bluuebunny/crossref_metadata_2025_split huggingface dataset:
Foxo1 Expressed in Osteoblasts Promotes the Leukemogenic Properties of β-Catenin By Activating Notch Signaling
Authors: Kode Aruna , Mosialou Ioanna , Manavalan Sanil J , Feldstein Julie Teruya , Bhagat Govind , Berman Ellin , Kousteni Stavroula 
Published: November 2019

Abstract
Osteoblasts affect self-renewal and expansion of hematopoietic stem cells (HSCs) and homing of healthy hematopoietic and tumor cells into the bone marrow. Moreover, in the mouse, constitutive activation of b-catenin (Ctnnb1CAosb mice) in osteoblasts is sufficient to alter the differentiation potential of myeloid and lymphoid progenitors and to trigger the development of acute myeloid leukemia (AML). Because the same genetic event is associated with AML development in humans we sought to better understand its molecular bases. For that purpose we examined in vivo whether FoxO1, a transcription factor known to interact with β-catenin, affects its AML inducing properties. Deleting one allele of FoxO1 mice from the osteoblasts of leukemic Ctnnb1CAosb mice prevented the appearance of anemia, peripheral monocytosis, neutrophilia and lymphocytopenia that are otherwise observed in Ctnnb1CAosb mice. Similarly, FoxO1 haploinsufficiency in osteoblasts of Ctnnb1CAosb mice prevented the shift in the differentiation of HSCs to the myeloid lineage and the increase in the LSK+/CD150+/CD48- subset of long term repopulating HSC progenitors (LT-HSCs). Histological analysis showed that myeloid and megakaryocyte dysplasias observed in Ctnnb1CAosb mice and associated with their AML phenotype were also rescued in Ctnnb1CAosb;FoxO1osb+/-animals. As a result FoxO1 haploinsufficiency in osteoblasts prevented the early lethality of Ctnnb1CAosb mice since Ctnnb1CAosb;FoxO1osb+/-mice lived and were healthy for at least one year, the entire time that they were observed. At the molecular level FoxO1 interacts with b-catenin in osteoblasts to induce expression of the Notch ligand Jagged-1 which initiates the dysmyelopoiesis leading to AML. ChiP/Re-ChiP assays and transient transfection experiments showed that this interaction requires the formation of a b-catenin/Foxo1 complex at two distinct locations in the regions of the Jagged-1 promoter. It also leads to subsequent activation of Notch signaling in LT- HSC progenitors. These events induce the leukemogenic transformation of HSCs. These findings identify FoxO1 through its expression in osteoblasts as a factor affecting hematopoiesis and provide a molecular mechanism whereby the FoxO1/ activated b-catenin interaction results in AML. They further support the notion that targeting the bone marrow niche may be a new strategy to treat leukemia and raise the prospect that FoxO1 oncogenic properties may occur in other tissues.

Disclosures
No relevant conflicts of interest to declare.
Generation and Characterization of FVIII-Specific CAR-Transduced Regulatory T Cells
Authors: Naumann Anja , Kim Yongchan , Königs Christoph , Scott David W 
Published: November 2019

Abstract
The development of inhibitory antibodies (inhibitors) against FVIII is the most critical complication in the treatment of hemophilia A patients as hemostasis can no longer be reestablished by FVIII replacement therapy. Immune tolerance induction by frequent FVIII infusions is demanding, costly and not successful in all treated patients leading to an urgent need for the development of new therapeutic approaches for the prevention or treatment of FVIII inhibitors. Regulatory T cells (Tregs) are important for the maintenance of tolerance and have a high therapeutic potential in the context of autoimmune or inflammatory immune disorders. As Tregs are polyclonal, treatment with Treg pools comprises the risk of a general immunosuppression. Thus, the establishment of antigen-specific Tregs could be of great benefit for a broad range of patients, including inhibitor positive hemophilia A patients. To create such specific Tregs, a FVIII-specific scFv isolated out of a synthetic phage display library was used to generate a second generation chimeric antigen receptor (CAR).
To verify the specificity of the CAR for FVIII and the functionality of the recombined cytoplasmic domain (CD28 and CD3zeta), naïve CD4 T cells were retrovirally transduced with the generated CAR construct and a proliferation assay was conducted in the presence of plate-bound or soluble FVIII, as well as soluble FVIII presented by autologous irradiated PBMCs. Proliferation of transduced cells was more effective when FVIII was presented plate-bound or by PBMCs. In a therapeutically-relevant setting, this would be very promising, as transduced T cells should not be activated by soluble FVIII in the bloodstream but rather by FVIII presented on antigen-presenting cells in lymphatic organs.
Next, functionality of the CAR construct in Tregs was addressed. Transduced Tregs showed extracellular expression of the scFv and could be stimulated MHC-independently with FVIII. Such stimulated cells showed increased expression of Treg activation markers LAP and GARP. Thus, by using the generated CAR for transduction of Tregs, it was possible to create FVIII-specific Tregs that can be stimulated MHC-independently, opening new possibilities for therapeutic approaches in hemophilia A patients with FVIII inhibitors.

