Wolf K. Hofmann

Long-Term Control of HIV by CCR5 Delta32/Delta32 Stem-Cell Transplantation

Infection with the human immunodeficiency virus type 1 (HIV-1) requires the presence of a CD4 receptor and a chemokine receptor, principally chemokine receptor 5 (CCR5). Homozygosity for a 32-bp deletion in the CCR5 allele provides resistance against HIV-1 acquisition. We transplanted stem cells from a donor who was homozygous for CCR5 delta32 in a patient with acute myeloid leukemia and HIV-1 infection. The patient remained without viral rebound 20 months after transplantation and discontinuation of antiretroviral therapy. This outcome demonstrates the critical role CCR5 plays in maintaining HIV-1 infection.

Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease

Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia.

HMG-CoA Reductase Inhibitors Reduce Senescence and Increase Proliferation of Endothelial Progenitor Cells via Regulation of Cell Cycle Regulatory Genes

Abstract— Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization of ischemic tissue. Ex vivo expansion of EPCs might be useful for potential clinical cell therapy of myocardial ischemia. However, cultivation of primary cells leads to cellular aging (senescence), thereby severely limiting the proliferative capacity. Therefore, we investigated whether statins might be able to prevent senescence of EPCs. EPCs were isolated from peripheral blood and characterized. After ex vivo cultivation, EPCs became senescent as determined by acidic &bgr;-galactosidase staining. Atorvastatin or mevastatin dose-dependently inhibited the onset of EPC senescence in culture. Moreover, atorvastatin increased proliferation of EPCs as assessed by BrdU incorporation and colony-forming capacity. Whereas geranylgeranylpyrophosphate or farnesylpyrophosphate reduced the senescence inhibitory effect of atorvastatin, NO synthase inhibition, antioxidants, or Rho kinase inhibitors had no effect. To get further insights into the underlying downstream effects of statins, we measured telomerase activity and determined the expression of various cell cycle regulatory genes by using a microarray assay. Whereas telomerase activity did not change, atorvastatin modulated expression of cell cycle genes including upregulation of cyclins and downregulation of the cell cycle inhibitor p27Kip1. Taken together, statins inhibited senescence of EPCs independent of NO, reactive oxygen species, and Rho kinase, but dependent on geranylgeranylpyrophosphate. Atorvastatin-mediated prevention of EPC senescence appears to be mediated by the regulation of various cell cycle proteins. The inhibition of EPC senescence and induction of EPC proliferation by statins in vitro may importantly improve the functional activity of EPCs for potential cell therapy.

Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization

Forkhead box O (Foxo) transcription factors are emerging as critical transcriptional integrators among pathways regulating differentiation, proliferation, and survival, yet the role of the distinct Foxo family members in angiogenic activity of endothelial cells and postnatal vessel formation has not been studied. Here, we show that Foxo1 and Foxo3a are the most abundant Foxo isoforms in mature endothelial cells and that overexpression of constitutively active Foxo1 or Foxo3a, but not Foxo4, significantly inhibits endothelial cell migration and tube formation in vitro. Silencing of either Foxo1 or Foxo3a gene expression led to a profound increase in the migratory and sprout-forming capacity of endothelial cells. Gene expression profiling showed that Foxo1 and Foxo3a specifically regulate a nonredundant but overlapping set of angiogenesis- and vascular remodeling-related genes. Whereas angiopoietin 2 (Ang2) was exclusively regulated by Foxo1, eNOS, which is essential for postnatal neovascularization, was regulated by Foxo1 and Foxo3a. Consistent with these findings, constitutively active Foxo1 and Foxo3a repressed eNOS protein expression and bound to the eNOS promoter. In vivo, Foxo3a deficiency increased eNOS expression and enhanced postnatal vessel formation and maturation. Thus, our data suggest an important role for Foxo transcription factors in the regulation of vessel formation in the adult.

Clinical Utility of Microarray-Based Gene Expression Profiling in the Diagnosis and Subclassification of Leukemia: Report From the International Microarray Innovations in Leukemia Study Group

PURPOSE The Microarray Innovations in Leukemia study assessed the clinical utility of gene expression profiling as a single test to subtype leukemias into conventional categories of myeloid and lymphoid malignancies. METHODS The investigation was performed in 11 laboratories across three continents and included 3,334 patients. An exploratory retrospective stage I study was designed for biomarker discovery and generated whole-genome expression profiles from 2,143 patients with leukemias and myelodysplastic syndromes. The gene expression profiling-based diagnostic accuracy was further validated in a prospective second study stage of an independent cohort of 1,191 patients. RESULTS On the basis of 2,096 samples, the stage I study achieved 92.2% classification accuracy for all 18 distinct classes investigated (median specificity of 99.7%). In a second cohort of 1,152 prospectively collected patients, a classification scheme reached 95.6% median sensitivity and 99.8% median specificity for 14 standard subtypes of acute leukemia (eight acute lymphoblastic leukemia and six acute myeloid leukemia classes, n = 693). In 29 (57%) of 51 discrepant cases, the microarray results had outperformed routine diagnostic methods. CONCLUSION Gene expression profiling is a robust technology for the diagnosis of hematologic malignancies with high accuracy. It may complement current diagnostic algorithms and could offer a reliable platform for patients who lack access to todays state-of-the-art diagnostic work-up. Our comprehensive gene expression data set will be submitted to the public domain to foster research focusing on the molecular understanding of leukemias.

