POT1a deficiency in mesenchymal niches perturbs B-lymphopoiesis.

Protection of telomeres 1a (POT1a) is a telomere binding protein. A decrease of POT1a is related to myeloid-skewed haematopoiesis with ageing, suggesting that protection of telomeres is essential to sustain multi-potency. Since mesenchymal stem cells (MSCs) are a constituent of the hematopoietic niche in bone marrow, their dysfunction is associated with haematopoietic failure. However, the importance of telomere protection in MSCs has yet to be elucidated. Here, we show that genetic deletion of POT1a in MSCs leads to intracellular accumulation of fatty acids and excessive ROS and DNA damage, resulting in impaired osteogenic-differentiation. Furthermore, MSC-specific POT1a deficient mice exhibited skeletal retardation due to reduction of IL-7 producing bone lining osteoblasts. Single-cell gene expression profiling of bone marrow from POT1a deficient mice revealed that B-lymphopoiesis was selectively impaired. These results demonstrate that bone marrow microenvironments composed of POT1a deficient MSCs fail to support B-lymphopoiesis, which may underpin age-related myeloid-bias in haematopoiesis.

Pan-PI3Ki targets multiple B-ALL microenvironment interactions that fuel systemic and CNS relapse.

The majority of adult patients with acute lymphoblastic leukemia (ALL) suffer relapse, and in patients with central nervous system (CNS) metastasis, prognosis is particularly poor. We recently demonstrated a novel route of ALL CNS metastasis dependent on PI3Kδ regulation of the laminin receptor integrin α6. B-ALL cells did not, however, rely on PI3Kδ signaling for growth. Here we show that broad targeting of PI3K isoforms can induce growth arrest in B-ALL, reducing systemic disease burden in mice treated with a single agent pan-PI3Ki, copanlisib. Moreover, we show that cellular stress activates PI3K/Akt-dependent survival pathways in B-ALL, exposing their vulnerability to PI3Kδ and pan-PI3Ki. The addition of a brief course of copanlisib to chemotherapy delivered the combined benefits of increased survival, decreased systemic disease, and reduced CNS metastasis. These data suggest the promising, multifaceted potential of pan-PI3Ki for B-ALL CNS prophylaxis, systemic disease control, and chemosensitization.

Mitochondrial Protein Synthesis Is Essential for Terminal Differentiation of CD45- TER119-Erythroid and Lymphoid Progenitors.

p32/C1qbp regulates mitochondrial protein synthesis and is essential for oxidative phosphorylation in mitochondria. Although dysfunction of p32/C1qbp impairs fetal development and immune responses, its role in hematopoietic differentiation remains unclear. Here, we found that mitochondrial dysfunction affected terminal differentiation of newly identified erythroid/B-lymphoid progenitors among CD45- Ter119- CD31- triple-negative cells (TNCs) in bone marrow. Hematopoietic cell-specific genetic deletion of p32/C1qbp (p32cKO) in mice caused anemia and B-lymphopenia without reduction of hematopoietic stem/progenitor cells. In addition, p32cKO mice were susceptible to hematopoietic stress with delayed recovery from anemia. p32/C1qbp-deficient CD51- TNCs exhibited impaired mitochondrial oxidation that consequently led to inactivation of mTORC1 signaling, which is essential for erythropoiesis. These findings uncover the importance of mitochondria, especially at the stage of TNCs during erythropoiesis, suggesting that dysregulation of mitochondrial protein synthesis is a cause of anemia and B-lymphopenia with an unknown pathology.

[Central nervous system involvement in acute lymphoblastic leukemia].

