bluuebunny/crossref_metadata_2025 huggingface dataset:
GATA1 Mutations in Newborns with Down Syndrome.
Authors: Sandoval Claudio , Pine Sharon R. , Druschel Charlotte , Jayabose Somasundaram , Guo Qianxu , Yin Changhong
Published: April 2020

Somatic mutations of the GATA1 gene have been detected in almost all cases of Down syndrome (DS)-associated acute megakaryoblastic leukemia (AMKL) and transient leukemia (TL). There is emerging evidence that the protein product of GATA1 mutations, GATA1s, directly contributes to leukemogenesis. Although an in utero origin of GATA1 mutations is established, a comprehensive study using a large number of cases is required to determine the overall incidence and clinical relevance of GATA1 mutations in DS newborns. We screened 575 DS infants born between January 1997 and December 1999 for GATA1 exon 2 mutations by single-strand conformation polymorphism analysis of PCR products. We used Gunthrie cards obtained from the New York Congenital Malformation Registry. Registry data was blinded until after the GATA1 mutation analyses were completed. Twenty-eight (4.9%) infants were identified as having a GATA1 mutation. There was no significant difference in the frequency of GATA1 mutations based on gender or maternal average age (p = 0.93 and 0.31, respectively). There was no significant difference in the GATA1 mutation frequency between those classified as black, white, or Asian, but Hispanics had a borderline non-significant increase in frequency of GATA1 mutations compared to non-Hispanics (8.5% compared to 4.0%, p=0.06). Based on data from the New York Cancer Registry reviewed in 2005, two of the patients with a GATA1 mutation subsequently developed leukemia; one patient developed AMKL and the other had a leukemia of unspecified phenotype. Out of the 547 GATA1-negative patients, there was only one case of leukemia, but it was AML excluding AMKL. These results confirm a pre-natal origin of GATA1 mutations in DS patients. The frequency in this study was lower than the 10 percent previously reported (2 of 21 DS blood spots). Obtaining Gunthrie card blood spots for GATA1 mutation analysis serves as a relatively non-invasive screening approach. The presence of a GATA1 mutation at the time of birth might serve as a biomarker for an increased risk of developing DS-related AMKL.
Alpha Hemoglobin Stabilizing Protein (AHSP) Optimizes Hemoglobin A Synthesis by Maintaining a Pool of Viable Alpha Globin Subunits.
Authors: Yu Xiang , Kong Yi , Dore Louis C. , Katein Anne M. , Choi John K. , Gow Andrew J. , Weiss Mitchell J.
Published: April 2020

AHSP binds alpha hemoglobin (Hb) to maintain its structure and limit its prooxidant activities. In addition, AHSP binds and stabilizes apo-alpha globin, which lacks heme. Previously, we demonstrated that Ahsp−/− mice exhibit hemolytic anemia with Hb precipitation in erythroid cells. Through interbreeding of mutant strains, we also showed that loss of AHSP exacerbates beta thalassemia. Together, these studies indicate that AHSP participates in Hb homeostasis and may act to neutralize potentially toxic excess alpha globin that is known to accumulate in normal erythroid precursors, and to a greater extent, in beta thalassemic ones. However, additional functions for AHSP may exist. In particular, AHSP-alpha globin complexes may also promote HbA synthesis. To test this, we depleted the pool of excess alpha globin in Ahsp−/− mice by interbreeding with alpha thalassemic ones. Compared to mice with either mutation alone, compound mutants missing both AHSP and one alpha globin allele (genotype Ahsp−/− //alpha globin*α/αα) exhibited more severe erythroid defects, including worsened anemia, hypochromia, Hb instability and ineffective erythropoiesis. Pulse-labeling of double-mutant reticulocytes showed that alpha to beta globin synthetic ratios were unaffected by loss of AHSP, but precipitation of both alpha and beta nascent chains into cell membranes was strongly enhanced. These data indicate that AHSP is important for erythropoiesis and Hb production even when alpha globin is not produced in excess. In vitro translation studies using wheat germ extracts showed that recombinant AHSP present during the synthesis of alpha globin improved its ability to become incorporated into HbA. Moreover, AHSP conferred protease resistance to nascent alpha globin, suggesting enhanced folding into the native state. Further supporting this interpretation, circular dichroism studies showed that AHSP accelerated refolding of purified denatured alpha Hb. Finally, through pulse labeling of reticulocytes followed by isoelectric focusing of soluble cytosolic fractions, we identified transient pools of free alpha Hb and AHSP-alpha Hb in vivo and showed that Ahsp gene ablation fully depleted both pools. Together, our studies indicate that AHSP acts as a molecular chaperone to promote alpha globin folding and stability prior to its incorporation into HbA. In addition, it is possible that alterations in AHSP gene function or expression could modulate alpha thalassemia severity in patients.
Intergenic Variants of HBS1L-MYB Are Responsible for a Major Quantitative Trait Locus on Chromosome 6q23 Influencing HbF Levels in Adults.
Authors: Thein Swee Lay , Menzel Stephan , Peng Xu , Best Steve , Jiang Jie , Close James , Silver Nicholas , Gerovasilli Ageliki , Ping Chen , Yamaguchi Masao , Wahlberg Karin , Muller Heike , Spector Tim D. , Garner Chad , Matsuda Fumihiko , Farrall Martin , Lathrop Mark
Published: April 2020

