Ina Nepstad

Characterisation and expression analysis of the Atlantic halibut (Hippoglossus hippoglossus L.) cytokines: IL-1β, IL-6, IL-11, IL-12β and IFNγ

Genes encoding the five Atlantic halibut (Hippoglossus hippoglossus L.) cytokines; interleukin (IL)-1β, IL-6, IL-11b, IL-12βc, and interferon (IFN) γ, were cloned and characterised at a molecular level. The genomic organisation of the halibut cytokine genes was similar to that seen in mammals and/or other fish species. Several mRNA instability motifs were found within the 3′-untranslated region (UTR) of all cytokine cDNA sequences. The putative cytokine protein sequences showed a low sequence identity with the corresponding homologues in mammals, avian and other fish species. Nevertheless, important structural features were presumably conserved such as the presence, or absence in the case of IL-1β, of a signal peptide, secondary structure and family signature motifs. The relative expression pattern of the cytokine genes was analyzed in several halibut organs, revealing a constitutive expression in both lymphoid and non-lymphoid organs. Interestingly, the gills showed a relatively high expression of IL-1β, IL-12βc and IFNγ. The real time RT-PCR data also showed that the mRNA level of IL-1β, IL-6, IL-12βc and IFNγ was high in the thymus, while IL-11b was relatively highly expressed in the posterior kidney and posterior gut. Moreover, the halibut brain showed a relatively high level of IL-6 transcripts. Anterior kidney leucocytes in vitro stimulated with imiquimod showed a significant increase in mRNA level of the five halibut cytokine genes. The sequence and characterisation data presented here will be useful for further investigation of both innate and adaptive immune responses in halibut, and be helpful in the design of vaccines for the control of various infectious diseases.

Co-activation of AMPK and mTORC1 Induces Cytotoxicity in Acute Myeloid Leukemia

AMPK is a master regulator of cellular metabolism that exerts either oncogenic or tumor suppressor activity depending on context. Here, we report that the specific AMPK agonist GSK621 selectively kills acute myeloid leukemia (AML) cells but spares normal hematopoietic progenitors. This differential sensitivity results from a unique synthetic lethal interaction involving concurrent activation of AMPK and mTORC1. Strikingly, the lethality of GSK621 in primary AML cells and AML cell lines is abrogated by chemical or genetic ablation of mTORC1 signaling. The same synthetic lethality between AMPK and mTORC1 activation is established in CD34-positive hematopoietic progenitors by constitutive activation of AKT or enhanced in AML cells by deletion of TSC2. Finally, cytotoxicity in AML cells from GSK621 involves the eIF2α/ATF4 signaling pathway that specifically results from mTORC1 activation. AMPK activation may represent a therapeutic opportunity in mTORC1-overactivated cancers.

2012 ECS-ECN JUNIOR REVIEW AWARD –The angioregulatory cytokine network in human acute myeloid leukemia – from leukemogenesis via remission induction to stem cell transplantation

Acute myeloid leukemia (AML) is characterized by bone marrow accumulation of immature leukemic blast cells. Conventional AML treatment includes induction chemotherapy to achieve disease control, followed by consolidation therapy with conventional chemotherapy or allogeneic/autologous stem cell transplantation (allo/auto-SCT) to eradicate residual disease. Even younger patients receiving the most intensive treatment have a median, long-term, AML-free survival of only 45-50%, highlighting the need for new treatment strategies. The important role of the bone marrow cytokine network during disease development and treatment is suggested by several observations, including: (i) the increased microvascular density (MVD) in leukemic bone marrow, (ii) experimental evidence of cytokine-mediated crosstalk between leukemic and microvascular endothelial cells, (iii) the prognostic impact of angioregulatory cytokine levels both in patients receiving conventional chemotherapy and allo-SCT, and (iv) the experimental evidence for an antileukemic effect of cytokine inhibition in human AML. Several cytokines are constitutively released by human AML cells, including interleukins, chemokines, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and angiopoietins. However, the cytokine system constitutes a functional, interacting network, and recent evidence suggests that analysis of serum cytokine profiles rather than the analysis of single cytokines should be used for prognostic evaluation of AML patients. We will discuss the role of angioregulatory cytokines in leukemogenesis, including their direct effects on the leukemic cells, as well as their indirect contribution to leukemogenesis through angioregulation and crosstalk between leukemic and neighboring stromal cells. We shall also discuss the possibility of targeting angioregulatory cytokines as a part of the treatment strategy in leukemia.

