ALBERT

Description: The mammalian target of rapamycin (mToR) plays a crucial role in cell growth due to its role as nutrient dependent regulator of important cytokine signalling pathways. In multiple myeloma (MM) mToR is involved in the AKT pathway which can be activated by the loss of the tumor suppressor PTEN or stimulation with growth and survival factors such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1). mToR can be blocked by rapamycin or everolimus. When various human plasmacytoma lines were tested, rapamycin and everolimus induced a dose-dependent growth inhibition. The addition of bone marrow stromal cells, IL-6 or IGF-1 was unable to prevent the inhibitory activity. Growth inhibition was mediated not only by G1 cell cycle arrest but also by an induction of apoptosis as measured by propidium iodide staining. Further analysis was performed to elucidate the mechanism of apoptosis in the particular sensitive plasma cell line INA-6. mToR inhibition induced exclusively cleavage of pro-caspase 8 (extrinsic pathway) but not of pro-caspase 9 (intrinsic pathway). To confirm this observation, INA-6 cells were transfected with BCL-XL, an inhibitor of the intrinsic pathway, and treated with rapamycin. The transfectants showed similar sensitivity to rapamycin as the paternal cell line. Rapamycin was also tested in the INA-6 SCID mouse xenograft model. Rapamycin was given orally for two weeks starting 24 h after tumor inoculation. A significant survival benefit of the rapamycin treated group compared to control animals (p= 0.0004) was observed. Six out of 14 treated mice did not develop plasmacytomas during the observation period of 149 days. In addition, even short term treatment of plasmacytoma bearing mice led to a significant shrinkage of the tumor mass. Histological examination revealed an increase of apoptotic plasma cells according to morphology after 72 h of treatment compared to controls. Induction of apoptosis in tumor cells was confirmed by immunohistological staining using an antibody specific for the human cleaved form of poly (ADP-ribose) polymerase. Since drugs inhibiting mToR are inducing apoptosis in human malignant plasma cells not only in vitro but also in vivo, the inhibition of mToR may represent an attractive new concept for multiple myeloma therapy.

Description: Treatment of chronic myeloid leukemia (CML) with the tyrosine kinase inhibitor imatinib represents a successful application of a molecularly targeted therapy. A rapid hematologic and cytogenetic response can be induced for the majority of patients even in advanced disease. However, the time course of disappearance of leukemia cells, characterized by the expression of the BCR-ABL fusion protein, varies between patients, and a complete eradication of the malignant cells is a rare event. The reasons for the heterogeneous response and the persistence of the malignant clone in many patients are currently not known. We propose a mathematical model which consistently explains short and long-term dynamics of BCR-ABL transcript levels in populations of CML patients under imatinib monotherapy. The model is based on the concept that normal and malignant cell clones compete for growth environments in which they behave slightly differently with regard to homing and cell cycle activation/deactivation. This concept has been successfully applied for understanding time-dependent chimerism in mice [Roeder et al.: Blood 105(2):609]. Applying the model to data sets from two independent cohorts of imatinib treated CML patients, we demonstrate the potential of our model to quantitatively describe the typical biphasic decline in BCR-ABL transcript levels during the first year of treatment. Besides the median transcript dynamics in the patient population the model is able to represent the heterogeneity in individual transcript time courses. Qualitative differences in the imatinib response are explained by small quantitative differences in the drug effects regarding proliferation inhibition and/or induction of apoptosis for BCR-ABL positive cells. As demonstrated by comparison with five years follow-up data of 69 unselected newly diagnosed CML patients recruited into the IRIS trial in Germany [Mueller et al.: Leukemia 17(12):2392] the model also correctly describes long-term BCR-ABL dynamics. The observed median BCR-ABL transcript levels, including the vanishing decline after year four of treatment, can quantitatively be explained by a decreasing treatment efficiency in a subset of patients, potentially caused by imatinib-resistant clones. Sensitivity analyses show that moderate functional differences of the resistance mutations can lead to remarkable differences in long-term treatment efficiency. On the other hand, in patients not developing resistance mutations our model predicts the general chance of an eradication of the malignant clone in the long run. This is supported by data in a patient subgroup showing a continued decline of BCR-ABL transcript levels over five years of treatment. Beyond the consistent description of the clinically observed BCR-ABL dynamics we provide testable predictions for effects of different combination treatments. Based on the explanation of CML as a clonal competition of malignant and normal hematopoietic stem cells, our model particularly predicts that the therapeutic benefit of imatinib can be augmented by a combination with proliferation stimulating treatment strategies. In addition the model permits to describe the heterogeneity of the effect of resistance mutations with respect to specific treatment strategies. In summary, our model describes CML dynamics under imatinib therapy with potential implications for the design of future treatment strategies.