Sickle cell disease and β-thalassemia demonstrate a remarkable diversity in phenotypic severity, and the innate ability to produce fetal hemoglobin (HbF, α 2γ 2) is a major ameliorating factor in these β-hemoglobinopathies. HbF and F cells (denoted FC; subset of erythrocytes that contain measurable HbF) are highly heritable quantitative traits; cis-acting variants in the β globin gene locus explain some of the variability but over 50% of the variance is unlinked to the β globin gene locus. We have previously mapped a quantitative trait locus (QTL) controlling FC in an Asian-Indian kindred with β-thalassemia to a 1.5 Mb interval on chromosome 6q23 that encompasses five known protein-coding genes (ALDH8A1, HBS1L, MYB, AHI1 and PDE7B), none of which harbored mutations. From the known genes within this interval, we selected HBS1L, a GTP-binding protein family member, and MYB, a transcription factor central to hematopoiesis, as candidates for detailed study. Two panels of twin-pairs (824 and 1217 individuals, respectively) of North European origin were recruited through the UK Adult Twin Registry. Age and sex, and XmnI-Gγ (−158C/T variant upstream of the Gγ globin gene which is associated with FC levels) show similar associations with FC levels in both panels. Altogether, 121 markers identified by re-sequencing and from public databases were genotyped in the HBS1L-MYB intergenic region. The high-resolution association study in Europeans identified genetic variants within and 5′ to HBS1L that are highly associated with F-cell levels (p=10−75). Additional HBS1L-MYB variants contribute independently to the QTL (p<10−3). Our study provides unequivocal evidence for the mapping of the chromosome 6q HbF / F-cell QTL to a 79 kb region flanked by HBS1L and MYB; the intergenic variants account for 16% of the trait variance. The biological effects of these intergenic variants appear to be complex as they may influence several genes or transcribed sequences in this genomic region. Our study illustrates the power of QTL mapping for positional identification of trans-acting genetic variants influencing fetal hemoglobin levels, which could lead to new diagnostic and therapeutic strategies.
Regulatory T Cells (Tregs) Are Highly Preserved in Multiple Myeloma Patients and Can Dominate Effector Functions.
Authors: Foglietta Myriam , Mariani Sara , Castella Barbara , Coscia Marta , Godio Laura , Zappia Serena , Boccadoro Mario , Massaia Massimo
Published: April 2020

Naturally occurring CD4+CD25+ T regulatory cells (Tregs) are a subpopulation of CD4+ T cells playing a key role in lymphocyte homeostasis and maintenance of tolerance. Tregs play a key role in hampering anti-tumor immune surveillance by regulating immune responses against tumor cells. Depletion of Tregs yields to improved anti-tumor immune responses in both preclinical and clinical settings. Contradictory data have been reported in the frequency and function of Tregs cells in multiple myeloma (MM). The aim of this study was to characterize MM Tregs from the phenotypic, molecular and functional standpoints. We studied both peripheral blood (PB) and bone marrow (BM) samples from patients with symptomatic MM and from healthy donors. CD4+CD25high Tregs were normally represented in both PB and BM of MM patients and expressed a memory and activated phenotype. No differences were observed between PB and BM Tregs of healthy donors and MM patients, based on the expression of CD45R0, HLADR, CD40L and CD69 cell surface antigens. Flow cytometry was also used to assess the intracellular expression of Foxp3, a transcription factor which is crucial for the inhibitory function of Tregs cells. More than 90% of CD4+CD25high T cells were Foxp3+ in the PB and BM of MM patients and healthy donors. CD4+CD25+ T cells were purified from PB of newly diagnosed MM by MACS sorting. These cells were anergic to the stimulation via TCR and as effective as normal donor-derived CD4+CD25+ cells in inhibiting the TCR-mediated proliferation of autologous CD4+CD25- counterparts. To investigate whether clonal restriction had occurred in MM Tregs, we studied TCR diversity of CD4+CD25+ and CD4+CD25- cells by determining the reciprocal usage of BV gene segments (TCRBV repertoire) with a novel multiplex polymerase chain reaction assay recently developed in our laboratory. Our results demonstrate an highly preserved polyclonal TCRBV repertoire, providing the first evidence in cancer patients that TCR diversity of Tregs is not skewed by the long lasting exposure to tumor cells. Based on these data, we propose that inhibitory signals delivered by the highly preserved Tregs subset in MM can easily overwhelm the effector mechanisms of antitumor immunosurveillance which are deeply compromised in MM. Thus, depletion or neutralization of Tregs should be considered in future trials aimed at controlling the disease in MM patients by immune intervention.
CS1 Is Expressed on Myeloma Cells from Early Stage, Late Stage, and Drug-Treated Multiple Myeloma Patients, and Is Selectively Targeted by the HuLuc63 Antibody.
Authors: Szmania Susann , Balasa Balaji , Malaviarachchi Priyangi , Zhan Fenguhuang , Huang Yongsheng , Draksharapu Aparna , Vexler Vladimir , Shaughnessy John , Barlogie Bart , Tricot Guido , Afar Daniel , Frits van Rhee
Published: April 2020

Introduction: One-third of multiple myeloma (MM) patients exhibit high-risk features such as abnormal cytogenetics, high LDH, amplification of CKS1-B or spiked expression of MAF, MAF-B or FGFR3. While not affecting complete response rates, median durations of event-free and overall survival, even with high-dose melphalan-based tandem autotransplants of such patients, do not exceed 24 mo and 36 mo, compared to 60 mo and ≥ 90 mo for the remainder. Monoclonal antibody (mAb)-mediated therapy may target a chemotherapy-resistant myeloma cell pool. CS1 (CD2 subset 1, CRACC, SLAMF7), a member of the CD2 family of cell surface glycoproteins, exhibits high-level expression on primary myeloma cells, indicating that CS1 is a potential target for treatment in MM.

Methods: Gene expression was assessed using an Affymetrix GeneChip array. Protein expression was measured by flow cytometry, and immunohistochemistry (IHC), using HuLuc63, a novel humanized anti-CS1 mAb. HuLuc63-mediated lysis of myeloma cells via antibody dependent cellular cytotoxicity (ADCC) was measured by 51Cr-release.

