil recruitment was significantly increased in CD146-deficient mice 12 h after peritonitis induction and associated with higher RANTES levels in the peritoneal cavity. In CD146-null macrophages, we also showed that increased RANTES production was dependent on constitutive inhibition of the p38-MAPK signaling pathway. Finally, Maraviroc, a RANTES receptor antagonist, was able to reduce atherosclerotic lesions and neutrophilia in CD146-deficient mice to the same level as that found in ApoE -/- mice. CONCLUSIONS: Our data indicate that CD146 deficiency is associated with the upregulation of RANTES production and increased inflammation of atheroma, which could influence the atherosclerotic plaque fate. Thus, these data identify CD146 agonists as potential new therapeutic candidates for atherosclerosis treatment.

Nidogen-1 Contributes to the Interaction Network Involved in Pro-B Cell Retention in the Peri-sinusoidal Hematopoietic Stem Cell Niche.

In the bone marrow, CXCL12 and IL-7 are essential for B cell differentiation, whereas hematopoietic stem cell (HSC) maintenance requires SCF and CXCL12. Peri-sinusoidal stromal (PSS) cells are the main source of IL-7, but their characterization as a pro-B cell niche remains limited. Here, we characterize pro-B cell supporting stromal cells and decipher the interaction network allowing pro-B cell retention. Preferential contacts are found between pro-B cells and PSS cells, which homogeneously express HSC and B cell niche genes. Furthermore, pro-B cells are frequently located in the vicinity of HSCs in the same niche. Using an interactome bioinformatics pipeline, we identify Nidogen-1 as essential for pro-B cell retention in the peri-sinusoidal niche as confirmed in Nidogen-1-/- mice. Finally, human pro-B cells and hematopoietic progenitors are observed close to similar IL-7+ stromal cells. Thus, a multispecific niche exists in mouse and human supporting both early progenitors and committed hematopoietic lineages.

Circadian Expression of Migratory Factors Establishes Lineage-Specific Signatures that Guide the Homing of Leukocyte Subsets to Tissues.

The number of leukocytes present in circulation varies throughout the day, reflecting bone marrow output and emigration from blood into tissues. Using an organism-wide circadian screening approach, we detected oscillations in pro-migratory factors that were distinct for specific vascular beds and individual leukocyte subsets. This rhythmic molecular signature governed time-of-day-dependent homing behavior of leukocyte subsets to specific organs. Ablation of BMAL1, a transcription factor central to circadian clock function, in endothelial cells or leukocyte subsets demonstrated that rhythmic recruitment is dependent on both microenvironmental and cell-autonomous oscillations. These oscillatory patterns defined leukocyte trafficking in both homeostasis and inflammation and determined detectable tumor burden in blood cancer models. Rhythms in the expression of pro-migratory factors and migration capacities were preserved in human primary leukocytes. The definition of spatial and temporal expression profiles of pro-migratory factors guiding leukocyte migration patterns to organs provides a resource for the further study of the impact of circadian rhythms in immunity.

Murine Bone Marrow Niches from Hematopoietic Stem Cells to B Cells.

After birth, the development of hematopoietic cells occurs in the bone marrow. Hematopoietic differentiation is finely tuned by cell-intrinsic mechanisms and lineage-specific transcription factors. However, it is now clear that the bone marrow microenvironment plays an essential role in the maintenance of hematopoietic stem cells (HSC) and their differentiation into more mature lineages. Mesenchymal and endothelial cells contribute to a protective microenvironment called hematopoietic niches that secrete specific factors and establish a direct contact with developing hematopoietic cells. A number of recent studies have addressed in mouse models the specific molecular events that are involved in the cellular crosstalk between hematopoietic subsets and their niches. This has led to the concept that hematopoietic differentiation and commitment towards a given hematopoietic pathway is a dynamic process controlled at least partially by the bone marrow microenvironment. In this review, we discuss the evolving view of murine hematopoietic⁻stromal cell crosstalk that is involved in HSC maintenance and commitment towards B cell differentiation.

Lack of junctional adhesion molecule (JAM)-B ameliorates experimental autoimmune encephalomyelitis.

