Hematology
Abstract
Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell–derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid–phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.
Authors
David A. Slatter, Charles L. Percy, Keith Allen-Redpath, Joshua M. Gajsiewicz, Nick J. Brooks, Aled Clayton, Victoria J. Tyrrell, Marcela Rosas, Sarah N. Lauder, Andrew Watson, Maria Dul, Yoel Garcia-Diaz, Maceler Aldrovandi, Meike Heurich, Judith Hall, James H. Morrissey, Sebastien Lacroix-Desmazes, Sandrine Delignat, P. Vincent Jenkins, Peter W. Collins, Valerie B. O’Donnell
Abstract
BACKGROUND. HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome is a severe variant of hypertensive disorders of pregnancy affecting approximately 1% of all pregnancies, and has significant maternal and fetal morbidity. Previously, we showed that upregulation of the alternative pathway of complement (APC) plays a role in HELLP syndrome. We hypothesize that HELLP syndrome follows a 2-hit disease model similar to atypical hemolytic uremic syndrome (aHUS), requiring both genetic susceptibility and an environmental risk factor. Our objective was to perform a comparative analysis of the frequency of APC activation and germline mutations in affected women and to create a predictive model for identifying HELLP syndrome. METHODS. Pregnant women with HELLP syndrome, and healthy controls after 23 weeks of gestation were recruited, along with aHUS and thrombotic thrombocytopenic purpura participants. We performed a functional assay, the mHam, and targeted genetic sequencing in all groups. RESULTS. Significantly more participants with rare germline mutations in APC genes were present in the HELLP cohort compared with controls (46% versus 8%, P = 0.01). In addition, significantly more HELLP participants were positive for the mHam when compared with controls (62% versus 16%, P = 0.009). Testing positive for both a germline mutation and the mHam was highly predictive for the diagnosis of HELLP syndrome. CONCLUSION. HELLP syndrome is characterized by both activation of the APC and frequent germline mutations in APC genes. Similar to aHUS, treatment via complement inhibition to mitigate maternal and fetal morbidity and mortality may be possible. FUNDING. National Heart Lung and Blood Institute grants T32HL007525 and R01HL133113.
Authors
Arthur J. Vaught, Evan M. Braunstein, Jagar Jasem, Xuan Yuan, Igor Makhlin, Solange Eloundou, Andrea C. Baines, Samuel A. Merrill, Shruti Chaturvedi, Karin Blakemore, C. John Sperati, Robert A. Brodsky
Abstract
Allogeneic hematopoietic cell transplantation (alloHCT) is a potentially curative treatment for myelodysplastic syndromes (MDS), but patients who relapse after transplant have poor outcomes. In order to understand the contribution of tumor clonal evolution to disease progression,we applied exome and error-corrected targeted sequencing coupled with copy number analysis to comprehensively define changes in the clonal architecture of MDS in response to therapy using 51 serially acquired tumor samples from 9 patients who progressed after an alloHCT. We show that small subclones before alloHCT can drive progression after alloHCT. Notably, at least one subclone expanded or emerged at progression in all patients. Newly acquired structural variants (SVs) were present in an emergent/expanding subclone in 8 of 9 patients at progression, implicating the acquisition of SVs as important late subclonal progression events. In addition, pretransplant therapy with azacitidine likely influenced the mutation spectrum and evolution of emergent subclones after alloHCT. Although subclone evolution is common, founding clone mutations are always present at progression and could be detected in the bone marrow as early as 30 and/or 100 days after alloHCT in 6 of 8 (75%) patients, often prior to clinical progression. In conclusion, MDS progression after alloHCT is characterized by subclonal expansion and evolution, which can be influenced by pretransplant therapy.