Description: We report the results of haplocompatible peripheral blood stem cell transplantation (PBSCT) utilizing CD34+ selection and T-cell depletion for 17 patients with severe combined immunodeficiency disease (SCID). Of these patients, 11 had T−B−NK+, 1 had T−B+NK+, and 5 had T−B+NK− phenotype. A total of 15 cell preparations were processed immediately after collection, in which two were shared between two twin siblings. Total viable nucleated cells (TVNC) in the original cell collections were between 5.9 and 9.13×1010 (median 7.0×1010) with 0.47–2.39% (median 0.9%) CD34+ cells. After Isolex 300i (Baxter Inc., n=14) or CliniMACS System (Miltenyi Biotec Inc., n=1) processing, a median number of 379×106 (89–970×106) TVNC were recovered, with a median viability of 98% (83–100%) and median purity of 96% (89–100%) CD34+ cells. All Isolex processed (n=14) cells were further T-cell depleted with OKT3 monoclonal antibody, yielding a median of 0.09% CD3+ cells (0.008–0.4%). One preparation that utilized the CliniMACS System yielded only 0.06% CD3+ cells, hence did not receive further T-cell depletion. Recovery of CD34+ cells after complete processing was from 13.3% to 60.2% (median 50.1%). Twelve patients (70.6%) are alive 2 months to 8.7 years post transplant. A total of five patients died from infections or transplant-related complications. Four patients suffered from autoimmune hemolytic anemia, which resulted in one death. Fourteen patients engrafted. One of three patients who did not engraft subsequently received a boost from the same donor but eventually died without engraftment, and 2 received a matched unrelated BMT with myeloablative conditioning and recovered T- and B-cell function. At last follow-up the median time for the recovery of T- and B-cell function was 8.5 months and 1 year, respectively. The dose of CD3 did not show any influence on T- or B-cell function recovery (p=0.48 and 0.09, respectively). And the dose of CD34+ cells did not influence T-cell function recovery (p=0.1), but did influence B-cell function recovery, which was statistically significant (p=0.02). The B− SCID phenotype is associated with a poorer outcome compared to the B+ SCID phenotype, with 50% and 100% survival rates, respectively. However, this result was not statistically significant (p=0.07). Of the 9 surviving patients followed for more than 2 years, most are in good general health. The body height growth curve is within the 5th and 10–25th percentiles in 3 and 4 patients, respectively. For body weight, the growth curve is within 10–25th and 50–75th percentiles in 5 and 2 patients, respectively. Five have achieved successful recovery of both T- and B-cell immunity and require no medication at last visit; however, 2 of these had graft failure following their initial haplocompatible transplant, and received a second BMT from a matched unrelated donor with conditioning. Three patients with X-linked SCID (3/5) didn’t achieve B-cell reconstitution and still require IVIG replacement therapy. Based on these results, we conclude that for SCID patients who lack an HLA-matched related donor, CD34+ selected T-cell depleted haplocompatible PBSCT is an effective treatment.

