In vitro testing of drug combinations employing nilotinib and alkylating agents with regard to pretransplant conditioning treatment of advanced-phase chronic myeloid leukemia.

Purpose: The prognosis of patients with advanced-phase chronic myeloid leukemia (CML) remains dismal despite the availability of targeted therapies and allogeneic stem cell transplantation (allo-SCT). Increasing the antileukemic efficacy of the pretransplant conditioning regimen may be a strategy to increase remission rates and duration. We therefore investigated the antiproliferative effects of nilotinib in combination with drugs that are usually used for conditioning: the alkylating agents mafosfamide, treosulfan, and busulfan.

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.

CXCR4 Expression and Treatment with SDF-1α or Plerixafor Modulate Proliferation and Chemosensitivity of Colon Cancer Cells.

Background: Signaling through stromal cell-derived factor-1α (SDF-1α), strongly secreted by bone marrow stromal cells and the CXC chemokine receptor 4 (CXCR4) exposed on tumor cells has pivotal roles in proliferation, metastasis, and tumor cell "dormancy." Dormancy is associated with cytostatic drug resistance and is probably a property of tumor stem cells and minimal residual disease. Thus, hampering the SDF-1α/CXCR4 cross talk by a CXCR4 antagonist like Plerixafor (AMD3100) should overcome tumor cell dormancy bymobilization of tumor cells from "sanctuary" niches.

A Lentiviral CXCR4 overexpression and knockdown model in colorectal cancer cell lines reveals plerixafor-dependent suppression of SDF-1α-induced migration and invasion.

Background: The development of distant metastasis is associated with poor outcome in patients with colorectal cancer (CRC). The stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) have pivotal roles in the chemotaxis of migrating tumor cells during metastasis. Thus, hampering the SDF-1/CXCR4 cross-talk is a promising strategy to suppress metastasis.

Overexpression of manganese superoxide dismutase does not increase clonogenic cell survival despite effect on apoptosis in irradiated lymphoblastoid cells.

Gene therapy-mediated overexpression of superoxide dismutases (SOD) appears to be a promising strategy for modulating radiosensitivity based on detoxification of superoxide radicals and suppression of apoptosis. Using recombinant lentiviral-based vectors, the effects of SOD overexpression on both were tested in human lymphoblastoid cells (TK6) that are sensitive to radiation-induced apoptosis. TK6 cells were transduced with vectors containing CuZnSOD, MnSOD or inverted MnSOD (MSODi) cDNA.

Efficient gene transfer with pseudotyped recombinant adeno-associated viral vectors into human chronic myelogenous leukemia cells.

Gene transfer into chronic myelogenous leukemia (CML) cells may become of relevance for overcoming therapy resistance. Single-stranded pseudotyped adeno-associated viruses of serotypes 2/1 to 2/6 (ssAAV2/1-ssAAV2/6) were screened on human CML cell lines and primary cells to determine gene transfer efficiency. Additionally, double-stranded self-complementary vectors (dsAAVs) were used to determine possible second-strand synthesis limitations.

Clonal inventory screens uncover monoclonality following serial transplantation of MGMT P140K-transduced stem cells and dose-intense chemotherapy.

Gene transfer of mutant O(6)-methylguanine-DNA-methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSCs) protects hematopoiesis from alkylating agents and allows efficient in vivo selection of transduced HSCs. However, insertional mutagenesis, high regenerative stress associated with selection, and the genotoxic potential of alkylating drugs represent considerable risk factors for clinical applications of this approach. Therefore, we investigated the long-term effect of MGMT(P140K) gene transfer followed by repetitive, dose-intensive treatment with alkylating agents in a murine serial bone marrow transplant model and assessed clonality of hematopoiesis up to tertiary recipients.

SNAI2 as a novel radioprotector of normal tissue by gene transfer using a lentiviral bicistronic SIN vector.

Tumor radiotherapy with large-field irradiation results in an increase of p53-dependent apoptosis of the radiosensitive hematopoietic stem cells. Proapoptotic PUMA is a transcriptional target of p53. Thus suppression of PUMA expression by gene therapy with the transcription repressor SNAI2 as transgene might be a potential approach for normal tissue protection during radiotherapy.

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.

Overexpression of caveolin-1 in lymphoblastoid TK6 cells enhances proliferation after irradiation with clinically relevant doses.

Background And Purpose: The transmembrane protein caveolin-1 (CAV1) is an essential component of caveolae, small membrane invaginations involved in vesicle formation. CAV1 plays a role in signal transduction, tumor suppression and oncogene transformation. Previous studies with CAV1 knockout mice and CAV1 knockdown in pancreatic tumor cells implicated CAV1 in mediating radioresistance.

Pseudotyped recombinant adeno-associated viral vectors mediate efficient gene transfer into primary human CD34(+) peripheral blood progenitor cells.

Background And Aims: Because of their pluripotency, human CD34(+) peripheral blood progenitor cells (PBPC) are targets of interest for the treatment of many acquired and inherited disorders using gene therapeutic approaches. Unfortunately, most current vector systems lack either sufficient transduction efficiency or an appropriate safety profile. Standard single-stranded recombinant adeno-associated virus 2 (AAV2)-based vectors offer an advantageous safety profile, yet lack the required efficiency in human PBPC.

