bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcitonin A Genotype Is Associated with Risk of Acute Graft-Versus-Host Disease Following Allogeneic Bone Marrow Transplantation for Children with Acute Myeloid Leukemia in First Remission.
Authors: Goyal Rakesh K. , Fairfull Liane , Gerbing Robert B. , Alonzo4 Todd A. , Meshinchi4 Soheil , Lange4 Beverly J. , Woods4 William J. , Ferrell Robert E.
Published: April 2020

Background: Procalcitonin (PCT) is a 13 kD 116-amino acid prohormone. Elevated serum levels of procalcitonin have been noted in patients with bacterial infections such as septicemia, pneumonia and immune-mediated conditions like graft-versus-host disease (GvHD) and organ rejection. In this study, we sought to examine if a single base pair polymorphism (a T to C transition) at position −624 of the calcitonin A gene (CALCA) was associated with risk of acute GvHD.

Subjects: 113 children (median age 10 y; range 0.38 – 19.0 y) with de novo acute myeloid leukemia (AML) treated on recent Children’s Oncology Group protocols 2891, 2941 or 2961. All of these patients received matched sibling bone marrow transplant (BMT) in first clinical remission. Ablative conditioning consisted of busulfan and cyclophosphamide. All patients received methotrexate until day 100 for GvHD prophylaxis.

Methods: All clinical DNA specimens were subjected to whole genome amplification. Genotyping was performed using the 5′-nuclease (TaqMan) assay on the ABI 7700 DNA analyzer (Applied Biosystems).

Results: Observed CALCA -624 T>C genotypes in 113 patients were: TT = 52 (46%), TC = 43 (38%) and CC = 18 (16%); T allele = 73 (65%) and C allele = 40 (35%). There were no significant differences in patient characteristics (age, gender, race, study 2891/2941/2961, white blood count at diagnosis, FAB type, or cytogenetics) between the TT vs. combined TC or CC groups. Overall, acute GvHD grade I–IV (Seattle criteria) was observed in 55/113 patients (49%). Nineteen out of 52 (37%) with TT, while 21 out of 43 (49%) with TC, and 15 out of 18 (83%) with CC CALCA genotypes developed acute GvHD. Alternatively, acute GvHD was observed in 37% patients with common TT genotype and in 59% patients with variant TC or CC genotype (p=0.023). The odds ratio for developing acute GvHD (grade I – IV) for children with TC or CC genotypes was 2.5 (CI 1.17, 5.33) compared with those with TT genotype. The difference in observed acute GvHD remained significant when the analyses were limited to more uniform subgroups: 54 patients on single trial, 2961 only (p=0.028) and 42 whites only on 2961 trial (p=0.028). Severe acute GvHD (grade III – IV) was seen in 6/52 (12%) patients with TT vs. 6/61 (10%) in those with TC or CC genotypes; this difference was not statistically significant (p =0.771). There were no significant differences in day +100 transplant related deaths, engraftment, relapse rate or overall survival. Plans: Association between CALCA genotypes and infectious complications will be also analyzed when clinical data abstraction is completed. CALCA −1752 C>G and 3740 A>G genotyping is in progress and clinical correlative analyses will be repeated at genotype and haplotype levels.
Outcome with the Hyper-CVAD and Imatinib Mesylate Regimen in Philadelphia (Ph) Positive Acute Lymphocytic Leukemia (ALL).
Authors: Thomas Deborah A. , Faderl Stefan , Cortes Jorge , O’Brien Susan , Giles Francis , Garcia-Manero Guillermo , Kornblau Steven , Andreeff Michael , Beran Miloslav , Verstovsek Srdan , Pierce Sherry , Letvak Laurie , Salvado August , Champlin Richard , Kantarjian Hagop
Published: April 2020

In Ph+ ALL, complete remission (CR) rates with intensive chemotherapy such hyper-CVAD is 90%, but most patients (pts) relapse within a median time of 16 months [Kantarjian et al, JCO18:547, 2000; Kantarjian et al, Cancer101:2788, 2004]. Single agent therapy with the tyrosine kinase inhibitor imatinib mesylate in relapsed or refractory Ph+ ALL or chronic myelogenous leukemia in lymphoid blast phase yielded CR rates of 20% with rapid disease recurrence. A phase II clinical trial of concurrent hyper-CVAD and imatinib was designed to improve these results, with the initial regimen of imatinib 400 mg orally daily days 1–14 of each course (fractionated cyclophosphamide, vincristine [VCR], doxorubicin and dexamethasone alternating with high dose methotrexate and cytarabine) followed by imatinib, VCR and prednisone maintenance with intensifications months 6 and 13. Allogeneic stem cell transplant (SCT) was performed in CR if feasible. Preliminary results of first 20 pts treated were encouraging [Thomas et al, Blood103:4396, 2004]. Recent modifications included increasing dosing of imatinib to 600 mg daily days 1–14 of course 1, then daily if tolerated with courses 2–8. Maintenance was extended to 24 months with imatinib indefinitely. To date, 43 pts with Ph+ ALL have been treated from April 2004 to July 2005. Thirty-six pts had active disease, either untreated (n=31) or refractory (n=1) to one induction course without imatinib; 7 pts were in CR after one induction course without imatinib. Of 35 evaluable pts, 33 (94%) achieved CR (1 induction death, 1 failed to meet platelet criteria for CR). Median days to response was 21 days. 13 pts underwent allogeneic SCT within a median of 3 months from start of therapy (range, 1–12). After a median follow-up of 3 yrs (range 1–48 months), 1 primary refractory pt relapsed at 12 months, 1 de novo pt had isolated CNS relapse, 2 pts relapsed after allogeneic SCT (no post SCT imatinib) and 2 pts changed therapy for persistent Ph+ metaphases (1 relapsing). Deaths in CR included 5 older pts without allogeneic SCT (1 osteomyelitis, 1 mucormycosis, 1 C. difficile colitis, 1 sudden death, 1 GNR sepsis) and 4 pts after allogeneic SCT (3 graft-versus-host disease, 1 GNR sepsis). Outcome with the hyper-CVAD and imatinib regimen continues to demonstrate favorable disease-free survival rates compared with hyper-CVAD alone, particularly for the de novo group. Use of higher dose imatinib concurrently appears to be feasible. Molecular response rates appear to be improved with the higher dose imatinib; additional accrual will be required to assess impact of modifications, including role of allogeneic SCT.
Myeloid Lineage-Restricted Progenitors Contribute to Vascular Endothelium.
Authors: Bailey Alexis S. , Willenbring Holger , Jiang Shuguang , Schroeder David A. , Anderson Daniel A. , Grompe Markus , Wong Melissa H. , Fleming William H.
Published: April 2020

