Leukotriene signaling via ALOX5 and cysteinyl leukotriene receptor 1 is dispensable for in vitro growth of CD34 (+)CD38(-) stem and progenitor cells in chronic myeloid leukemia

Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL(+)CD34(+)CD38(-) cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34(+)CD38(-) cells from 7 CML patients. The majority of the single leukemic BCR-ABL(+)CD34(+)CD38(-) cells expressed cysteinyl leukotriene receptors CYSLTI and CYSLT2. However, montelukast, an inhibitor of CYSLTI, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLTI signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients. (C) 2017 The Author(s). Published by Elsevier Inc.Peer reviewe

Leukocyte activation in newborns in relation to prenatal stress

The aim of this project is to describe the pattern of innate immunity responses at birth with focus on the recruitment of leukocytes such as eosinophils and neutrophils and their content of cathelicidin antimicrobial peptide LL-37 associated to different degrees of fetal stress and delivery modes. We know that delivery is associated to fetal stress including release of cortisol and cathecolamines. Stress has profound effects on the immune system, also by acting on the trafficking of leukocytes, a process in which adhesion and chemotaxis are primordial and critical events for the development of effective antimicrobial defences. Birth and postnatal adaptation are associated with leukocyte recruitment and activation, an acute phase response and inflammatory skin reactions in the human newborn, all together indicating an enhanced innate immunity at that time-point in life. We found that with a progressive increase in fetal stress, there are significant elevations in total white blood count, in particular neutrophils and monocytes, as well as an enhanced IL-8 and soluble ESelectin level. Assisted delivery, associated with the highest degree of fetal stress in addition has an increased Interferon-gamma level. The direct correlation between specific leukocytes and Interferon-gamma respectively, with stress markers such as cortisol, beta-endorphin and cathecolamines reflects the impact of stress on fetal immunity. Also, after normal delivery cord plasma LL-37 levels were 3 times higher compared to C-section indicating fetal peptide release. Neutrophils from cord blood after normal delivery contain 10 times more cytoplasmatic cathelicidin peptide compared to corresponding cells after C-section where a strict granular localization is found, a possible sign of cell activation. A highly significant correlation was observed between maternal and cord plasma LL-37 levels, most probably reflects maternal cathelicidin placental transfer. We also could show that cord eosinophils contain and to some degree release LL-37. Interesting, cord eosinophils contain 3 times more LL-37 compared to adults. Thus, it seems reasonable to deduce that these cells may exert important regulatory functions during the neonatal period, also including antimicrobial activity and immunomodulatory effects by peptide LL-37. Our data analysis shows gene expression differences in eosinophils between the two delivery modes. We have identified the genes with most significant expression difference in C-section eosinophils compared to normal delivery eosinophils, here, being IFN-gamma receptor and STAT1. Our RT-PCR data confirms the upregulation of these genes in eosinophils from cord blood compared to normal delivery. This indicates a more enhanced immunity in eosinophils after normal delivery. This study assembles a picture of newborn infant s ability to mount a powerful inflammatory immune response at birth and that the stress of normal labor and terrestrial adaptation induces profound structural and functional changes in fetal leukocytes

IFN gamma directly counteracts imatinib-induced apoptosis of primary human CD34+CML stem/progenitor cells potentially through the upregulation of multiple key survival factors

Tyrosine kinase inhibitors (TKIs) have dramatically improved the survival in chronic myeloid leukemia (CML), but residual disease typically persists even after prolonged treatment. Several lines of evidence suggest that TKIs administered to CML patients upregulate interferon gamma (IFN gamma) production, which may counteract the anti-tumorigenic effects of the therapy. We now show that activated T cell-conditioned medium (TCM) enhanced proliferation and counteracted imatinib-induced apoptosis of CML cells, and addition of a neutralizing anti-IFN gamma antibody at least partially inhibited the anti-apoptotic effect. Likewise, recombinant IFN gamma also reduced imatinib-induced apoptosis of CML cells. This anti-apoptotic effect of IFN gamma was independent of alternative IFN gamma signaling pathways, but could be notably diminished by STAT1-knockdown. Furthermore, IFN gamma upregulated the expression of several anti-apoptotic proteins, including MCL1, PARP9, and PARP14, both in untreated and imatinib-treated primary human CD34+ CML stem/progenitor cells. Our results suggest that activated T cells in imatinib-treated CML patients can directly rescue CML cells from imatinib-induced apoptosis at least partially through the secretion of IFN gamma, which exerts a rapid, STAT1-dependent anti-apoptotic effect potentially through the simultaneous upregulation of several key hematopoietic survival factors. These mechanisms may have a major clinical impact, when targeting residual leukemic stem/progenitor cells in CML.Funding Agencies|Cancer Research Funds of Radiumhemmet [174283]; Thorsmans stiftelse for preleukemiforskning; Cathrine Everts Research Foundation; Lars Hierta Memorial Foundation; Swedish Research Council [2013-08807]; Karolinska Institute Foundation and Funds; Gunnar Grimfors Gavofond for Hematologisk Forskning; Emil Andersson Fund for Medical Research</p

Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.Funding Agencies|Cancer Research Funds of Radiumhemmet [174283]; Thorsmans Stiftelse for Pre-leukemi Forskning; Gunnar Grimfors Gavofond for Hematologisk Forskning; Cathrine Everts Research Foundation; Lars Hierta Memorial Foundation; Emil Anderson Fund for Medical Research; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2013-08807]; Karolinska Institute Foundation and Funds; Karolinska InstituteKarolinska Institutet</p