ABSTRACT
Prior studies by our group and others have demonstrated that platelets from patients with myeloproliferative neoplasms (MPNs) exhibit a hyperreactive phenotype. However, a complete understanding of platelet alterations in MPNs remains lacking, and the mechanisms by which platelets contribute to MPN-related thrombosis, as well as other MPN disease features, are incompletely understood. In this study, utilizing multiomic approaches to interrogate platelet phenotypes in patient samples, in conjunction with relevant MPN animal models, we aim to investigate mechanisms of dysregulated platelet activity in MPNs, and explore how these findings may be leveraged to uncover novel therapeutic strategies.
We initially studied platelet activation in peripheral blood from patients with essential thromobocythemia (ET) and compared to age-, sex-matched healthy controls. We found significantly increased P-selectin exposure in conjunction with increased platelet-leukocyte aggregates indicating activation of platelets from ET patients. To investigate the transcriptional signature of PBMCs and platelets at the single cell level, we performed single cell RNA-seq (scRNA-seq) in PBMCs from ET patients and healthy controls. Monocytes were increased in ET patients and displayed the highest inflammation index, implicating monocytes as the primary source of inflammation in ET. Aside from the quantitative increases of platelets in ET patients, we demonstrated strong enrichment of platelet activation, mTOR and oxidative phosphorylation (OXPHOS) genes in platelets from ET patients. Proteomic data confirmed the activation of OXPHOS and mTOR pathways in platelets from MPN patients. Metabolomics analysis revealed distinct metabolic phenotypes consisting of elevated tricarboxylic acid (TCA) cycle components, ATP generation and lower alpha-ketoglutarate (α-KG), in platelets from MPN patients, all consistent with enhanced OXPHOS and mTOR activities. Enhanced mitochondrial respiration at both baseline and after ex vivo stimulation with the platelet agonist thrombin receptor activator peptide (TRAP6) by extracellular flux analysis confirmed the bioenergetic alterations in platelets from MPN patients.
α-KG is a key TCA cycle intermediate, which inhibits PI3K/AKT/mTOR pathway signaling via suppression of ATP synthase. α-KG supplementation drastically reduced oxygen consumption and ATP generation in platelets from MPN patients. We further investigated the effects of α-KG on MPN platelet activation. Ex vivo incubation of platelets from both MPN patients and Jak2 V617F knock-in mice with α-KG significantly reduced platelet surface P-selectin and integrin α2bβ3 activation. Additionally, α-KG inhibited the spreading and adhesion of platelets from Jak2 V617F knock-in mice to fibrinogen-coated surfaces. Platelet phosphoblots demonstrated significant downregulation of p-AKT and p-ERK after treatment with α-KG, suggesting the involvement of PI3K/AKT/mTOR and MAPK pathways in the inhibitory effects of α-KG on platelet activation. Thus, α-KG inhibited platelet hyperreactivities in both human and mouse MPN samples.
To test the therapeutic impact of α-KG on MPN disease features, we treated Jak2 V617F knock-in mice with α-KG for 6 weeks. Oral α-KG supplementation decreased splenomegaly and reduced elevated platelets and hematocrit. Additionally, monocytes were significantly decreased as early as 2 weeks after α-KG treatment in Jak2 V617F knock-in mice. Consistently, RNA-seq of bone marrow samples from α-KG treated mice revealed inhibition of heme metabolism, OXPHOS and mTOR signaling pathways. In ex vivo studies with MPN patient CD34+ cells, α-KG treatment for 10 days led to a decrease in CD41+ CD61+ cells, suggesting decreased megakaryocyte commitment. We further observed that α-KG incubation significantly decreased the secretion of proinflammatory cytokines from sorted CD14+ human monocytes. Mass cytometry analysis of whole blood from MPN patients demonstrated inhibition of MAPK pathway signaling after α-KG treatment. Taken together, these results suggest that α-KG supplementation may exert therapeutic effects through both direct inhibition of MPN platelet activity and via quenching of monocyte hyper-inflammation.
In summary, these studies reveal a previously unrecognized metabolic disorder in platelets from MPN patients and highlight a prominent role for α-KG and mTOR signalling in aberrant MPN platelet activity and monocyte-driven inflammation. These findings have potential relevance for novel therapeutic approaches for MPN patients.
Competing Interest Statement
S.T.O. has served as a consultant for Kartos Therapeutics, CTI BioPharma, Celgene/Bristol Myers Squibb, Disc Medicine, Blueprint Medicines, PharmaEssentia, Constellation, Geron, Abbvie, Sierra Oncology, and Incyte. All other authors disclose no competing interests.