Cathepsin L is required for endothelial progenitor cell-induced neovascularization

Infusion of endothelial progenitor cells (EPC), but not of mature endothelial cells, promotes neovascularization after ischemia. We performed gene expression profiling of EPC and endothelial cells to identify genes that might be important for the neovascularization capacity of EPC. Notably, the protease cathepsin L (CathL) was highly expressed in EPC as opposed to endothelial cells and was essential for matrix degradation and invasion by EPC in vitro. CathL-deficient mice showed impaired functional recovery following hind limb ischemia, supporting the concept of a crucial role for CathL in postnatal neovascularization. Infused CathL-deficient progenitor cells neither homed to sites of ischemia nor augmented neovascularization. Forced expression of CathL in mature endothelial cells considerably enhanced their invasive activity and sufficed to confer their capacity for neovascularization in vivo. We concluded that CathL has a critical role in the integration of circulating EPC into ischemic tissue and is required for EPC-mediated neovascularization.

FOXO-dependent expression of the proapoptotic protein Bim: pivotal role for apoptosis signaling in endothelial progenitor cells

Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization. Risk factors for coronary artery disease reduce the number of EPCs in humans. Since EPC apoptosis might be a potential mechanism to regulate the number of EPCs, we investigated the effects of oxidative stress and HMG‐CoA‐reductase inhibitors (statins) on EPC apoptosis. Atorvastatin, mevastatin, or VEGF prevented EPC apoptosis induced by H2O2. The antiapoptotic effect was reversed by inhibition of the PI3K/Akt pathway. Forkhead transcription factors (FOXO1, FOXO3a, FOXO4) exert proapoptotic effects and are phosphorylated and, thereby, inactivated by Akt. Therefore, we elucidated the involvement of forkhead transcription factors. Atorvastatin induced the phosphorylation of the predominant forkhead factor FOXO4 in EPCs. In addition, atorvastatin reduced the expression of the proapoptotic forkhead‐regulated protein Bim in a PI3K‐dependent manner. Consistently, overexpression of FOXO4 activated the Bim promoter as determined by reporter gene expression and stimulated the expression of Bim, resulting in an increased EPC apoptosis. Statins failed to prevent EPC apoptosis induced by overexpression of Bim or nonphosphorylatable FOXO4, suggesting that the protective effects of statins depend on this pathway. In summary, our results show that FOXO‐dependent expression of Bim plays a pivotal role for EPC apoptosis. Statins reduce oxidative stress‐induced EPC apoptosis, inactivate FOXO4, and down‐regulate Bim.

Myelodysplastic Cells in Patients Reprogram Mesenchymal Stromal Cells to Establish a Transplantable Stem Cell Niche Disease Unit

Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin(-)CD34(+)CD38(-) stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.

BCL6-mediated repression of p53 is critical for leukemia stem cell survival in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is induced by the oncogenic BCR-ABL1 tyrosine kinase and can be effectively treated for many years with tyrosine kinase inhibitors (TKIs). However, unless CML patients receive life-long TKI treatment, leukemia will eventually recur; this is attributed to the failure of TKI treatment to eradicate leukemia-initiating cells (LICs). Recent work demonstrated that FoxO factors are critical for maintenance of CML-initiating cells; however, the mechanism of FoxO-dependent leukemia initiation remained elusive. Here, we identified the BCL6 protooncogene as a critical effector downstream of FoxO in self-renewal signaling of CML-initiating cells. BCL6 represses Arf and p53 in CML cells and is required for colony formation and initiation of leukemia. Importantly, peptide inhibition of BCL6 in human CML cells compromises colony formation and leukemia initiation in transplant recipients and selectively eradicates CD34+ CD38− LICs in patient-derived CML samples. These findings suggest that pharmacological inhibition of BCL6 may represent a novel strategy to eradicate LICs in CML. Clinical validation of this concept could limit the duration of TKI treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation.

Gene Expression Profile of Serial Samples of Transformed B-Cell Lymphomas

Follicular lymphoma (FL) is characterized by a continuous rate of relapse and transformation to a high-grade lymphoma, usually diffuse large B-cell lymphoma (DLBCL), associated with a dismal prognosis and a poor response to conventional chemotherapy. The progression of indolent to aggressive FL is accompanied by the successive accumulation of recurrent chromosomal defects, but the resultant alterations of gene expression are largely unknown. To expand the understanding of the pathogenesis of FL transformation, we initially performed oligonucleotide microarray analyses using Affymetrix HuFL chips on five cases with matched snap-frozen lymph nodes before and after transformation. Expression data were analyzed using the Affymetrix Microarray Suite 4.0 and Genespring 4.0. Thirty-six genes with increased expression and 66 genes with decreased expression associated with transformation were identified and functionally classified. The expression of differentially expressed genes was confirmed by real-time quantitative RT-PCR (QRT-PCR) using a total of seven matched pairs and an additional five FL and five unrelated DLBCL. In addition, selected genes were further analyzed by QRT-PCR or immunohistochemistry using a large, unrelated series of FL (grades 1 to 3) as well as transformed and de novo DLBCL (total of 51 samples). The microarray results correlated with the protein expression data obtained from samples at the time of initial diagnosis and transformation. Furthermore, the expression of 25 candidate genes was evaluated by QRT-PCR with a 78% confirmation rate. Some of the identified genes, such as nucleobindin, interferon regulatory factor 4, and tissue inhibitor of metalloproteinases 1, are already known to be associated with high-grade non-Hodgkins lymphoma. Novel candidate genes with confirmed increased and decreased expression in transformed DLBCL include ABL2 and NEK2, and PDCD1 and VDUP1, respectively. In summary, this study shows that transformation of FL to DLBCL is associated with a distinct set of differentially expressed genes of potential functional importance.