Autophagy in Myelodysplastic Syndromes: The Role of HIF-1a/REDD1 Molecular Pathway

Abstract

Background: Hypoxia is a prominent feature of the BM microenvironment, influencing both normal and malignant hematopoiesis. HIF-1α, which is a key regulator of hypoxia responses by mediating the transition to glycolytic metabolism, serves as a cell cycle checkpoint of HSC quiescence and function. It has been proposed that differential HIF-1α protein expression between hypoxic endosteal and less hypoxic vascular niche finely regulates normal hematopoiesis by promoting both quiescence and survival of HSCs, as well as proliferation and differentiation of HPCs. DNA damage response 1 gene (REDD1) is a direct transcriptional target of HIF-1α linking hypoxia to energy regulation and autophagy. Recent evidence suggests that metabolism and autophagy are developmentally programmed and essential for effective hematopoiesis. Aims: To study the implication of HIF-1α/REDD1/autophagy/metabolism axis in differentiation/maturation of hematopoietic BM cells of MDS patients. Methods: BM aspiration and biopsy samples were collected from 15 untreated MDS patients from all subtypes except MDS-RARS and 7 age-matched controls with non-malignant hematologic disorder. Demographic, clinical, laboratory and karyotypic parameters were recorded. BM biopsies were immunohistochemicallly stained by fluorescent-labeled 2-nitroimidazole to assess hypoxic areas in BM. CD34 and myeloid lineage cells were isolated using magnetic beads and ficoll double-layer protocol, respectively. BM cell populations were determined by FACS analysis using standard gating strategies. HIF-1α and REDD1 gene and protein expression was evaluated by qRT-PCR and FACS analysis, respectively. Autophagy was determined by immunofluorescence for LAMP-1/LC3B and immunoblotting for LC3B/p62 (SQSTM1), whereas mitophagy by immunofluorescence for LC3B/TOMM20. Mitochondrial membrane potential (ΔΨ) and mitochondrial mass were analyzed by FACS analysis using mitotrackers. Metabolomic analysis of myeloid lineage cells was performed by liquid chromatography mass spectrometry (LC-MSn). Raw data files were processed using several chemo-informatics tools. Results: We found a preferential strong accumulation of 2-nitroimidazole in intrasinusoidal regions of MDS BM, indicating that hypoxia is a fundamental feature of BM in MDS. We demonstrated a statistically significant REDD1 gene overexpression and an increased intracellular protein co-expression of HIF-1α and REDD1 protein levels in both CD34 and myeloid cells from MDS compared to controls, as determined by RT-qPCR and FACS analysis, respectively. Higher REDD1 protein expression was shown in patients with high grade dysplasia as assessed by the Ogata classification system. Moreover, both CD34 and myeloid cells from MDS demonstrated increased LC3B puncta compared to controls with concurrent staining for CD34 and MPO. The quantitative evaluation of LC3B by Western blot revealed high level of expression of LC3B-II in the MDS myeloid cells compared to controls indicating increased autophagic activity. The observed p62/SQSTM1 degradation along with the colocalization pattern of LC3B/LAMP-1 suggest increased autophagic flux. Metabolomic analysis of MDS myeloid lineage cells compared to controls revealed excessive glycolysis, defective oxidative phosphorylation and increased reductive carboxylation glutaminolysis associated with elevated level of intracellular 2-hydroxyglutarate, all indicative of HIF-1α driven metabolism. The co-localization between TOMM20 marker and autophagosomes in MDS myeloid cells was compatible with increased mitophagy whereas, MDS myeloid cells, were characterized by a reduction of mitochondrial mass and membrane potential in comparison to controls, as determined by FACS analysis. Conclusion: Our results provide evidence for the first time of the hypoxia-driven HIF-1α/REDD1/autophagy axis in the pathophysiology of MDS. Our study suggests that this deregulated pathway is responsible for the production of 2-hydroxyglutarate, an oncometabolite, which is implicated in dysregulated epigenetic homeostasis. All the above may lead to the dysregulated metabolism and differentiation potential of the myeloid cells, thus unraveling a new pathogenetic mechanism for the MDS development. Disclosures No relevant conflicts of interest to declare.

Bioenergetic Profiling of the Differentiating Human MDS Myeloid Lineage with Low and High Bone Marrow Blast Counts

“…and K.K. unpublished data, 2020) [12]. In these cells, mitophagy is featured as the most prominent metabolic feature, probably due to dysfunctional mitochondria (I.E.S.…”

“…and K.K. unpublished data, 2020) [12]. A failing TCA cycle, namely an increased NAD/NADH and FAD/FADH2 ratio with markedly increased ADP/ATP levels, leads to FA accumulation due to the failure of effective adequate beta oxidation [34,36].…”

“…and K.K. unpublished data, 2020) [12], indicated that metabolism may indeed play a crucial role in myelodysplastic syndromes (MDS) and MDS-related AML pathogenesis [13]. HIF1-induced metabolic reprogramming consists of a finely tuned network of extramitochondrial catabolism of glucose, despite adequate oxygen perfusion (the Warburg effect), coupled with an adequate mitochondrial function to reinstate redox balance [14].…”

Myelodysplastic Syndromes and Metabolism

“…Epigenetic modifications are regulated by metabolites, such as acetyl-coenzyme A (CoA), S-adenosylmethionine, α-ketoglutarate (α-KG), 2-hydroxyglutarate (2-HG), and butyrate, which act as substrates, cofactors, or antagonists in this context [ 36 ]. For example, excessive glycolysis coupled with defective OXPHOS and increased reductive carboxylation of glutamine in MDS is associated with elevated levels of the oncometabolite 2-HG [ 37 ] ( Figure 1 ). This effect is particularly prominent in IDH1 -mutated MDS and AML, where the defective IDH1 enzyme results in a very high accumulation of 2-HG rather than α-KG [ 38 ].…”

Integrative metabolic profile of myelodysplastic syndrome based on UHPLC–MS

“…In patients with MDS, the level of 2‐hydroxyglutarate in myeloid lineage cells is increased. Besides, glycolysis, oxidative phosphorylation, and reductive carboxylation glutaminolysis were associated with the occurrence of MDS (Stergiou et al, 2018).…”