bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted α particle immunotherapy for myeloid leukemia
Authors: Jurcic Joseph G. , Larson Steven M. , Sgouros George , McDevitt Michael R. , Finn Ronald D. , Divgi Chaitanya R. , Ballangrud Åse M. , Hamacher Klaus A. , Ma Dangshe , Humm John L. , Brechbiel Martin W. , Molinet Roger , Scheinberg David A.
Published: October 2019

Unlike β particle–emitting isotopes, α emitters can selectively kill individual cancer cells with a single atomic decay. HuM195, a humanized anti-CD33 monoclonal antibody, specifically targets myeloid leukemia cells and has activity against minimal disease. When labeled with the β-emitters 131I and 90Y, HuM195 can eliminate large leukemic burdens in patients, but it produces prolonged myelosuppression requiring hematopoietic stem cell transplantation at high doses. To enhance the potency of native HuM195 yet avoid the nonspecific cytotoxicity of β-emitting constructs, the α-emitting isotope 213Bi was conjugated to HuM195. Eighteen patients with relapsed and refractory acute myelogenous leukemia or chronic myelomonocytic leukemia were treated with 10.36 to 37.0 MBq/kg 213Bi-HuM195. No significant extramedullary toxicity was seen. All 17 evaluable patients developed myelosuppression, with a median time to recovery of 22 days. Nearly all the 213Bi-HuM195 rapidly localized to and was retained in areas of leukemic involvement, including the bone marrow, liver, and spleen. Absorbed dose ratios between these sites and the whole body were 1000-fold greater than those seen with β-emitting constructs in this antigen system and patient population. Fourteen (93%) of 15 evaluable patients had reductions in circulating blasts, and 14 (78%) of 18 patients had reductions in the percentage of bone marrow blasts. This study demonstrates the safety, feasibility, and antileukemic effects of 213Bi-HuM195, and it is the first proof-of-concept for systemic targeted α particle immunotherapy in humans.
Regulation of vasculogenesis and angiogenesis by EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells
Authors: Oike Yuichi , Ito Yasuhiro , Hamada Koichi , Zhang Xiu-Qin , Miyata Keishi , Arai Fumio , Inada Tomohisa , Araki Kimi , Nakagata Naomi , Takeya Motohiro , Kisanuki Yaz Y. , Yanagisawa Masashi , Gale Nicholas W. , Suda Toshio
Published: October 2019

Although the cellular and molecular mechanisms governing angiogenesis are only beginning to be understood, signaling through endothelial-restricted receptors, particularly receptor tyrosine kinases, has been shown to play a pivotal role in these events. Recent reports show that EphB receptor tyrosine kinases and their transmembrane-type ephrin-B2 ligands play essential roles in the embryonic vasculature. These studies suggest that cell-to-cell repellent effects due to bidirectional EphB/ephrin-B2 signaling may be crucial for vascular development, similar to the mechanism described for neuronal development. To test this hypothesis, we disrupted the precise expression pattern of EphB/ephrin-B2 in vivo by generating transgenic (CAGp-ephrin-B2 Tg) mice that express ephrin-B2 under the control of a ubiquitous and constitutive promoter, CMV enhancer-β-actin promoter-β-globin splicing acceptor (CAG). These mice displayed an abnormal segmental arrangement of intersomitic vessels, while such anomalies were not observed in Tie-2p-ephrin-B2 Tg mice in which ephrin-B2 was overexpressed in only vascular endothelial cells (ECs). This finding suggests that non-ECs expressing ephrin-B2 alter the migration of ECs expressing EphB receptors into the intersomitic region where ephrin-B2 expression is normally absent. CAGp-ephrin-B2 Tg mice show sudden death at neonatal stages from aortic dissecting aneurysms due to defective recruitment of vascular smooth muscle cells to the ascending aorta. EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells plays an essential role in vasculogenesis, angiogenesis, and vessel maturation.
Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils
Authors: Iwabuchi Kazuhisa , Nagaoka Isao
Published: October 2019

This study is focused on the functional significance of neutrophil lactosylceramide (LacCer)–enriched microdomains, which are involved in the initiation of a signal transduction pathway leading to superoxide generation. Treatment of neutrophils with anti-LacCer antibody, T5A7 or Huly-m13, induced superoxide generation from the cells, which was blocked by PP1, a Src kinase inhibitor; wortmannin, a phosphatidylinositol-3 kinase inhibitor; SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor; and H7, an inhibitor for protein kinase C. When promyelocytic leukemia HL-60 cells were differentiated into neutrophilic lineage by dimethyl sulfoxide (DMSO) treatment, they acquired superoxide-generating activity but did not respond to anti-LacCer antibodies. Density gradient centrifugation revealed that LacCer and Lyn were recovered in detergent-insoluble membrane (DIM) of neutrophils and DMSO-treated HL-60 cells. However, immunoprecipitation experiments indicated that LacCer was associated with Lyn in neutrophils but not in DMSO-treated HL-60 cells. Interestingly, T5A7 induced the phosphorylation of Lyn in neutrophils but not in DMSO-treated HL-60 cells. Moreover, T5A7 induced the phosphorylation of p38 MAPK in neutrophils. T5A7-induced Lyn phosphorylation in neutrophil DIM fraction was significantly enhanced by cholesterol depletion or sequestration with methyl-β-cyclodextrin or nystatin. Collectively, these data suggest that neutrophils are characterized by the presence of cell surface LacCer-enriched glycosphingolipid signaling domain coupled with Lyn and that the ligand binding to LacCer induces the activation of Lyn, which may be suppressibly regulated by cholesterol, leading to superoxide generation through the phosphatidylinositol-3 kinase–, p38 MAPK–, and protein kinase C–dependent signal transduction pathway.
HLA-DR15 (DR2) is overrepresented in myelodysplastic syndrome and aplastic anemia and predicts a response to immunosuppression in myelodysplastic syndrome
Authors: Saunthararajah Yogen , Nakamura Ryotaro , Nam Jun-Mo , Robyn Jamie , Loberiza Fausto , Maciejewski Jaroslaw P. , Simonis Toni , Molldrem Jeffrey , Young Neal S. , Barrett A. John
Published: October 2019

