Bone marrow recovery by morphometry during induction chemotherapy for acute lymphoblastic leukemia in children

Bone marrow architecture is grossly distorted at the diagnosis of ALL and details of the morphological changes that accompany response to Induction chemotherapy have not been reported before. While marrow aspirates are widely used to assess initial response to ALL therapy and provide some indications, we have enumerated marrow components using morphometric analysis of trephine samples with the aim of achieving a greater understanding of changes in bone marrow niches. Morphometric analyses were carried out in the bone marrow trephine samples of 44 children with ALL, using a NanoZoomer HT digital scanner. Diagnostic samples were compared to those of 32 control patients with solid tumors but without marrow involvement. Samples from patients with ALL had significantly increased fibrosis and the area occupied by bony trabeculae was lower than in controls. Cellularity was higher in ALL samples due to leukemic infiltration while the percentage of normal elements such as megakaryocytes, adipocytes, osteoblasts and osteoclasts were all significantly lower. During the course of Induction therapy, there was a decrease in the cellularity of ALL samples at day 15 of therapy with a further decrease at the end of Induction and an increase in the area occupied by adipocytes and the width of sinusoids. Reticulin fibrosis decreased throughout Induction. Megakaryocytes increased, osteoblasts and osteoclasts remained unchanged. No correlation was found between clinical presentation, early response to treatment and morphological changes. Our results provide a morphological background to further studies of bone marrow stroma in ALL

mTORC1 plays an important role in osteoblastic regulation of B-lymphopoiesis

Skeletal osteoblasts are important regulators of B-lymphopoiesis, serving as a rich source of factors such as CXCL12 and IL-7 which are crucial for B-cell development. Recent studies from our laboratory and others have shown that deletion of Rptor, a unique component of the mTORC1 nutrient-sensing complex, early in the osteoblast lineage development results in defective bone development in mice. In this study, we now demonstrate that mTORC1 signalling in pre-osteoblasts is required for normal B-lymphocyte development in mice. Targeted deletion of Rptor in osterix-expressing pre-osteoblasts (Rptor; ob; -/-; ) leads to a significant reduction in the number of B-cells in the bone marrow, peripheral blood and spleen at 4 and 12 weeks of age. Rptor; ob; -/-; mice also exhibit a significant reduction in pre-B and immature B-cells in the BM, indicative of a block in B-cell development from the pro-B to pre-B cell stage. Circulating levels of IL-7 and CXCL12 are also significantly reduced in Rptor; ob; -/-; mice. Importantly, whilst Rptor-deficient osteoblasts are unable to support HSC differentiation to B-cells in co-culture, this can be rescued by the addition of exogenous IL-7 and CXCL12. Collectively, these findings demonstrate that mTORC1 plays an important role in extrinsic osteoblastic regulation of B-cell development

mTORC1 plays an important role in osteoblastic regulation of B-lymphopoiesis

Skeletal osteoblasts are important regulators of B-lymphopoiesis, serving as a rich source of factors such as CXCL12 and IL-7 which are crucial for B-cell development. Recent studies from our laboratory and others have shown that deletion of Rptor, a unique component of the mTORC1 nutrient-sensing complex, early in the osteoblast lineage development results in defective bone development in mice. In this study, we now demonstrate that mTORC1 signalling in pre-osteoblasts is required for normal B-lymphocyte development in mice. Targeted deletion of Rptor in osterix-expressing pre-osteoblasts (Rptor; ob; -/-; ) leads to a significant reduction in the number of B-cells in the bone marrow, peripheral blood and spleen at 4 and 12 weeks of age. Rptor; ob; -/-; mice also exhibit a significant reduction in pre-B and immature B-cells in the BM, indicative of a block in B-cell development from the pro-B to pre-B cell stage. Circulating levels of IL-7 and CXCL12 are also significantly reduced in Rptor; ob; -/-; mice. Importantly, whilst Rptor-deficient osteoblasts are unable to support HSC differentiation to B-cells in co-culture, this can be rescued by the addition of exogenous IL-7 and CXCL12. Collectively, these findings demonstrate that mTORC1 plays an important role in extrinsic osteoblastic regulation of B-cell development

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival

Extent: 14 p.The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.Daniel Thomas, Jason A. Powell, Benjamin D. Green, Emma F. Barry, Yuefang Ma, Joanna Woodcock, Stephen Fitter, Andrew C.W. Zannettino, Stuart M. Pitson, Timothy P. Hughes, Angel F. Lopez, Peter R. Shepherd, Andrew H. Wei, Paul G. Ekert and Mark A. Guthridg

Application of Autologous Bone Marrow Derived Mesenchymal Stem Cells to an Ovine Model of Growth Plate Cartilage Injury

