The classical model of hematopoiesis predicts a dichotomous lineage restriction of multipotent hematopoietic progenitors (MPPs) into common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). However, this idea has been challenged by the identification of lymphoid progenitors retaining partial myeloid potential (e.g., LMPPs), implying that granulocytes can arise within both the classical lymphoid and the myeloid branches. Here, we resolve this issue by using cell-surface CD133 expression to discriminate functional progenitor populations. We show that eosinophilic and basophilic granulocytes as well as erythrocytes and megakaryocytes derive from a common erythro-myeloid progenitor (EMP), whereas neutrophilic granulocytes arise independently within a lympho-myeloid branch with long-term progenitor function. These findings challenge the concept of a CMP and restore dichotomy to the classical hematopoietic model.

We here describe a novel xenograft model of chronic lymphocytic leukemia (CLL) generated by infusion of human primary CLL cells into immunodeficient nonobese/severe combined immunodeficient (NOD/SCID) mice. Combined i.v. and i.p. injection of peripheral blood mononuclear cells (PBMC) from 39 patients with CLL resulted in highly reproducible splenic (37 of 39) and peritoneal (35 of 39) engraftment, which remained stable over a time span of 4 to 8 weeks. By comparison, recovery of leukemic cells from bone marrow (21 of 39) or peripheral blood (8 of 22) was substantially lower. The engraftment pattern of CLL PBMC 4 weeks posttransplant was correlated with clinical disease activity: infusion of PBMC from donors with Binet stage A, lymphocyte doubling time of >12 months, and normal lactate dehydrogenase (LDH) serum levels led to marked engraftment of T cells whereas comparably few tumor cells could be detected. In contrast, NOD/SCID mice receiving PBMC from donors with advanced stage Binet C, lymphocyte doubling time of <12 months, and elevated LDH serum levels exhibited predominant engraftment of tumor cells and comparably low numbers of T cells. These results suggest that this model reflects the heterogeneity and important clinical characteristics of the disease, and thus may serve as a tool for preclinical drug testing and investigation of the pathophysiology of CLL. [Cancer Res 2007;67(18):8653-61]

The olfactory receptor (OR) family was found to be expressed mainly in the nasal epithelium. In the last two decades members of the OR family were detected to be functional expressed in different parts of the human body such as in liver, prostate or intestine cancer cells. Here, we detected the expression of several ORs in the human chronic myelogenous leukemia (CML) cell line K562 and in white blood cells of clinically diagnosed acute myeloid leukemia (AML) patients by RT-PCR and next-generation sequencing. With calcium-imaging, we characterized in greater detail the cell biological role of one OR (OR2AT4) in leukemia. In both cell systems, the OR2AT4 agonist Sandalore-evoked strong Ca2+ influx via the adenylate cyclase-cAMP-mediated pathway. The OR2AT4 antagonist Phenirat prevented the Sandalore-induced intracellular Ca2+ increase. Western blot and flow cytometric experiments revealed that stimulation of OR2AT4 reduced the proliferation by decreasing p38-MAPK phosphorylation and induced apoptosis via phosphorylation of p44/42-MAPK. Furthermore, Sandalore increased the number of hemoglobin-containing cells in culture. We described for the first time an OR-mediated pathway in CML and AML that can regulate proliferation, apoptosis and differentiation after activation. This mechanism offers novel therapeutic options for the treatment of AML.

The Wnt signalling pathway, one of the core de-regulated pathways in chronic lymphocytic leukaemia (CLL), is activated in only a subset of patients through somatic mutations. Here we describe alternative, microenvironment-dependent mechanisms of Wnt activation in malignant B cells. We show that tumour cells specifically induce Notch2 activity in mesenchymal stromal cells (MSCs) required for the transcription of the complement factor C1q. MSC-derived C1q in turn inhibits Gsk3-β mediated degradation of β-catenin in CLL cells. Additionally, stromal Notch2 activity regulates N-cadherin expression in CLL cells, which interacts with and further stabilises β-catenin. Together, these stroma Notch2-dependent mechanisms induce strong activation of canonical Wnt signalling in CLL cells. Pharmacological inhibition of the Wnt pathway impairs microenvironment-mediated survival of tumour cells. Similarly, inhibition of Notch signalling diminishes survival of stroma-protected CLL cells in vitro and disease engraftment in vivo. Notch2 activation in the microenvironment is a pre-requisite for the activation of canonical Wnt signalling in tumour cells.

