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.

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.

Examination of galectin-induced lattice formation on early B-cell development.

Galectin-1 (GAL1) is a pre-B cell receptor (pre-BCR) ligand that induces pre-BCR clustering and leads to efficient pre-B cell proliferation and differentiation in the bone marrow. To study pre-BCR-GAL1 interactions and its functional consequence on the early steps of the B cell development, we combine structural nuclear magnetic resonance (NMR) approaches and B cell biology techniques. NMR is applied to identify the residues involved in pre-BCR-GAL1 interactions by monitoring chemical shift perturbations when the complex is formed. This structural information is then used at the cellular level to target specifically the complex formation during GAL1-induced pre-BCR clustering and lattice formation, using immunofluorescence techniques. Moreover, an in vivo assay was set up to study the consequence of synapse formation on the early steps of B cell development.

Germinal center reentries of BCL2-overexpressing B cells drive follicular lymphoma progression.

It has recently been demonstrated that memory B cells can reenter and reengage germinal center (GC) reactions, opening the possibility that multi-hit lymphomagenesis gradually occurs throughout life during successive immunological challenges. Here, we investigated this scenario in follicular lymphoma (FL), an indolent GC-derived malignancy. We developed a mouse model that recapitulates the FL hallmark t(14;18) translocation, which results in constitutive activation of antiapoptotic protein B cell lymphoma 2 (BCL2) in a subset of B cells, and applied a combination of molecular and immunofluorescence approaches to track normal and t(14;18)(+) memory B cells in human and BCL2-overexpressing B cells in murine lymphoid tissues. BCL2-overexpressing B cells required multiple GC transits before acquiring FL-associated developmental arrest and presenting as GC B cells with constitutive activation-induced cytidine deaminase (AID) mutator activity. Moreover, multiple reentries into the GC were necessary for the progression to advanced precursor stages of FL. Together, our results demonstrate that protracted subversion of immune dynamics contributes to early dissemination and progression of t(14;18)(+) precursors and shapes the systemic presentation of FL patients.

Human t(14;18)positive germinal center B cells: a new step in follicular lymphoma pathogenesis?

Follicular lymphoma (FL) is a B-cell neoplasm resulting from the transformation of germinal center (GC) B cells. Although t(14;18) and ectopic B-cell lymphoma 2 (BCL2) expression constitute the genetic hallmark of FL, t(14;18)(pos) B cells bearing genotypic and phenotypic features of FL cells can be found in the blood of most healthy individuals. Nevertheless, the localization of these FL-like cells (FLLCs) in nonmalignant GC-rich tissues and the functional consequences of BCL2 overexpression have not been evaluated thus far. Among 85 reactive lymph node (RLN) samples, 14% were found to contain high levels of t(14;18) by quantitative polymerase chain reaction. In t(14;18)(hi) RLNs, CD20(pos)BCL2(pos)CD10(pos) FLLCs consistently accumulated within the GC, essentially as nonproliferative CXCR4(neg) centrocytes. Moreover, they displayed a reduced response to proliferative stimuli in vitro. Altogether, our findings provide new insights into in situ FLLC functional properties and suggest that these cells have not acquired the ultimate genetic events leading to FL transformation.

The BLNK adaptor protein has a nonredundant role in human B-cell differentiation.

BACKGROUND: Expression of the pre-B-cell receptor (pre-BCR) by pre-BII cells constitutes a crucial checkpoint in B-cell differentiation. Mutations that affect the pre-B-cell receptor result in early B-cell differentiation blockades that lead to primary B-cell immunodeficiencies. BLNK adaptor protein has a key role in the pre-B-cell receptor signaling cascade, as illustrated by the abnormal B-cell development in the 4 patients with BLNK gene defects reported to date. However, the BLNK protein's precise function in human B-cell differentiation has not been completely specified. METHODS: B-cell development, including IgVH and Vk chain repertoires analysis, was studied in the bone marrow of a new case of BLNK deficiency in vitro and in vivo. RESULTS: Here, we report on a patient with agammaglobulinemia, with a total absence of circulating B cells. We detected a homozygous mutation in BLNK, which leads to the complete abrogation of BLNK protein expression. In the bone marrow, we identified a severe differentiation blockade at the pre-BI- to pre-BII-cell transition. IgVH gene rearrangements and selection of the IgH repertoire were normal, whereas the patient's pre-BI cells showed very restricted usage of the IgVκ repertoire. Complementation of bone marrow progenitors from the patient with the BLNK gene and transplantation into NOD/SCID/γcko mice allowed the complete restoration of B-cell differentiation and a normal usage of the IgVκ genes.

