We retrospectively studied 232 patients with cold agglutinin disease (CAD) at 24 centers in 5 countries. In Norway and a northern region of Italy, the study was close to being population-based. For the first time, we demonstrate fourfold differences between cold and warmer climates regarding prevalence (20 vs 5 cases/million) and incidence (1.9 vs 0.48 cases/million per year). Mean baseline hemoglobin level was 9.3 g/dL, but 27% had hemoglobin <8 g/dL. Identification of typical features of CAD-associated lymphoproliferative disorder in the bone marrow was greatly increased by centralized biopsy assessment. CAD seems to be associated with a slightly increased risk of venous thrombosis. This work includes a follow-up study of therapies, focusing on the long-term outcomes of the rituximab plus bendamustine and rituximab plus fludarabine regimens. Rituximab plus bendamustine therapy resulted in responses in 35 (78%) of 45 patients; 24 (53%) achieved complete response. Interestingly, these rates were still higher than observed in the original (2017) prospective trial, and we also found a shift toward deeper responses with time. This is explained by the prolonged time to response seen in many patients, probably related to long-lived plasma cells. In patients responding to rituximab-bendamustine, median response duration was not reached after 88 months, and estimated 5-year sustained remission was 77%. The regimen appeared safe regarding late-occurring malignancies. Rituximab plus fludarabine therapy seems to carry a higher risk of long-term adverse effects.

Although the serum-abundant metal-binding protein transferrin (encoded by the Trf gene) is synthesized primarily in the liver, its function in the liver is largely unknown. Here, we generated hepatocyte-specific Trf knockout mice (Trf-LKO), which are viable and fertile but have impaired erythropoiesis and altered iron metabolism. Moreover, feeding Trf-LKO mice a high-iron diet increased their susceptibility to developing ferroptosis-induced liver fibrosis. Importantly, we found that treating Trf-LKO mice with the ferroptosis inhibitor ferrostatin-1 potently rescued liver fibrosis induced by either high dietary iron or carbon tetrachloride (CCl4) injections. In addition, deleting hepatic Slc39a14 expression in Trf-LKO mice significantly reduced hepatic iron accumulation, thereby reducing ferroptosis-mediated liver fibrosis induced by either a high-iron diet or CCl4 injections. Finally, we found that patients with liver cirrhosis have significantly lower levels of serum transferrin and hepatic transferrin, as well as higher levels of hepatic iron and lipid peroxidation, compared with healthy control subjects. Taken together, these data indicate that hepatic transferrin plays a protective role in maintaining liver function, providing a possible therapeutic target for preventing ferroptosis-induced liver fibrosis.

Drug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding of obvious cause are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood, which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that affect the biosynthesis of active vitamin K-dependent (VKD) coagulation factors with possible adverse off-target results. The National Institutes of Health (NIH) Clinical Collection (NCC) library containing 727 drugs was screened, and 9 drugs were identified, including the most commonly prescribed anticoagulant warfarin. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), whereas idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other 3 drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs that target VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based, high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.

Polyclonal anti-D is a first-line therapy for immune thrombocytopenia (ITP). Monoclonal antibodies are desirable alternatives, but none have yet proven successful despite their ability to opsonize erythrocytes (or red blood cells, RBCs) and cause anemia. Here, we examined 12 murine erythrocyte-specific antibodies of different specificity and subtypes and found that 8 of these antibodies could induce anemia in antigen-positive mice. Of these 8 antibodies, only 5 ameliorated ITP. All antibodies were examined for their in vitro ability to support macrophage-mediated phagocytosis of erythrocytes. Antibodies which supported erythrocyte phagocytosis in vitro successfully ameliorated ITP in vivo. To examine the ability of each antibody to inhibit phagocytosis of platelets, the antibodies were used to sensitize erythrocytes in vitro and these were added to a platelet phagocytosis assay. Antibodies that inhibited platelet phagocytosis in vitro also all ameliorated ITP in vivo. We conclude that inducing anemia is not a sufficient condition for amelioration of ITP but that the antibody's ability to prevent platelet phagocytosis in vitro predicted its ability to ameliorate ITP. We suggest that inhibition of in vitro platelet phagocytosis may prove to be a valuable tool for determining which erythrocyte antibodies would likely be candidates for clinical use in ITP.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.