Disclosures
No relevant conflicts of interest to declare.
Engineered Antigen-Specific Human Regulatory T Cells Suppress Both FVIII-Specific T- and B-Cell Responses
Authors: Kim Yong Chan , Zhang Aihong , Pratt Kathleen P. , Shevach Ethan , Scott David William 
Published: November 2019

Abstract
Expansion of human regulatory T cells (Tregs) for clinical applications offers great promise for the treatment of undesirable immune responses in autoimmunity, transplantation, allergy and anti-drug antibody responses, including inhibitor responses in hemophilia A patients. However, polyclonal Tregs are “nonspecific” and therefore their therapeutic application could potentially cause global immunosuppression. To avoid this undesirable outcome, the generation of antigen-specific Tregs would represent a significant advance. In this study, we demonstrate the production and properties of engineered antigen-specific Tregs created by transduction of a recombinant T-cell receptor into expanded human FoxP3+ Tregs. The DNA sequence of this T cell receptor was obtained from a FVIII-specific T cell clone that had been expanded from blood donated by a hemophilia A subject. The resulting engineered FVIII-specific Tregs efficiently suppressed both proliferation and cytokine production of FVIII-specific CD4 T effector cells. Moreover, studies with an HLA-transgenic, FVIII-deficient mouse model demonstrated that antibody production from FVIII-primed spleen cells in vitro was profoundly inhibited in the presence of these FVIII-specific Tregs, suggesting that this novel approach has translational potential for the prevention and/or reversal of inhibitor responses in hemophilia A patients. Furthermore, these FVIII-specific Tregs successfully cross-suppressed unrelated, bystander T effector cells (specific for a myelin peptide) only in the presence of FVIII and the unrelated, stimulatory myelin peptide. These data strongly imply that such FVIII-specific Tregs would be effective at suppressing multiple effector cells, e.g., CD4 T effector cells specific for other FVIII epitopes besides that recognized by the Tregs expressing the engineered TCR, in the local milieu when FVIII is present. Thus, we propose that FVIII-specific Tregs have potential as a therapeutic tool to induce tolerance to FVIII, by preventing or reversing inhibitor formation in hemophilia A patients.
Supported by NIH RO1-HL061883 (DWS), funding from Bayer and CSL Behring (KPP) and intramural support from NIAID (EMS). We thank the Puget Sound Blood Center for enrolling hemophilia A patients and we thank all blood donors.

Disclosures
Kim: Henry Jackson Foundation: YCK and DWS have a provisional patent filing via the Henry Jackson Foundation on Design and use of specific regulatory T cells to induce immune tolerance Patents & Royalties. Scott:Henry Jackson Foundation: YCK and DWS have a provisional patent filing via the Henry Jackson Foundation on Design and use of specific regulatory T cells to induce immune tolerance Patents & Royalties.
Cytotoxic T-Cell Responses (CTL) Against Several Leukemia-Associated-Antigens (LAA) Related to the Detected Cytokine Profile in the Course of Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) and Donor Lymphocyte Infusion (DLI) in Patients with Different Hematological Diseases
Authors: Hofmann Susanne , Götz Marlies , Herbst Cornelia , Schneider Vanessa , Wiesneth Markus , Bunjes Donald , Döhner Hartmut , Greiner Jochen 
Published: November 2019

