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During ontogeny, the transcription factor RUNX1 governs the emergence of definitive hematopoietic cells from specialized endothelial cells called hemogenic endothelium (HE). The ultimate consequence of this endothelial-to-hematopoietic transition is the concomitant activation of the hematopoietic program and downregulation of the endothelial program. However, due to the rare and transient nature of the HE, little is known about the initial role of RUNX1 within this population. We, therefore, developed and implemented a highly sensitive DNA adenine methyltransferase identification-based methodology, including a novel data analysis pipeline, to map early RUNX1 transcriptional targets in HE cells. This novel transcription factor binding site identification protocol should be widely applicable to other low abundance cell types and factors. Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion- and migration-associated genes within the HE. This suggests that RUNX1 orchestrates HE cell positioning and integration prior to the release of hematopoietic cells. Overall, our genome-wide analysis of the RUNX1 binding and transcriptional profile in the HE provides a novel comprehensive resource of target genes that will facilitate the precise dissection of the role of RUNX1 in early blood development.

The membrane-dependent interaction of factor Xa (FXa) with factor Va (FVa) forms prothrombinase and drives thrombin formation essential for hemostasis. Activated platelets are considered to provide the primary biological surface to support prothrombinase function. However, the question of how other cell types may cooperate within the biological milieu to affect hemostatic plug formation remains unaddressed. We used confocal fluorescence microscopy to image the distribution of site-specific fluorescent derivatives of FVa and FXa after laser injury in the mouse cremaster arteriole. These proteins bound to the injury site extend beyond the platelet mass to the surrounding endothelium. Although bound FVa and FXa may have been present on the platelet core at the nidus of the injury, bound proteins were not evident on platelets adherent even a small distance from the injury site. Manipulations to drastically reduce adherent platelets yielded a surprisingly modest decrease in bound FXa and FVa with little impact on fibrin formation. Thus, platelets adherent to the site of vascular injury do not play the presumed preeminent role in supporting prothrombinase assembly and thrombin formation. Rather, the damaged/activated endothelium and possibly other blood cells play an unexpectedly important role in providing a procoagulant membrane surface in vivo.

Atypical hemolytic-uremic syndrome (aHUS) is associated with genetic complement abnormalities/anti–complement factor H antibodies, which paved the way to treatment with eculizumab. We studied 44 aHUS patients and their relatives to (1) test new assays of complement activation, (2) verify whether such abnormality occurs also in unaffected mutation carriers, and (3) search for a tool for eculizumab titration. An abnormal circulating complement profile (low C3, high C5a, or SC5b-9) was found in 47% to 64% of patients, irrespective of disease phase. Acute aHUS serum, but not serum from remission, caused wider C3 and C5b-9 deposits than control serum on unstimulated human microvascular endothelial cells (HMEC-1). In adenosine 5'-diphosphate–activated HMEC-1, also sera from 84% and 100% of patients in remission, and from all unaffected mutation carriers, induced excessive C3 and C5b-9 deposits. At variance, in most patients with C3 glomerulopathies/immune complex-associated membranoproliferative glomerulonephritis, serum-induced endothelial C5b-9 deposits were normal. In 8 eculizumab-treated aHUS patients, C3/SC5b-9 circulating levels did not change posteculizumab, whereas serum-induced endothelial C5b-9 deposits normalized after treatment, paralleled or even preceded remission, and guided drug dosing and timing. These results point to efficient complement inhibition on endothelium for aHUS treatment. C5b-9 endothelial deposits might help monitor eculizumab effectiveness, avoid drug overexposure, and save money considering the extremely high cost of the drug.