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) autoaggressive CD4+ T cells cross the blood-brain barrier (BBB) and cause neuroinflammation. Therapeutic targeting of CD4+ T-cell trafficking into the CNS by blocking α4-integrins has proven beneficial for the treatment of MS but comes with associated risks, probably due to blocking CD8+ T cell mediated CNS immune surveillance. Our recent observations show that CD8+ T cells also rely on α4ß1-integrins to cross the BBB. Besides vascular cell adhesion molecule-1 (VCAM-1), we identified junctional adhesion molecule-B (JAM-B) as a novel vascular α4ß1-integrin ligand involved in CD8+ T-cell migration across the BBB. This prompted us to investigate, if JAM-B also mediates CD4+ T-cell migration across the BBB. We first ensured that encephalitogenic T cells can bind to JAM-B in vitro and next compared EAE pathogenesis in JAM-B-/- C57BL/6J mice and their wild-type littermates. Following immunization with MOGaa35-55 peptide, JAM-B-/- mice developed ameliorated EAE compared to their wild-type littermates. At the same time, we isolated higher numbers of CD45+ infiltrating immune cells from the CNS of JAM-B-/- C57BL/6J mice suffering from EAE. Immunofluorescence staining revealed that the majority of CD45+ inflammatory cells accumulated in the leptomeningeal and perivascular spaces of the CNS behind the BBB but do not gain access to the CNS parenchyma. Trapping of CNS inflammatory cells was not due to increased inflammatory cell proliferation. Neither a loss of BBB integrity or BBB polarity potentially affecting local chemokine gradients nor a lack of focal gelatinase activation required for CNS parenchymal immune cell entry across the glia limitans could be detected in JAM-B-/- mice. Lack of a role for JAM-B in the effector phase of EAE was supported by the observation that we did not detect any role for JAM-B in EAE pathogenesis, when EAE was elicited by in vitro activated MOG aa35-55-specific CD4+ effector T cells. On the other hand, we also failed to demonstrate any role of JAM-B in in vivo priming, proliferation or polarization of MOGaa35-55-specific CD4+ T cells in peripheral immune organs. Finally, our study excludes expression of and thus a role for JAM-B on peripheral and CNS infiltrating myeloid cells. Taken together, although endothelial JAM-B is not required for immune cell trafficking across the BBB in EAE, in its absence accumulation of inflammatory cells mainly in CNS leptomeningeal spaces leads to amelioration of EAE.

Junctional adhesion molecules JAM-B and JAM-C promote autoimmune-mediated liver fibrosis in mice.

Fibrosis remains a serious health concern in patients with chronic liver disease. We recently reported that chemically induced chronic murine liver injury triggers increased expression of junctional adhesion molecules (JAMs) JAM-B and JAM-C by endothelial cells and de novo synthesis of JAM-C by hepatic stellate cells (HSCs). Here, we demonstrate that biopsies of patients suffering from primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) or autoimmune hepatitis (AIH) display elevated levels of JAM-C on portal fibroblasts (PFs), HSCs, endothelial cells and cholangiocytes, whereas smooth muscle cells expressed JAM-C constitutively. Therefore, localization and function of JAM-B and JAM-C were investigated in three mouse models of autoimmune-driven liver inflammation. A PBC-like disease was induced by immunization with 2-octynoic acid-BSA conjugate, which resulted in the upregulation of both JAMs in fibrotic portal triads. Analysis of a murine model of PSC revealed a role of JAM-C in PF cell-cell adhesion and contractility. In mice suffering from AIH, endothelial cells increased JAM-B level and HSCs and capsular fibroblasts became JAM-C-positive. Most importantly, AIH-mediated liver fibrosis was reduced in JAM-B-/- mice or when JAM-C was blocked by soluble recombinant JAM-C. Interestingly, loss of JAM-B/JAM-C function had no effect on leukocyte infiltration, suggesting that the well-documented function of JAMs in leukocyte recruitment to inflamed tissue was not effective in the tested chronic models. This might be different in patients and may even be complicated by the fact that human leukocytes express JAM-C. Our findings delineate JAM-C as a mediator of myofibroblast-operated contraction of the liver capsule, intrahepatic vasoconstriction and bile duct stricture. Due to its potential to interact heterophilically with endothelial JAM-B, JAM-C supports also HSC/PF mural cell function. Together, these properties allow JAM-B and JAM-C to actively participate in vascular remodeling associated with liver/biliary fibrosis and suggest them as valuable targets for anti-fibrosis therapies.