Megakaryocytes Research Articles

Immunological face of megakaryocytes.

Megakaryocytes (MKs), which are traditionally known for their role in platelet production, are now emerging as unique immune cells with diverse capabilities. They express immune receptors, participate in pathogen recognition and response, phagocytose pathogens, contribute to antigen presentation, and interact with various immune cell types. When encountering inflammatory challenges, MKs exhibit intricate immune functions that can either promote or inhibit inflammation. These responses are mediated through mechanisms, such as the secretion of either anti-inflammatory or pro-inflammatory cytokines and release of immunomodulatory platelets according to specific conditions. This intricate array of responses necessitates a detailed exploration to determine whether the immune functions of MKs are carried out by the entire MK population or by a specific subpopulation. Breakthroughs in single-cell RNA sequencing have uncovered a unique "immune MK" subpopulation, revealing its distinct characteristics and immunoregulatory functions. This review provides latest insights into MKs' immune attributes and their roles in physiological and pathological contexts and emphasizes the discovery and functions of "immune MKs".

LMAN1 serves as a cargo receptor for thrombopoietin.

Thrombopoietin (TPO) is a plasma glycoprotein that binds its receptor on megakaryocytes (MK) and MK progenitors, resulting in enhanced platelet production. The mechanism by which TPO is secreted from hepatocytes remains poorly understood. LMAN1 and MCFD2 form a complex at the endoplasmic reticulum membrane, recruiting cargo proteins into COPII vesicles for secretion. In this study, we showed that LMAN1 deficient mice (with complete germline LMAN1 deficiency) exhibited mild thrombocytopenia, whereas the platelet count was entirely normal in mice with approximately 7% Lman1 expression. Surprisingly, mice deleted for Mcfd2 did not exhibit thrombocytopenia. Analysis of peripheral blood from LMAN1 deficient mice demonstrated normal platelet size and normal morphology of dense and alpha granules. LMAN1 deficient mice exhibited a trend toward reduced MK and MK progenitors in the bone marrow. We next showed that hepatocyte-specific but not hematopoietic Lman1 deletion results in thrombocytopenia, with plasma TPO level reduced in LMAN1 deficient mice, despite normal Tpo mRNA levels in LMAN1 deficient livers. TPO and LMAN1 interacted by co-immunoprecipitation in a heterologous cell line and TPO accumulated intracellularly in LMAN1 deleted cells. Altogether, these studies confirmed the hepatocyte as the cell of origin for TPO production in vivo and were consistent with LMAN1 as the endoplasmic reticulum cargo receptor that mediates the efficient secretion of TPO. To our knowledge, TPO is the first example of an LMAN1-dependent cargo that is independent of MCFD2.

The role of platelets and megakaryocytes in sepsis and ARDS.

Since the global COVID-19 pandemic, there has been a renewed focus on lung injury during infection. Systemic inflammatory responses such as acute respiratory distress syndrome (ARDS) and sepsis are a leading cause of morbidity and mortality for both adults and children. Improvements in clinical care have improved outcomes but mortality remains ∼40% and significant morbidity persists for those patients with severe disease. Mechanistic studies of the underlying biological processes remain essential to identifying therapeutic targets. Furthermore, methods for identifying the underlying drivers of organ failure are key to treating and preventing tissue injury. In this review, we discuss the contribution of megakaryocytes (MKs) and platelets to the pathogenesis of systemic inflammatory syndromes. We explore the role of MKs and the new identification of extramedullary MKs during sepsis. We describe the alterations in the platelet transcriptome during sepsis. Lastly, we explore platelet function as defined by aggregation, activation and the formation of heterotypic aggregates. Much more work is necessary to explore the contribution of platelets to these heterogenous syndromes, but the foundation of platelets as key contributors to inflammation has been laid.

