Structurally diverse MDM2-p53 antagonists act as modulators of MDR-1 function in neuroblastoma.

BACKGROUND: A frequent mechanism of acquired multidrug resistance in human cancers is overexpression of ATP-binding cassette transporters such as the Multi-Drug Resistance Protein 1 (MDR-1). Nutlin-3, an MDM2-p53 antagonist, has previously been reported to be a competitive MDR-1 inhibitor. METHODS: This study assessed whether the structurally diverse MDM2-p53 antagonists, MI-63, NDD0005, and RG7388 are also able to modulate MDR-1 function, particularly in p53 mutant neuroblastoma cells, using XTT-based cell viability assays, western blotting, and liquid chromatography-mass spectrometry analysis. RESULTS: Verapamil and the MDM2-p53 antagonists potentiated vincristine-mediated growth inhibition in a concentration-dependent manner when used in combination with high MDR-1-expressing p53 mutant neuroblastoma cell lines at concentrations that did not affect the viability of cells when given alone. Liquid chromatography-mass spectrometry analyses showed that verapamil, Nutlin-3, MI-63 and NDD0005, but not RG7388, led to increased intracellular levels of vincristine in high MDR-1-expressing cell lines. CONCLUSIONS: These results show that in addition to Nutlin-3, other structurally unrelated MDM2-p53 antagonists can also act as MDR-1 inhibitors and reverse MDR-1-mediated multidrug resistance in neuroblastoma cell lines in a p53-independent manner. These findings are important for future clinical trial design with MDM2-p53 antagonists when used in combination with agents that are MDR-1 substrates.

Gene therapy for paediatric leukaemia.

Improvements in the chemotherapeutic and transplant regimens have had a significant impact in improving survival rates for paediatric leukaemia. However, there are still important problems to address including what options are available for patients with chemoresistant disease and what strategies are available to avoid the concerns regarding the toxicity associated with highly cytotoxic treatment regimens. Gene therapy and immunotherapy protocols hold great promise. Using gene transfer of a marker gene, a number of biological issues in the therapy of leukaemia have been addressed. For example, by gene marking autologous bone marrow grafts it has been possible to demonstrate that infused marrow contributes to relapse in acute and chronic myeloid leukaemias. In the allogeneic transplant setting, genetically modified T-cells have proven valuable for the prophylaxis and treatment of viral diseases and may have an important role in preventing or treating disease relapse. Gene transfer is also being used to modify tumour function, enhance immunogenicity, and confer drug-resistance to normal haematopoietic stem cells. With the continued scientific advancements in this field, gene therapy will almost certainly have a major impact on the treatment of paediatric leukaemia in the future.

Cancer vaccines.

Attempts to generate an anticancer immune response in vivo in patients with cancer have taken several forms. Although to date there have been relatively few published studies describing the effects of the approach in hematologic malignancy, that circumstance is expected to change rapidly during the next few years. In solid tumors, it is not known which, if any, of the approaches being explored will be able to produce responses of sufficient effectiveness and duration to be of general clinical value. Despite the documented increase in survival of patients developing an immune response to tumor immunization, no randomized clinical trial has been entirely convincing. As knowledge of the molecular basis of the immune response and of the immune defenses used by cancer cells improves, it is reasonable to expect to see increasing benefits from tumor vaccines, which are likely to complement, long before they replace, conventional therapies.

Transgenic expression of CD40L and interleukin-2 induces an autologous antitumor immune response in patients with non-Hodgkin's lymphoma.

The malignant B cells of non-Hodgkin's lymphoma (B-NHL cells) express peptides derived from tumor-specific antigens such as immunoglobulin idiotypes, and also express major histocompatibility complex antigens. However, they do not express co-stimulatory molecules, which likely contributes to their protection from host antitumor immunity. To stimulate NHL-specific immune responses, we attempted to transfer the human CD40 ligand (hCD40L) gene to B-NHL cells and enhance their co-stimulatory potential. We found that an adenoviral vector encoding human CD40L (AdhCD40L) was ineffective at transducing B-NHL cells because these cells lack the coxsackievirus B-adenovirus receptor and alpha(v) integrins. However, preculture of the B-NHL cells with the human embryonic lung fibroblast line, MRC-5, significantly up-regulated expression of integrin alpha(v)beta 3 and markedly increased their susceptibility to adenoviral vector transduction. After prestimulation, transduction with AdhCD40L increased CD40L expression on B-NHL cells from 1.3+/-0.2% to 40.8+/-11.9%. Transduction of control adenoviral vector had no effect. Expression of transgenic human CD40L on these CD40-positive cells was in turn associated with up-regulation of other co-stimulatory molecules including B7-1/-2. Transduced B-NHL cells were now able to stimulate DNA synthesis of autologous T cells. However, the stimulated T cells were unable to recognize unmodified lymphoma cells, a requirement for an effective tumor vaccine. Based on previous results in an animal model, we determined the effects of combined use of B-NHL cells transduced with AdhCD40L and AdhIL2 vectors. The combination enhanced initial T-cell activation and generated autologous T cells capable of specifically recognizing and killing parental (unmodified) B-NHL cells via major histocompatibility complex--restricted cytotoxic T lymphocytes. These findings suggest that the combination of CD40L and IL2 gene-modified B-NHL cells will induce a cytotoxic immune response in vivo directed against unmodified tumor cells.

Autologous antileukemic immune response induced by chronic lymphocytic leukemia B cells expressing the CD40 ligand and interleukin 2 transgenes.

Although the B cells of chronic lymphocytic leukemia (B-CLL cells) express both tumor-specific peptides and major histocompatibility complex (MHC) class I antigens, they lack the capacity for costimulatory signaling, contributing to their protection against host antitumor immunity. To stimulate CLL-specific immune responses, we sought to transfer the human CD40 ligand (hCD40L) gene to B-CLL cells, using an adenoviral vector, in order to upregulate costimulating factors on these cells. Because efficient gene transduction with adenoviral vectors requires the expression of virus receptors on target cells, including the coxsackievirus B-adenovirus receptors (CAR) and alpha(v) integrins, we cocultured B-CLL cells with human embryonic lung fibroblasts (MRC-5 line). This exposure led to increased expression of integrin alpha(v)beta3 on B-CLL cells, which correlated with higher transduction rates. Using this novel prestimulation system, we transduced B-CLL cells with the hCD40L gene. The Ad-hCD40L-infected cells had higher expression of B7 molecules and induced activation of autologous T cells in vitro, but these T cells could not recognize parental leukemic cells. By contrast, an admixture of Ad-hCD40L-positive cells and leukemic cells transduced with the human interleukin 2 (IL-2) gene produced greater T cell activation than did either immunostimulator population alone. Importantly, this combination generated autologous T cells capable of specifically recognizing parental B-CLL cells. These findings suggest that the combined use of genetically modified CD40L-expressing B-CLL cells in combination with IL-2-expressing B-CLL cells may induce therapeutically significant leukemia-specific immune responses.