bluuebunny/crossref_metadata_2025 huggingface dataset:
Survivin Is Required for Proliferation, but Not Polyploidization of Megakaryocytes.
Authors: Wen Jeremy Q , Leung Cindy , Huang Zan , Small Sara , Crispino John
Published: April 2020

Survivin is a member of chromosome passenger complex, which plays an important role in chromosome alignment, separation and cytokinesis. We recently reported that survivin is necessary for the proliferation and survival of hematopoietic stem and progenitor cells. Furthermore, we previously showed that reduced levels of survivin expression facilitates megakaryocyte development, whereas elevated levels of survivin inhibit their maturation and polyploidization. However, the extent to which survivin is necessary for polyploidization and terminal differentiation of committed megakaryocytes remains unclear. To determine whether survivin is required for megakaryocyte and platelet biogenesis, we mated mice with floxed alleles of survivin (sur fl/fl) to mice that express Cre recombinase under the control of the PF4 promoter. Compound mutant animals appeared grossly normal and harbored normal platelet counts. Furthermore, survivin deleted and control littermates displayed similar expression of CD41 and CD42, as well as similar DNA content within the CD41+ population. The only significant difference detected was an increase in annexin V staining of CD41+ cells within the bone marrow of the mice with survivin deletion. Analysis of DNA extracted from these bone marrows showed no evidence of the survivin deletion, indicating that the surviving cells all escaped excision. These in vivo findings are consistent with a requirement for survivin in the survival or proliferation of megakaryocyte progenitors. Next, to induce megakaryocyte development ex vivo, we cultured bone marrow from surv fl/fl mice in vitro in the presence of TPO. Using this approach, we were able to induce survivin deletion in 75% of the cells as evidenced by PCR. Despite the deletion of survivin, polyploidization of the ex vivo generated megakaryocytes was unaffected. Finally, we induced deletion of survivin by retroviral infection of surv fl/fl progenitors with MSCV-Cre and found that megakaryocyte polyploidization was actually increased in the excised population. Taken together, our results suggest that survivin is not required for polyploidization, but is necessary for proliferation of megakaryocyte progenitors.
Role of Lipocalin 24p3 in Apoptosis and Leukemia.
Authors: Devireddy L.R.
Published: April 2020

Programmed cell death or Apoptosis, is a critical aspect of normal physiology as well as the genesis and treatment of cancer. Certain apoptotic pathways are transcriptionally regulated; in these cases, apoptosis is induced by the transcriptional activation of genes encoding proapoptotic proteins. We originally identified lipocalin 24p3 as the gene undergoing maximum transcriptional stimulation following induction of apoptosis by cytokine-deprivation of interleukin 3 (IL-3) dependent cells. 24p3 is a member of the lipocalin family of carrier proteins – a group of small-secreted molecules that bind and transport low-molecular weight ligands. By delivering this cargo via cell-surface receptors they are known to influence many responses. 24p3 is a secreted lipocalin, which we have found induces apoptosis when added to a variety of lymphoid cells. These and other results revealed a model in which IL-3 deprivation activates 24p3 transcription, leading to synthesis and secretion of 24p3, which induces apoptosis through an autocrine/paracrine pathway. We have isolated the 24p3 cell surface receptor (24p3R) and found that 24p3 induces apoptosis through a novel pathway culminating in a decrease in intracellular iron levels (a biological iron chelator). Interestingly, iron chelators inhibit cellular proliferation and induce apoptosis, and are under active investigation as chemotherapeutic agents. The basis by which decreased intracellular iron induces apoptosis is not well understood. We performed expression-profiling experiments to identify differentially regulated genes in 24p3 and as a control in Deferoxamine (DFO), a synthetic iron chelator, treated cells. Our preliminary results suggest that 24p3 activates the expression of a novel gene, ING-2 (inhibitor of growth-2). ING-2 prevents cell growth by inducing cell cycle arrest at the G2/M phase. In contrast, the synthetic iron chelator, DFO activates the expression of NDRG1 (n-Myc downstream-regulated gene 1), which induces cell cycle arrest at G0/G1 phase. These results suggest that 24p3 induces cell death by activating regulators of the cell cycle. Finally, we have also found that the oncogene BCR-ABL counteracts the 24p3 proapoptotic pathway by misregulating expression of 24p3 and 24p3R. To study the contribution of 24p3 apoptotic pathway in the progression of CML, we have performed CML modeling experiments in mice. BCR-ABL transformed 24p3 deficient bone marrow cells failed to induce myeloproliferative disease in recipients upon transplantation. However, wild-type bone marrow cells when transduced with BCR-ABL oncogene readily induced CML-like disease in transplanted mice. Therefore, the secretion of 24p3 by BCR-ABL transformed cells facilitates the progression of CML. We have also demonstrated that 24p3 plays an important role in Gleevec-induced cell death in BCR-ABL transformed cells. These studies have therapeutic implications for Gleevec resistant CML.
Acidic Leucine-Rich Nuclear Phosphoprotein 32 Family Member B (ANP32B), a Novel Caspase-3 Substrate, Exerts Anti-Apoptotic Effects on Acute Myeloid Leukemic Cells.
Authors: Yu Yun , Shen Shao-Ming , Wang Li-Shun , Zhao Qian , Chen Guo-Qiang
Published: April 2020

