The cell of origin and the leukemia stem cell in acute myeloid leukemia.

There is experimental and observational evidence that the cells of the leukemic clone in acute myeloid leukemia (AML) have different phenotypes even though they share the same somatic mutations. The organization of the malignant clone in AML has many similarities to normal hematopoiesis, with leukemia stem cells (LSCs) that sustain leukemia and give rise to more differentiated cells. LSCs, similar to normal hematopoietic stem cells (HSCs), are those cells that are able to give rise to a new leukemic clone when transplanted into a recipient. The cell of origin of leukemia (COL) is defined as the normal cell that is able to transform into a leukemia cell. Current evidence suggests that the COL is distinct from the LSC. Here, we will review the current knowledge about LSCs and the COL in AML.

Selective NFAT targeting in T cells ameliorates GvHD while maintaining antitumor activity.

Graft-versus-host disease (GvHD) is a life-threatening immunological complication after allogenic hematopoietic stem cell transplantation (allo-HCT). The intrinsic graft-versus-leukemia (GvL) effect, however, is the desirable curative benefit. Patients with acute GvHD are treated with cyclosporine A (CsA) or tacrolimus (FK506), which not only often causes severe adverse effects, but also interferes with the anticipated GvL. Both drugs inhibit calcineurin, thus at first suppressing activation of the nuclear factor of activated T cells (NFAT). Therefore, we explored the specific contribution of individual NFAT factors in donor T cells in animal models of GvHD and GvL. Ablation of NFAT1, NFAT2, or a combination of both resulted in ameliorated GvHD, due to reduced proliferation, target tissue homing, and impaired effector function of allogenic donor T cells. In contrast, the frequency of Foxp3(+) regulatory T (Treg) cells was increased and NFAT-deficient Tregs were fully protective in GvHD. CD8(+) T-cell recall response and, importantly, the beneficial antitumor activity were largely preserved in NFAT-deficient effector T cells. Thus, specific inhibition of NFAT opens an avenue for an advanced therapy of GvHD maintaining protective GvL.

Blocking TWEAK-Fn14 interaction inhibits hematopoietic stem cell transplantation-induced intestinal cell death and reduces GVHD.

Inhibition of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) system reduces intestinal cell death and disease development in several models of colitis. In view of the crucial role of TNF and intestinal cell death in graft-versus-host disease (GVHD) and the ability of TWEAK to enhance TNF-induced cell death, we tested here the therapeutic potential of Fn14 blockade on allogeneic hematopoietic cell transplantation (allo-HCT)-induced intestinal GVHD. An Fn14-specific blocking human immunoglobulin G1 antibody variant with compromised antibody-dependent cellular cytotoxicity (ADCC) activity strongly inhibited the severity of murine allo-HCT-induced GVHD. Treatment of the allo-HCT recipients with this monoclonal antibody reduced cell death of gastrointestinal cells but neither affected organ infiltration by donor T cells nor cytokine production. Fn14 blockade also inhibited intestinal cell death in mice challenged with TNF. This suggests that the protective effect of Fn14 blockade in allo-HCT is based on the protection of intestinal cells from TNF-induced apoptosis and not due to immune suppression. Importantly, Fn14 blockade showed no negative effect on graft-versus-leukemia/lymphoma (GVL) activity. Thus, ADCC-defective Fn14-blocking antibodies are not only possible novel GVL effect-sparing therapeutics for the treatment of GVHD but might also be useful for the treatment of other inflammatory bowel diseases where TNF-induced cell death is of relevance.

Interleukin-2 critically regulates bone marrow erythropoiesis and prevents anemia development.

Mice deficient in IL-2 signaling develop severe anemia indicating a defect in erythropoiesis. However, why deficiency in IL-2, an essential growth factor for lymphocytes, or in IL-2 signaling components should result in defective erythropoiesis is unclear. Here, we have analyzed the mechanism of IL-2 signaling deficiency induced anemia in mice and show that IL-2 plays an indispensable role in bone marrow (BM) erythropoiesis via maintenance of regulatory T (Treg) cells. In absence of IL-2 signaling, IFN-γ produced by the activated T cells suppressed klf1 expression, resulting in an early block in erythrocyte differentiation. Anemia, in IL-2 or IL-2 signaling deficient mice always developed prior to the manifestation of other autoimmune complications such as colitis, suggesting that anemia in these mice might be a contributing factor in inducing other pathological complications in later stages. Our study shows, how essential cytokines of lymphoid cells could exert critical influence on the development of erythrocytes and thus expanding our understanding of the complex regulation of hematopoiesis in the BM. Besides, our findings might facilitate the use of IL-2 and anti-IFN-γ as a clinical remedy against anemia that arise in cancer patients following radiotherapy or chemotherapy, a context which simulates the situation of IL-2 deficiency.