This prospective, observational study investigated the utility of Fibtem A5 and Clauss fibrinogen as predictors of progression of postpartum hemorrhage (PPH). A consecutive cohort of 356 women experiencing 1000 to 1500 mL PPH was recruited. Fibtem and fibrinogen were measured and subsequent transfusions, invasive procedures, and bleed volume recorded. Women progressing to 8 U blood products (red blood cells [RBCs] + fresh frozen plasma [FFP] + platelets) had a median (interquartile range) fibrinogen and Fibtem A5 of 2.1 (1.8-3.4) g/L and 12 (7-17) mm, respectively, compared with 3.9 (3.2-4.5) and 19 (17-23) for those not progressing. On multivariate analysis, Fibtem was an independent predictor for progression to bleeds >2500 mL (95% confidence interval [CI], 0.85 [0.77-0.95]). Receiver operating characteristic area under the curve (95% CI) for progression to RBC transfusion was 0.67 (0.60-0.74) for fibrinogen and 0.61 (0.54-0.68) for Fibtem, and progression to >2500 mL was 0.71 (0.61-0.81) and 0.75 (0.66-0.85) for fibrinogen and Fibtem, respectively. Fibtem A5 <10 mm was associated with more prolonged bleeds (median [95% CI], 127 [44-210] compared with 65 [59-71] minutes; P = .018) and longer stay in the high-dependency unit (23.5 [18.4-28.5] compared with 10.8 [9.7-11.8] hours). Fibtem is a rapidly available early biomarker for progression of PPH.

Overexpression of HOXB4 in hematopoietic stem cells (HSCs) leads to increased self-renewal without causing hematopoietic malignancies in transplanted mice. The molecular basis of HOXB4-mediated benign HSC expansion in vivo is not well understood. To gain further insight into the molecular events underlying HOXB4-mediated HSC expansion, we analyzed gene expression changes at multiple time points in LinSca1+c-kit+ cells from mice transplanted with bone marrow cells transduced with a MSCV-HOXB4-ires-YFP vector. A distinct HOXB4 transcriptional program was reproducibly induced and stabilized by 12 weeks after transplant. Dynamic expression changes were observed in genes critical for HSC self-renewal as well as in genes involved in myeloid and B-cell differentiation. Prdm16, a transcription factor associated with human acute myeloid leukemia, was markedly repressed by HOXB4 but upregulated by HOXA9 and HOXA10, suggesting that Prdm16 downregulation was involved in preventing leukemia in HOXB4 transplanted mice. Functional evidence to support this mechanism was obtained by enforcing coexpression of sPrdm16 and HOXB4, which led to enhanced self-renewal, myeloid expansion, and leukemia. Altogether, these studies define the transcriptional pathways involved in HOXB4 HSC expansion in vivo and identify repression of Prdm16 transcription as a mechanism by which expanding HSCs avoid leukemic transformation.

Acute inflammation is traditionally characterized by polymorphonuclear leukocytes (PMN) influx followed by phagocytosing macrophage (Ms) that clear injurious stimuli leading to resolution and tissue homeostasis. However, using the peritoneal cavity, we found that although innate immune-mediated responses to low-dose zymosan or bacteria resolve within days, these stimuli, but not hyperinflammatory stimuli, trigger a previously overlooked second wave of leukocyte influx into tissues that persists for weeks. These cells comprise distinct populations of tissue-resident Ms (resMs), Ly6chi monocyte-derived Ms (moMs), monocyte-derived dendritic cells (moDCs), and myeloid-derived suppressor cells (MDSCs). Postresolution mononuclear phagocytes were observed alongside lymph node expansion and increased numbers of blood and peritoneal memory T and B lymphocytes. The resMs and moMs triggered FoxP3 expression within CD4 cells, whereas moDCs drive T-cell proliferation. The resMs preferentially clear apoptotic PMNs and migrate to lymph nodes to bring about their contraction in an inducible nitric oxide synthase-dependent manner. Finally, moMs remain in tissues for months postresolution, alongside altered numbers of T cells collectively dictating the magnitude of subsequent acute inflammatory reactions. These data challenge the prevailing idea that resolution leads back to homeostasis and asserts that resolution acts as a bridge between innate and adaptive immunity, as well as tissue reprogramming.

Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The role of altered sialylation in multiple myeloma (MM) cell trafficking has not been previously investigated. In the present study we identified high expression of β-galactoside α-2,3-sialyltransferase, ST3GAL6, in MM cell lines and patients. This gene plays a key role in selectin ligand synthesis in humans through the generation of functional sialyl Lewis X. In MRC IX patients, high expression of this gene is associated with inferior overall survival. In this study we demonstrate that knockdown of ST3GAL6 results in a significant reduction in levels of α-2,3–linked sialic acid on the surface of MM cells with an associated significant reduction in adhesion to MM bone marrow stromal cells and fibronectin along with reduced transendothelial migration in vitro. In support of our in vitro findings, we demonstrate significantly reduced homing and engraftment of ST3GAL6 knockdown MM cells to the bone marrow niche in vivo, along with decreased tumor burden and prolonged survival. This study points to the importance of altered glycosylation, particularly sialylation, in MM cell adhesion and migration.

Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML.

Somatic cohesin mutations have been reported in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). To account for the morphologic and cytogenetic diversity of these neoplasms, a well-annotated cohort of 1060 patients with myeloid malignancies including MDS (n = 386), myeloproliferative neoplasms (MPNs) (n = 55), MDS/MPNs (n = 169), and AML (n = 450) were analyzed for cohesin gene mutational status, gene expression, and therapeutic and survival outcomes. Somatic cohesin defects were detected in 12% of patients with myeloid malignancies, whereas low expression of these genes was present in an additional 15% of patients. Mutations of cohesin genes were mutually exclusive and mostly resulted in predicted loss of function. Patients with low cohesin gene expression showed similar expression signatures as those with somatic cohesin mutations. Cross-sectional deep-sequencing analysis for clonal hierarchy demonstrated STAG2, SMC3, and RAD21 mutations to be ancestral in 18%, 18%, and 47% of cases, respectively, and each expanded to clonal dominance concordant with disease transformation. Cohesin mutations were significantly associated with RUNX1, Ras-family oncogenes, and BCOR and ASXL1 mutations and were most prevalent in high-risk MDS and secondary AML. Cohesin defects were associated with poor overall survival (27.2 vs 40 months; P = .023), especially in STAG2 mutant MDS patients surviving >12 months (median survival 35 vs 50 months; P = .017).

Rare missense mutations in the von Willebrand factor (VWF) A3 domain that disrupt collagen binding have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been limited. Here, we have developed mouse models of collagen-binding mutants and analyzed the function of the A3 domain using comprehensive in vitro and in vivo approaches. Five loss-of-function (p.S1731T, p.W1745C, p.S1783A, p.H1786D, A3 deletion) and 1 gain-of-function (p.L1757A) variants were generated in the mouse VWF complementary DNA. The results of these various assays were consistent, although the magnitude of the effects were different: the gain-of-function (p.L1757A) variant showed consistent enhanced collagen binding whereas the loss-of-function mutants showed variable degrees of functional deficit. We further analyzed the impact of direct platelet-collagen binding by blocking glycoprotein VI (GPVI) and integrin α2β1 in our ferric chloride murine thrombosis model. The inhibition of GPVI demonstrated a comparable functional defect in thrombosis formation to the VWF–/– mice whereas α2β1 inhibition demonstrated a milder bleeding phenotype. Furthermore, a delayed and markedly reduced thrombogenic response was still evident in VWF–/–, GPVI, and α2β1 blocked animals, suggesting that alternative primary hemostatic mechanisms can partially rescue the bleeding phenotype associated with these defects.