The extent and importance of autoimmune mechanisms in myelodysplastic syndrome (MDS) and the role of immunosuppression in the treatment of this disease are not well defined. We report overrepresentation of HLA-DR2 and its serologic split HLA-DR15 in both MDS and aplastic anemia (AA). Four clinically and ethnically defined patient groups were analyzed. The HLA-DR15 antigen frequencies among North American white MDS patients (n = 72) and AA patients (n = 59), who received immunosuppressive treatment at the National Institutes of Health (NIH), were 36% and 42%, respectively. These antigen frequencies were significantly higher than that of the control population of 240 North American white NIH blood donors typed for HLA antigens by the same molecular technique (HLA-DR15, 21.3%,P = .01 for MDS, P < .001 for AA). Among North American white patients reported in the International Bone Marrow Transplant Registry (IBMTR), 30% of 341 MDS patients and 33% of 364 AA patients were positive for HLA-DR2. These antigen frequencies were higher than those reported for the general North American white population (HLA-DR2, 25.3%, P = .089 for MDS,P = .01 for AA). The DR15 and DR2 frequencies were significantly increased in MDS refractory anemia (RA) (P = .036 and P = .01, respectively) but not MDS refractory anemia with excess blasts. In the NIH MDS patients, HLA-DR15 was significantly associated with a clinically relevant response to antithymocyte globulin (ATG) or cyclosporine immunosuppression (multivariate analysis, P = .008). In MDS with RA, DR15 may be useful as a guide to pathophysiology, prognosis, and treatment.
Efficient gene transfer of CD40 ligand into primary B-CLL cells using recombinant adeno-associated virus (rAAV) vectors
Authors: Wendtner Clemens-Martin , Kofler David M. , Theiss Hans D. , Kurzeder Christian , Buhmann Raymund , Schweighofer Carmen , Perabo Luca , Danhauser-Riedl Susanne , Baumert Jens , Hiddemann Wolfgang , Hallek Michael , Büning Hildegard
Published: October 2019

B cells of chronic lymphocytic leukemia (B-CLL) are resistant to transduction with most currently available vector systems. Using an optimized adenovirus-free packaging system, recombinant adeno-associated virus (rAAV) vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and CD40 ligand (AAV/CD40L) were packaged and highly purified resulting in genomic titers up to 3 × 1011/mL. Cells obtained from 24 patients with B-CLL were infected with AAV/EGFP or AAV/CD40L at a multiplicity of infection (MOI) of 100 resulting in transgene expression in up to 97% of cells as detected by flow cytometry 48 hours after infection. Viral transduction could be specifically blocked by heparin. Transduction with AAV/CD40L resulted in up-regulation of the costimulatory molecule CD80 not only on infected CLL cells but also on noninfected bystander leukemia B cells, whereas this effect induced specific proliferation of HLA-matched allogeneic T cells. Vaccination strategies for patients with B-CLL using leukemia cells infected ex vivo by rAAV vectors now seems possible in the near future.
Mixed peripheral blood stem cell transplantation for autoimmune disease in BXSB/MpJ mice
Authors: Yamamoto Yoshihisa , Wang Bingyan , Fukuhara Shirou , Ikehara Susumu , Good Robert A.
Published: October 2019

We examined whether mixed allogeneic transplantation with syngeneic plus allogeneic peripheral blood stem cells (PBSCs) is sufficient to interrupt autoimmune processes in BXSB mice and confer a potential therapeutic option for the treatment of patients with autoimmune diseases. Eight-week-old BXSB mice were lethally irradiated and reconstituted with BALB/c (H-2d)+BXSB (H-2b) PBSCs, in which the number of injected allogeneic progenitor cells was 5 times that of syngeneic progenitor cells. The survival of mixed PBSC chimeras (BALB/c+BXSB→BXSB) was 80% at the age of 48 weeks, whereas that of full chimeras (BALB/c→BXSB) was 90%. Mixed PBSC transplantation (PBSCT) prevented the production of anti-DNA antibodies and the development of lupus nephritis in BXSB recipients and induced tolerance to both allogeneic and syngeneic antigens. Moreover, mixed chimeras exhibited immunological functions superior to fully allogeneic chimeras. On the other hand, increases in the number of BXSB PBSCs resulted in the transfer of lupus nephritis in BXSB+BALB/c→BALB/c mice. Thus, the number of hematopoietic progenitor cells from normal mice proved critical to the prevention of autoimmune diseases. We propose that mixed allogeneic PBSCT for the interruption of the autoimmune process can be carried out by injecting increased numbers of allogeneic normal hematopoietic progenitor cells to prevent the relapse of autoimmune diseases, although it is necessary to decide upon a minimum dose of syngeneic PBSCs to achieve the desired beneficial effects on autoimmunity.
Unsupervised Analysis of Human Leukemia Cells Identifies the Valosin-Containing Protein as a Putative Marker for Glucocorticoid Resistance.
Authors: Lauten Melchior , Neuhoff Nils v. , Welte Karl , Schlegelberger Brigitte , Schrappe Martin
Published: October 2019

