SF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells ...with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of ∼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5′ UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non–rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation.
•Induced pluripotent stem cell model of SF3B1-mutant MDS develops RS.•Coordinated mis-splicing of TMEM14C and ABCB7 causes RS formation.
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Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the ...dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion.
Platelets from 12-LOX
(wild-type WT) and 12-LOX
mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry-multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice.
Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX
mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbβ3 integrin activation in 12-LOX
platelets than in WT platelets. Stored platelets from 12-LOX
mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX
than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX
platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion.
Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1-dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.
Abnormal nuclear morphology is a hallmark of malignant cells widely used in cancer diagnosis. Pelger-Huët anomaly (PHA) is a common abnormality of neutrophil nuclear morphology of unknown molecular ...etiology in myeloid neoplasms (MNs). We show that loss of nuclear lamin B1 (LMNB1) encoded on chromosome 5q, which is frequently deleted in MNs, induces defects in nuclear morphology and human hematopoietic stem cell (HSC) function associated with malignancy. LMNB1 deficiency alters genome organization inducing in vitro and in vivo expansion of HSCs, myeloid-biased differentiation with impaired lymphoid commitment, and genome instability due to defective DNA damage repair. Nuclear dysmorphology of neutrophils in patients with MNs is associated with 5q deletions spanning the LMNB1 locus, and lamin B1 loss is both necessary and sufficient to cause PHA in normal and 5q-deleted neutrophils. LMNB1 loss thus causes acquired PHA and links abnormal nuclear morphology with HSCs and progenitor cell fate determination via genome organization.
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•LMNB1 gene is commonly deleted in myeloid malignancies•Loss of LMNB1 promotes self-renewal and myeloid-biased hematopoiesis•Loss of LMNB1 causes acquired Pelger-Huët neutrophil nuclear anomaly•Loss of LMNB1 alters 3D genome organization in HSPCs and neutrophils
Abnormal nuclear morphology is a hallmark of cancerous cells. Here, Reilly et al. demonstrate that deletion of lamin B1, which is common in myeloid malignancies, causes acquired Pelger-Huët nuclear anomaly and links aberrant nuclear morphology with HSC fate determination via 3D genome organization.
Megakaryocyte endoreplication, maturation and platelet production have all been reported to be affected by developmental stage (Elagib, Exp Hematol 2018). Additionally, we have recently found that ...P-selectin expression and function are altered in fetal and neonatal murine platelets (Stolla, Blood Adv 2019), suggesting that both megakaryopoiesis and platelet function are developmentally regulated. The developmental regulation of megakaryopoiesis can have important implications regarding platelet production in premature and full-term neonates, platelet regeneration following cord blood transplantation, and the in vitro generation of platelets from iPS cells (Elagib, Exp Hematol 2018). To further explore differences between fetal and adult megakaryocytes, we analyzed primary murine megakaryocytes in the fetal liver and adult bone marrow, and found that fetal megakaryocytes have lower average ploidy as well as increased cell size and initial accumulation of surface GP1bbeta expression at 2N and 4N stages. These latter findings indicate that cytoplasmic maturation begins at lower ploidy stages in fetal versus adult murine megakaryocytes. Consistent with findings in cultured human cord blood versus adult blood-derived progenitors (Liu, Blood 2015), we found lower ploidy and earlier cytoplasmic maturation in cultures of fetal versus adult megakaryocytes. These findings suggest the existence of a cell intrinsic developmental program of megakaryocyte maturation. Global gene expression studies of cultured megakaryocytes derived from fetal liver versus adult bone marrow progenitors revealed differential expression of Gata1, Cyclin D1, Cyclin D2, and Igf2bp3, supporting their proposed roles in fetal versus adult megakaryopoiesis (Klusmann, Genes Dev 2010; Liu, Blood 2011; Elagib, J Clin Invest 2017). Igf1R transcripts were increased in fetal megakaryocytes consistent with their sensitivity to IGF signaling (Klusmann, Genes Dev 2010). In addition, we found increased expression of Lin28b, a negative regulator of Let7 miRNAs, as well as multiple downstream targets of Let7 miRNAs in fetal liver-derived megakaryocytes. Induction of Lin28b expression in adult mice (Zhu, Cell 2011) led to increased expression of Gata1, Cyclin D1, Igf2bp3 and IGF1R in bone marrow-derived megakaryocytes. Interestingly, adult megakaryocyte average ploidy was decreased and the sensitivity to IGF1R inhibition was increased by Lin28b induction, suggesting that the Lin28b axis regulates key aspects of fetal versus adult megakaryopoiesis.
The largest category of upregulated genes in adult versus fetal megakaryocyte transcriptomes was immune-related genes, including PPBP, a neutrophil chemotactic cytokine, and beta2M, which we have recently shown to mediate platelet-monocyte interactions (Hilt, JCI Insight 2019). Consistent with the transcriptomic data, neonatal platelets express less MHCI on their surface (an indirect indication of less platelet beta2M protein expression) and release less PPBP post-activation when compared to adult platelets. Activated neonatal platelets induce less neutrophil migration and activation when compared to activated adult platelets. In addition, the releasates from activated neonatal versus adult platelets differentially altered the balance of proinflammatory versus proreparative signaling in monocytes. Finally, the induction of Lin28b expression in adult mice resulted in platelets that express less MHCI, release less PPBP, and induce less neutrophil chemotaxis. Taken together, our studies support the hypothesis that megakaryocyte maturation and resulting platelet-immune cell interactions are developmentally regulated, in part, through a Lin28b-mediated program.