Neutrophil extravasation requires opening of the endothelial barrier but does not necessarily cause plasma leakage. Leaks are prevented by contractile actin filaments surrounding the diapedesis pore, keeping this opening tightly closed around the transmigrating neutrophils. We have identified the receptor system that is responsible for this. We show that silencing, or gene inactivation, of endothelial Tie-2 results in leak formation in postcapillary venules of the inflamed cremaster muscle at sites of neutrophil extravasation, as visualized by fluorescent microspheres. Leakage was dependent on neutrophil extravasation, because it was absent upon neutrophil depletion. We identified the Cdc42 GTPase exchange factor FGD5 as a downstream target of Tie-2 that is essential for leakage prevention during neutrophil extravasation. Looking for the Tie-2 agonist and its source, we found that platelet-derived angiopoietin-1 (Angpt1) was required to prevent neutrophil-induced leaks. Intriguingly, blocking von Willebrand factor (VWF) resulted in vascular leaks during transmigration, indicating that platelets interacting with endothelial VWF activate Tie-2 by secreting Angpt1, thereby preventing diapedesis-induced leakiness.

Chemotherapy and irradiation cause DNA damage to hematopoietic stem cells (HSCs), leading to HSC depletion and dysfunction and the risk of malignant transformation over time. Extrinsic regulation of HSC DNA repair is not well understood, and therapies to augment HSC DNA repair following myelosuppression remain undeveloped. We report that epidermal growth factor receptor (EGFR) regulates DNA repair in HSCs following irradiation via activation of the DNA-dependent protein kinase-catalytic subunit (DNA-PKcs) and nonhomologous end joining (NHEJ). We show that hematopoietic regeneration in vivo following total body irradiation is dependent upon EGFR-mediated repair of DNA damage via activation of DNA-PKcs. Conditional deletion of EGFR in hematopoietic stem and progenitor cells (HSPCs) significantly decreased DNA-PKcs activity following irradiation, causing increased HSC DNA damage and depressed HSC recovery over time. Systemic administration of epidermal growth factor (EGF) promoted HSC DNA repair and rapid hematologic recovery in chemotherapy-treated mice and had no effect on acute myeloid leukemia growth in vivo. Further, EGF treatment drove the recovery of human HSCs capable of multilineage in vivo repopulation following radiation injury. Whole-genome sequencing analysis revealed no increase in coding region mutations in HSPCs from EGF-treated mice, but increased intergenic copy number variant mutations were detected. These studies demonstrate that EGF promotes HSC DNA repair and hematopoietic regeneration in vivo via augmentation of NHEJ. EGF has therapeutic potential to promote human hematopoietic regeneration, and further studies are warranted to assess long-term hematopoietic effects.

The bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition. Primary HLH occurs early in life as a result of monogenic biallelic mutations affecting lymphocyte cytotoxicity. Secondary HLH occurs mostly in adults secondary to infection, lymphoma, or rheumatic disease. In this latter setting, lymphocyte cytotoxicity status is not known. We conducted a systematic evaluation of natural killer (NK) cell cytotoxicity in adult patients with secondary HLH. Adult patients with secondary HLH were prospectively studied ex vivo for total lymphocyte count and subtype, NK cell phenotype, perforin expression and degranulation, and natural or antibody-dependent cell cytotoxicity, in comparison with patients affected by the same underlying disease without HLH (disease controls [DCs]) and with healthy controls (HCs). Screening for variants of cytotoxity genes was systematically performed. 68 patients were included in the HLH group and 34 each in the DC and HC groups. In HLH patients, severe and transient lymphopenia, activated NK cell phenotype (eg, increased CD69, ICAM-1, HLADR, and CCR5 expression), and decreased capacity of interferon γ production were observed; mean perforin expression was normal; and degranulation tests and NK cell cytotoxicity were not different from those in DCs. A monoallelic variant of uncertain significance affecting a lymphocyte cytotoxicity gene or the perforin variant A91V was observed in almost 50% of the patients. We detected no major intrinsic cytotoxicity dysfunction in secondary HLH patients compared with DCs and no predicted pathogenic gene variant. The activated NK phenotype profile associated with decreased interferon γ production seems similar to those of other hyperinflammatory diseases such as sepsis or systemic juvenile idiopathic arthritis.

