Boris Fehse

Reversal of P-glycoprotein–Mediated Multidrug Resistance by the Murine Double Minute 2 Antagonist Nutlin-3

Murine double minute 2 (MDM2) negatively regulates the activity of the tumor suppressor protein p53. Nutlin-3 is a MDM2 inhibitor under preclinical investigation as nongenotoxic activator of the p53 pathway for cancer therapy. Here, nutlin-3 was evaluated for its activity alone or in combination with established chemotherapeutic drugs for antitumor action in chemosensitive and chemoresistant neuroblastoma and rhabdomyosarcoma cell lines. Effects of nutlin-3 single treatment were much more pronounced in p53 wild-type cell lines (IC(50)s <3 micromol/L) than in p53-mutated cell lines (IC(50)s >17 micromol/L). In sharp contrast to the expectations, nutlin-3 concentrations that did not affect viability of p53-mutated cell lines strongly increased the efficacy of vincristine in p53-mutated, P-glycoprotein (P-gp)-overexpressing cell lines (decrease in IC(50)s 92- to 3,434-fold). Similar results were obtained for other P-gp substrates. Moreover, nutlin-3 reduced efflux of rhodamine 123 and other fluorescence dyes that are effluxed by P-gp. Investigation of Madin-Darby canine kidney (MDCK) II cells stably transfected with plasmids encoding for P-gp (MDCKII MDR1) or multidrug resistance protein 1 (MRP-1, MDCKII MRP1) revealed that nutlin-3 not only interferes with P-gp but also affects MRP-1-mediated efflux. Kinetic studies and investigation of P-gp-ATPase activity showed that nutlin-3 is likely to act as a P-gp transport substrate. Examination of the nutlin-3 enantiomers nutlin-3a and nutlin-3b revealed that, in contrast to MDM2-inhibitory activity that is limited to nutlin-3a, both enantiomers similarly interfere with P-gp-mediated drug efflux. In conclusion, nutlin-3-induced inhibition of P-gp and MRP-1 was discovered as a novel anticancer mechanism of the substance in this report.

Quantitative monitoring of NPM1 mutations provides a valid minimal residual disease parameter following allogeneic stem cell transplantation

BACKGROUNDnMinimal residual disease (MRD) diagnostics in acute myeloid leukemia (AML) gain increasing importance after allogeneic stem cell transplantation (SCT). Nucleophosmin (NPM1) mutations, with their high frequency in AML, were suggested to represent suitable MRD markers, but so far no study has evaluated their usefulness in the posttransplantation period.nnnMATERIALS AND METHODSnWe evaluated the validity of this MRD marker in the posttransplantation period in a cohort of 13 patients with an NPM1A mutation (NPM1Amut). For this most frequent NPM1A subtype, quantitative real-time polymerase chain reaction (qPCR) was retrospectively performed on bone marrow/peripheral blood samples that had been taken before and after SCT.nnnRESULTSnNPM1Amut was retrospectively followed up in 13 patients who received 14 transplantations. One-hundred and thirty-nine qPCR analyses were performed (median: 7 time points; median follow-up: 216 days; range, 35-1825 days). After SCT, 10 of 14 NPM1Amut cases (71%) became PCR-negative, of which four achieved stable remissions. All four patients (29%) who remained NPM1Amut-positive after SCT relapsed. In all nine relapse cases, increases of NPM1Amut were seen that preceded morphological relapse and the decrease of molecular chimerism with mean intervals of 24 days (range, 12-38 days) and 15 days (range, 1-36 days), respectively.nnnCONCLUSIONSnQuantitative assessment of NPM1Amut seems to provide a reliable MRD marker in the posttransplantation period, predicting relapse earlier than morphology or molecular chimerism, which should be confirmed in larger studies.