Palis:Rubius Therapeutics: Consultancy.
Platelets have numerous well-defined roles during adult hemostasis, however little is known about whether these functions are developmentally regulated. In the adult, P-selectin is constitutively ...expressed in megakaryocytes and localized to the membrane of alpha granules. Activation of platelets results in granule secretion and P-selectin surface exposure allowing for recruitment of leukocytes to sites of vascular injury and the formation of leukocyte-platelet aggregates. Here, we examined the expression and function of P-selectin in platelets during murine embryogenesis. Platelets were isolated from timed pregnant outbred-ICR mice at embryonic day 12.5 (E12.5), E15.5, postnatal day 1 (PN1), PN7, PN21, and were assayed for thrombin induced integrin activation and P-selectin translocation. While no significant differences were seen in the ability of embryonic platelets to be activated by thrombin, P-selectin surface expression after thrombin stimulation was significantly reduced in embryonic compared to adult platelets (E15.5 p=.0006, E12.5 p=.0004, PN1 p=.01, n=3, unpaired t-test). Surprisingly, P-selectin expression began to appear on the surface of activated platelets only after birth, approaching 50% and 75% of maternal expression at PN7 and PN21, respectively. No significant difference was seen in VEGF secretion after thrombin activation of E12.5, E15.5 and adult platelets (n=5), suggesting that the lack of P-selectin surface expression was not due to a loss of alpha granule secretion. To further characterize expression of P-selectin, real-time qPCR was performed on mRNA harvested from platelets at E12.5, E15.5, PN1 and adult stages. P-selectin transcript levels were significantly lower in embryonic and PN1 platelets, suggesting that P-selectin expression is transcriptionally regulated. Consistent with this concept, Imagestream analysis indicates that P-selectin protein is absent in megakaryocytes derived from the fetal liver. P-selectin expression on platelets is an important mediator of platelet-leukocyte interactions; therefore, we assayed the ability of platelets to bind to leukocytes in embryonic whole blood. Preliminary data suggest that embryonic platelets do not form leukocyte-platelet aggregates upon ex vivo stimulation with AYPGKF, a Par4 agonist. Taken together, our data indicate that P-selectin expression is developmentally regulated in primary mouse platelets. The absence of P-selectin expression in embryonic platelets correlates with an impaired ability to interact with leukocytes.
No relevant conflicts of interest to declare.
Autophagy is a self-degradation pathway that is essential for erythropoiesis. During erythroid differentiation, autophagy facilitates the degradation of macromolecules and the programmed clearance of ...mitochondria. Impaired mitochondrial clearance results in anemia and alters the lifespan of red blood cells in vivo. While several essential autophagy genes contribute to autophagy in erythropoiesis, little is known about erythroid-specific mediators of this pathway. Genetic analysis of primary human erythroid and nonerythroid cells revealed the selective upregulation of the core autophagy gene ATG4A in maturing human erythroid cells. Because the function of ATG4A in erythropoiesis is unknown, we evaluated its role using an ex vivo model of human erythropoiesis. Depletion of ATG4A in primary human hematopoietic stem and progenitor cells selectively impaired erythroid but not myeloid lineage differentiation, resulting in reduced red cell production, delayed terminal differentiation, and impaired enucleation. Loss of ATG4A impaired autophagy and mitochondrial clearance, giving rise to reticulocytes with retained mitochondria and autophagic vesicles. In summary, our study identifies ATG4A as a cell type–specific regulator of autophagy in erythroid development.
•ATG4A regulates terminal erythroid maturation.•ATG4A promotes autophagosome maturation and mitochondrial clearance.
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Platelets are essential for hemostasis; however, several studies have identified age-dependent differences in platelet function. To better understand the origins of fetal platelet function, we have ...evaluated the contribution of the fetal-specific RNA binding protein Lin28b in the megakaryocyte/platelet lineage. Because activated fetal platelets have very low levels of P-selectin, we hypothesized that the expression of platelet P-selectin is part of a fetal-specific hematopoietic program conferred by Lin28b. Using the mouse as a model, we find that activated fetal platelets have low levels of P-selectin and do not readily associate with granulocytes in vitro and in vivo, relative to adult controls. Transcriptional analysis revealed high levels of Lin28b and Hmga2 in fetal, but not adult, megakaryocytes. Overexpression of LIN28B in adult mice significantly reduces the expression of P-selectin in platelets, and therefore identifies Lin28b as a negative regulator of P-selectin expression. Transplantation of fetal hematopoietic progenitors resulted in the production of platelets with low levels of P-selectin, suggesting that the developmental regulation of P-selectin is intrinsic and independent of differences between fetal and adult microenvironments. Last, we observe that the upregulation of P-selectin expression occurs postnatally, and the temporal kinetics of this upregulation are recapitulated by transplantation of fetal hematopoietic stem and progenitor cells into adult recipients. Taken together, these studies identify Lin28b as a new intrinsic regulator of fetal platelet function.
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