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Myeloid leukaemias share the common characteristics of being stem cell-derived clonal diseases, characterised by excessive proliferation of one or more myeloid lineage. Chronic myeloid leukaemia (CML) arises from a genetic alteration in a normal haemopoietic stem cell (HSC) giving rise to a leukaemic stem cell (LSC) within the bone marrow (BM) 'niche'. CML is characterised by the presence of the oncogenic tyrosine kinase fusion protein breakpoint cluster region-abelson murine leukaemia viral oncogene homolog 1 (BCR-ABL), which is responsible for driving the disease through activation of downstream signal transduction pathways. Recent evidence from our group and others indicates that important regulatory networks involved in establishing primitive and definitive haemopoiesis during development are reactivated in myeloid leukaemia, giving rise to an LSC population with altered self-renewal and differentiation properties. In this review, we explore the role the bone morphogenic protein (BMP) signalling plays in stem cell pluripotency, developmental haemopoiesis, HSC maintenance and the implication of altered BMP signalling on LSC persistence in the BM niche. Overall, we emphasise how the BMP and Wnt pathways converge to alter the Cdx-Hox axis and the implications of this in the pathogenesis of myeloid malignancies., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Telomere shortening, a well-known marker of aging and cellular stress, occurs under several conditions in the hematopoietic compartment, including aplastic anemia and following iatrogenic noxae. We decided to verify whether pathological telomere erosion also arises in restored Philadelphia-negative (Ph-negative) hematopoiesis following successful treatment of chronic myeloid leukemia (CML). Eighty-one CML patients in complete cytogenetic remission were compared to 76 age-matched healthy subjects. Myeloid cells of CML patients had shorter telomeres than controls (6521 bp vs 7233 bp, p<0.001). This difference was specific for the myeloid compartment, since it was not observed in lymphoid cells (6774 bp vs 6909 bp, p=0.620). Acquired Ph-negative cytogenetic abnormalities (p=0.010), lack of complete molecular remission (p=0.016) and age (p=0.013) were independent predictors of telomere shortening. Telomere dynamics were assessed over a median follow-up period of 22 months. We documented accelerated non-physiological ongoing telomere shortening in 17/59 CML patients (28%). Patients experiencing grade 2-4 hematological toxicity, during CML remission possessed significantly shorter telomeres compared to those lacking toxicity (p=0.005 for any toxicity, p=0.007 for anemia). CML patients suffer from significant and often ongoing telomere stress resulting in premature and selective aging of the myeloid compartment which might have long-term consequences on function and integrity of Ph-negative hematopoiesis., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Hedgehog (Hh) is a developmental signaling pathway in which Hh ligands bind Patched (Ptch), which relieves its inhibition of Smoothened (Smo), allowing the Gli family of transcription factors to translocate to the nucleus and activate Hh target genes. The role of Hh signaling in hematopoiesis is controversial and ill defined. Although some groups observed self-renewal defects with decreased replating and reduced efficiency of secondary murine transplants, other groups reported no hematopoietic phenotypes, which may be related to the timing of Hh abrogation. In malignant hematopoiesis, most attention has been focused on the role of Hh signaling in chronic myeloid leukemia (CML), considered by many to be a stem cell disorder that bears the constitutively active BCR-ABL tyrosine kinase. Despite the elimination of most leukemia cells through BCR-ABL inhibition, most patients remain PCR positive, suggesting that the putative CML stem cell may be resistant to kinase antagonism. Groups are now exploring the Hh pathway as an alternate pathway supporting CML stem cell survival. Knockdown or inhibition of Smo abrogates or delays the appearance of CML in several in vitro and in vivo models. These data have lead to clinical trials using BCR-ABL kinase and novel Smo inhibitors in combination.

