Platelet transcriptome identifies progressive markers and potential therapeutic targets in chronic myeloproliferative neoplasms

Shen, Zhu; Du, Wenfei; Perkins, Cecelia; Fechter, Lenn; Natu, Vanita; Maecker, Holden T.; Rowley, Jesse W.; Gotlib, Jason; Zehnder, James L.; Krishnan, Anandi

doi:10.1016/j.xcrm.2021.100425

Cited by 30 publications

(35 citation statements)

References 93 publications

(172 reference statements)

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“…The copyright holder for this preprint this version posted December 11, 2022. ; https://doi.org/10.1101/2022.12.08.519689 doi: bioRxiv preprint A recent large-scale gene expression profiling of platelets from MPN patients suggested the activation of OXPHOS and mTORC1 signaling pathways in all three subtypes of MPNs 33 . Importantly, these differentially expressed genes robustly predict MPN progression, highlighting the importance of platelet biology in MPNs 33 . Our proteomics data validated the activation of OXPHOS and mTORC1 proteins in platelet from MPN.…”

Section: Discussionmentioning

confidence: 99%

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Multiomic Profiling Reveals Metabolic Alterations Mediating Aberrant Platelet Activity and Inflammation in Myeloproliferative Neoplasms

Laranjeira

Kong

et al. 2022

Preprint

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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 (alpha-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. alpha-KG is a key TCA cycle intermediate, which inhibits PI3K/AKT/mTOR pathway signaling via suppression of ATP synthase. alpha-KG supplementation drastically reduced oxygen consumption and ATP generation in platelets from MPN patients. We further investigated the effects of alpha-KG on MPN platelet activation. Ex vivo incubation of platelets from both MPN patients and Jak2 V617F knock-in mice with alpha-KG significantly reduced platelet surface P-selectin and integrin gpIIb/IIIa activation. Additionally, alpha-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 alpha-KG, suggesting the involvement of PI3K/AKT/mTOR and MAPK pathways in the inhibitory effects of alpha-KG on platelet activation. Thus, alpha-KG inhibited platelet hyperreactivities in both human and mouse MPN samples. To test the therapeutic impact of alpha-KG on MPN disease features, we treated Jak2 V617F knock-in mice with alpha-KG for 6 weeks. Oral alpha-KG supplementation decreased splenomegaly and reduced elevated platelets and hematocrit. Additionally, monocytes were significantly decreased as early as 2 weeks after alpha-KG treatment in Jak2 V617F knock-in mice. Consistently, RNA-seq of bone marrow samples from alpha-KG treated mice revealed inhibition of heme metabolism, OXPHOS and mTOR signaling pathways. In ex vivo studies with MPN patient CD34+ cells, alpha-KG treatment for 10 days led to a decrease in CD41+ CD61+ cells, suggesting decreased megakaryocyte commitment. We further observed that alpha-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 alpha-KG treatment. Taken together, these results suggest that alpha-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 alpha-KG and mTOR signaling in aberrant MPN platelet activity and monocyte-driven inflammation. These findings have potential relevance for novel therapeutic approaches for MPN patients.

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Section: Discussionmentioning

confidence: 99%

“…A recent large-scale gene expression profiling of platelets from MPN patients suggested the activation of OXPHOS and mTORC1 signaling pathways in all three subtypes of MPNs 33 . Importantly, these differentially expressed genes robustly predict MPN progression, highlighting the importance of platelet biology in MPNs 33 .…”

Section: Discussionmentioning

confidence: 99%

Multiomic Profiling Reveals Metabolic Alterations Mediating Aberrant Platelet Activity and Inflammation in Myeloproliferative Neoplasms

Laranjeira

Kong

et al. 2022

Preprint

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“…Gorbenko [117] JAK2 V617F allele burden qRT-PCR 24 -Correlation between leukocyte DNA and platelet mRNA: R = 0.79, R 2 = 0.62; changes in platelet RNA preceded changes in leukocyte DNA Shen [118] 3326 genes RNA-seq 95 21 The progressive transcriptome model in the classification of 24 essential thrombocythemias, 33 polycythemia vera, and 40 myelofibrosis: AUC = 0.97 Thyroid neoplasm Shen [119] 765 genes RNA-seq 42 27 Ac = 97%; AUC = 0.998. The average Ac for the diagnosis of thyroid adenomas, papillary thyroid cancer and metastasized papillary thyroid cancer was 80.5% Prostate cancer…”