Results: CS1 mRNA was detected in >95% of CD138+ purified plasma cells from >95% of healthy donors, newly diagnosed myeloma patients, and those with relapsed myeloma (Fig. 1a). CS1 remained highly expressed in patients following VDTPACE treatment, albeit at a reduced level. CS1 expression was also high following bortezomib (Velcade®) treatment, with a subset of patients showing increased expression post-treatment. CS1 protein expression on primary myeloma cells was confirmed by flow cytometry, while IHC analysis of normal tissues revealed anti-CS1 staining primarily on CD138+ tissue plasma cells. Finally, we determined that HuLuc63 could induce killing of myeloma cells using purified allogeneic NK cells (Fig. 1b). Blocking the Fc receptor greatly reduced this activity indicating an ADCC mechanism. Killing of myeloma targets was also observed in autologous systems suggesting that HuLuc63 can overcome KIR-mediated NK cell inhibition of autologous NK cells. In summary, we observed high mRNA and protein expression of CS1 in myeloma from early stage, late stage, and treated patients, and showed enhanced lysis of myeloma cells in vitro with HuLuc63. Our data support the potential clinical utility of CS1-targeted therapy. HuLuc63 will be entering a phase I clinical trial for advanced myeloma patients.

Figure 1a. CS1 mRNA is highly expressed in CD138+ purified plasa cells. Figure 1a. CS1 mRNA is highly expressed in CD138+ purified plasa cells. Figure 1b. CS1 antibody enhances killing of myeloma cells by allo - NK cells. Figure 1b. CS1 antibody enhances killing of myeloma cells by allo - NK cells.
Xenotransplantation of Primary CD34+CD38- Hematopoietic Stem Cells Results in an In Vivo Model of Idiopathic Myelofibrosis.
Authors: Saito Noriyuki , Ishikawa Fumihiko , Shimoda Kazuya , Yoshida Shuro , Saito Yoriko , Kawano Noriaki , Shulz Leonard D. , Akashi Koichi , Harada Mine
Published: April 2020

Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These abnormal cells secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and lead to extramedullary hematopoiesis and the appearance of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate at the level of hematopoietic stem cell (HSC), this has not been demonstrated directly in primary human IMF. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we used the newborn NOD/SCID/IL2rg-null xenotransplantation model. We purified PB CD34+ cells from six IMF patients, transplanted 1–10 x10e4 cells intravenously into newborn NOD/SCID/IL2rg-null recipients and analyzed PB and BM human CD45+ hematopoietic cell chimerism, degree of suppression of murine hematopoiesis, presence of hallmark BM fibrosis and plasma TGF-b1 levels in the recipients at 6 months post-transplantation. Primary IMF PB CD34+ cells from five out of six patients engrafted in twelve out of twelve recipients. BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of murine fibroblasts, the presence of human megakaryocytes and elevated plasma TGF-b1 levels, recapitulating the clinical features of IMF. Three distinct patterns of human hematopoietic reconstitution were observed among the engrafted recipients:

Predominantly malignant myelomonocytic engraftment in the PB and BM (n=4), Reconstitution of both normal human hematopoiesis (with mature B and T cells, myeloid cells and platelets) and malignant myelomonocytic cells (n=6) and Development of acute leukemia (n=2).

Fibrotic change was seen even in the BM of recipients that showed normal human hematopoietic reconstitution, showing that in IMF, there is co-existence of both normal and malignant hematopoietic stem/progenitor cells in the PB CD34+ fraction. Furthermore, when 5–10 x 10e3 sorted PB CD34+CD38– cells from three patients were transplanted into six newborn NOD/SCID/IL2rg-null recipients, reconstitution with human myelomonocytic cells associated with BM fibrosis was demonstrated in all recipients, with compatible level of PB and BM chimerism with those transplanted with PB CD34+ cells. These findings demonstrate that the IMF-initiating cells are contained within the HSC fraction. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and may be useful for development of novel therapeutic agents for IMF.
Mice Lacking the Signaling Molecule, CalDAG-GEFI, Represent a Mouse Model for Leukocyte Adhesion Deficiency Type III.
Authors: Bergmeier Wolfgang , Goerge Tobias , Wang Hong-Wei , Cifuni Stephen M. , Crittenden Jill R. , Graybiel Ann M. , Wagner Denisa D.
Published: April 2020

Defective inside-out activation of β 1, β 2, and β 3 integrins in platelets and leukocytes is a main characteristic of patients with leukocyte adhesion deficiency (LAD)-III syndrome. We have recently shown that CalDAG-GEFI, a member of the CalDAG-GEF/RasGRP family of intracellular signaling molecules that catalyzes the exchange of GTP for GDP bound to Rap1, plays a key role for the activation of α IIbβ 3 in murine platelets. Here we studied the role of CalDAG-GEFI for neutrophil function as well as the activation of β 1 integrins in platelets. Neutrophils from CalDAG-GEFI−/ − mice showed normal surface expression of key adhesion receptors such as L-selectin, PSGL-1, or β 1/β 2 integrins. Calcium flux, degranulation, and oxygen radical formation were similar in wild-type (WT) and mutant cells. In contrast, β 2 integrin-mediated adhesion to fibrinogen was significantly reduced in cells lacking CalDAG-GEFI when compared to controls. In vivo, CalDAG-GEFI-deficient neutrophils showed normal rolling along stimulated venules, while firm adhesion was almost completely inhibited. A similar defect in firm adhesion was observed in WT mice pre-treated with blocking antibodies against β 2 integrins. To determine the role of CalDAG-GEFI in neutrophil emigration, inflammation was induced in the peritoneum or the skin. In both models, neutrophil infiltration was significantly reduced in CalDAG-GEFI−/ − mice when compared to controls. We further demonstrate that CalDAG-GEFI regulates the activation of β 1 integrins in platelets and that CalDAG-GEFI-deficiency leads to a complete inhibition of arterial thrombus formation in mice. Due to its central role in the activation of β 1, β 2, and β 3 integrins, we propose CalDAG-GEFI as a candidate gene defective in LAD-III patients.
Balance of Transcription Factors Downstream of Notch Signaling Determines the Fate of Myeloid Progenitors toward Differentiation to Mast Cells or Immortalization without Differentiation.
Authors: Sakata-Yanagimoto Mamiko , Nakahara Fumio , Yamaguchi-Nakagami Etsuko , Kumano Keiki , Saito Toshiki , Kurokawa Mineo , Ogawa Seishi , Chiba Shigeru
Published: April 2020