Bone marrow engraftment in the context of hematopoietic stem cell and progenitor (HSC/P) transplantation is based on the ability of intravenously administered cells to lodge in the medullary cavity and be retained in the appropriate marrow space, a process referred to as homing. It is likely that homing is a multistep process, encompassing a sequence of highly regulated events that mimic the migration of leukocytes to inflammatory sites. In leukocyte biology, this process includes an initial phase of tethering and rolling of cells to the endothelium via E- and P-selectins, firm adhesion to the vessel wall via integrins that appear to be activated in an "inside-out" fashion, transendothelial migration, and chemotaxis through the extracellular matrix (ECM) to the inflammatory nidus. For HSC/P, the cells appear to migrate to the endosteal space of the bone marrow. A second phase of engraftment involves the subsequent interaction of specific HSC/P surface receptors, such as alpha(4)beta(1) integrin receptors with vascular cell-cell adhesion molecule-1 and fibronectin in the ECM, and interactions with growth factors that are soluble, membrane, or matrix bound. We have utilized knockout and conditional knockout mouse lines generated by gene targeting to study the role of Rac1 and Rac2 in blood cell development and function. We have determined that Rac is activated via stimulation of CXCR4 by SDF-1, by adhesion via beta(1) integrins, and via stimulation of c-kit by the stem cell factor-all of which involved in stem cell engraftment. Thus Rac proteins are key molecular switches of HSC/P engraftment and marrow retention. We have defined Rac proteins as key regulators of HSC/P cell function and delineated key unique and overlapping functions of these two highly related GTPases in a variety of primary hematopoietic cell lineages in vitro and in vivo. Further, we have begun to define the mechanisms by which each GTPase leads to specific functions in these cells. These studies have led to important new understanding of stem cell bone marrow retention and trafficking in the peripheral circulation and to the development of a novel small molecule inhibitor that can modulate stem cell functions, including adhesion, mobilization, and proliferation. This chapter describes the biochemical footprint of stem cell engraftment and marrow retention related to Rho GTPases. In addition, it reviews abnormalities of Rho GTPases implicated in human immunohematopoietic diseases and in leukemia/lymphoma.

Telomeres both reflect and limit the replicative lifespan of normal somatic cells. Immature sub-populations of human CD34+38- hematopoietic stem cell (HSC) can be identified in vitro based on their growth kinetics and telomere length. Fluorescence in situ hybridization and flow cytometry (flow-FISH) has been used to characterize telomere length dynamics as a surrogate marker for HSC turnover in vivo. Investigations in normal steady-state hematopoiesis provided the basis for follow-up studies in model scenarios characterized by increased HSC turnover. Disorders with underlying malignant transformation of HSC (e.g., chronic myeloid leukemia (CML)) can be discriminated from disease states with increased HSC turnover rates secondary to depletion of the stem cell compartment, for example, as in defined bone marrow failure syndromes. In some of these model scenarios, the degree of telomere shortening can be correlated with disease duration, disease stage and severity as well as with response to disease-modifying treatment strategies. Whether increased telomere shortening represents a causal link between HSC turnover, replicative senescence and/or the induction of genetic instability in acquired HSC disorders remains to be shown. However, data from congenital disorders, like dyskeratosis congenita (DKC), suggest that disturbed telomere maintenance may play a role for replicative exhaustion of the HSC pool in vivo.

Several studies have shown defective progenitor-stromal interactions in chronic myeloid leukemia (CML), and adhesive defects induced by BCR/ABL have been described. However, controversial results have been reported, and the role of the stroma in abnormal development of the hematopoietic system is not clear. In this study, CML hematopoietic and irradiated stromal cells were co-cultured in different combinations for 10 or 21 days. Maintenance of viable cells was dependent both on the sources of hematopoietic progenitors and stromal adherent layers, with normal cells performing better than their leukemic counterparts. The frequency of CD34(+) CD38(-) cells in the non-adherent fraction was more related to the source of hematopoietic cells than of stroma, and hematopoietic cells from normal subjects showed better performance. The simultaneous analysis of different combinations of normal and leukemic precursor cells and stromal layers, as done in the present work, suggests that the outcome of the interaction depends on characteristics of both compartments. This hematopoietic system development is influenced by intrinsic qualities of both hematopoietic stem cells and the supportive stroma.