Abstract
T-cell responses play a central role in maintaining remission and prolonging overall-survival in patients with hematologic malignancies after allo-HSCT and delayed DLI. The role of LAA and mHag has to be elucidated although there is evidence that the graft-versus-leukemia-effect observed after DLI is based on CTL-mediated immunity which is reactive against mHag and LAA.
In this study, we analysed peripheral blood and serum samples of 10 patients with AML, T-NHL, CML and Multiple Myeloma in the course of allo-HSCT and before and after DLI for specific CTL responses against several LAA and established a cytokine profile. Epitopes derived from PRAME, NPM1mut, RHAMM, WT-1 and other LAA were tested. Immune reactions of CTL were measured in ELISpot assays (INF-gamma and granzyme B). In addition tetramer assays were performed. The corresponding cytokine profile of several cytokines was analysed for further interpretation of the clinical data.
In all patients we could detect a CTL response against at least one of the tested LAA in the course of DLI. Two patients were AML patients with NPM1-mutation (NPM1mut) who received preemptive DLI in molecular relapse and showed a polyspecific CTL-response against several epitopes including those derived from the mutated region of NPM1. The frequency of CTL-responses changed during the course. All of the analysed cytokines IL18, CD40L, IL1ra, IL6, IL7, IL10, IL15, IL17, IL18, IL2, IFNg and TGFß were detected in at least one of the samples of the patients, but the pattern differed dependent on the time point after allo-HSCT and DLI, immune-suppression, immune-reconstitution, GvHD and disease status. Some patients showed an increase in cytokine levels in the course of therapy, e.g. in one patient with AML with NPM1mut who received preemptive DLI in molecular relapse we detected significant higher levels of several cytokines after DLI. The patient was in molecular remission again and had developed GvHD.
Taken together, we detected CTL responses against several LAA after allo-HSCT with an increase of polyspecific T cell responses after DLI in some tested patients. These might support the role of CTL against LAA in the control of disease. Especially patients with AML with NPM1mut seem to be a very interesting population with T-cell activation after DLI. Moreover, cytokine profiles might be important and need to be correlated to the cellular responses as well as the clinical data, to further characterize the mechanisms of DLI.

Disclosures
No relevant conflicts of interest to declare.
Longer Procedure Duration and Large Volume Harvests Reduce the Likelihood of Achieving the Requested Cell Dose from Unrelated Bone Marrow Donors
Authors: Anthias Chloe , Arkwright Rebecca , Billen Annelies , Madrigal J. Alejandro , Shaw Bronwen E. 
Published: November 2019

Abstract
Introduction
The factors influencing the quality of bone marrow (BM) harvests and total nucleated cell count (TNC) achieved are poorly understood. As the number of BM harvest procedures falls (due to an increasing proportion of harvest requests for peripheral blood stem cells), concerns are raised around maintaining expertise in this area. We investigate, in four Anthony Nolan collection centers, the procedure- and donor -related factors influencing the quality of harvests, and likelihood of achieving the requested TNC.
Materials & Method
110 consecutive BM harvests of healthy unrelated donors (UD)s were performed according to standard Anthony Nolan protocols. Harvest quality was defined as TNC/ml volume collected. Univariate analyses of factors influencing the TNC and harvest quality were performed using Chi squared or Fisher’s test where appropriate.
Results
110 BM harvests were performed in 80 male and 30 female donors. The median donor age was 28 years (range 18-56 years).
The median TNC requested was 4x108/kg, and median harvested TNC 4.2x108/kg recipient weight, with the requested TNC dose achieved in 50% of harvests. Higher volume harvests (greater than the median volume 1200 mls) were significantly less likely to achieve TNC> 4x108/kg recipient weight (38% vs 70% p=0.001), as were harvests with a procedure time greater than 30 minutes (p=0.037). We investigated donor factors influencing the harvest yield and found that donor/recipient weight discrepancy significantly impacted the TNC achieved: with only 18% of BM harvests from donors who weighed less than their recipient achieving TNC >4x108/kg compared to 62% harvests from donors who were heavier than their recipient (p=0.001). No other donor factor had a significant influence on the TNC.
The median TNC/ml collected was 18.2x10^6/ml and harvests in donors>40 years were more likely to be of lower quality (p=0.036). No other donor factors affected harvest quality, however a significant variation between the 4 collection centers was observed: both the harvest quality (p=0.014) and volume collected (p=0.035) varied significantly between centers.
Conclusions
Donor weight is a strong determinant of bone marrow harvest yield; we recommend that when multiple matched UDs are available, a donor with high body weight should be selected for BM harvest. Procedure factors including large volume harvests and long procedure times reduce the likelihood of achieving the requested TNC, and significant variation was seen between centers. Given the declining number of requests for BM harvests, it is crucial these are performed by the most experienced centers and that systems exist to ensure operators retain their expertise.

