Expression analysis of inflamation factors S100A4, S100A8/9, S100A12 in myeloproliferative neoplasms: a connection with the presence of mutation in the JAK2 gene

Abstract

Myeloproliferative neoplasms (MPNs) belong to the group of malignant diseases of the blood, characterized by a clonal expansion of neoplastically transformed hematopoietic progenitors of the myeloid lineage in the bone marrow. MPN in the narrow sense includes polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In patients with MPN, the underlying disorder is the JAK2V617F mutation, which is a driving mutation in more than 95% of patients with PV and 50-60% of patients with ET and PMF. The JAK2V617F mutation leads to constitutive activation of JAK2 kinase and cytokine-independent cell growth. As a consequence, downstream signaling pathways (JAK2 / STAT3, PI3K-AKT, MAPK), which are involved in myeloproliferation, are activated. The presence of the JAK2V617F mutation in MPN in hematopoietic cells alters the activity of inflammatory signaling pathways, altering the production of reactive oxygen species in neutrophils, altering serum inflammatory cytokine levels, and altering reactivity to these cytokines. The main feature of chronic inflammation is the high level of circulating cytokines and chemokines, as well as the accumulation of reactive oxygen species, which leads to genetic instability and may be suitable for the formation and progression of neoplasms. In patients with MPN, a significant increase in many inflammatory mediators has been observed, including a group of calcium-binding proteins, called S100 proteins. Due to their tumorigenic and pro-inflammatory activity, the S100 family of proteins that potentially have the greatest effect on the formation and development of myeloproliferative neoplasms are S100A4, S100A8, S100A9 and S100A12, as several studies have shown their elevated levels in MPN. The aim of this dissertation was to investigate the gene expression and levels of S100A4, S100A8, S100A9 and S100A12 factors in MPN patients, study the effect of pro-inflammatory cytokine IL-6, anti-inflammatory cytokine IL-10, as well as JAK inhibitors on the levels of these proteins on human HEL 92.1.7 model cell line with a JAK2 mutation, as well as an examination of the signaling mechanisms by which IL-6 and IL-10 exert their effects on peripheral blood cells of patients with MPN and the human HEL 92.1.7 model. cell line. The results of this study showed that inflammation factors show an increase in levels, which is dependent on the presence of the JAK2V617F mutation in individual proteins. This increase in the level of inflammatory factors indicates that patients with MPN have chronic inflammation. Treatment with hydroxyurea and a combination of hydroxyurea and a specific JAK2 inhibitor decreases the levels of inflammatory factor in the HEL 92.1.7 cell line, indicating that the S100 proteins examined are expressed in a JAK2-dependent manner. The pro-inflammatory cytokine IL-6 decreased S100 protein levels in mononuclear cells of patients with MPN, and this decrease in MPN was mediated by NF-κB and PI3K signaling pathways. The anti-inflammatory cytokine IL-10 also reduced S100 protein levels, especially S100A8 and S100A9 in patients with ET, but mediated by the PI3K-AKT signaling pathway. IL-6 / IL-10-mediated regulation of S100 protein levels by MPNs is dominated by NF-κB and PI3K signaling pathways, whereas activation of the JAK-STAT signaling pathway is constitutive.

Mijeloproliferativne neoplazme (MPN) spadaju u grupu malignih oboljenja krvi, za koje je karakteristična klonalna ekspanzija neoplastično transformisanih hematopoetskih progenitora mijeloidne loze u kostnoj srži. U MPN u užem smislu spadaju policitemija vera (PV), esencijalna trombocitemija (ET) i primarna mijelofibroza (PMF). Kod pacijenata sa MPN osnovni poremećaj predstavlja mutacija JAK2V617F, koja je pokretačka mutacija u više od 95% pacijenata sa PV i 50-60% pacijenata sa ET i PMF. JAK2V617F mutacija vodi konstitutivnoj aktivaciji JAK2 kinaze i rastu ćelija nezavisnom od citokina. Kao posledica, aktivirani su nishodni signalni putevi (JAK2/STAT3, PI3K-AKT, MAPK), koji su uključeni u mijeloproliferaciju. Prisustvo JAK2V617F mutacije kod MPN u hematopoetskim ćelijama menja aktivnost signalnih puteva značajnih za inflamaciju, pri čemu dolazi do promene nastajanja reaktivnih vrsta kiseonika kod neutrofila, promene nivoa inflamatornih citokina u serumu i izmenjene reaktivnosti na ove citokine. Glavna odlika hronične inflamacije jeste visok nivo citokina i hemokina u cirkulaciji, kao i nagomilavanje reaktivnih kiseoničnih vrsta, što dovodi do genetičke nestabilnosti i može biti pogodno za nastanak i progresiju neoplazmi. Kod pacijenata sa MPN uočeno je značajno povećanje brojnih medijatora inflamacije, među kojima je i grupa kalcijum vezujućih proteina, pod nazivom S100 proteini. Zbog svog tumorigenog i proinflamatornog delovanja, proteini S100 familije koji potencijalno imaju najveći efekat na nastanak i razvoj mijeloproliferativnih neoplazmi su S100A4, S100A8, S100A9 i S100A12, jer je u nekoliko studija pokazan njihov povišen nivo kod MPN. Cilj istraživanja ove disertacije bilo je ispitivanje genske ekspresije i nivoa S100A4, S100A8, S100A9 i S100A12 faktora kod MPN pacijenata, ispitivanje uticaja proinflamatornog citokina IL-6, antiinflamatornog citokina IL-10, kao i JAK inhibitora na nivoe ovih proteina na modelu humane HEL 92.1.7 ćelijske linije sa JAK2 mutacijom, kao i ispitivanje signalnih mehanizama putem kojih IL-6 i IL-10 ostvaruju svoje efekte na ćelije periferne krvi pacijenata sa MPN i modelu humane HEL 92.1.7. ćelijske linije. Rezultati ove studije su pokazali da faktori inflamacije pokazuju povećanje nivoa, koje zavisi od prisustva JAK2V617F mutacije kod pojedinačnih proteina. Navedeno povećanje nivoa faktora inflamacije ukazuje da kod obolelih od MPN postoji hronična inflamacija. Tretman hidroksiureom i kombinacijom hidroksiuree i specifičnog JAK2 inhibitora smanjuje nivoe faktora inflamacije u HEL 92.1.7 ćelijskoj liniji, što ukazuje da se ispitivani S100 proteini eksprimiraju na JAK2-zavistan način. Proinflamatorni citokin IL-6 smanjivao je nivoe S100 proteina u mononuklearima pacijenata sa MPN, a ovo smanjenje kod MPN posredovano je NF-κB i PI3K signalnim putevima. Antiinflamatorni citokin IL-10 takođe je smanjivao nivoe S100 proteina, pogotovo S100A8 i S100A9 kod pacijenata sa ET, ali posredovano PI3K-AKT signalnim putem. U IL-6 / IL-10 posredovanoj regulaciji nivoa S100 proteina kod MPN dominiraju NF-κB i PI3K signalni putevi, dok je aktivacija JAK-STAT signalnog puta konstitutivna.