Different inflammatory, fibrotic, and immunologic signatures between pre-fibrotic and overt primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloid proliferative neoplasm (MPN) characterized by bone marrow (BM) fibrosis. Pre-fibrotic PMF (pre-PMF) progresses to overt PMF. Megakaryocytes (MKs) play a primary role in PMF; however, the functions of MK subsets and those of other hematopoietic cells during PMF progression remain unclarified. Therefore, we analyzed BM aspirates in pre-PMFs, overt PMFs, and other MPNs using single-cell RNA sequencing (scRNA-seq). We identified 14 cell types with subsets, including hematopoietic stem and progenitor cells (HSPCs) and MKs. HSPCs in overt PMF were MK-biased and inflammation/fibrosis-enriched. Among MKs, the epithelial-mesenchymal transition (EMT)-enriched subset was abruptly increased in overt PMF. MKs in non-fibrotic/non-PMF MPN were MK differentiation-enriched, whereas those in fibrotic/non-PMF MPN were inflammation/fibrosis-enriched. Overall, the inflammation/fibrosis signatures of the HSPC, MK, and CD14+ monocyte subsets increased from pre-PMF to overt PMF. Cytotoxic and dysfunctional scores also increased in T and NK cells. Clinically, MK and HSPC subsets with high inflammation/fibrosis signatures were frequent in the patients with peripheral blood blasts ≥1%. scRNA-seq predicted higher cellular communications of MK differentiation, inflammation/fibrosis, immunologic effector/dysfunction, and tumor-associated signaling in overt PMF than pre-PMF. However, no decisive subset emerged during PMF progression. Our study demonstrated that HSPCs, monocytes, and lymphoid cells contribute to PMF progression, and subset specificity existed regarding inflammation/fibrosis and immunologic dysfunction. PMF progression may depend on multiple cell types' alterations, and EMTenriched MKs may be potential targets for the diagnosis and therapy of the progression.

LRRC8 complexes are adenosine nucleotide release channels regulating platelet activation and arterial thrombosis.

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in Leucine-Rich Repeat Containing 8 (LRRC8) protein subunits that form the Volume-Regulated Anion Channel (VRAC) which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume, adhesion, and agonist-stimulated activation, aggregation, ATP secretion and calcium mobilization. MK-specific LRRC8A cKO mice have reduced arteriolar thrombus formation and prolonged arterial thrombosis without affecting bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP/ADP release to amplify agonist-stimulated calcium and PI3K-AKT signaling via P2X1, P2Y 1 and P2Y 12 receptors. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo . These studies identify the mechanoresponsive LRRC8 channel complex as an ATP/ADP release channel in platelets which regulates platelet function and thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

Deciphering Transcriptomic Variations in Hematopoietic Lineages: HSCs, EBs, and MKs.

In the realm of hematopoiesis, hematopoietic stem cells (HSCs) serve as pivotal entities responsible for generating various blood cell types, initiating both the myeloid and lymphoid branches within the hematopoietic lineage. This intricate process is marked by genetic variations that underscore the crucial role of genes in regulating cellular functions and interactions. Recognizing the significance of genetic factors in this context, this article delves into a genetic perspective, aiming to unravel the biological factors that govern the transition from one cell's fate to another within the hematopoietic system. To gain deeper insights into the genetic traits of three distinct blood cell types-HSCs, erythroblasts (EBs), and megakaryocytes (MKs)-we conducted a comprehensive transcriptomic analysis. Leveraging diverse hematopoietic cell datasets from healthy individuals, sourced from The BLUEPRINT consortium, our investigation targeted the identification of genetic variants responsible for changes in gene expression levels and epigenetic modifications across the entire human genome in each of these cell types. The total number of normalized expressed transcripts includes 14,233 novel trinity lncRNAs, 13,749 mRNAs, and 3092 lncRNAs. This scrutiny revealed a total of 31,074 transcripts, with a notable revelation that 14,233 of them were previously unidentified or novel lncRNAs, highlighting a substantial reservoir of genetic information yet to be explored. Examining their expression across distinct lineages further unveiled 2845 differentially expressed (DE) mRNAs and 354 DE long noncoding RNAs (lncRNAs) notably enriched among the three distinct blood cell types: HSCs, EBs, and MKs. Our investigation extended beyond mRNA to focus on the dynamic expression of lncRNAs, revealing a well-defined pattern that played a significant role in regulating differentiation and cell-fate specification. This coordination of lncRNA dynamics extended to aberrations in both mRNA and lncRNA transcriptomes within HSCs, EBs, and MKs. We specifically characterized lncRNAs with preferential expression in HSCs, as well as in various downstream differentiated lineage progenitors of EBs and MKs, providing a comprehensive perspective on lncRNAs in human hematopoietic cells. Notably, the expression of lncRNAs exhibited substantial cell-to-cell variation, a phenomenon discernible only through single-cell analysis. The comparative analysis undertaken in this study provides valuable insights into the distinctive genetic signatures guiding the differentiation of these crucial hematopoietic cell types.