Aldehyde dehydrogenase activity as a marker for the quality of hematopoietic stem cell transplants

Summary:Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH), hematopoietic stem cells (HSC) were defined as SSCloALDHbr – reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon, we investigated the usefulness of ALDH activity for characterizing HSC graft quality, particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time, and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48u2009h. We then retrospectively related ALDH and CD34 expression as well as granulocyte–macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly, in all six patients who received markedly decreased numbers of SSCloALDHbr cells, this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism, whereas all other patients showed early complete donor chimerism. In conclusion, we suggest to measure ALDH activity as a surrogate marker for HSC activity, and to transport and store PBSC under controlled cooling conditions.

Stem Cell Marking With Promotor-deprived Self-inactivating Retroviral Vectors Does Not Lead to Induced Clonal Imbalance

Stable genetic modification of stem cells holds great promise for gene therapy and marking, but commonly used gamma-retroviral vectors were found to influence growth/survival characteristics of hematopoietic stem cells (HSCs) by insertional mutagenesis. In this article, we show that promoter-deprived gamma-retroviral self-inactivating (pd-SIN) vectors allow stable genetic marking of serially reconstituting murine HSC. In contrast to findings with gamma-retroviral long terminal repeat (LTR) vectors, serial transplantation of pd-SIN-marked HSC in a sensitive mouse model was apparently not associated with induced clonal imbalance of gene-marked HSC. Furthermore, insertions of pd-SIN into protooncogenes, growth-promoting and signaling genes occurred significantly less frequent than in control experiments with LTR vectors. Also, transcriptional dysregulation of neighboring genes potentially caused by the pd-SIN insertion was rarely seen and comparatively weak. The integration pattern of promotor-deprived SIN vectors in reconstituting HSC seems to depend on the transcriptional activity of the respective gene loci reflecting the picture described for LTR vectors. In conclusion, our data strongly support the use of SIN vectors for gene-marking studies and suggest an increased therapeutic index for vectors lacking enhancers active in HSC.

Screening and monitoring of MPL W515L mutation with real-time PCR in patients with myelofibrosis undergoing allogeneic-SCT

Monitoring of minimal residual disease (MRD) after allogeneic (allo)-SCT for myelofibrosis (MF) allows recognizing the depth of remission and thus guides application of appropriate therapeutic interventions. MPL W515L/K mutations, which are detected in 5–10% of JAK2V617F-negative patients, may be useful for this purpose. Using a highly sensitive quantitative PCR method, we tested 90 patients with MF who underwent allo-SCT for the presence of MPL W515L/K mutations. Two patients with primary MF were found to harbor MPLW515L while no patient was positive for MPLW515K mutation. Both patients were JAK2V617F negative and cleared the mutation rapidly after allo-SCT and remained negative for a median follow-up of 19 months. The results of molecular monitoring correlated well with other remission parameters such as normalization of peripheral blood counts and morphology and complete donor chimerism. We conclude that MPLW515L can be cleared after allo-SCT and hence may be used as an MRD marker in a proportion of JAK2V617F-negative MF patients.

Monitoring of minimal residual disease in multiple myeloma after allo-SCT : flow cytometry vs PCR-based techniques

Monitoring of minimal residual disease in multiple myeloma after allo-SCT: flow cytometry vs PCR-based techniques

Transportation and cryopreservation may impair haematopoietic stem cell function and engraftment of allogeneic PBSCs, but not BM

Recent data suggest that the practice of using frozen allogeneic grafts is becoming increasingly common among transplant centres. Therefore, we retrospectively analysed 31 frozen allogeneic PBSC and 8 BM grafts by flow cytometry with regard to their CD34+ content, membrane integrity (7-AAD) and stem cell-specific enzyme activity (aldehyde dehydrogenase, ALDH) in relation to individual transplantation results. Membrane integrity of CD34+ cells was significantly impaired in cryopreserved PBSC but not in BM compared to unfrozen allografts. In 9 out of 31 frozen PBSC (but none of the BM) grafts numbers of SSCloALDHbr cells per kg body weight (BW) were significantly reduced while in the same grafts the numbers of CD34+ cells per kg BW were close to normal. Overall, 9 out of 33 patients (27%) who received unrelated PBSC allografts cryopreserved after transportation did not achieve engraftment. For comparison, primary graft failure was observed in our centre in only 7 out of 493 recipients (1.4%) of fresh allogeneic PBSC grafts. Moreover, we did not see any graft failure in patients receiving frozen/thawed BM or autologous PBSC transplants. We, therefore, conclude that PBSC grafts become much more sensitive to cryopreservation after transport and/or storage. Importantly, the engraftment potential of frozen HSC grafts may reliably be predicted by measuring ALDH activity.

