Most cancers evolve over time as patients initially responsive to therapy acquire resistance to the same drugs at relapse. Cancer stem cells have been postulated to represent a therapy-refractory reservoir for relapse, but formal proof of this model is lacking. We prospectively characterized leukemia stem cell populations (LSCs) from a well-defined cohort of patients with acute myelogenous leukemia (AML) at diagnosis and relapse to assess the effect of the disease course on these critical populations. Leukemic samples were collected from patients with newly diagnosed AML before therapy and after relapse, and LSC frequency was assessed by limiting dilution analyses. LSC populations were identified using fluorescent-labeled cell sorting and transplantation into immunodeficient NOD/SCID/interleukin 2 receptor γ chain null mice. The surface antigen expression profiles of pretherapy and postrelapse LSCs were determined for published LSC markers. We demonstrate a 9- to 90-fold increase in LSC frequency between diagnosis and relapse. LSC activity at relapse was identified in populations of leukemic blasts that did not demonstrate this activity before treatment and relapse. In addition, we describe genetic instability and exceptional phenotypic changes that accompany the evolution of these new LSC populations. This study is the first to characterize the evolution of LSCs in vivo after chemotherapy, identifying a dramatic change in the physiology of primitive AML cells when the disease progresses. Taken together, these findings provide a new frame of reference by which to evaluate candidate AML therapies in which both disease control and the induction of more advanced forms of disease should be considered.

To test the hypothesis that skeletal muscle myosins can directly influence blood coagulation and thrombosis, ex vivo studies of the effects of myosin on thrombogenesis in fresh human blood were conducted. Addition of myosin to blood augmented the thrombotic responses of human blood flowing over collagen-coated surfaces (300 s-1 shear rate). Perfusion of human blood over myosin-coated surfaces also caused fibrin and platelet deposition, evidencing myosin's thrombogenicity. Myosin markedly enhanced thrombin generation in both platelet-rich plasma and platelet-poor plasma, indicating that myosin promoted thrombin generation in plasma primarily independent of platelets. In purified reaction mixtures composed only of factor Xa, factor Va, prothrombin, and calcium ions, myosin greatly enhanced prothrombinase activity. The Gla domain of factor Xa was not required for myosin's prothrombinase enhancement. When binding of purified clotting factors to immobilized myosin was monitored using biolayer interferometry, factors Xa and Va each showed favorable binding interactions. Factor Va reduced by 100-fold the apparent Kd of myosin for factor Xa (Kd ∼0.48 nM), primarily by reducing koff, indicating formation of a stable ternary complex of myosin:Xa:Va. In studies to assess possible clinical relevance for this discovery, we found that antimyosin antibodies inhibited thrombin generation in acute trauma patient plasmas more than in control plasmas (P = .0004), implying myosin might contribute to acute trauma coagulopathy. We posit that myosin enhancement of thrombin generation could contribute either to promote hemostasis or to augment thrombosis risk with consequent implications for myosin's possible contributions to pathophysiology in the setting of acute injuries.

The first definitive hematopoietic stem cells (dHSCs) in the mouse emerge in the dorsal aorta of the embryonic day (E) 10.5 to 11 aorta-gonad-mesonephros (AGM) region. Notch signaling is essential for early HSC development but is dispensable for the maintenance of adult bone marrow HSCs. How Notch signaling regulates HSC formation in the embryo is poorly understood. We demonstrate here that Notch signaling is active in E10.5 HSC precursors and involves both Notch1 and Notch2 receptors, but is gradually downregulated while they progress toward dHSCs at E11.5. This downregulation is accompanied by gradual functional loss of Notch dependency. Thus, as early as at final steps in the AGM region, HSCs begin acquiring the Notch independency characteristic of adult bone marrow HSCs as part of the maturation program. Our data indicate that fine stage-dependent tuning of Notch signaling may be required for the generation of definitive HSCs from pluripotent cells.

