W. Jens Zeller

Plerixafor inhibits chemotaxis toward SDF-1 and CXCR4-mediated stroma contact in a dose-dependent manner resulting in increased susceptibility of BCR-ABL+ cell to Imatinib and Nilotinib.

Despite Imatinibs remarkable success in chronic myelogenous leukemia treatment, monotherapy frequently causes resistance, underlining the rationale for combination chemotherapy. A potential approach would be interrupting the SDF-1/CXCR4 axis using the selective CXCR4 antagonist Plerixafor (previously AMD3100), as this axis has been reported to provide survival-enhancing effects to myeloid progenitor cells. By efficient CXCR4 blocking in the CXCR4+/BCR-ABL+ cell line BV-173, plerixafor (1–100 μM) significantly inhibits SDF-1α-mediated chemotaxis and cell migration toward the murine stroma cell line FBMD-1. Furthermore, plerixafor also significantly (10–100 μM) increased the detachment rate of SDF-1-mediated/VCAM-1-associated cell adherence under shear stress. Using a stroma-dependent coculture assay, plerixafor sensitized BCR-ABL+ cells toward tyrosine kinase inhibitor therapy. Because the level of cell killing nearly reached that of samples cultured without stroma, a cell–cell interaction disruption seems to improve the efficacy of BCR-ABL-targeting drugs. In addition, we could show that exposure of BCR-ABL+ cells to Imatinib or Nilotinib induced an increase in surface CXCR4 expression. Our data suggest that for BCR-ABL+ leukemia, the selective blocking of the SDF-1/CXCR4 axis by plerixafor is a potential mechanism to overcome the protective effect of the bone marrow environment, thereby increasing the therapeutic potency of anti-BCR-ABL drugs and the therapeutic window.

Multidrug Resistance 1 Gene Transfer Can Confer Chemoprotection to Human Peripheral Blood Progenitor Cells Engrafted in Immunodeficient Mice

Myelosuppression is the main side effect of cancer chemotherapy. An improved rate of retroviral vector-mediated gene transfer to hematopoietic stem cells, shown in more recent clinical trials, has created the basis to test the concept of myeloprotective gene therapy. We transplanted clinical-scale human peripheral blood progenitor cell grafts (n = 2) transduced with retroviral vector SF91m3, which contains the human multidrug resistance 1 gene (MDR1), into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Engrafted mice of one cohort were protected from paclitaxel toxicity (p < 0.05) and we noted a similar trend in the second cohort. In paclitaxel-treated mice that had received gene-transduced cells we found a significant increase in gene marking (p < 0.05 - p < 0.01) or P-glycoprotein expression (p < 0.01) compared with their chemotherapy-naive counterparts. This is the first report showing that cytostatic drug resistance gene therapy can mediate chemoprotection of human clinically relevant stem cell populations with marrow engraftment potential.

Favorable therapeutic index of a p210BCR-ABL-specific tyrosine kinase inhibitor; activity on lineage-committed and primitive chronic myelogenous leukemia progenitors

