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.

DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene DNAJB1-PRKACA (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long noncoding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and in vivo disease models. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, which indicates mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease.

Rare loss of function variants in the hepatokine gene INHBE protect from abdominal obesity.

Identifying genetic variants associated with lower waist-to-hip ratio can reveal new therapeutic targets for abdominal obesity. We use exome sequences from 362,679 individuals to identify genes associated with waist-to-hip ratio adjusted for BMI (WHRadjBMI), a surrogate for abdominal fat that is causally linked to type 2 diabetes and coronary heart disease. Predicted loss of function (pLOF) variants in INHBE associate with lower WHRadjBMI and this association replicates in data from AMP-T2D-GENES. INHBE encodes a secreted protein, the hepatokine activin E. In vitro characterization of the most common INHBE pLOF variant in our study, indicates an in-frame deletion resulting in a 90% reduction in secreted protein levels. We detect associations with lower WHRadjBMI for variants in ACVR1C, encoding an activin receptor, further highlighting the involvement of activins in regulating fat distribution. These findings highlight activin E as a potential therapeutic target for abdominal obesity, a phenotype linked to cardiometabolic disease.

Germline ETV6 mutation promotes inflammation and disrupts lymphoid development of early hematopoietic progenitors.

Germline mutations in ETV6 are associated with a syndrome of thrombocytopenia and leukemia predisposition, and ETV6 is among the most commonly mutated genes in leukemias, especially childhood B-cell acute lymphoblastic leukemia. However, the mechanisms underlying disease caused by ETV6 dysfunction are poorly understood. To address these gaps in knowledge, using CRISPR/Cas9, we developed a mouse model of the most common recurrent, disease-causing germline mutation in ETV6. We found defects in hematopoiesis related primarily to abnormalities of the multipotent progenitor population 4 (MPP4) subset of hematopoietic progenitor cells and evidence of sterile inflammation. Expression of ETV6 in Ba/F3 cells altered the expression of several cytokines, some of which were also detected at higher levels in the bone marrow of the mice with Etv6 mutation. Among these, interleukin-18 and interleukin-13 abrogated B-cell development of sorted MPP4 cells, but not common lymphoid progenitors, suggesting that inflammation contributes to abnormal hematopoiesis by impairing lymphoid development. These data, along with those from humans, support a model in which ETV6 dysfunction promotes inflammation, which adversely affects thrombopoiesis and promotes leukemogenesis.

The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation.

Tyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate lonesome kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet α-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology but have significant changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets exhibit a significant decrease in several tyrosine phosphobands. Results of functional testing of VLK-deficient platelets show decreased protease-activated receptor 4-mediated and collagen-mediated platelet aggregation but normal responses to adenosine 5'-diphosphate. Dense granule and α-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased protease-activated receptor 4-mediated Akt (S473) and Erk1/2 (T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets display strongly reduced platelet accumulation and fibrin formation after laser-induced injury of cremaster arterioles compared with control mice but with normal bleeding times. These studies show that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.

Megakaryocytes package contents into separate α-granules that are differentially distributed in platelets.

In addition to their primary roles in hemostasis and thrombosis, platelets participate in many other physiological and pathological processes, including, but not limited to inflammation, wound healing, tumor metastasis, and angiogenesis. Among their most interesting properties is the large number of bioactive proteins stored in their α-granules, the major storage granule of platelets. We previously showed that platelets differentially package pro- and antiangiogenic proteins in distinct α-granules that undergo differential release upon platelet activation. Nevertheless, how megakaryocytes achieve differential packaging is not fully understood. In this study, we use a mouse megakaryocyte culture system and endocytosis assay to establish when and where differential packaging occurs during platelet production. Live cell microscopy of primary mouse megakaryocytes incubated with fluorescently conjugated fibrinogen and endostatin showed differential endocytosis and packaging of the labeled proteins into distinct α-granule subpopulations. Super-resolution microscopy of mouse proplatelets and human whole-blood platelet α-granules simultaneously probed for 2 different membrane proteins (VAMP-3 and VAMP-8), and multiple granular content proteins (bFGF, ENDO, TSP, VEGF) confirmed differential packaging of protein contents into α-granules. These data suggest that megakaryocytes differentially sort and package α-granule contents, which are preserved as α-granule subpopulations during proplatelet extension and platelet production.

