Megakaryocytic Differentiation Research Articles

Rectifying the impairment of immune thrombocytopenia plasmas through photobiomodulation

<p>Immune thrombocytopenia (ITP) is an autoimmune hemorrhage disorder. The first-line treatment of this disorder is corticosteroids, followed by thrombopoietin (TPO) receptor agonists such as Nplate, and/or splenectomy. Yet, the extended usage of corticosteroids or the expensive Nplate, coupled with the implications of splenectomy, raises concerns due to the array of associated side effects and an escalated vulnerability to subsequent complications. The current investigation shows that while anti-platelet antibodies and ITP plasmas hinder megakaryocyte differentiation and maturation and impair proplatelet and platelet formation in ex vivo culture of umbilical cord human CD34+ stem cells (cHSCs), low-level laser (LLL) treatment or photobiomodulation (PBM) effectively mitigates these detrimental impacts. PBM reinstated megakaryocyte differentiation and maturation, bolstering proplatelet and platelet formation in the presence of auto-platelet antibodies or ITP plasmas. The mitigating effects of PBM appear to pivot on its capacity to uphold cellular mitochondrial functionality and rectify the mitochondrial impairments engendered by anti-platelet antibodies or ITP plasmas. These findings underscore the potential of PBM as a safe and cost-efficient alternative for the management of a specific subset of ITP patients.</p>

Thrombopoietin-mediated regulation of hematopoietic stem cells

Sustaining lifelong hematopoiesis requires maintenance, proliferation, and differentiation of hematopoietic stem cells. Thrombopoietin is a cytokine essential for regulation of hematopoietic stem cells as well as differentiation and maturation of megakaryocytes required for platelet production. Due to these properties, thrombopoietin agonists have been used to treat bone marrow failure syndromes such as aplastic anemia. Through analysis of thrombopoietin gene-deficient mice, my colleagues and I have demonstrated the mechanism of action of thrombopoietin receptor agonists in hematopoietic stem cell maintenance and differentiation. This review focuses on governance of homeostasis in the hematopoietic system by thrombopoietin signaling.

Genistin: A Novel Estrogen Analogue Targeting ERβ to Alleviate Thrombocytopenia.

Thrombocytopenia, a prevalent hematologic challenge, correlates directly with the mortality of numerous ailments. Current therapeutic avenues for thrombocytopenia are not without limitations. Here, we identify genistin, an estrogen analogue, as a promising candidate for thrombocytopenia intervention, discovered through AI-driven compound library screening. While estrogen's involvement in diverse biological processes is recognized, its role in thrombopoiesis remains underexplored. Our findings elucidate genistin's ability to enhance megakaryocyte differentiation, thereby augmenting platelet formation and production. In vivo assessments further underscore genistin's remedial potential against radiation-induced thrombocytopenia. Mechanistically, genistin's efficacy is attributed to its direct interaction with estrogen receptor β (ERβ), with subsequent activation of both ERK1/2 and the Akt signaling pathways membrane ERβ. Collectively, our study positions genistin as a prospective therapeutic strategy for thrombocytopenia, shedding light on novel interplays between platelet production and ERβ.

Chlorogenic acid compounds from sweetpotato (Ipomoea batatas L.) leaves facilitate megakaryocyte differentiation and thrombocytopoiesis via PI3K/AKT pathway.

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by antiplatelet autoantibodies, thrombocytopenia, and bleeding, however, its treatment options are limited. In this study, a kind of active component, chlorogenic acid compounds (CGAs) from sweetpotato leaves was extracted out to explore its medicinal value and provide novel therapeutic strategies for the treatment of ITP. CGAs was isolated by ionic liquids-ultrasound (IL-UAE), which contains six isomers of chlorogenic acid with total purity of 95.69%. The thrombopoietic effect and mechanism of CGAs were investigated using in silico prediction and experimental validation. The changes of HEL cells morphology in volume and the increase in the total cell percentage of polyploid cells indicated that CGAs could promote megakaryocyte differentiation. Meanwhile, CGAs could promote platelet formation in a murine model of ITP, which was established by injection of antiplatelet antibody. Further quantitative proteomics analysis and Western blot verification revealed that CGAs could activate PI3K/AKT signaling pathway, which confirmed the mechanism prediction. It suggested that CGAs may provide a novel therapeutic strategy that relies on the PI3K/AKT pathway to facilitate megakaryocyte differentiation and platelet production.

GATA1 in Normal and Pathologic Megakaryopoiesis and Platelet Development.

GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N)terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1(friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor(MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed.

A novel GATA1 variant p.G229D causing the defect of procoagulant platelet formation

IntroductionGATA1 is one of the master transcription factors in hematopoietic lineages development which is crucial for megakaryocytic differentiation and maturation. Previous studies have shown that distinct GATA1 variants are associated with varying severities of macrothrombocytopenia and platelet dysfunction. ObjectiveTo determine the underlying pathological mechanisms of a novel GATA1 variant (c. 686G > A, p. G229D) in a patient with recurrent traumatic muscle hematomas. MethodsComprehensive phenotypic analysis of the patient platelets was performed. Procoagulant platelet formation and function were detected using flow cytometry assay and thrombin generation test (TGT), respectively. The ANO6 expression was measured by qPCR and western blot. The intracellular supramaximal calcium flux was detected by Fluo-5N fluorescent assay. ResultsThe patient displayed mild macrothrombocytopenia with defects of platelet granules, aggregation, and integrin αIIbβ3 activation. The percentage of the procoagulant platelet formation of the patient upon the stimulation of thrombin plus collagen was lower than that of the healthy controls (40.9 % vs 49.0 % ± 5.1 %). The patient platelets exhibited a marked reduction of thrombin generation in platelet rich plasma TGT compared to the healthy controls (peak value: ∼70 % of the healthy controls; the endogenous thrombin potential: ∼40 % of the healthy controls). The expression of ANO6 and intracellular calcium flux were impaired, which together with abnormal granules of the patient platelets might contribute to defect of procoagulant platelet function. ConclusionsThe G229D variant could lead to a novel platelet phenotype characterized by defective procoagulant platelet formation and function, which extended the range of GATA1 variants associated platelet disorders.