Purpose: In order to assess the effect of the tyrosine kinase inhibitor CGP57148B on lineage-committed and primitive chronic myelogenous leukemia (CML) progenitor cells, peripheral blood progenitor cells (PBPC) mobilized in chronic phase CML were exposed to this compound in vitro. Methods: Both short-term (≤1 week) and long-term exposure (≥2 weeks) to CGP57148B were investigated using suspension culture, semisolid (methylcellulose) assay or stroma-dependent long-term culture (LTC). The proportion of bcr/abl-positive progenitors was determined after direct plating [2 weeks in colony-forming cell (CFC) assay] as well as after 2 or 6 weeks LTC (LTC always followed by CFC replates). Results: Incubation of CML PBPC over 48 h in suspension culture with 100 μM CGP57148B reduced the proportion of bcr/abl-positive colonies to 4.4 ± 4.3% (n = 5) after direct plating, 6.6 ± 4.2% (n = 5) after 2 weeks LTC and 5 ± 5.6% (n = 2) after 6 weeks LTC. At this dose, survival of drug-exposed normal PBPC was 53 ± 4.2%, 51 ± 2.8% and 54.5 ± 4.9% (n = 2), respectively. Incubation with CGP57148B at a concentration of 10 μM over 1 week under LTC conditions reduced the number of bcr/abl-positive colonies to 11.8 ± 6.1% (n = 5) after direct plating, 12 ± 6.4% (n = 4) after 2 weeks LTC and 14.3 ± 11.4% (n = 3) after 6 weeks LTC; survival of normal PBPC was 84.5 ± 2.1%, 93 ± 4.2% and 86 ± 1.4% (n = 2), respectively. Following long-term exposure to CGP57148B at a concentration of 1 μM, the proportion of remaining bcr/abl-positive colonies was 35%, 9% and 25% of untreated CML samples after direct plating as well as after 2 and 6 weeks LTC, respectively. The respective values for 10 μM CGP57148B were 10%, 11% and 19%. Long-term exposure of normal progenitors to CGP57148B yielded a survival of 98%, 100% and 93% (1 μM) or 77%, 86% and 80% (10 μM), respectively. Conclusion: The results support the use of CGP57148B either for short-term exposure in vitro (e.g. purging) or for continuous treatment of CML in vivo.

Lentiviral vector integration sites in human NOD/SCID repopulating cells.

Recent observations of insertional mutagenesis in preclinical and clinical settings emphasize the relevance of investigating comprehensively the spectrum of integration sites targeted by specific vectors.

Peripheral blood progenitor cell (PBPC) counts during steady-state haemopoiesis enable the estimation of the yield of mobilized PBPC after granulocyte colony-stimulating factor supported cytotoxic chemotherapy: an update on 100 patients

Peripheral blood progenitor cells (PBPC) can be mobilized using chemotherapy and granulocyte colony‐stimulating factor (G‐CSF). We and others previously reported a correlation of steady‐state PBPC counts and the PBPC yield during mobilization in a small group of patients. Here we present data on 100 patients (patients: 25 non‐Hodgkins lymphoma (NHL), five Hodgkins disease, 35 multiple myeloma (MM), 35 solid tumour) which enabled a detailed analysis of determinants of steady‐state PBPC levels and of mobilization efficiency in patient subgroups. Previous irradiation (P = 0.0034) or previous chemotherapy in patients with haematological malignancies (P = 0.0062) led to a depletion of steady‐state PB CD34+ cells. A correlation analysis showed steady‐state PB CD34+ cells (all patients: r = 0.52, P < 0.0001; NHL patients, r = 0.69, P = 0.0003; MM patients: r = 0.66, P = 0.0001) and PB colony‐forming cells can reliably assess the CD34+ cell yield in mobilized PB. In patients with solid tumour a similar trend was observed in mobilization after the first chemotherapy cycle (r = 0.51, P = 0.05) but not if mobilization occurred after the second or further cycle of a sequential dose‐intensified G‐CSF‐supported chemotherapy regimen, when premobilization CD34+ counts were 18‐fold elevated (P = 0.004). When the patients with MM (r = 0.63, P = 0.0008) or with NHL (r = 0.65, P = 0.006) were analysed separately, a highly significant correlation of the steady‐state PB CD34+ cell count to the mean leukapheresis CD34+ cell yield was found, whereas no correlation was observed for patients with a solid tumour. For patients with haematological malignancies estimates could be calculated which, at a specific steady‐state PB CD34+ cell count, could predict with a 95% probability a defined minimum progenitor cell yield. These results enable recognition of patients who mobilize PBPC poorly and may assist selection of patients for novel mobilization regimens.

Good manufacturing practice-grade production of unrestricted somatic stem cell from fresh cord blood