New Insights Into the Differentiation of Megakaryocytes From Hematopoietic Progenitors.

Megakaryocytes are hematopoietic cells, which are responsible for the production of blood platelets. The traditional view of megakaryopoiesis describes the cellular journey from hematopoietic stem cells, through a hierarchical series of progenitor cells, ultimately to a mature megakaryocyte. Once mature, the megakaryocyte then undergoes a terminal maturation process involving multiple rounds of endomitosis and cytoplasmic restructuring to allow platelet formation. However, recent studies have begun to redefine this hierarchy and shed new light on alternative routes by which hematopoietic stem cells are differentiated into megakaryocytes. In particular, the origin of megakaryocytes, including the existence and hierarchy of megakaryocyte progenitors, has been redefined, as new studies are suggesting that hematopoietic stem cells originate as megakaryocyte-primed and can bypass traditional lineage checkpoints. Overall, it is becoming evident that megakaryopoiesis does not only occur as a stepwise process, but is dynamic and adaptive to biological needs. In this review, we will reexamine the canonical dogmas of megakaryopoiesis and provide an updated framework for interpreting the roles of traditional pathways in the context of new megakaryocyte biology. Visual Overview- An online visual overview is available for this article.

Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia.

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell-precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ets variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.

A novel mutation in PLP1 causes severe hereditary spastic paraplegia type 2.

Hereditary spastic paraplegia (HSP) type 2 is a proteolipid protein (PLP1)-related genetic disorder that is characterized by dysmyelination of the central nervous system resulting primarily in limb spasticity, cognitive impairment, nystagmus, and spastic urinary bladder of varying severity. Previously reported PLP1 mutations include duplications, point mutations, or whole gene deletions with a continuum of phenotypes ranging from severe Pelizaeus-Merzbacher disease (PMD) to uncomplicated HSP type 2. In this manuscript we report a novel PLP1 missense mutation (c.88G>C) in a family from Argentina. This mutation is in a highly conserved transmembrane domain of PLP1 and the mutant protein was found to be retained in the endoplasmic reticulum when expressed in vitro. Due to the variable expressivity that characterizes these disorders our report contributes to the knowledge of genotype-phenotype correlations of PLP1-related disorders.

Exercise performance in thalassemia major: correlation with cardiac iron burden.

Exercise performance is decreased in patients with Thalassemia major (TM), but the relative impact of anemia and iron overload on exercise capacity is unknown. We assessed the cardiopulmonary function of 71, well-transfused TM patients via graded treadmill exercise stress test. All patients underwent MRI of the heart, pancreas, and liver and diagnostic phlebotomy. Patients ranged in age from 13 to 46 years of age. Fifteen patients were excluded from analysis due to submaximal effort. Mean Vo2 max was 83.0% of predicted and was limited by abnormal cardiovascular mechanisms, consisting of a decreased O2 pulse (86.6% of predicted) in men and decreased maximum heart rate (HR) response (85% of predicted) in women. Patients with hemoglobin less than 12 g/dL had lower O2 pulse and Vo2 max, regardless of sex. Cardiac iron was negatively associated with maximum HR response and Vo2 max (r2 = 0.10 and 0.08, respectively, P < 0.05). Vo2 max was correlated with cardiac R2*, hs-CRP, sex and hemoglobin in decreasing strength of association. In thalassemia, exercise performance is limited by impaired stroke-volume reserve in men and blunted HR response in women. Iron toxicity may be mediated through vascular inflammation and direct modulation of HR response to exercise.

Pituitary iron and volume predict hypogonadism in transfusional iron overload.