Recently, we showed that ADAMTS13 circulates in an open conformation during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Although the cause of this conformational change remains elusive, ADAMTS13 is primarily closed in iTTP patients in remission with ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, as well as after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, immunoglobulin G from 18 acute iTTP patients was purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14 of 18 (78%) samples, proving that anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n = 197) that also included plasma samples from iTTP patients in remission in whom ADAMTS13 activity was <50%. The open ADAMTS13 conformation was found during acute iTTP, as well as in patients in remission with ADAMTS13 activity <50% and in half of the patients with ADAMTS13 activity >50%, although free anti-ADAMTS13 autoantibodies were not always detected. Thus, open ADAMTS13 is a hallmark of acute iTTP, as well as a novel biomarker that can be used to detect subclinical iTTP in patients in remission. Finally, a long-term follow-up study in 1 iTTP patient showed that the open conformation precedes a substantial drop in ADAMTS13 activity. In conclusion, we have shown that anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation. Most importantly, an open ADAMTS13 conformation is a biomarker for subclinical iTTP and could become an important tool in TTP management.

The field of malignant hematology has experienced extraordinary advancements with survival rates doubling for many disorders. As a result, many life-threatening conditions have since evolved into chronic medical ailments. Paralleling these advancements have been increasing rates of complex hematologic pain syndromes, present in up to 60% of patients with malignancy who are receiving active treatment and up to 33% of patients during survivorship. Opioids remain the practice cornerstone to managing malignancy-associated pain. Prevention and management of opioid-related complications have received significant national attention over the past decade, and emerging data suggest that patients with cancer are at equal if not higher risk of opioid-related complications when compared with patients without malignancy. Numerous tools and procedural practice guides are available to help facilitate safe prescribing. The recent development of cancer-specific resources directing algorithmic use of validated pain screening tools, prescription drug monitoring programs, urine drug screens, opioid use disorder risk screening instruments, and controlled substance agreements have further strengthened the framework for safe prescribing. This article, which integrates federal and organizational guidelines with known risk factors for cancer patients, offers a case-based discussion for reviewing safe opioid prescribing practices in the hematology setting.

Anemic stress induces stress erythropoiesis, which rapidly generates new erythrocytes to restore tissue oxygenation. Stress erythropoiesis is best understood in mice where it is extramedullary and occurs primarily in the spleen. However, both human and mouse stress erythropoiesis use signals and progenitor cells that are distinct from steady-state erythropoiesis. Immature stress erythroid progenitors (SEPs) are derived from short-term hematopoietic stem cells. Although the SEPs are capable of self-renewal, they are erythroid restricted. Inflammation and anemic stress induce the rapid proliferation of SEPs, but they do not differentiate until serum erythropoietin (Epo) levels increase. Here we show that rather than directly regulating SEPs, Epo promotes this transition from proliferation to differentiation by acting on macrophages in the splenic niche. During the proliferative stage, macrophages produce canonical Wnt ligands that promote proliferation and inhibit differentiation. Epo/Stat5-dependent signaling induces the production of bioactive lipid mediators in macrophages. Increased production of prostaglandin J2 (PGJ2) activates peroxisome proliferator-activated receptor γ (PPARγ)-dependent repression of Wnt expression, whereas increased production of prostaglandin E2 (PGE2) promotes the differentiation of SEPs.

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.