The acidic leucine-rich nuclear phosphoprotein 32B (ANP32B, also called APRIL) is a member of a conserved superfamily of nuclear proteins that includes ANP32A/pp32, a factor that binds histones and inhibits their acetylation and regulates cell growth and differentiation in a tissue-specific manner. Recently, ANP32B was identified as a novel histone chaperone, and it can interact with the transcription factor KLF5, leading to transcriptional repression of a KLF5-downstream gene through stimulation of promoter region-specific histone incorporation and inhibition of histone acetylation. Additionally, ANP32B and/or ANP32A also serve as adaptor molecules linking the HuR nucleocytoplasmic shuttle protein and the nuclear export receptor CRM1 to regulate the cytoplasmic accumulation of some transcripts such as c-fos and CD83. However, its biological activity is still poorly understood. By the two-dimensional electrophoresis plus MALDI-TOF/TOF tandem mass spectrometry-based analysis of subcellular protein expression profiles, we identified ANP32B protein to become a small fragment in the cytosols of apoptotic leukemic cell line induced by NSC606985, a camptothecin analog. The ongoing immunoblot analyses confirmed that ANP32B protein was cleaved during cellular context-independent and caspase-3 activation-dependent apoptosis induced by etoposide, doxorubin and arsenic trioxide besides NSC606985. Further in vitro proteolytic experiments supported that ANP32B is a direct substrate of caspase-3, and the site-directed mutagenesis analysis identified the unclassical aspartate (AEVD163) of ANP32B sequence to be the caspase-3 targeted sites. Thus, we investigated the potential role of ANP32B in apoptosis induction. Our results showed that the suppression of ANP32B expression by siRNA in acute myeloid leukemic cell line U937 cells strongly enhances NSC606985 and etoposide-induced apoptosis. Based on these findings, this work also analyzed molecular mechanism of anti-apoptotic effect of ANP32B, and some interesting findings were confirmed.
In Childhood ALL, Both Blasts with a CD20−/Low and a CD20High Immunophenotype Have the Ability to Transfer the Leukemia Onto Immune-Deficient NOD/Scid Y−/− Mice.
Authors: Wilson Kerrie , Rehe Klaus , Bomken Simon , Case Marian , Shultz Leonard , Irving Julie , Vormoor Josef
Published: April 2020

There is an ongoing controversy as to whether cancer is always maintained by a rare population of highly specialized cancer stem cells or whether cancer-propagating cells may be more abundant in some cancer types. We have previously shown that in a heterogeneous group of childhood ALL different blast populations, regardless of their expression of the progenitor/stem cell marker CD34 or the lymphoid differentiation antigen CD19, contain leukemia-initiating activity (Cancer Cell 2008, 14(1), 47–58). By profiling B cell transcription factor expression, these different populations appeared to mirror stages of normal B cell development. Here we extend our experiments to another lymphoid differentiation marker, CD20, to provide further evidence that ALL blasts at different stages of maturation possess the ability to re-initiate the leukemia.

Patient Transplant Mice Population Cell dose Engrafted L736 Secondary 9 CD20High 10.000–100.000 6 11 CD20−/Low 10 Tertiary 4 CD20High 10.000 4 4 CD20−/Low 1 L754 Primary 4 CD20High 100.000 2 4 CD20−/Low 3 Secondary 11 CD20High 5.000-100.000 9 11 CD20−/Low 7 A67 Secondary 6 CD20High 9.000-20.000 6 6 CD20−/Low 6

Unsorted bone marrow cells from 3 different ALL patients (L736, L754 and A67) were transplanted into 12 primary mice. Bone marrow was harvested from leukemic mice and flow sorted candidate populations (CD19+CD20−/Low and CD19+CD20High) were re-transplanted into 52 secondary and 8 tertiary mice. As expected from our previous experiments both CD19+CD20−/Low and CD19+CD20High cells were able to re-establish the disease in unconditioned NOD/scid y−/− mice (see table). Leukemic engraftment ranged from 0.5 to 73% as determined by flow cytometry on bone marrow aspirates. Both populations re-established the complete phenotype of the original leukemia including CD20−/Low and CD20High blasts. These results were confirmed by directly sorting primary ALL blasts from L754 without prior passage in the mice. Cell purity after flow sorting was high (81–99%) and low numbers of cells engrafted (5000 for both CD19+CD20−/Low and CD19+CD20High). The three patients reflected different ALL subtypes (L736 intermediate risk ALL: high WBC/MRD low risk, L754:high hyperdiploid/MRD high risk; and A67: high risk ALL/t(9;22)).

In conclusion, these results confirm our previous observation that ALL blasts irrespective of the expression of lymphoid differentiation markers are able to engraft immune-deficient mice. Therefore, leukemia-propagating cells in childhood ALL may be more abundant than previously thought.
A Cell-Autonomous Defect of Bone Marrow Hematopoietic Stem/Progenitor Cells and Impaired Stromal Function in FA-a Patients
Authors: Zhang Xiaoling , Shang Xun , Guo Fukun , Murphy Kim , Kirby Michelle , Kelly Patrick , Reeves Lilith , Smith Franklin O , Williams David A. , Zheng Yi , Pang Qishen
Published: April 2020

