Unit:
Faculty of MedicineLibrary of the School of Health Sciences
Dissertation committee:
Μιχαήλ Βουλγαρέλης, Καθηγητής, Ιατρική Σχολή, ΕΠΚΑ
Αθανάσιος Τζιούφας, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Σταυρούλα Γιαννοούλη, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Παναγιώτης Βλαχογιαννόπουλος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Μενέλαος Μανουσάκης, Καθηγητής, Ιατρικη Σχολή, ΕΚΠΑ
Ευσταθία Καψογεωργού, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Ανδρέας Γουλές, Επίκουρος Καθηγήτης, Ιατρική Σχολή, ΕΚΠΑ
Original Title:
Αυτοφαγία στα μυελοδυσπλαστικά σύνδρομα: ο ρόλος του HIF-1α/REDD1 μοριακού μονοπατιού
Translated title:
Autophagy in myelodysplastic syndromes: the role of HIF-1α/REDD1 molecular pathway
Summary:
Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells (HPCs) disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor Hypoxia-Inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target.
In the first section of the study, we examine the aberrant HIF-1 stabilization in BMs from MDS patients.
Methods: BΜ aspiration samples, as well as BM biopsies, were collected from 16 newly diagnosed previously untreated MDS patients and 7 controls with non-malignant hematologic disorder. qRT-PCR and flow cytometry analysis were respectively used for the assessment of mRNA and protein levels of HIF-1α and HIF-1 direct transcriptional target, regulated in development and DNA damage responses 1 (REDD1), in BM aspiration samples A nitroimidazole-indocyanine conjugate was used for the study of the hypoxic phenotype in BM biopsies, as well as in cultured CD34+ BM cells. Autophagy and mitophagy were examined by means of immunofluorescence and electron microscopy. We finally performed in vitro cultures of human BM derived CD34+ HSCs under myeloid priming using a functional HIF-1α inhibitor.
Results: We show that HIF-1 aberrant expression and transcription activity is oxygen independent establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and control aspirates given that both phenomena are HIF-1 dependent. We show that mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite cultured under 21% O2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in controls or in >5%-intra-BM blasts-MDS that uniformly died within 3 days of culture.
Conclusions: We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology and their manipulation has some potential in the therapeutics of benign MDS.
In the second section of the study, we examine the different metabolic profile of MDS myeloid lineage with low and high BM blast percentage.
Methods: We performed an untargeted metabolomic analysis of differentiating myeloid lineage cells from MDS BM aspirates that exhibited <5% (G1) or ≥5% (G2) blasts, in order to delineate its role in MDS severity and malignant potential. BM aspirates were collected from 14 previously untreated MDS patients (G1, n = 10 and G2, n = 4) and age matched controls (n = 5). Following myeloid lineage cell isolation, untargeted mass spectrometry-based metabolomics analysis was performed. Data were processed and analyzed using Metabokit. Enrichment analysis was performed using Metaboanalyst v4 employing pathway-associated metabolite sets.
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Results: We established a bioenergetic profile coordinated by the Warburg phenomenon in both groups, but with a massively different outcome that mainly depended upon each group mitochondrial function and redox state. G1 cells are overwhelmed by glycolytic intermediate accumulation due to failing mitochondria, while the functional electron transport chain and improved redox in G2 compensate for Warburg disruption. Both metabolomes reveal the production and abundance of epigenetic modifiers. G1 and G2 metabolomes differ and eventually determine the MDS clinical phenotype, as well as the potential for malignant transformation.
Conclusion: Our findings suggest that a perturbed metabolism underlies the syndrome’s pathogenesis and also determines the disease severity. We also propose that these bioenergetic alterations are essentially featured in and indeed drive the process of leukemic transformation. Our data not only offer novel insight into the elusive MDS pathophysiology, but also change our viewpoint on MDS-related acute myeloid leukemia biology.
Main subject category:
Health Sciences
Keywords:
Myelodysplastic syndromes, Hypoxia, Autophagy, Metabolism, HIF-1α
Number of references:
463