Endothelial progenitors of hematopoietic origin have a role in promoting recovery from vascular injury. Recently we have shown that following radiation damage, transplanted hematopoietic stem cells (HSCs) give rise to endothelial cell outcomes at the clonal level. This raises the question of whether prospectively identifiable HSC progeny have endothelial cell activity. Using β-galactosidase and EGFP expression as donor markers, we show that lineage-restricted myeloid progenitors can give rise to endothelial cells. Transplanted common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs) gave rise to similar numbers of endothelial cells in liver portal vein branches. Donor-derived endothelial cells expressing functional endothelial markers including CD31, von Willebrand factor (vWF) and Tie-2 were clearly distinguished from mature hematopoietic cells by the absence of CD45 and F4/80 expression. Confocal microscopy showed the co-localization of donor and endothelial cell marker expression in individual cells so that endothelial cells could be discriminated from perivascular cells. To evaluate cell fusion as a potential mechanism for the emergence of endothelial cells of hematopoietic origin, HSCs transgenic for EGFP and Tie-2-Cre recombinase were transplanted into ROSA26 reporter (R26R) mice. Activation of β-galactosidase reporter gene expression, indicative of cell fusion with a donor-derived hematopoietic cell, could be detected in hepatocytes but not in endothelial cells. However, due to the tissue-specificity of the Tie-2 promoter, endothelial fusion products in which the donor nucleus had failed to activate the Tie-2-driven Cre recombinase would have also lacked detectable reporter gene expression. To ensure that the absence of β-galactosidase expression in the donor-derived endothelial cells was not due to insufficient nuclear reprogramming, HSCs transgenic for EGFP and R26R were transplanted into Tie-2-Cre recipients. Donor-derived endothelial cells were again negative for both β-galactosidase expression and activity. To determine whether radiation injury is required for the incorporation of donor cells into the endothelium, a parabiosis model was utilized. Both long-term donor-derived endothelial cell outcomes and multilineage hematopoiesis were detected in parabiotic mice. These results demonstrate that CMPs and further lineage-restricted GMPs are capable of differentiating into endothelial cells in the absence of cell fusion. Importantly, radiation injury is not required for endothelial cell engraftment, indicating that circulating progenitors contribute to angiogenesis during steady-state conditions.
CUL-4A Short Hairpin RNA (shRNA) Impairs Normal Hematopoiesis of Human Cord Blood CD34+ Cells In Vitro and In Vivo.
Authors: Shieh Jae Hung , Zhang Jianxuan , Lu Xiaoling , Zhou Pengbo , Moore Malcolm A.S.
Published: April 2020

The CUL-4A ubiquitination machinery regulates the stability of HOXA9 by promoting its ubiquinitation and proteasome-dependent degradation. Our previous study showed the perturbation of CUL-4A biosysthesis in 32D cells by RNA-mediated interference increased an accumulation of HOXA9, and impaired G-CSF-induced terminal differentiation to granulocytes. HOXA9 is preferentially expressed in primitive hematopoietic cells and downregulates with differentiation. In this study, a lentiviral vector expressed both CUL-4A shRNA and GFP was constructed and was used to transduce cord blood (CB) CD34+ cells (GFP-shCUL-4A-CD34+ cells). The ability of GFP-shCUL-4A-CD34+ cells to form CFC colonies, to differentiate into myeloid, erythroid, B-lymphoid and T-lymphoid cells in vitro, and to engraft in irradiated NOD/SCID mice was evaluated. Our results showed that knockdown of CUL-4A m-RNA dramatically inhibited hematopoisis both in vivo and in vitro. In the CFC assay, FACS-sorted GFPbright-shCUL-4A-CD34+ cell formed fewer colonies (5%) that were smaller size (<500 cells/colonies) relative to progeny of GFPbright-CD34+ cells. In contrast, FACS-sorted GFPdim-shCUL-4A-CD34+ cells produced only 50% less colonies than the control GFPdim-CD34+ cells. However, GFPdim-shCUL-4A-CD34+ cells had 30% more colonies with <500 cells/colony but 82% less colonies with >500 cells/colony comparing to that of GFPdim-CD34+ cells. After transplanting the GFP-shCUL-4A-CD34+ cells into irradiated NOD/SCID mice for 6 weeks, GFP-shCUL-4A-CD45+ cells decreased more than 80% in the mouse bone marrow. These human GFP-shCUL-4A CD45 + cells were capable of forming GFP+-CFC but with >80% reduction in their potency. No reduction in % of GFP-CD45+ cells and GFP+-CFC was observed in the GFP-CD34+ cells group pre- and post-transplantation. The in vitro culture systems showed that knockdown of CUL-4A mRNA in CB CD34+ cells resulted in the developmental impairment in monocytic, megakaryocytic, erythroid, B lymphoid and T-lymphoid lineages. This impairment is a time- and shCUL-4A dose-dependent process. Further study using cell cycle analysis indicated that transduction of CUL-4A shRNA to HL-60 or CD34+ cells induced apoptosis. Our results, for the first time, demonstrate an essential role of the CUL-4A ubiquitin ligase in maintaining cell survival to ensure proper differentiation of normal human stem and progenitor cells into different hematopoietic lineages both in vivo and in vitro.
Hematopoietic Defects in Cited2 Mutant Fetal Liver.
Authors: Chen Yu , Yang Yu-Chung
Published: April 2020