Src-kinase inhibitors sensitize human cells of myeloid origin to Toll-like-receptor-induced interleukin 12 synthesis.

Src-kinase inhibitors hold great potential as targeted therapy against malignant cells. However, such inhibitors may also affect nonmalignant cells and cause pronounced off-target effects. We investigated the role of the dual kinase inhibitor dasatinib on human myeloid cells. Dasatinib is clinically used for the treatment of bcr/abl⁺ leukemias because it blocks the mutated tyrosine kinase abl. To understand its effect on the development of antigen-specific T-cell responses, we assessed antigen-specific priming of human, naïve T cells. In surprising contrast to the direct inhibition of T-cell activation by dasatinib, pretreatment of maturing dendritic cells (DCs) with dasatinib strongly enhanced their stimulatory activity. This effect strictly depended on the activating DC stimulus and led to enhanced interleukin 12 (IL-12) production and T-cell responses of higher functional avidity. Src-kinase inhibitors, and not conventional tyrosine kinase inhibitors, increased IL-12 production in several cell types of myeloid origin, such as monocytes and classical or nonclassical DCs. Interestingly, only human cells, but not mouse or macaques DCs, were affected. These data highlight the potential immunostimulatory capacity of a group of novel drugs, src-kinase inhibitors, thereby opening new opportunities for chemoimmunotherapy. These data also provide evidence for a regulatory role of src kinases in the activation of myeloid cells.

Generation of highly effective and stable murine alloreactive Treg cells by combined anti-CD4 mAb, TGF-ß, and RA treatment.

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long-term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti-CD4 antibody (aCD4). Here, we investigated whether adding TGF-ß and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg-cell generation and function. Murine CD4(+) T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF-ß+RA or aCD4+Rapa. Addition of TGF-ß+RA or Rapa resulted in an increase of CD25(+)Foxp3(+)-expressing T cells. Expression of CD40L and production of IFN-γ and IL-17 was abolished in aCD4+TGF-ß+RA aTreg cells. Additionally, aCD4+TGF-ß+RA aTreg cells showed the highest level of Helios and Neuropilin-1 co-expression. Although CD25(+)Foxp3(+) cells from all culture conditions displayed complete demethylation of the Treg-specific demethylated region, aCD4+TGF-ß+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF-ß+RA aTreg cells suppressed effector T-cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF-ß+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.

Tumor necrosis factor receptor 2-dependent homeostasis of regulatory T cells as a player in TNF-induced experimental metastasis.

The cytokine tumor necrosis factor (TNF) has pleiotropic functions both in normal physiology and disease. TNF signals by the virtue of two cell surface receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Exogenous TNF promotes experimental metastasis in some models, yet the underlying mechanisms are poorly understood. To study the contribution of host TNFR1 and TNFR2 on tumor cell progression and metastasis, we employed a syngeneic B16F10 melanoma mouse model of lung metastasis combined with in vivo bioluminescence imaging. Treatment of tumor-bearing mice with recombinant human TNF resulted in a significant increase in tumor burden and metastatic foci. This correlated with an increase in pulmonary regulatory CD4(+)/Foxp3(+) T cells. TNF caused an expansion of regulatory T (Treg) cells in vitro in a TNFR2-dependent manner. To assess the contribution of immune cell expression of endogenous TNF and its two receptors on B16F10 metastasis, we generated bone marrow chimeras by reconstituting wild-type mice with bone marrow from different knockout mice. Loss of either TNF or TNFR2 on immune cells resulted in decreased B16F10 metastasis and lower numbers of Treg cells within the lungs of these animals. Selective depletion of Treg cells attenuated metastasis even in conjunction with TNF treatment. We propose a novel mechanism in which TNF activates TNFR2 on Treg cells and thereby expands this immunosuppressive immune cell population. Loss of either TNF or TNFR2 prevents the accumulation of Treg cells and results in a less tolerogenic environment, enabling the immune system to control B16F10 tumor metastasis and growth.