It is well known that acute lymphoblastic leukemia (ALL) cells can invade the central nervous system (CNS), but the underlying mechanism of such invasion is still unclear. We discovered the direct routes taken by ALL cells when migrating into CNS in ALL model mice. We observed that ALL cells migrate along the external surface of vessels that pass directly between the vertebral or calvarial bone marrow and the subarachnoid space. The basement membrane of these bridging vessels is enriched in laminin. The laminin is recognized by integrin α6, which is expressed by ALL cells. The interaction between integrin α6 and laminin mediated the invasion of ALL cells. Furthermore, the expression of integrin α6 depends on PI3Kδ activity. Mice with ALL xenografts were treated with a PI3Kδ inhibitor, which decreased integrin α6 expression on ALL cells. This resulted in significant reductions in blast counts in the cerebrospinal fluid and in CNS disease symptoms. Our data suggest that the PI3Kδ inhibitor has potential to prevent CNS involvement in ALL.

Leukaemia hijacks a neural mechanism to invade the central nervous system.

Acute lymphoblastic leukaemia (ALL) has a marked propensity to metastasize to the central nervous system (CNS). In contrast to brain metastases from solid tumours, metastases of ALL seldom involve the parenchyma but are isolated to the leptomeninges, which is an infrequent site for carcinomatous invasion. Although metastasis to the CNS occurs across all subtypes of ALL, a unifying mechanism for invasion has not yet been determined. Here we show that ALL cells in the circulation are unable to breach the blood-brain barrier in mice; instead, they migrate into the CNS along vessels that pass directly between vertebral or calvarial bone marrow and the subarachnoid space. The basement membrane of these bridging vessels is enriched in laminin, which is known to coordinate pathfinding of neuronal progenitor cells in the CNS. The laminin receptor α6 integrin is expressed in most cases of ALL. We found that α6 integrin-laminin interactions mediated the migration of ALL cells towards the cerebrospinal fluid in vitro. Mice with ALL xenografts were treated with either a PI3Kδ inhibitor, which decreased α6 integrin expression on ALL cells, or specific α6 integrin-neutralizing antibodies and showed significant reductions in ALL transit along bridging vessels, blast counts in the cerebrospinal fluid and CNS disease symptoms despite minimally decreased bone marrow disease burden. Our data suggest that α6 integrin expression, which is common in ALL, allows cells to use neural migratory pathways to invade the CNS.

Multiple myeloma cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-ß/Smad pathway activation.

The emergence of new molecular targeting agents has improved the prognosis of patients with multiple myeloma (MM). However, MM remains incurable because MM stem cells are likely resistant to these agents. Thus, it is important to further investigate the biology of MM stem cells, which reside in the hypoxic bone marrow niche. In this study, we established and investigated the characteristics of hypoxia-adapted MM (HA-MM) cells, which could proliferate for more than six months under hypoxic conditions (1% O2). The G0 fraction of HA-MM cells was larger than that of parental MM cells under normoxic conditions (20% O2). HA-MM cells possess enhanced tumorigenicity in primary and secondary transplantation studies. HA-MM cells also exhibited increased mRNA levels of stem cell markers and an enhanced self-renewal ability, and thus demonstrated characteristics of MM stem cells. These cells overexpressed phosphorylated Smad2, and treatment with a transforming growth factor (TGF)-ß/Smad signaling inhibitor decreased their clonogenicity in a replating assay. In conclusion, MM cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-ß/Smad pathway activation.

Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles.

Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34(+) hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation.

The role of growth differentiation factor 15 in the pathogenesis of primary myelofibrosis.