Hypogonadism is the most common morbidity in patients with transfusion-dependent anemias such as thalassemia major. We used magnetic resonance imaging (MRI) to measure pituitary R2 (iron) and volume to determine at what age these patients develop pituitary iron overload and volume loss. We recruited 56 patients (47 with thalassemia major, five with chronically transfused thalassemia intermedia and four with Blackfan-Diamond syndrome) to have pituitary MRIs to measure pituitary R2 and volume. Hypogonadism was defined clinically based on the timing of secondary sexual characteristics or the need for sex hormone replacement therapy. Patients with transfusional iron overload begin to develop pituitary iron overload in the first decade of life; however, clinically significant volume loss was not observed until the second decade of life. Severe pituitary iron deposition (Z > 5) and volume loss (Z < -2.5) were independently predictive of hypogonadism. Pituitary R2 correlated significantly with serum ferritin as well as liver, pancreatic, and cardiac iron deposition by MRI. Log pancreas R2* was the best single predictor for pituitary iron, with an area under the receiving operator characteristic curve of 0.88, but log cardiac R2* and ferritin were retained on multivariate regression with a combined r(2) of 0.71. Pituitary iron overload and volume loss were independently predictive of hypogonadism. Many patients with moderate-to-severe pituitary iron overload retained normal gland volume and function, representing a potential therapeutic window. The subset of hypogonadal patients having preserved gland volumes may also explain improvements in pituitary function observed following intensive chelation therapy.

Pancreatic iron and glucose dysregulation in thalassemia major.

Pancreatic iron overload and diabetes mellitus (DM) are common in thalassemia major patients. However, the relationship between iron stores and glucose disturbances is not well defined. We used a frequently sampled oral glucose tolerance test (OGTT), coupled with mathematical modeling, and magnetic resonance imaging (MRI) to examine the impact of pancreatic, cardiac, and hepatic iron overload on glucose regulation in 59 patients with thalassemia major. According to OGTT results, 11 patients had DM, 12 had impaired glucose tolerance (IGT), 8 had isolated impaired fasting glucose (IFG), and 28 patients had normal glucose tolerance (NGT). Patients with DM had significantly impaired insulin sensitivity and insulin release. Insulin resistance was most strongly associated with markers of inflammation and somatic iron overload, while disposition index (DI) (a measure of beta cell function) was most strongly correlated with pancreas R2*. Patients with DM and IGT had significantly worse DI than those with NGT or IFG, suggesting significant beta cell toxicity. One-third of patients having elevated pancreas R2* had normal glucose regulation (preclinical iron burden), but these patients were younger and had lower hepatic iron burdens. Our study indicates that pancreatic iron is the strongest predictor of beta cell toxicity, but total body iron burden, age, and body habitus also influence glucose regulation. We also demonstrate that MRI and fasting glucose/insulin are complementary screening tools, reducing the need for oral glucose tolerance testing, and identify high-risk patients before irreversible pancreatic damage.

Electrocardiographic consequences of cardiac iron overload in thalassemia major.

Iron cardiomyopathy is a leading cause of death in transfusion-dependent thalassemia major (TM) patients and MRI (T2*) can recognize preclinical cardiac iron overload, but, is unavailable to many centers. We evaluated the ability of 12-lead electrocardiography to predict cardiac iron loading in TM. 12-lead electrocardiogram and cardiac T2* measurements were performed prospectively, with a detectable cardiac iron cutoff of T2*less than 20 ms. Patients with and without cardiac iron were compared using two-sample statistics and against population norms using age and gender-matched Z-scores. 45/78 patients had detectable cardiac iron. Patients having cardiac iron were older and more likely female but had comparable liver iron burdens and serum ferritin. Increased heart rate (HR) and prolonged corrected QT interval (QT(c)) were present, regardless of cardiac iron status. Repolarization abnormalities were the strongest predictors of cardiac iron, including QT/QT(c) prolongation, left shift of T-wave axis, and interpretation of ST/T-wave morphology. Recursive partitioning of the data for females using T-axis and HR and for males using QT, HR, and T-axis produced algorithms with AUROC's of 88.3 and 87.1, respectively. Bradycardia and repolarization abnormalities on 12-lead electrocardiography were the most specific markers for cardiac iron in thalassemia major. Changes in these variables may be helpful to stratify cardiac risk when cardiac MRI is unavailable. However, diagnostic algorithms need to be vetted on larger and more diverse patient populations and longitudinal studies are necessary to determine reversibility of the observed abnormalities.

Pulmonary function in thalassaemia major and its correlation with body iron stores.

This study compared pulmonary function tests (PFTs) with cardiac, pancreatic and liver iron in 76 thalassemia major (TM) patients. Restrictive lung disease was observed in 16%, hyperinflation in 32% and abnormal diffusing capacity in 3%. While no patients met Global Initiative for Chronic Lung Disease criteria for airways obstruction, there were indicators of small airways disease and air trapping. PFTs did not correlate with somatic iron burden, blood counts or haemolysis. Restrictive lung disease was associated with inflammation. We conclude that TM patients have pulmonary abnormalities consistent with small airways obstruction. Restrictive disease and impaired diffusion are less common.

