bluuebunny/crossref_metadata_2025 huggingface dataset:
Global Gene Expression Changes during Arsenic Trioxide -Induced Apoptosis in Myeloma Cell Lines Expressing Mutant or w.t p53.
Authors: Gazitt Yair , Akay Cagla , Kircelli Fatih
Published: October 2019

Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging primarily the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. These findings were further supported by a recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32°C and mutant p53 phenotype at 37°C (Akay et al. Akay et al., AACR; Abstract #5344, 2005). Furthermore, myeloma cells expressing w.t. p53 transfected with SiRNA for p53 or p21 behaved like cells with mutant p53 (Kircelli et al. ASH presentation,2005). In order to identify new genes affected by ATO we used the Affymetrix Microarray technology to compare global gene expression in IM9 myeloma cells (w.t. p53) and U266 myeloma cells (mutant p53) following 0, 1,5 and 10 hours of treatment with ATO. We found ≥2 fold increase in gene expression by Affymetrix Microarray Suite Software (MAS) in 94 genes at 5 h and 455 genes at 10 h with an increase in 134, at both 5 and 10 h of treatment. By similar analysis, 263 genes were decreased at 5 h and 679 genes were decreased at 10 h with 204 genes decreased at both time points. Similar analysis with GeneSpring (GS) software revealed an increase in 202 genes and a decrease in 233 genes at both 5 and 10 h. Combination of the 2 analysis methods yielded 90 consistent increasers and 64 consistent decreasers. A great number of these genes in whom we detected changes in this study are genes that were previously identified by us and by others using Western immunoblotting. In addition, we observed differential effect of ATO in IM9 and U266 myeloma cells in apoptosis-related genes (HRK, BID, MCL1); cell cycle-related genes (GADD45, Cyclin D1, cyclin D2 and cyclin D3); signal transduction proteins (ERK, NFkB, ATM, ATR, CHK2, TRAILR2, TNF-R5/6 and VEGF); in chaperon proteins; cyclophyllin B; SAT, ToPoIIA and others. Systematic validation of these changes on the protein levels is ongoing.
Ikaros 6 Immortalizes Murine Hematopoietic Precursors In Vitro.
Authors: Ruiz Anna , Brady Hugh J.M.
Published: October 2019

The Ikaros transcription factor has been shown to play an important role in the differentiation of both the myeloid and lymphoid lineages. The ikaros gene encodes for a zinc finger protein containing seven exons that can be alternatively spliced generating several isoforms with differing functional properties. Isoforms with less than three DNA binding domains act as dominant negative (DN) by forming complexes with longer isoforms and interfering with their DNA binding and transcriptional activation ability. Mice heterozygous for a DN ikaros isoform develop T cell leukemia and lymphoma with 100% penetrance. Overexpression of DN Ikaros isoforms has been found in some forms of leukemias. We have previously reported overexpression of the DN Ikaros6 (Ik6) isoform in a subset of leukemia patients harboring t(4;11) translocations. In addition, we inducibly expressed Ik6 in BaF3 cells and found that Ik6 overexpression delayed cell death after IL-3 withdrawal.

To further investigate the leukemogenic properties of Ik6 overexpression, we have transduced murine hematopoietic precursors with a retroviral Ik6 expression vector and have analysed the effects on proliferation and differentiation of these precursors by in vitro colony formation assays. We have found that Ik6 can immortalize murine hematopopietic precursors in these in vitro assays. We are currently analysing the leukemogenic potential of Ik6 in vivo by transplanting Ik6 expressing cell lines into NOD/SCID mice.
Aldehyde Dehydrogenase (ALDH) Activity in Fresh (Pre-Freezing) and Post-Thawing Leukapheresis and Cord Blood Collections.
Authors: Perotti Cesare , Viarengo Gianluca , Del Fante Claudia , Bellotti Laura , Parisi Cristina , Marchesi Andrea , Salvaneschi Laura
Published: October 2019

Introduction: Primitive hematopoietic progenitor cells may be characterized by the detection of the intracellular enzymatic activity of aldehyde dehydrogenase (ALDH ). The use of a fluorescent substrate for ALDH (Aldefluor) permits the cytofluorimetric study of different stem cell populations in the functional way rather then as markers of cell surface. Aim of this study was to evaluate the ALDH activity of the stem cells in pre-freezing (fresh) leukapheresis (LKF) colllections and in cord blood (CB) units, to verify the feasibility of detecting the ALDH activity in the post-thawing units and finally to compare the results between the fresh and post thawed stem cells to be transplanted.