Growth differentiation factor 15 (GDF15) is a pleiotropic cytokine that belongs to the transforming growth factor-ß superfamily. Elevated serum concentrations of this cytokine have been reported in patients with various malignancies. To assess the potential roles of GDF15 in hematologic malignancies, we measured its serum levels in patients with these diseases. We found that serum GDF15 levels were elevated in almost all these patients, particularly in patients with primary myelofibrosis (PMF). Immunohistochemical staining of bone marrow (BM) specimens revealed that GDF15 was strongly expressed by megakaryocytes, which may be sources of increased serum GDF15 in PMF patients. Therefore, we further assessed the contribution of GDF15 to the pathogenesis of PMF. Recombinant human (rh) GDF15 enhanced the growth of human BM mesenchymal stromal cells (BM-MSCs), and it enhanced the potential of these cells to support human hematopoietic progenitor cell growth in a co-culture system. rhGDF15 enhanced the growth of human primary fibroblasts, but it did not affect their expression of profibrotic genes. rhGDF15 induced osteoblastic differentiation of BM-MSCs in vitro, and pretreatment of BM-MSCs with rGDF15 enhanced the induction of bone formation in a xenograft mouse model. These results suggest that serum levels of GDF15 in PMF are elevated, that megakaryocytes are sources of this cytokine in BM, and that GDF15 may modulate the pathogenesis of PMF by enhancing proliferation and promoting osteogenic differentiation of BM-MSCs.

Isolation of mesenchymal stromal/stem cells from small-volume umbilical cord blood units that do not qualify for the banking system.

The clinical application of mesenchymal stromal/stem cells (MSCs) has been extensively explored. In this study, we examined the availability of freshly donated umbilical cord blood (UCB) units that do not qualify for the Japanese banking system for transplantation because of their small volume as a source of MSCs. Forty-five UCB units were used. The median volume of each UCB unit and number of nucleated cells per unit were 40 mL and 5.39 × 10(8), respectively. MSCs were successfully isolated from 18 of 45 units (40 %). The MSC isolation rate was not affected by cell processing method or the interval between delivery and cell processing. The volume of the UCB unit and the mononuclear cell count were predictive factors of the MSC isolation rate. MSCs were effectively isolated by selecting UCB units with a volume of ≥54 mL and containing ≥1.28 × 10(8) mononuclear cells, yielding a MSC isolation rate of >70 %. UCB-derived MSCs were similar to bone marrow-derived MSCs in terms of their morphology, surface marker expression, and differentiation potential, apart from adipogenesis. Our data indicate that UCB units that are currently discarded due to inadequate volume should be reconsidered as a source of MSCs using the well-established UCB banking system.

Parathyroid hormone enhances hematopoietic expansion via upregulation of cadherin-11 in bone marrow mesenchymal stromal cells.

Parathyroid hormone (PTH) stimulates hematopoiesis in mouse models. The involvement of osteoblasts in this process has been well investigated; however, the effects of PTH on human hematopoiesis and bone marrow mesenchymal stromal cells (BM-MSCs) are unclear. Here, we show that BM-MSCs contribute to the hematopoiesis-stimulating effects of PTH via upregulation of cadherin-11 (CDH11). When culture-expanded human BM-MSCs were stimulated with PTH, their ability to expand cocultured CD34(+) hematopoietic progenitor cells (HPCs) was enhanced. Furthermore, when PTH-treated BM-MSCs were subcutaneously implanted into NOD/SCID mice, the induction of hematopoietic cells was enhanced. Culture-expanded human BM-MSCs expressed CDH11, and the level of CDH11 expression increased following PTH stimulation. Depletion of CDH11 expression in BM-MSCs using small interfering RNA abolished the enhancement of HPC expansion by PTH-treated BM-MSCs. In lethally irradiated mice that underwent BM transplantation, CDH11 expression in BM-MSCs was higher and survival was better in PTH-treated mice than in control mice. The number of hematopoietic cells in BM and the number of red blood cells in peripheral blood were higher in PTH-treated mice than in control mice. Our results demonstrate that PTH stimulates hematopoiesis through promoting the upregulation of CDH11 expression in BM-MSCs, at least in part. PTH treatment may be an effective strategy to enhance the ability of BM-MSCs to support hematopoiesis.

CCAAT/enhancer-binding protein ß expressed by bone marrow mesenchymal stromal cells regulates early B-cell lymphopoiesis.

The transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) regulates the differentiation of a variety of cell types. Here, the role of C/EBPß expressed by bone marrow mesenchymal stromal cells (BMMSCs) in B-cell lymphopoiesis was examined. The size of the precursor B-cell population in bone marrow was reduced in C/EBPß-knockout (KO) mice. When bone marrow cells from C/EBPß-KO mice were transplanted into lethally irradiated wild-type (WT) mice, which provide a normal bone marrow microenvironment, the size of the precursor B-cell population was restored to a level equivalent to that generated by WT bone marrow cells. In coculture experiments, BMMSCs from C/EBPß-KO mice did not support the differentiation of WT c-Kit(+) Sca-1(+) Lineage(-) hematopoietic stem cells (KSL cells) into precursor B cells, whereas BMMSCs from WT mice did. The impaired differentiation of KSL cells correlated with the reduced production of CXCL12/stromal cell-derived factor-1 by the cocultured C/EBPß-deficient BMMSCs. The ability of C/EBPß-deficient BMMSCs to undergo osteogenic and adipogenic differentiation was also defective. The survival of leukemic precursor B cells was poorer when they were cocultured with C/EBPß-deficient BMMSCs than when they were cocultured with WT BMMSCs. These results indicate that C/EBPß expressed by BMMSCs plays a crucial role in early B-cell lymphopoiesis.

Chimerism of bone marrow mesenchymal stem/stromal cells in allogeneic hematopoietic cell transplantation: is it clinically relevant?

Multipotent mesenchymal stem/stromal cells (MSCs) have been extensively used as a transplantable cell source for regenerative medicine and immunomodulatory therapy. Specifically in allogeneic hematopoietic stem cell transplantation (HSCT), co-transplantation or post-transplant infusion of MSCs derived from bone marrow (BM) of non-self donors has been implicated in accelerating hematopoietic recovery, ameliorating graft-vs.-host disease, and promoting tissue regeneration. However, irrespective of the use of MSC co-administration, post-transplant chimerism of BM-derived MSCs after allogeneic HSCT has been reported to remain of host origin, suggesting that the infused donor MSCs are immunologically rejected or not capable of long-term engraftment in the host microenvironment. Also, hematopoietic cell allografts currently used for HSCT do not seem to contain sufficient amount of MSCs or their precursors to reconstitute host BM microenvironment. Since the toxic conditioning employed in allo-HSCT may impair the function of host MSCs to maintain hematopoietic/regenerative stem cell niches and to provide a local immunomodulatory milieu, we propose that new directions for enhancing immunohematopoietic reconstitution and tissue repair after allogeneic HSCT include the development of strategies to support functional replenishment of residual host MSCs or to support more efficient engraftment of infused donor MSCs. Future areas of research should include in vivo tracking of infused MSCs and detection of their microchimeric presence in extra-marrow sites as well as in BM.

C/EBPß is involved in the amplification of early granulocyte precursors during candidemia-induced "emergency" granulopoiesis.

Granulopoiesis is tightly regulated to meet host demands during both "steady-state" and "emergency" situations, such as infections. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) plays critical roles in emergency granulopoiesis, but the precise developmental stages in which C/EBPß is required are unknown. In this study, a novel flow cytometric method was developed that successfully dissected mouse bone marrow cells undergoing granulopoiesis into five distinct subpopulations (#1-5) according to their levels of c-Kit and Ly-6G expression. After the induction of candidemia, rapid mobilization of mature granulocytes and an increase in early granulocyte precursors accompanied by cell cycle acceleration was followed by a gradual increase in granulocytes originating from the immature populations. Upon infection, C/EBPß was upregulated at the protein level in all the granulopoietic subpopulations. The rapid increase in immature subpopulations #1 and #2 observed in C/EBPß knockout mice at 1 d postinfection was attenuated. Candidemia-induced cell cycle acceleration and proliferation of hematopoietic stem/progenitors were also impaired. Taken together, these data suggest that C/EBPß is involved in the efficient amplification of early granulocyte precursors during candidemia-induced emergency granulopoiesis.

Identification of IL-7-producing cells in primary and secondary lymphoid organs using IL-7-GFP knock-in mice.