Retroviral insertion site analysis in dominant haematopoietic clones.

Identification of retroviral vector insertion sites in single, dominating cell clones has become an important tool for the investigation of cellular signalling pathways involved in clonal expansion and malignant transformation. Also, recent severe adverse events in clinical trials resulting from retroviral vector-mediated insertional mutagenesis underline the need of well-designed safety studies including integration site analyses to estimate cost/benefit ratios in gene therapy. We have recently described a modified ligation-mediated PCR (LM PCR) method allowing preferential retrieval of insertion sites causally linked to clonal dominance of an affected clone. In the first part of the given work we focus on particularities of the LM PCR procedure to be taken into account when working with self-inactivating as compared to classical retrovectors. In the following sections we focus on data acquisition, processing, organisation, and analysis. Thus the protocol presented here should be helpful in establishing and utilising databases of retroviral integration sites.

Influence of mannose-binding lectin genotypes and serostatus in allo-SCT: analysis of 131 recipients and donors

Mannan-binding lectin (MBL) deficiency is determined by MBL gene polymorphisms and is associated with an increased infection risk. To clarify the role of MBL in Allo-SCT, 131 recipients–donors were analysed. MBL genotypes were determined by PCR and heteroduplex analyses, MBL serum levels by ELISA, and MBL oligomers by western blotting. MBL levels <400u2009ng/ml were associated with increased susceptibility to fungal pneumonia (7/12 vs 35/111; P=0.04, adjusted P=0.002), HSV/VZV (7/12 vs 26/111; P=0.03), CMV reactivation and acute GVHD. Donor genotypes had no influence. Pre-SCT MBL levels corresponded to recipients’ genotypes (P<0.001), changed significantly post-SCT, but were not influenced by donors’ genotypes. MBL oligomer profiles were similar pre-/post-SCT. Cultured CD34+ cells were found not to synthesise MBL. In conclusion, low MBL levels pre-transplant predisposed patients to sepsis, fungal and viral infection. Donors’ MBL genotypes did not influence infection rates. Prospective studies should clarify the importance of MBL as a prelude for MBL replacement after SCT.

Retroviral insertional mutagenesis can contribute to immortalization of mature T lymphocytes.

Several cases of T-cell leukemia caused by gammaretroviral insertional mutagenesis in children treated for x-linked severe combined immunodeficiency (SCID) by transplantation of autologous gene-modified stem cells were reported. In a comparative analysis, we recently showed that mature T cells, on the contrary, are highly resistant to transformation by gammaretroviral gene transfer. In the present study, we observed immortalization of a single T-cell clone in vitro after gammaretroviral transduction of the T-cell protooncogene LMO2. This clone was CD4/CD8 double-negative, but expressed a single rearranged T-cell receptor. The clone was able to overgrow nonmanipulated competitor T-cell populations in vitro, but no tumor formation was observed after transplantation into Rag-1 deficient recipients. The retroviral integration site (RIS) was found to be near the IL2RA and IL15RA genes. As a consequence, both receptors were constitutively upregulated on the RNA and protein level and the immortalized cell clone was highly IL-2 dependent. Ectopic expression of both, the IL2RA chain and LMO2, induced long-term growth in cultured primary T cells. This study demonstrates that insertional mutagenesis can contribute to immortalization of mature T cells, although this is a rare event. Furthermore, the results show that signaling of the IL-2 receptor and the protooncogene LMO2 can act synergistically in maligniant transformation of mature T lymphocytes.