Notch signaling represents one of the fundamental communication channels in various types of cells. While Notch activation has been shown to inhibit myeloid differentiation in a subset of hematopoietic progenitors, the role of Notch signaling in mast cell differentiation is not clear. When common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) purified from mouse bone marrow cells were stimulated with Delta1-Fc, a soluble form of Notch ligand, in the presence of stem cell factor, IL-3, IL-6, and thrombopoietin, granulocyte and macrophage differentiation, which is observed at day 7 of culture in the absence of Delta1-Fc, was markedly inhibited. Instead, Lin-c-Kit+FcεR+ mast cells dominated in the culture. Delta1-Fc did not increase mast cell generation from either CMPs or GMPs of the bone marrow of pI:pC-treated Mx-Cre x Notch2 flox/flox (N2-MxcKO) mice, in contrast to littermate Notch2 flox/flox mice treated with pI:pC, which suggests that Notch2 is responsible for the Delta1-Fc-augmented mast cell generation from CMPs and GMPs in culture. Retroviral transfer of constitutive active form of Notch2 (aN2) into CMPs and GMPs resulted in the complete loss of granulocyte-macrophage colony-forming cells and the emergence of basophilic granules-containing blast like cells, indicating the cell fate instruction. Real-time PCR analysis revealed that Delta1-Fc stimulation and aN2 introduction up-regulated the expression of Hes1, a transcriptional suppressor that is known to be a direct target of Notch activation in several cell types, within 12 h. Moreover, among GATA genes, Delta1-Fc stimulation and aN2 introduction resulted in increase of GATA3 mRNA, while expression levels of GATA1 and GATA2, which have been suggested to play a role in regulating mast cell differentiation, were unchanged. Next, we retrovirally expressed Hes1 and/or a GATA gene into CMPs and GMPs to see if the same effects were observed. Mast cells were increased only when both genes were expressed. On the other hand, when Hes1 alone was transduced, we observed rapid growth and immortalization of these cells without differentiation. C/EBPa, which is known to be suppressed in mast cell differentiation and upregulated in myeloid cell differentiation, was down-regulated within 48 h from the initiation of Hes1 retroviral transduction, suggesting that C/EBPa is a downstream target of Hes1 in this myeloid cell fate determination. Theses results indicate that, at the downstream of Notch activation, there are a C/EBPa down-regulation pathway that is Hes1-dependent and a GATA3 up-regulation pathway. Balanced regulation of these pathways should play a physiological role in myeloid and mast cell differentiation, while imbalance between these two pathways might provide a new model of myeloid transformation.
The Identification and Characterization of Zebrafish Hematopoietic Stem Cells.
Authors: Ma Dongdong , Lin Hui-feng , Handin Robert Irving
Published: April 2020

Hematopoietic stem cells (HSCs) are defined by their ability to self-renew and maintain hematopoiesis throughout the lifespan of an organism. Because of the optical clarity of their embryos and the ease of genetic manipulation, the zebrafish (Danio rario) is an excellent model for studying hematopoiesis. To date, though, there have been no published studies characterizing zebrafish HSCs. We have engineered transgenic zebrafish strains with green fluorescent protein (GFP+) thrombocytes by driving expression of the GFP reporter with the platelet/thrombocyte-specific CD41 promoter (Lin et. al. Blood. 2005 Dec 1; 106: 3803). We have identified two populations of CD41-GFP+ cells in suspensions of whole kidney marrow by flow cytometry. The majority of the cells strongly express GFP (GFPhi) and are mature thrombocytes. There is also a small population of immature cells, which express GFP weakly (GFPlo), that are of uncertain lineage. Peripheral blood and splenic cell suspensions contain only GFPhi cells. Studies in humans and mice have shown that CD41 is transiently expressed on the earliest hematopoietic progenitors and is then silenced, suggesting that the GFPlo cells might be hematopoietic progenitors or HSCs. To test this hypothesis, we transplanted flow-sorted GFPhi and GFPlo cells into gamma-irradiated adult AB strain zebrafish. The GFPhi cells were no longer detected 7 days after transplantation. In contrast, we have observed long-term engraftment and multi-lineage reconstitution for over 6 months after transplantation of GFPlo cells into zebrafish conditioned with gamma irradiation at 25 Gray or 40 Gray. Multi-lineage reconstitution persisted following serial transplantation of the GFPlo cells. Initial transplants were carried out with 105 GFPlo cells. To test the self renewal properties of GFPlo cells, transplants were carried out with injections of 1000 to 1 GFPlo cell(s)/fish. Multi-lineage reconstitution was seen with all doses and was achieved in 5 of 54 fish after transplantation of a single GFPlo cell. Flow cytometric analysis of whole kidney marrow from fish examined six months after transplantation demonstrated the persistence of GFPlo cells. Taken together, these studies suggest that GFPlo cells are capable of long-term hematopoietic reconstitution and are true HSCs. We believe that the zebrafish HSC transplant model holds great promise for the in vivo study of HSCs and may provide a valuable tool to track HSC homing, proliferation and differentiation.
α4-Integrin+ Endothelium Derived from Primate Embryonic Stem Cells Generates Both Primitive and Definitive Hematopoietic Cells.
Authors: Shinoda Gen , Umeda Katsutsugu , Heike Toshio , Arai Masato , Niwa Akira , Ma Feng , Suemori Hirofumi , Luo Hong Yuan , Chui David H.K. , Torii Ryuzo , Shibuya Masabumi , Nakatsuji Norio , Nakahata Tatsutoshi
Published: April 2020