IL-7 is a cytokine crucial for development and maintenance of lymphocytes and other hematopoietic cells. However, how IL-7-expressing cells are distributed in lymphoid organs is not well known. To address this question, we established and analyzed IL-7-GFP knock-in mice. Thymic epithelial cells (TECs) expressed high GFP levels in the cortex and medulla, as detected with an anti-GFP Ab. Thymic mesenchymal cells also expressed GFP. Flow cytometry analysis suggested that cortical TECs expressed higher GFP levels than did medullary TECs. In bone marrow, immunohistochemistry indicated high levels of GFP in many VCAM-1(+) mesenchymal stromal cells and in some VCAM-1(-) cells. Additionally, half of the VCAM-1(+)CD31(-) stromal cells and some platelet-derived growth factor receptor α(+) stromal cells were GFP(+), as detected by flow cytometry. Moreover, we detected GFP expression in fibroblastic reticular cells in the T cell zone and cortical ridge of lymph nodes. Remarkably, lymphatic endothelial cells (LECs) expressed GFP at high levels within the lymph node medulla, skin epidermis, and intestinal tissues. Additionally, we detected abundant IL-7 transcripts in isolated LECs, suggesting that LECs produce IL-7, a heretofore unknown finding. Furthermore, GFP is expressed in a subpopulation of intestinal epithelial cells, and that expression was markedly upregulated in a dextran sulfate sodium-induced acute colitis model. Overall, IL-7-GFP knock-in mice serve as a unique and powerful tool to examine the identity and distribution of IL-7-expressing cells in vivo.

Rapid automated detection of ABL kinase domain mutations in imatinib-resistant patients.

ABL tyrosine kinase inhibitor (TKI), imatinib is used for BCR-ABL(+) leukemias. We developed an automatic method utilizing guanine-quenching probes (QP) to detect 17 kinds of mutations frequently observed in imatinib-resistance. Results were obtained from 100µL of whole blood within 90min by this method. Detected mutations were almost identical between QP method and direct sequencing. Furthermore, the mutation-biased PCR (MBP) was added to the QP method to increase sensitivity, resulting earlier detection of T315I mutation which was insensitive to any ABL TKIs. Thus, the QP and MBP-QP may become useful methods for the management of ABL TKI-treated patients.

Growth inhibition of imatinib-resistant CML cells with the T315I mutation and hypoxia-adaptation by AV65--a novel Wnt/ß-catenin signaling inhibitor.

We investigated the effect of a novel Wnt/ß-catenin signaling inhibitor, AV65 on imatinib mesylate (IM)-sensitive and -resistant human chronic myeloid leukemia (CML) cells in vitro. AV65 inhibited the proliferation of various CML cell lines including T315I mutation-harboring cells. AV65 reduced the expression of ß-catenin in CML cells, resulting in the induction of apoptosis. Moreover, AV65 inhibited the proliferation of hypoxia-adapted primitive CML cells that overexpress ß-catenin. The combination of AV65 with IM had a synergistic inhibitory effect on the proliferation of CML cells. These findings suggest that AV65 could be a novel therapeutic agent for the treatment of CML.

Targeting the Wnt/ß-catenin signaling pathway in human cancers.

INTRODUCTION: The Wnt/ß-catenin signaling pathway plays a pivotal role in the regulation of cell growth, cell development and the differentiation of normal stem cells. Constitutive activation of the Wnt/ß-catenin signaling pathway is found in many human cancers, and is thus an attractive target for anti-cancer therapy. Specific inhibitors of this pathway have been keenly researched and developed. AREAS COVERED: This review discusses the potential of inhibiting the Wnt/ß-catenin signaling pathway, as a therapeutic approach for cancer, along with an overview of the development of specific inhibitors. EXPERT OPINION: Cancer stem cells (CSCs) play a significant role in the development and recurrence of several cancers, and Wnt/ß-catenin signaling is important for the proliferation of CSCs. Inhibition of Wnt/ß-catenin signaling is therefore a promising treatment approach. Progress has been made in the development of screening methods to identify Wnt/ß-catenin signaling inhibitors. Biomarker-based screening is an effective and promising method for the identification of compounds of interest.