Fanconi anemia (FA) is a devastating blood disease associated with bone marrow (BM) failure. Currently the effective treatment for FA is BM transplantation. We and others showed that FA murine hematopoietic stem and progenitor cells (HSC/Ps) have defective hematopoietic reconstitution after BM transplantation. In addition, several clinical gene therapy trials in FA patients have failed to show sustained engraftment of FA HSC/Ps complemented with a functional FA gene. The mechanism underlying this engraftment defect remains unknown. To determine whether the poor engraftment of FA HSC/Ps observed in mouse knockout models and clinical gene therapy trials in FA patients results from an intrinsic defect of the FA HSC/Ps or an impaired microenvironment in the FA bone marrow, we have assessed the self-renewal ability of HSC/Ps and hematopoietic supportive capacity of BM stromal cells from FA-A patients. First, we performed quantitative cobblestone area-forming cell (CAFC) assays using MS-5 stromal layer. CAFCs were enumerated at 2 and 5 weeks of cocultures between BM mononuclear cells from a normal donor and three FA-A patients. Early-appearing CAFCs (week 2) represent transient repopulating cells equivalent to progenitor colony-forming units (CFUs), whereas late-appearing CAFCs (week 5) are representative of long-term repopulating stem cells. The frequency of FA-A CAFCs was 15–20 folds decreased compared with that of the normal donor at both week 2 and week 5. Second, we carried out long-term culture-initiating cell (LTC-IC) assays using BM mononuclear cells and BM-derived stromals from normal donors or FA-A patients. Compared to coculture with normal stromal cells, the recovery of colony forming cells (CFCs) that grew on FA-A stromal layer after 5–6 weeks incubation was significantly decreased for either normal or FA-A HSC/Ps (41.4% ± 3.1 CFC in normal HSC/Ps - normal stroma compared to 14.3% ± 1.6 CFC in normal HSC/Ps – FA-A stroma and 2.4 ± 0.6 CFC in FA-A HSC/Ps- FA-A stroma). Finally, we conducted CFC and cell-cell adhesion assays using BM-derived stromal cells from normal donors and FA-A patients. We observed that FA-A BM stroma failed to support HSC/P cells from either normal donors or FA-A patients. Specifically, severe adhesion defect was found in adhesion of normal and FA-A HSC/Ps with FA-A stromal layer (5.5% ± 0.6 CFC adhesion in normal HSC/Ps - normal stroma compared to 2.4% ± 0.5 CFC adhesion in normal HSC/Ps – FA-A stroma and 1.0 ± 0.7 CFC adhesion in FA-A HSC/Ps- FA-A stroma). These results suggest that both a cell-autonomous defect of FA HSC/Ps and an impaired FA BM microenvironment may contribute to the abnormal hematopoiesis in FA.
Phosphorylation of Runx1 by Cyclin-Dependent Kinases Regulates Its Interaction with HDAC1 and HDAC3.
Authors: Guo Hong , Friedman Alan D.
Published: April 2020

Runx1/AML1 is a key regulator of hematopoiesis and leukemic transformation, as RUNX1(−/−) mice do not develop definitive hematopoietic stem cells, and sever alleukemic oncogenes, e.g. AML1-ETO, CBFβ-SMMHC, or TEL-AML1, inhibit Runx1activities. We have investigated regulation of cell cycle progression by Runx1. Runx1stimulates G1 to S cell cycle progression in hematopoietic cell lines and in transduced myeloid progenitors, and inhibition of Runx1 by CBFβ-SMMHC or AML1-ETO slows G1 progression. Runx1 induces cdk4 and cyclin D3 transcription, and exogenous cdk4, cyclin D2, or c-Myc overcomes inhibition of G1 progression by CBF oncoproteins. In addition to regulating cell cycle progression, Runx1 protein levels are themselves increased as hematopoietic cells progress from G1 to S to G2/M, though mRNA levels remain constant. Runx1 contains three consensus cdk sites, (S/T)PX(R/K), S48, S303, and S424, and using phospho-specific antisera we find that each of these is modified in hematopoietic cells. Mutation of these serines to aspartic acid, mimicking phosphorylation, increases trans-activation of a reporter containing four CBF sites or the TCRβ promoter, whereas mutation to alanine reduces trans-activation. p300 interacts similarly with Runx1(tripleA) and Runx1(tripleD). We have now evaluated interaction of HDACs1–8 with these variants and Runx1 and find that both HDAC1 and HDAC3 have reduced affinity for RUNX1(tripleD), as assessed by co-immunoprecipitation from transiently transfected 293T cells. Evaluation of single serine residue mutants (S48D, S303D, and S424D) demonstrates reduced affinity of HDAC1 or HDAC3 specifically for the Runx1(S424D) mutant, consistent with previous mapping of the Runx1:HDAC1 and Runx1:HDAC3 interactions to this region of Runx1. Thus, cdk phosphorylation of Runx1 S424 reduces affinity for HDAC1 and HDAC3, increasing Runx1 trans-activation potency. Regulation of Runx1 activity by cdks may control key developmental processes, including expansion of definitive HSC during development and regulation of the balance between adult HSC quiescence and proliferation.
Cannabinoid Receptors CB1 and CB2 Regulate Mobilization of Hematopoietic Stem and Progenitor Cells.
Authors: Avraham Hava , Zagozdzon Radoslaw , Fu Yigong , Groopman Jerome E. , Jiang Shuxian
Published: April 2020

Endocannabinoids are lipid mediators that modulate central and peripheral neural functions as well as immune responses and bind to specific G-protein-coupled receptors:CB1 and CB2. Here, we investigated the role of CB1/CB2 and cognate cannabinoid ligands in hematopoietic stem and progenitor cell (HSPC) mobilization. CB1 and CB2receptor expression was observed in human and murine HSPCs. Cannabinoid ligands induced the chemotaxis of the human CD34+ cells and caused mobilization of murine HSPC in vivo. Inhibition of degradation of the endocannabinoid anandamide by URB597induced mobilization of HSPC and significantly augmented mobilization of these cells when URB597 was administered with G-CSF-, AMD3100- and with the combination of G-CSF+AMD3100. Moreover, the cannabinoid- or G-CSF-induced mobilization of HSPC was significantly impaired by cannabinoid receptor inhibitors and in CB1- andCB2-knockout mice. Administration of exogenous cannabinoid agonists in mice induced mobilization of progenitors as well as stem cells in vivo with long-term repopulating ability. These results demonstrate a novel pathway by which the cannabinoid system regulates HSPC cell trafficking, and may be therapeutically applied in clinical conditions, such as bone marrow transplantation.
Deletion of Fak in Hematopoietic Stem Cells Leads to Enhanced Engraftment.
Authors: Lu Jiayun , Nombela-Arrieta Cesar , Park Shin-Young , Le Yi , Chai Li , Silberstein Leslie E.
Published: April 2020