The involvement of CD146 and its novel ligand Galectin-1 in apoptotic regulation of endothelial cells.

CD146 is a highly glycosylated junctional adhesion molecule, expressed on human vascular endothelial cells and involved in the control of vessel integrity. Galectin-1 is a lectin produced by vascular cells that can binds N- and O-linked oligosaccharides of cell membrane glycoproteins. Because both CD146 and Galectin-1 are involved in modulation of cell apoptosis, we hypothesized that Galectin-1 could interact with CD146, leading to functional consequences in endothelial cell apoptosis. We first characterized CD146 glycosylations and showed that it is mainly composed of N-glycans able to establish interactions with Galectin-1. We demonstrated a sugar-dependent binding of recombinant CD146 to Galectin-1 using both ELISA and Biacore assays. This interaction is direct, with a K(D) of 3.10(-7) M, and specific as CD146 binds to Galectin-1 and not to Galectin-2. Moreover, co-immunoprecipitation experiments showed that Galectin-1 interacts with endogenous CD146 that is highly expressed by HUVEC. We observed a Galectin-1-induced HUVEC apoptosis in a dose-dependent manner as demonstrated by Annexin-V/7AAD staining. Interestingly, both down-regulation of CD146 cell surface expression using siRNA and antibody-mediated blockade of CD146 increase this apoptosis. Altogether, our results identify Galectin-1 as a novel ligand for CD146 and this interaction protects, in vitro, endothelial cells against apoptosis induced by Galectin-1.

JAM-B regulates maintenance of hematopoietic stem cells in the bone marrow.

In adult mammals, hematopoietic stem cells (HSCs) reside in the bone marrow (BM) and are maintained in a quiescent and undifferentiated state through adhesive interactions with specialized microenvironmental niches. Although junctional adhesion molecule-C (JAM-C) is expressed by HSCs, its function in adult hematopoiesis remains elusive. Here, we show that HSCs adhere to JAM-B expressed by BM stromal cells in a JAM-C dependent manner. The interaction regulates the interplay between HSCs and BM stromal cells as illustrated by the decreased pool of quiescent HSCs observed in jam-b deficient mice. We further show that this is probably because of alterations of BM stromal compartments and changes in SDF-1α BM content in jam-b(-/-) mice, suggesting that JAM-B is an active player in the maintenance of the BM stromal microenvironment.

Galectin-1-expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow.

In the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7-producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1(+) reticular cells. We also determined that IL-7(+) and GAL1(+) cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7(+) to GAL1(+) supportive BM niches during their development.

Osteoclast activity modulates B-cell development in the bone marrow.

B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.

Galectin-1 is a powerful marker to distinguish chondroblastic osteosarcoma and conventional chondrosarcoma.

The clinical management of osteosarcoma differs significantly from that of chondrosarcoma; therefore, it is extremely important to diagnose these 2 types of bone tumor accurately. In the absence of a specific marker, differential diagnosis by histochemistry is sometimes impossible, especially between chondroblastic osteosarcoma and conventional chondrosarcoma. We analyzed 165 bone sarcomas by immunohistochemical staining of tissue microarrays for expression of the galectin-1 (GAL1) lectin and by Western blot experiments. We found that GAL1 was abundant in normal human osteoblasts from benign proliferations and in osteosarcomas, including chondroblastic osteosarcomas, but not in chondrosarcomas. There was a highly significant statistical difference in the percentage of stained cells (P < 10(-4)) and in the staining intensity (P < 10(-3)) of chondroblastic osteosarcomas compared to conventional chondrosarcomas. This discriminatory potential of GAL1 staining for osteosarcoma-derived tumors was confirmed by Western blotting. We propose a diagnostic test for bone tumors that takes into account the optimal discriminative values for the percentage of cells stained and the intensity of staining. The positive and negative predictive values were 85.7% (trust interval of 63.7%-97%) and 90% (trust interval of 80%-95.9%), respectively, demonstrating the pertinence of the test. Altogether, our data indicate that GAL1 is a powerful diagnostic marker that distinguishes chondroblastic osteosarcomas from conventional chondrosarcomas.

Conditional TPM3-ALK and NPM-ALK transgenic mice develop reversible ALK-positive early B-cell lymphoma/leukemia.

NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) and TPM3-ALK (nonmuscular tropomyosin 3-anaplastic lymphoma kinase) are oncogenic tyrosine kinases implicated in the pathogenesis of human ALK-positive lymphoma. We report here the development of novel conditional mouse models for ALK-induced lymphomagenesis, with the use of the tetracycline regulatory system under the control of the EmuSRalpha enhancer/promoter. The expression of either oncogene resulted in the arrest of the differentiation of early B cells and lymphomagenesis. We also observed the development of skin keratoacanthoma lesions, probably because of aberrant ALK expression in keratinocytes. The inactivation of the ALK oncogene on doxycycline treatment was sufficient to induce sustained regression of both hematopoietic tumors and skin disease. Importantly, treatment with the specific ALK inhibitor (PF-2341066) also reversed the pathologic states, showing the value of these mouse models for the validation of ALK tyrosine kinase inhibitors. Thus, our results show (1) that NPM-ALK and TPM3-ALK oncogenes are sufficient for lymphoma/leukemia development and required for tumor maintenance, hence validating ALK as potentially effective therapeutic target; and (2) for the first time, in vivo, the equal tumorigenic potential of the NPM-ALK and TPM3-ALK oncogenic tyrosine kinases. Our models offer a new tool to investigate in vivo the molecular mechanisms associated with ALK-induced lymphoproliferative disorders.

Follicular lymphoma-like B cells in healthy individuals: a novel intermediate step in early lymphomagenesis.

Follicular lymphoma is one of the most common adult lymphoma, and remains virtually incurable despite its relatively indolent nature. t(14;18)(q32;q21) translocation, the genetic hallmark and early initiating event of follicular lymphoma (FL) pathogenesis, is also present at low frequency in the peripheral blood of healthy individuals. It has long been assumed that in healthy individuals t(14;18) is carried by circulating quiescent naive B cells, where its oncogenic potential would be restrained. Here, we question this current view and demonstrate that in healthy individuals, t(14;18) is actually carried by an expanding population of atypical B cells issued from germinal centers, displaying genotypic and phenotypic features of FL, and prone to constitute potent premalignant FL niches. These findings strongly impact both on the current understanding of disease progression and on the proper handling of t(14;18) frequency in blood as a potential early biomarker for lymphoma.

Clustering of pre-B cell integrins induces galectin-1-dependent pre-B cell receptor relocalization and activation.

Interactions between B cell progenitors and bone marrow stromal cells are essential for normal B cell differentiation. We have previously shown that an immune developmental synapse is formed between human pre-B and stromal cells in vitro, leading to the initiation of signal transduction from the pre-BCR. This process relies on the direct interaction between the pre-BCR and the stromal cell-derived galectin-1 (GAL1) and is dependent on GAL1 anchoring to cell surface glycosylated counterreceptors, present on stromal and pre-B cells. In this study, we identify alpha(4)beta(1) (VLA-4), alpha(5)beta(1) (VLA-5), and alpha(4)beta(7) integrins as major GAL1-glycosylated counterreceptors involved in synapse formation. Pre-B cell integrins and their stromal cell ligands (ADAM15/fibronectin), together with the pre-BCR and GAL1, form a homogeneous lattice at the contact area between pre-B and stromal cells. Moreover, integrin and pre-BCR relocalizations into the synapse are synchronized and require actin polymerization. Finally, cross-linking of pre-B cell integrins in the presence of GAL1 is sufficient for driving pre-BCR recruitment into the synapse, leading to the initiation of pre-BCR signaling. These results suggest that during pre-B/stromal cell synapse formation, relocalization of pre-B cell integrins mediated by their stromal cell ligands drives pre-BCR clustering and activation, in a GAL1-dependent manner.

The chemosensitivity to therapy of childhood early B acute lymphoblastic leukemia could be determined by the combined expression of CD34, SPI-B and BCR genes.

We have identified genes differentially expressed in childhood early B acute lymphoblastic leukemia at diagnosis, according to chemosensitivity. Chemosensitive (M1) and chemoresistant (M3) patients present <5% and >25% of residual leukemic blasts at 21 days of treatment, respectively. The expression profiles of 4205 genes for 32 patients included in the FRALLE93 protocol have been determined using microarray. From differential analysis, CD34, SPI-B and BCR distinguished M1 from M3 patients using microarray and RT-PCR data. Linear discriminant analysis (LDA) and cross-validation show that the combined expression of these three genes classify and predict correctly around 90% and 80% of patients, respectively.