Materials and methods: Samples containing 2 x106 total nucleated cells obtained from 5 LKF and 5 CB units immediately after collection and from the same units after thawing, were incubated with Aldeflour (StemCo Biomedical, Durham, NC, USA) at 37°C for 45 min. and successively for 15 min. with anti CD34 PE, (Beckman Coulter, Fullerton, CA, USA) and anti CD45 PerCP (BD Biosciences, San Jose, CA, USA). Viability of CD34+ cells was evaluated using 7AAD, (Beckman Coulter, Fullerton, CA, USA). The setting of thawed samples was carried out immediately after thawing, taking care to make the sample dilution very quickly, in order to avoid the detrimental action on the cells exerted by DMSO at 37°C and at room temperature. All samples were analized using a BD Biosciences FACSCalibur flow cytometer device (San Jose, CA, USA).

Results: The results of the ALDH and CD34+ cell analysis and stem cell viability on fresh and thawed samples are detailed in table 1.

Conclusions: Our results demonstrate the feasibility of ALDH determination even in post thawed samples on condition that the test is conducted with accuracy with particular attention at the dilution step that must be completed in a very short time. The ALDH activity tends to decline in thawed stem cells demonstrating the detrimental action on stem cell functionality of the entire cryopreservation and thawing processes. In particular the contact of the stem cell with cryoprotectant mixture (DMSO) at room temperature has a negative, time dependent, impact on stem cell quality, confirming that the reinfusion phase of the graft must be carried out as soon as possible. The introduction of a functional test to evaluate the graft quality may be helpful in transplant clinical practice.

Table 1: Results of ALDH assayes on pre-freezing and post-thawing leukapheresis and cord blood collections. CD34+ (%) ALDH+ (%) CD34+/ALDH+ (%) ALDH+/CD34+ (%) Viability (%) Fresh LKF n=5 0.56 0.12 64.6 97.8 99.2 Fresh CB n=5 0.28 0.23 76.7 99.8 98.1 Thawed LKF n=5 0.48 0.09 65.5 94.4 81.2 Thawed CB n=5 0.24 0.19 71.5 96.8 77.3
Usefulness of Recombinant Human Erythropoietin during Induction-Consolidation Chemotherapy for Chronic Hematological Lymphoid Malignancies: Improving Stem Cell Harvest and Reducing Blood-Product Support for Patients Receiving Autologous Stem-Cell Transplantation.
Authors: Bijou Fontanet D. , Tabrizi Reza , Legay Thibaut , Melot Cyril , Bouabdallah Krimo , Pigneux Arnaud , Boiron Jean-Michel , Marit Gerald
Published: October 2019

Many discrepancies remain on how to use the combination of G-CSF and Epo in patients (pts) with hematological malignancies. Some studies have shown efficacy of this combination in autologous stem cell transplantation (ASCT) when they are used throughout the procedure, while other have demonstrated no benefit when this combination is used after ASCT particularly for Epo. We reviewed retrospectively 30 pts (NHL= 6, HD= 2, MM= 22)who received ASCT for myeloma and lymphoma between September 2003 and May 2005. The main goal of this observation was to evaluate the impact of Epo combination (epoetin beta, alfa or darbepoetin) with G-CSF during chemotherapy administered before ASCT in order to achieve a better hemoglobin (Hb) level before the ASCT procedure. We also evaluated a possible efficacy of this combination upon hematopoietic recovery, transfusion support and stem cell harvest for this population of pts. We used G-CSF (5 μg/kg) alone after stem cell reinfusion in our therapeutic scheme. Patients characteristics were F/M 19/11; 6 pts were in complete response and 24 in partial response of their disease. Conditioning regimen preparations were standard with Melphalan at doses between 140–200 mg/m2 (22 pts), BEAM (7 pts), Cyclophosphamide and Total Body Irradiation (1 pt). After at least 12 weeks of treatment, median Hb level before ASCT was 11.6 g/dl (8.5–14.8) and epoetin beta was used in most patients. Median CD34 cells reinfused were 4.4 (1.2–13.7) with a median number of leukapheresis of 3 (2–9). Hematopoietic reconstitution was fast according to published data and local experience, with a median duration of neutropenia (absolute neutrophils count < 0.5 x 109/l) of 7 days (5–11); the median number of days with platelets counts < 20 x 109 /l and 50 x 109/l was 3 (0–14) and 7 (2–17) respectively. Median transfusion requirement was 1 red cells unit (0–6) and 2 platelets units (0–8) respectively. Median duration of hospitalization was 18 days (15–26).