Description: Acute myelogenous leukemia (AML) is often associated with mutations in transcription factors that are essential for normal hematopoietic development and differentiation. The basic leucine zipper (bZIP) protein C/EBPalpha is mutated in 7–10% of AML. Two different classes of C/EBPalpha mutations are found in AML: 1) mutations that introduce a stop codon between the primary translational initiation site and a downstream ATG, resulting in translation of an N-terminally truncated protein (p30) 2) mutations that disrupt the basic region of the bZIP domain in the C-terminus, abolishing DNA binding. Of note, biallellic mutations are often but not always observed. It has been proposed that p30 is dominant-negative over wt C/EBPalpha (p42), blocking its transcriptional activity. We have previously shown that p30 blocks erythrocytic commitment and inhibits terminal granulocytic differentiation when expressed in human but not murine primary hematopoietic cells. To identify the molecular mechanism by which the p30 inhibits differentiation and to evaluate the role of homo- and heterodimerization between p30 and p42 in leukemogenesis, we created a p30 that carries point mutations in bZIP (p30-L12V) that disrupt dimerization. Expression of p30-L12V had no effect on the differentiation of human CD34+ cells, arguing for the requirement of dimerization. To inhibit potential heterodimerization between p30 and p42 but permit p30 homodimerization, we generated a p30-GZ, in which the leucine zipper was replaced by an artificial dimerization domain. Expression of p30-GZ induced a similar differentiation block as the normal p30, arguing against a dominant-negative function of p30 over p42 mediated by dimerization. In a second approach to understand the mechanism by which p30 inhibits differentiation, we wanted to identify gene targets of p30. For this purpose, p30 was fused to the modified ligand-binding domain of the estrogen receptor (p30-ERtm). Expression of p30-ERtm in CD34+ cells in the presence of tamoxifen showed a phenotype very similar to the one observed after the expression of p30, while in the absence of tamoxifen no effect was observed. We used this system of inducible p30 expression in human CD34+ cells to evaluate gene expression patterns. If p30 is dominant-negative and interferes with the transcriptional activation of p42, reciprocal expression patterns of target genes should be observed for wt C/EBPalpha and p30. Our data show that homodimerization of p30 is necessary and sufficient to block erythrocytic commitment and terminal granulocytic differentiation of human CD34+ cells and to induce known C/EBPalpha target genes. These data argue that the formation of p30/p42 heterodimers is not required for the effect of C/EBPalpha p30, questioning the proposed dominant-negative function.

Description: Preclinical animal models are important for evaluating the safety and therapeutic efficacy of new therapeutic modalities such as gene therapy. From the different large animal models, nonhuman primate models have emerged over the last decades as highly desirable experimental systems from both a pathophysiologic and pharmacokinetic viewpoint and the study of nonhuman primates has provided important information on the efficacy and safety of gene therapy systems in vivo prior to human trials. The common marmoset (Callithrix jacchus) has the advantage that it is a small, and thus relatively inexpensive nonhuman primate model. Currently, very little data on the transduction efficiency of foamyviral vectors for gene transfer into marmoset stem cells exists. We therefore performed a direct comparison using identically designed gammaretroviral, lentiviral and foamyviral vector constructs expressing the enhanced green fluorescent protein (EGFP) from the spleen focus forming virus (SFFV) promoter pseudotyped with either the modified human foamy virus (HFV) envelope EM140 or the G-protein of vesicular stomatitis virus (VSV-G) for the transduction of common marmoset embryonic stem cells (CMES) as well as marmoset CD34+ hematopoietic progenitor cells. Virus stocks of these vectors were prepared by polyethyleneimine-mediated transfection of 293T cells and concentrated approximately 10-fold by centrifugation for 4 hours at 10.000 g at 4°C. Three different target cell populations were transduced: common marmoset embryonic stem cells (CMES) or cryopreserved CD34-enriched cells from bone marrow of a common marmoset either after a two-day prestimulation in the presence of IL-6, FLT3L, cSCF and TPO at a concentration of 100 ng/mL each, or after overnight incubation with 100 ng/mL SCF only. Equal numbers of cells were exposed to the four different vector preparations for 14 hours in 12-well dishes coated with CH-296. The read-out was based on fluorescence microscopy of colonies plated in methyl cellulose as well as flow cytometry (FACS). Foamyviral vectors with the foamyviral envelope were the most efficient gene transfer tool for marmoset hematopoietic CD34-positive cells with stable transduction rates of over 80% as assessed by flow cytometry at both 2 or 7 days after the end of transduction and on average 88% transduction efficiency into colony forming cells (CFU-C). Transduction of CFU-C with all other vector preparations was below 60%. In CMES, initial gene transfer rates of over 80% were achieved with the VSV-G pseudotype lentiviral vector, however, expression decreased to 13% after 7 days. In contrast, the foamyviral vector pseudotyped with the foamyviral envelope decreased only from 49% to 24% after 7 days. In conclusion, we achieved stable viral gene transfer and expression in CMES cells as well as highly efficient gene transfer into common marmoset hematopoietic CD34 positive cells using foamyviral vectors. These results suggest that foamyviral vectors may be highly feasible vectors for stem cell gene transfer and thus set the stage for a more detailed analysis of this vector system in transplantation studies in this nonhuman primate model.