Linear podosomes display low Cdc42 activity for proplatelet elongation by megakaryocytes

Blood platelets result from differentiation of megakaryocytes (MKs) into the bone marrow. It culminates with the extension of proplatelets (PPT) through medullar sinusoids and release of platelets in the blood stream. Those processes are regulated by contact with the microenvironment mediated by podosomes. We previously demonstrated that contact of megakaryocytes to Collagen I fibers initiated the formation of linear podosomes required for proplatelets extension and release of mature platelets. MKs linear podosomes have the particularity of displaying mechanical pulling activity but, unlike other linear podosomes, they lack the ability of digesting the extracellular matrix (ECM), as we recently demonstrated. The Cdc42 small GTPase is required for actomyosin-dependent maturation of the demarcation membrane system (DMS), a membrane reservoir for PPT and platelets components. Cdc42 is a known protein of the podosomes core, and is instrumental to accurate platelets release into the sinusoids. Indeed, FRET analysis showed that Cdc42 activity was very high and central to DMS formation. Unexpectedly, even though we found the protein in linear podosomes, almost undetectable Cdc42 activity was detected in those structures. This observation suggests that Cdc42 could also act as scaffold to assemble proteins required for PPT formation/elongation along Collagen I fibers in MKs. Eventually, we demonstrated that linear podosomes appear as points of contact between Collagen I fibers and DMS membranes, to mechanically extend PPT along Collagen bundles, independently of Cdc42 activity.

Genetic engineering of megakaryocytes from blood progenitor cells using messenger RNA lipid nanoparticles

BackgroundPlatelets are an essential component of hemorrhage control and management, and engineering platelets to express therapeutic proteins could expand their use as a cell therapy. Genetically engineered platelets can be achieved by modifying the platelet precursor cells, megakaryocytes (MKs). Current strategies include transfecting MK progenitors ex vivo with viral vectors harboring lineage-driven transgenes and inducing the production of in vitro modified platelets. The use of viruses, however, poses challenges in clinical implementation, and no methods currently exist to genetically modify MKs with nonviral techniques. Lipid nanoparticles (LNPs) are a nonviral delivery system that could enable a facile strategy to modify MKs with a variety of nucleic acid payloads. ObjectivesTo investigate whether LNP can transfect cultured hematopoietic stem/progenitor cell–derived MKs to express exogenous proteins and induce functional changes. MethodsMK and MK progenitors differentiated from cord blood–derived hematopoietic stem/progenitor cells were treated with LNP formulations containing messenger RNA and resembling the clinically approved LNP formulations. Transfection efficiency was assessed through flow cytometry by expression of enhanced green fluorescent protein. Functional changes to the MKs were assessed through rotational thromboelastometry by expression of exogenous coagulation factor (F)VII, a representative physiologically relevant protein. ResultsLNP enabled transfection efficiencies of 99% in MKs and did not impair MK maturation, viability, and morphology. MKs engineered to express exogenous FVII decreased clotting time in FVII-deficient plasma following clot initiation. ConclusionThis approach provides an easy-to-use modular platform to genetically modify MK and MK progenitors, which can be potentially extended to producing genetically modified cultured platelets.