Injury to growth plate cartilage in children can lead to bone bridge formation and result in bone growth deformities, a significant clinical problem currently lacking biological treatment. Mesenchymal stem/stromal cells (MSC) offer a promising therapeutic option for regeneration of damaged cartilage, due to their self renewing and multi-lineage differentiation attributes. Although some small animal model studies highlight the therapeutic potential of MSC for growth plate repair, translational research in large animal models, which more closely resemble the human condition, are lacking. Our laboratory has recently characterised MSCs derived from ovine bone marrow, and demonstrated these cells form cartilage-like tissue when transplanted within the gelatin sponge, Gelfoam, in vivo. In the current study, autologous bone marrow MSC were seeded into Gelfoam scaffold containing TGF-β1, and transplanted into a surgically created defect of the proximal ovine tibial growth plate. Examination of implants at 5 week post-operatively revealed transplanted autologous MSC failed to form new cartilage structure at the defect site, but contributed to an increase in formation of a dense fibrous tissue. Importantly, the extent of osteogenesis was diminished, and bone bridge formation was not accelerated due to transplantation of MSCs or the gelatin scaffold. The current study represents the first work that has utilised this ovine large animal model to investigate whether autologous bone marrow derived MSC can be used to initiate regeneration at the injured growth plate

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

Extent: 10p.INTRODUCTION: TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro. METHODS: TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry. RESULTS: TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts. CONCLUSIONS: The expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.Anak A. S. S. K. Dharmapatni, Malcolm D. Smith, Tania N. Crotti, Christopher A. Holding, Cristina Vincent, Helen M. Weedon, Andrew C. W. Zannettino, Timothy S. Zheng, David M. Findlay, Gerald J. Atkins and David R. Hayne

SAMSN1 is a tumor suppressor gene in multiple myeloma

Multiple myeloma (MM), a hematological malignancy characterized by the clonal growth of malignant plasma cells (PCs) in the bone marrow, is preceded by the benign asymptomatic condition, monoclonal gammopathy of undetermined significance (MGUS). Several genetic abnormalities have been identified as critical for the development of MM; however, a number of these abnormalities are also found in patients with MGUS, indicating that there are other, as yet unidentified, factors that contribute to the onset ofMMdisease. In this study, we identify a Samsn1 gene deletion in the 5TGM1/C57BL/KaLwRij murine model of myeloma. In addition, SAMSN1 expression is reduced in the malignant CD138+ PCs of patients with MM and this reduced expression correlates to total PC burden. We identify promoter methylation as a potential mechanismthrough which SAMSN1 expression is modulated in human myeloma cell lines.Notably, re-expression of Samsn1 in the 5TGM1murinePCline resulted in complete inhibition ofMMdisease development in vivo and decreased proliferation in stromal cell–PC co-cultures in vitro. This is the first study to identify deletion of a key gene in the C57BL/KaLwRij mice that also displays reduced gene expression in patients withMMand is therefore likely to play an integral role in MM disease development.Jacqueline E. Noll, Duncan R. Hewett, Sharon A. Williams, Kate Vandyke, Chung Kok, Luen B. To, and Andrew C.W. Zannettin

Large-scale exome-wide association analysis identifies loci for White Blood Cell Traits and Pleiotropy with Immune-Mediated Diseases

White blood cells play diverse roles in innate and adaptive immunity. Genetic association analyses of phenotypic variation in circulating white blood cell (WBC) counts from large samples of otherwise healthy individuals can provide insights into genes and biologic pathways involved in production, differentiation, or clearance of particular WBC lineages (myeloid, lymphoid) and also potentially inform the genetic basis of autoimmune, allergic, and blood diseases. We performed an exome array-based meta-analysis of total WBC and subtype counts (neutrophils, monocytes, lymphocytes, basophils, and eosinophils) in a multi-ancestry discovery and replication sample of ∼157,622 individuals from 25 studies. We identified 16 common variants (8 of which were coding variants) associated with one or more WBC traits, the majority of which are pleiotropically associated with autoimmune diseases. Based on functional annotation, these loci included genes encoding surface markers of myeloid, lymphoid, or hematopoietic stem cell differentiation (CD69, CD33, CD87), transcription factors regulating lineage specification during hematopoiesis (ASXL1, IRF8, IKZF1, JMJD1C, ETS2-PSMG1), and molecules involved in neutrophil clearance/apoptosis (C10orf54, LTA), adhesion (TNXB), or centrosome and microtubule structure/function (KIF9, TUBD1). Together with recent reports of somatic ASXL1 mutations among individuals with idiopathic cytopenias or clonal hematopoiesis of undetermined significance, the identification of a common regulatory 3 UTR variant of ASXL1 suggests that both germline and somatic ASXL1 mutations contribute to lower blood counts in otherwise asymptomatic individuals. These association results shed light on genetic mechanisms that regulate circulating WBC counts and suggest a prominent shared genetic architecture with inflammatory and autoimmune diseases