Eike C. Buss

A crosstalk between intracellular CXCR7 and CXCR4 involved in rapid CXCL12‐triggered integrin activation but not in chemokine‐triggered motility of human T lymphocytes and CD34+ cells

The chemokine CXCL12 promotes migration of human leukocytes, hematopoietic progenitors, and tumor cells. The binding of CXCL12 to its receptor CXCR4 triggers Gi protein signals for motility and integrin activation in many cell types. CXCR7 is a second, recently identified receptor for CXCL12, but its role as an intrinsic G‐protein‐coupled receptor (GPCR) has been debated. We report that CXCR7 fails to support on its own any CXCL12‐triggered integrin activation or motility in human T lymphocytes or CD34+ progenitors. CXCR7 is also scarcely expressed on the surface of both cell types and concentrates right underneath the plasma membrane with partial colocalization in early endosomes. Nevertheless, various specific CXCR7 blockers get access to this pool and attenuate the ability of CXCR4 to properly rearrange by surface‐bound CXCL12, a critical step in the ability of the GPCR to trigger optimal CXCL12‐mediated stimulation of integrin activation in T lymphocytes as well as in CD34+ cells. In contrast, CXCL12‐triggered CXCR4 signaling to early targets, such as Akt as well as CXCR4‐mediated chemotaxis, is insensitive to identical CXCR7 blocking. Our findings suggest that although CXCR7 is not an intrinsic signaling receptor for CXCL12 on lymphocytes or CD34+ cells, its blocking can be useful for therapeutic interference with CXCR4‐mediated activation of integrins.

Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells

Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine stromal cell derived factor-1 (SDF-1) to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4+ human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, whereas β2-adrenergic antagonist inhibited both steady state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady-state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.

Enhanced c-Met activity promotes G-CSF–induced mobilization of hematopoietic progenitor cells via ROS signaling

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1(+)/c-Kit(+)/Lin(-) cells and interfered with their enhanced chemotactic migration to stromal cell-derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met-mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.

Human haematopoietic stem cell lineage commitment is a continuous process

Blood formation is believed to occur through stepwise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a ‘continuum of low-primed undifferentiated haematopoietic stem and progenitor cells’ (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady-state haematopoiesis downstream of HSCs and provide a basis for the understanding of haematopoietic malignancies.

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

BACKGROUND AIMS Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. METHODS This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. RESULTS The strategy for quality control testing depends on the products cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. CONCLUSIONS This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Imatinib combined with mitoxantrone/etoposide and cytarabine is an effective induction therapy for patients with chronic myeloid leukemia in myeloid blast crisis.

Despite advances in drug therapy and allogeneic stem cell transplantation (allo‐SCT), the prognosis of patients with chronic myeloid leukemia (CML) in blast crisis remains poor. Imatinib has demonstrated synergistic effects in vitro with mitoxantrone, etoposide, and cytarabine.

Kinetics of Peripheral Blood Stem Cell Mobilization Following G‐CSF‐Supported Chemotherapy

It would be a clinical and economical advantage if the optimal time point of peripheral blood stem cell (PBSC) mobilization following G‐CSF‐supported chemotherapy (CT) was known in advance. Therefore, we retrospectively analyzed mobilization parameters in 113 adult tumor patients treated in our institution within 1 year. The start of apheresis was guided by CD34+ cell measurements in the PB and occurred on or after day 11 after start of mobilization CT in 97% of patients. The median peak (p)CD34+ cell count in PB uniformly occurred on day 14‐15 (range: 6‐32 days) after the start of CT, irrespective of the diagnosis (multiple myeloma n = 76, other histology n = 37), the type, but not the amount, of premobilization CT or radiotherapy (RT), the mobilization regimen, or the G‐CSF dosage administered. Among more heavily pretreated patients (>six cycles of prior CT or RT), a higher proportion mobilized late (pCD34+ cell count later than day 20 in 12% and 13%, respectively, versus 2%‐5% in the other groups). Therefore, we propose to start measuring CD34+ cells in the PB on day 11 after the start of mobilization therapy. The wide range of optimal mobilization time points argues for an individualized rather than a preset start of apheresis.

Targeting CDK9 by wogonin and related natural flavones potentiates the anti-cancer efficacy of the Bcl-2 family inhibitor ABT-263

Tumor initiation, progression and resistance to therapies are tightly associated with over‐expression of anti‐apoptotic proteins Bcl‐2, Bcl‐xL, Bcl‐w and Mcl‐1. ABT‐263 (Navitoclax), an orally bio‐available small‐molecule mimetic of the Bcl‐2 homology domain 3, inhibits Bcl‐2, Bcl‐xL, and Bcl‐w and has shown anti‐cancer effects mainly on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of ABT‐263 in patients is dose‐limited due to causing thrombocytopenia via inhibition of Bcl‐xL in platelets. ABT‐199 specifically inhibits Bcl‐2; however, its use is limited to tumors over‐expressing only Bcl‐2. Besides, many tumors resist treatment due to high levels of Mcl‐1 expression or develop resistance via up‐regulation of Mcl‐1 during long‐term exposure. These obstacles highlight the demand to improve the ABT‐263‐based therapy. In this study, we show that anti‐cancer flavones, e.g., wogonin, baicalein, apigenin, chrysin and luteolin enhance ABT‐263‐induced apoptosis in different cancer cell lines and in primary AML and ALL cells by down‐regulation of Mcl‐1 expression. Importantly, wogonin does not enhance the toxicity of ABT‐263 to proliferating normal T cells and thrombocytes. Wogonin also potentiates the lethality of ABT‐263 in cancer cells which have acquired resistance to ABT‐263. Furthermore, we show that combination of wogonin with ABT‐263 promotes in vivo tumor regression in a human T‐cell leukemia xenograft mouse model. Our study demonstrates that wogonin (and related flavones) reduce the effective dose of ABT‐263 thereby possibly decreasing the risk of adverse side effects.

The rarity of ALDH+ cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH+ cells; <1.9%; ALDH‐rare AML), whereas 24 patients had relatively numerous ALDH+ cells (≥1.9%; ALDH‐numerous AML). In patients with ALDH‐rare AML, normal HSC could be separated by their CD34+ALDH+ phenotype, whereas LSC were exclusively detected among CD34+ALDH− cells. For patients with ALDH‐numerous AML, the CD34+ALDH+ subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH+ cells from ALDH‐numerous AML were quiescent, refractory to ARA‐C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long‐term outcome were also characteristic for patients with ALDH‐numerous AML providing an additional risk‐stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible.

Identifying leukemia stem cells – Is it feasible and does it matter?

Present evidence indicates that acute myeloid leukemia (AML) is a stem cell disease. Leukemia stem cells (LSC) might originate from malignant transformation of normal hematopoietic stem cells (HSC), or alternatively, from progenitors in which the acquired mutations have re-installed a dysregulated self-renewal program. Since LSC, similar to their normal counterparts, divide extreme slowly, this might account for the ineffectiveness of conventional chemotherapy in inducing long-term cure. The present review will focus on the detection of LSC, their cellular and molecular biology, their genetic heterogeneity and on correlative studies that have demonstrated the clinical significance of estimating LSC burden. For long-term cure of AML, it is of importance to define LSC candidates and to understand their biology compared to normal HSC. Finally, we will discuss the perspectives of developing treatment strategies for eradication of LSC.