The prednisone response (PR) is a clinical phenomenon in childhood acute lymphoblastic leukemia (ALL) indicating in vivo glucocorticoid (GC) resistance. For patients treated within the ALL-Berlin-Frankfurt-Muenster treatment protocols, the PR is the strongest predictor for therapy outcome. PR is defined by the number of peripheral leukemic blasts on day eight of initial prednisone treatment. The threshold value for the distinction between good and poor prednisone response is 1000 blasts/μl. In trial ALL-BFM 90, prednisone good responders (PGR) had an estimated probability of 6-years event free survival (6y-pEFS) of 82% in contrast to prednisone poor responders (PPR), who showed a 6y-pEFS of only 34% (Schrappe et al., Blood 95(11):3310–3322, 2000). Despite various attempts to elucidate the cause of glucocorticoid resistance, its biological background still remains unclear. Therefore, we performed a case-control study for PR using two-dimensional electrophoresis (2-DE) as screening method to broadly investigate proteome differences in leukemic blasts from in vivo glucocorticoid sensitive and resistant childhood B-cell precursor ALL patients. Producing virtual average gels and difference maps for the proteomes of each responder group by software analysis we identified differentially expressed proteins in glucocorticoid sensitive as well as in glucocorticoid resistant patients. Among the differentially expressed proteins, the valosin-containing protein (VCP) appeared to be overexpressed in PPR. VCP was identified after excision of the respective spot from the 2-DE gel, tryptic digestion and analysis by surface enhanced laser desorption and ionisation (SELDI) mass spectrometry. In previous studies VCP had already been associated with prognosis in different solid tumour entities. This was the reason, why we chose the VCP for validation and quantification by Western Blot analysis. For the Western Blot analysis, a second independent case-control-group of B-cell precursor ALL patients was investigated (cases: 10 PPR, controls: 20 PGR), which confirmed the 2-DE results: median VCP expression (P25-P75) in all ALL samples was 0.19 (0.12 – 0.40) compared to 0.15 (0.11 – 0.28) in PGR and 0.34 (0.14 – 0.99) in PPR patients (P>0.05). This is the first study detecting the association of VCP and GC resistance in two different sets of closely matched patient samples representing one of the leading disease entities treated with GCs.
Immunoglobulin (Ig) Variable Gene Usage Identifies a Subset of Unmutated Chronic Lymphocytic Leukemia (CLL) Patients Expressing 51p1-Like alleles and a Particularly Unfavorable Prognostic Outcome.
Authors: Ghia Emanuela M. , Guarini Anna , Mauro Francesca R. , Gentile Massimo , Starza Irene Della , Capello Daniela , Gaidano Gianluca , Foa Robert
Published: October 2019

Great interest has recently focused on a biased use of the immunoglobulin (Ig) heavy chain variable region genes (VH genes) and on a preferential light chain (VL gene) usage in patients with chronic lymphocytic leukemia (CLL). VH / VL bias maybe correlated with a possible response to a common antigen epitope. To further investigate the Ig VH gene usage in CLL, DNA was extracted from peripheral blood mononuclear cells of 137 consecutive unrelated and untreated CLL cases. PCR amplification was performed using VH family specific and consensus JH primers, followed by automated sequencing. The sequences were aligned to Ig sequences from the GenBank and V-BASE databases. The VH family gene usage was as follows: VH1 19.7%, VH2 4.4%, VH3 49.6%, VH4 21.9%, VH5 2.2% and VH6 2.2%. Mutated VH genes (>2% mutations) were found in 58.4% of the cases analyzed and unmutated VH genes (<2% mutations) were detected in 41.6% of samples. Within the VH1 gene family, 88.9% of cases were unmutated and the most frequently expressed alleles were related to 51p1 (a major VH1-69 allele), that accounted for 10.2% of all VH rearranged genes and for 51.9% of rearranged VH1 genes. Among all patients expressing unmutated 51p1-like alleles, a preferential use of certain D segments was observed: D2-2 accounted for 28% of cases, followed by D3-3 and D3-16 that accounted for 21.4% and 14.3%, respectively; D1-20, D3-10, D3-22, D5-18 and D6-6 were less frequently expressed (1 case each). The most frequently used JH gene segment was JH6 (64.3%), whereas JH4 and JH3 accounted for 21.4% and 14.3%, respectively. All patients expressing 51p1-like alleles showed an identical CDR2 region. The CDR3 region was characterized by similarities in a subset of cases: the amino acid motifs DIVVVPAA(I/M) and GGXYDY(I/V)WGSYRPNDAFDI were the most common and accounted each for 14.3% of CLL samples that expressed 51p1-like alleles: 79% of all patients expressing 51p1-like alleles had a long CDR3 region of 19.4 ± 2.9 codons. In contrast to what has been observed in other diseases, e.g. mixed cryoglobulinemia and non-Hodgkin lymphoma, all VH1-69 expressing CLL cases analyzed were not associated with HCV infection. The overall median survival for this subset of patients was 40 months, considerably shorter than the reported median survival of unmutated CLL cases; furthermore, to date 36% of patients have succumbed of their disease. The results of this study confirm, on the one hand, that restricted CDR3 features and a biased VH gene repertoire are suggestive of a role of antigen selection operational in this disease and have allowed, on the other hand, to identify a subset of patients with a particularly unfavorable survival compared to the overall population of unmutated CLL cases.
DNA Microarray Analysis of Stage Progression Mechanism in Adult T-Cell Leukemia.
Authors: Choi Young Lim , Tsukasaki Kunihiro , Onimaru Yasuyuki , Yamada Yasuaki , Kamihira Shimeru , Tomonaga Masao , Mano Hiroyuki
Published: October 2019