Hematopoietic stem and progenitor cells (HSPC) can be induced to leave the bone marrow (BM) and such mobilized hematopoietic stem cell (HSC) enriched cell populations when infused intravenously rapidly home back to the marrow and transmigrate into the extravascular compartment where they lodge in specialized niches. Niche induced signals coupled to intrinsic factors, promote HSC maintenance and regulate HSC localization, migration and/or differentiation. A better understanding of molecular pathways governing these HSC fate decisions could identify new targets for ex vivo HSC expansion and improvement of transplantation. Focal adhesion kinase (Fak) is a non-receptor protein tyrosine kinase, whose function has been well studied in fibroblasts, where it plays an important role in cell adhesion, survival and motility. Fak is also differentially expressed in HSPC, with highest expression in long-term HSCs (LT-HSCs, Lin-Sca-1+c-Kit +CD34-). Its functional role in hematopoiesis has not been well studied due to early embryonic lethality (E.8) of Fak−/− mice. We have generated conditional Fak deleted mice (Fakfl/flMx1-Cre+) to define how Fak signaling contributes to different components of HSC transplantation. We observed that the total cellularity in BM was comparable between Fak knockout (Fak−/−Mx1-Cre+) and control (Fakfl/fl) mice. The frequencies of both Lin-Sca-1+c-Kit + cells (LSKs) and short-term HSCs (ST-HSCs, Lin-Sca-1+c-Kit +CD34+) increased 2-fold in BM of Fak knockout mice compared to WT controls (n=5; p<0.05), while LT-HSCs appeared unaffected. Moreover, the number of LSKs in the spleens of Fak knockout mice increased by 3-fold relative to controls (n≥7, P<0.01). In vitro colony-forming assay also showed that the functionally defined progenitor cell numbers significantly increased in Fak knockout mice spleen. However, Fak deletion did not affect HPC populations including common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), megakaryocyte erythrocyte progenitors (MEP) and common lymphoid progenitors (CLP). Importantly, in the competitive repopulation assay Fak−/− HSCs exhibited 2-fold enhanced engraftment for 20 weeks post-transplant, while lineage reconstitution in peripheral blood (PB) was similar between wild-type and knockout donor-derived cells suggesting Fak deletion did not affect PB mature B, T and myeloid cell survival under non-stress conditions. These findings indicate that FAK may play an important role in HSC homeostasis and that targeting the FAK pathway ex vivo may lead to innovative approaches in human HSPC transplantation.
Intercommunication Between C-Myc and PTEN in the Regulation of Hematopoietic Stem Cell-Niche Interactions and Hematopoietic Progenitor Cell Lineage Commitment.
Authors: Guo Yinshi , Niu Chao , Breslin Peter , Zhang Shubin , Wei Wei , Tang Minghui , Zhang Jiwang
Published: April 2020

It was shown that c-Myc is required for the proliferation and differentiation of hematopoietic stem cells and progenitors. Mice with c-Myc deletions develop severe anemia and cytopenia. However, hematopoietic stem cells (HSCs) accumulate in significant numbers in the bone marrow (BM) of mutant mice, probably due to an increase in adhesiveness of the mutant HSCs to BM osteoblastic niche cells. Previously, we demonstrated that PTEN might play a critical role in the process of adhesion of HSCs to BM niche cells. Deletion of PTEN results in the proliferation and peripheral mobilization of HSCs, followed by a decline in these cells. PTEN mutant mice proceed to develop myeloproliferative disorders.

Here we report that c-Myc also has an essential role to play in regulating the lineage commitment of HSCs and progenitors (HSC/Ps). HSC/Ps in which c-Myc is deleted are biased toward megakaryocytic lineage differentiation at the expense of other lineages. c-Myc knockout mice develop significant thrombocytosis (3- to 5-fold increase in peripheral platelet numbers) due to an obviously increased megakaryopoiesis in BM and spleen. PTEN deletion causes down-regulation of expression of adhesive molecules, including integrins and N-cadherin, in HSCs, resulting not only in an increased mobilization of c-Myc-mutant HSCs from the BM niche but also mobilization of c-Myc-mutant megakaryocytic progenitors to the spleen. We found that HSCs and megakaryocytic progenitors are significantly reduced in BM but dramatically increased in the spleens of PTEN/c-MYC double-knockout mice, compared to c-Myc single-gene knockout mice. In addition, PTEN deletion further promotes megakaryocytic progenitor cell proliferation, as well as infiltration of these cells into the liver. PTEN/c-Myc double-gene knockout mice consistently develop megakaryocytic proliferative disorders.

We conclude that the ability of c-Myc to regulate HSC-BM niche interactions is at least partially accomplished through inhibition of PTEN function by c-Myc. In addition, c-Myc controls the lineage commitment of HSC/Ps. Deletion of c-Myc converts the myeloproliferative disorder seen in PTEN knockout mice to a megakaryocytic proliferative disorder. Whether PTEN and c-Myc mutations are likewise etiologically involved in human megakaryocytic proliferative disorders and megakaryocytic leukemia is currently a topic of active investigation in our laboratory.
Wiskott-Aldrich Syndrome Protein (WASP) Is An Effector of Kit Signaling.
Authors: Mani Maheswaran , Venkatasubrahmanyam Shivkumar , Sanyal Mrinmoy , Yang Yujun , Huang Jing , Levy Shoshana , Butte Atul , Weinberg Kenneth I. , Jahn Thomas
Published: April 2020

Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive immunodeficiency disorder with eczema, thrombocytopenia and high susceptibility to opportunistic and pyogenic infections. The gene product of the WAS locus, WAS protein (WASP), is expressed in a hematopoietic-specific fashion and regulates cytoskeletal actin reorganization via Cdc42 and Arp2/3 interactions. Non-random inactivation of the X chromosome carrying the defective WASP gene in all peripheral blood cells from obligate female carriers of WAS suggests a selective advantage of hematopoietic stem cells or immature progenitor cells expressing the intact WASP gene. Because such progenitor cells are dependent on the Kit receptor/Kit ligand (KL) pathway, we tested whether or not WASP plays a role in signaling responses through Kit. WASP and interacting proteins WIP and Arp2/3 were strongly phosphorylated in response to KL stimulation of Mo7e cells. Time kinetics revealed onset of tyrosine phosphorylation of WASP as early as 1 min and a maximum at 5 min after KL stimulation. Although real-time KL-induced actin reorganization and KL-mediated spreading of bone marrow-derived mast cells (BMMC) on fibronectin-coated surfaces were grossly normal, KL-induced formation of filopodia was significantly decreased in number and size in the absence of WASP. In addition, KL-induced calcium-flux in BMMCs was aberrant in the absence of WASP suggesting that KL-dependent calcium signals and cytoskeletal rearrangement are linked through WASP. When BMMC cultures were established from WASP heterozygous female mice using KL as a growth factor, the cultures initially contained a mixture of WASP positive and negative populations. KL-driven differentiation into mature BMMCs eventually resulted in homogenous WASP positive cultures derived from the WASP positive progenitors. Thus, WASP expression conferred a selective advantage to the development of Kit-dependent hematopoiesis consistent with the selective advantage of WASP positive blood cells observed in WAS heterozygous female humans. Finally, KL-mediated gene expression in BMMCs derived from WASP negative mice or WT controls was compared and revealed in summary that at least 30% of all changes are WASP-dependent. The results indicate that WASP is downstream of Kit signaling and necessary for Kit-mediated filopodia formation, cell survival and gene expression.
Elevated Circulating Stromal Derived Factor-1 Levels in Sickle Cell Disease.
Authors: Landburg Precious , Nur Erfan , Maria Naomi , Biemond Bart J. , Brandjes Dees P.M. , Schnog John-John , Duits Ashley
Published: April 2020

Inflammation and angiogenesis are of importance in the pathophysiology of sickle cell disease (SCD). Recently, the chemokine Stromal Derived Factor-1 (SDF-1) has been shown to be a key mediator of angiogenesis and inflammation. In this study we determined serum SDF-1 levels in consecutive adult sickle cell patients during the clinically asymptomatic state as well as during painful crisis. Serum SDF-1 levels were significantly elevated in HbSS/HbSβ0-thalassemia patients ([n=39], 3805 pg/mL (2121–6790)) as opposed to HbSC/HbSβ+-thalassemia patients ([n=18], 2405 pg/mL (1365–3047)) and healthy HbAA controls (n=25, 2623 pg/mL (2435–2988)) (p=0.04). During painful crisis SDF-1 levels increased significantly in both HbSS/HbSβ0-thalassemia ([n=24]) 26040 pg/mL (20135–27960)) and HbSC/HbSβ+-thalassemia patients ([n=5], 12666 pg/mL (3936–24453)) (p<0.001 and p=0.01, respectively). Paired sample analysis was possible in 8 patients (SDF-1 determined in steady state and during painful crisis) and all patients demonstrated a strong SDF-1 increment from steady state levels (p=0.01) and serial measurements, available from 11 patients, showed persistently elevated SDF-1 levels well beyond crisis abatement (data not shown). Furthermore, SDF-1 levels were significantly higher in sickle cell patients with pulmonary hypertension as compared to patients without pulmonary hypertension in both HbSS/HbSβ0-thalassemia (PHT+ [n=8]: 6549 pg/mL (3893–7159), PHT- [n=24]: 2945 pg/mL (1560–6627)) and HbSC/HbSβ+-thalassemia (PHT+ [n=4] 3316 pg/mL (2900–6417) and PHT- [n=12] 2146 pg/mL (1138–2592)) patients (p=0.02 and p=0.01, respectively). SDF-1 levels were not related to other forms of organ damage, nor were they significantly associated with hemoglobin levels, the percentage of fetal hemoglobin or leukocyte counts in the clinically asymptomatic state (data not shown). Taken together, elevated circulating SDF-1 levels occur in patients with SCD and may play a role in the pathophysiology of acute and specific chronic disease related complications.
WIP Is a Chaperone for Wiskott-Aldrich Syndrome Protein.
Authors: Sasahara Yoji , de la Fuente Miguel A , Ramesh Narayanaswamy , Tsuchiya Shigeru , Geha Raif S
Published: April 2020

WASP, the product of the gene mutated in Wiskott-Aldrich syndrome (WAS), plays a critical role in T cell activation and actin reorganization. WIP is a WASP-interacting protein that binds to Ena-VASP homology 1 (EVH1) domain of WASP. We previously reported functional roles of WIP in the recruitment and activation of WASP in T cells. To investigate a role of WIP in WASP degradation and understand the reason why most missense mutations in WAS patients are in EVH1 domain of WASP, we examined WASP levels in WIP knockout (WIP−/−) T cells and mechanisms of WASP degradation following T cell receptor (TCR) ligation. WASP protein levels, but not mRNA levels, were severely diminished in T cells from WIP−/− mice and were restored by introduction of full-length or WASP binding domain of WIP. A fraction of WASP was cleaved by Ca-dependent protease calpain, ubiquitinated and degraded by the proteasome following TCR ligation. Inducible expression of WASP binding domain of WIP increased WASP levels in vivo and recombinant WIP protein protected WASP from degradation by calpain in vitro. Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP protein levels in T cells from WIP−/− mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupted WIP binding. The calpain inhibitor calpeptin also increased WASP protein levels in activated T cells and B cells from the WAS patients. Despite its ability to increase WASP levels, proteasome inhibitors failed to correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient (published in PNAS, 104, p926–931, 2007). These results demonstrate that WIP stabilizes WASP in normal T cells and that WIP deficiency or impaired WASP-WIP interaction in WAS patients with missense mutation in WIP binding site results in WASP degradation.
Notch Pathway Activation by a Combination of Activating NOTCH1-Receptor and Inactivating FBXW7 Mutations Robustly Predicts Excellent Early Treatment Response in Pediatric T-ALL.
Authors: Kox Corinne , Leible Svenja , Breit Stephen , Stanulla Martin , Schrappe Martin , Muckenthaler Martina Ulrike , Kulozik Andreas
Published: April 2020