Wnt/β-Catenin-Signaling Modulates Megakaryopoiesis at the Megakaryocyte-Erythrocyte Progenitor Stage in the Hematopoietic System.

The bone marrow (BM) hematopoietic system (HS) gives rise to blood cells originating from hematopoietic stem cells (HSCs), including megakaryocytes (MKs) and red blood cells (erythrocytes; RBCs). Many steps of the cell-fate decision remain to be elucidated, being important for cancer treatment. To explore the role of Wnt/β-catenin for MK and RBC differentiation, we activated β-catenin signaling in platelet-derived growth factor b (Pdgfb)-expressing cells of the HS using a Cre-lox approach (Ctnnb1BM-GOF). FACS analysis revealed that Pdgfb is mainly expressed by megakaryocytic progenitors (MKPs), MKs and platelets. Recombination resulted in a lethal phenotype in mutants (Ctnnb1BM-GOFwt/fl, Ctnnb1BM-GOFfl/fl) 3 weeks after tamoxifen injection, showing an increase in MKs in the BM and spleen, but no pronounced anemia despite reduced erythrocyte counts. BM transplantation (BMT) of Ctnnb1BM-GOF BM into lethally irradiated wildtype recipients (BMT-Ctnnb1BM-GOF) confirmed the megakaryocytic, but not the lethal phenotype. CFU-MK assays in vitro with BM cells of Ctnnb1BM-GOF mice supported MK skewing at the expense of erythroid colonies. Molecularly, the runt-related transcription factor 1 (RUNX1) mRNA, known to suppress erythropoiesis, was upregulated in Ctnnb1BM-GOF BM cells. In conclusion, β-catenin activation plays a key role in cell-fate decision favoring MK development at the expense of erythroid production.

Prothymosin α accelerates dengue virus-induced thrombocytopenia

Thrombocytopenia is the hallmark finding in dengue virus (DENV) infection. Prothymosin α (ProT) has both intracellular and extracellular functions involved in cell cycle progression, cell differentiation, gene regulation, oxidative stress response, and immunomodulation. In this study, we found that ProT levels were elevated in dengue patient sera as well as DENV-infected megakaryoblasts and their culture supernatants. ProT transgenic mice had reduced platelet counts with prolonged bleeding times. Upon treatment with DENV plus anti-CD41 antibody, they exhibited severe skin hemorrhage. Furthermore, overexpression of ProT suppressed megakaryocyte differentiation. Infection with DENV inhibited miR-126 expression, upregulated DNA (cytosine-5)-methyltransferase 1 (DNMT1), downregulated GATA-1, and increased ProT expression. Upregulation of ProT led to Nrf2 activation and reduced reactive oxygen species production, thereby suppressing megakaryopoiesis. We report the pathophysiological role of ProT in DENV infection and propose an involvement of the miR-126-DNMT1-GATA-1-ProT-Nrf2 signaling axis in DENV-induced thrombocytopenia.

Genomic landscape of patients with germline RUNX1 variants and familial platelet disorder with myeloid malignancy

AbstractFamilial platelet disorder with associated myeloid malignancies (FPDMM) is caused by germline RUNX1 mutations and characterized by thrombocytopenia and increased risk of hematologic malignancies. We recently launched a longitudinal natural history study for patients with FPDMM. Among 27 families with research genomic data by the end of 2021, 26 different germline RUNX1 variants were detected. Besides missense mutations enriched in Runt homology domain and loss-of-function mutations distributed throughout the gene, splice-region mutations and large deletions were detected in 6 and 7 families, respectively. In 25 of 51 (49%) patients without hematologic malignancy, somatic mutations were detected in at least 1 of the clonal hematopoiesis of indeterminate potential (CHIP) genes or acute myeloid leukemia (AML) driver genes. BCOR was the most frequently mutated gene (in 9 patients), and multiple BCOR mutations were identified in 4 patients. Mutations in 6 other CHIP- or AML-driver genes (TET2, DNMT3A, KRAS, LRP1B, IDH1, and KMT2C) were also found in ≥2 patients without hematologic malignancy. Moreover, 3 unrelated patients (1 with myeloid malignancy) carried somatic mutations in NFE2, which regulates erythroid and megakaryocytic differentiation. Sequential sequencing data from 19 patients demonstrated dynamic changes of somatic mutations over time, and stable clones were more frequently found in older adult patients. In summary, there are diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in patients with FPDMM. Monitoring changes in somatic mutations and clinical manifestations prospectively may reveal mechanisms for malignant progression and inform clinical management. This trial was registered at www.clinicaltrials.gov as #NCT03854318.

Iguratimod Could Correct the Defective IL-35-Mediated Megakaryopoiesis in Immune Thrombocytopenia