The mechanism of commencement of hematopoiesis in blood islands of the yolk sac and the aorta-gonad-mesonephros (AGM) region during primate embryogenesis remains elusive. We previously showed the development of both primitive and definitive hematopoiesis when cynomolgus monkey embryonic stem cells were co-cultured with OP9 stromal cells. In this study, we examined the hematopoietic potential of endothelial cells developing in our coculture system and demonstrated that VE-cadherin+CD45− endothelial cells derived from embryonic stem cells were able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. All floating erythrocytes which emerged initially expressed ε- and ζ-globins, while β-globin expression was hardly detected. The percentage of floating erythrocytes positive for β-globin gradually increased thereafter, and almost all erythrocytes were positive by day 40. Meanwhile, expression of ε- and ζ-globins declined gradually. Clonal analysis revealed that single bipotential cells for hematopoietic and endothelial lineages were included in this endothelial cell population. Hemogenic activity of endothelial cells was observed exclusively in the α4-integrin+ subpopulation. RT-PCR data showed that Runx1, a transcriptional factor associated with definitive hematopoiesis, was expressed in the hemogenic α4-integrin+ subpopulation, but not the non-hemogenic α4-integrin− subpopulation among embryonic stem cell-derived endothelial cells. The kinetics of this hemogenic subpopulation was similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo, in that they emerged only for a limited time. On the other hand, VE-cadherin−CD45−α4-integrin+ cells gave rise to more primitive erythrocytes than VE-cadherin+CD45−α4-integrin+ cells, but hardly contributed to definitive hematopoiesis. These results indicate that VE-cadherin+CD45−α4-integrin+ endothelial cells generate primitive and definitive hematopoietic cells sequentially, while VE-cadherin−CD45−α4-integrin+ cells are primary sources for primitive hematopoiesis. It seems that precursors of primitive and definitive erythropoiesis arise simultaneously but that the definitive precursors require a period of maturation before they differentiated into blood cells. We suggest that a subset of endothelial cells is involved in primitive as well as definitive hematopoiesis during primate embryogenesis, and that α4-integrin marks the hemogenic subpopulation in primates.
Active Immunotherapy with FavId® (Id/KLH) Following Rituximab Induction: Long-Term Follow-Up of Response Rate Improvement (RRI) and Disease Progression in Follicular Lymphoma Patients (pts).
Authors: Koc Omer N. , Redfern Charles , Wiernik Peter H. , Rosenfelt Fred , Winter Jane N. , Guthrie Troy H. , Kaplan Lawrence , Holman Peter , Densmore John , Hainsworth John , Lin Thomas S. , Castillo Rene A. , Janakiraman Nalini , Melink Teresa , Liu Song , Bender John
Published: April 2020

Background: Studies of Id/KLH immunotherapy in treatment naïve (TN) fNHL pts following first remission from chemotherapy have demonstrated a prolonged duration of remission. We expanded this approach in a PhII trial to include TN and relapsed/refractory (R/R) pts with stable disease (SD) or PR or CR/CRu following 4 wkly doses of rituximab. We report here long term follow up of RRI, defined as the % of pts who converted from SD to PR or PR to CR/CRu following initiation of Id/KLH treatment, and analysis of disease progression.

Treatment: Pts received rituximab (375mg/m2 i.v. wkly x 4 during wk1–4) and those with stable or responsive disease received Id/KLH (1 mg s.q. mo x 6) starting on wk 12 along with GM-CSF (250 mcg, s.q.) on days 1–4. Pts continued to receive booster injections on a reduced schedule until disease progression. Radiological scans, performed every 3 mos, were reviewed centrally. Disease progression and response were assessed using modified Cheson criteria. This data analysis was performed 32 mo from end of enrollment.

Results: 103 pts were enrolled and received rituximab; 89 pts had ≥SD and received Id/KLH + GM-CSF of whom, 54 were R/R, and 35 were TN. The overall response rate (ORR) was 47% (42/89) at mo 3. After the initiation of Id/KLH dosing, ORR improved to 63% (56/89). In RRI pts, median time to best response was 9.1 mos from start of rituximab treatment (5.2 – 29.6 mos). 12 of 39 PR pts (31%) converted to CR/Cru. 14 of 43 SD pts (33%) converted to PR.