Adult T-cell leukemia (ATL) is an intractable malignancy of peripheral CD4-positive T cells, in which human T-cell leukemia virus type I plays a pathological role. Clinical course of ATL can be subdivided into several phases; indolent “smoldering” and “chronic” stages, and aggressive “acute” stage. Individuals at the former usually undergo a stage progression toward the latter within several years, and ATL cells at the acute stage are highly refractory to current chemotherapeutic reagents. Molecular mechanisms underlying this stage progression are poorly understood yet. DNA microarray enables us to quantitate the mRNA amount for tens of thousands of genes simultaneously, and may provide novel insights into the pathogenesis as well as the stage progression mechanism of ATL. However, since the proportion of ATL cells within mononuclear cells (MNCs) of peripheral blood (PB) varies among different stages, it would be desirable to purify and directly compare ATL cells for an accurate profiling of gene expression. Given the fact that most PB MNCs are occupied by ATL cells in both chronic and acute stages, we purified CD4-positive T cells from PB of ATL individuals at chronic (n = 19) or acute (n = 22) stage. As a normal control, surface marker-matched T cells were purified from PB of healthy volunteers, and stimulated in vitro or not with PHA (n = 3 for each). A total of 47 specimens were thus subject to DNA microarray analysis with Affymetrix HGU133 A&B array sets, measuring the transcriptional level of ~33,000 human genes. Unsupervised hierarchical clustering based on the whole genes indicated that normal CD4-positive T cells, irrespective of PHA stimulation, had a molecular signature distinct from that of CD4-positive ATL cells, while the acute and chronic stages of ATL were not clearly separated from each other. Combination of a t-test (Welch’s ANOVA, P<0.001) and an effect size selection (>150 U) identified 46 genes, expression of which contrasted the two clinical stages of ATL. Correspondence analysis of such stage-associated genes also demonstrated visually that both phases of ATL have a distinct molecular signature. Additionally, a very high accuracy was obtained by an artificial neural network in a trial of gene expression-based stage diagnosis. Further, we tried to screen, from our data set, novel “acute stage-specific gene markers”, resulting in the identification of a gene for a cytokine receptor. Intriguingly, the serum concentration of its ligand protein was elevated in ATL patients, especially at the acute stage. This autocrine loop for cell growth would be an interesting candidate for the transforming events which triggers the stage-progression in ATL, and also be a candidate for the targets of novel ATL therapies.
Gene Expression Profiling of Leukemic Cell Lines and Primary Leukemias Reveals Conserved Molecular Signatures among Subtypes with Specific Genetic Aberrations: Identification of Fusion Gene-Specific Transcriptional Profiles and Expression Pattern of Tyrosine Kinase-Encoding Genes.
Authors: Andersson Anna , Edén Patrik , Lindgren David , Nilsson Jens , Lassen Carin , Heldrup Jesper , Fontes Magnus , Borg Åke , Mitelman Felix , Johansson Bertil , Höglund Mattias , Fioretos Thoas
Published: October 2019

Hematologic malignancies are characterized by fusion genes of biological/clinical importance. Immortalized cell lines with such aberrations are today widely used to model different aspects of leukemogenesis. Using cDNA microarrays, we determined the gene expression profiles of 40 cell lines as well as of primary leukemias harboring 11q23/MLL-rearrangements, t(1;19)[TCF3/PBX1], t(12;21)[ETV6/RUNX1], t(8;21)[RUNX1/CBFA2T1], t(8;14)[IGH@/MYC], t(8;14)[TRA@/MYC], t(9;22)[BCR/ABL1], t(10;11)[PICALM/MLLT10], t(15;17)[PML/RARA], or inv(16)[CBFB/MYH11]. Unsupervised classification revealed that hematopoietic cell lines of diverse origin, but with the same primary genetic changes, segregated together, suggesting that pathogenetically important regulatory networks remain conserved despite numerous passages. Moreover, primary leukemias co-segregated with cell lines carrying identical genetic rearrangements, further supporting that critical regulatory pathways remain intact in hematopoietic cell lines. Transcriptional signatures correlating with clinical subtypes/primary genetic changes were identified and annotated based on their biological/molecular properties and chromosomal localization. Furthermore, the expression profile of tyrosine kinase-encoding genes was investigated, identifying several differentially expressed members, segregating with primary genetic changes, which may be targeted with tyrosine kinase inhibitors. The identified conserved signatures are likely to reflect regulatory networks of importance for the transforming abilities of the primary genetic changes and offer important pathogenetic insights as well as a number of targets for future rational drug design.
Comparison of Gene Expression by Normal and Leukemic Progenitor Cells in Childhood B Cell Precursor ALL - Are There ALL Stem Cells? .
Authors: Anthony J. , George A. , Senadheera S. , Barsky L. , McCarthy C. , Schaub B. , Triche T. , Schofield D. , Franklin J. , Parkman R. , Crooks G. , Marjoram P. , Weinberg K.
Published: October 2019