Cited2 [cAMP-responsive element-binding protein (CBP)/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2] is a newly identified transcriptional modulator. Knockout of Cited2 gene is embryonic lethal because of heart and neural tube defects. Cited2 binds directly to CBP and p300, which have been shown to be crucial for hematopoietic stem cell self-renewal and proper hematopoietic differentiation, respectively. Cited2 also induces the expression of a polycomb-group gene, Bmi-1, which is essential for self-renewal of adult hematopoietic stem cells. These connections provided rationale to study the potential role of Cited2 in hematopoiesis. Mouse fetal liver is the major hematopoietic organ from day 10 postcoitus until right before birth. The smaller sized Cited2−/− fetal liver and significantly decreased fetal liver cellularity strongly suggest the potential defect in hematopoiesis. In vitro colony formation assay in methycellulose-based medium was used to characterize the hematopoietic progenitors. We found that fetal liver cells from E13.5, 14.5 and E15.5 Cited2−/− embryos gave rise to much less colonies, which reflects the decreased number and proliferative ability of hematopoietic progenitors due to Cited2 deficiency. Immunostaining of lineage-specific cell surface markers followed by flow cytometry was performed to characterize different hematopoietic populations in E14.5 and E15.5 fetal liver of wild type and Cited2−/− embryos. Cited2−/− fetal liver cells displayed a significant reduction in numbers throughout the hematopoietic hierarchy including hematopoietic stem cells (Lin− c-Kit+ Sca-1+), progenitor cells (Lin− c-Kit+), and differentiated cells of different lineages (CD45+, Ter119+, Mac-1+, Gr-1+), thus revealing a multi-level hematopoietic deficiency of Cited2−/− embryos. Long-term reconstitution experiment was then carried out to measure the ability of hematopoietic stem cells from Cited2−/− fetal liver cells to engraft and reconstitute hematopoietic system of congenic recipient mice. Mice transplanted with Cited2−/− fetal liver cells showed reconstitution of T cells whereas a 2-fold decrease in the reconstitution of B cell and myeloid lineages was observed, indicating a compromised ability of Cited2−/− fetal liver hematopoietic stem cells to maintain hematopoiesis. The results suggest an important role of Cited2 in hematopoietic differentiation and a selective function of Cited2 in B lymphoid &myeloid induction. The underlying mechanisms responsible for these defects will be pursued by microarray analysis of gene expression profile of Cited2−/− fetal liver cells, followed by more detailed phenotypic analyses of B and myeloid lineage markers plus in vitro and in vivo functional assays.
The Vent-Like Homeobox Gene Ventx2 Is a Novel Candidate for a Human Hematopoietic Regulatory Protein.
Authors: Arseni Natalia , Ahmed Farid , Sadlon Timothy J. , d’Andrea Richard J. , Hiddemann Wolfgang , Buske Christian , Feuring-Buske Michaela
Published: April 2020

Identification of the genes which are critically involved in normal and leukemic hematopoiesis is a major goal for experimental and clinical hematology. Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. Since it was shown, that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus it is of particular interest to examine the expression profile and function of the human homologue Ventx2 in hematopoietic development. We first analysed Ventx2 expression by RT-PCR in CD3, CD19 and CD33 cells highly purified by FACS sort from peripheral blood of healthy donors. Expression of Ventx2 was detected in T- and B- as well as differentiated myeloid cells indicating that Ventx2 is expressed in multiple hematopoietic lineages. Furthermore, VENTX2 expression was recurrently detected in bone marrow samples from AML patients at diagnosis as determined by RT-PCR (n=6). In an attempt to characterize the functional relevance of Ventx2 expression for hematopoietic development we retrovirally engineered murine hematopoietic progenitor cells to constitutively express the gene using a MSCV-based retroviral construct with an IRES-EGFP cassette. Successfully transduced cells were injected into lethally irradiated mice or used for in vitro experiments. At the level of the clonogenic progenitor VENTX2 induced a 3fold increase in the number of CFU-G (n=5; p<0.001) compared to the GFP control (62 versus 25 CFU-G, respectively, per 1000 initially plated cells) without increasing the total number of colonies, indicating that VENTX2 promoted granulocytic differentiation in vitro. Re-plating assays confirmed the effect of the homeobox gene with an over 9fold increase in the number of secondary CFU-G (511 vs. 54, respectively, per 1000 initially plated cells). When the effect of VENTX-2 on the frequency of LTC-IC was determined by limiting dilution assay (n=2), no major differences were detected between the homeobox gene and the control arm (453 LTC-IC vs. 801 LTC-IC per 1x106 cells, respectively, p = n.s.). Furthermore, the number of colonies generated per LTC-IC did not significantly differ between the two arms (17 colonies for VENTX2 and 26 colonies for the control). In NOD/SCID mice VENTX2 induced a 2.9fold increase in the proportion of CD15+ mature myeloid cells within the GFP-positive compartment (n=7) compared to the control (n=9)(6.4 % vs. 2.2 %, respectively; p<0.02), translating into 4 x 106 (± 1 x 106) human CD15+ /GFP+ cells per mouse in the VENTX2 group compared to 2x106 cells (± 6 x 105) in the control. These findings characterize VENTX2 as a novel regulatory protein in human hematopoiesis and add information about the role of non-clustered homeobox genes in early blood development.
Identification of Two Src Recruitment Sites in the Juxtamembrane Region of Flt3 with Opposing Effects on Flt3-Ligand-Induced Signaling.
Authors: Ronnstrand Lars , Heiss Elke , Sundberg Christina , Masson Kristina , Pedersen Malin , Sun Jianmin , Bengtsson Susanne
Published: April 2020