Objective: Transforming growth factor beta3 (TGF-beta3) is a potent suppressor of human hematopoietic progenitor cells. In this article, we compare the activity of TGF-beta3 on highly purified CD34+ cells and more immature CD34-DR(-) cells from chronic myelogenous leukemia (CML) patients in chronic phase and normal donors., Materials and Methods: Primitive hematopoietic progenitors were stimulated in liquid cultures and clonogenic assays by early-acting growth factors such as stem cell factor (SCF) and interleukin 11 (IL-11) and the intermediate-late-acting stimulating factors IL-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Molecular analysis of bcr/abl mRNA was performed on single CML colonies by nested reverse transcriptase polymerase chain reaction. Moreover, cell cycle analysis and assessment of apoptosis of normal and leukemic CD34+ cells were performed by propidium iodide (PI) alone and simultaneous staining with annexin V and PI, respectively., Results: The colony-forming efficiency of CML CD34+ cells was generally inhibited by more than 90% regardless of whether the colony-stimulating factors were used alone or combined. When compared to normal CD34+ cells, leukemic cells were significantly more suppressed in 6 of 8 culture conditions. The inhibitory effect of TGF-beta3 on CD34+ cells was exerted within the first 24 hours of incubation as demonstrated by short-term preincubation followed by IL-3-and SCF-stimulated colony assays. Evaluation of bcr/abl transcript on residual CML colonies incubated with TGF-beta3 demonstrated a small subset of neoplastic CD34+ cells unresponsive to the inhibitory effect of the study cytokine. TGF-beta3 demonstrated a greater inhibitory activity on primitive CD34+DR cells than on more mature CD34+ cells. Again, CML CD34+DR(-) cells were significantly more inhibited by TGF-beta3 than their normal counterparts in 3 of 8 culture conditions. Kinetic analysis performed on CD34+ cells showed that TGF-beta induces cell cycle arrest in G(1) phase. However, this mechanism of action is shared by normal and leukemic cells. Conversely, TGF-beta3 preferentially triggered the programmed cell death of CML CD34-cells without increasing the proportion of leukemic cells coexpressing CD95 (Fas receptor), and this effect was not reversed by functional blockade of Fas receptor. Conclusion. We demonstrate that TGF-beta3 exerts a potent suppressive effect on CML cells that is partly mediated by Fas-independent apoptosis.

In the majority of newly diagnosed patients with chronic myeloid leukaemia (CML), the bone marrow contains consistent numbers of normal Ph-negative surrogate stem cells (LTC-IC) which seem to decline rapidly with time. This is confirmed by mobilization studies showing that early after diagnosis is the optimal time to collect Ph-negative progenitor to be utilized for restoring Ph-negative haematopoiesis. In the marrow of the majority of CML patients at diagnosis Ph-positive LTC-IC are found at a lower frequency than Ph-negative LTC-IC and, unexpectedly, they do not show a tendency to increase with time as long as patients remain in chronic phase. Therefore, the decline of normal haematopoiesis does not seem related to a parallel increase in Ph-positive stem cells.

In normal adult mammals, blood cell production, hemopoiesis, takes place within the medullary cavity. There, hemopoietic cell proliferation and differentiation are regulated by a network of stromal/accessory cells and their products (ie cytokines and extracellular matrix molecules), known as the hemopoietic microenvironment. Recent in vitro studies indicate that both cell composition and functional abnormalities of the hemopoletic microenvironment are present in a proportion of patients with myeloid leukemia, both chronic (CML) and acute (AML). Cell composition abnormalities have been primarily observed in a subset of patients with AML; these abnormalities include reduced numbers of fibroblast progenitors and, in some cases, reduced numbers of macrophages and adipocytes. In terms of function, it has been shown that the marrow stromal cells from a significant number of both CML and AML patients, possess a deficient hemopoletic supportive capacity in vitro. This seems to be related to the presence of functionally abnormal, malignant macrophages. The mechanisms by which these macrophages alter the hemopoietic function of the marrow stroma, as a whole, are still not fully understood. Whereas in AML, a macrophage-derived soluble inhibitory activity (containing tumor necrosis factor alpha) has been described; in CML, a direct, macrophage-mediated cell-to-cell contact mechanism for hemopoietic inhibition seems to be involved. To date, however, it is not clear whether the abnormalities in the hemopoietic microenvironment are secondary to myeloid leukemia or if they precede clinical CML/AML. Furthermore, it is not known to what extent the functional abnormalities observed in vitro contribute to the hematologic dysfunction that characterizes myeloid leukemia and to the in vivo progression of the disease.