Introduction: IL-35 is an inhibitory cytokine of the IL-12 family, and comprises an EBI3 subunit and a p35 subunit. Previous data indicated that serum levels of IL-35 were significantly lower in immune thrombocytopenia (ITP) patients than in healthy controls. Moreover, the concentration of plasma IL-35 in ITP patients achieving complete response returned to normal, but was not corrected in those who failed to achieve response. However, the underlying pathological function of IL-35 in impaired megakaryopoiesis in ITP remains unexplored. Our study aimed to evaluate the immunoregulatory role of IL-35 in megakaryocytes (MKs) differentiation. Iguratimod, a traditional therapy for inflammatory disorders, has been demonstrated to promote megakaryopoiesis. The effect of iguratimod on the treatment of ITP was retrospectively reviewed. Methods: Bone marrow samples were obtained from newly-diagnosed primary ITP patients and healthy donors. CD34+ cells from ITP patients and healthy donors were differentiated into MKs with and without IL-35 to determine the mechanism of IL-35 in megakaryopoiesis. Iguratimod was added to the culture medium of MKs to determine its function in MKs differentiation. Additionally, ITP mouse model was established to further validate the function of IL-35. Results: In comparison with healthy controls, ITP patients showed lower bone marrow levels of IL-35. IL-35 receptor expression was progressively induced on bone marrow MKs from healthy controls during the differentiation of CD34+ progenitors. In contrast, MKs from ITP patients exhibited defective expression of the IL-35 receptor. We then determined the effect of IL-35 on the megakaryocytic potential of CD34+ progenitor cells in culture medium containing TPO. IL-35 increased the number of medium-sized and large-sized colonies, and increased the total number of CD41+ cells. In IL-35-treated MKs, higher levels of Akt phosphorylation were observed. Pretreatment with LY294002, an inhibitor of Akt phosphorylation, blocked the differentiation of MKs induced by IL-35. LY294002 inhibited proplatelet formation and aggregation of β1-tubulin in MKs stimulated by IL-35. Flow cytometry analysis revealed that platelet production decreased in the LY294002 pretreatment group. These results demonstrated that IL-35 can enhance the proliferation and differentiation of MKs through Akt pathways. The decreased level of bone marrow IL-35 may partially explain the defective megakaryopoiesis in ITP. We then explored the potential causes of the decreased IL-35 levels in ITP. Flow cytometry analysis showed that the percentage of bone marrow iTr35 cells, the primary sources of IL-35, was significantly lower in ITP patients. Furthermore, we cocultured CD4+ T cells with MSCs from healthy controls and ITP patients, respectively. The percentage of iTr35 cells was lower when cultured with ITP-MSCs. These results showed that ITP-MSCs exhibited an impaired capability of inducing iTr35 cells, which might contribute to the reduced level of bone marrow IL-35 in ITP patients. We further investigated the effects of iguratimod on the differentiation of CD34+ progenitors into MKs. When CD34+ cells were cultured with TPO alone, the addition of iguratimod upregulated the expression of IL-35 receptors on MKs from ITP patients and a murine model of this disease, and consequently promoted the formation of MK colonies. In the retrospective study of iguratimod, a total of 68 adult patients with corticosteroid-resistant primary ITP were included. Initial response was achieved in 42/68 (62%) patients, and complete response was achieved in 25/68 (37%) patients. In patients who achieved initial response, the median time to response was 28 days. Sustained response at 6 months was seen in 35/68 (51%). Bleeding symptoms were reduced from 37/68 (54%) patients at baseline to 14/68 (21%) patients at 6 months. Most adverse events were mild (grade 1-2), and there were no grade 4 or worse adverse events in our patients. The most common adverse events were elevated ALT, gastrointestinal disorders, and nausea. Conclusions: ITP-MSCs exhibited an impaired capability of inducing iTr35 cells, which contributed to the reduced level of bone marrow IL-35 in ITP. IL-35 promoted MKs differentiation through the Akt pathway. Iguratimod promoted megakaryopoiesis by inducing the expression of IL-35 receptors on MKs. Iguratimod had good efficacy and safety in the treatment of ITP.

GATA-1 and BCL11A Collaboratively Regulate Erythroid Gene Expression through HDAC1 Dependent Manner

Transcription factor GATA-1 plays important roles in hematopoiesis through interaction with other key transcription factors and epigenetic modulators. Previously, we established that GATA-1 can physically interact with HDAC1 at two arginine residues of the linker region between two zinc fingers. Arginine to alanine mutation at these sites blocks the HDAC1 interaction and results in constitutive GATA-1 acetylation, and reduced GATA-1 chromatin binding at the gene regulatory regions. In addition, the loss of HDAC1 interaction causes the blockage of erythroid and megakaryocytic differentiation in mice. Having established the importance of GATA-1 and HDAC1 interaction in GATA-1 function, next we aim to further investigate whether HDAC1-associated proteins are involved in regulating the function of GATA-1. We performed LC-MS/MS analysis using GATA-1 wild type and mutant expressing cells and identified that BCL11A was one of the major proteins that associates with GATA-1 through HDAC1. BCL11A is a master regulator of γ-globin gene silencing through association with the NuRD complex and blocking of NFYA recruitment to γ-globin gene promoters. The role of BCL11A in regulating gene expression during erythroid differentiation is not well studied. HDAC1 mediated GATA-1 and BCL11A association was validated by pull down and immunoprecipitation assay. Next, the BCL11A and HDAC1 interacting site was determined. The N-terminal region of BCL11A is found to be important for HDAC1 interaction by pull down assay. Through analysis of the secondary structure of BCL11A N-terminal, L12/K14 are predicted to be important for protein-protein interaction. Indeed, L12A/K14A mutation significantly reduced HDAC1 interaction as well as HDAC1 mediated GATA-1 and NuRD complex association. To assess the effects of the L12A/K14A mutation of BCL11A on gene expression, we performed RNA-seq analysis using BCL11A knock down (KD) or L12A/K14A mutant overexpressed in HUDEP-2 cells. The result shows that the L12A/K14A mutation de-repressed γ-globin expression and positively affects genes for erythrocyte development that is similar to the expression profile of BCL11A KD. Therefore, L12/K14 residues of BCL11A might have an important role in γ-globin silencing and erythropoiesis by mediating HDAC1 associated GATA-1 and NuRD complex. The Cut&Run assay was performed to study the recruitment of BCL11A, GATA-1, HDAC1, NuRD complex, and NFYA in WT, BCL11A KD, and Flag-BCL11A L12A/K14A mutant cells. There are over 7000 GATA-1 and BCL11A co-binding peaks detected in wild type cells. Within these peaks, about 2000 peaks are at BCL11A binding motif site. GATA-1, HDAC1 and NuRD binding were abolished at these sites in BCL11A KD or L12A/K14A mutant cells, while BCL11A binding was not affected. There are about 3000 peaks at GATA-1 binding motif sites. While GATA-1 binding is not affected at these sites in BCL11A KD or L12A/K14A mutant cells, BCL11A, HDAC1 and NuRD complex binding were significantly reduced. This study demonstrates that there are two modes of BCL11A and GATA-1 co-recruitment: BCL11A can recruit GATA-1 and GATA-1 can recruit BCL11A to their specific binding motifs, respectively. Interestingly, these two modes regulate differential gene targets. BCL11A recruits GATA-1 at genes mainly involved in chromatin organization and cell cycle, while GATA-1 recruits BCL11A to genes mainly involved in transcription, erythrocyte and megakaryocyte differentiation. At the β-globin locus, BCL11A KD or L12A/K14A mutation reduced GATA-1 and NuRD binding and enhanced NFYA binding at the γ-globin promoter, suggesting that BCL11A cooperates with GATA-1, HDAC1, and NuRD complex and prevents NFYA binding at the distal binding region to suppress γ-globin expression. At LCR hypersensitive site 2 (HS2) region, we also observed a reduction of GATA-1 and NuRD recruitment with BCL11A KD or L12A/K14A mutation while GATA-1 recruitment at other promoters or LCR regions were not changed, suggesting that the HS2 region may be involved in reactivating γ-globin in BCL11A KD or L12A/K14A mutation cells. Future study will continue to analyze how BCL11A KD or mutation affects BCL11A and GATA-1 co-binding and target gene expression, and NFYA or other factors recruitment. In summary, our study shows that BCL11A interacts with GATA-1 in a HDAC1 dependent manner and this interaction is important in regulating fetal globin expression and erythroid development.