Trogocytosis: A potential pathogenic mechanism in immune thrombocytopenia leading to impaired megakaryocyte maturation and reduced platelet production

Immune thrombocytopenia (ITP) is an autoimmune disease due to increased peripheral platelets destruction and inappropriate bone marrow production of platelets by megakaryocytes (MKs). The pathophysiology of ITP is complex and multifactorial, which remains not fully understood. However, the production of antiplatelet autoantibodies and the destruction of antibody-coated platelets through antibody-dependent cellular phagocytosis by macrophages (Mθ) expressing Fc gamma receptor (FcγR) are regarded as the key mechanisms. Glycoprotein (GP) Ⅱb/Ⅲa is the main target of antiplatelet antibodies leading to platelet phagocytosis by splenic Mθ, through interactions with FcγR. As GPⅡb/Ⅲa is normally expressed both on platelets and the MKs from which they are derived, we inferred that partial phagocytosis (definitely trogocytosis) of MKs by bone marrow Mθ might exist, leading to the increased amount of granular MKs (or non-platelets-producing MKs) in ITP. And we further hypothesized that the “bitten” MKs cannot sustain their normal function of maturation. Such trogocytosis mechanism may shed new light on the understanding of morphology alterations, maturation defects and decreased platelets production of MKs in ITP, and provide appealing implications in therapeutic modifications targeting the interplay of MKs and Mθ in bone marrow.

YAP1 regulates thrombopoiesis by binding to MYH9 in immune thrombocytopenia

AbstractImmune thrombocytopenia (ITP) is a complicated bleeding disease characterized by a sharp platelet reduction. As a dominating element involved in ITP, megakaryocytes (MKs) are responsible for thrombopoiesis. However, the mechanism underlying the dysregulation of thrombopoiesis that occurs in ITP remains unidentified. In this study, we examined the role of Yes-associated protein 1 (YAP1) in thrombopoiesis during ITP. We observed reduced YAP1 expression with cytoskeletal actin misalignment in MKs from patients with ITP. Using an experimental ITP mouse model, we showed that reduced YAP1 expression induced aberrant MK distribution, reduced the percentage of late MKs among the total MKs, and caused submaximal platelet recovery. Mechanistically, YAP1 upregulation by binding of GATA-binding protein 1 to its promoter promoted MK maturation. Phosphorylated YAP1 promoted cytoskeletal activation by binding its WW2 domain to myosin heavy chain 9, thereby facilitating thrombopoiesis. Targeting YAP1 with its activator XMU-MP-1 was sufficient to rescue cytoskeletal defects and thrombopoiesis dysregulation in YAP1+/− mice with ITP and patients. Taken together, these results demonstrate the crucial role of YAP1 in thrombopoiesis, providing potential for the development of diagnostic markers and therapeutic options for ITP.

Lung megakaryocytes engulf inhaled airborne particles to promote intrapulmonary inflammation and extrapulmonary distribution

Many lung immune cells are known to respond to inhaled particulate matter. However, current known responses cannot explain how particles induce thrombosis in the lung and how they translocate to distant organs. Here, we demonstrate that lung megakaryocytes (MKs) in the alveolar and interstitial regions display location-determined characteristics and act as crucial responders to inhaled particles. They move rapidly to engulf particles and become activated with upregulation in inflammatory responses and thrombopoiesis. Comprehensive in vivo, in vitro and ex vivo results unraveled that MKs were involved in particle-induced lung damages and shed particle-containing platelets into blood circulation. Moreover, MK-derived platelets exhibited faster clotting, stronger adhesion than normal resting platelets, and inherited the engulfed particles from parent MKs to assist in extrapulmonary particle transportation. Our findings collectively highlight that the specific responses of MKs towards inhaled particles and their roles in facilitating the translocation of particles from the lungs to extrapulmonary organs for clearance.