The number and activity of osteoclasts (OCs) are strongly enhanced by myeloma cells, leading to significant bone lesions in patients with multiple myeloma (MM). Mechanisms remain elusive as to whether myeloma-supporting OCs also induce suppressive immune bone marrow (BM) microenvironment. Here, we first show that OCs significantly protect MM cells against T-cell-mediated cytotoxicity via direct inhibition of proliferating CD4(+) and CD8(+) T cells. The immune checkpoint molecules programmed death ligand 1 (PD-L1), Galectin-9, herpesvirus entry mediator (HVEM), and CD200, as well as T-cell metabolism regulators indoleamine 2, 3-dioxygenase (IDO), and CD38 are significantly upregulated during osteoclastogenesis. Importantly, the levels of these molecules, except CD38, are higher in OCs than in MM cells. Anti-PD-L1 monoclonal antibody (mAb) and IDO inhibitor partly overcome OC-inhibited T-cell responses against MM cells, confirming their roles in OC-suppressed MM cell lysis by cytotoxic T cells. In addition, Galectin-9 and a proliferation-induced ligand (APRIL), secreted by OCs, are significantly upregulated during osteoclastogenesis. Galectin-9 specifically induces apoptosis of T cells while sparing monocytes and MM cells. APRIL induces PD-L1 expression in MM cells, providing additional immune inhibition by OCs. Moreover, CD38 is significantly upregulated during osteoclastogenesis. When targeted by an anti-CD38 mAb, suppressive T-cell function by OCs is alleviated, associated with downregulation of HVEM and IDO. Taken together, these results define the expression of multiple immune proteins and cytokines in OCs essential for suppressive MM BM milieu. These results further support the combination of targeting these molecules to improve anti-MM immunity.

Follicular lymphoma (FL) is a clinically and molecularly heterogeneous disease. Posttreatment surrogate end points, such as progression of disease within 24 months (POD24) are promising predictors for overall survival (OS) but are of limited clinical value, primarily because they cannot guide up-front treatment decisions. We used the clinical and molecular data from 2 independent cohorts of symptomatic patients in need of first-line immunochemotherapy (151 patients from a German Low-Grade Lymphoma Study Group [GLSG] trial and 107 patients from a population-based registry of the British Columbia Cancer Agency [BCCA]) to validate the predictive utility of POD24, and to evaluate the ability of pretreatment risk models to predict early treatment failure. POD24 occurred in 17% and 23% of evaluable GLSG and BCCA patients, with 5-year OS rates of 41% (vs 91% for those without POD24, P < .0001) and 26% (vs 86%, P < .0001), respectively. The m7-FL International Prognostic Index (m7-FLIPI), a prospective clinicogenetic risk model for failure-free survival, had the highest accuracy to predict POD24 (76% and 77%, respectively) with an odds ratio of 5.82 in GLSG (P = .00031) and 4.76 in BCCA patients (P = .0052). A clinicogenetic risk model specifically designed to predict POD24, the POD24-PI, had the highest sensitivity to predict POD24, but at the expense of a lower specificity. In conclusion, the m7-FLIPI prospectively identifies the smallest subgroup of patients (28% and 22%, respectively) at highest risk of early failure of first-line immunochemotherapy and death, including patients not fulfilling the POD24 criteria, and should be evaluated in prospective trials of precision medicine approaches in FL.

Programmed cell death ligand 1 (PD-L1) is expressed on both tumor and tumor-infiltrating nonmalignant cells in lymphoid malignancies. The programmed cell death 1 (PD-1)/PD-L1 pathway suppresses host antitumor responses, although little is known about the significance of PD-1/PD-L1 expression in the tumor microenvironment. To investigate the clinicopathological impact of PD-L1 expression in adult T-cell leukemia/lymphoma (ATLL), we performed PD-L1 immunostaining in 135 ATLL biopsy samples. We observed 2 main groups: 1 had clear PD-L1 expression in lymphoma cells (nPD-L1(+), 7.4% of patients), and the other showed minimal expression in lymphoma cells (nPD-L1(-), 92.6%). Within the nPD-L1(-) group, 2 subsets emerged: the first displayed abundant PD-L1 expression in nonmalignant stromal cells of the tumor microenvironment (miPD-L1(+), 58.5%) and the second group did not express PD-L1 in any cell (PD-L1(-), 34.1%). nPD-L1(+) ATLL (median survival time [MST] 7.5 months, 95% CI [0.4-22.3]) had inferior overall survival (OS) compared with nPD-L1(-) ATLL (MST 14.5 months, 95% CI [10.1-20.0]) (P = .0085). Among nPD-L1(-) ATLL, miPD-L1(+) ATLL (MST 18.6 months, 95% CI [11.0-38.5]) showed superior OS compared with PD-L1(-) ATLL (MST 10.2 months, 95% CI [8.0-14.7]) (P = .0029). The expression of nPD-L1 and miPD-L1 maintained prognostic value for OS in multivariate analysis (P = .0322 and P = .0014, respectively). This is the first report describing the clinicopathological features and outcomes of PD-L1 expression in ATLL. More detailed studies will disclose clinical and biological significance of PD-L1 expression in ATLL.