Treatment with Hetrombopag for Poor Platelet Engraftment after Allo-HSCT-a Single Center Retrospective, Observational Study

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a common and important treatment for most hematopoietic diseases. Some patients may experience poor platelet engraftment after allo-HSCT, which may lead to transplant failure and is also an important influencing factor for post-HSCT complications such as graft versus host disease (GVHD) and non-relapse mortality (NRM). Thus, exploring methods to improve platelet engraftment is very important. Hetrombopag is a small-molecule thrombopoietin-receptor agonist (TPO-RA) produced in China, stimulates the proliferation and differentiation of megakaryocytes and promotes platelet production through binding to thrombopoietin receptor to stimulate multiple intracellular signaling pathways. It has been reported that hetrombopag was effective in promoting platelet engraftment and decreasing platelet transfusion after allo-HSCT. Here, we studied the efficacy and safety of hetrombopag for poor platelet engraftment after allo-HSCT, of whom did not respond to rhTPO treatment. Aims: To evaluate the efficacy of hetrombopag in patients with poor platelet engraftment after allogeneic hematopoietic stem cell transplantation. Methods: In this retrospective study, data from patients with poor platelet engraftment after allo-HSCT who had been treated firstly with rhTPO and then with hetrombopag at Henan Cancer Hospital from July 1, 2021 to July 1, 2023 were collected. Patients should meet the following criteria to enroll in the study: experienced poor platelet engraftment who had been treated with rhTPO from day +4 post-HSCT. Here we defined poor platelet engraftment as platelet count (PLT) < 20 × 10 9/L beyond day +28 post-HSCT. We analyzed 57 patients mentioned above and divided them into two groups in a ratio of 1:2, 19 of them were given hetrombopag until their PLT exceeded 50 × 10 9/L and maintained for more than 7 days without platelet transfusion (hetrombopag group), another 38 patients were treated without hetrombopag (control group). The initial dose of hetrombopag was 5, or 7.5 mg/d according to the patient's conditions and characteristics (e.g., age, PLT, and bleeding risk). After allo-HSCT, patients administered rhTPO at a dose of 300U/kg·d from day +4. We defined rhTPO as ineffective when PLT was still below 20×10 9/L beyond +21 days post-HSCT after rhTPO administration. At this time, rhTPO was replaced with hetrombopag, or IL-11, decitabine, platelet transfusion, etc. Results: The median ages of the two groups of patients were 38 years (hetrombopag group) and 41 years (control group), respectively. The median baseline platelet count in both groups were 20 × 10 9/L. The proportion of patients with PLT recovery to 50×10 9/L was significantly higher in the hetrombopag group than in the control group (47.3% vs 7.9%, χ2=26.03, P<0.0001). The median time to PLT recovery to 50×10 9/L was 10 (7-48) days in the hetrombopag group and 33 (21-94) days in the control group ( P=0.004). The mean units of platelet transfusion in the hetrombopag group was significantly lower than that in the control group (2.7 vs 6.3 U, P=0.001), and no thrombotic events occurred. There was no statistical difference in the incidence of aGVHD (42.0% vs 44.7%, P=0.850) and NRM (5.2% vs 7.9%, P=0.714) between the two groups, suggesting that hetrombopag had no significant effect on aGVHD and NRM. Conclusion: For allo-HSCT patients who experienced poor platelet engraftment after rhTPO treatment, hetrombopag can rapidly promote PLT recovery, reduce the units of platelet transfusion, and have no risk of the increase of aGVHD and NRM. These results still need further validation in a larger sample prospective confirmatory study in the future.

STK10 Mutation Block Erythropoiesis in Acquired Pure Red Cell Aplasia Via Down-Regulated the Ribosome Biosynthesis

Background: Acquired pure red cell aplasia (PRCA) is anemia associated with the absence of erythroblasts and characterized by persistent and easy recurrence. However, the underline mechanisms of acquired PRCA remain obscure, and the role of gene mutation in the pathogenesis of acquired PRCA was not elucidated yet. Aims: To identify gene mutations in acquired PRCA patients and their possible effects on the origin of the disease. Methods: Blood and buccal genome DNA extracted from thirty newly diagnosed patients with acquired PRCA were detected with whole exome sequencing. The candidate genes with high frequency in acquired PRCA but the low frequency in 1000 normal genomes which may affect protein function were selected. The pGreen-CMV-puro system was employed to generate lentivirus expressing short hairpin RNAs to target the candidate genes, and stable transfected K562 cell lines with silenced candidate genes were constructed. The erythroid and megakaryocytic differentiation was evaluated for the transfected K562 cell lines either by benzidine staining hemoglobin and CD235a expression or CD41 expression on the cell surface. STK10 gene was selected which affects the erythropoiesis. The influence of the STK10 gene on the expression of p53 mRNA/ protein was also detected. The RNA sequencing in STK10 silenced K562 cells was performed and KEGG enrichment was analyzed. Next, ribosome RNA synthesis was detected, and ribosome proteins and p53 signaling pathway were also detected by western blotting. Results: Through whole exome sequencing in patients with acquired PRCA, we confirmed that STK10 gene mutation is common in acquired PRCA patients, the mRNA/protein expression of STK10 was reduced and p53 increased in the bone marrow of the patients in which the gene mutated. Furthermore, the silence of the STK10 gene through the lentiviral vector harboring short hairpin RNAs in K562 cells could inhibit the erythropoiesis after being induced by Hemin. Whereas, CD41 expression was similar in STK10 silenced K562 cells to control K562 cells after being inducted by phorbol 12-myristate 13-acetate. We performed RNA sequencing in STK10 silenced K562 cells. KEGG enrichment analysis of differentially expressed ribosome biosynthesis pathway and p53 signaling pathway was affected. 28S and 18S in ribosome RNA synthesis impaired in these STK10 silenced K562 cells through RNA electrophoresis. Further, through the western blotting test in STK10 silenced K562 cells, we found ribosome proteins expression down-regulated and p53, phospho-p53 and p21 expression up-regulated due to STK10 mutated. Conclusion: STK10 gene mutation is common in patients with acquired PRCA and causes the reduction of mRNAs and protein expression in the mutated patients. The underlying research revealed STK10 gene mutation could affect the ribosome biosynthesis pathway and down-regulated the ribosome protein level, contributing to the abnormal erythropoiesis. This research elucidated the pathogenesis of PRCA, which may provide evidence for precise diagnosis and exploring potential therapeutic targets in patients with acquired PRCA. Keywords: pure red cell aplasia, mutation, erythropoiesis, STK10, ribosome biosynthesis