Abstract Tu032: LRRC8 complex regulates platelet activation and thrombosis

During activation, platelets undergo both shape and volumetric changes which are mechanical events essential for thrombosis. Volume-Regulated Anion Channels (VRACs) are ubiquitously expressed mechanoresponsive, heterohexameric anion channels comprised of leucine-rich repeat containing protein 8 (LRRC8) A (essential for channel activity) in combination with B, C, D and/or E subunits. In this study, we identify single-nucleotide polymorphisms (SNPs) in three LRRC8 subunits (LRRC8A, C, and D) which are expressed in human platelets and are independently associated with altered mean platelet volume in human genetic studies – implicating the volume-sensing LRRC8 channel complex as a regulator of platelet function in humans. We show that LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout (cKO) mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist-stimulated P-selectin exposure and aIIbb3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces and reduced agonist-stimulated aggregation and ATP secretion. Additionally, VRAC in MKs mediate ATP currents which are abolished in LRRC8A-null MKs. In vivo, MK-specific LRRC8A cKO mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis, and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP release to amplify agonist-stimulated calcium influx and platelet aggregation. Small-molecule LRRC8 channel inhibitors largely recapitulate the defects observed in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which regulates platelet function and arterial thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

Cell cycle-dependent centrosome clustering precedes proplatelet formation.

Platelet-producing megakaryocytes (MKs) primarily reside in the bone marrow, where they duplicate their DNA content with each cell cycle resulting in polyploid cells with an intricate demarcation membrane system. While key elements of the cytoskeletal reorganizations during proplatelet formation have been identified, what initiates the release of platelets into vessel sinusoids remains largely elusive. Using a cell cycle indicator, we observed a unique phenomenon, during which amplified centrosomes in MKs underwent clustering following mitosis, closely followed by proplatelet formation, which exclusively occurred in G1 of interphase. Forced cell cycle arrest in G1 increased proplatelet formation not only in vitro but also in vivo following short-term starvation of mice. We identified that inhibition of the centrosomal protein kinesin family member C1 (KIFC1) impaired clustering and subsequent proplatelet formation, while KIFC1-deficient mice exhibited reduced platelet counts. In summary, we identified KIFC1- and cell cycle-mediated centrosome clustering as an important initiator of proplatelet formation from MKs.

Clinical and Molecular Characteristics of Megakaryocytes in Myelodysplastic Syndrome.

Objective Myelodysplastic syndrome (MDS) is a malignant clonal disorder of hematopoietic stem cells which is characterized by morphologic dysplasia. However, the pathological characteristics of megakaryocytes (MKs) in MDS patients with gene mutation are not well established. Methods Bone marrow MK specimens from 104 patients with primary MDS were evaluated, and all patients were distributed into two groups according to gene mutation associated with functional MKs. The morphologic and cellular characteristics of MKs and platelets were recorded and compared. Results The more frequently mutated genes in MDS patients were TUBB1 (11.54%), VWF (8.65%), NBEAL2 (5.77%), and the most common point mutation was TUBB1 p.(R307H) and p.(Q43P). Patients with MK mutation showed a decrease in adenosine diphosphate-induced platelet aggregation, high proportion of CD34 + CD61 + MKs (10.00 vs. 4.00%, p = 0.012), and short overall survival (33.15 vs. 40.50 months, p = 0.013). Further, patients with a higher percent of CD34 + CD61 + MKs (≧20.00%) had lower platelet counts (36.00 × 10 9 /L vs. 88.50 × 10 9 /L, p = 0.015) and more profound emperipolesis ( p = 0.001). By analyzing RNA-sequencing of MKs, differentially expressed mRNA was involved in physiological processes including platelet function and platelet activation, especially for MDS patients with high percent of CD34 + CD61 + MKs. The high levels of expression of CD62P, CXCL10, and S100A9 mRNA, shown by RNA sequencing, were validated by PCR assay. Conclusion High proportion of CD34 + CD61 + MKs was a poor prognostic factor in MDS patients with MK mutation. CD62P, CXCL10, and S100A9 may be the potential targets to evaluate the molecular link between gene defects and platelet function.

