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B-cell acute lymphoblastic leukemia (B-ALL) occurs most commonly in children, whereas chronic myeloid leukemia is more frequent in adults. The myeloid bias of hematopoiesis in elderly individuals has been considered causative, but the age of the bone marrow microenvironment (BMM) may be contributory. Using various murine models of B-ALL in young vs old mice, we recapitulated B-ALL preponderance in children vs adults. We showed differential effects of young vs old BM macrophages on B-ALL cell function. Molecular profiling using RNA- and ATAC-sequencing revealed pronounced differences in young vs old BMM-derived macrophages and enrichment for gene sets associated with inflammation. In concordance with the role of C-X-C motif chemokine (CXCL) 13 for disease-associated B-cell chemoattraction, we found CXCL13 to be highly expressed in young macrophages on a translational compared with a transcriptional level. Inhibition of CXCL13 in BM macrophages impaired leukemia cell migration and decreased the proliferation of cocultured B-ALL cells, whereas recombinant CXCL13 increased pAKT and B-ALL cell expansion. Pretreatment of B-ALL-initiating cells with CXCL13 accelerated B-ALL progression. Deficiency of Cxcr5, the receptor for CXCL13, on B-ALL-initiating cells prolonged murine survival, whereas high expression of CXCR5 in pediatric B-ALL may predict central nervous system relapse. CXCL13 staining was increased in bone sections from pediatric compared with adult patients with B-ALL. Taken together, our study shows that the age of the BMM and, in particular, BM macrophages influence the leukemia phenotype. The CXCR5-CXCL13 axis may act as prognostic marker and an attractive novel target for the treatment of B-ALL., (© 2021 by The American Society of Hematology.)

We recently described a positive feedback loop connecting c-MYC, NAMPT, DBC1 and SIRT1 that contributes to unrestricted cancer cell proliferation. Here we determine the relevance of the loop for serrated route intestinal tumorigenesis using genetically well-defined Braf V600E and K-ras G12D mouse models. In both models we show that c-MYC and SIRT1 protein expression increased through progression from hyperplasia to invasive carcinomas and metastases. It correlated with high NAMPT expression and was directly associated to activation of the oncogenic drivers. Assessing functional and molecular consequences of pharmacological interference with factors of the loop, we found that inhibition of NAMPT resulted in apoptosis and reduced clonogenic growth in human BRAF-mutant colorectal cancer cell lines and patient-derived tumoroids. Blocking SIRT1 activity was only effective when combined with a PI3K inhibitor, whereas the latter antagonized the effects of NAMPT inhibition. Interfering with the positive feedback loop was associated with down-regulation of c-MYC and temporary de-repression of TP53, explaining the anti-proliferative and pro-apoptotic effects. In conclusion we show that the c-MYC-NAMPT-DBC1-SIRT1 positive feedback loop contributes to murine serrated tumor progression. Targeting the feedback loop exerted a unique, dual therapeutic effect of oncoprotein inhibition and tumor suppressor activation. It may therefore represent a promissing target for serrated colorectal cancer, and presumably for other cancer types with deregulated c-MYC., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Vitamin K antagonists (VKAs) have been used in 1% of the world's population for prophylaxis or treatment of thromboembolic events for 64 years. Impairment of osteoblast function and osteoporosis has been described in patients receiving VKAs. Given the involvement of cells of the bone marrow microenvironment (BMM), such as mesenchymal stem cells (MSCs) and macrophages, as well as other factors such as the extracellular matrix for the maintenance of normal hematopoietic stem cells (HSCs), we investigated a possible effect of VKAs on hematopoiesis via the BMM. Using various transplantation and in vitro assays, we show here that VKAs alter parameters of bone physiology and reduce functional HSCs 8-fold. We implicate impairment of the functional, secreted, vitamin K-dependent, γ-carboxylated form of periostin by macrophages and, to a lesser extent, MSCs of the BMM and integrin β3-AKT signaling in HSCs as at least partly causative of this effect, with VKAs not being directly toxic to HSCs. In patients, VKA use associates with modestly reduced leukocyte and monocyte counts, albeit within the normal reference range. VKAs decrease human HSC engraftment in immunosuppressed mice. Following published examples that alteration of the BMM can lead to hematological malignancies in mice, we describe, without providing a causal link, that the odds of VKA use are higher in patients with vs without a diagnosis of myelodysplastic syndrome (MDS). These results demonstrate that VKA treatment impairs HSC function via impairment of the BMM and the periostin/integrin β3 axis, possibly associating with increased MDS risk., (© 2019 by The American Society of Hematology.)