Targeting of polo-like kinases and their cross talk with Aurora kinases – possible therapeutic strategies in human acute myeloid leukemia?

Introduction: Five human polo-like kinases (PLKs) have been identified, and PLK1 – 4 seem to interact with Aurora kinases and act as cell cycle regulators in both normal and malignant human cells. Areas covered: The present review describes i) experimental evidence for a role for PLKs and Aurora kinases in human leukemogenesis and ii) the results from clinical studies of PLK and Aurora kinase inhibitors in the treatment of human acute myeloid leukemia (AML). The review was based on searches in the PubMed and the ClinicalTrials.gov databases. These inhibitors have antiproliferative and proapoptotic effects in AML cells. Hematological and gastrointestinal toxicities are frequently dose limiting, and this may limit the use of these agents in combination with conventional AML therapy. Aurora kinase inhibitors seem to be most effective for patients with high expression of the target kinases, and the same may be true for PLK inhibitors. Expert opinion: PLK inhibition is a promising strategy for the treatment of AML. Future clinical studies have to clarify i) whether this strategy is most effective for certain subsets of patients; ii) whether multikinase inhibitors targeting several cell cycle regulators should be preferred; and iii) how this therapeutic strategy eventually should be combined with conventional antileukemic chemotherapy.

STAT3 as a possible therapeutic target in human malignancies: lessons from acute myeloid leukemia

STAT3 is important for transcriptional regulation in human acute myeloid leukemia (AML). STAT3 has thousands of potential DNA binding sites but usually shows cell type specific binding preferences to a limited number of these. Furthermore, AML is a very heterogeneous disease, and studies of the prognostic impact of STAT3 in human AML have also given conflicting results. A more detailed characterization of STAT3 functions and the expression of various isoforms in human AML will therefore be required before it is possible to design clinical studies of STAT3 inhibitors in this disease, and it will be especially important to investigate whether the functions of STAT3 differ between patients. Several other malignancies also show extensive biological heterogeneity, and the present discussion and the suggested scientific approaches may thus be relevant for other cancer patients.

Increased antileukemic effects in human acute myeloid leukemia by combining HSP70 and HSP90 inhibitors

Background: Heat shock proteins (HSPs) are molecular chaperones that assist proteins in their folding to native structures. HSP90, and more recently HSP70, have emerged as possible therapeutic targets in human malignancies, including acute myeloid leukemia (AML). Design and methods: The authors investigated the effects of the HSP70 inhibitor VER-155008 tested alone or in combination with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) on proliferation, viability, constitutive cytokine release and intracellular HSP levels of primary human AML cells. Results: VER-155008 caused a dose-dependent inhibition of cytokine-dependent AML cell proliferation both in suspension cultures and in a colony formation assay, and the drug also had a proapoptotic effect. HSP70 and HSP90 inhibition had additive antiproliferative and proapoptotic effects. VER-155008 caused a strong inhibition of the constitutive AML cell release of several growth factors/regulators of hematopoiesis (i.e., TNF-α, VEGF, IL-3, IL-1β, IL-1 receptor antagonist), but had relatively weak effects on the constitutive chemokine release. HSP70 inhibition did not induce any compensatory increase of other HSPs. Conclusion: HSP70 inhibition has antileukemic effects when tested alone, and the combination of HSP70 and HSP90 inhibition seems to have additive antileukemic effects for primary human AML cells in vitro.

An Extended Study of Seroprevalence of Anti-Anisakis simplex IgE Antibodies in Norwegian Blood Donors

During the last decade, cases of the fish parasite Anisakis simplex infection and allergy in human have increased in countries with high fish consumption. Our aim was to perform an extended seroprevalence study of anti‐IgE antibodies against this parasite in Norway, one of the high fish‐consuming countries. At the Department of Immunology and Transfusion Medicine and the Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway, two main groups of anonymized serum samples were collected; the first (n = 993) from recently recruited blood donors (designated ‘BDO’) and the second (n = 414) from patient with total IgE levels ≥1000 kU/l (designated ‘IGE+’). The sera were analysed by the ImmunoCAP® method for total IgE and IgE antibodies against A. simplex, house dust mite (HDM), shrimp, cod, crab, brine shrimp and shrimp tropomyosin. The A. simplex positive sera were further tested by an enzyme‐linked immunosorbent assay (ELISA) method, which uses 2 recombinant (r) major allergens, rAni s 1 and rAni s 7 as target antigens. SDS‐PAGE and Western immunoblotting analyses were also performed. Whereas the prevalences by ImmunoCAP® were 0.4% and 16.2% in the BDO and IGE+ groups, respectively, analyses with recombinant allergens showed only 0.0% and 0.2%. Cross‐reactivity and immunoblotting analyses suggested that most of the ImmunoCAP® positive sera were probably false‐positive due to cross‐sensitization to shrimp and HDM. However, positivity due to other A. simplex antigens should also be considered. Compared with other high fish‐consuming countries, we observed a very low seroprevalence of anti‐Anisakis IgE antibodies in a Norwegian population.