Pt Groups # of Pts % Prog Free Median TTP Est by K-M (mo) Pts w/RRI 26 69% 37.9 TN rituximab responders 23 70% Not Reached (NR) Rituximab responders (All) 42 60% NR Non Responders (All) 47 36% 14.9 All Pts 89 47% 18.2 TN Pts (All) 35 66% NR Relapsed Pts (All) 54 37% 16.8

Conclusion: RRI following FavId is seen in nearly one third of pts. The magnitude of this improvement, the number of conversions to CR/CRu and median time to best response (9 mo and as late as 29.6 mo) suggests this is likely to be a FavId effect rather than late response to rituximab. Responders and TN pts show more durable responses compared to previously published data for rituximab therapy. FavId may also confer a benefit for nonresponders and R/R pts relative to rituximab alone. To confirm benefit of FavId following rituximab induction, a placebo controlled PhIII study was initiated; it completed enrollment in Jan 06. RRI data from this study is expected to be available in Nov 06.
Regulation of Platelet PLC-β2 Expression by NF-κB: Studies in Human Platelet PLC-β2 Deficiency.
Authors: Mao Guangfen , Kunapuli Satya P. , Rao A. Koneti
Published: April 2020

We have previously described a patient with platelet phospholipase C (PLC)-β2 deficiency characterized by impaired platelet responses to activation with multiple G-protein coupled receptor agonists. The PLC-β2 coding sequence was normal and platelet PLC-β2 mRNA levels were decreased in the patient (Blood, 2002, 99:905). Very little is currently known regarding the transcriptional regulation of PLC-β2. PCR-amplification of patient leukocyte DNA and sequencing of the PLC-β2 5′-upstream region revealed a heterozygous 13-bp deletion (−1645 to −1633 bp from ATG) that encompasses a consensus binding site (GGGAATTCCC) for nuclear factor-κB, NF-κB. This deletion was present in the propositus and her affected son, but not in control subjects. PCR amplification of region −1791 to −1606 bp of genomic DNA revealed one band in 5 control subjects (size ~186 bp) on agarose gel electrophoresis but 2 bands in the patient and her son, consistent with a heterozygous defect. Luciferase reporter gene studies were performed in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA, 30 nM) to induce megakaryocytic transformation. Genomic fragment (−1648/−23 nt) of PLC-β2 5′-upstream sequence and its truncated form without the 13 nt region (−1633/−23 nt) were inserted upstream of luciferase gene in a promoterless expression vector PGL3-basic (Promega) and transiently transfected into HEL cells. Truncation of the wild-type −1648/−23 fragment at 1631 bp resulted in a consistent decrease in promoter activity by ~ 25% (6 experiments, p<0.05). Protein binding assay (EMSA) was performed using PMA-treated HEL cell nuclear extracts and oligonucleotide probes (−1652/−1628 bp) with wild-type and mutated NF-κB consensus sites. Specific protein binding to the wild-type oligonucleotide was abolished when the NF-κB consensus sequence was deleted or mutated. Protein binding to wild-type probe was not competed by the unlabeled mutant oligonucleotide lacking NF-κB consensus sequence. In supershift assay, antibody targeted against the p65 subunit of NF-κB abolished protein binding, indicating a role for NF-κB. In summary, our studies demonstrate in the 5′-upstream region of PLC-β2 gene of the patient a 13-bp deletion that has a consensus site for NF-κB. Luciferase gene promoter assays demonstrate loss of activity when the 13-bp site is truncated. These studies provide evidence that impaired regulation of PLC-β2 gene by NF-κB may be the basis for the PLC-β2 deficiency in our patient. They show for the first time that PLC-β2, the most abundant β-PLC in platelets, is regulated by NF-κB. These findings are highly relevant because of the important role of PLC-β2 in platelet function, and of NF-κB in megakaryocytic differentiation and atherosclerosis.
Pharmacogenetics of Oral Anticoagulation - VKORC1-Haplotypes Determine the Inter-Individual and Inter-Ethnical Variability.
Authors: Geisen Christof , Watzka Matthias , Sittinger Katja , Luxembourg Beate , Steffens Michael , Müller Clemens R. , Wienker Thomas F. , Lindhoff-Last Edelgard , Seifried Erhard , Oldenburg Johannes
Published: April 2020

Purpuse: Coumarins are known for their high inter-individual and inter-ethnical variability of the dose-response association (i.e. significant higher maintenance dose in Africans and lower dose in Chinese). Recently, common SNPs of the VKORC1 gene (encoding the molecular target of coumarins, vitamin K epoxide reductase) were shown to be associated with lower warfarin dose requirement (c.-1639G>A [rs17878363], c.173+1000C>T [rs9934438]). In order to clarify whether these SNPs are part of more extended haplotypes, we established a comprehensive haplotype map of the entire VKORC1 gene locus. The association of VKORC1-haplotypes with coumarin dose requirement was analysed in different ethnical populations and patient cohorts.

Methods: In 200 German blood donors the VKORC1 gene locus was analysed by direct sequencing. VKORC1 genotype data in 7 additional populations from Africa, Asia and Europe were retrieved from internet databases. Haplotype frequencies were inferred using EHplus and FAMHAP. Unselected patients receiving phenprocoumon (n=61) and patient cohorts of European origin with either increased coumarin sensitivity (n=14, weekly phenprocoumon dose <6mg) or partial coumarin resistance (n=36, weekly phenprocoumon dose >42 mg or warfarin dose>70mg) were typed for VKORC1 haplotypes.

Results: In 200 controls we identified 28 SNPs within the VKORC1 gene. 6 SNPs formed 3 main haplotypes covering more than 99% of the genetic variability of VKORC1 (VKORC1*2: 42%, VKORC1*3: 38%, and VKORC1*4: 20%). SNPs associated with low warfarin dose (rs17878363, rs9934438) were in complete linkage disequilibrium with the VKORC1*2 haplotype. Haplotype frequency in Africans and Chinese differed significantly from the European sample (for VKORC1*2: Europeans 42%, Chinese 95%, Africans 14%). In 61 unselected patients receiving phenprocoumon c.-1639AA genotype patients (homozygous VKORC1*2) required less phenprocoumon (1.40 mg/d) than AG (2.12 mg/d) and GG patients (3.02 mg/d).13 of 14 patients (93%) with increased coumarin sensitivity but none of the patients with partial coumarin resistance were c.-1639AA. Vice versa, the c.-1639G allele (present in the non VKORC1*2 haplotypes) was found homozygous in 31 patients (86%) with partial coumarin resistance but in none of the patients with increased coumarin sensitivity.