A Cytochrome c Variant Enhances Megakaryocytic Maturation

Mutations in genes which play a role in the processes of megakaryopoiesis and platelet production cause thrombocytopenia. Five pathogenic variants in the gene encoding cytochrome c ( CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. The role of cytochrome c in megakaryopoiesis and platelet formation is unknown, and it is unclear how mutations in cytochrome c cause thrombocytopenia. The goal of this project is to understand how CYCS mutations cause thrombocytopenia. We are using the human leukemic K562 cell line as a model system to study the impact of CYCS mutations on megakaryocyte differentiation and mitochondrial function. One of the CYCS pathogenic variants (c.145T>C; p.Tyr49His) was introduced into K562 cells using the CRISPR-Cas9 base-editing method. K562 cells have three copies of CYCS-containing chromosome 7 and we isolated CYCS WT/WT/WT, CYCS WT/WT/Y49H, CYCS WT/Y49H/Y49H, CYCS Y49H/Y49H/Y49H clonal lines, confirmed by sequencing and mass spectrometry. K562 cells undergo megakaryocyte differentiation in response to PMA. We monitored the expression of megakaryocyte markers CD61 and CD41a by flow cytometry in response to PMA in all clonal lines. The K562- CYCS Y49H/Y49H/Y49H line showed faster maturation compared to all other clonal lines and the parental K562 cells. We assessed mitochondrial function using the Seahorse XFe96 Analyzer. Our results show that the mutation does not impact mitochondrial respiratory function. Finally, we have introduced the Y49H mutation into WTC11 iPSCs. Investigations of the impact of the Y49H variant on apoptosis showed that there is no significant differences in apoptosis between WT and the clonal line carrying the Y49H variant. Our results suggest that the pathogenic c.145T>C; p.Tyr49His CYCS variant enhances megakaryocyte maturation.

Proteomics Landscape and Machine Learning Predictor of Response to Splenectomy in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an auto-immune disease characterized by antibody-mediated platelet destruction. Splenectomy remains the effective curative treatment for patients with ITP who failure to respond to TPO-RAs and/or rituximab (Mageau et al, Am J Hematol. 2022). The effectiveness rate of splenectomy for ITP is reported to be roughly 80%, with a 5-year remission rate ranging from 50% to 75% (Chaturvedi et al, Blood. 2018). For subjects presenting with severely low platelet counts or active bleeding, splenectomy represents the primary approach for rapidly increasing platelet levels and potentially saving lives. To achieve personalized and precise treatment, the prediction efficacy on splenectomy remains an urgent problem. Here, we have characterized immune features of ITP patients with splenectomy, and developed a predictive model for assessing the response to splenectomy. Methods Bone marrow plasma samples before splenectomy were collected and analyzed by high-resolution liquid chromatography-tandem mass spectrometry. Weight protein co-expression network analysis (WPCNA) was applied to generate modules from all proteins and mapped them to clinical characteristics. We used least absolute shrinkage and selection operator (Lasso) regression, recursive feature elimination (RFE) for predictors selection. Next, multivariable logistic regression (LR), support vector machine (SVM), and decision tree algorithms were used to develop a predictive model. Results 60 patients with ITP undergoing splenectomy treatment were enrolled into our study, and divided into three groups according to the response [patients with CR (n=30), NR (n=7), and Relapse (n=23)]. A pathway enrichment analysis of WPCNA modules by ClueGO revealed that megakaryocyte differentiation, humoral immune response mediated by circulating immunoglobulin, Fc receptor signaling pathway, and cGMP-PKG signaling pathway were correspondingly enriched in MEtan, MEturquoise, MEblue, and MEpink modules, which were associated with treatments with TPO-RA, IVIG, rhTPO, and post-operation platelet level, respectively. We then performed KEGG pathway enrichment analyses on the different expression proteins (DEPs) to deciphering the immune landscape among each group. For the relapse group, DEPs were primarily enriched in ribosome, spliceosome, neutrophil extracellular trap formation, and necroptosis pathways, comparing with the CR group. For the NR group, most DEPs were enriched in natural killer cell mediated cytotoxicity, NF-kappa B signaling, Fc gamma R-mediated phagocytosis pathways, comparing with the CR group. The models were based on a multi-step predictor pipeline. Inside the pipeline, features were first filtered by lasso regression and RFE algorithm. We identified five proteomic features present in the bone marrow plasm that associated with relapsed to therapy, which included CD40, COL4A3, GALNT6, GSTM3, and UBE2A (Figure). Each ensemble consisted of three algorithms acting in parallel: LR, SVM and decision tree. The AUC values of LR, SVM, and decision tree were 0.962 (95%CI: 0.915-1), 0.957 (95%CI: 0.897-1), and 0.875 (95%CI: 0.782-0.967), respectively. The three algorithms were then tested for performance. The sensitivity, specificity, positive predictive value, and negative predictive value analysis revealed that LR model reached the best performance. Targeting CD40 represents a promising therapeutic approach for enhancing clinical outcomes in patients with ITP who have shown inadequate response following splenectomy. Conclusion Our analysis revealed that ITP patients with splenectomy showed distinct immune characteristics when achieving different response. Protein features extracted from bone marrow plasma can be selected as predictors of response to splenectomy.