Viruses are present in the intestinal microflora and are currently discussed as a potential causative mechanism for the development of inflammatory bowel disease. A number of viruses, such as Human Herpesvirus-8, express homologs to cellular FLIPs, which are major contributors for the regulation of epithelial cell death. In this study we analyzed the consequences of constitutive expression of HHV8-viral FLIP in intestinal epithelial cells (IECs) in mice. Surprisingly, expression of vFlip disrupts tissue homeostasis and induces severe intestinal inflammation. Moreover vFlip IEC-tg mice showed reduced Paneth cell numbers, associated with excessive necrotic cell death. On a molecular level vFlip expression altered classical and alternative NFκB activation. Blocking of alternative NFκB signaling by deletion of Ikka in vivo largely protected mice from inflammation and Paneth cell loss induced by vFLIP. Collectively, our data provide functional evidence that expression of a single viral protein in IECs can be sufficient to disrupt epithelial homeostasis and to initiate chronic intestinal inflammation.

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34 + CD38 - ) as well as the leukemic bulk (CD34 + CD38 + ) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR., (© 2018 by The American Society of Hematology.)

Competing Interests: Conflict-of-interest disclosure: The author declares no competing financial interests.

Similar to their healthy counterpart, malignant hematopoietic stem cells in myeloid malignancies, such as myeloproliferative neoplasms, myelodysplastic syndromes, and acute myeloid leukemia, reside in a highly complex and dynamic cellular microenvironment in the bone marrow. This environment provides key regulatory signals for and tightly controls cardinal features of hematopoietic stem cells (HSCs), including self-renewal, quiescence, differentiation, and migration. These features are essential to maintaining cellular homeostasis and blood regeneration throughout life. A large number of studies have extensively addressed the composition of the bone marrow niche in mouse models, as well as the cellular and molecular communication modalities at play under both normal and pathogenic situations. Although instrumental to interrogating the complex composition of the HSC niche and dissecting the niche remodeling processes that appear to actively contribute to leukemogenesis, these models may not fully recapitulate the human system due to immunophenotypic, architectural, and functional inter-species variability. This review summarizes several aspects related to the human hematopoietic niche: (1) its anatomical structure, composition, and function in normal hematopoiesis; (2) its alteration and functional relevance in the context of chronic and acute myeloid malignancies; (3) age-related niche changes and their suspected impact on hematopoiesis; (4) ongoing efforts to develop new models to study niche-leukemic cell interaction in human myeloid malignancies; and finally, (5) how the knowledge gained into leukemic stem cell (LSC) niche dependencies might be exploited to devise novel therapeutic strategies that aim at disrupting essential niche-LSC interactions or improve the regenerative ability of the disease-associated hematopoietic niche., (© 2017 by The American Society of Hematology.)

Background Aims: Natural killer (NK) cells can rapidly respond to transformed and stressed cells and represent an important effector cell type for adoptive immunotherapy. In addition to donor-derived primary NK cells, continuously expanding cytotoxic cell lines such as NK-92 are being developed for clinical applications., Methods: To enhance their therapeutic utility for the treatment of B-cell malignancies, we engineered NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors (CARs) that target CD19 and contain human CD3ζ (CAR 63.z), composite CD28-CD3ζ or CD137-CD3ζ signaling domains (CARs 63.28.z and 63.137.z)., Results: Exposure of CD19-positive targets to CAR NK-92 cells resulted in formation of conjugates between NK and cancer cells, NK-cell degranulation and selective cytotoxicity toward established B-cell leukemia and lymphoma cells. Likewise, the CAR NK cells displayed targeted cell killing of primary pre-B-ALL blasts that were resistant to parental NK-92. Although all three CAR NK-92 cell variants were functionally active, NK-92/63.137.z cells were less effective than NK-92/63.z and NK-92/63.28.z in cell killing and cytokine production, pointing to differential effects of the costimulatory CD28 and CD137 domains. In a Raji B-cell lymphoma model in NOD-SCID IL2R γ null mice, treatment with NK-92/63.z cells, but not parental NK-92 cells, inhibited disease progression, indicating that selective cytotoxicity was retained in vivo., Conclusions: Our data demonstrate that it is feasible to generate CAR-engineered NK-92 cells with potent and selective antitumor activity. These cells may become clinically useful as a continuously expandable off-the-shelf cell therapeutic agent., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Unresolved chronic inflammation is implicated in all stages of cancer development and an inflammatory tumor microenvironment is considered a hallmark of cancer. Signaling pathways involved in normal tissue regeneration and repair are dysregulated both in chronic inflammation and cancer. Here, we review some of the recently identified signaling cascades and unexpected functions of stromal cells that affect both tissue regeneration and tumorigenesis in colon and pancreatic cancer, which may pave the way for the development of novel therapeutic strategies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Clonal evolution is believed to be a main driver for progression of various types of cancer and implicated in facilitating resistance to drugs. However, the hierarchical organization of malignant clones in the hematopoiesis of myelodysplastic syndromes (MDS) and its impact on response to drug therapy remain poorly understood. Using high-throughput sequencing of patient and xenografted cells, we evaluated the intratumoral heterogeneity (n= 54) and reconstructed mutational trajectories (n = 39) in patients suffering from MDS (n = 52) and chronic myelomonocytic leukemia-1 (n = 2). We identified linear and also branching evolution paths and confirmed on a patient-specific level that somatic mutations in epigenetic regulators and RNA splicing genes frequently constitute isolated disease-initiating events. Using high-throughput exome- and/or deep-sequencing, we analyzed 103 chronologically acquired samples from 22 patients covering a cumulative observation time of 75 years MDS disease progression. Our data revealed highly dynamic shaping of complex oligoclonal architectures, specifically upon treatment with lenalidomide and other drugs. Despite initial clinical response to treatment, patients' marrow persistently remained clonal with rapid outgrowth of founder-, sub-, or even fully independent clones, indicating an increased dynamic rate of clonal turnover. The emergence and disappearance of specific clones frequently correlated with changes of clinical parameters, highlighting their distinct and far-reaching functional properties. Intriguingly, increasingly complex mutational trajectories are frequently accompanied by clinical progression during the course of disease. These data substantiate a need for regular broad molecular monitoring to guide clinical treatment decisions in MDS., (© 2016 by The American Society of Hematology.)