We report a 33-year-old man with Ph chromosome-positive CML who underwent an allogeneic BMT from an unrelated donor. DNA microsatellite studies showed complete donor chimaerism immediately after BMT followed by mixed chimaerism; by day + 45 haematopoiesis was exclusively of recipient origin. Throughout the first year post-transplant all marrow metaphases were Ph negative but with non-clonal rearrangements consistent with autologous recovery. Cytogenetic relapse of leukaemia was first detected 15 months post-transplant. This case is unusual in that non-malignant stem cells of recipient origin survived the transplant and reconstituted haematopoiesis very early after BMT. Later the leukaemic cells reasserted their 'proliferative' advantage.

CD44 is a widely expressed, multifunctional, cell-surface glycoprotein that has been implicated in the regulation of normal hematopoiesis. In addition, expression of particular isoforms of CD44 has been associated with malignant transformation and/or the acquisition of metastatic potential. In this study, we used two recently developed monoclonal anti-CD44 antibodies, one reactive with an epitope shared by many CD44 isoforms and the other with an epitope unique to CD44 isoforms containing amino acids encoded by the alternatively spliced exon v10, to compare the expression of CD44 on primitive hematopoietic cells from the marrow of normal individuals and their neoplastic counterparts present in the peripheral blood of patients with chronic myeloid leukemia (CML). Multiparameter fluorescence-activated cell sorter (FACS) analysis and cell sorting studies showed that CD44 is normally expressed at high to very high levels on both long-term culture-initiating cells (LTC-IC) and granulopoietic colony-forming cells (granulocyte-macrophage colony-forming units [CFU-GM]). In contrast, primitive erythropoietic progenitors (burst-forming units-erythroid [BFU-E]) in normal marrow were more homogeneous in their expression of CD44, and very few (less than 5%) showed the very high levels of CD44 seen on 20% to 25% of LTC-IC and CFU-GM. Antibody staining showed the expression of exon v10-containing CD44 isoforms to be restricted to a small subpopulation (4% to 8%) of morphologically recognizable mature (CD34-) myeloid cells within the light-density fraction of normal marrow cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the presence of two exon v10-containing mRNA species. In CML, a significantly greater proportion of the circulating neoplastic CFU-GM expressed very high levels of CD44, and these CFU-GM were accompanied by an increased number of light density v10+ cells, including some that coexpressed CD34. Nonmalignant hematopoietic progenitors mobilized by prior chemotherapy and growth factor treatment of patients with Hodgkin's disease or acute myeloid leukemia in remission showed no changes in CD44 expression relative to normal marrow progenitors. These results provide evidence of early differentiation-associated changes in CD44 expression during normal hematopoiesis in vivo that may be deregulated in the neoplastic clone of patients with CML.