Mutational Analysis of the Genome Stability Factor BOD1L1 in Myeloproliferative Neoplasm

The myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders that include essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) which arise from somatic mutations in JAK2, MPL or CALR, but which can evolve to acute myeloid leukemia (AML) by the acquisition of additional somatic mutations. To investigate the contribution that mutations might make to the pathogenesis or clinical course of MPNs, 261 genes recurrently mutated in myeloid malignancies were sequenced in DNA from germline and blood samples from patients with AML, ET and MF enrolled in studies of the LSD1 inhibitor bomedemstat. Earlier work identified somatic mutations inthegene BOD1L1 in isolated megakaryocytes in 6 of 12 patients with MPN with mutations suggestive of loss-of-function (Guo et al., 2017). BOD1L1 plays a key role in multiple pathways of cellular DNA repair in conjunction with the histone methyltransferase SETD1A, which regulates transcription and DNA repair (Higgs et al. 2018; Hoshii et al. 2018. We previously demonstrated that BOD1L1 prevents uncontrolled resection of damaged DNA forks under conditions of replication stress, and that its loss leads to severe genome instability (Higgs et al., 2015, 2018). In addition, BOD1L1 promotes repair of DNA double-strand breaks after ionizing radiation through its interaction with the DNA repair protein RIF1 (Bayley et al., 2022). This sequencing-based analysis identified specific germline mutations in BOD1L1 that were 3-6 times more prevalent in MPN patients compared to a control population (gnomAD). This enrichment of BOD1L1 mutations was confirmed in whole-exome sequencing from an independent data set (EGA) of 183 patients with MPNs. We predicted these highly deleterious mutations, or at least a subset, may compromise the DNA repair functions of BOD1L1 and potentially influence the development and/or progression of MPN. To investigate this prediction, we disrupted BOD1L1 using a doxycycline-inducible CRISPR-Cas9 system in HeLa cells and complemented them with gRNA-resistant wild-type BOD1L1,or one of six mutants distributed throughout the protein. None of the mutants affected BOD1L1 protein expression; however, two of the six variants were defective for RIF1 binding by co-immunoprecipitation/proximity ligation, failed to recruit this key DNA repair factor to radiation-induced double-strand breaks, and compromised repair of these breaks. Moreover, single molecule DNA fiber analyses revealed that these same two mutants were unable to protect stalled replication forks against resection, leading to elevated levels of genomic damage. To explore the functional consequences of BOD1L1 loss-of-function on megakaryocytes, the gene was silenced using shRNA in human megakaryoblastic JAK2 V617F SET-2 cells, differentiated with thrombopoietin, and analyzed by flow cytometry. These cells were CD41-positive, consistent with megakaryocytic differentiation, and showed increased DNA damage (γH2AX) compared to controls. Depletion of BOD1L1 also lead to a greater number of cells with 2n nuclear content, and fewer with ≥8n, compared with controls, suggesting that the loss of BOD1L1 led to impaired polyploidization likely due to unrepaired DNA damage and subsequent cell death. In summary, we have shown that MPN patients have a high prevalence of mutations in the key genome instability factor BOD1L1 and that a subset of these mutations compromised its DNA repair functions and increased genome instability. The presence of BOD1L1 somatic mutations in megakaryocytes in MF patients may deregulate polyploidization, altering megakaryocyte function that may contribute to the development of bone marrow fibrosis. That no patients with BOD1L1 germline mutations progressed to AML in these clinical studies, coupled with the fact that these variants were not enriched in patients with AML, invites the possibility that BOD1L1 mutations may be synthetically lethal with other somatic mutations that result in genome instability and, hence, protect against progression to AML.

Role of Tubulin Detyrosination in Platelet Biogenesis

Introduction. Microtubules are hollow cylindrical polymers that are made of α/β-tubulin heterodimers. They are involved in a broad range of essential functions such as cell division, intracellular trafficking or cell motility. Their functional diversity is thought to be regulated through the expression of distinct α- and β-tubulin isotypes together with several post-translational modifications (PTMs); a concept known as tubulin code. In the megakaryocytic lineage, while tubulin isotypes are well-known regulators of platelet biogenesis (TUBB1, TUBA4A, TUBA8) 1, the temporal regulation and biological significance of tubulin PTMs remain mostly unknown. Aims. This project aims to characterize the kinetics and to evaluate the functional importance of tubulin detyrosination, one of the main α-tubulin PTM, in platelet biogenesis. Methods. Analyzes were performed in megakaryocytes and platelets obtained from cultured human CD34 + hematopoietic stem and progenitor cells. To characterize the kinetics of tubulin detyrosination, the PTM and its modifying enzymes were monitored at the proteomic (Western Blot) and transcriptional (qRT-PCR) levels, at several defined stages of megakaryocyte differentiation. To evaluate the function of tubulin detyrosination, differentiation of CD34 + hematopoietic stem and progenitor cells was performed in the presence of the SVBP/VASH-detyrosinase inhibitor EpoY 2 or following CRISPR-mediated inactivation of the VASH detyrosinase co-factor SVBP. Results. Tubulin detyrosination levels progressively increased as megakaryocytes matured. This was accompanied by a gradual increase in the transcript levels of the detyrosinase VASH1, while those of its cofactor SVBP remained stable. Transcripts of the newly identified detyrosinase MATCAP 3 mirrored those of VASH1. Together, these results suggest that tubulin detyrosination occurring during platelet biogenesis could be mediated by both the SVBP-VASH1 complex and/or MATCAP, and prompt the question of their functional importance in the process. To evaluate this, we first focused on the SVBP-VASH1 complex with the inhibitor EpoY. Interestingly, while this compound had no impact on early megakaryocyte differentiation, the final stage of platelet biogenesis, proplatelet formation, was perturbed. In particular, proplatelets obtained in the presence of EpoY showed morphological alterations with thinner unbranched shafts, suggesting potential repercussions on platelet production. Preliminary data obtained with SVBP-inactivated megakaryocytes confirmed these observations. Follow-up work will aim to evaluate the downstream consequences on platelet production as well as the relative contribution of MATCAP. Conclusion. These results suggest that tubulin detyrosination could act as a potential regulator of terminal megakaryocyte maturation, and further highlight the importance of the microtubule cytoskeleton and the tubulin code in platelet biogenesis. References. 1. Kimmerlin, Q., Strassel, C., Eckly, A., and Lanza, F. (2023). The tubulin code in platelet biogenesis. Semin Cell Dev Biol 137, 63-73. 10.1016/j.semcdb.2022.01.010. 2. Hotta, T., Haynes, S.E., Blasius, T.L., Gebbie, M., Eberhardt, E.L., Sept, D., Cianfrocco, M., Verhey, K.J., Nesvizhskii, A.I., and Ohi, R. (2021). Parthenolide Destabilizes Microtubules by Covalently Modifying Tubulin. Curr Biol 31, 900-907 e906. 10.1016/j.cub.2020.11.055. 3. Landskron, L., Bak, J., Adamopoulos, A., Kaplani, K., Moraiti, M., van den Hengel, L.G., Song, J.Y., Bleijerveld, O.B., Nieuwenhuis, J., Heidebrecht, T., et al. (2022). Posttranslational modification of microtubules by the MATCAP detyrosinase. Science 376, eabn6020. 10.1126/science.abn6020.