BACKGROUND AIMS The discovery of unrestricted somatic stem cells (USSC), a non-hematopoietic stem cell population, brought cord blood (CB) to the attention of regenerative medicine for defining more protocols for non-hematopoietic indications. We demonstrate that a reliable and reproducible method for good manufacturing practice (GMP)-conforming generation of USSC is possible that fulfils safety requirements as well as criteria for clinical applications, such as adherence of strict regulations on cell isolation and expansion. METHODS In order to maintain GMP conformity, the automated cell processing system Sepax (Biosafe) was implemented for mononucleated cell (MNC) separation from fresh CB. After USSC generation, clinical-scale expansion was achieved by multi-layered CellSTACKs (Costar/Corning). Infectious disease markers, pyrogen and endotoxin levels, immunophenotype, potency, genetic stability and sterility of the cell product were evaluated. RESULTS The MNC isolation and cell cultivation methods used led to safe and reproducible GMP-conforming USSC production while maintaining somatic stem cell character. CONCLUSIONS Together with implemented in-process controls guaranteeing contamination-free products with adult stem cell character, USSC produced as suggested here may serve as a universal allogeneic stem cell source for future cell treatment and clinical settings.

Delineation of cell cycle state and correlation to adhesion molecule expression of human CD34+ cells from steady-state bone marrow and peripheral blood mobilized following G-CSF-supported chemotherapy

Treatment with a combination of chemotherapy and G‐CSF leads to the release of hematopoietic stem cells from the bone marrow (BM) to the peripheral blood (PB), where they can be harvested for transplantation. Premobilization BM CD34+ cells were reported to proliferate actively, while virtually none of the mobilized PB CD34+ cells were in the S/G2M phase. We were interested in elucidating the cell cycle state further and in investigating the role of adhesion molecule expression on marrow‐adherent and circulating CD34+ cells during different phases of the cell cycle. Consecutive premobilization BM and leukapheresis product (LP) samples were obtained from 14 patients following G‐CSF‐supported chemotherapy. Steady‐state BM and LP CD34+ selected cells were triple‐stained for CD34, for DNA using the intercalating dye 7‐aminoactinomycin D, and for Ki‐67, cyclins, or adhesion antigens. Ki‐67 is expressed in all phases of the cell cycle except G0 and was found in 69.14% ± 3.46% (mean ± standard error [SE]) of BM CD34+ cells and 62.78% ± 3.37% of LP CD34+ cells, while in BM significantly more CD34+/Ki‐67+ cells were in the S/G2M phase of the cell cycle than in LP (8.6% ± 0.9% versus 1.8% ± 0.3%, respectively, p = 0.0001). Therefore, most circulating mobilized CD34+ cells are in the G1 phase, similar to their steady‐state BM counterparts. Cyclin A became detectable in the 2n DNA peak. As expected, a higher proportion of CD34+/cyclin A+/S/G2M cells was found in BM than in LP (p < 0.05). Antigen density of the cyclins D3 and D2 tended to be higher on LP than on BM CD34+ cells, while D1 was found at low levels in similar density. The adhesion antigens CD18, CD49b, CD49d, CD49e, CD58, and CD62L were expressed in a significantly higher proportion of S/G2M‐phase than in G0/G1‐phase CD34+ cells. The strongest association to the proliferative status was observed for CD49d, which was coexpressed by 85.9% ± 2.6% (BM) or 90.8% ± 2.5% (LP) of CD34+/S/G2M cells, whereas a distinct CD34+/CD49d−/S/G2M population could not be detected. The average coexpression of the other antigens was 57% (CD49e, CD18) or lower. Our results demonstrate that the majority of PB CD34+ cells mobilized following G‐CSF‐supported chemotherapy and steady‐state BM CD34+ cells are in the late G1 phase of the cell cycle and show a correlation between the expression of adhesion receptors and cell cycle status of CD34+ cells in both BM and LP.

The Disparate Twins: A Comparative Study of CXCR4 and CXCR7 in SDF-1α–Induced Gene Expression, Invasion and Chemosensitivity of Colon Cancer

Purpose: In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor-1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7–induced intracellular communication triggering malignancy is still only marginally understood. Experimental Design: In tumor tissue of 52 patients with colorectal cancer, we observed that expression of CXCR7 and CXCR4 increased with tumor stage and tumor size. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α. Results: Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Differentially regulated genes were assigned by gene ontology to migration, proliferation, and lipid metabolic processes. Expressions of AKR1C3, AXL, C5, IGFBP7, IL24, RRAS, and TNNC1 were confirmed by quantitative real-time PCR. Using the in silico gene set enrichment analysis, we showed that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. Functionally, exposure to SDF-1α increased invasiveness of CXCR4 and CXCR7 cells, AXL knockdown hampered invasion. Compared with controls, CXCR4 cells showed increased sensitivity against 5-FU, whereas CXCR7 cells were more chemoresistant. Conclusions: These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists such as plerixafor. Clin Cancer Res; 20(3); 604–16. ©2013 AACR.

