Brenda Kusler

Calcineurin Negatively Regulates TLR-Mediated Activation Pathways

In innate immunity, microbial components stimulate macrophages to produce antimicrobial substances, cytokines, other proinflammatory mediators, and IFNs via TLRs, which trigger signaling pathways activating NF-κB, MAPKs, and IFN response factors. We show in this study that, in contrast to its activating role in T cells, in macrophages the protein phosphatase calcineurin negatively regulates NF-κB, MAPKs, and IFN response factor activation by inhibiting the TLR-mediated signaling pathways. Evidence for this novel role for calcineurin was provided by the findings that these signaling pathways are activated when calcineurin is inhibited either by the inhibitors cyclosporin A or FK506 or by small interfering RNA-targeting calcineurin, and that activation of these pathways by TLR ligands is inhibited by the overexpression of a constitutively active form of calcineurin. We further found that IκB-α degradation, MAPK activation, and TNF-α production by FK506 were reduced in macrophages from mice deficient in MyD88, Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF), TLR2, or TLR4, whereas macrophages from TLR3-deficient or TLR9 mutant mice showed the same responses to FK506 as those of wild-type cells. Biochemical studies indicate that calcineurin interacts with MyD88, TRIF, TLR2, and TLR4, but not with TLR3 or TLR9. Collectively, these results suggest that calcineurin negatively regulates TLR-mediated activation pathways in macrophages by inhibiting the adaptor proteins MyD88 and TRIF, and a subset of TLRs.

Calcineurin inactivation leads to decreased responsiveness to LPS in macrophages and dendritic cells and protects against LPS-induced toxicity in vivo

Microbial components such as lipopolysaccharide (LPS) bind to Toll-like receptors (TLRs) and activate innate and inflammatory responses. Responses to LPS and other microbial components are limited by the activation of negative feedback mechanisms that reduce responsiveness to subsequent LPS exposure, often termed LPS tolerance. Our laboratory has previously shown that calcineurin, a phosphatase known for its activation of T cells via NFAT, negatively regulates the TLR pathway in macrophages; consequently, calcineurin inhibitors (FK506 and cyclosporin A) mimic TLR ligands in activating the TLR pathway, NF-KB, and associated innate and inflammatory responses. This study investigated the physiological consequences of calcineurin inactivation for LPS-induced inflammatory responses in vitro and in vivo using two models: calcineurin inhibition by FK506 (tacrolimus) and myeloid cell-specific calcineurin deletion. Activation of dendritic cells and macrophages with FK506 in vitro was shown to induce a state of reduced responsiveness to LPS (i.e. a form of LPS tolerance). Similarly, macrophages from FK506-treated mice or from mice in which the calcineurin B1 (CnB1) subunit was conditionally knocked out in myeloid cells were found to have diminished LPS-induced inflammatory responses. In addition, mice with CnB1-deficient myeloid cells and mice undergoing FK506 treatment showed improved survival and recovery when challenged with high doses of systemic LPS compared to controls. These results demonstrate that inactivation of calcineurin in macrophages and other myeloid cells by inhibition or deletion can induce a form of LPS tolerance and protect the host from LPS toxicity in vivo.

Impact of TET2 mutations on mRNA expression and clinical outcomes in MDS patients treated with DNA methyltransferase inhibitors

Despite a common lineage derivation, the clonal myeloid disorders acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN) are phenotypically distinct, with few known shared recurrent genetic abnormalities. The recent recognition that the ten-eleven translocation 2 (TET2) gene located on the long arm of chromosome four is mutated with high frequency across all myeloid malignancies is therefore a compelling unifying feature [1]. As many as 30% of MDS patients have a variety of TET2 abnormalities including deletions, insertions and nonsense or missense mutations throughout the nine coding exons of the 150 kB gene, many of which are predicted to result in truncated translation [2]. Based on analyses from a small number of patient samples, TET2 mutations may confer a favourable outcome in MDS patients [3], but a worse outcome in those with chronic myelomonocytic leukemia (CMML) [4] and AML [5]. TET2 belongs to a three-member family that also includes TET1 and TET3; TET1 was originally identified as a partner for the MLL gene within t(10;11)(p12;q23) translocations in AML [6]. All three paralogs share two highly conserved regions, BOX1 and BOX2 (Figure 1A). A highly homologous catalytic domain shared among the three proteins catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine in TET1 [7], which could epigenetically regulate gene expression by altering methylation-driven gene silencing. Given its homology to TET1, TET2 is hypothesized to act as a tumour suppressor gene (TSG) by similarly regulating DNA methylation and epigenetic control of gene expression at critical loci important for myelopoeisis and leukemogenesis [8]. The DNA methyltransferase inhibitors (DNMTIs) 5-azacytidine and decitabine, approved agents with activity in MDS, are incorporated into DNA and/or RNA, inhibiting DNA methyltransferase and preventing promoter methylation and TSG silencing. The aim of this study was to determine whether the presence of TET2 mutations is associated with prognosis in MDS patients treated with DNMTIs. Bone marrow aspirates from 12 patients with MDS who received DNMTIs and had documented clinical follow up were obtained with informed consent in accordance with the Declaration of Helsinki and approval by the Institutional Review Board at the Stanford University School of Medicine (SUMC). DNA and RNA were extracted using a Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA). All patients were diagnosed according to World Health Organization criteria and confirmed by the clinical pathology department at SUMC.

Reactive Oxygen Species Regulate Nucleostemin Oligomerization and Protein Degradation

Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H2O2) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H2O2 impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-l-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.

Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice.

Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.

Effect of nucleophosmin1 haploinsufficiency on hematopoietic stem cells

Nucleophosmin1 (NPM1), located at 5q35.1, is frequently mutated, deleted, or translocated in a number of hematopoietic malignancies (1). Deletion of 5q encompassing the NPM1 locus occurs in approximately 40% of high risk MDS/AML with complex karyotypes (2), although the common 5q interstitial deletion that occurs in MDS does not include this region (3). Mice genetically engineered to harbor heterozygous loss of Npm1 (Npm1+/−) were shown to have a mild MDS-like phenotype characterized by dyserythropoiesis and dysplastic megakaryocytes with macrocytic anemia, but without significant cytopenias or alterations in lineage commitment. These mice also exhibited increased susceptibility to hematologic malignancies with age (4, 5). While these studies confirmed NPM1’s role as a tumor suppressor, they did not characterize a role for NPM1 in hematopoietic stem or progenitor cell function, a likely phenotype alteration given the propensity of NPM1 mutant mice to develop hematologic neoplasms. In order to better understand the effects of loss of a single NPM1 allele on hematopoiesis, we obtained Npm1 heterozygous deficient mice (#11744-UCD) from the Mutant Mouse Regional Resource Centre (UC Davis, CA). An embryonic stem cell clone with a single gene trap event in the Npm1 locus was used to generate the Npm1+/− line (genetic background 129/SvEvBrd X C57BL/6J).