Single-Cell RNA Sequencing Uncovers Abnormal Differentiation of Neutrophil and Megakaryocyte Might Mediated Ruxolitinib Resistance for Patinets with PMF

Background: Primary myelofibrosis (PMF ) is a heterogeneous disorder characterized by bone marrow fibrosis, megakaryocyte hyperplasia, systemic inflammation and extramedullary hematopoiesis. Ruxolitinib (Rux）have shown clinical benefit with reduction of splenomegaly and related symptoms. However, about 50% of patients discontinued ruxolitinib for resistance and/or intolerance，and the mechanism of resistance to ruxolitinib is still unclear. Recently studies found that immune microenvironment and megakaryocyte promote PMF progression, whether the abnormalities of immune microenvironment and megakaryocyte associated with Rux resistance needs to be further elucidated. M ethods: In this study, 8 participants (Newly Diagnosed PMF =3, Resistance to Rux PMF =3, Healthy Donors =2) were included. Peripheral blood was taken and single-cell transcriptome sequencing technology was used. Combined with pseudo-temporal analysis, SCENIC analysis, Single Cell Lineage Inference Using Cell Expression Similarity and Entropy (SLICE), cellular interaction analysis, gene ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) pathway analysis, Gene Set Variation Analysis (GSVA), L-R receptor coexpression analysis and GEPIA online tool for data analysis, Use R language or Python for data processing. Results: In PMF patients, the proportion of neutrophils and monocytes was significantly increased, while the proportion of T cells was significantly decreased in Resistance to Rux group (P<0.05). Further subdivision of neutrophils showed that neutrophils were divided into 6 subgroups. Compared with healthy control group, neutrophils increased in Resistance to Rux group and decreased in Neutrophil_1, among which Neutrophil_6 was only enriched in PMF patients. Neutrophil_6 significantly up-regulated tumor necrosis factor (TNF) superfamily product pathway, IL-1 signaling pathway, Toll-like Receptor 4 (TLR4) signaling pathway, peroxide-producing NADPH oxidase activator activity pathway, MAPK pathway and IL-8 product signaling pathway. Neutrophil_4-6 significantly up-regulates MYB, EZH2, SUZ12, YBX1, POLR3G, E2F7, E2F8 and other transcription factors. L-R up-regulation between Neutrophil_4 and Neutrophil_6 and T cells mediated T cell exhaustion by LGALS9/Tim3 relate to resistance to Rux. We have identified a subset of HSCs in PMF that exhibits a bias towards megakaryocytic/platelet differentiation (HSCs_2) in Rux resistant group. This subset is characterized by the upregulation of specific genes, such as GATA1, PRKAR2B, ITGA2B, PLEK, and KLF1.CellPhoneDB revealed that the regulation of platelets-platelets involves CD40L-CD40 ligand receptors. Notably, the heightened reactivity of platelets may be closely associated with the risk of PMF-related thrombosis. Additionally, we observed an upregulation of the MIF-CD74 cytokine ligand receptor between the immune platelet subsets and HSC subsets in Rux resistant group. Conclusion: In Rux resistant group, Up-regulation of inflammatory and tumor-related signaling pathways promotes the occurrence of PMF inflammation and amplification of malignant clones; Up-regulation of LGALS9 in neutrophil band to receptor TIM3 mediates T cell depletion; the regulation of platelets-platelets in PMF is mediated by CD40L-CD40 ligand receptors; MIF-CD74 might be the relationship between PMF-HSC and PMF-PLT in the context of primary myelofibrosis.

Identification of the Role of SEL1L in Platelet Function with Implications for Atypical Equine Thrombasthenia in Thoroughbred Horses