Early signal relay steps upon ligand-binding to the receptor tyrosine kinase Flt3, i.e. sites of Flt3-autophosphorylation and subsequent docking partners, are mainly unresolved. Here we demonstrate for the first time identification of ligand-induced in vivo phosphorylation sites in Flt3. By immunoprecipitation of specific tryptic peptides contained in the juxtamembrane region of human Flt3 and subsequent radiosequencing we identified the tyrosine residues 572, 589, 591 and 599 as in vivo autophosphorylation sites. Focusing on Y589 and Y599, we examined Flt3-ligand-mediated responses in WT-Flt3, Y589F-Flt3 and Y599F-Flt3 expressing 32D cells. Compared to WT-Flt3-32D cells, 32D-Y589F-Flt3 showed upon ligand-stimulation enhanced Erk activation as well as proliferation/survival whereas 32D-Y599F-Flt3 cells displayed substantially diminished responses. Both pY589 and pY599 were identified as association sites for multiple signal relay molecules including Src family kinases. Consistently, 32D-Y589F-Flt3 and 32D-Y599F-Flt3 showed decreased FL-triggered Src activation, impaired phosphorylation of the adapter molecules Cbl and ShcA and deficient receptor ubiquitination and degradation. Interference with the Src-dependent negative regulation of Flt3 signaling may account for the enhanced mitogenic response of Y589F-Flt3. pY599 was additionally found to interact with the protein tyrosine phosphatase Shp2. As Y599F-Flt3-32D lacked ligand-induced Shp2 phosphorylation and since silencing of Shp2 in WT-Flt3-expressing cells mimicked the Y599F-Flt3-phenotype we hypothesize that recruitment of Shp2 to pY599 contributes to FL-mediated Erk activation and proliferation.

To summarize, our work presents novel insights in Flt3-mediated signal transduction. We have identified the in vivo autophosphorylation sites of the juxtamembrane region of Flt3, revealed Src family kinases and Shp2 as binding partners of pY589 and/or pY599, respectively, as well as their potential impact on FL-mediated signaling in Flt3-32D cells. Future work will now focus on elucidation of additional and possibly novel interaction partners of the found phosphorylation sites by employing an unbiased proteomics approach. With this gained knowledge it will be of interest to see whether ITDs differing in the nature of the duplicated tyrosines also confer distinct signaling behavior. If so, these tyrosines might serve as a diagnostic marker and point towards a successful combinatorial therapy consisting of a receptor tyrosine kinase inhibitor and an inhibitor for the specifically affected signal transduction pathway.
CrkL Mediates SDF-1-Induced Chemotaxis through Ras, Rac, and Erk Activation in Hematopoietic Cells.
Authors: Arai Ayako , Yan Weihua , Aoki Mae , Mizuchi Daisuke , Kurosu Tetsuya , Miki Tohru , Miura Osamu
Published: April 2020

The chemokine SDF-1 was originally identified as a growth promoting factor for pre-B cells and has been recognized to be indispensable for hematopoiesis from the findings on both its knock-out mice and those of its receptor CXCR4. SDF-1 is secreted by the bone marrow stromal cells and plays a crucial role for retention and homing of hematopoietic cells in the bone marrow through chemotaxis. In addition, SDF-1 regulates not only motility but also survival, proliferation, differentiation, and adhesion of the cells. Furthermore it was recently reported that leukemic cells show different responses to SDF-1 stimulation from normal hematopoietic cells. Despite its important roles in hematopoiesis, however, intracellular signaling pathways from CXCR4, especially those regulating chemotaxis of the hematopoietic cells, have largely remained to be clarified. We investigated the molecular mechanisms regulating chemotaxis induced by SDF-1 in hematopoietic cells and found that the enforced expression of adaptor molecule CrkL enhanced SDF-1-induced chemotaxis of murine hematopoietic cell lines, BAF3 and 32Dcl3, expressing CXCR4 on the cell surface. To examine the pathways up- and downstream of CrkL, we performed transient and stable transfection of CrkL and its mutants into BAF3 cell. Overexpression of CrkL enhanced SDF-1-induced Erk activation through Raf-1 and Mek. CrkL also enhanced SDF-1-induced activation of the Ras family GTPases Ras and Rap1 as well as the Rho family GTPase Rac. Among the dominant negative mutants of these small GTPases, those of Ras and Rac significantly suppressed CrkL-enhanced Erk activation. In addition, we also performed in vitro kinase assays using GST-CrkL as a substrate and found that SDF-1 induced phosphorylation of CrkL through the Src family tyrosine kinase Lyn. However, pretreatment with PP1, an inhibitor of Lyn, did not suppress activation of Ras, Rap1, Rac, and Erk at the concentration that suppressed the phosphorylation of CrkL. In addition, overexpression of a CrkL mutant that lacks the tyrosine targeted by Lyn did not reduce SDF-1-induced Erk activation. These results show that the phosphorylation of CrkL was not necessary for activation of the small GTPases and Erk. On the other hand, overexpression of a CrkL mutant lacking the N-terminal SH3 domain (CrkLdSH3N) required for the constitutive association with C3G, the guanine nucleotide exchange factor for Ras family GTPases, significantly reduced SDF-1-induced Erk activation. Therefore, this domain was required for the enhancement of SDF-1-induced Erk activation by CrkL through Ras. Finally we performed chemotaxis assays with transiently expressed mutants of these molecules. CrkLdSH3N as well as dominant negative forms of Ras and Rac reduced SDF-1-induced chemotaxis. Together, these results indicate that CrkL mediates SDF-1-induced chemotaxis of hematopoietic cells through activation of Ras, Rac, and the Raf/MEK/Erk pathway.
Novel Mechanism of Activated FLT3-Associated Leukemogenisis: FLT3 Regulates Wnt Signaling by Phosphorylating β-Catenin and Promoting β-Catenin Nuclear Localization.
Authors: Kajiguchi Tomohiro , Chung Eun Joo , Kiyoi Hitoshi , Naoe Tomoki , Neckers Len , Trepel Jane
Published: April 2020