Synergistic activity of nilotinib and established chemotherapeutic drugs in imatinib-sensitive and -resistant BCR-ABL-positive cells.

We investigated various combination treatment regimens employing nilotinib with established chemotherapeutic agents (daunorubicin, mitoxantrone, etoposide and cytarabine) in imatinib-sensitive and -resistant BCR-ABL-positive cells. Mitoxantrone or cytarabine showed synergism (CI < 1) in combination with nilotinib in imatinib-sensitive LAMA84 cells, whereas in imatinib-resistant LAMA84-R cells synergistic effects could be assessed for daunorubicin, mitoxantrone and etoposide when combined with nilotinib. In both imatinib-sensitive and -resistant K562 cells daunorubicin, mitoxantrone and etoposide demonstrated synergism in combination with nilotinib.

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 Imatinib's 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 muM) significantly inhibits SDF-1alpha-mediated chemotaxis and cell migration toward the murine stroma cell line FBMD-1.

Normal-tissue radioprotection by overexpression of the copper-zinc and manganese superoxide dismutase genes.

Background And Purpose: Protection of normal tissue against radiation-induced damage may increase the therapeutic ratio of radiotherapy. A promising strategy for testing this approach is gene therapy-mediated overexpression of the copper-zinc (CuZnSOD) or manganese superoxide dismutase (MnSOD) using recombinant adeno-associated viral (rAAV2) vectors. The purpose of this study was to test the modulating effects of the SOD genes on human primary lung fibroblasts (HPLF) after irradiation.

Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line.

Background: For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low.

Generation of efficient human blood progenitor-targeted recombinant adeno-associated viral vectors (AAV) by applying an AAV random peptide library on primary human hematopoietic progenitor cells.

Objective: Currently standard recombinant adeno-associated virus serotype 2(rAAV2)-based vectors lack the efficiency for gene transfer into primary human CD34(+) peripheral blood progenitor cells (PBPC).

Materials And Methods: An advancement in vector development now allows the generation of rAAV capsid mutants that offer higher target cell efficiency and specificity. To increase the gene transfer into hematopoietic progenitor cells, we applied this method for the first time on primary human CD34(+) PBPC cells.

MDR1 gene transfer using a lentiviral SIN vector confers radioprotection to human CD34+ hematopoietic progenitor cells.

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.

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

Background: 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.

Methods: We followed the engraftment of lentivirally transduced human cord blood (CB) progenitor cells after transplantation into NOD/SCID mice using a self-inactivating HIV-1-derived vector expressing the enhanced green fluorescent protein (EGFP).

Results: The mean of transduction of CD34(+) CB cells was 41%, as deduced from the percentage of EGFP(+) cells before transplantation.

Overexpression of MDR1 using a retroviral vector differentially regulates genes involved in detoxification and apoptosis and confers radioprotection.

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.

Viral determinants of integration site preferences of simian immunodeficiency virus-based vectors.

Preferential integration into transcriptionally active regions of genomes has been observed for retroviral vectors based on gamma-retroviruses and lentiviruses. However, differences in the integration site preferences were detected, which might be explained by differences in viral components of the preintegration complexes. Viral determinants of integration site preferences have not been defined.

The CXCR4 antagonist AMD3100 releases a subset of G-CSF-primed peripheral blood progenitor cells with specific gene expression characteristics.

Objective: AMD3100 is a new CXCR4 antagonist that induces a rapid release of hematopoietic progenitors from the bone marrow to the peripheral blood. We conducted a clinical study where patients with multiple myeloma and non-Hodgkin's lymphoma were treated with AMD3100 (A) to increase the number of peripheral blood progenitor cells (PBPCs) when given a mobilization regimen of granulocyte colony-stimulating factor (G-CSF, G). Because experimental data suggest that A+G-mobilized PBPCs are functionally different from G-mobilized PBPCs, we were interested in an intraindividual comparison of the gene expression profile of CD34+ cells in the two different settings.

Combination treatment of imatinib-sensitive and -resistant BCR-ABL-positive CML cells with imatinib and farnesyltransferase inhibitors.

Background: Resistance to imatinib monotherapy frequently emerges in advanced stages of chronic myelogenous leukemia (CML), supporting the rationale for combination drug therapy. In the present study, the activities of the farnesyltransferase inhibitors (FTIs) L744,832 and LB42918, as single agents and in combination with imatinib, were investigated in different imatinib-sensitive and -resistant BCR-ABL-positive CML cells.

Materials And Methods: Growth inhibition of the cell lines and primary patient cells was assessed by MTT assays and colony-forming cell assays, respectively.

Retroviral integration sites correlate with expressed genes in hematopoietic stem cells.

In this study, we analyzed whether retroviral integration sites in repopulating hematopoietic cells correlate with genes expressed in fractions enriched in hematopoietic stem cells (HSCs). We have previously described microarray studies of two populations enriched in HSCs: CD34+/CD38- and the slow dividing fraction of CD34+/CD38- cells (SDF). Furthermore, we demonstrated that oncoretroviral integrations in severe combined immunodeficient repopulating cells are preferentially located near the transcription start.