In contrast to pre B-ALL, robust DNA-based biomarkers with prognostic and predictive value are not available in T-ALL. We have previously shown that common activating mutations of the NOTCH1 receptor predict good early treatment response and excellent long term outcome in T-ALL (Breit et al., Blood 2006). We now aimed at analyzing if recently described inactivating mutations of FBXW7, the E3 ubiquitin ligase of NOTCH, contribute to the predictive power of the Notch1 pathway activation in pediatric T-ALL.

Figure Figure

We genotyped FBXW7 in 137 T-ALL and found inactivating mutations in 22 patients. We next analyzed the influence of this mutation on treatment response and found that most patients with FBXW7 mutations showed a prednisone good response (PGR; 19/22), whereas PGR was observed in only 58/115 with no FBWX7 mutation (p=0.0006). This beneficial effect of FBXW7 inactivating mutations is enhanced when the NOTCH1 mutational status was considered. Among 17 patients with NOTCH1 and FBXW7 mutations, only 1 showed a prednisone poor response (PPR; p=0.0003). These results suggest a synergistic effect of NOTCH1 and FBXW7 mutations in early treatment response in pediatric T-ALL.

Notably, FBXW7 mutations occur in the group of leukemias with NOTCH1 heterodimerisation (HD) domain mutations only and do not occur in those with truncations of the PEST domain, where FBXW7 physiologically targets NOTCH for degradation. One might thus predict that patients with NOTCH1-HD/PEST double mutations would be biologically similar to those with NOTCH1-HD/FBXW7 double mutations. A comparison of early treatment response between these groups indeed showed PGR in 10/13 patients with NOTCH1-HD/PEST and 16/17 with NOTCH1-HD/FBXW7 status. Furthermore, these groups taken together exhibit a significantly better prednisone response than both, the patients with single mutations in either NOTCH1 or in FBXW7 or those with detectable mutations in neither NOTCH1 nor FBWX7 (see bar diagram).

These data indicate that activation of the Notch1 pathway by a combination of receptor gain of function mutations and inactivating mutations of its E3 ubiquitin ligase has a strong favorable effect on in-vivo prednisone sensitivity. Notch pathway activation may thus define a novel group of biomarkers that predict early treatment response and thus help to stratify risk of children and adolescents with T-ALL.
Negative Effect of KIR Alloreactivity in Recipients of Umbilical Cord Blood Transplantation Depends on Transplantation Conditioning Intensity
Authors: Brunstein Claudio , Wagner John E. , Weisdorf Daniel , Cooley Sarah , Noreen Harriet , Barker Juliet , DeFor Todd , Verneris Michael , McGlave Philip B. , Blazar Bruce R. , Miller Jeffrey S.
Published: April 2020

Transplant strategies involving natural killer (NK) cell alloreactivity (KIR-ligand mismatch [KIR-L mismatch]) have demonstrated superior outcomes for patients receiving T cell depleted HLA haploidentical hematopoietic stem cell allografts. It is unknown whether KIR-L mismatch has a similar effect in recipients of partially HLA-matched umbilical cord blood (UCB) grafts which contain comparatively few T-cells. We examined the clinical impact of KIR-L mismatch in 257 UCB recipients treated with either a myeloablative (n=155) or reduced intensity (n=102) regimen. After myeloablative conditioning, KIR-L mismatch had no demonstrable effect on grades III–IV acute GVHD (17% [CI, 6–28%] vs. 17% [CI, 10–24], p=.97), transplant-related mortality (TRM) (27% [CI, 14–40%] vs. 18% [CI, 11–25%], p=.19), relapse at 2 yrs (18% [95%CI, 6–30%] vs. 28% [95%CI, 19–27%], p=.37) and survival at 3 yrs (50% [CI, 32–68%] vs. 57% [CI, 47–67%], p=.46). In contrast, following reduced intensity conditioning when the engrafting unit was KIR-L mismatched there was a significantly higher incidence of grades III–IV acute GVHD (42% [CI, 27–59) vs. 13% [CI, 5–21], p < .01). Multivariate analysis confirmed NK cell alloreactivity as the only predictive factor associated with severe acute GVHD was (RR 1.8, CI [1.1–2.9]; p=.02). TRM was higher (27% [CI, 12–42%] vs. 12% [CI, 5–19%], p=.03) and three year survival was poorer (32% [CI, 15–59%] vs. 52% [CI, 47–67%], p=.03) when the engrafting unit was KIR-L mismatched, but relapse was unaffected (39% [95%CI, 21–57%] vs. 47% [95%CI, 34–60%], p=.72). KIR-L mismatch in recipients of a RIC UCB transplant was associated with a significant increased risk of death (RR 1.8, 95%CI, 1.0–3.1, p=.05). This data identify divergent effects of NK cell alloreactivity which are unmasked when comparing myeloablative versus RIC transplant platforms. We conclude that KIR-L mismatch should be avoided in recipients of a reduced intensity UCB transplant.
Clinical Implications of Killer Cell Immunoglobulin-Like Receptor (KIR) Genotypes on Autologous Peripheral Blood Stem Cell Transplantation for Acute Myelogenous Leukemia Patients.
Authors: Kim Heeje , Choi Young , Lee Tai-Hyang , Hahn Dong-Hoon , Gang Hoei-Sung , Min Woo-Sung
Published: April 2020