CTLA-4 is a critical inhibitory "checkpoint" molecule of immune activation. Several recent reports have described patients with immune dysregulation and lymphoproliferative disease resulting from 2 different genetic diseases that directly or indirectly cause CTLA-4 deficiency. Numerous articles have also been published describing CTLA-4 blockade in cancer immunotherapy and its side effects, which are ultimately the consequence of treatment-induced CTLA-4 deficiency. Here, we review these 2 diseases and CTLA-4 blockade therapy, emphasizing the crucial role of CTLA-4 in immune checkpoint regulation.

In this issue of Blood, Assouline et al report the clinical activity of the oral histone deacetylase inhibitor, panobinostat, in 40 patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) not preselected on the basis of molecular subtype as well as transformed lymphomas. The authors found a modest overall response rate (ORR) of 28% in the entire cohort with no benefit from the addition of rituximab. Importantly, a subset of responders achieved relatively durable remissions, including 6 responders (55%) who had yet to progress.

Therapies with novel mechanisms of action are needed for multiple myeloma (MM). B-cell maturation antigen (BCMA) is expressed in most cases of MM. We conducted the first-in-humans clinical trial of chimeric antigen receptor (CAR) T cells targeting BCMA. T cells expressing the CAR used in this work (CAR-BCMA) specifically recognized BCMA-expressing cells. Twelve patients received CAR-BCMA T cells in this dose-escalation trial. Among the 6 patients treated on the lowest 2 dose levels, limited antimyeloma activity and mild toxicity occurred. On the third dose level, 1 patient obtained a very good partial remission. Two patients were treated on the fourth dose level of 9 × 10(6) CAR(+) T cells/kg body weight. Before treatment, the first patient on the fourth dose level had chemotherapy-resistant MM, making up 90% of bone marrow cells. After treatment, bone marrow plasma cells became undetectable by flow cytometry, and the patient's MM entered a stringent complete remission that lasted for 17 weeks before relapse. The second patient on the fourth dose level had chemotherapy-resistant MM making up 80% of bone marrow cells before treatment. Twenty-eight weeks after this patient received CAR-BCMA T cells, bone marrow plasma cells were undetectable by flow cytometry, and the serum monoclonal protein had decreased by >95%. This patient is in an ongoing very good partial remission. Both patients treated on the fourth dose level had toxicity consistent with cytokine-release syndrome including fever, hypotension, and dyspnea. Both patients had prolonged cytopenias. Our findings demonstrate antimyeloma activity of CAR-BCMA T cells. This trial was registered to www.clinicaltrials.gov as #NCT02215967.

Cardiovascular disease resulting from iron accumulation is still a major cause of death in patients with thalassemia major (TM). Voltage-gated calcium-channel blockade prevents iron entry into cardiomyocytes and may provide an adjuvant treatment to chelation, reducing myocardial iron uptake. We evaluated whether addition of amlodipine to chelation strategies would reduce myocardial iron overload in TM patients compared with placebo. In a multicenter, double-blind, randomized, placebo-controlled trial, 62 patients were allocated to receive oral amlodipine 5 mg/day or placebo in addition to their current chelation regimen. The main outcome was change in myocardial iron concentration (MIC) determined by magnetic resonance imaging at 12 months, with patients stratified into reduction or prevention groups according to their initial T2* below or above the normal human threshold of 35 ms (MIC, 0.59 mg/g dry weight). At 12 months, patients in the reduction group receiving amlodipine (n = 15) had a significant decrease in MIC compared with patients receiving placebo (n = 15) with a median of -0.26 mg/g (95% confidence interval, -1.02 to -0.01) vs 0.01 mg/g (95% confidence interval, -0.13 to 0.23), P = .02. No significant changes were observed in the prevention group (treatment-effect interaction with P = .005). The same findings were observed in the subgroup of patients with T2* <20 ms. Amlodipine treatment did not cause any serious adverse events. Thus, in TM patients with cardiac siderosis, amlodipine combined with chelation therapy reduced cardiac iron more effectively than chelation therapy alone. Because this conclusion is based on subgroup analyses, it needs to be confirmed in ad hoc clinical trials. This trial was registered at www.clinicaltrials.gov identifier as #NCT01395199.

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.

Overcoming the silencing of the fetal γ-globin gene has been a long-standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the β-globin locus, called the locus control region (LCR), dynamically interacts with the developmental stage-appropriate β-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells, the LCR can be redirected from the adult β- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom-designed zinc finger (ZF) proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacologic reactivation of fetal hemoglobin (HbF), hematopoietic cells from patients with SCD were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double that observed with decitabine and pomalidomide; butyrate had an intermediate effect whereas tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle hemoglobin (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared with the drug regimens support its therapeutic potential for the treatment of SCD.