The number of colony-forming units of granulocytes/monocytes (CFU-GM) in the peripheral blood of 7 patients with chronic myeloid leukemia (CML) in chronic phase (CP) receiving standard doses of busulfan (BSF, 0.1-0.2 mg/kg/day p.o.) was compared to that found in 8 patients with CML in CP not previously treated in order to establish if non-myeloablative chemotherapy mobilizes committed granulomonocytic progenitor cells into the circulation. The number (mean +/- SEM) of spontaneous CFU-GM in untreated patients was similar to that recorded in 10 sex- and age-matched controls, 2.6 +/- 1.9 and 3.5 +/- 2.1, respectively. BSF-treated patients showed significantly more spontaneous CFU-GM (13.9 +/- 7.5) than controls and untreated patients. Addition of recombinant human granulocyte/monocyte colony-stimulating factor to cultures promoted colony growth in controls but not in untreated and BSF-treated patients. These data seemingly indicate that: 1) administration of standard non-myeloablative doses of BSF to patients with CML in CP mobilizes CFU-GM into the circulation and 2) BSF therapy selects a granulomonocytic colony-forming progenitor cell population with increased autonomous growth potential. These findings may contribute to the understanding of the therapeutic role of BSF in CML.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a negative regulator of normal haemopoietic stem cell proliferation. Insensitivity to MIP-1 alpha of progenitor cells in chronic myeloid leukaemia (CML) could, therefore, explain myeloid expansion in this disease. We compared the effects of MIP-1 alpha on progenitor cells in normal marrow and in the blood and marrow of patients with chronic phase CML. Plastic-adherent precursors of granulocyte-macrophage colony-forming cells (P delta progenitors) are very primitive progenitor cells and are detected by incubating them for 1 week in liquid culture and assaying the CFU-GM released into the supernatant. Direct CFU-GM assays were also used in this study. Daily addition of 300 ng/ml/day of MIP-1 alpha to P delta progenitor assays of normal marrow cells suppressed CFU-GM production by 50% and in CML bone marrow P delta cultures by 20-30%. The response of CML blood P delta progenitors was heterogeneous. In five of nine cases, CFU-GM production was doubled in the presence of MIP-1 alpha and in four of nine cases, it was reduced. Addition of 100-500 ng MIP-1 alpha to direct assays of CFU-GM stimulated colony formation by normal marrow and CML blood cells to a similar extent.

We investigated the expression of p53 in paraformaldehyde-lysine-periodate fixed normal and chronic myelogenous leukemia (CML) hemopoietic cells with flow cytometry and two monoclonal antibodies, PAb1801 and the mutant-conformation-associated PAb240. With both antibodies p53 proteins were detected in more than 50% of CD34+ cells and in more than 95% neutrophils but were undetectable in the CD34- myeloid precursors. The expression of a p53 protein reactive with PAb240 was closely associated with CD34+/HLA-DR+ cells and with cells in active cell cycle, while the p53 protein recognized by PAb1801 was mainly found in CD34+/HLA-DR- cells and in cells in the G0/G1 phases of the cell cycle. Treatment of chronic-phase CML cells with p53 antisense oligonucleotides resulted in significantly increased numbers of granulocyte-macrophage colony-forming unit colonies in 12 of 17 cases studied. Slightly reduced granulocyte-macrophage colony-forming unit colony numbers were observed in one case and no change in the four others. In eight samples of normal bone marrow cells, treatment with antisense oligonucleotides showed no consistent changes in granulocyte-macrophage colony-forming unit numbers. Our data suggest that the expression of the tumor suppressor p53 is involved in the regulation of both normal and CML hemopoiesis and that the inhibition of p53 expression could modulate the proliferation of CML hemopoietic cells and possibly of normal cells.

We studied the effects of several cytokines on the development of granulocyte-macrophage (GM) progenitors using the serum-deprived culture. SCF plays an important role in the GM-CSF- or IL-3-dependent production of neutrophils and macrophages. In vitro colony assay also suggests an increase in sensitivity of GM progenitors to cytokines (GM-CSF, IL-3, G-CSF and/or SCF) in a patient with juvenile chronic myelogenous leukemia. A high level of serum IFN-gamma was associated with leukopenia and thrombocytopenia in a patient with hemophagocytic syndrome. Based on the evidence that IFN-gamma significantly inhibited the proliferation of GM progenitors, IFN-gamma-mediated suppression was suggested as one of the mechanisms causing cytopenia. In patients with aplastic anemia and neutropenia, an increase of serum G-CSF levels was observed when neutrophils decreased remarkably in number. However, the serum SCF levels were constant in these patients. A failure of SCF to enhance colony growth in some patients with aplastic anemia implies qualitative abnormalities of hematopoietic progenitors.