Key Points• CD44 expression in CLL is micromilieu instructed and promotes leukemic cell survival, which can be antagonized by CD44 antibodies.• As a surface coreceptor, CD44 supports leukemogenesis by modulating stimuli of MCL1 expression (eg, B-cell receptor signals).The cell-surface glycoprotein CD44 is expressed in chronic lymphocytic leukemia (CLL), but its functional role in this disease is poorly characterized. We therefore investigated the contribution of CD44 to CLL in a murine disease model, the Em-TCL1 transgenic mouse, and in CLL patients. Surface CD44 increased during murine CLL development. CD44 expression in human CLL was induced by stimulation with interleukin 4/soluble CD40 ligand and by stroma cell contact. Engagement of CD44 by its natural ligands, hyaluronic acid or chondroitin sulfate, protected CLL cells from apoptosis, while anti-CD44 small interfering RNAs impaired tumor cell viability. Deletion of CD44 during TCL1-driven murine leukemogenesis reduced the tumor burden in peripheral blood and spleen and led to a prolonged overall survival. The leukemic cells from these CD44 knockout animals revealed lower levels of antiapoptotic MCL1, a higher propensity to apoptosis, and a diminished B-cell receptor kinase response. The inhibitory anti-CD44 antibodies IM7 and A3D8 impaired the viability of CLL cells in suspension cultures, in stroma contact models, and in vivo via MCL1 reduction and by effector caspase activation. Taken together, CD44 expression in CLL is mediated by the tumor microenvironment. As a coreceptor, CD44 promotes leukemogenesis by regulating stimuli of MCL1 expression. Moreover, CD44 can be addressed therapeutically in CLL by specific antibodies. (Blood. 2013;121(20):4126-4136)

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SummaryHematopoietic stem and progenitor cells (HSPCs) can self-renew and create committed progenitors, a process supposed to involve asymmetric cell divisions (ACDs). Previously, we had linked the kinetics of CD133 expression with ACDs but failed to detect asymmetric segregation of classical CD133 epitopes on fixed, mitotic HSPCs. Now, by using a novel anti-CD133 antibody (HC7), we confirmed the occurrence of asymmetric CD133 segregation on paraformaldehyde-fixed and living HSPCs. After showing that HC7 binding does not recognizably affect biological features of human HSPCs, we studied ACDs in different HSPC subtypes and determined the developmental potential of arising daughter cells at the single-cell level. Approximately 70% of the HSPCs of the multipotent progenitor (MPP) fraction studied performed ACDs, and about 25% generated lymphoid-primed multipotent progenitor (LMPP) as wells as erythromyeloid progenitor (EMP) daughter cells. Since MPPs hardly created daughter cells maintaining MPP characteristics, our data suggest that under conventional culture conditions, ACDs are lineage instructive rather than self-renewing.

Progranulin (Pgrn) is a 88 kDa secreted protein with pleiotropic functions including regulation of cell cycle progression, cell motility, wound repair and tumorigenesis. Using microarray based gene expression profiling we have recently demonstrated that the gene for Pgrn, granulin (GRN), is significantly higher expressed in aggressive CD38+ZAP-70+ as compared to indolent CD38−ZAP-70− chronic lymphocytic leukemia (CLL) cases. Here, we measured Pgrn plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in the Essen CLL cohort of 131 patients and examined Pgrn for association with established prognostic markers and clinical outcome. We found that high Pgrn plasma levels were strongly associated with adverse risk factors including unmutated IGHV status, expression of CD38 and ZAP-70, poor risk cytogenetics (11q-, 17p-) as detected by flourescence in situ hybridization (FISH) and high Binet stage. Pgrn as well as the aforementioned risk factors were prognostic for time to first treatment and overall survival in this series. Importantly, these results could be confirmed in the independent multicentric CLL1 cohort of untreated Binet stage A patients (n = 163). Here, multivariate analysis of time to first treatment revealed that high risk Pgrn (HR = 2.06, 95%-CI = 1.13–3.76, p = 0.018), unmutated IGHV status (HR = 5.63, 95%-CI = 3.05–10.38, p<0.001), high risk as defined by the study protocol (HR = 2.06, 95%-CI = 1.09–3.89, p = 0.026) but not poor risk cytogenetics were independent prognostic markers. In summary our results suggest that Pgrn is a novel, robust and independent prognostic marker in CLL that can be easily measured by ELISA.