Conclusions: Three main haplotypes represent more than 99% of VKORC1’s genetic variability in Europeans. VKORC1 haplotypes are strongly associated with the inter-individual and inter-ethnical variability of oral anticoagulation. In future studies, VKORC1-haplotype testing may provide a clinically relevant predictor of coumarin dosing and bleeding risk in oral anticoagulation.
Dasatinib (SPRYCEL®) 140 mg Once Daily (QD) vs 70 mg Twice Daily (BID) in Patients (pts) with Advanced Phase Chonic Myeloid Leukemia (ABP-CML) or Ph(+) ALL Who Are Resistant or Intolerant to Imatinib (im): Results of the CA180-035 Study.
Authors: Kantarjian Hagop , Ottmann O. , Pasquini R. , Goh Y.T. , Kim D.W. , VanTornout J. , Damokosh A. , Shah N.
Published: April 2020

Dasatinib (SPRYCEL®, formerly BMS-354825), an oral multi-targeted kinase inhibitor of BCR-ABL and SRC kinases, has been shown to be safe and effective at 70 mg BID in pts with CML and Ph(+) ALL who are resistant or intolerant to im. Based on a previous phase I study, in which QD dosing was as effective as BID dosing, a dose-optimization study was performed. CA180035 is a randomized, global multicenter, open-label trial of dasatinib 140 mg QD versus 70 mg BID in pts with ABP-CML (accelerated or blastic phase) or Ph(+)ALL that were resistant to or intolerant of im. Patients were stratified by phase of disease (accelerated, myeloid blast, or lymphoid blast/Ph(+)ALL) and by prior im (resistant or intolerant). The primary objective of the study was to compare the major hematologic response (MaHR) rate between the 2 regimens. Dose escalation to 180 mg QD or 90 mg BID was allowed for inadequate response, and dose reduction to 100 or 80 mg QD or 50 or 40 mg BID for drug toxicity. Evaluations consisted of weekly blood counts for the first 12 wks, and bone marrow cytogenetics every 3 months (mo) in patients with complete hematologic response (CHR) and BCR-ABL mutation analysis at baseline and at end of study. From June 2005 through March 2006, 612 pts were randomized: 56% of the pts were male with median (med) age of 55 years and med time from CML diagnosis of 58 mo. All pts received prior im: 42% had >600 mg/day, and 37% were treated >3 years. Other prior therapy included interferon in 42% of pts, chemotherapy in 57% of pts and stem cell transplant in 14% of pts. With a minimum follow-up of 4 mo, 42% of pts have been discontinued, mostly due to disease progression. The MaHR rate was 35% including 21% CHR and the major cytogenetic response (MCyR) rate was 33%, including 23% complete cytogenetic response (CCyR). The most common non-hematologic drug-related toxicities included diarrhea (24%, gr 3–4: 3%), headache (17%, gr 3–4: 1%), nausea (17%, gr 3–4: 2%), pleural effusion (15%, gr 3–4: 4%), and fatigue (12%, gr 3–4: 3%). Hematologic toxicity included neutropenia gr 3 and gr 4 in 22% and 40% of pts, respectively, and thrombocytopenia gr 3 and gr 4 in 16% and 50% of pts, respectively. Dose reductions were required in 23% of pts and dose interruptions were required in 43% of pts, both due mostly to non-hematologic toxicity. This is the first direct comparison of the 70 mg BID vs 140 mg QD dasatinib schedules in pts with ABP-CML or Ph(+)ALL. Updated efficacy and safety data, including response by baseline mutation analysis, on all pts with a minimum of 6-months follow-up will be presented at the meeting.
A Randomized Trial of Rabbit Anti-Thymocyte Globulin, Given on Day+7 after Alternative Donor Transplants.
Authors: Bacigalupo Andrea , Lamparelli Teresa , Ciceri Fabio , Peccatori Fedro , Locasciulli Anna , Majolino Ignazio , Di Bartolomeo Paolo , Milone Giuseppe , Mazza Patrizio , Lacopino Pasquale , Sacchi Nicoletta , van Lint Maria Teresa
Published: April 2020

Background. We have shown that the risk of transplant mortality (TRM) can be predicted by using laboratory values, on day+7 after an allogeneic hemopoietic stem cell transplant (HSCT) (Sormani et al, Bone Marrow Transpl.2003). TRM in patients with low, intermediate and high risk on day+7, is 15%, 40% and 69%: increased mortality is mainly, but not exclusively, due to increased acute graft versus host disease (GvHD). In a pilot study we have shown that a small dose of rabbit anti-thymocyte globulin (rATG) (Thymoglobulin, Genzyme), was feasible and reduced GvHD and TRM.

Aim of the study. We have therefore opened a randomized trial testing this hypothesis in patient undergoing an unrelated donor HSCT, with rATG (7.5 mg/kg) in the conditioning regimen.

Patients. 170 were eligible: all patients received rATG 3.75 mg/kg x2 pre-transplant, and on day+7 they were scored and randomized to receive an additional dose of rATG (1,25 mg/kg day +7 and day+9) (n=84) or to receive no further treatment (n=86) (control arm). The two groups were balanced for age, disease phase and day+7 score.

Results. The predictive value of day+7 score on TRM was confirmed in the control arm (18% vs 42% for intermediate and high risk patients, p=0.03), whereas in the day+7 ATG arm, there was no difference (29% vs 29%, p=1.0). TRM was overall reduced from 35% in the control arm to 29% in the ATG day+7 arm (p=0.37) : the difference was more pronounced in patients with early disease and high risk on day 7 (35% vs 15% p=0.08). Acute GvHD grade III–IV was reduced overall from 16% to 6% (p=0.04), and chronic GvHD was reduced 32% to 14% (p=0.02). In multivariate COX analysis TRM and overall survival was predicted by risk score day7 and disease phase.