Natural killer (NK) cell alloreactivity after allogeneic hematopoietic stem cell transplantation (HSCT) in various settings is introduced by the regulatory role of killer cell immunoglobulin-like receptors (KIR). However, no influence of NK cells on relapse or survival after autologous HSCT for patients with acute myelogenous leukemia (AML) has been demonstrated. Since it is still debated whether autologous NK cells are involved in the eradication of leukemia cells, we evaluated the role of NK cells as a susceptibility factor for long-term survival. All 67 enrolled patients who received autologous HSCT were typed for the presence or absence of 19 different KIR genes and pseudogenes using a polymerase chain reaction-based sequence-specific primer method with the Pel-Freez kit, according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). The median follow-up was 21 months (range 2–55). All patients were in first complete remission (CR), as assessed immediately before the elective transplantation. The median age of the enrolled patients was 42 years (range 16–68). There were 33 females and 34 males. All patients received our modified TAM conditioning, which consisted of fractionated total body irradiation (12 Gy, five fractions in 3 days) from day −8 to −6, followed by intermediate-dose cytarabine (Ara-C; 1.5 g/m2 over 3 h every 12 h for six doses) from day −5 to −3, and melphalan (100 mg/m2 over 30 min) on day −2 only. The peripheral blood stem cells were collected after administering granulocyte colony-stimulating factor, following consolidation chemotherapy. The patients had 7–13 KIR genes, which is similar to the range in the normal population. The median infused cell dose was 4.2 × 106/kg (range 1.7–6.3) CD34+ cells. Fifty patients were alive (75%). Of the 67 patients, 43 were in continuous CR (64%) and 14 died in relapse (21%), while 3 (4%) died due to non-relapse causes. The Kaplan-Meier estimated 4-year disease-free survival (DFS) rate was 55% (95% CI 47–62%). We evaluated whether the presence or absence of any single or combination of inhibitory or activating KIR genes in the patients affected DFS and eventfree survival (EFS). The presence of an activating KIR 2DS1 gene was critical for the DFS and EFS, as shown in a previous study of allogeneic HSCT (Kim et al., Transplantation 2007). Additionally, the presence of two activating genes in the combinations 2DS3-2DS4*003 or 2DS4*003-2DS5 tended to be associated with DFS (P=0.0866). Since our findings are based on a single disease entity, i.e., adult AML, they suggest that the presence of activating KIR genes may, at least in some ways, be critical for autologous NK immunomodulation to influence the anti-leukemic effects, especially considering the impact of activating KIR genotypes on long-term survival.
Dissection of the Human Pre-B Cell Receptor (pre-BCR) Signalling Cascade.
Authors: Lassoued Kaiss , Ibata Stella , Piec Isabelle , Alleaume Celine , Brassard Bertrand , Nyga Remy , Fuentes Vincent , Bouhlal Hicham
Published: April 2020

Pre-BCR acts as a critical checkpoint guards in pre-B cell selection and expansion. There is also an increasing body of evidence suggesting a role for this receptor in leukemogenesis. However, the molecular events that follow its stimulation remain largely unknown. Most of the functions that have been so far attributed to this receptor are speculative or deduced from genetic studies conducted in mice, using either knock out or transgenic mice, which makes difficult to figure out whether these functions are directly or indirectly triggered via pre-BCR stimulation. Moreover, one should also be cautious in extrapolating data from mice to humans, as many discrepancies in B cell development have been reported between both species. We extensively analyzed herein the pre-BCR signalling machinery using the human 697 and Nalm6 pre-B cell lines and F(ab′)2 anti-human heavy chain μ to stimulate pre-BCR. We showed that stimulation of this receptor results in a rapid phosphorylation of Lyn, Blk, Syk, BLNK, LAB as well as adaptors that were so far considered as T-cell specific, together with Vav, Btk, PLCγ2 and Akt, which in turns activates FKHRL and GSK3. It also induces activation of Ras and the 3 major downstream arms of the Ras/MAPKinase pathway as well as number of transcription factors that will be further discussed. Its stimulation is followed by cell cycle progression and proliferation that are associated with p27kip1 degradation and Rb phosphorylation and with c-Myc upregulation. Moreover, pre-BCR aggregation induces phosphorylation of Bcl2 on residue serine 70 suggesting that this receptor mediates anti-apoptotic signal. In addition, CD19 coligation was found to potentiate pre-BCR signalling capabilities. c-Cbl and SHIP1 are rapidly phosphorylated upon pre-BCR aggregation, probably acting as negative regulators of this receptor signalling. We bring also evidence that Syk plays a pivotal role in pre-BCR signalling cascade and functions. Altogether these findings demonstrate that pre-BCR is directly implied in the control of proliferation and survival signals in pre-B cells and describes the multiple molecular events involved in these processes. We also show that pre-BCR stimulation induces downmodulation of rag1/2 and zap70 gene transcription but not of λ5, vpre-b, CD79a, E2A and EBF. Besides its major interest in understanding the regulation of the first steps of B cell development, analysis of pre-BCR associated-signalling pathways in leukemic B cell progenitors would certainly be helpful to better characterize the ongogenic events and identify new therapeutic targets in acute lymphoblastic leukemia.
Functional IDO Is Expressed on CD34+- and Monocyte-Derived Dendritic Cells According to Differentiation Status.
Authors: Curti Antonio , Trabanelli Sara , Onofri Chiara , Rutella Sergio , De Cristofaro Raimondo , Baccarani Michele , Lemoli Roberto M
Published: April 2020