Mutations of the gene encoding the FLT3 tyrosine kinase are the most common mutation in AML. Recently Wnt-dependent signaling has been suggested to cooperate with FLT3 in myeloid transformation. In the work presented here we have examined the mechanism of the interaction of FLT3 and the Wnt effector β-catenin in FLT3 leukemia cells. In comparison to cells with wild-type FLT3, cells with activating mutations of FLT3 including internal tandem duplication (ITD) of the juxtamembrane domain of FLT3 and the FLT3 D835Y mutation had elevated levels of tyrosine-phosphorylated β-catenin. Although β-catenin was located mainly in the cytoplasm in WT-FLT3 cell lines, it was primarily nuclear in cells with FLT-3 activating mutations. Treatment of activated FLT3 cells with the FLT3 kinase inhibitor AG1296 decreased tyrosine-phoshorylation of β-catenin and downregulated the level of nuclear β-catenin. Treatment of WT-FLT3 cell lines with FLT3 ligand increased tyrosine-phosphorylation of β-catenin and upregulated its nuclear accumulation. Immunoprecipitation experiments demonstrated that FLT3-ITD binds to β-catenin and treatment with AG1296 decreased interaction between FLT3-ITD and β-catenin. In vitro kinase assays using recombinant FLT3 and β-catenin showed that an active FLT3 kinase phosphorylates tyrosine residues of β-catenin directly and AG1296 decreased the activated FLT3-induced tyrosine-phosphorylation of β-catenin. The data demonstrate that activated FLT3 induces tyrosine phosphorylation of β-catenin and accumulation of nuclear β-catenin. We propose that increased β-catenin nuclear signaling is a potential mechanism of activated FLT-3-associated malignant transformation.
YM477, a Novel Orally-Active Thrombopoietin Receptor Agonist.
Authors: Suzuki Ken-ichi , Abe Masaki , Fukushima-Shintani Mari , Sugasawa Keizo , Hirayama Fukushi , Kawasaki Tomihisa , Hiramatsu Hidefumi , Nakahata Tatsutoshi
Published: April 2020

Thrombopoietin (TPO) is the principal physiologic regulator of platelet production. The search for an orally-active nonpeptidyl small molecule TPO receptor agonist has resulted in the discovery of YM477. YM477 acted specifically on the TPO receptor and stimulated megakaryocytopoiesis throughout the development and maturation of megakaryocytes just as TPO does. YM477, however, was shown to have high species specificity, effective in only humans and chimpanzees. Recently, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were characterized as an efficient engraftment model for human hematopoietic stem cells, as this model results in the production of human platelets. In this way, we examined the in vivo platelet-increasing effect of YM477 in human platelet-producing NOD/SCID mice in which human hematopoietic stem cells were transplanted. In this study, we used commercially available cryopreserved human fetal liver CD34+ cells as a source of human hematopoietic stem cells. The cells were transplanted into sublethally irradiated (240 cGy) NOD/SCID mice. Human platelets started to appear in peripheral blood of these mice 4 weeks after transplantation. The production of human platelets continued up to one year post-transplant. Various doses of YM477 (0, 0.3, 1, and 3 mg/kg/day) were orally administered for 14 days to NOD/SCID mice that had been confirmed to produce human platelets stably. Oral administration of YM477 dose-dependently increased the number of human platelets produced by these mice, with significance at 1 mg/kg/day and above. The increase in the human platelet count reached about 2.7-fold at 1 mg/kg/day and about 3.0-fold at 3 mg/kg/day on day 14. Withdrawal of YM477 administration caused the human platelet count to return to the pretreatment level. The number of murine platelets did not change during the study period. Next, to evaluate the function of human platelets produced in peripheral blood of these mice, the expression of activation-dependent marker CD62P (P-selectin) on human platelets stimulated with thrombin receptor agonist peptide (TRAP) were examined. CD62P expression on human platelets was induced by the stimulation of blood from transplanted mice with TRAP, suggesting that human platelets produced in NOD/SCID mice were functional. Furthermore, the maximum response of CD62P expression on human platelets induced by TRAP was evaluated before and after administration of YM477 at 3 mg/kg/day for 14 days. CD62P expression was not changed by administration of YM477, which was similar to the results obtained with a vehicle group. These results suggest that YM477 is an orally active TPO receptor agonist useful for treating patients with thrombocytopenia.
Expression of the Human Germinal Center Associated Lymphoma (HGAL) Protein Identifies a Subset of Classical Hodgkin Lymphoma of Germinal Center Derivation and Improved Outcome.
Authors: Natkunam Yasodha , His Eric D. , Zhao Shuchun , Elson Paul , Pohlman Brad , Levy Ronald , Lossos Izidore S.
Published: April 2020