Physical activity (PA) impact on metabolome and immunity of oncogene addicted non-small cell lung cancer (NSCLC): The EXcellenT trial.

e20582 Background: Today patients (pts) with oncogene addicted advanced NSCLC receive molecular targeted therapies as first-line treatment ensuring high compliance to therapy, few side effects and long survival time. These pts are usually young and with a strong wish to regain the pre-diagnosis quality of life (QoL), including the ability to perform PA. It’s known that PA can impact significantly on metabolomic and immunity status, but to date only few studies on its role in NSCLC have been performed. The aim of the EXcellenT trial is to evaluate if a tailored PA program may have an impact on QoL and performances of this homogeneous subgroup of pts. An exploratory objective was to explore the complex interplay between PA and immune status and the influence of PA on metabolome. Methods: EXcellenT (Exercise in Extended oncogene addicted Lung Cancer in Active Treatment - NCT05306652) is an Italian monocentric prospective study enrolling 40 pts with oncogene-addicted advanced NSCLC in active treatment with tyrosine kinase inhibitors (TKI). Pts are randomized in 2 arms: an interventional arm of a personalized supervised PA program or a counselling group of home-based exercise. We explored peripheral variations of T cells subsets during TKI treatment combined with exercise training to better define the immuno-dependence of these tumors. Fatty acid composition (FA) of plasma has been carried out using gas chromatography-flame ionization detection (GC-FID) and liquid chromatography-mass spectrometry (LC-MS). Immature and mature neutrophils, monocytes, CD34+ cells and megakaryocytes (MK) precursors have been determined through flow cytometry analysis. Results: A preliminary exploratory analysis on the first patients enrolled with 3 complete time point (baseline; week 4 and 12) was performed. To date 23 out of 40 pts have been enrolled, 46% of them with pre-diagnosis leisure-time physical activity. The oncogene identified are: ALK 52%, EGFR 26%, BRAF 9%, RET 4%, ROS1 9%. 35% had stable brain metastasis (mts) and 39% bone mts not at risk of skeletal-related events. FA analysis (4 pts) showed significant increase of monounsaturated fatty acids (p = 0.0329) during the study. According to immunophenotype, we observed (3 pts) a significant reduction of CD34+ cancer stem cell (p 0.0112) and a consistent increase in mature CD66b+CD10+ neutrophils with a trend of decrease in CD66b+CD10- cells. Conclusions: The ExcellenT trial is showing that PA programs are feasible and well accepted by patients with oncogene-addicted NSCLC. The QoL analysis will be performed at the end of enrolment. The preliminary analysis on metabolomic and immunity status shows an improvement of fatty acid composition and an increase of mature cell precursors compared to immature counterpart suggesting a potential role of PA in hindering tumor immune escape strategies. Clinical trial information: NCT05306652 .

Abstract 119: LRRC8 Complex Is An ATP Release Channel Regulating Platelet Activation And Thrombosis

Platelet shape and volume changes are early mechanical events in platelet activation proposed to contribute to arterial thrombosis. Here, we identify single-nucleotide polymorphisms (SNPs) in four leucine-rich repeat containing protein subunits (LRRC8A, B, C, and D) expressed in human platelets that are independently associated with increased mean platelet volume in human genetic studies - implicating the volume-sensing LRRC8 channel complex as regulating platelet function in humans. We show LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist -stimulated P-selectin exposure, and αIIbβ3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces, agonist-induced platelet aggregation and ATP secretion. MK-specific LRRC8A conditional knockout mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, LRRC8A mediates swelling-induced ATP release to amplify agonist-stimulated calcium influx and aggregation in platelets. Treatment of platelets with small-molecule LRRC8 channel inhibitors recapitulate the defects in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as a regulator of platelet function and thrombosis - providing a proof-of-concept for a novel anti-thrombotic drug target.

Activating SRC/MAPK signaling via 5-HT1A receptor contributes to the effect of vilazodone on improving thrombocytopenia.