Atypical Equine Thrombasthenia (AET) is a recessive heritable platelet disorder in horses due to aberrant platelet signaling after thrombin stimulation, preventing platelets from fully activating or efficiently binding to fibrinogen, leading to prolonged bleeding. To date, AET has only been identified in the Thoroughbred horse breed. A prevalence study performed at one breeding farm found that one in every 150 Thoroughbreds was affected. Despite the negative effects on horse health and racing performance, the underlying etiology of this unique platelet disorder is unknown. Here we show that a variant in SEL1L, a gene which encodes a protein known to play a pivotal role in endoplasmic reticulum-associated protein degradation (ERAD) and not previously known to be involved in platelet function, leads to AET. In order to identify the underlying genetic mechanism, we performed whole genome sequencing on five affected, one equivocal, and eleven control Thoroughbred horses. A whole-genome association study was conducted, and associated variants were filtered by determining if they were in or near genes known to be expressed in platelets. This evaluation identified three putative loci: a missense variant in SEL1L (p.Ile604Val), a 1.6kb deletion in a long non-coding RNA ( AL355838.1) and a 73bp intronic deletion in VIPAS39. The long non-coding RNA AL355838.1 did not show expression in horse platelets, and there was no differential expression of SEL1L or VIPAS39 mRNA identified between affected and unaffected individuals. However, a significant difference in SEL1L protein expression was observed in AET-affected horses' platelets. Flow cytometry and immunofluorescence studies demonstrated that SEL1L is located intracellularly in equine platelets, but not in the α-granule, and moves to the surface of the platelet upon activation with thrombin. Although the template bleeding time was normal, platelets from horses that were homozygous for the SEL1L variant showed defective spreading on collagen. To support SEL1L as the causative gene and dissect the underlying mechanisms, we undertook studies in three additional models. Differentiation of human megakaryocytes revealed the presence of two SEL1L protein isoforms, p100 and p38, and both showed increased expression during megakaryopoiesis, although only p100 was delivered to mature platelets. We perfused whole blood from Mx1-Cre +; Sel1l fl/fl conditional knockouts and controls through a collagen-coated microfluidic chamber. We found that significantly fewer platelets from knockout mice bound to the collagen as compared to control siblings. Finally, a knockout zebrafish line was developed by deleting exons 3-20 using CRISPR/Cas9 mediated genome editing. Similar to the mouse, laser-mediated arterial endothelial injury of 5-day old mutant larvae resulted in significantly fewer thrombocytes (the fish platelet equivalent) adhering to the injured vessel wall as compared to wild-type siblings. Overall, these data support the conclusion that the SEL1L missense variant leads to AET, and that SEL1L has a previously undescribed and conserved role in platelet function, particularly the ability of platelets/thrombocytes to adhere to exposed collagen at sites of endothelial injury. These results will allow the Thoroughbred industry to easily diagnose AET-affected horses and make appropriate decisions for their racing and breeding careers. These data also provide an excellent introduction to further investigation into the understudied and potential role of ERAD proteins in human platelet disorders.

Interplay between Inflammatory Microenvironment and RUNX1-Mediated Transcriptomic Changes Drives Defective Hematopoiesis in Familial Platelet Disorder

Background: Familial platelet disorder (FPD) is caused by germline loss-of-function mutations in RUNX1 an essential transcription factor regulating hematopoiesis. The absence of functional RUNX1 leads to mild-to-moderate thrombocytopenia, platelet dysfunction, and increased bleeding in FPD patients. In ~40% of cases, the patients progress to overt leukemia at median age of 33 years through unknown mechanisms. FPD patients also present with skin allergy and eczema which are associated with systematic elevation of cytokines. Given the well-established connection between inflammation and leukemia initiation and progression, we sought to investigate whether inflammatory stress play a role in regulating abnormal hematopoiesis and eventually cause clonal evolution in FPD. Methods: We acquired bone marrow cells from FPD patients who had not developed leukemia yet. We then compared FPD bone marrow hematopoietic stem and progenitors (HSPCs) with healthy progenitors using in vitro differentiation and colony formation assays. For comprehensive mechanistic evaluation, we performed 10x single-cell RNA sequencing (scRNA-seq) on FPD (n=10) and healthy (n=4) bone marrow cells, analyzing >100,000 cells using Seruat package. We evaluated cytokine levels in bone marrow fluid and peripheral blood using 65-plex Luminex assay. The impact of the inflammatory cytokines and intervention strategies were tested using in vitro differentiation and colony formation assays. The results were validated in vivo using mice with germline Runx1 mutation. Results: We identified that FPD progenitors are impaired in megakaryocytic (>2.0-fold, p<0.05) and erythroid differentiation with increased myeloid differentiation (>1.7-fold, p<0.05) compared to healthy control. In addition, FPD HSPCs show increased myeloid colony formation (>1.6-fold, p<0.05) and serial replating ability. scRNA-seq identified 18 cell clusters and pseudotime analysis revealed higher percentage of FPD cells differentiating towards monocytes, supporting enhanced myeloid differentiation. The pathway enrichment analysis of differentially expressed genes in HSC cluster revealed upregulation of inflammatory (TNFα via NF-kB) and pro-survival pathways (PI3K/AKT/mTOR). Accordingly, cytokine profiling revealed upregulation of numerous cytokines and chemokines in FPD compared to healthy samples including TNFα, CXCL8, CCL24, CCL2, and IL1β (>2.5 to 1000-fold, p<0.05), secreted by progenitors, monocytes, and MSCs. Further, under inflammatory stress mediated by TNFα, CXCL8, CCL24, CCL2, and IL1β, FPD HSPCs exhibited resistant to growth suppression and have increased myeloid colony formation. However, pharmacological inhibition of individual cytokines only partially rescued the differentiation defects, possibly due to compensatory effects of each cytokine. Phospho-flow analysis revealed that increased cytokine levels leads to the activation of PI3K/mTOR and JAK signaling that we also found upregulated using scRNA-seq analysis. Interestingly, inhibition of mTORi (rapamycin and AZD2014), PI3Ki (idelalisib), and JAKi (ruxolitinib) showed an increase in percentage of megakaryocytes (up to 2-fold, p<0.05) and a decrease in monocytes (up to 0.6-fold change, p<0.05). Also, inhibition of these pathways in FPD reduced cytokines levels. In line with human FPD data, in vivo treatment of Runx1 R188Q/+ mouse with ruxolitinib suppressed myeloid differentiation with no effects on megakaryocytes, while rapamycin resulted in significantly increased platelet activation compared to untreated mice (>1.7-fold, p<0.05) and a reduction of monocytes (<0.5-fold, p<0.05), suggesting mTOR inhibition might be an effective intervention strategy. Conclusion: FPD HSPCs manifest defective megakaryopoiesis with increased myelopoiesis. The elevated autocrine and paracrine inflammatory stress is an early event in FPD, driving myeloid differentiation. This inflammatory stress mediates activation of PI3K/mTOR and JAK signaling, leading to augmented myeloid differentiation. Consequently, the observed FPD phenotypes are the result of combined transcriptional changes and inflammatory stress. Targeting inflammatory and pro-survival signaling at an early stage might mitigate hematopoietic defects and delay disease evolution in FPD. Our results advance mechanistic understanding and hold significant clinical implications.