Classical Hodgkin lymphoma (CHL) exhibit crippled somatic mutations in rearranged Ig genes and an immunophenotype unlike any other hematolymphoid malignancy. Expression profiles of CHL cell lines show that CHL is most closely related to EBV-transformed peripheral blood B-cells with marked loss of B lineage-specific gene expression. The Human Germinal center-Associated Lymphoma (HGAL) gene was identified as a candidate molecule that predicted improved outcome in diffuse large B-cell lymphoma. Previously, we showed that HGAL mRNA and protein are expressed specifically in germinal centers (GC), GC-derived lymphomas and a substantial proportion of CHL. Here, we sought to investigate whether HGAL protein expression could serve to distinguish biologically distinct subgroups of CHL. Ninety informative cases of CHL on a tissue microarray of 1.0 mm representative cores of paraffin embedded tissue were used. Staining was performed and the percent of stained Hodgkin cells was scored on a 3-tiered scale as negative (0%), weak (<30%), and strong (>30%), as previously described (Natkunam et al. Blood 2005, 105:3979–86). These cutoffs were chosen before results were correlated with clinical endpoints. Twenty-five patients (28%) showed no staining for HGAL and 25 patients (28%) had weak staining; HGAL was strongly expressed in 40 patients (44%). Thirteen of the 90 patients had died and 19 had failed (i.e. relapsed or died). The overall 5-year survival (OS) and failure-free survival (FFS) rates were 87% + 4% and 79% + 4%, respectively. HGAL expression was associated with improved OS in univariate analysis (p=0.04, no vs weak/strong staining), as were age < 45 years (p<0.001), stage I or II (p=0.02), and possibly histology (nodular sclerosing vs other, p=0.09). However, in multivariate analysis with age, stage and histology, HGAL expression no longer had an impact (p=0.93). HGAL did not impact FFS in either univariate (p=0.13) or multivariate analysis (p=0.87). The expression of the GC-specific marker HGAL in a subset of CHL suggests that a substantial proportion of CHL retain characteristics of lymphomas of GC derivation. The association with improved OS in univariate but not multivariate analysis suggests HGAL may be a biologic marker related to known clinical parameters of improved OS and FFS. Comparative studies with well-characterized GC markers, BCL6 and CD10, and with BCL2, MUM1 and Blimp-1 are underway to further define biologically distinct subsets of CHL. Analysis of additional cases of CHL is also on-going to substantiate these findings in multivariate analysis.
Potential Role of Immunosuppression in Therapeutic Effects Conferred by Cord Blood- Derived Mesenchymal Stem Cells.
Authors: El-Badri (Dajani) Nagwa S. , English Denis , Hakki Amal , Mudhusoodanan Sriram , Sanberg Cyndy D. , Sanberg Paul R.
Published: April 2020

Our previous studies consistently demonstrate enhanced neural protective effects of cord blood in comparison to stem cells from adult marrow. Similarly, cord blood possesses diminished immuno-stimulatory activity, and the basis of this effect has not been defined. Since neural damage may be potentiated by immune activation of inflammatory cascades, we examined the effects of cord blood-derived MSCs (mesenchymal stem cells) on immune responses. We isolated and characterized a population of cord blood MSCs. These cells maintained their pluripotency in culture. Progeny generated in the absence of differentiation were strongly adherent, did not express CD34, CD45, CD3, CD19 antigens, and did not generate hematopoietic colonies in methylcellulose. However, cultured cord blood MSCs possessed a remarkable ability to support the proliferation as well as the differentiation of hematopoietic cells in vitro. In addition, supernatants from cultured cord blood MSCs promoted survival of peripheral blood mononuclear cells cultured under conditions designed to induce cell stress and limit protein synthesis. We examined immune modulation by cord blood MSCs after co-culture with murine splenocytes. While spleen cells from normal C57Bl/6 mice exhibited a prominent IgM response after immunization with the T-cell dependent antigen, SRBCs, this response was significantly decreased after incubation with cord blood MSCs. Consistently, cord blood MSCs mitigated the enhanced mixed lymphocyte proliferative response C57Bl/6 T-cells exhibit when exposed to lymphocytes from non-related animals. To investigate whether these immune suppressive properties could be therapeutically useful in a transplantation model for autoimmune disease, cord blood MSCs were transplanted into sublethally irradiated BXSB mouse model for systemic lupus. Thirty days after infusion, defective IgM humoral immune responses of splenocytes of these mice normalized. This normalization paralleled normalization of the disrupted lymphoid cellularity observed in the spleens of diseased animals. Our results are consistent with the hypothesis that immune regulation is involved in the therapeutic utility of MSCs.
Stro-1 Positive and Stro-1 Negative Human Mesenchymal Stem Cells Express Different Levels of Immunosuppression.
Authors: Francois Sabine , Zhang Yizhuo , Bensidhoum Morad , Mazurier Christelle , Nasef Aisha , Bouchet Sandrine , Mathieu Noelle , Gorin Norbert-Claude , Lopez Manuel , Bourgeade Michel , Thierry Dominique , Chapel Alain , Fouillard Loic
Published: April 2020

Mesenchymal stem cells (MSC) have been shown to elicit immunosuppressive effect on allogeneic lymphocyte response. However, MSC are heterogeneous and data on the inhibitory abilities of different MSC subsets are lacking. The Stro-1 antigen potentially defines a more pure and more primitive MSC subset