Authors
Meagan A. Jacoby, Eric J. Duncavage, Gue Su Chang, Christopher A. Miller, Jin Shao, Kevin Elliott, Joshua Robinson, Robert S. Fulton, Catrina C. Fronick, Michelle O’Laughlin, Sharon E. Heath, Iskra Pusic, John S. Welch, Daniel C. Link, John F. DiPersio, Peter Westervelt, Timothy J. Ley, Timothy A. Graubert, Matthew J. Walter
Abstract
Mutations in KIT and TET2 are associated with myeloid malignancies. We show that loss of TET2-induced PI3K activation and -increased proliferation is rescued by targeting the p110α/δ subunits of PI3K. RNA-Seq revealed a hyperactive c-Myc signature in Tet2–/– cells, which is normalized by inhibiting PI3K signaling. Loss of TET2 impairs the maturation of myeloid lineage–derived mast cells by dysregulating the expression of Mitf and Cebpa, which is restored by low-dose ascorbic acid and 5-azacytidine. Utilizing a mouse model in which the loss of TET2 precedes the expression of oncogenic Kit, similar to the human disease, results in the development of a non–mast cell lineage neoplasm (AHNMD), which is responsive to PI3K inhibition. Thus, therapeutic approaches involving hypomethylating agents, ascorbic acid, and isoform-specific PI3K inhibitors are likely to be useful for treating patients with TET2 and KIT mutations.
Authors
Lakshmi Reddy Palam, Raghuveer Singh Mali, Baskar Ramdas, Sridhar Nonavinkere Srivatsan, Valeria Visconte, Ramon V. Tiu, Bart Vanhaesebroeck, Axel Roers, Alexander Gerbaulet, Mingjiang Xu, Sarath Chandra Janga, Clifford M. Takemoto, Sophie Paczesny, Reuben Kapur
Abstract
BACKGROUND. DC-based tumor vaccines have had limited clinical success thus far. SOCS1, a key inhibitor of inflammatory cytokine signaling, is an immune checkpoint regulator that limits DC immunopotency. METHODS. We generated a genetically modified DC (gmDC) vaccine to perform immunotherapy. The adenovirus (Ad-siSSF) delivers two tumor-associated antigens (TAAs), survivin and MUC1; secretory bacterial flagellin for DC maturation; and an RNA interference moiety to suppress SOCS1. A 2-stage phase I trial was performed for patients with relapsed acute leukemia after allogenic hematopoietic stem cell transplantation: in stage 1, we compared the safety and efficacy between gmDC treatment (23 patients) and standard donor lymphocyte infusion (25 patients); in stage 2, we tested the efficacy of the gmDC vaccine for 12 acute myeloid leukemia (AML) patients with early molecular relapse. RESULTS. gmDCs elicited potent TAA-specific CTL responses in vitro, and the immunostimulatory activity of gmDC vaccination was demonstrated in rhesus monkeys. A stage 1 study established that this combinatory gmDC vaccine is safe in acute leukemia patients and yielded improved survival rate. In stage 2, we observed a complete remission rate of 83% in 12 relapsed AML patients. Overall, no grade 3 or grade 4 graft-versus-host disease incidence was detected in any of the 35 patients enrolled. CONCLUSIONS. This study, with combinatory modifications in DCs, demonstrates the safety and efficacy of SOCS1-silenced DCs in treating relapsed acute leukemia. TRIAL REGISTRATION. ClinicalTrials.gov NCT01956630. FUNDING. National Institute of Health (R01CA90427); the Key New Drug Development and Manufacturing Program of the “Twelfth Five-Year Plan” of China (2011ZX09102-001-29); and Clinical Application Research of Beijing (Z131107002213148).
Authors
Danhong Wang, Xue F. Huang, Bangxing Hong, Xiao-Tong Song, Liangding Hu, Min Jiang, Bin Zhang, Hongmei Ning, Yuhang Li, Chen Xu, Xiao Lou, Botao Li, Zhiyong Yu, Jiangwei Hu, Jianlin Chen, Fan Yang, Haiyan Gao, Guoliang Ding, Lianming Liao, Lisa Rollins, Lindsey Jones, Si-Yi Chen, Hu Chen
Abstract
Vertebrates possess 2 proteins with vitamin K oxidoreductase (VKOR) activity: VKORC1, whose vitamin K reduction supports vitamin K–dependent (VKD) protein carboxylation, and VKORC1-like 1 (VKORC1L1), whose function is unknown. VKD proteins include liver-derived coagulation factors, and hemorrhaging and lethality were previously observed in mice lacking either VKORC1 or the γ-glutamyl carboxylase (GGCX) that modifies VKD proteins. Vkorc1–/– mice survived longer (1 week) than Ggcx–/– mice (midembryogenesis or birth), and we assessed whether VKORC1L1 could account for this difference. We found that Vkorc1–/–;Vkorc1l1–/– mice died at birth with severe hemorrhaging, indicating that VKORC1L1 supports carboxylation during the pre- and perinatal periods. Additional studies showed that only VKORC1 sustains hemostasis beyond P7. VKORC1 expression and VKOR activity increased during late embryogenesis and following birth, while VKORC1L1 expression was unchanged. At P0, most (>99%) VKOR activity was due to VKORC1. Prothrombin mRNA, protein, and carboxylation also increased during this period, as did mRNA levels of coagulation factors encoding genes F7, F9, and F10. VKORC1L1 levels in Vkorc1–/– mouse liver may therefore be insufficient for supporting carboxylation beyond day 7. In support of this conclusion, VKORC1L1 overexpression in liver rescued carboxylation and hemostasis in adult Vkorc1–/– mice. These findings establish that VKORC1L1 supports VKD protein carboxylation in vivo.