A diagnostic window for the treatment of acute graft-versus-host disease prior to visible clinical symptoms in a murine model.

BACKGROUND: Acute graft-versus-host disease (aGVHD) poses a major limitation for broader therapeutic application of allogeneic hematopoietic cell transplantation (allo-HCT). Early diagnosis of aGVHD remains difficult and is based on clinical symptoms and histopathological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestation. Therefore, for improved disease prevention it is important to develop predictive assays to identify patients at risk of developing aGVHD. Here we address whether insights into the timing of the aGVHD initiation and effector phases could allow for the detection of migrating alloreactive T cells before clinical aGVHD onset to permit for efficient therapeutic intervention. METHODS: Murine major histocompatibility complex (MHC) mismatched and minor histocompatibility antigen (miHAg) mismatched allo-HCT models were employed to assess the spatiotemporal distribution of donor T cells with flow cytometry and in vivo bioluminescence imaging (BLI). Daily flow cytometry analysis of peripheral blood mononuclear cells allowed us to identify migrating alloreactive T cells based on homing receptor expression profiles. RESULTS: We identified a time period of 2 weeks of massive alloreactive donor T cell migration in the blood after miHAg mismatch allo-HCT before clinical aGVHD symptoms appeared. Alloreactive T cells upregulated α4ß7 integrin and P-selectin ligand during this migration phase. Consequently, targeted preemptive treatment with rapamycin, starting at the earliest detection time of alloreactive donor T cells in the peripheral blood, prevented lethal aGVHD. CONCLUSIONS: Based on this data we propose a critical time frame prior to the onset of aGVHD symptoms to identify alloreactive T cells in the peripheral blood for timely and effective therapeutic intervention.

Dioxin toxicity, aryl hydrocarbon receptor signaling, and apoptosis-persistent pollutants affect programmed cell death.

Exogenous ligands of the aryl hydrocarbon receptor (AhR) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related substances are highly toxic pollutants ubiquitously present in the environment. They cause a variety of toxic effects to different organs and tissues. Among other effects, TCDD exposure to laboratory animals leads to thymus atrophy and immunosuppression on the one hand, and to tumor formation on the other. Apoptosis appears to be involved in both these toxic effects: AhR activation by TCDD was discussed to induce apoptosis of immune cells, leading to the depletion of thymocytes and ultimately immunosuppression. This mechanism could help to explain the highly immunotoxic actions of TCDD but it is nevertheless under debate whether this is the mode of action for immunosuppression by this class of chemical substances. In other cell types, especially liver cells, TCDD inhibits apoptosis induced by genotoxic treatment. In initiation-promotion studies, TCDD was shown to be a potent liver tumor promoter. Among other theories it was hypothesized that TCDD acts as a tumor promoter by preventing initiated cells from undergoing apoptosis. The exact mechanisms of apoptosis inhibition by TCDD are not fully understood, but both in vivo and in vitro studies consistently showed an involvement of the tumor suppressor p53 in this effect. Various strings of evidence have been established linking apoptosis to the detrimental effects of exogenous activation of the AhR. Within this article, studies elucidating the effects of TCDD and related substances on apoptosis signaling, be it inducing or repressing, is to be reviewed.

Characterization of ochratoxin A-induced apoptosis in primary rat hepatocytes.