We compared the suppressive property of expanded Stro-1+ and Stro-1− MSC when add to mixed lymphocyte reactions (MLR) or to mitogen response assays. Results showed that T lymphocyte proliferation was significantly more inhibited by expanded Stro-1+ (11.0%~63.7% of maximal response) than by expanded stro-1− MSC (35.5%~106% of maximal response) (p<0.01). Results indicated that as few as 1,000 expanded Stro-1+ MSC could induce a similar inhibition of lymphocyte proliferation than 10 times more (10,000 cells) expanded Stro-1− MSC −. Stro-1+ MSC was the main population responsible for T cell inhibition. The expression of genes coding for inhibitors of T-cell activation and proliferation (IL-10, LIF, IDO, TGF-b1, HGF, HLA-G), for adhesion molecules (VCAM, LFA-3), and for chemotactic factors (IL-8, SDF-1) was analysed by semi-quantitative real time RT-PCR in the Stro1+ and Stro1- MSC. Results are expressed as a fold increase in the mRNA level in expanded Stro-1+ compared to Stro-1− MSC. IL-10 (0.24 fold p=0.002) and TGF-β1 (0.43 fold, p=0.03) were down regulated in expanded Stro-1+ compared to Stro-1− MSC. LIF (106 fold, p=0.025), IDO (6130 fold, p=0.04), HGF (24 fold, p=0.01), and HLA-G (260 fold, p= 0.02) were up regulated in expanded Stro-1+ compared to Stro-1− MSC. VCAM-1 and LFA-3 were up regulated in expanded Stro-1+ compared to Stro-1− MSC (28.2 fold, p=0.03 and 298.4 fold, p=0.04 for VCAM-1 and LFA-3 respectively): IL-8 was over expressed in expanded Stro-1+ (4.9 fold, p=0.0015). There was no significant difference between expanded Stro-1+ and Stro-1− MSC for SDF-1. These results suggest that LIF, IDO, HGF, HLA-G inhibitory factors and VCAM1, LFA-3 adhesion molecules are mainly responsible for the different T cell inhibition observed between Stro-1+ and Stro-1− MSC.

These findings suggest that in clinics a Stro-1+ pre-selection of MSC might produce a more effective immunosuppression especially for the prevention and the treatment of graft versus host disease.
Association of HLA-E Polymorphism with the Incidence of Severe Bacterial Infections in Sickle Cell Anemia.
Authors: Tamouza Ryad , Fortier Catherine , Diagne Ibrahima , Diallo Dapa A. , Labie Dominique , Girot Robert , Charron Dominique
Published: April 2020

While it is commonly admitted that the susceptibility to severe infections (septicemia, meningitis, or osteomyelitis) in Sickle Cell Anemia (SCA) patients is a major cause of mortality and morbidity, these complications remains poorly explained. However, several lines of evidence highlight the involvement of an immunogenetic modulation, including both innate and adaptive immune responses. HLA-E belongs to the HLA-class I non-classical family and is ubiquitously expressed. HLA-E molecules bind either to self-peptides from the leader sequences of various HLA-class I molecules and thus modulate NK (Natural killer) cell activation/inhibition, or to the microbial derived-peptides from human viruses or bacteria to induce T cell responses. Only two functional alleles, HLA-E*0101 and HLA-E*0103, differing by a single amino acid substitution in the α2 heavy chain domain and by their cell surface expression level, have been reported so far. In order to evaluate whether the HLA-E polymorphism could influence the incidence of severe bacterial infections in SCA, we analyzed a cohort of 99 SCA patients living in Paris, but for the majority, originating from black Africa. All patients were older than 5 years of age at inclusion. Among the cohort, 51 presented at least one severe bacterial infection and 48 did not disclose any major infectious episode in their clinical history. Statistical analysis showed that the incidence of severe bacterial infections are high when the patient’s genotype was HLA-E*0101/E*0101 (47% in the first group versus 21% in the second group; χ2 = 7.54 p = 0.006, pc = 0.01, OR: 3.28, [95%CI = 1.28–9.07]), supporting a negative effect of this genotype. The genetic association herein found between HLA-E polymorphism and severe bacterial infections is of relevance, given the emerging evidence for the involvement of HLA-E molecules in pathogen-derived peptide presentation to human CD8+ T cells and hence in host response to pathogens. Furthermore, these data are in agreement with our previous findings in bone marrow transplantation settings, were the homozygous state for HLA-E*0101 allele is also a risk factor for early severe bacterial infections. Both situations by constitutively lowering the overall threshold of resistance to infection, might unmask anti infectious genetic factors normally silent.
Elevated Plasma Arginase Levels in Hemoglobinopathies.
Authors: Larkin Sandra K. , Morris Claudia R. , Styles Lori A. , Kuypers Frans A.
Published: April 2020

The decreased bioavailability of arginine (Arg) and the resulting lower nitric oxide (NO) production has been shown to be an important factor in the pathology of sickle cell disease (SCD) and thalassemia. Vascular alterations leading to pulmonary hypertension are important factors of heart failure and death in these hemoglobinopathies. Red blood cell (RBC) hemolysis in these patients will release arginase into the circulation and contribute to the reduction of plasma Arg levels, change the Arg-to-ornithine ratio, and increase other downstream amino acid metabolites. Such compounds, including proline and polyamines, may contribute to vascular and airway remodeling. To study the contribution of arginase released from RBC, we measured the arginase activity and arginase protein concentration in the plasma and RBC lysates of normal controls, SCD patients, and thalassemia patients.

Arginase activity was determined by the conversion of 14C-labeled guanidine-L-arginine to 14C-labeled urea, then to 14CO2 by urease and trapped as Na214CO3 for scintillation counting. Arginase and hemoglobin concentration were measured by ELISA (BioVendor, Candler, NC and Bethyl Labs Inc., Montgomery, TX respectively). Mean cellular hemoglobin concentration was measured with a Coulter Counter (Beckman Coulter, Fullerton, CA) or Technicon H3 analyzer (Tarrytown, NY). RBC were separated by density gradient centrifugation with OptiPrep (Axis Shield, Dundee, UK). Arginase specific activity (SA) was calculated by dividing the activity by the concentration to yield mole of Arg metabolized per g of arginase per hour. We found a higher level of arginase in the plasma and RBC of SCD and thalassemia patients as compared to normal controls. These levels correlated with cell-free hemoglobin content (plasma) or reticulocyte count (RBC). The light density fraction (<1.077 g/ml) of RBC, enriched in reticulocytes, showed higher arginase levels confirming increased arginase levels in young RBC. Interestingly, as the plasma arginase protein and activity increased with cell-free hemoglobin, the SA decreased. Similarly in RBC lysates, the SA decreases with hemoglobin concentration. This suggests that a factor exists within the RBC that negatively modulates arginase activity. Due to the high SA of arginase at low hemoglobin concentrations, low levels of intravascular hemolysis can generate relatively high levels of Arg breakdown.