Thrombocytopenia caused by long-term radiotherapy and chemotherapy exists in cancer treatment. Previous research demonstrates that 5-Hydroxtrayptamine (5-HT) and its receptors induce the formation of megakaryocytes (MKs) and platelets. However, the relationships between 5-HT1A receptor (5-HTR1A) and MKs is unclear so far. We screened and investigated the mechanism of vilazodone as a 5-HTR1A partial agonist in promoting MK differentiation and evaluated its therapeutic effect in thrombocytopenia. We employed a drug screening model based on machine learning (ML) to screen the megakaryocytopoiesis activity of Vilazodone (VLZ). The effects of VLZ on megakaryocytopoiesis were verified in HEL and Meg-01 cells. Tg (itga2b: eGFP) zebrafish was performed to analyze the alterations in thrombopoiesis. Moreover, we established a thrombocytopenia mice model to investigate how VLZ administration accelerates platelet recovery and function. We carried out network pharmacology, Western blot, and immunofluorescence to demonstrate the potential targets and pathway of VLZ. VLZ has been predicted to have a potential biological action. Meanwhile, VLZ administration promotes MK differentiation and thrombopoiesis in cells and zebrafish models. Progressive experiments showed that VLZ has a potential therapeutic effect on radiation-induced thrombocytopenia in vivo. The network pharmacology and associated mechanism study indicated that SRC and MAPK signaling are both involved in the processes of megakaryopoiesis facilitated by VLZ. Furthermore, the expression of 5-HTR1A during megakaryocyte differentiation is closely related to the activation of SRC and MAPK. Our findings demonstrated that the expression of 5-HTR1A on MK, VLZ could bind to the 5-HTR1A receptor and further regulate the SRC/MAPK signaling pathway to facilitate megakaryocyte differentiation and platelet production, which provides new insights into the alternative therapeutic options for thrombocytopenia.

Activating SRC/MAPK signaling via 5-HT1A receptor contributes to the effect of vilazodone on improving thrombocytopenia

Thrombocytopenia caused by long-term radiotherapy and chemotherapy exists in cancer treatment. Previous research demonstrates that 5-Hydroxtrayptamine (5-HT) and its receptors induce the formation of megakaryocytes (MKs) and platelets. However, the relationships between 5-HT1A receptor (5-HTR1A) and MKs is unclear so far. We screened and investigated the mechanism of vilazodone as a 5-HTR1A partial agonist in promoting MK differentiation and evaluated its therapeutic effect in thrombocytopenia. We employed a drug screening model based on machine learning (ML) to screen the megakaryocytopoiesis activity of Vilazodone (VLZ). The effects of VLZ on megakaryocytopoiesis were verified in HEL and Meg-01 cells. Tg (itga2b: eGFP) zebrafish was performed to analyze the alterations in thrombopoiesis. Moreover, we established a thrombocytopenia mice model to investigate how VLZ administration accelerates platelet recovery and function. We carried out network pharmacology, Western blot, and immunofluorescence to demonstrate the potential targets and pathway of VLZ. VLZ has been predicted to have a potential biological action. Meanwhile, VLZ administration promotes MK differentiation and thrombopoiesis in cells and zebrafish models. Progressive experiments showed that VLZ has a potential therapeutic effect on radiation-induced thrombocytopenia in vivo. The network pharmacology and associated mechanism study indicated that SRC and MAPK signaling are both involved in the processes of megakaryopoiesis facilitated by VLZ. Furthermore, the expression of 5-HTR1A during megakaryocyte differentiation is closely related to the activation of SRC and MAPK. Our findings demonstrated that the expression of 5-HTR1A on MK, VLZ could bind to the 5-HTR1A receptor and further regulate the SRC/MAPK signaling pathway to facilitate megakaryocyte differentiation and platelet production, which provides new insights into the alternative therapeutic options for thrombocytopenia.