Disclosures
No relevant conflicts of interest to declare.
An SCF-FBXW7 Ubiquitin Ligase Mediated Feedback Loop Facilitates GATA Factor Switching and Reinforces Commitment to Terminal Erythroid Maturation
Authors: Ghamari Alireza , Jaensch Elizabeth , Giadone Richard , Akie Thomas E , Xu Jian , Bauer Daniel , Orkin Stuart H. , Cantor Alan B. 
Published: November 2019

Abstract
The GATA family transcription factors GATA2 and GATA1 play reciprocal roles during terminal erythroid and megakaryocytic maturation. GATA2 is expressed early in hematopoiesis and is required for early progenitor cell proliferation and survival. It must be down regulated in order for terminal maturation to occur. In contrast, GATA1 increases in expression during erythropoiesis and megakaryopoiesis, and is required for terminal maturation of these lineages. During this process, GATA1 displaces GATA2 at a large number of chromatin loci, typically leading to repression of early progenitor genes and activation of terminal maturation genes. This “GATA factor switch” is facilitated by the considerably shorter half-life of GATA2 (~30 min) compared to GATA1 (>4-6 hours). GATA2’s short half-life is mediated by ubiquitin-proteosomal degradation mechanisms. Yet the ubiquitin complex(es) responsible for GATA2-specific clearance has not been identified. In this study, we show that the Skip1-Cullin-Fbox (SCF) substrate recognition factor Fbxw7 is involved in GATA2 ubiquitin-mediated degradation. Proteomic and co-immunoprecipitation experiments show physical association of Fbxw7 with GATA2, but not GATA1. CRISPR/Cas9 deletion of Fbxw7 in G1ER4 cells results in elevated GATA2 steady-state protein levels, a prolonged GATA2 half-life, delayed GATA2 clearance upon estradiol induction, and impaired erythroid terminal maturation. Importantly, Fbxw7 mRNA and protein levels normally increase during terminal erythroid maturation and this occurs in a GATA1 dependent manner. We identified a key cis-regulatory element upstream of the Fbxw7 gene that is occupied by GATA2 during early erythropoiesis, but becomes bound by GATA1/TAL1 during late erythroid maturation in induced G1-ER4 cells and human CD34+ in vitro differentiated erythroblasts. This is accompanied by the acquisition of active enhancer histone marks at this site. Deletion of this regulatory element in G1ER4 cells blocks the GATA1 dependent increase in Fbxw7 mRNA transcript levels during erythroid cell maturation. Lastly, we identified a family with congenital hypoplastic anemia (lacking mutations in all known Diamond Blackfan Anemia genes) who harbor a germline Fbxw7missense mutation. Collectively, these findings identify GATA2 as a novel Fbxw7 substrate and uncover an ubiquitin-mediated post-transcriptional GATA factor feedback loop that reinforces GATA factor switching and commitment to terminal erythroid maturation.

Disclosures
Cantor: Amgen: Membership on an entity's Board of Directors or advisory committees.
G-CSF-Primed Bone Marrow As a Source of Hematopoietic Stem Cells for Allogeneic Transplantation in Malignant and Non-Malignant Hematological Disorders
Authors: León-Rodríguez Eucario , Guzmán-Uribe Patricia , Deffis Court Marcela , Perez-Alvarez Sandra Ileana , Rivera Franco Monica M 
Published: November 2019

