Thomas Licht

Histone Deacetylase Inhibitors Induce a Very Broad, Pleiotropic Anticancer Drug Resistance Phenotype in Acute Myeloid Leukemia Cells by Modulation of Multiple ABC Transporter Genes

Purpose: Histone deacetylase inhibitors (HDACi) are being studied in clinical trials with the aim to induce cellular differentiation, growth arrest, and apoptosis of tumor cells. Recent reports suggest that the multidrug resistance-1 (MDR1) gene is regulated by epigenetic mechanisms. To investigate whether additional drug transporters are regulated by HDACi and how this affects cytotoxicity, acute myeloid leukemia (AML) cells were examined. Experimental Design: AML cells were cultured in the presence of phenylbutyrate, valproate, suberoylanilide hydroxamic acid, or trichostatin A and analyzed for drug transporter expression and function as well as sensitivity to anticancer drugs. Results:MDR1, breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRP) 7 and 8 were induced in a dose- and time-dependent manner as shown by semiquantitative PCR. The pattern of gene induction was cell line specific. Phenylbutyrate induced P-glycoprotein and BCRP expression and the efflux of drugs as determined with labeled substrates. KG-1a cells treated with phenylbutyrate developed resistance to daunorubicin, mitoxantrone, etoposide, vinblastine, paclitaxel, topotecan, gemcitabine, and 5-fluorouracil; as a result drug-induced apoptosis was impaired. Chromatin immunoprecipitation revealed the hyperacetylation of histone proteins in the promoter regions of MDR1, BCRP, and MRP8 on valproate treatment. Furthermore, an alternative MRP8 promoter was induced by HDACi treatment. Conclusions: Exposure of AML cells to HDACi induces a drug resistance phenotype broader than the “classic multidrug resistance,” which might negatively affect treatment effectiveness.

Drug selection with paclitaxel restores expression of linked IL-2 receptor γ-chain and multidrug resistance (MDR1) transgenes in canine bone marrow

Unstable expression of transferred genes is a major obstacle to successful gene therapy of hematopoietic diseases. We have investigated in a canine large-animal model whether expression of transduced genes can be recovered in vivo. Mixed-breed dogs had undergone autologous bone marrow transplantation (BMT) with stem cell factor and granulocyte–colony-stimulating factor-mobilized retrovirally marked hematopoietic cells. The bicistronic retroviral vector construct allowed for coexpression of MDR1 and human IL-2 receptor common γ-chain cDNAs. The latter gene is deficient in X-linked severe combined immunodeficiency. After initial high-level expression, P-glycoprotein and the γ-chain were undetectable in blood and bone marrow 17 months post-BMT. Six months later, one dog was treated i.v. with 125 mg/m2 paclitaxel. Three administrations restored expression of the two linked genes to high levels in blood and bone marrow. Two dogs treated with higher paclitaxel doses died from myelosuppression after the first administration. As determined by flow cytometry, both genes were expressed in granulocytes, monocytes, and lymphocytes of the surviving animal. PCR analysis of DNA from peripheral blood confirmed that the retroviral cDNA was increased after paclitaxel treatment, suggesting enrichment of transduced cells. P-glycoprotein was detectable for more than 1 year after cessation of paclitaxel. Repeated analyses of blood and bone marrow aspirates gave no indication of hematopoietic disturbance after BMT with transduced cells and paclitaxel treatment. In summary, we have shown that with the use of a drug-selectable marker gene, chemotherapy can select for cells that express an otherwise nonselected therapeutic gene in blood and bone marrow.

Chemoprotection of Hematopoietic Cells by a Mutant P-Glycoprotein Resistant to a Potent Chemosensitizer of Multidrug-Resistant Cancers

Cancers are frequently chemoresistant because of overexpression of P-glycoprotein. Two different approaches to improve cancer treatment are currently being investigated in clinical trials: inhibition of P-glycoprotein function by reversing agents, and alleviation of leukocytopenia by MDR1 gene transfer to normal bone marrow of patients. We report here that retroviral vectors encoding a mutant P-glycoprotein (MDR1-F983A) protect hematopoietic cells from anticancer drugs even in the presence of trans-(E)-flupentixol, an inhibitor of P-glycoprotein. Transfer of either mutant or wild-type MDR1 to K562 erythroleukemia cells or primary murine bone marrow resulted in reduced accumulation of daunomycin and vinblastine because of increased drug efflux.trans-(E)-Flupentixol at concentrations up to 10 microM failed to reverse drug efflux mediated by the product of the mutant MDR1 while wild-type P-glycoprotein was inhibited. In the presence of 2 microM trans-(E)-flupentixol chemoresistance to daunomycin was circumvented only in K562 cells transduced with wild-type, but not with mutant, MDR1. Moreover, drug resistance of KB-8-5 epidermoid cancer cells, which express the wild-type MDR1 gene at levels comparable to clinical specimens from multidrug-resistant cancers, was fully overcome in the presence of trans-(E)-flupentixol. Vectors expressing mutant P-glycoprotein may help improve chemotherapy by allowing safe dose intensification under conditions in which multidrug-resistant cancers are rendered drug sensitive by reversing agents.