Maintaining cellular redox balance is vital for cell survival and tissue homoeostasis because imbalanced production of reactive oxygen species (ROS) may lead to oxidative stress and cell death. The antioxidant enzyme glutathione peroxidase 4 (Gpx4) is a key regulator of oxidative stress-induced cell death. We show that mice with deletion of Gpx4 in hematopoietic cells develop anemia and that Gpx4 is essential for preventing receptor-interacting protein 3 (RIP3)-dependent necroptosis in erythroid precursor cells. Absence of Gpx4 leads to functional inactivation of caspase 8 by glutathionylation, resulting in necroptosis, which occurs independently of tumor necrosis factor α activation. Although genetic ablation of Rip3 normalizes reticulocyte maturation and prevents anemia, ROS accumulation and lipid peroxidation in Gpx4-deficient cells remain high. Our results demonstrate that ROS and lipid hydroperoxides function as not-yet-recognized unconventional upstream signaling activators of RIP3-dependent necroptosis., (© 2016 by The American Society of Hematology.)

Background Aims: Human cytomegalovirus (CMV) infection and reactivation is a leading complication of allogeneic hematopoietic stem cell transplantation (HSCT). In addition to drug treatment, the adoptive transfer of virus-specific T cells to restore cellular immunity has become a standard therapy after allogeneic HSCT. We recently demonstrated potent anti-leukemic activity of interleukin (IL)-15-activated cytokine-induced killer (CIK) cells. With the use of the same expansion protocol, we asked whether concurrent CMV antigen-pulsing might generate CIK cells with anti-leukemic and anti-CMV activity., Methods: CIK cells expanded in the presence of interferon-γ, IL-2, IL-15 and anti-CD3 antibody were pulsed once with CMV(pp65) peptide pool. CMV-specific CIK (CIK(pp65)) and conventional CIK cells were phenotypically and functionally characterized according to their cytokine secretion pattern, degranulation capacity and T-cell receptor (TCR)-mediated and NKG2D-mediated cytotoxicity., Results: We demonstrated that among CIK cells generated from CMV-seropositive donors, a single stimulation with CMV(pp65) protein co-expanded cytotoxic CMV-specific cells without sacrificing anti-tumor reactivity. Cells generated in this fashion lysed CMV(pp65)-loaded target cells and CMV-infected fibroblasts but also leukemic cells. Meanwhile, the alloreactive potential of CIK(pp65) cells remained low. Interestingly, CMV reactivity was TCR-mediated and CMV-specific cells could be found in CD3(+)CD8(+)CD56(+/-) cytotoxic T-cell subpopulations., Conclusions: We provide an efficient method to generate CIK(pp65) cells that may represent a useful cell therapy approach for preemptive immunotherapy in patients who have both an apparent risk of CMV and impending leukemic relapse after allogeneic stem cell transplantation., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The activity of antagonizing transcription factors represents a mechanistic paradigm of bidirectional lineage-fate control during hematopoiesis. At the megakaryocytic/erythroid bifurcation, the cross-antagonism of krueppel-like factor 1 (KLF1) and friend leukemia integration 1 (FLI1) has such a decisive role. However, how this antagonism is resolved during lineage specification is poorly understood. We found that runt-related transcription factor 1 (RUNX1) inhibits erythroid differentiation of murine megakaryocytic/erythroid progenitors and primary human CD34(+) progenitor cells. We show that RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation by epigenetic repression of the erythroid master regulator KLF1. RUNX1 binding to the KLF1 locus is increased during megakaryocytic differentiation and counterbalances the activating role of T-cell acute lymphocytic leukemia 1 (TAL1). We found that corepressor recruitment by RUNX1 contributes to a block of the KLF1-dependent erythroid gene expression program. Our data indicate that the repressive function of RUNX1 influences the balance between erythroid and megakaryocytic differentiation by shifting the balance between KLF1 and FLI1 in the direction of FLI1. Taken together, we show that RUNX1 is a key player within a network of transcription factors that represses the erythroid gene expression program., (© 2015 by The American Society of Hematology.)