The main target organ of the mycotoxin ochratoxin A (OTA) in mammals is the kidney but OTA has also been shown to be hepatotoxic in rats and to induce tumors in mouse liver. Even at very low concentrations, OTA causes perturbations of cellular signaling pathways as well as enhanced apoptosis. OTA has been extensively studied in kidney cell systems. Since this substance also affects liver health, we focused our work on apoptosis-related events induced by OTA in primary rat hepatocytes. We performed pathway-specific polymerase chain reaction arrays to assess the expression of genes involved in apoptosis. Treatment with 1 microM OTA for 24 h caused marked changes in apoptosis-related gene expression. Genes as apaf1, bad, caspase 7, polb (DNA polymerase beta, performs base excision repair), and p53, which are marker genes for DNA damage, were upregulated. FAS and faslg were also markedly induced by treatment with OTA. Treatment of hepatocytes with OTA led to a concentration-dependent inhibition of protein biosynthesis. Apoptosis-inducing factor was released from mitochondria following OTA treatment; the mycotoxin induced the activity of caspases 8, 9, and 3/7 and caused chromatin condensation and fragmentation. Caspase inhibition led to a significant but not complete reduction of OTA-induced apoptosis. Our data suggest that not only OTA leads to p53-dependent apoptosis in rat hepatocytes but it also hints to other mechanisms, independent of caspase activation or protein biosynthesis, being involved.

Inhibition of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin depends on protein biosynthesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic persistent organic pollutant. Most of the toxic effects of TCDD are believed to be mediated by high-affinity binding to the aryl hydrocarbon receptor (AhR) and subsequent effects on gene transcription and protein expression. TCDD causes cancer in multiple tissues in different animal species and is classified as a class 1 human carcinogen. In initiation-promotion studies, TCDD was shown to be a potent liver-tumor promotor. Among other theories it has been hypothesized that TCDD promotes tumor growth by preventing initiated cells from correctly executing apoptosis. In this study, we examined the effects of TCDD on apoptosis induced by UV-C light, ochratoxin A (OTA), and cycloheximide (CHX) in primary rat hepatocytes. Both UV-C light and OTA caused caspase activation and nuclear apoptotic effects. CHX did not activate caspases but nevertheless caused DNA fragmentation and chromatin condensation. TCDD inhibited UV-C light-induced apoptosis and this effect seemed to be dependent on AhR-activation as was shown by employing an AhR antagonist. In contrast to UV-C light-induced apoptosis, TCDD failed to protect primary rat hepatocytes from OTA- or CHX-induced apoptosis. Since both of these compounds inhibit protein biosynthesis as was demonstrated by measuring the incorporation of radiolabeled leucin and protein expression of cytochrome P450 1A1, we propose that the inhibition of apoptosis by TCDD depends on protein biosynthesis. Either TCDD induces some anti-apoptotic protein in an AhR-dependent manner or inhibits pro-apoptotic proteins induced by UV irradiation.

N-Methyl-D-Aspartate Receptor Hypofunction in Meg-01 Cells Reveals a Role for Intracellular Calcium Homeostasis in Balancing Megakaryocytic-Erythroid Differentiation.

The release of calcium ions (Ca2+) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 -/- cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca2+ levels were higher in Meg-01-GRIN1 -/- cells, but ER Ca2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca2+. Microarray analysis revealed that Meg-01-GRIN1 -/- cells had deregulated expression of transcripts involved in Ca2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.

A T-Cell Surface Marker Panel Predicts Murine Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (aGvHD) is a severe and often life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). AGvHD is mediated by alloreactive donor T-cells targeting predominantly the gastrointestinal tract, liver, and skin. Recent work in mice and patients undergoing allo-HCT showed that alloreactive T-cells can be identified by the expression of α4ß7 integrin on T-cells even before manifestation of an aGvHD. Here, we investigated whether the detection of a combination of the expression of T-cell surface markers on peripheral blood (PB) CD8+ T-cells would improve the ability to predict aGvHD. To this end, we employed two independent preclinical models of minor histocompatibility antigen mismatched allo-HCT following myeloablative conditioning. Expression profiles of integrins, selectins, chemokine receptors, and activation markers of PB donor T-cells were measured with multiparameter flow cytometry at multiple time points before the onset of clinical aGvHD symptoms. In both allo-HCT models, we demonstrated a significant upregulation of α4ß7 integrin, CD162E, CD162P, and conversely, a downregulation of CD62L on donor T-cells, which could be correlated with the development of aGvHD. Other surface markers, such as CD25, CD69, and CC-chemokine receptors were not found to be predictive markers. Based on these preclinical data from mouse models, we propose a surface marker panel on peripheral blood T-cells after allo-HCT combining α4ß7 integrin with CD62L, CD162E, and CD162P (cutaneous lymphocyte antigens, CLA, in humans) to identify patients at risk for developing aGvHD early after allo-HCT.