Abstract 1733: SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia

Abstract The SET-NUP214 (S/N) fusion gene, which results from either cryptic t(9:9) (q34;q34) or del(9) (q34.11q34.13), is found in a very narrow spectrum of patients diagnosed with Acute Myeloid Leukemia (AML) or T-cell acute lymphoblastic leukemia (T-ALL). AML patients with S/N fusion were reported to have relatively poor prognostic outcomes despite receiving myeloablative conditioning. While previously, the fusion has been described to overrepresent HOX cluster genes, the underlying mechanism of gene regulation that contributes to the pathology remains elusive due to the rare incidence of the disease. The current study used the MEGAL (ACC719, DSMZ) cell line, identified as the solo megakaryocytic AML line expressing the S/N-fusion, for determining genome-wide DNA methylation (DNAm) and gene expression using 935K Infinium MethylationEPIC v2.0 BeadChip array and RNA-sequencing, respectively. The differential DNAm and expression data were analyzed and compared to the hematopoietic stem progenitor cells (HSPC) or normal megakaryocytes (MK). The ChIP-sequencing data from the S/N positive T-ALL cell line LOUCY has been used to represent the coincidence of regulatory chromatin marks and differentially methylated CpG (mC) sites. We observed n=1934 genes upregulated and n=2396 downregulated in MEGAL, compared to both HSPC and MK. The direction of expression changes between MEGAL and HSPC and MEGAL and MK showed a high correlation (r=0.92, p< 0.01). Per the previous literature, we observed an overrepresentation of HOX genes (n=19) in MEGAL. Based on DNAm profiling, we noticed a propensity of hypermethylation in MEGAL across the promoters (93%±0.6), bodies (93%±0.8), and intergenic (85%±0.3) regions, compared to HSPC/MK. Next, we integrated the mC and expression at the promoters and identified that the majority (75%±3) of the genes belong to the hyper-down (n=2563 against HPSC; n=1907 against MK) or hyper-up (n=965 against HPSC; n=923 against MK) clusters. We further investigated the HOXB (HOXB7, HOXB8, HOXB9) and HOXC (HOXC10, HOXC12, HOXC13, HOXC13-AS, HOXC4, HOXC5, HOC6, HOXC8, HOXC9, HOXC-AS1, HOXC-AS2, and HOXC-AS3) cluster genes, where hypermethylated CpGs mainly were concentrated at the CpG islands and overlapped with active promoter (H3K4me3, H3K4me1) signatures or enhancer-like (CTCF bound) elements. We also observed frequent overlaps between mC and H3K36me3 across the observed genes. The differentially expressed genes in this AML subtype were enriched for the hippo-signaling pathway, calcium signaling, focal adhesion, and PI3-Akt pathways. Collectively, our data showed that S/N favors genome-wide hypermethylation, which, in conjunction with the cis-regulatory elements, induces overrepresentation of the HOX cluster genes. These novel epigenetic vulnerabilities are worth pursuing for investigating the cellular function and pathophysiology of the disease. Citation Format: Samrat Roy Choudhury, Akhilesh Kaushal. SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia [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 1733.

A let-7 microRNA-RALB axis links the immune properties of iPSC-derived megakaryocytes with platelet producibility

We recently achieved the first-in-human transfusion of induced pluripotent stem cell-derived platelets (iPSC-PLTs) as an alternative to standard transfusions, which are dependent on donors and therefore variable in supply. However, heterogeneity characterized by thrombopoiesis-biased or immune-biased megakaryocytes (MKs) continues to pose a bottleneck against the standardization of iPSC-PLT manufacturing. To address this problem, here we employ microRNA (miRNA) switch biotechnology to distinguish subpopulations of imMKCLs, the MK cell lines producing iPSC-PLTs. Upon miRNA switch-based screening, we find imMKCLs with lower let-7 activity exhibit an immune-skewed transcriptional signature. Notably, the low activity of let-7a-5p results in the upregulation of RAS like proto-oncogene B (RALB) expression, which is crucial for the lineage determination of immune-biased imMKCL subpopulations and leads to the activation of interferon-dependent signaling. The dysregulation of immune properties/subpopulations, along with the secretion of inflammatory cytokines, contributes to a decline in the quality of the whole imMKCL population.