Description: Interactions between tumorigenic cells and the microenvironment are increasingly recognized as integral to tumor progression in a range of human malignancies. However, the specific cellular mechanisms that are required to initiate these multistage processes are incompletely understood. Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1, a pandemic autosomal dominant genetic disorder of the nervous system characterized by the development of neurofibromas. Neurofibromas are complex tumors composed of Schwann cells, fibroblasts, endothelial cells, and high concentrations of degranulating mast cells. Though neurofibromas are generally benign, plexiform neurofibromas can progress to malignancy. Genetic studies in cre/lox mice indicate that nullizygous loss of Nf1 in the tumorigenic Schwann cells (Krox20; Nf1flox/flox) is necessary, but not sufficient for neurofibroma formation when the microenvironment is wildtype. However, neurofibromas form with 100% penetrance in Krox20; Nf1flox/− mice that are heterozygous at Nf1 in all lineages of the tumor microenvironment (Science, 2002). Here, we addressed the role of the hematopoietic system in the tumor microenvironment by using adoptive transfer. Syngeneic Nf1+/− or wildtype (WT) bone marrow was transplanted into lethally irradiated Krox20;Nf1flox/flox mice. Krox20; Nf1flox/flox recipients transplanted with WT bone marrow (n=25) did not develop plexiform neurofibromas and had a normal lifespan. In contrast, Krox20; Nf1flox/flox mice transplanted with Nf1+/− bone marrow (n=25) consistently developed neurofibromas infiltrated with Nf1+/− mast cells. These mice had a 90% mortality at 14 months following transplantation. In complementary experiments, WT bone marrow was transplanted into irradiated Krox20; Nf1flox/− mice. Despite the remainder of the tumor microenvironment being heterozygous, WT bone marrow was sufficient to prevent tumor progression in Krox20; Nf1flox/− mice. To specifically assess the role of the mast cell compartment in tumor progression, Nf1+/− mice were intercrossed with two strains of naturally occurring W mutant mice that have variably diminished c-kit activity and mast cell function. Mice homozygous at the Wv locus have a greater than 90% reduction in c-kit activity, while W41/W41 mutants have approximately a 65–75% reduction in c-kit activity. Importantly, while Krox20;Nf1flox/flox mice transplanted with Nf1+/− bone marrow consistently develop plexiform neurofibromas, adoptive transfer of Nf1+/−; Wv/Wv or Nf1+/−; W41/W41 bone marrow cells into Krox20; Nf1flox/flox mice was sufficient to prevent neurofibroma formation. Collectively, these studies provide genetic evidence that the hematopoietic system and specifically mast cells are integral to plexiform neurofibroma formation in genetically engineered mice. These studies have therapeutic implications for NF1 since molecular therapies directed at the haploinsufficient hematopoietic cells, particularly the c-kit receptor tyrosine kinase, may have an important role in treating or preventing plexiform neurofibromas.