Immunotoxin BL22 induces apoptosis in mantle cell lymphoma (MCL) cells dependent on Bcl-2 expression

Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL has an even worse prognosis and new treatment options are clearly needed. We analysed the effects of BL22, an immunotoxin composed of the Fv portion of an anti‐ CD22 antibody fused to a 38‐kDa Pseudomonas exotoxin‐A fragment on four MCL cell lines as well as on primary cells of four MCL patients. Apoptosis induction by BL22 was much more pronounced in MCL cell lines with low Bcl‐2 expression (NCEB‐1, JeKo‐1 and JVM‐2) compared to Granta‐519 cells with high Bcl‐2 expression. While the expression of the antiapoptotic protein Mcl‐1 declined (NCEB‐1, Granta‐519), Bcl‐2 levels remained unchanged in Granta‐519 cells. However transfection of BCL2 cDNA into NCEB‐1, JeKo‐1 and JVM‐2 cells significantly reduced BL22‐mediated toxicity. Accordingly we examined the effects of Bcl‐2 inactivation in Granta‐519 cells using siRNA. Indeed, apoptosis induction was strongly enhanced in Granta‐519 cells with silenced Bcl‐2. Our results were confirmed in freshly isolated MCL‐cells from patients with leukaemic MCL. We conclude that Bcl‐2 expression is important for mediating resistance against the immunotoxin BL22 in MCL cells.

Efficacy of Consolidation High-Dose Chemotherapy with Ifosfamide, Carboplatin and Etoposide (HD-ICE) Followed by Autologous Peripheral Blood Stem Cell Rescue in Chemosensitive Patients with Metastatic Soft Tissue Sarcomas

Background: Prognosis of patients with metastatic soft tissue sarcomas (MSTS) is poor even after response to doxorubicin-based chemotherapy. We report phase II data of high-dose chemotherapy and peripheral blood stem cell (PBSC) rescue in patients with MSTS responding to AI-G chemotherapy. Patients and Methods: From 1997 to 2002, 55 patients with MSTS were prospectively treated with 4 cycles of AI-G (doxorubicin 75 mg/m2, ifosfamide 6 g/m2 with G-CSF support). Responders received 2 further cycles of AI-G with collection of PBSCs. High-dose chemotherapy consisted of ifosfamide 12 g/m2, carboplatin 1.2 g/m2 and etoposide 1.2 g/m2 (HD-ICE) followed by reinfusion of PBSCs. Results: Twenty-one of 55 patients (38%) were assessed as responders (3 complete response, 18 partial response). All but 2 patients refusing treatment received high-dose chemotherapy with PBSC rescue leading to grade IV hematologic toxicity without severe infections in all patients. No toxic death occurred. After a median follow-up time of 30 months, the median progression-free time was 12 months and survival time was 22 months for the entire group. By intent-to-treat analysis the probability of 5-year progression-free survival was significantly higher for patients allocated to HD-ICE compared to patients receiving second-line chemotherapy after failure of AI-G (14 vs. 3%; p = 0.003). The estimated 5-year overall survival between the 2 groups was different (27% vs. not reached) but did not reach significance (p = 0.08). Conclusion: HD-ICE is feasible and promising in patients with chemosensitive MSTS. A randomized phase III trial is warranted to further define the role of HD-ICE as consolidation treatment in these patients.

Selection and maintenance of multidrug-resistant cells.

Publisher Summary This chapter discusses the selection and maintenance of multidrug-resistant cells, including specialized techniques, such as transfection and transduction of multidrug resistance genes and the introduction of multidrug resistance genes into bone marrow to form drug-resistant bone marrow colony-forming units (cfus). In the former approach, a cell line is carefully cloned so that all the cells in the population derive from a single cell; these cloned cells are exposed to a single cytotoxic agent in doses that kill the great majority of the cells in the population. This approach results in single-step resistance to the agent and individual surviving clones may be screened for cross-resistance to other drugs. The second approach is to expose an uncloned cell population to a selective drug in gradually increasing doses, allowing the population to die back and reexpand after each increase in the stringency of selection. This approach may result in highly multidrug-resistant populations in which the mechanisms of multidrug resistance may be relatively unique, particularly if a potent mechanism is responsible for resistance at an early stage of selection and the derivatives of the original resistant cell have overgrown the population. The advantage of this approach is that it casts a wide net for many different possible kinds of drug resistance.

Retroviral transfer of human MDR1 gene to hematopoietic cells: effects of drug selection and of transcript splicing on expression of encoded P-glycoprotein.