The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Background Aims: Cytokine-induced killer (CIK) cells may offer a novel therapeutic approach for patients with malignancies relapsing after allogeneic stem cell transplantation. Although CIK cells display negligible alloreactivity and cause minimal graft versus-host-disease (GVHD), high CIK cell doses required during relapse may pose a risk for severe GVHD, specifically in the mismatched or haploidentical transplantation setting. Manipulation of CIK cells may reduce risk for GVHD without affecting the anti-tumor potential., Methods: In this pre-clinical study, we provide a detailed functional comparison of conventional and irradiated, CD56-enriched or T-cell receptor α/β-depleted CIK cells., Results: In vitro analysis showed retained anti-leukemic and anti-tumor potential after CIK cell manipulation. Even being sequentially infused into immunodeficient mice grafted with malignant cells, cytotoxic effects were fewest after irradiation but were improved by CD56 enrichment and were best with conventional CIK cells. Hence, considering the proliferative capacity of inoculated malignancies and effector cells, a single dose of conventional CIK cells resulted in prolonged disease-free survival and elimination of rhabdomyosarcoma cells, whereas sequential infusions were needed to achieve comparable results in leukemia-bearing mice. However, this mouse model has limitations: highly effective conventional CIK cells demonstrated both limited xenogenic GVHD and low alloreactive potential in vitro., Conclusions: Our study revealed that conventional CIK cells demonstrate no significant alloreactive potential but provide the strongest anti-tumor efficacy compared with manipulated CIK cells. Conventional CIK cells may therefore be tested in high numbers and short-term intervals in patients with impending relapse even after mismatched transplantation., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Fas ligand (FasL) not only induces apoptosis in Fas receptor-bearing target cells, it is also able to transmit signals into the FasL-expressing cell via its intracellular domain (ICD). Recently, we described a Notch-like proteolytic processing of FasL that leads to the release of the FasL ICD into the cytoplasm and subsequent translocation into the nucleus where it may influence gene transcription. To study the molecular mechanism underlying such reverse FasL signaling in detail and to analyze its physiological importance in vivo, we established a knockout/knockin mouse model, in which wild-type FasL was replaced with a deletion mutant lacking the ICD. Our results demonstrate that FasL ICD signaling impairs activation-induced proliferation in B and T cells by diminishing phosphorylation of phospholipase C γ, protein kinase C, and extracellular signal-regulated kinase 1/2. We also demonstrate that the FasL ICD interacts with the transcription factor lymphoid-enhancer binding factor-1 and inhibits lymphoid-enhancer binding factor-1-dependent transcription. In vivo, plasma cell numbers, generation of germinal center B cells, and, consequently, production of antigen-specific immunoglobulin M antibodies in response to immunization with T cell-dependent or T cell-independent antigen are negatively affected in presence of the FasL ICD, suggesting that FasL reverse signaling participates in negative fine-tuning of certain immune responses.

RUNX1/ETO, the fusion protein resulting from the chromosomal translocation t(8;21), is one of the most frequent translocation products in acute myeloid leukemia. Several in vitro and in vivo studies have shown that the homo-tetramerization domain of ETO, the nervy homology region 2 (NHR2), is essential for RUNX1/ETO oncogenic activity. We analyzed the energetic contribution of individual amino acids within the NHR2 to RUNX1/ETO dimer-tetramer transition and found a clustered area of 5 distinct amino acids with strong contribution to the stability of tetramers. Substitution of these amino acids abolishes tetramer formation without affecting dimer formation. Similar to RUNX1/ETO monomers, dimers failed to bind efficiently to DNA and to alter expression of RUNX1-dependent genes. RUNX1/ETO dimers do not block myeloid differentiation, are unable to enhance the self-renewal capacity of hematopoietic progenitors, and fail to induce leukemia in a murine transplantation model. Our data reveal the existence of an essential structural motif (hot spot) at the NHR2 dimer-tetramer interface, suitable for a molecular intervention in t(8;21) leukemias.

Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in patients treated for X-linked severe combined immunodeficiency. Here, we analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental "worst case scenario," we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gamma retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1, or DeltaTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1-deficient recipients (Ly5.2), animals that received stem cell transplants developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated polymerase chain reaction analysis revealed monoclonality or oligoclonality of the malignancies. In striking contrast, none of the mice that received T-cell transplants transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. Thus, our data provide direct evidence that mature T cells are less prone to transformation than hematopoietic progenitor cells.