Description: The RUNX1 gene encodes an alpha subunit of the core-binding factor (CBF), an important heterodimeric transcription factor in hematopoietic ontogeny and development, and is one of the most frequently disrupted genes in acute leukemia. In addition to its involvement in several translocations, the RUNX1 gene is often subject to deletions or point mutations in acute myelogenous leukemia (AML). Interestingly, in addition to complete loss-of-function mutations, many of the alterations involve missense point mutations within the Runt domain that disrupt DNA binding activity (DB-mutants). In vitro assays have suggested that these DB mutants have a dominant-negative (DN) activity, presumably due to their ability to bind and sequester CBF beta but inability to bind DNA. A strict correlation between the type of mutation and its monoallelic or biallelic incidence is not apparent even though a DN mutant should only affect one allele while a loss of function mutation should affect both alleles. It has been hypothesized that loss of one allele (haploinsufficiency) is sufficient for loss of tumor suppressor activity but the relative high incidence of specific DB mutations suggests a more complex scenario. We thus sought to determine if expression of DB mutants in murine bone marrow (BM) resulted in a similar phenotype as the loss of Runx1, or if these mutations are associated with a gain-of-function. Two RUNX1-DB mutants were thus evaluated using the established retroviral transduction/transplantation mouse model. Between 3 and 6 months after transplantation, peripheral blood, spleen and BM cells were analyzed. Long-term repopulating cells expressing RUNX1 DB-mutants were able to contribute normally to both myeloid and lymphoid compartments, although a disproportionate increase in the B-cell compartment was observed in 3 out of 10 mice. Surprisingly, and inconsistent with a DN activity, disruption of normal T-cell or megakaryocytic development was not observed in the mice, in contrast to Runx1−/+ mice. Significantly, however, replating assays in vitro demonstrated that RUNX1-DB mutants lead to a significant increase in self-renewal activity, in contrast to BM cells of floxed Runx1 mice expressing the Cre recombinase, which showed a less dramatic effect on self-renewal. Colonies derived from CFU-Cs expressing RUNX1-DB mutants were composed of dysplastic granulocytic and monocytic cells, with an increasing number of immature blasts after multiple replatings (〉7), whereas residual colonies from Runx1fl/− BM receiving CRE showed a different morphology with more mature cells. Thus our data suggest that RUNX1-DB mutants do not act in a dominant negative fashion to inhibit normal RUNX1 function, but impart a gain-of-function that results in impaired myeloid differentiation and increased self-renewal potential, consistent with its association with AML.

Description: Signal transducer and activator of transcription 3 (Stat3) is implicated in the pathogenesis of many malignancies and essential for IL-6–dependent survival and growth of multiple myeloma cells. Here, we demonstrate that the gene encoding oncogenic microRNA-21 (miR-21) is controlled by an upstream enhancer containing 2 Stat3 binding sites strictly conserved since the first observed evolutionary appearance of miR-21 and Stat3. MiR-21 induction by IL-6 was strictly Stat3 dependent. Ectopically raising miR-21 expression in myeloma cells in the absence of IL-6 significantly reduced their apoptosis levels. These data provide strong evidence that miR-21 induction contributes to the oncogenic potential of Stat3.

Description: Emerging data suggest a critical role for bone marrow angiogenesis in hematologic malignancies. The angiopoietin/Tie ligand-receptor system is an essential regulator of this process. We evaluated whether circulating angiopoietin-2 (Ang-2) is a predictor for the probability of disease-free survival (DFS) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for high-risk acute myeloid leukemia or myelodysplastic syndrome. Ang-2 was measured by enzyme-linked immunosorbent assay in serum from 20 healthy controls and 90 patients with acute myeloid leukemia or myelodysplastic syndrome before conditioning for HSCT. Circulating Ang-2 was elevated in patients (median, 2.21 ng/mL; range, 0.18-48.84 ng/mL) compared with controls (median, 0.87 ng/mL; range, 0.27-4.51 ng/mL; P 〈 .001). Multivariate analyses confirmed the independent prognostic impact of Ang-2 (hazard ratio [HR] = 2.46; 95% confidence interval [CI], 1.27-4.76, P = .005), percentage of bone marrow infiltration (HR = 1.14; 95% CI, 1.01-1.29, P = .033), and chemotherapy cycles before HSCT (HR = 1.38; 95% CI, 1.01-1.08, P = .048). Regression tree analysis detected optimal cutoff values for Ang-2 and recursively identified bone marrow blasts and Ang-2 as the best predictors for DFS. Because few predictors for DFS exist in the setting of allo-HSCT, Ang-2 may be used as a readily available powerful biomarker to pre-estimate DFS and may open new perspectives for risk-adapted treatment of high-risk myeloid malignancies.