B-cell precursor ALL (B-ALL) are generally believed to express genes, including those encoding surface antigens like CD19, CD22 and CD38, which correspond to those normally expressed by B-cell progenitors. Our previous efforts to immunophenotypically isolate normal HSC from B-ALL marrow for autologous transplantation demonstrated that patients’ putative normal CD34+ CD38− HSC populations contained leukemic cells, even though the B-ALL was positive for CD38 expression. These results raised the question of whether B-ALL stem cells, like normal HSC and ANLL stem cells, might be CD34+ CD38− . Therefore, gene expression was analyzed in rare CD34+ CD38− and more common CD34+ CD38+ B-ALL cells, and in normal CD34+ CD38− HSC and CD34+ CD38+ CD19+ B cell progenitors (BCP). RNA from stringently FACS isolated cells from marrow samples of >10 different B-ALL patients were amplified and hybridized to Affymetrix U133A microarrays. CD34+ CD38+ B-ALL cells expressed numerous genes expected of either B-ALL or normal BCP, including CD19, CD22, CD79a/b, Tdt, RAGs, Pax5 which were not expressed by normal CD34+ CD38− HSC. The expression pattern of the CD34+ CD38− B-ALL cells was different from that of the CD34+ CD38+ B-ALL cells, and from normal HSC or BCP. Compared to normal CD34+ CD38− HSC, the CD34+ CD38− B-ALL cells also expressed several B cell differentiation markers including CD19, CD22 and CD79, but underexpressed CD59, a modulator of responses to decay accelerating factor. There were also differences between the CD34+ CD38− B-ALL cells and the CD34+ CD38+ B-ALL cells. As predicted, CD34+ CD38− B-ALL cells underexpressed CD38, but unexpectedly lacked Tdt. The CD34+ CD38− B-ALL cells expressed multiple genes involved in cell cycle checkpoint control (cullin, MAD2, BUB3, MCMs, DDA3), survival and cellular/genomic maintenance (IAPs, translin), and immortalization (prohibitin), which were not expressed by the CD38+ B-ALL cells. FACS analyses of newly diagnosed patients with B-ALL demonstrated a rare population of CD34+ CD38− CD19+ leukemia cells, confirming that the CD19 mRNA found by array was translated. B-ALL consists of at least two subpopulations, one of which (CD34+ CD38− CD19+) has features that distinguish it both from normal HSC and the majority of B-ALL cells in the marrow. Besides their unique immunophenotype, the CD34+ CD38− CD19+ B-ALL cells express genes likely to be important for processes needed to maintain a malignant stem cell population. Potential implications of a phenotypically identifiable B-ALL stem cell include improved understanding of the biology of ALL and its therapy, the confounding results of MRD analyses, and the limitations of array analyses of unfrationated leukemic cells.
High Glucose Activates AKT Signaling and Induces Upregulation of Genes Encoding Glycolytic Enzymes Glut-1 and HK-2 in Acute Lymphocytic Leukemia.
Authors: Buglio Daniela , Schober Wendy , Contractor Rooha , McQueen Teresa , Zeng Zhihong , Kantarjian Hagop , Weiser Mary A. , Andreeff Michael , Konopleva Marina
Published: October 2019

Background: Hyperglycemia represents a common side effect of steroid therapy and is not uncommon during treatment of patients with acute lymphocytic leukemia (ALL). Our recent retrospective study in 278 adult patients with ALL demonstrated that hyperglycemia during induction chemotherapy occurred in up to 37% of patients and was associated with a shorter median complete remission duration and a shorter median survival (Cancer 2004 Mar 15;100(6):1179–85). The serine/threonine AKT, a major downstream PI3K target, promotes continued cell growth and metabolism by increasing glucose uptake, stimulating glycolysis and ATP production. Recent data in transgenic models demonstrated that AKT activation resulted in transcriptional upregulation of enzymes essential for glycolysis including hexokinase (HK) and Glut-1 (Nature Med., 2004, 10:594). The purpose of this study was to determine whether high AKT activity in leukemic blasts in the setting of hyperglycemia promotes survival and chemoresistance of leukemic cells by stimulation of glycolysis.

Methods and Results: A 24-hour culture of REH cells in serum-free medium supplemented with 14mM glucose resulted in induction of AKT phosphorylation (by Western blot analysis) and increase in mRNA levels of Glut-1 and HK-2 (by quantitative TaqMan PCR). PI3K inhibitor LY294002 (15 μM) prevented AKT phosphorylation and decreased mRNA expression of Glut-1 and HK-2 in high-glucose conditions and enhanced doxorubicin- and dexamethasone-induced killing. We next investigated expression of AKT 1, Glut1, and HK2 mRNA in bone marrow samples from 14 adult patients with ALL (9 pre-B-, 2 B-, 3 pre-T-ALL). The results demonstrated elevated (> 2-fold compared with normal bone marrow or peripheral blood) levels of AKT in 7/14 patients, 6 of those showed increased levels of Glut1 and 5 of HK2. In addition, in samples from 2 patients with low baseline AKT expression high HK-2 mRNA levels were observed.

Conclusions: High expression of AKT, glucose transporter Glut-1 and the glycolytic enzyme HK-2 is prevalent in primary ALL blast cells. Our results suggest that high glucose further stimulates AKT signaling in ALL blast cells resulting in upregulation of glucose transporter Glut-1 and of the glycolytic enzyme HK-2. This effect was abrogated by inhibition of PI3K/AKT signaling, resulting in chemosensitization. These data provide rationale for use of PI3K/AKT inhibitors in the therapy of ALL. The hypothesis that tight blood glucose control improves outcome in ALL patients is currently being tested in a Phase III clinical trial at M.D. Anderson Cancer Center.
CXCR4+ CD45− Tissue-Committed Stem Cells (TCSC) for Myocardium Reside in the Bone Marrow, Are Mobilized into the Peripheral Blood during Myocardial Infarction, and “Home” to Infarcted Myocardium in CXCR4-SDF-1 and HGF/SF-c-Met Dependent Manner.
Authors: Kucia Magda , Dawn Buddhadeb , Guo Yiru , Hunt Greg , Wysoczynski Marcin , Majka Marcin , Zuba Ewa , Rezzoug Francine , Ildstad Suzanne T. , Bolli Roberto , Ratajczak Mariusz Z.
Published: October 2019