The p53-inducible and death domain-containing PIDD/LRDD protein has been described as an adaptor protein, which forms large protein complexes with RAIDD, another death domain-containing protein, leading to recruitment, and activation of the initiator caspase-2, and p53-mediated apoptosis. Here, we describe in further detail the proteolytic processing of PIDD/LRDD that occurs in healthy cells before induction of apoptosis. We could demonstrate that the C-terminal fragment containing the PIDD death domain shuttles into the nucleoli. This translocation is mediated by or leads to the interaction of the PIDD death domain with nucleolin, a protein important for rRNA processing within nucleoli and possibly involved in the DNA damage response. Ectopically expressed LRDD and endogenous nucleolin co-localized within the nucleoli, and overexpression of both full-length LRDD and the LRDD death domain sensitized cells for UV-induced apoptosis. When expressed alone, the PIDD/LRDD death domain tended to form large filamentous structures resembling so-called death filaments. The functional consequences of the identified PIDD/nucleolin interaction remain to be elucidated, but may be related to a recently discovered new role for PIDD in the activation of NF-kappaB upon genotoxic stress.

Previous clinical trials in HIV-infected patients involving infusion of T cells protected by an antiviral gene have failed to show any therapeutic benefit. The value of such a treatment approach is thus still highly controversial. In this study, the anticipated effects of gene-modified cells on virus and T-cell kinetics are analysed by mathematical modeling. Because technically only a small fraction of all T cells in a patient can be manipulated ex vivo, therapeutic success will depend on the accumulation of gene-modified cells after infusion into the patient by in vivo selection. Our simulations predict that a significant therapeutic benefit is conferred only by antiviral genes that inhibit HIV replication before virus integration (class I genes). Genes that inhibit viral protein expression (class II, used in previous clinical trials), require a much higher inhibitory activity than class I genes to promote the regeneration of T cells and reduce the viral load. Inhibition of virus assembly and release alone (class III) confers no selective advantage to the T cell and is therefore ineffective unless combined with class I (or, possibly, class II) genes. Also crucial in determining the clinical outcome are the regenerative capacity of the gene-modified cells and the level of HIV replication in the patient. These results can be important for guiding future strategies in the field of gene therapy for HIV infection.

Persistent activation of T-lymphocytes requires two signals: one is initiated by T-cell receptor binding to antigenic peptide presented by MHC molecules. In addition, binding of the B7 family members CD80 or CD86 on professional antigen presenting cells to CD28 on T cells is considered to provide an important costimulatory signal. Activation without costimulation induces T-cell unresponsiveness or anergy. To selectively localize costimulatory activity to the surface of tumor cells and enhance activation of tumor-specific T cells, we have developed a novel molecular design for bispecific costimulatory proteins with antibody-like structure. Within a single polypeptide chain we have assembled the IgV-like, CD28-binding domain of human CD86 (CD86(111)) together with hinge, CH2 and CH3 domains of human IgG1, and the scFv(FRP5) antibody fragment which recognizes the ErbB2 (HER2) protooncogene present at high levels on the surface of many human tumor cells. Upon expression in the yeast Pichia pastoris, the resulting CD86(111)-IgG-scFv(FRP5) protein could be purified as a homodimeric, tetravalent molecule from culture supernatants using single-step affinity chromatography. Bispecific binding of the molecule to ErbB2 on the surface of tumor cells and to the B7 counter receptor CTLA-4 was demonstrated by FACS analysis. Potent costimulatory activity of chimeric CD86(111)-IgG-scFv(FRP5) was confirmed by its ability to stimulate the proliferation of primary human lymphocytes pre-activated by low concentrations of anti-CD3 antibody. Our results suggest that such multivalent soluble proteins which combine specific targeting to tumor cells with costimulatory activity may become useful tools to elicit and/or improve T-cell mediated, tumor-specific immune responses.

The insulin-like growth factor 1 receptor (IGF-1R) is an important signaling molecule in cancer cells and plays an essential role in the establishment and maintenance of the transformed phenotype. Inhibition of IGF-1R signaling thus appears to be a promising strategy to interfere with the growth and survival of cancer cells. Different classes of molecules, e.g., antisense RNA, monoclonal antibodies and dominant negative IGF-1R gene variants, have been employed towards this aim. These agents have been able to reverse the transformed phenotype in several rodent and human cancer cell lines. The application of peptide aptamers specifically binding to the IGF-1R represents a novel approach to target IGF-1R signaling. The integration of peptide aptamers into targeted protein degradation vehicles and their transduction into cells allows the temporary elimination of the receptor protein. This review summarizes recently published data about inhibition of IGF-1R signaling and provides a perspective on upcoming possibilities.