Exogenous TNFR2 activation protects from acute GvHD via host T reg cell expansion.

Donor CD4(+)Foxp3(+) regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT [allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2- and T reg cell-dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo.

Disturbing the histone code in leukemia: translocations and mutations affecting histone methyl transferases.

Leukemia is characterized by increased numbers of blasts originating from transformed early hematopoietic stem and progenitor cells. Genetic alterations are widely recognized as the main drivers of oncogenic transformation. Of considerable interest are mutations affecting the writers of epigenetic marks. In this review, we focus on histone methyltransferases--enzymes that catalyze the methylation of lysine residues in core histones. Histone methylation is a tightly controlled mechanism that is responsible for both activating as well as repressing gene expression in a site-specific manner, depending on which lysine residue is methylated. Histone methyltransferases, including MLL1, DOT1L, EZH2, and SETD2 are recurrently deregulated in human leukemia, either directly by gene mutations or balanced translocations, or indirectly as components of protein complexes that are disturbed in leukemia due to alterations of the other components in these complexes. Several small molecule inhibitors of histone methyltransferases are currently being clinically evaluated for their therapeutic potential in human leukemia. These drugs reverse some of the adverse effects of aberrant histone methylation, and can induce differentiation and cell death in leukemic blasts.

A novel llama antibody targeting Fn14 exhibits anti-metastatic activity in vivo.

Expression of fibroblast growth factor (FGF)-inducible 14 (Fn14), a member of the tumor necrosis factor receptor superfamily, is typically low in healthy adult organisms, but strong Fn14 expression is induced in tissue injury and tissue remodeling. High Fn14 expression is also observed in solid tumors, which is why this receptor is under consideration as a therapeutic target in oncology. Here, we describe various novel mouse-human cross-reactive llama-derived recombinant Fn14-specific antibodies (5B6, 18D1, 4G5) harboring the human IgG1 Fc domain. In contrast to recombinant variants of the established Fn14-specific antibodies PDL192 and P4A8, all three llama-derived antibodies efficiently bound to the W42A and R56P mutants of human Fn14. 18D1 and 4G5, but not 5B6, efficiently blocked TNF-like weak inducer of apoptosis(TWEA K) binding at low concentrations (0.2­2 µg/ml). Oligomerization and Fcγ receptor (FcγR) binding converted all antibodies into strong Fn14 agonists. Variants of 18D1 with enhanced and reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity were further analyzed in vivo with respect to their effect on metastasis. In a xenogeneic model using human colon carcinoma cancer cells, both antibody variants were effective in reducing metastasis to the liver. In contrast, only the 18D1 variant with enhanced ADCC activity, but not its ADCC-defective counterpart, suppressed lung metastasis in the RE NCA model. In sum, this suggests that Fn14 targeting might primarily act by triggering of antibody effector functions, but also by blockade of TWEA K-Fn14 interaction in some cases

Identification and characterization of OSTL (RNF217) encoding a RING-IBR-RING protein adjacent to a translocation breakpoint involving ETV6 in childhood ALL.

Genomic aberrations involving ETV6 on band 12p13 are amongst the most common chromosomal abnormalities in human leukemia. The translocation t(6;12)(q23;13) in a childhood B-cell acute lymphoblastic leukemia (ALL) cell line fuses ETV6 with the putative long non-coding RNA gene STL. Linking STL properties to leukemia has so far been difficult. Here, we describe a novel gene, OSTL (annotated as RNF217 in Genbank), which shares the first exon and a CpG island with STL but is transcribed in the opposite direction. Human RNF217 codes for a highly conserved RING finger protein and is mainly expressed in testis and skeletal muscle with different splice variants. RNF217 shows regulated splicing in B cell development, and is expressed in a number of human B cell leukemia cell lines, primary human chronic myeloid leukemia, acute myeloid leukemia with normal karyotype and acute T-ALL samples. Using a yeast two-hybrid screen, we identified the anti-apoptotic protein HAX1 to interact with RNF217. This interaction could be mapped to the C-terminal RING finger motif of RNF217. We propose that some of the recurring aberrations involving 6q might deregulate the expression of RNF217 and result in imbalanced apoptosis signalling via HAX1, promoting leukemia development.