We conclude that low levels of intravascular hemolysis of RBC with increased arginase content in SCD and thalassemia, play a role in the altered Arg metabolism and decreased NO production. While the reason for high levels of arginase in the RBC as well as complete clinical impact of elevated arginase activity in plasma remain to be determined, dysregulated Arg metabolism and a shift toward Arg catabolic products, may be associated with the development of pulmonary hypertension and mortality in SCD and thalassemia.
Gene Expression Profiling Can Identify Novel MRD Markers for AML.
Authors: Woodward Eleanor L. , Gilkes Amanda F. , Walsh Val , Austin Steve J. , Daly Sarah B. , Lin J. A. , Grimwade David , Burnett Alan K. , Mills Kenneth I.
Published: April 2020

In AML, the majority of patients <60 years of age will enter remission but at least 50% will subsequently relapse, therefore the monitoring of minimal residual disease (MRD) during treatment has become an important issue. Current molecular markers for MRD are mainly limited to the RT-PCR detection of the fusion genes resulting from recurrent translocations which paradoxically are mostly limited to favourable risk groups who are least likely to relapse leaving the majority of the patients, including those with a normal karyotype, without a molecular marker suitable for monitoring.

Two hundred and twenty patients have been assessed by gene expression profiling using Affymetrix U133A chips and the data analysed with the aim of identifying novel MRD markers for patients who do not currently have a suitable marker. As an initial “proof of principle”, we have identified possible MRD markers for patients with either t(15;17), t(8;21) or inv(16) and correlated with changes in expression of these markers with clinical changes as measured by established molecular MRD markers (PML-RARα or WT1).

Of the expression profile from 22,283 probe sets in 29 cases of t(15;17), 20 genes were identified which had at least a two fold over expression which was unique to the t(15;17) subgroup. Of these several of the probe sets were related to the same gene, but from the reduced gene list 2 (HGF and ILGF binding protein) were selected for quantitation by quantitative PCR. Similarly the expression profile identified 20 genes which were unique to the 15 cases of t(8;21), and 20 genes which were unique to the 19 cases of inv(16). These included ETO and MYH11 representing the respective 3′ end of the respective fusion transcript. Three other genes (PRAME, POU4F1, and IL5RA) were selected for the t(8;21) cases and ST18, CLIP-170 and MNI for the inv(16) cases. When the relative quantitative expression of each of these “unique” genes was correlated with the expression of the established markers of minimal residual disease (PML-RARα or WTI) there was good correlation. These data suggest that gene expression profiling can identify ‘unique’ genes which can be used to develop specific markers for minimal residual disease monitoring for a larger proportion of cases of AML than is currently available.
High Incidence of t(7;12)(q36;p13) with Involvement of HLXB9 in Infant AML.
Authors: von Bergh Anne R.M. , van Drunen Ellen , van Wering Elisabeth R. , Meijerink Jules P. , Pieters Rob , Beverloo Berna
Published: April 2020

The ETV6/TEL gene (12p13) is rearranged in a spectrum of hematological malignancies resulting in the formation of a functional fusion gene or an ETV6 promoter enforced transcription of over 25 different partner genes. The t(7;12)(q36;p13) is a recurrent translocation with a heterogeneous breakpoint distribution in 7q36. We have shown a fusion transcript between HLXB9 and ETV6 in infant AML with t(7;12), however a fusion transcript is not consistently found. To study the incidence of t(7;12) and the involvement of HLXB9 in infant and childhood acute leukemia we screened 320 infants and pediatric patients younger than 36 months by FISH. We studied the presence of an HXLB9-ETV6 fusion and quantified the expression of various genes in the proximity of the 7q36 breakpoints. Cases studied by FISH included 1) 179 ALL and 54 AML patients younger than 36 months diagnosed in the Netherlands between 1991–1997 or 2003–2004, obtained from the DCOG; (ALL: 14 age 0–12 months, 55 age 12–24 months, 110 age 24–36 months; AML: 16 age 0–12 months, 22 age 12–24 months, 16 age 24–36 months); and 2) 85 infant-ALL from the Interfant-99 study and 2 additional infant-AML. In total, six AML patients were found to carry the t(7;12)(q36;p12) of which 5 were infants and 1 was 18-months old. This indicates that the t(7;12) encompasses 30% of infant AML. In ALL the t(7;12) seems to be uncommon, only one of the 264 patients harbored the t(7;12). The t(7;12) was associated with a poor clinical outcome. A fusion of ETV6 to HLXB9 was found in four AML with t(7;12). The expression of 7q36 genes (HLXB9, NOM1, Lmbr1, RNF32 and SHH) was determined by real-time PCR in several groups: t(7;12)+ cases, t(7;12)- AML, t(7;12)- ALL and healthy individuals. NOM1, Lmbr1, RNF32 and SHH were equally expressed among t(7;12)+ and other groups. However, the HLXB9 expression was highly increased in t(7;12)+ compared to every control group, including those with an HLXB9-ETV6 fusion, suggesting that ectopic expression of HLXB9 is commonly involved in the t(7;12)(q36;p13).