The continuously growing natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origins without affecting normal human cells. Based on this selectivity, the potential of NK-92 cells for adoptive therapy is currently being investigated in phase I clinical studies. To further enhance the antitumoral activity of NK-92 cells and expand the range of tumor entities suitable for NK-92-based therapies, here by transduction with a retroviral vector we have generated genetically modified NK-92 cells expressing a chimeric antigen receptor specific for the tumor-associated ErbB2 (HER2/neu) antigen, which is overexpressed by many tumors of epithelial origin. The chimeric antigen receptor consists of the ErbB2-specific scFv(FRP5) antibody fragment, a flexible hinge region derived from CD8, and transmembrane and intracellular regions of the CD3 zeta chain. Transduced NK-92-scFv(FRP5)-zeta cells express high levels of the fusion protein on the cell surface as determined by fluorescence-activated cell-scanning (FACS) analysis. In europium release assays, no difference in cytotoxic activity of NK-92 and NK-92-scFv(FRP5)-zeta cells toward ErbB2-negative targets was found. However, even at low effector-to-target ratios, NK-92-scFv(FRP5)-zeta cells specifically and efficiently lysed established and primary ErbB2-expressing tumor cells that were completely resistant to cytolytic activity of parental NK-92 cells. These results demonstrate that efficient retargeting of NK-92 cytotoxicity can be achieved and might allow the generation of potent cell-based therapeutics for the treatment of ErbB2-expressing malignancies.

Extracellular hormones, growth factors or cytokines relay their effects on the transcription of genes through the recognition of specific receptors and intracellular signaling molecules. Stat (signal transducers and activators of transcription) have been recognized as crucial intracellular signaling molecules. The cytokine receptor associated Jak kinases convert the latent monomeric form of the Stat molecules to the activated dimeric form through tyrosine phosphorylation. The dimers bind to specific DNA response elements and are able to induce transcription. The transcription factor Stat5 is a central determinant of mammary gland development and function. It is activated during pregnancy by prolactin and contributes to the growth and alveolar differentiation of the epithelial cells. During lactation it governs milk protein gene expression and contributes to cell survival. Negative regulatory potential is exerted by the expression of the short form of the molecule, lacking the transactivation domain, but associating with nuclear co-repressors. This form is activated through tyrosine phosphorylation and dimerisation similarly to the full-length form, but is impeded in dephosphorylation and co-activator recruitment. Positive enhancement of Stat5 transactivation potential is provided by the glucocorticoid receptor (GR). Ligand activation of the receptor causes complex formation with Stat5 and deviation to the Stat5 DNA binding site. An additional regulatory loop is provided by the reactivation of the short form of Stat5 through GR association.

The transcription factor Stat5 mediates the cellular response to activation of multiple cytokine receptors involved in the regulation of proliferation and differentiation of hematopoietic cells. Recently, the human Stat5 gene was found to be translocated to the RARalpha gene in a patient with acute promyelocytic leukemia indicating that Stat5 might also play a role in cellular transformation. We investigated the mechanism by which Stat5 might exert this function and studied the biochemical and cellular functions of fusion proteins comprising Stat5 and RARalpha. The expression of Stat5-RARalpha causes the transcriptional repression of gene transcription, a process that requires the coiled-coil domain of Stat5 (amino acid positions 133-333). Oligomerization of this domain in the Stat5-RARalpha fusion protein leads to stable binding of the corepressor SMRT independent of all-trans retinoic acid (ATRA) stimulation and is accompanied by an impaired response to differentiation signals in hematopoietic cells. This inhibitory effect on myeloid differentiation cannot be overcome by simultaneous coexpression of RARalpha. We conclude that Stat5 is capable of interacting with a corepressor complex that alters the pattern of corepressor binding to RARalpha and its dissociation in response to ATRA stimulation, leading to enhanced repressor activity and a block of hematopoietic differentiation.

Extracellular hormones, growth factors and cytokines relay their effects on the transcription of genes through the recognition of specific receptors and intracellular signalling molecules. Signal transducers and activators of transcription (STATs) have been recognized as crucial intracellular signalling molecules. The cytokine receptor-associated Janus kinases (JAKs) convert the latent monomeric form of the STAT molecules to the activated dimeric form through tyrosine phosphorylation. The dimers bind to specific DNA response elements and are able to induce transcription. This induction requires the full-length form of the STAT molecules. Negative regulatory potential is exerted by the short form of the molecule, which lacks the trans-activation domain. This short form is activated and dimerized, but dephosphorylation is impaired. The short form of STAT occupies the DNA-binding sites in a stable fashion and acts as a strong suppressor of wild-type action. Positive enhancement of STAT5 trans-activation potential is provided by the glucocorticoid receptor. Ligand activation of this receptor causes the formation of a complex with STAT5 and deviation to the STAT5 DNA-binding site. An additional regulatory loop is provided by the reactivation of the short form of STAT5 through glucocorticoid receptor association. Conversely, classical glucocorticoid-responsive genes are negatively affected by STAT5 activation.

Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34(+) cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45(+) cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions., (Copyright 1998 by The American Society of Hematology)

The expression of many cytokines is dysregulated in individuals infected with the human immunodeficiency virus-1 (HIV-1). To determine the effects of HIV-1 infection on cytokine expression in individual cells (at the single cell level), we investigated the intracellular levels of proinflammatory cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, and IL-8) and hematopoietic growth factors (granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF]) in monocyte-derived macrophages, mock-infected, or infected with HIV-1 by immunocytochemical staining for cytokine protein and compared this with secreted cytokine levels as determined by specific enzyme-linked immunosorbent assay (ELISA). No difference in the frequency or intensity of cell-associated immunocytochemical cytokine staining could be observed between HIV-1 and mock-infected cells even though the level of secreted proinflammatory cytokines increased and the hematopoietic growth factors decreased in HIV-1-infected cultures. Furthermore, equal expression of cytokine mRNA was observed in all cells in the culture regardless of whether the cells were productively infected with HIV-1 as determined by double-labelling immunocytochemical staining for HIV-1 p24 antigen and in situ hybridization for cytokine mRNA expression. These results indicate that HIV-1 infection results in dysregulation of intracellular cytokine mRNA expression and cytokine secretion not only in HIV-1-infected cells, but also through an indirect way(s) affecting cells not producing virus.

PLK (polo-like kinase) belongs to a family of serine/threonine kinases and represents the human counterpart of polo in Drosophila melanogaster and of CDC5 in Saccharomyces cerevisiae. It is strongly involved in spindle formation and chromosome segregation during mitosis. We have shown previously that PLK mRNA expression correlates with the mitotic activity of cells and the prognosis of lung cancer patients. In this report, the level of PLK protein was analyzed using immunohistochemical techniques. PLK protein was found expressed in the nuclei of tumor cells from lung and breast cancer as well as in several tumor cell lines. Furthermore, in peripheral lymphocytes treated with phytohemagglutinin, elevated proliferative activity of the cells correlated with the up-regulation of PLK protein expression. In contrast, in U937 and HL-60 cells after induction of differentiation with phorbol ester, PLK immunostaining disappeared under conditions of terminal differentiation. Most of the PLK protein was found in the nucleus of proliferating cells with diffuse but distinct staining also in the cytoplasm. Taken together, high levels of PLK protein are associated with cellular proliferation. Combined with other proliferative and oncogene markers, PLK may be useful for improved prediction of the clinical prognosis of cancer patients and for early cancer diagnosis. Due to its activity late in the cell cycle, it may be a target for cancer chemotherapy.

Cells of the macrophage lineage (MAC) play an important role in human immunodeficiency virus (HIV) infection. However, the knowledge on the extent of macrophage involvement in the pathogenesis of HIV infection is still incomplete. In this study we examined the secretory repertoire of HIV-infected MAC with respect to the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6, IL-8, and the hematopoietic growth factors M-, G- and granulocyte-macrophage colony stimulating factor (GM-CSF). Using a culture system on hydrophobic teflon membranes, blood-derived MO from healthy donors were infected with a monocytotropic HIV-1 isolate (HIV-1D117IIII). We analyzed the constitutive and lipopolysaccharides-stimulated secretion of MO/MAC early after infection as well as in long-term cultured, virus-replicating cells. The release of proinflammatory mediators and hematopoietic growth factors were differentially regulated after infection with HIV: the secretion of TNF-alpha, IL-1 beta, IL-6, IL-8 was upregulated, whereas a down-regulation of M-, G-, and GM-CSF could be observed. These results may provide some explanation for the immunological dysfunction, the hematopoietic failure and the chronic inflammatory disease occurring in HIV-infected patients.

RNA was specifically extracted from tumor and peritumor tissue of a hepatitis C virus (HCV)-associated hepatocellular carcinoma after microdissection. RT-PCR products of the HCV 5'-noncoding (NC), core and nonstructural (NS)-5 gene were examined. Nucleotide sequences of the core region derived from the tumor tissue revealed deletions and mutations resulting in truncated proteins. Peritumor and serum HCV sequences were unaffected.

Monocytes of healthy donors were infected with HIV1 in vitro: 14-21 days after infection 50-70% of the cells produced p24 HIV1 antigen as detected with anti-p24 immunostaining; infected cultures showed enhanced secretion of interleukin-6 (IL6), interleukin-8 (IL8) and tumour necrosis factor alpha (TNF-alpha). The expression of cytokines on the single-cell level was further analysed by in situ hybridization using nonradioactive digoxigenin for detection. HIV1 (p24+) -producing cells were compared with non-HIV (p24-) -producing cells. All morphological subtypes of macrophages showed HIV production; no difference in cytokine expression was observed. Immunocytochemistry of HIV-infected and uninfected cultures also showed no difference in the pattern of IL1-beta, IL6, IL8 and TNF-alpha protein expression in the cells.