Indoleamine 2,3-dioxigenase (IDO) is the rate-limiting enzyme in tryptophan catabolism along the kynurenine pathway. IDO expression by different cell subsets inhibits T-cell activation, proliferation and survival and induces regulatory T cells (Tregs). Although human monocyte-derived dendritic cells (Mo-DCs) have been shown to express IDO, little is known about the expression of IDO in other subsets of human DCs, including those generated from CD34+ hematopoietic progenitors (CD34+-derived DCs) In the present study, we performed a full characterization of IDO expression and function by human dendritic cells including Mo-DCs and CD34+-derived DCs. Mo-DCs were generated from purified CD14+ monocytes after culture with GM-CSF and IL-4 and then matured with CD40L, LPS alone, LPS plus IFN-gamma and a cytokine cocktail (IL-1beta, TNF-alpha, IL-6, PGE2). CD34+-derived DCs were generated from purified CD34+ cells after 7 days of culture with GM-CSF and TNF-alpha, followed by 7 day-treatment with GM-CSF and IL-4 and then matured with the same stimuli used for MoDCs. After culture, DCs were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogenic proliferation and Tregs induction. Monocytes lack IDO expression. Immature Mo-DCs have little if any expression of IDO. During maturation, the cytokine cocktail is the most effective in up-regulating IDO, both at mRNA and protein level, which is paralleled with higher kynurenine production and inhibition of allogeneic T cell proliferation. PGE2 has a crucial role in inducing IDO expression, while IL6 has an opposite effect. Mature Mo-DCs are shown to generate a population of CD4+CD25+FOXP3+ Tregs which are capable of suppressing allogeneic T-cell proliferation. This inhibitory effect is abrogated by the addition of the IDO inhibitor 1-methyl tryptophan (1-MT). CD34+ cells lack IDO mRNA expression. During DC differentiation, IDO expression is observed at day 7, but not at day 14. Flow cytometry analysis of day 7 cells reveal a population of CD34−, CD14+, CD1a+/− cells. Similarly to Mo-DCs, maturation stimuli induce a marked up-regulation of IDO mRNA. Similar results are observed when IDO protein is measured. Production of kynurenine, inhibition of allogeneic T cell proliferation and generation of Tregs from normal CD3+ T-cells are also paralleled with IDO expression. In conclusion, in human DCs the maturation status is associated with a differential expression of IDO both in Mo-DCs and CD34+-derived DCs. Differentiation of CD34+ cells into DCs results in a transient and early expression of IDO at precursor level. Given its functional capacity to inhibit T-cell-mediated immune response, the expression of IDO along DC differentiation may indicate a protective role of the pool of precursor cells which are committed to DC lineage.
Human Langerhans-Type Dendritic Cells Break Tolerance against the Tumor Antigen, WT1, by a Largely IL-15-Dependent Mechanism.
Authors: Romano Emanuela , Silva Rosa Barreira da , Chandrasekar Sharanya , Münz Christian , Heller Glenn , Young James W.
Published: April 2020

The Wilms’ tumor 1 (WT1) gene encodes a transcription factor essential for cell growth and development, but overexpression in many leukemias, myelodysplastic syndromes, and solid tumors correlates with poor prognosis. The WT1 protein could therefore serve as a therapeutic target, if tolerance could be broken against this self-differentiation, tumor-associated antigen (TAA). Vaccination protocols using peptide-pulsed dendritic cells (DCs) to induce anti-tumor responses present only defined epitopes restricted to certain human leukocyte antigens (HLA). In contrast, vaccination with mRNA-electroporated DCs supports processing and presentation of multiple TAA epitopes tailored to patient-specific HLA. Human Langerhans-type dendritic cells (LCs) derived from CD34+ hematopoietic progenitors are superior to other conventional DC subtypes, like monocyte-derived DCs (moDCs), in producing IL-15 to generate MHC-restricted, Ag-specific cytolytic T lymphocytes (CTL). Mature LCs transcribe 3 to 5-fold higher amounts of IL-15R-alpha mRNA and express the highest levels of IL-15R-alpha protein compared with other conventional DCs as measured by real-time RT-PCR, Western blot, and immunofluorescence microscopy (P<0.001 to 0.05, n=3–4 independent expts). Anti-IL-15R-alpha blockade also significantly reduces but does not completely eliminate the capacity of LCs to stimulate CTL. This indicates that LCs have a greater capacity than moDCs for presenting IL-15 in trans to stimulate lytic T cell responses. We therefore compared the ability of LCs and moDCs electroporated with WT1 mRNA to break tolerance against this self-differentiation TAA by generating CTLs against WT1+ tumor cells. After a single 7-day round of stimulation at T cell:LC ratio of only 10:1 to 30:1 and in the absence of exogenous IL-15, T cells stimulated by WT-1-electroporated LCs lyse 85–95% of a WT-1+ tumor cell line. MoDCs are completely incapable of generating WT-1-specific CTL under comparable conditions and require exogenous IL-15 10ng/ml to generate 70–80% specific lysis of WT-1+ targets. Lysis of a WT-1- HLA-A*0201+ target cell line or NK cell-sensitive LCL721.221 targets is at background levels (<5%) in all cases. Most importantly, WT-1-electoporated LCs also induce 63% lysis (+/− 11 SEM) of primary WT1+ HLA-A*0201+ blasts from a patient with AML after 7 days’ coculture at a T cell:LC ratio of 10:1 without exogenous IL-15. These results demonstrate a major role for the IL-15R-alpha-IL-15 complex on LCs in expanding anti-tumor cytolytic T cells and breaking tolerance against self-differentiation Ags like WT1. These data also support the use of IL-15-producing LCs in active immunization or expansion of T cells ex vivo for adoptive immunotherapy.