Authors
Julie Lacombe, Mark A. Rishavy, Kathleen L. Berkner, Mathieu Ferron
Abstract
The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention.
Authors
Wilson I. Gonsalves, Vijay Ramakrishnan, Taro Hitosugi, Toshi Ghosh, Dragan Jevremovic, Tumpa Dutta, Dhananjay Sakrikar, Xuan-Mai Petterson, Linda Wellik, Shaji K. Kumar, K. Sreekumaran Nair
Abstract
Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2–/–) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2–/– cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2–/– cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.
Authors
Lindsay A.M. Rein, James W. Wisler, Jihee Kim, Barbara Theriot, LiYin Huang, Trevor Price, Haeyoon Yang, Minyong Chen, Wei Chen, Dorothy Sipkins, Yuri Fedoriw, Julia K.L. Walker, Richard T. Premont, Robert J. Lefkowitz
Abstract
Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury–induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.
Authors
Frédéric Torossian, Bernadette Guerton, Adrienne Anginot, Kylie A. Alexander, Christophe Desterke, Sabrina Soave, Hsu-Wen Tseng, Nassim Arouche, Laetitia Boutin, Irina Kulina, Marjorie Salga, Beulah Jose, Allison R. Pettit, Denis Clay, Nathalie Rochet, Erica Vlachos, Guillaume Genet, Charlotte Debaud, Philippe Denormandie, François Genet, Natalie A. Sims, Sébastien Banzet, Jean-Pierre Levesque, Jean-Jacques Lataillade, Marie-Caroline Le Bousse-Kerdilès
Abstract
Sickle cell disease (SCD) results from a point mutation in the β-globin gene forming hemoglobin S (HbS), which polymerizes in deoxygenated erythrocytes, triggering recurrent painful vaso-occlusive crises and chronic hemolytic anemia. Reactivation of fetal Hb (HbF) expression ameliorates these symptoms of SCD. Nuclear factor (erythroid derived-2)–like 2 (Nrf2) is a transcription factor that triggers cytoprotective and antioxidant pathways to limit oxidative damage and inflammation and increases HbF synthesis in CD34+ stem cell–derived erythroid progenitors. We investigated the ability of dimethyl fumarate (DMF), a small-molecule Nrf2 agonist, to activate γ-globin transcription and enhance HbF in tissue culture and in murine and primate models. DMF recruited Nrf2 to the γ-globin promoters and the locus control region of the β-globin locus in erythroleukemia cells, elevated HbF in SCD donor–derived erythroid progenitors, and reduced hypoxia-induced sickling. Chronic DMF administration in SCD mice induced HbF and increased Nrf2-dependent genes to detoxify heme and limit inflammation. This improved hematological parameters, reduced plasma-free Hb, and attenuated inflammatory markers. Chronic DMF administration to nonanemic primates increased γ-globin mRNA in BM and HbF protein in rbc. DMF represents a potential therapy for SCD to induce HbF and augment vasoprotection and heme detoxification.
Authors
Sriram Krishnamoorthy, Betty Pace, Dipti Gupta, Sarah Sturtevant, Biaoru Li, Levi Makala, Julia Brittain, Nancy Moore, Benjamin F. Vieira, Timothy Thullen, Ivan Stone, Huo Li, William E. Hobbs, David R. Light
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