Several recent studies in animals as well as humans support the notion that bone marrow (BM)-derived cells participate in myocardial regeneration. However, this subject remains highly controversial and the identity of the specific cell type responsible for regeneration remains unknown. Recent work from our laboratory revealed that BM contains a highly mobile population of CXCR4+ cells that express mRNA for various markers of early tissue-committed stem cells (TCSC) and which are distinct from hematopoietic stem cells (HSC) (Leukemia2004:18;29–40). In the current study we investigated whether BM also contains a mobile pool of TCSC destined to differentiate into cardiomyocytes. Our data demonstrate that TCSC for cardiomyocytes (i) are present in significant amounts in BM harvested from young (1–2 month-old) while being barely detectable in older (1-year-old) mice; ii) reside in populations of murine BM-derived non-hematopoietic Sca-1+ CD45− cells and in population of human CXCR4+ CD34+ AC133+ CD45− BMMNC, iii) are mobilized from BM into peripheral blood (PB) during pharmacological mobilization or myocardial infarction; iv) the identified by us chemoattractants for these cells: stromal derived factor -1 (SDF-1), and hepatocyte growth factor/scatter factor (HGF/SF) are upregulated in infarcted myocardium, and v) blocking experiments with T140 (CXCR4 antagonist) and K252a (c-MET antagonist) confirmed that TCSC for cardiomyocytes are chemoattracted to the damaged myocardium in SDF-1-CXCR4 and SF/HGF-c-Met dependent manner. Thus, we conclude that the bone marrow is a potential source of TCSC for heart repair and since purified CD45+ HSC neither express cardiac markers nor differentiate in vitro into cardiomyocytes, we provide for the first time evidence that cardiac TCSC residing in bone marrow but not “plastic” HSC may account for cardiac differentiation of BM-derived cells. These observations provide rationale for further studies aimed at optimizing therapeutic cardiac regeneration by BM-derived non-hematopoietic cardiac TCSC. Finally, our observation that the number of marrow derived mobile/circulating cardiac TCSC is the highest in BM of young animals and decreases with age provides a novel insight into aging and may explain why the heart regeneration process becomes less effective in older individuals.
Imatinib Mesylate (STI571) Abrogates the Resistance to Doxorubicin in K562 Chronic Myeloid Leukemia Cells by Inhibition of BCR/ABL Kinase-Mediated DNA Repair.
Authors: Majsterek Ireneusz , Blasiak Janusz , Slupianek Artur , Skorski Tomasz
Published: October 2019

Imatinib mesylate (STI571), a specific inhibitor of the BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanisms by which inhibition of BCR/ABL activity results in pharmacological responses remain unknown. BCR/ABL-positive human CML cells resistant to doxorubicin K562doxoR and their sensitive K562doxoS counterparts were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. We examined kinetics of DNA repair after cell treatment with the drug by the alkaline comet assay. MTT assay was used to estimate resistance against doxorubicin and Western Blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562doxoR cells exhibited accelerated kinetics of DNA repair in comparison to doxorubicin-sensitive K562doxoS cells. Inhibition of BCR/ABL kinase in K562doxoR cells with 1 μM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA repair pathways, which in turn may enhance drug sensitivity of leukemia cells.
Transcriptional Programming in Quiescent Human Hematopoietic Stem Cells - Implications for Regulation of Lineage Commitment Decisions.
Authors: Pina Cristina , Enver Tariq
Published: October 2019

Human cord blood-derived CD133+G0 cells are a primitive population highly enriched in hematopoietic stem cells (HSC). We used this population to investigate the molecular characteristics of primitive human HSC, and, in particular, to unveil how different cell fates of quiescence, self-renewal and lineage commitment and differentiation are regulated at the molecular level. We isolated cord blood CD133+ cells in the G0 and G1 compartments of the cell cycle on the basis of low or high RNA content, respectively, as detected by Pyronin Y staining. More than 98% of the CD133+G0 cells were Ki67-negative, and at least 90% did not express CD38, thus confirming the quiescent and primitive status of the cells. Consistent with earlier findings showing that CD133+G0 cells have the highest reported frequency of LTC-IC (1), our quiescent population presented a significantly higher frequency of LTC-IC when compared to CD133+G1 cells. We further showed that CD133+G0 cells had significantly higher colony-forming capacity in progenitor assays and a higher CFU-Mix content. Initial RT-PCR analysis revealed that while both compartments express the erythroid marker beta-globin, myeloid MPO and lymphoid IL7R can only be observed in CD133+G1 cells. This suggests a hierarchy of commitment decisions in relation to cell cycle that places the erythroid signature upstream of myelo-lymphoid differentiation and may be in agreement with revised models of the hematopoietic differentiation tree recently proposed in mouse (2). This hypothesis is currently being assessed at the single-cell level. We next compared the overall gene expression programmes of CD133+G0 and G1 populations using global profiling. Consistent with the sorting criteria, CD133+G1-enriched transcripts have a comparatively higher frequency of cell cycle, protein synthesis and RNA processing, and metabolism-associated genes, which underlines the robustness of the data. The CD133+G1 population is associated with a lymphoid signature, including immunoglobulin heavy and light chains, and the SLAM family member CD48, which is consistent with the revised hierarchy of commitment decisions discussed above. Functional categories comparatively over-represented amongst CD133+G0-enriched genes include transcriptional regulation and signal transduction, suggesting that primitive quiescent HSC are ready to respond to a variety of cues that modulate alternative fate decisions. Since the transcriptional profile of a given population of cells may not reflect the transcriptional profile of each individual cell, we are currently analyzing the expression patterns of CD133+G0-enriched transcription factors (TF) at the single-cell level. This approach may help define subpopulations of cells with unique molecular signatures and suggest functional subcompartments within an otherwise heterogeneous population of primitive hematopoietic cells.
Antagonistic Effect of C/EBPα and Pax5 in Myeloid or Lymphoid Lineage Choice in Common Lymphoid Progenitors.
Authors: Fleischman Angela G. , Hsu Chia-Lin , Lai Anne Y. , Matsumoto Yukie , Weissman Irving L. , Kondo Motonari
Published: October 2019