Abstract

Introduction: To date, worldwide, the main source of stem cells is peripheral blood (PBSCs). However, its use has been associated with a higher incidence of graft versus host disease (GVHD) when compared with the use of bone marrow. It has been demonstrated that the fast engraftment using G-CSF-primed bone marrow as a source of stem cells, compares to that seen with PBSCs, and it is also associated with a decreased incidence of GVHD.
Objective: To describe the results obtained from allogeneic, G-CSF-primed bone marrow transplantations, at INCMNSZ, from November 1998 to March 2013.
Material and methods: A retrospective analysis was performed in patients who underwent allogeneic, G-CSF-primed bone marrow transplantation. Clinical characteristics, frequency of GVHD, and survival, were conducted, using the Statistical Software Package SPSS v21.0.
Results: Forty-nine patients who underwent allogeneic, G-CSF-primed bone marrow transplantation, from November 1998 to March 2013, were included. Patients (male, 63%) had a median age of 29 years (range 16-59). The patients had a following range of underlying diseases: aplastic anemia (n=15, 30.6%), myelodysplastic syndrome (n=12, 24.5%), chronic myeloid leukemia (CML, n=9, 18.4%), acute lymphoblastic leukemia (LLA, n=7, 14.3%), acute myeloid leukemia (AML, n=3, 6.1%), or others (n=3, 6.1%). Patients achieved a bilineage engraftment with a median time of 20 days (range, 11-40) for absolute neutrophil count and 15 days (range, 5-45) for platelets. Acute GVHD was observed in 4 patients (8.2%), reported as grade I and II; and 14 patients (28.6%) experienced a limited form of chronic GVHD. Nine out of 49 (18.3%) patients relapsed: CML (n=5), AML (n=1), AA (n=1), ALL (n=1), others (n=1). All patients with relapsed CML were treated with donor lymphocyte infusions, but only 2 responded. Treatment related mortality (TRM) was 8.16%, including infectious complications in 2 patients, cGVHD in one patient, and veno-oclussive disease (VOD) in one patient. Overall mortality was 26.5%; the majority of deaths (69.2%) were caused by progressive and relapsing disease. With a follow up of 43 months (0-149), the median overall survival (OS) has not been reached; however, the estimated 10-year OS is 69%.
Conclusion: According to our data, it seems that G-CSF-primed bone marrow is a better source of hematopoietic stem cells for allogeneic transplantation in malignant and non-malignant hematological disorders, when compared with PBSCs, due to the similar engraftment time, and a decreased incidence and severity of acute and chronic GVHD. Nevertheless, this strategy does not appear to be valid as an approach for diseases were graft versus tumor effect (GVT) is important to eradicate the malignancy, such as in CML.

Disclosures
No relevant conflicts of interest to declare.
IP3 3-Kinase B Controls Hematopoietic Stem Cell Homeostasis and Prevents Lethal Hematopoietic Failure in Mice
Authors: Sauer Karsten 
Published: November 2019

Tight regulation of hematopoietic stem cell (HSC) homeostasis ensures life-long hematopoiesis and prevents blood cancers. The mechanisms balancing HSC quiescence with expansion and differentiation into hematopoietic progenitors are incompletely understood. Here, we identify inositoltrisphosphate (IP3) 3-kinase B (Itpkb) as a novel essential regulator of HSC quiescence and function. Young Itpkb-/- mice accumulated phenotypic HSC which were less quiescent and proliferated more than wildtype controls. Itpkb-/- HSC downregulated quiescence and stemness associated mRNAs, but upregulated activation, oxidative metabolism, protein synthesis and lineage associated transcripts. Although they showed no significant homing defects and had normal to elevated viability, Itpkb-/- HSC had a severely reduced competitive long-term repopulating potential. Aging Itpkb-/- mice lost hematopoietic stem and progenitor cells and died with severe anemia. Wildtype HSC normally repopulated Itpkb-/- hosts, indicating a HSC-intrinsic Itpkb requirement. In vitro, Itpkb-/- HSC had reduced cobblestone-area forming cell activity and showed increased stem cell factor activation of the phosphoinositide 3-kinase (PI3K) effector Akt. This was reversed by exogenous provision of the Itpkb product IP4, a known PI3K/Akt antagonist. Itpkb-/- HSC also showed transcriptome changes consistent with hyperactive Akt/mTOR signaling. Thus, we propose that Itpkb ensures HSC quiescence and function in part by limiting cytokine-induced PI3K signaling in HSC.

Disclosures
No relevant conflicts of interest to declare.