Pancreatic iron loading in chronically transfused sickle cell disease is lower than in thalassaemia major.

Iron endocrinopathy and cardiomyopathy are common in chronically transfused thalassaemia major patients, but relatively rare in chronically transfused patients with sickle cell disease. Since magnetic resonance imaging can demonstrate preclinical organ iron deposition, we hypothesized that pancreas and cardiac R2* would likewise be lower in sickle cell disease patients than thalassaemia major patients having comparable transfusional burdens. To test this hypothesis, we examined pancreatic and cardiac iron in a convenience sample of 100 chronically-transfused sickle cell disease and 131 thalassaemia major patients. Cardiac R2* (30 ± 9·2 vs. 73 ± 53 Hz, P < 0·0001) and pancreatic R2* (52 ± 62 vs. 253 ± 224 Hz, P < 0·0001) were significantly lower in sickle cell disease than thalassaemia major. Liver iron concentration was similar in both groups (14·9 ± 9·8 vs. 12·3 ± 8·4 mg/g dry weight, P = 0·101). The observed disparity in pancreatic and cardiac iron loading between sickle cell disease and thalassaemia major patients mirrors prior observations of organ toxicity in these patients. Greater cumulative transfusional iron exposure in thalassaemia major patients partially explains these observations but our data also suggest innate differences in labile iron handling between the two diseases.

Pancreatic iron loading predicts cardiac iron loading in thalassemia major.

Diabetes mellitus and cardiomyopathy are common in chronically transfused thalassemia major patients, occurring in the second and third decades of life. We postulated that pancreatic iron deposition would precede cardiac iron loading, representing an environment favorable for extrahepatic iron deposition. To test this hypothesis, we examined pancreatic and cardiac iron in 131 thalassemia major patients over a 4-year period. Cardiac iron (R2* > 50 Hz) was detected in 37.7% of patients and pancreatic iron (R2* > 28 Hz) in 80.4% of patients. Pancreatic and cardiac R2* were correlated (r(2) = 0.52), with significant pancreatic iron occurring nearly a decade earlier than cardiac iron. A pancreatic R2* less than 100 Hz was a powerful negative predictor of cardiac iron, and pancreatic R2* more than 100 Hz had a positive predictive value of more than 60%. In serial analysis, changes in cardiac iron were correlated with changes in pancreatic iron (r(2) = 0.33, P < .001), but not liver iron (r(2) = 0.025, P = .25). As a result, pancreatic R2* measurements offer important early recognition of physiologic conditions suitable for future cardiac iron deposition and complementary information to liver and cardiac iron during chelation therapy. Staging abdominal and cardiac magnetic resonance imaging examinations could significantly reduce costs, magnet time, and need for sedation in young patients.

Patterns of hepatic iron distribution in patients with chronically transfused thalassemia and sickle cell disease.

Patients with sickle cell disease (SCD) appear to be at lower risk of endocrinopathies and cardiac dysfunction than those with thalassemia major (TM). Circulating redox active iron is lower in these patients, possibly due to increased systemic inflammation and circulating cytokines. Hepcidin synthesis is upregulated during chronic inflammation, reducing intestinal iron absorption and promoting retention of iron in the reticuloendothelial cells. Hence, we hypothesized that livers of patients with SCD would exhibit greater iron deposition in sinusoidal spaces relative to hepatocytes and less in portal tracts when compared to patients with TM. To test this hypothesis, iron scoring analysis was performed on 70 clinically indicated liver biopsy specimens from children and young adults with the two syndromes. Sinusoidal scores were lower in around 1 of 4 patients with TM but the relative iron loading in hepatocytes, and portal tracts was identical in both diseases. Sinusoidal iron burdens saturated at low hepatic iron concentration (HIC) while hepatocyte and portal iron depots increased proportionally to HIC. Liver fibrosis was increased in patients with TM regardless of their chronic hepatitis status. Overall, liver iron distribution was relatively insensitive to differences in disease type and to the presence or absence of hepatitis.