Lymphoid lineage committed progenitors, such as common lymphoid progenitors (CLPs), maintain a latent myeloid differentiation potential, which can be initiated by stimulation through exogenously expressed cytokine receptors including IL-2 receptors. The transcription factor C/EBPa is promptly upregulated in CLPs upon ectopic IL-2 stimulation. We show that enforced C/EBPa expression is sufficient to initiate myeloid differentiation from CLPs, as well as from proT and proB cells, even though proB cells do not give rise to myeloid cells following ectopic IL-2 stimulation. Expression of Pax5, a B lymphoid affiliated transcription factor, is completely suppressed by enforced C/EBPa but not by ectopic IL-2 stimulation in proB cells. Introduction of Pax5 blocks ectopic IL-2 receptor-mediated myeloid lineage conversion in CLPs. These data suggest that C/EBPa is a proximal target of cytokine induced lineage conversion in lymphoid progenitors. Furthermore, complete loss of Pax5 expression triggered by upregulation of C/EBPa is a critical event for lineage conversion from lymphoid to myeloid lineage in CLPs and proB cells.
Negative Regulation of c-Kit Is Dependent on Direct Binding of Cbl to Tyrosines 568 and 936.
Authors: Masson Kristina , Heiss Elke , Ronnstrand Lars
Published: October 2019

C-Kit belongs to the family of type III receptor tyrosine kinases and is mainly expressed in hematopoietic precursor cells. Binding of stem cell factor (Kit ligand) leads to receptor dimerization, activation of intrinsic tyrosine kinase activity and cross-autophosphorylation of receptor tyrosine residues which provide specific docking sites for molecules that transduce the signal further downstream. Signaling through c-Kit results in several different cellular responses, most of which are directly involved in growth control and survival. Negative regulation of the receptor activity is therefore of much importance concerning pathogenic processes such as tumorigenesis in which activated c-Kit often plays a role. Here we present data showing that the ubiquitin E3 ligase Cbl must be activated by the Src family kinases and recruited to c-Kit in order to trigger receptor degradation. We demonstrate that the autophosphorylation sites Y568 and Y936 in c-Kit, previously reported to be binding sites for APS, Src or Grb2, respectively, are involved in Cbl recruitment and activation, possibly in a direct manner. I571 and L939 of c-Kit hereby determine the specificity for binding the N-terminal tyrosine kinase binding domain of Cbl. We show that activation of Cbl by Src family kinase members on one hand and direct or indirect recruitment of active Cbl to the pY568/I571 and pY936/L939 motifs of c-Kit on the other, are separable events and are both necessary for Cbl-mediated c-Kit degradation. Consistently, both the Y568/570F double mutant lacking Src kinase activity and the I571A/L939A double mutant lacking Cbl-specific interaction sites fail to ubiquitinate and degrade c-Kit upon ligand stimulation in stably transfected PAE and Ba/F3 cells. Thus, we could reveal novel aspects of the dynamic process of docking and activating signaling molecules at receptor phosphotyrosines that negatively regulate c-Kit-mediated responses. Moreover, we demonstrate that Cbl mediates monoubiquitination of c-Kit and that the receptor subsequently is targeted for lysosomal degradation. Taken together, our findings reveal novel insights on how c-Kit -mediated signal transduction is negatively regulated by Cbl.
T Cells Response and Autoantibodies to Human Heat Shock Proteins in Aplastic Anemia Patients.
Authors: Cheng Yunfeng , Tang Yong , Young Neal S.
Published: October 2019

Heat shock proteins (HSP) have been implicated in autoimmune diseases such as type I diabetes mellitus, systemic lupus erythematosus, and multiple sclerosis, in which T cell proliferative responses or autoantibodies towards endogenous HSP have been detected (Journal of Autoimmunity2003;20:313). HSP70 can function as an endogenous ‘danger’ signal, acting on antigen-presenting cells and critically influencing the decision between induction of tolerance and immunity upon antigen encounter (Millar et al. Nature Medicine2003; 9:1469). We studied T-cell proliferative responses and auto-antibodies to human HSP60, HSP70 and HSP90 proteins in 20 newly diagnosed aplastic anemia patients, peripheral blood was obtained (11 females, 9 males; age average 36.1±19 years). A non-isotopic immunoassay was used for BrdU incorporation into proliferative T cells and ELISA to measure HSP antibody titer. T cell proliferation was measured as the Eu-fluorescence in a time-resolved fluorometer. A positive result was defined as > 2 standard deviations (SD) from the mean of the controls. T-cell responses to HSP70 in the patient group (N=20; mean±SD Eu-fluorescence= 47,129±36,248) were significantly greater than those of the control group (N=18 healthy adult; mean Eu-fluorescence= 23,941±12,996; p=0.01). Fifty percent of the patients showed increased T cell proliferation after HSP70 stimulation compared to 5% in the control group (p=0.03). T-cell responses of the patient group to HSP90 and HSP60 were similar to those of the control group. Twenty percent of patients showed increased T cell proliferation to HSP 60 and HSP 90 stimulation compared to 5% in the control group (p=0.363). HSP antibody (IgG/A/M) seropositivity was 25% to HSP60, 50% to HSP70, and 5% to HSP90 in patients and 8% to HSP60, 0% to HSP70, and 0% to HSP90 in controls. Heightened autoimmunity to HSP70, but not to HSP60 and HSP90, is a feature of acquired aplastic anemia at presentation.
Mechanism of Selectivity of Arsenic Trioxide for Acute Promyelocytic Leukemia Cells from Screening a Genome Wide Set of Saccharomyces cerevisiae Deletion Strains.
Authors: Dilda Pierre J. , Perrone Gabriel G. , Dawes Ian W. , Hogg Philip J.
Published: October 2019

