Abstract 1381: Characterization of the 12-lipoxygenase expression in the megakaryoblastic cell line, Dami

Abstract

Abstract Megakaryocytes are myeloid cells produced primarily in the bone marrow and are best known for releasing platelets in the blood stream. In the platelet production process, megakaryocytes transfer their bioactive content, including the 12-lipoxygenase (12-LO) enzyme, into newly formed platelets. The 12-LO has been shown to be implicated in platelet activation and is overexpressed in several chronic inflammatory conditions, including several types of cancers. The 12-LO is responsible for the conversion of the arachidonic acid into the 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a key lipid mediator implicated in several steps of the metastatic cascade. While 12-LO is expressed in mature human megakaryocytes, we recently identified two megakaryoblast cell lines isolated from leukemia patients lacking 12-LO expression. However, only one of the two cell lines expresses functional 12-LO protein upon megakaryocyte maturation. The goal of this study is to characterize the 12-LO expression during the megakaryocyte maturation process. We hypothesize that 12-LO expression is increased during cell maturation and that inhibition of the 12-LO expression will result in dysfunctional platelets. Both the Dami cell and MEG-01 cells were induced with the co-incubation of both phorbol myristate acetate and eltrombopag, a thrombopoietin receptor agonist. The maturation and viability of the cells were confirmed by flow cytometry. Expression of the 12-LO was assessed by RT-qPCR and immunoblot. Quantification of the 12(S)-HETE was performed by reversed-phase high performance liquid chromatography. Platelet release was quantified by flow cytometry using CD41 fluorescent labelled antibodies. We observed a 3-fold increase in 12-LO protein expression following cell maturation in the Dami cell line. Interestingly, mRNA expression between the undifferentiated Dami cells and mature Dami cells remained unchanged. Furthermore, we did not detect any 12-LO protein expression in the MEG-01 after the cell’s maturation. Finally, we identified a specific 12-LO antisens non-coding RNA that is implicated in the regulation of the protein expression in megakaryocytes. Our study reports the first mechanistic regulation of 12-LO expression implicated in the megakaryocyte maturation process. Since both platelets and platelet-specific 12-LO play an important role in cancer biology, it is important to better understand how megakaryocytes package the enzyme inside these small cells. It remains elusive whether 12-LO enrichment in platelet correlates with the disease severity. Citation Format: Luc H. Boudreau, Vanessa L. Gauvin, Jaël Richard, Marie-France N. Soucy, Mathieu P.A. Hébert, Eric P. Allain. Characterization of the 12-lipoxygenase expression in the megakaryoblastic cell line, Dami [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1381.