Mesenchymal Stem Cells Support Survival and Proliferation of Primary Human Acute Myeloid Leukemia Cells through Heterogeneous Molecular Mechanisms

Acute myeloid leukemia (AML) is a bone marrow malignancy, and various bone marrow stromal cells seem to support leukemogenesis, including osteoblasts and endothelial cells. We have investigated how normal bone marrow mesenchymal stem cells (MSCs) support the in vitro proliferation of primary human AML cells. Both MSCs and primary AML cells show constitutive release of several soluble mediators, and the mediator repertoires of the two cell types are partly overlapping. The two cell populations were cocultured on transwell plates, and MSC effects on AML cells mediated through the local cytokine/soluble mediator network could thus be evaluated. The presence of normal MSCs had an antiapoptotic and growth-enhancing effect on primary human AML cells when investigating a group of 51 unselected AML patients; this was associated with increased phosphorylation of mTOR and its downstream targets, and the effect was independent of cytogenetic or molecular-genetic abnormalities. The MSCs also supported the long-term proliferation of the AML cells. A subset of the patients also showed an altered cytokine network with supra-additive levels for several cytokines. The presence of cytokine-neutralizing antibodies or receptor inhibitors demonstrated that AML cells derived from different patients were heterogeneous with regard to effects of various cytokines on AML cell proliferation or regulation of apoptosis. We conclude that even though the effects of single cytokines derived from bone marrow MSCs on human AML cells differ among patients, the final cytokine-mediated effects of the MSCs during coculture is growth enhancement and inhibition of apoptosis.

Predicting effects of kinase inhibitor in therapy for myeloid malignancies – the challenges in capturing disease heterogeneity

Protein kinase inhibitors have proved to be effective and well tolerated in special form of malignant diseases in which targeted kinases play a central role in the development and progression of the malignant clone. In addition, the development of acquired drug resistance, due to new mutations or clonal evolution, during treatment is common. Methods for measuring the activity and predicting the efficacy of such compounds are warranted for evaluating individual responses to treatment, particularly in a context of widespread preclinical and clinical studies of protein kinase inhibitors in patients with heterogeneous myeloid malignancies.

Antileukemic effects of midostaurin in acute myeloid leukemia – the possible importance of multikinase inhibition in leukemic as well as nonleukemic stromal cells

ABSTRACT Introduction: Midostaurin is a multikinase inhibitor that inhibits receptor tyrosine kinases (Flt3, CD117/c-kit, platelet-derived growth factor receptor, vascular endothelial growth factor receptor 2) as well as non-receptor tyrosine kinases (Frg, Src, Syk, Protein kinase C). Combination of midostaurin with conventional intensive chemotherapy followed by one year maintenance monotherapy was recently reported to improve the survival of acute myeloid leukemia (AML) patients with Flt3 mutations. Areas covered: Relevant publications were identified through literature searches in the PubMed database. We searched for (i) original articles describing the results from clinical studies; (ii) published articles describing the importance of midostaurin-inhibited kinases for leukemogenesis and chemosensitivity. Expert opinion: Midostaurin monotherapy is well tolerated, combined with conventional chemotherapy gastrointestinal toxicity increases significantly. Midostaurin alters anthracycline pharmacokinetics. Furthermore, its antileukemic effects may not only be mediated through Flt3 inhibition alone; the inhibition of other kinases may also be important for the overall antileukemic effect. Midostaurin may then have direct effects on the leukemic cells but also indirect antileukemic effects through inhibition of the AML-supporting effects of neighboring stromal cells in the bone marrow microenvironment. Midostaurin may thus be used in combination with intensive chemotherapy, as maintenance treatment or as disease-stabilizing treatment for elderly unfit patients.