We studied the effect of recombinant interferon-alpha 2a (IFN alpha) on the interaction between stromal cells and granulocyte-macrophage colony-forming units (CFU-GM) from the marrow of normal individuals and chronic myeloid leukemia (CML) patients in chronic phase in long-term bone marrow cultures using preformed stromal layers. These stromal layers were established with marrow cells from normal allogeneic donors and grown to confluence in the presence or absence of IFN alpha at low concentration (100 U/ml). The number of CML CFU-GM localized within IFN alpha-treated stromal layers was significantly greater than the corresponding number localized within control stromal layers. Conversely, the distribution of normal CFU-GM between the adherent and nonadherent compartments was unaffected by IFN alpha treatment of the stromal layer. Preincubation of CML marrow cells with IFN alpha did not alter this distribution, so the observed effect of IFN alpha must be due to a primary action on stromal layers. Thus, in addition to its well known antiproliferative effect, IFN alpha enhances the capacity of marrow stromal cells to bind and/or retain CML progenitor cells, and the resulting restoration of normal regulatory control may be the basis for the selectivity of IFN alpha in CML.

Fractionated total body irradiation was given to a patient with chronic myeloid leukemia as part of a conditioning regimen for bone marrow transplantation. The radiation treatment was discontinued after the third fraction (total dose: 385 cGy) and no bone marrow graft was given because of the patient's refusal. Peripheral blood lymphocyte levels were monitored for 3 months and lymphocyte subsets up to 50 days post-irradiation. Lymphocyte numbers reached the nadir 48 h after the last fraction and lymphopenia was still present 3 months later. All lymphocyte subsets (T, B, CD4 and CD8 cells) showed a similar decrease except for natural killer cells which exhibited a larger decline. The regenerative capacity of T cells, CD4 subset and natural killer cells was less than that of B cells and CD8 lymphocyte subsets.

Human trabecular bone that encloses the bone marrow (BM) is covered by a single layer of thin, sometimes inconspicuous, flat, elongated (spindle-shaped) endothelium-like cells with a round or oval nucleus. These "bone lining" cells, or endosteal cells (EC), form a continuous membrane (endosteum) over the trabecular bone surfaces. In most cases, the composition and thickness of these cells do not vary unless the cells are in intimate contact with hematopoietic tissue. In that instance, they are seen as a single layer adjacent to hematopoietic tissue or as a zone of tightly packed or loosely arranged mononuclear (hematopoietic) cells, some apparently originating from the endosteum. In a reparative process, such as following BM harvest, during which bony trabeculae (BT) are mechanically fractured, these cells are seen giving rise to osteoprogenitor (osteoblasts and osteoclasts) cells. Occasionally, the EC appear similar to BM stromal cells (morphologically and by their association with collagen/reticulin fibers) and are best seen at or near the BT that are cut tangentially. Short processes extending from the EC towards the underlying osteocytes have also been observed, suggesting that a channel of communication exists between them and osteocytes. Our observations, coupled with the experimental findings of others (i.e., that hematopoietic stem cells are concentrated near the endosteum, that cells responsible for BM and stroma regeneration are derived from the endosteal layer, and that high concentrations of hematopoietic colony-stimulating factors are produced there), indicate that, in addition to functioning as a simple membranous covering layer for BT, the endosteum helps to support osteocytes and maintains mineral homeostasis.(ABSTRACT TRUNCATED AT 250 WORDS)

The bone-marrow microenvironment has a decisive role in maintaining haemopoietic stem cells and regulating their differentiation. In diseases of the haemopoietic system, viz. anaemias and leukemias, the microenvironment has been shown to play a key role. In this paper we show that leukemic cell conditioned medium and sera from CML patients, interact with the in vitro bone-marrow environment and bring about changes which affect the maintenance of normal stem cells.