Protection of hematopoietic cells of patients undergoing anticancer chemotherapy by MDR1 gene transfer is currently being studied in clinical trials. From animal studies, it has been suggested that aberrant splicing due to cryptic donor and acceptor sites in the MDR1 cDNA could be a major reason for failure to obtain high-level expression of P-glycoprotein in bone marrow. We investigated effects of drug selection on protein expression levels and on splicing of MDR1 transcripts in murine bone marrow cells (BMCs) in vitro. To this end, retroviruses were generated through an identical plasmid, pHaMDR1/A, introduced into different packaging cells. GP + E86- but not PA317-derived producer cells were found to express truncated in addition to full-length message. In BMCs transduced with GP + E86-derived viruses, both messages were increased after treatment with colchicine or daunomycin. Similar results were obtained with NIH 3T3 fibroblasts. However, transduced and drug-selected BMCs displayed the spliced transcript even if the respective PA317-derived producer cells contained no truncated RNA as detected in transduced NIH 3T3 fibroblasts. Short-term drug selection in BMCs transduced with either ecotropic or amphotropic retroviruses resulted in a striking increase in P-glycoprotein expression. Thus, aberrant splicing failed to abrogate P-glycoprotein expression in BMCs. We also studied a vector in which MDR1 was coexpressed with glucocerebrosidase, using an internal ribosomal entry site. Although chemoprotection was less efficient than with pHaMDR1/A, augmentation of protein expression was observed at low selecting drug concentrations. Our study shows that drug selection can partially compensate for inefficient transduction of hematopoietic cells, and may help to develop strategies by which unstable expression of transduced genes can be overcome.

Expression of multidrug resistance-associated ABC transporters in B-CLL is independent of ZAP70 status

PurposeTo assess whether the poor prognosis of ZAP70-positive B-cell chronic lymphocytic leukemia (CLL) is associated with the overexpression of ABC transporter genes that are responsible for pleiotropic drug resistance.Materials and methodsThe transcript level of ten drug transporters was analyzed using semiquantitative and quantitative RT-PCR in control hematopoietic cells, in 41 CLL patient samples and in 5 lymphoma cell lines. ZAP70 status was determined by immunoblotting.ResultsOf all analyzed transporters, MDR1, MDR2, MRP1, MRP4, MRP5, and MRP7 were expressed at a significantly higher level in B lymphocytes when compared with other hematopoietic cells in peripheral blood. A subgroup of 41 CLL patient samples showed similar or higher expression of these genes than control B cells, and CLL cells exhibited high expression when compared with multiple lymphoma cell lines. No significant correlation between ZAP70 expression and ABC transporter expression was observed.ConclusionThe ZAP70 status is independent of the multidrug resistance phenotype in CLL.

Ifosfamide, epirubicin and etoposide rituximab in refractory or relapsed B-cell lymphoma: Analysis of remission induction and stem cell mobilization

Chemotherapy with ifosfamide, epirubicin and etoposide (IEV) is an effective treatment regimen for refractory/relapsed non-Hodgkin lymphoma (NHL). Rituximab has been shown to improve response rates, progression-free survival and overall survival in B-cell NHL. This study included 85 patients who were treated with IEV or rituximab-IEV (R-IEV) for refractory/relapsed B-cell NHL. The overall response rate was 40.7% (IEV) versus 68.8% (R-IEV). Fever occurred after 23.4% of IEV and 19.4% of R-IEV cycles. 94.9% of patients mobilized sufficient numbers of CD34+ cells (IEV) versus 93.8% (R-IEV). Fifty-five patients (64.7%) proceeded to high-dose therapy after IEV±rituximab. The median survival time was 60.0 months (IEV) and 19.5 months (R-IEV), and has not been reached for patients who received high-dose therapy. The addition of rituximab to IEV salvage chemotherapy increases the response rates in B-cell NHL without affecting stem cell mobilization, but overall survival for patients proceeding to high-dose chemotherapy is not improved.

RETROVIRAL TRANSFER OF MULTIDRUG TRANSPORTER TO MURINE HEMATOPOIETIC STEM CELLS

Publisher Summary This chapter describes a cell preparation procedure and a protocol for transfer of a multidrug resistance gene ( MDR1 ) cDNA that permits expression of functional human P-glycoprotein in mouse stem cells and their progeny in the bone marrow. High numbers of hematopoietic progenitor cells with the capacity of engrafting bone marrow of recipient SCID mice are generated in coculture by stimulation with growth factors. The transfer of the multidrug transporter to hematopoietic stem cells may be useful to ameliorate the adverse effects of anticancer drug treatment. Polycistronic vectors have been constructed in which an MDR1 cDNA is combined with genes that correct genetically determined diseases. Transduction with such vectors might improve low transgene expression, which hampers the clinical gene therapy of hematopoietic disorders.