Rakicidin A effectively induces apoptosis in hypoxia adapted Bcr-Abl positive leukemic cells.

Treatment with Abl tyrosine kinase inhibitors (TKI) drastically improves the prognosis of chronic myelogenous leukemia (CML) patients. However, quiescent CML cells are insensitive to TKI and can lead to relapse of the disease. Thus, research is needed to elucidate the properties of these quiescent CML cells, including their microenvironment, in order to effectively target them. Hypoxia is known to be a common feature of solid tumors that contributes to therapeutic resistance. Leukemic cells are also able to survive and proliferate in severely hypoxic environments. The hypoxic conditions in the bone marrow (BM) allow leukemic cells that reside there to become insensitive to cell death stimuli. To target leukemic cells in hypoxic conditions, we focused on the hypoxia-selective cytotoxin, Rakicidin A. A previous report showed that Rakicidin A, a natural product produced by the Micromonospora strain, induced hypoxia-selective cytotoxicity in solid tumors. Here, we describe Rakicidin A-induced cell death in hypoxia-adapted (HA)-CML cells with stem cell-like characteristics. Interestingly, apoptosis was induced via caspase-dependent and -independent pathways. In addition, treatment with Rakicidin A in combination with the TKI, imatinib, resulted in synergistic cytotoxicity against HA-CML cells. In conclusion, Rakicidin A is a promising compound for targeting TKI-resistant quiescent CML stem cells in the hypoxic BM environment.

Activity of the multitargeted kinase inhibitor, AT9283, in imatinib-resistant BCR-ABL-positive leukemic cells.

Despite promising clinical results from imatinib mesylate and second-generation ABL tyrosine kinase inhibitors (TKIs) for most BCR-ABL(+) leukemia, BCR-ABL harboring the mutation of threonine 315 to isoleucine (BCR-ABL/T315I) is not targeted by any of these agents. We describe the in vitro and in vivo effects of AT9283 (1-cyclopropyl-3[5-morpholin-4yl methyl-1H-benzomidazol-2-yl]-urea), a potent inhibitor of several protein kinases, including Aurora A, Aurora B, Janus kinase 2 (JAK2), JAK3, and ABL on diverse imatinib-resistant BCR-ABL(+) cells. AT9283 showed potent antiproliferative activity on cells transformed by wild-type BCR-ABL and BCR-ABL/T315I. AT9283 inhibited proliferation in a panel of BaF3 and human BCR-ABL(+) cell lines both sensitive and resistant to imatinib because of a variety of mechanisms. In BCR-ABL(+) cells, we confirmed inhibition of substrates of both BCR-ABL (signal transducer and activator of transcription-5) and Aurora B (histone H3) at physiologically achievable concentrations. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3/BCR-ABL, human BCR-ABL(+) cell lines, or primary patient samples expressing BCR-ABL/T315I or glutamic acid 255 to lysine, another imatinib-resistant mutation. These data together support further clinical investigation of AT9283 in patients with imatinib- and second-generation ABL TKI-resistant BCR-ABL(+) cells, including T315I.

Osteoclasts are involved in the maintenance of dormant leukemic cells.

Osteoclasts (OCs) are specialized cells for the resorption of bone matrix that have also been recently reported to be involved in the mobilization of hematopoietic progenitor cells. When Ba/F3 cells expressing wild-type bcr-abl were co-cultured with osteoblasts (OBs), OCs, and bone slices, their proliferation was significantly suppressed, and the Ki-67 negative population, which is believed to be in G(0) phase, was increased. The results of our in vitro experiments suggest that OCs could be involved in the maintenance of dormant leukemic cells in the bone marrow (BM) microenvironment through the release of soluble factors, one of which could be TGF-beta.