Overexpression of MDR1 Using a Retroviral Vector Differentially Regulates Genes Involved in Detoxification and Apoptosis and Confers Radioprotection

Abstract Maier, P., Fleckenstein, K., Li, L., Laufs, S., Zeller, W. J., Baum, C., Fruehauf, S., Herskind, C. and Wenz, F. Overexpression of MDR1 Using a Retroviral Vector Differentially Regulates Genes Involved in Detoxification and Apoptosis and Confers Radioprotection. Radiat. Res. 166, 463–473 (2006). Overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, might complement chemotherapy and radiotherapy in the treatment of tumors. However, for safety and mechanistic reasons, it is important to know whether MDR1 overexpression influences the expression of other genes. Therefore, we analyzed differential gene expression in cells of the human lymphoblast cell line TK6 retrovirally transduced with MDR1 using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). Sixty-one annotated genes showed a significant change in expression (P < 10−4) in MDR1-overexpressing cells compared to untransduced cells and cells transduced with a control virus expressing the neomycin phosphotransferase gene. Several genes coding for proteins involved in detoxification and exocytosis showed ∼1.4– 4-fold increases in transcript levels (e.g. ALDH1A, UNC13). Additionally, pro-apoptosis genes were down-regulated (e.g. twofold for CASP1, 2.5-fold for NALP7) with concomitant increased expression of the potential anti-apoptosis gene AKT3. In functional assays the influence of MDR1 overexpression on apoptosis signaling was further corroborated by showing reduced rates of apoptosis in response to irradiation in TK6 cells transduced with MDR1. In conclusion, the resistant phenotype of MDR1-mediated P-gp-overexpressing cells is associated with differential expression of genes coding for metabolic and apoptosis-related proteins. These results have important implications for understanding the mechanisms by which MDR1 gene therapy can protect normal tissues from radiation- or chemotherapy-induced damage during tumor treatment.

MDR1 Gene Transfer Using a Lentiviral SIN Vector Confers Radioprotection to Human CD34+ Hematopoietic Progenitor Cells

Abstract Maier, P., Herskind, C., Fleckenstein, K., Spier, I., Laufs, S., Jens Zeller, W., Fruehauf, S. and Wenz, F. MDR1 Gene Transfer Using a Lentiviral SIN Vector Confers Radioprotection to Human CD34+ Hematopoietic Progenitor Cells. Radiat. Res. 169, 301–310 (2008). Tumor radiotherapy with large-field irradiation results in an increase in apoptosis of the radiosensitive hematopoietic stem cells (CD34+). The aim of this study was to demonstrate the radioprotective potential of MDR1 overexpression in human CD34+ cells using a lentiviral self-inactivating vector. Transduced human undifferentiated CD34+ cells were irradiated with 0–8 Gy and held in liquid culture under myeloid-specific maturation conditions. After 12 days, MDR1 expression was determined by the rhodamine efflux assay. The proportion of MDR1-positive cells in cells from four human donors increased with increasing radiation dose (up to a 14-fold increase at 8 Gy). Determination of expression of myeloid-specific surface marker proteins revealed that myeloid differentiation was not affected by transduction and MDR1 overexpression. Irradiation after myeloid differentiation also led to an increase of MDR1-positive cells with escalating radiation doses (e.g. 12.5–16% from 0–8 Gy). Most importantly, fractionated irradiation (3 × 2 Gy; 24-h intervals) of MDR1-transduced CD34+ cells resulted in an increase in MDR1-positive cells (e.g. 3–8% from 0–3 × 2 Gy). Our results clearly support a radioprotective effect of lentiviral MDR1 overexpression in human CD34+ cells. Thus enhancing repopulation by surviving stem cells may increase the radiation tolerance of the hematopoietic system, which will contribute to widening the therapeutic index in radiotherapy.