Conclusions. We confirm that patients with different risk of TRM can be identified on day+7 after HSCT. Administration of rATG on day+7 can influence the risk of TRM, by reducing acute and chronic GvHD. Additional intensified supportive care (including anti-infectious therapy) may further reduce TRM in patients with a high risk score on day+7.
Von Willebrand Factor Protects Factor VIII from Endocytosis by Human Monocytes−Derived Dendritic Cells and Subsequent Presentation to Immune Effectors.
Authors: Repesse Yohann , Dasgupta Suryasarathi , Navarette Ana Maria , Delignat Sandrine , Wootla Bharath , Borel-Derlon Annie , Kaveri Srinivas V. , Lacroix-Desmazes Sebastien
Published: April 2020

The role of Von Willebrand Factor (VWF) as a chaperone molecule for procoagulant factor VIII (FVIII) has been extensively documented. Under physiological conditions, VWF binds to FVIII after its release in the circulation. VWF protects FVIII from proteolysis by lipid−bound proteases, stabilizes FVIII and further regulates its elimination by lipoprotein receptors. We investigated whether VWF might modulate FVIII endocytosis by professionnal antigen presenting cells (APCs). Immature dendritic cells (DCs), utilised as model of APCs, were generated from circulating monocytes of healthy blood donors and incubated with FITC−labeled FVIII (FVIII−FITC). T cell assays were performed by co−culturing the human FVIII−specific CD4+ T cell clone, D9E9, with immature DCs from MHC−matched donors in presence of unconjugated FVIII and increasing concentrations of VWF (1:1 to 1:130). Preincubation of FVIII−FITC with VWF at 1 to 130−fold molar excess resulted in a dose−dependent inhibition of FVIII endocytosis (12 ± 2% to 94 ± 18%, respectively). Human serum albumin used in equivalent molar ratios did not prevent FVIII internalization. In contrast, the protective effect of VWF on FVIII uptake was significantly restored by co−incubation of FVIII and VWF in the presence of F(ab’)2 fragments of BO2C11, an anti−FVIII IgG, and Ac418, an anti−VWF IgG, that both disrupt the interaction between FVIII and VWF. In addition, blocking DC entry of FVIII by VWF resulted in a dose−dependent reduction of the activation of D9E9 (up to 75%). Interestingly, D9E9 activation by DCs loaded with a synthetic FVIII−derived peptide, I2144–T2161, was not altered in the presence of equivalent concentrations of VWF, indicating that VWF does not have a direct inhibitory effect on T cell activation. The administration of exogenous FVIII to patients with hemophilia A, results in up to 30% of the cases, in the developement of FVIII−specific antibodies that inhibit FVIII procoagulant activity. In vivo experimental evidences and clinical observations have documented that the presence of VWF in FVIII therapeutic concentrates may be associated with a lower incidence of FVIII inhibitors. Here, we demonstrate that VWF may reduce the immunogenicity of FVIII by preventing its internalization by professionnal APCs, and reducing the subsequent presentation of FVIII−derived peptides to T lymphocytes.
Purification of an MLL Partner Associated Complex (MPAC) Suggests a Common Role for MLL Fusion Partners in Transcriptional Elongation.
Authors: Slany Robert K. , Mueller Dorothee , Bach Christian , Zeisig Deniz , Garcia-Cuellar Maria-Paz , Monroe Sara , Sreekumar Arun , Zhao Ron , Nesvizhskii Alexey , Chinnaiyan Arul , Hess Jay L.
Published: April 2020

Chromosomal translocations at 11q23 are characteristic for a subset of particularly aggressive acute lymphoid and myeloid leukemias. This event fuses the mixed lineage leukemia (MLL) gene to a puzzling variety of seemingly unrelated fusion partners to create novel transcription factors with potent oncogenic activity. Despite the molecular heterogeneity, MLL−associated leukemias show similar clinical presentations and an almost identical gene expression pattern regardless of the translocation partner. We and others previously identified interactions between individual MLL translocation partners suggesting that these might share a common mechanism for transformation. To characterize these interactions in more detail we generated a HEK293−derived producer line expressing a FLAG−tagged derivative of the frequent fusion partner ENL. Double affinity purification of nuclear extracts on FLAG and ENL antibody resins reproducibly yielded a macromolecular complex of 10 proteins we term MPAC, for MLL Partner Associated Complex, which in addition to ENL includes the MLL fusion partners AF4, AF9 and AF5q31, and two components involved in transcriptional elongation: pTEFb and DOT1L. The pTEFb dimer consists of cyclin T1/2 and CDK9 and is responsible for serine−2 phosphorylation of the C−terminal repeat domain of elongating RNA Polymerase II. DOT1L is the only known histone methyltransferase that catalyzes dimethylation of H3K79, a modification introduced during elongation of actively transcribed transcription units. Surprisingly, PC3 and RING1b, members of the polycomb repressive complex I were also identified in the MPAC complex. Further examination demonstrated a direct interaction of ENL with DOT1L through C−terminal interaction domains by yeast 2−hybrid, pulldown and colocalization experiments. Knock−down of ENL diminished global H3K79 dimethylation and also impaired various promoters in transient reporter assays suggesting the MPAC complex is involved in transcriptional regulation mediated by DOT1L, even in the absence of MLL fusion proteins. In summary, our results indicate that the most common MLL fusion partners are associated in an hitherto undescribed complex involved in transcriptional elongation.