All-trans retinoic acid (ATRA) targets the underlying molecular lesion in acute promyelocytic leukaemia (APL) and leads to differentiation of leukemic blasts into mature granulocytes. Treatment with ATRA, however, is associated with the retinoic acid syndrome and relapse is also a problem. In relapsed patients, arsenic trioxide is considered the treatment of choice. Arsenic trioxide is a trivalent arsenical that selectively kills APL cells. To better understand how arsenic trioxide perturbs cellular functions we chose to screen a genome-wide set of Saccharomyces cerevisiae deletion strains for sensitivity or resistance to the drug. The idea was that genes whose loss conferred sensitivity to the drug would reflect mechanisms of cell death, while genes whose loss conferred resistance would suggest mechanisms of selectivity. 7.6% of the 4,564 mutants were more sensitive to arsenic trioxide than the wild-type strain, while 1.5% was more resistant. In accordance with published studies in mammalian cells, yeast mutants lacking genes required for cytoskeleton stability, response to oxidative stress and DNA repair were prominent in the sensitive list. The most prominent genes in the resistant list were that encoding the plasma membrane transporter, Fps1, Hog1 and repressors of Hog1. Fps1 is an aquaglyceroporin that mediates plasma membrane glycerol flux and uptake of inorganic arsenic in yeast. Its activity is controlled by the high osmolarity MAPK signalling or HOG pathway. There was a clear correlation between genes involved in modulating HOG pathway activity and altered arsenic trioxide tolerance in yeast. In mammalian cells, aquaglyceroporin 9 is the human homolog of yeast Fps1 and the p38 MAPK signalling cascade constitutes the analogous pathway to HOG. APL is characterized by chromosomal rearrangements of 17q21 leading to the formation of fusion proteins involving retinoic acid receptor α. Notably, the fusion protein interacts with p38 kinase and modulates its activity. Our findings imply that modulation of p38 by the APL fusion protein leads to increased arsenic trioxide uptake via aquaglyceroporin 9 in APL cells. This mechanism may account for the selectivity of arsenic trioxide for APL cells.
A Novel Bcr-Abl Mediated Pro-Survival Pathway: Reduction of Releasable Calcium Levels in the Endoplasmic Reticulum Inhibits Calcium Dependent Apoptotic Signaling.
Authors: Vejda Susanne , Piwocka Katarzyna , Cotter Thomas G. , O’Sullivan Gerald C. , McKenna Sharon L.
Published: October 2019

The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia (CML). Several studies have suggested that the expression levels of Bcr-Abl are elevated at disease progression to blast crisis (BC) and that this plays significant role in the achievement of drug resistance. We have established cell lines expressing low (C2 cells) and high levels (C4 cells) of Bcr-Abl to study the molecular mechanisms involved in disease progression especially with regard to drug resistance. Microarray analysis revealed that Bcr-Abl expressing cells exhibit increased expression of various endoplasmic reticulum (ER) resident proteins indicating that the ER is a novel target of Bcr-Abl. Recently, this organelle has been described as an important apoptotic gateway. Indeed, by measuring calcium levels (steady state and following drug treatment) in the ER and cytoplasm we could elucidate that the calcium dependent apoptotic signaling is disturbed in Bcr-Abl transformed cells. Moreover, inhibition of Bcr-Abl activity by Imatinib reversed this phenotype indicating that Bcr-Abl is directly modulating calcium homeostasis by yet unknown mechanisms. Two Bcr-Abl expressing human cell lines, K562 and BV173, also showed reduced amounts of calcium in the ER compared to the Bcr-Abl negative cell lines HL60 and Jurkat. Etoposide was chosen for apoptosis induction as this drug was classified to target not only the mitochondria (classical mitochondrial pathway) but also the ER (calcium dependent apoptotic pathway). Cell death was induced in the parental and Bcr-Abl transformed cell lines and calcium changes in the cytosol and the mitochondria were measured using organelle specific calcium probes. In C2 and C4 cells an early mitochondrial calcium overload was absent possibly due to the decreased amount of free releasable calcium in the ER. Moreover, other ER/calcium activated pathways were greatly diminished, such as cytosolic calcium elevation and calpain activation during apoptosis. It has been shown that decreased levels of releasable calcium in the ER is associated with cell survival. It is possible therefore that Bcr-Abl may exert some of its pro-survival or anti-apoptotic effects at the ER, by directly influencing the levels of releasable calcium. This study demonstrates a novel downstream consequence of Bcr-Abl signaling. The ability to negate calcium dependent apoptotic signaling is likely to be a major pro-survival mechanism in Bcr-Abl transformed cells.