Hemostatic thrombi develop a characteristic architecture in which a core of highly activated platelets is covered by a shell of less-activated platelets. Here we have used a systems biology approach to examine the interrelationship of this architecture with transport rates and agonist distribution in the gaps between platelets. Studies were performed in mice using probes for platelet accumulation, packing density, and activation plus recently developed transport and thrombin activity probes. The results show that intrathrombus transport within the core is much slower than within the shell. The region of slowest transport coincides with the region of greatest packing density and thrombin activity, and appears prior to full platelet activation. Deleting the contact-dependent signaling molecule, Sema4D, delays platelet activation, but not the emergence of the low transport region. Collectively, these results suggest a timeline in which initial platelet accumulation and the narrowing gaps between platelets create a region of reduced transport that facilitates local thrombin accumulation and greater platelet activation, whereas faster transport rates within the shell help to limit thrombin accumulation and growth of the core. Thus, from a systems perspective, platelet accumulation produces an altered microenvironment that shapes thrombus architecture, which in turn affects agonist distribution and subsequent thrombus growth.

Hemostatic thrombi formed after a penetrating injury have a heterogeneous architecture in which a core of highly activated, densely packed platelets is covered by a shell of less-activated, loosely packed platelets. In the first manuscript in this series, we show that regional differences in intrathrombus protein transport rates emerge early in the hemostatic response and are preserved as the thrombus develops. Here, we use a theoretical approach to investigate this process and its impact on agonist distribution. The results suggest that hindered diffusion, rather than convection, is the dominant mechanism responsible for molecular movement within the thrombus. The analysis also suggests that the thrombus core, as compared with the shell, provides an environment for retaining soluble agonists such as thrombin, affecting the extent of platelet activation by establishing agonist-specific concentration gradients radiating from the site of injury. This analysis accounts for the observed weaker activation and relative instability of platelets in the shell and predicts that a failure to form a tightly packed thrombus core will limit thrombin accumulation, a prediction tested by analysis of data from mice with a defect in clot retraction.

Hemostatic thrombi formed after a penetrating injury have a distinctive structure in which a core of highly activated, closely packed platelets is covered by a shell of less-activated, loosely packed platelets. We have shown that differences in intrathrombus molecular transport emerge in parallel with regional differences in platelet packing density and predicted that these differences affect thrombus growth and stability. Here we test that prediction in a mouse vascular injury model. The studies use a novel method for measuring thrombus contraction in vivo and a previously characterized mouse line with a defect in integrin αIIbβ3 outside-in signaling that affects clot retraction ex vivo. The results show that the mutant mice have a defect in thrombus consolidation following vascular injury, resulting in an increase in intrathrombus transport rates and, as predicted by computational modeling, a decrease in thrombin activity and platelet activation in the thrombus core. Collectively, these data (1) demonstrate that in addition to the activation state of individual platelets, the physical properties of the accumulated mass of adherent platelets is critical in determining intrathrombus agonist distribution and platelet activation and (2) define a novel role for integrin signaling in the regulation of intrathrombus transport rates and localization of thrombin activity.

Acute graft-versus-host disease (GVHD) is induced by alloreactivity of donor T cells toward host antigens presented on antigen-presenting cells (APCs). Apoptotic cells are capable of inducing tolerance by altering APC maturation. Apoptosis can be induced by extracorporeal photopheresis (ECP). We demonstrate that the use of ECP as a prophylaxis prior to conditioning significantly improves survival (P < .0001) after bone marrow transplantation (BMT) by inhibiting the initiation phase of acute GVHD in a murine BMT model. ECP-treated autologous splenocytes resulted in immune tolerance in the host, including reduced dendritic cell activation with decreased nuclear factor-B engagement, increased regulatory T-cell (Treg) numbers with enhanced expression of cytolytic T lymphocyte-associated antigen 4, potentiating their suppressive function. The protective effect required host production of interleukin-10 and host Tregs. Conventional T cells that entered this tolerant environment experienced reduced proliferation, as well as a reduction of tissue homing and expression of activation markers. The induction of this tolerant state by ECP was obviated by cotreatment with lipopolysaccharide, suggesting that the inflammatory state of the recipient prior to treatment would play a role in potential clinical translation. The use of prophylactic ECP may provide an alternative and safe method for immunosuppression in the bone marrow transplant setting.