Conditioned media from cell cultures derived from AIDS-associated Kaposi's sarcoma (KS) promote the growth of KS cells, fibroblasts and endothelial cells. Application of KS cells to the chicken chorion allantoic membrane induces neo-angiogenesis similar to that caused by fibroblast growth factors (FGF). Testing for known factors of the FGF family by Northern blotting does not reveal any difference from control fibroblasts negative for such an activity in their supernatants. This indicates secretion of an unknown FGF-like factor by KS cells possessing autocrine (KS cells) and paracrine (fibroblasts, endothelial cells) activity. It was reported that transforming growth factor-beta (TGF beta) is secreted by HIV-infected lymphocytes in AIDS patients. By testing this growth factor on KS cell cultures, growth-promoting activity was observed 5 to 20 times greater than that of the control value without TGF beta.

We studied the response of monocytes/macrophages (MO/MAC) to lipopolysaccharide (LPS) and interferon-gamma (IFN gamma) stimulation with respect to the expression of macrophage-specific products, i.e. macrophage-colony-stimulating factor (M-CSF), c-fms, c-sis, tissue factors, transforming growth factor-beta (TGF beta) and interleukin-8 (IL8) after in vitro infection with HIV. The expression of IL8 was strongly elevated in HIV-infected cells, peaking at 4 h after stimulation with LPS. At that time, the uninfected control showed only weak expression of IL8. Other products, e.g. tissue factor, c-fms, M-CSF and TGF beta were not modulated after stimulation. In contrast to IL8, the expression of c-cis was significantly lower in infected cells after stimulation with IFN gamma compared to uninfected control cells.

Human blood-borne monocytes (MO) differentiating into mature macrophages (MAC) were cultured on hydrophobic Teflon membranes. The cells were infected with two different monocytotropic HIV isolates: HIV1D117III obtained from a perinatally infected child, and HIV2D194 obtained from an AIDS patient who suffered exclusively from neurological symptoms. Virus production monitored by reverse transcriptase activity and HIV-antigen ELISA in cell-free supernatant was of a high level and continued for several weeks. To investigate possible modulatory pathways interfering with HIV infection in MAC we tested various recombinant cytokines as well as bacterial lipopolysaccharides (LPS) in our culture system. Whereas interleukin-1 (IL1) accelerated and increased HIV replication in MO/MAC, the interferons (IFN) alpha, beta and gamma effectively suppressed or delayed infection depending on the concentration used. Suppression was seen at concentrations as low as 0.3 U/ml and was most effective when the IFN were given prior to infection. No effect was observed with IL6 up to 2,000 U/ml. LPS affected virus infection in a complex manner: at 1-100 ng/ml virus replication was inhibited, but it was enhanced at subnanogram concentrations (25-100 pg/ml).

Because of the very important role of the mononuclear phagocyte system in the immunopathogenesis of HIV infection, a culture system for in vitro studies of infection of monocytes/macrophages with HIV was developed. A method is described for the infection of human monocytes/macrophages cultured on hydrophobic membranes (Teflon) with different strains of HIV. The HIV isolates can be characterized according to their replication potential on monocytes/macrophages cultures. The biological properties of some HIV1 and HIV2 isolates are compared in lymphocyte and monocyte/macrophage cultures.

Due to the important role of monocytes/macrophages in the pathogenesis of AIDS, potential drugs with anti-HIV activity in lymphocytes must also be effective in monocytes/macrophages. For testing the efficacy of antiviral substances, monocytes/macrophages from peripheral blood were infected, respectively, with highly replicating HIV1 and HIV2 strains, thereby providing an extremely sensitive system of testing. Azidothymidine was found to inhibit both HIV types at 0.04 microgram/ml. The polysulphated polyxylan, Hoe/Bay-946 (MW 6,000 Daltons), which acts through a different mechanism and is being tested in clinical pilot studies in Germany, was also found to be effective against HIV1 and HIV2 in macrophages at concentrations of 10-50 micrograms/ml.

HIV2 strains were isolated from a Gambian with neuro-AIDS (HIV2D194) and from an asymptomatic Ghanian (HIV2D205). Like HIV1 biological subtype c, both isolates grew slowly and induced few or no syncytia, but eventually produced high levels of particle-associated reverse transcriptase (RT) in cultures of fresh peripheral blood lymphocytes. Each produced even higher levels of RT in fresh human macrophages, especially HIV2D194, where maximal RT values of 1,800,000 cpm/ml supernatant of approximately 30,000 cells were measured. The viruses were molecularly cloned after a single passage in culture. Restriction site analysis showed heterogeneity within each isolate. Nucleotide sequence analysis of HIV2D194 revealed that, genetically, it is a member of the prototypic HIV2 family, displaying 12% divergence vs. HIV2ROD and HIV2NIHZ. In contrast, HIV2D205 is the most highly divergent HIV2 strain yet described: it is equidistant in relation between the known HIV2 strains and the SIVMAC isolates (23-24% nucleotide sequence divergence).