Section: Myeloproliferative Neoplasmsmentioning

confidence: 99%

A narrative review for platelets and their RNAs in cancers: New concepts and clinical perspectives

Xiang

Xiang²,

Zhang

et al. 2022

Medicine

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Recent years have witnessed a growing body of evidence suggesting that platelets are involved in several stages of the metastatic process via direct or indirect interactions with cancer cells, contributing to the progression of neoplastic malignancies. Cancer cells can dynamically exchange components with platelets in and out of blood vessels, and directly phagocytose platelets to hijack their proteome, transcriptome, and secretome, or be remotely regulated by metabolites or microparticles released by platelets, resulting in phenotypic, genetic, and functional modifications. Moreover, platelet interactions with stromal and immune cells in the tumor microenvironment lead to alterations in their components, including the ribonucleic acid (RNA) profile, and complicate the impact of platelets on cancers. A deeper understanding of the roles of platelets and their RNAs in cancer will contribute to the development of anticancer strategies and the optimization of clinical management. Encouragingly, advances in high-throughput sequencing, bioinformatics data analysis, and machine learning have allowed scientists to explore the potential of platelet RNAs for cancer diagnosis, prognosis, and guiding treatment. However, the clinical application of this technique remains controversial and requires larger, multicenter studies with standardized protocols. Here, we integrate the latest evidence to provide a broader insight into the role of platelets in cancer progression and management, and propose standardized recommendations for the clinical utility of platelet RNAs to facilitate translation and benefit patients.

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“…However, this study was primarily in mice and remains to be confirmed in independent additional MPN models and patient-derived specimens. Our recent investigation, profiling the blood platelet transcriptome in all three subtypes of MPN patients 20 identified high expression of genes and pathways associated with impaired proteostasis, ER stress and unfolded protein response (UPR) well-recognized in other age-related disorders 21-25 . Here, we extend our prior MPN patient platelet transcriptomic data with a focus on genes associated with proteostasis, cell proliferation, ER stress or calcium signaling at high statistical significance (FDR <0.01), and representing four types of trends in platelet RNA expression across MPN subtypes: progressively downregulated, progressively upregulated, consistently upregulated, and uniquely upregulated in MF alone.…”

Section: Introductionmentioning

confidence: 99%

Enkurin: A novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens

Sumanth

Liu

et al. 2023

Preprint

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Impaired protein homeostasis, though well established in age-related disorders, has been linked in recent research with the pathogenesis of myeloproliferative neoplasms (MPNs). As yet, however, little is known about MPN-specific modulators of proteostasis, thus impeding our ability for increased mechanistic understanding and discovery of additional therapeutic targets. Loss of proteostasis, in itself, is traced to dysregulated mechanisms in protein folding and intracellular calcium signaling at the endoplasmic reticulum (ER). Here, using ex vivo and in vitro systems (including CD34+ cultures from patient bone marrow, and healthy cord/peripheral blood specimens), we extend our prior data from MPN patient platelet RNA sequencing, and discover select proteostasis-associated markers at RNA and/or protein levels in each of platelets, parent megakaryocytes, and whole blood specimens. Importantly, we identify a novel role in MPNs for enkurin (ENKUR), a calcium mediator protein, implicated originally only in spermatogenesis. Our data reveal consistent ENKUR downregulation at both RNA and protein levels across MPN patient specimens and experimental models, with a concomitant upregulation of a cell cycle marker, CDC20. Silencing of ENKUR by shRNA in CD34+ derived megakaryocytes further confirm this association with CDC20 at both RNA and protein levels; and indicate a likely role for the PI3K/Akt pathway. The inverse association of ENKUR and CDC20 expression was further confirmed upon treatment with thapsigargin (an agent that causes protein misfolding in the ER by selective loss of calcium) in both megakaryocyte and platelet fractions at RNA and protein levels. Together, our work sheds light on enkurin as a novel marker of MPN pathogenesis beyond the genetic alterations; and indicates further mechanistic investigation into a role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation.

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Platelet transcriptome identifies progressive markers and potential therapeutic targets in chronic myeloproliferative neoplasms

Cited by 30 publications

References 93 publications

Multiomic Profiling Reveals Metabolic Alterations Mediating Aberrant Platelet Activity and Inflammation in Myeloproliferative Neoplasms

Multiomic Profiling Reveals Metabolic Alterations Mediating Aberrant Platelet Activity and Inflammation in Myeloproliferative Neoplasms

A narrative review for platelets and their RNAs in cancers: New concepts and clinical perspectives

Enkurin: A novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens

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