In conclusion, the use of combined G-CSF and Epo has probably improved clinical course of ASCT by reducing transfusion requirement, duration of hospitalization and neutropenia even when it is used before ASCT. Attempts have to be made to identify the place of Epo administration during ASCT procedure. Randomized prospective study might bring some important information about influence of this combination upon stem cell harvest particularly when it is used before the ASCT during induction and consolidation chemotherapy.
Does Early Lymphocyte Recovery Predict Superior Survival in Children and Young Adults Undergoing Hematopoietic Stem Cell Transplantation?.
Authors: Imran Hamayun , Khan Shakila P. , Arndt Carola A.S. , Rodriguez Vilmarie , Allen Julia A. , Trotz Barbara A.
Published: October 2019

Introduction: Early lymphocyte recovery predicts outcome in adults undergoing high dose chemotherapy followed by autologous stem cell transplantation for a variety of malignancies (Porrata et al; 2001, 2002, 2005). Whether rate of lymphoid recovery is predictive of outcome post-hematopoietic stem cell transplantation (HSCT) in children and young adults has not been studied.

Objective: To explore the association between early ALC recovery and survival in children and young adults undergoing allogeneic and autologous (HSCT) for leukemia or lymphoma.

Methods: Retrospective chart review was performed for all consecutive patients (age 6 months to 21 years) undergoing first HSCT for hematologic malignancies and lymphomas at Mayo Clinic, Division of Pediatric Hematology/Oncology from 1995 to 2004. Day 15 and day 28 ALC were calculated for all patients. The primary end point was 1-year relapse free (RFS) and overall survival (OS). To explore the association between ALC and primary outcome, patients were stratified in 2 groups: ≥ or < than median ALC and Kaplan Meier survival curves were generated for both the groups and compared with the log-rank test. Matched related donor (MRD), matched unrelated donor (MUD) and haploidentical (as one group) and autologous transplantations were analyzed separately.

Results: There were a total of 50 patients. 28 received MRD, 2 MUD, 10 haploidentical and 10 autologous transplant. The underlying malignancies were AML n =25, Biphenotypic Leukemia n=1, ALL n =7, NHL n=3, HD n=7, JMML n=5, CML n=2, CLL n=1). No patient was lost to follow up during that time and data was available for all except (day 28 ALC) for one patient. The 1-year RFS and OS rate was the same; 81% for the MRD, 50% for MUD and haploidentical and 70% for autologous transplant populations, respectively. In patients receiving MRD transplants, higher ALC at either day 15 or day 28 post transplantation was associated with better, albeit statistically non-significant, RFS/OS rates at 1 year (85% for ≥ median (0.23) vs. 78% for < median p=0.69 and 86% for ≥ median (0.47) vs. 77% for < median p= 0.59 respectively). Opposite results were observed for MUD and Haploidentical transplantation group (33% for ALC ≥ median (0.05) at day 15 vs. 67% for < median p=0.2, 33% for ALC ≥ median (0.35) at day 28 vs. 66% for < median p=0.28) and for autologous transplantations (67% for ALC ≥ median (0.23) at day 15 vs. 75% for < median p=0.68, 40% for ALC ≥ median (0.48) at day 28 vs. 100% for < median p=0.07).

Conclusions: Our data generated conflicting findings of the association between ALC and RFS/OS in different patient populations undergoing HSCT. However, our results are limited by the small sample size. Further collaborative studies are needed to explore this association.
Co-Transplantation Haploidentical Mesenchymal Stem Cells and Hematoietic Stem Cells with Nonmyeloablative Condition for Treatment of Relapsed or Refractory Acute Leukaemia.
Authors: Ai Hui-Sheng , Guo Mei , Yu Chang-Lin , Wang Dan-Hong , Qiao Jian-Hui , Sun Qi-Yun , Liu Li-Hui , Shi Zhang , Zhao Robert Chunhua , Sun Zhao
Published: October 2019

Background The major problems of haploidentical stem cell transplantation is severe graft versus host disease (GVHD) and graft rejection because of MHC barrier. Recently, some studies shown that mesenchymal stem cells (MSCs) which can be differentiat into various cells of MSCs origin has some good ability of immunosuppressive activity which may be used for enhance hematopoietic engraftment and lessen GVHD after transplant. So we desinged the protocole of co-transplantation haploiddentical MSCs and perpheral blood stem cell with nonmyeloablative conditions based on our past clinical trails of NST with HLA-march donor and haploid transplant experiment of Rhesus and want to improve the effective of haploidentical transplant in relapsed or refractory acute leukaemia

Methods Eleven parients(year range 8–33) with HLA-mismarch donor(3/6=6, 4/6=3,5/6=1) in relapsed or refractory acute leukaemia were received reduced toxicity condition(cyclophosphamide, fludarabine, anti-thymocyte gloubin, and low dose totol body irradiation(2.0GY) following a MSCs injection of bone marrow and perpheral blood stem cells with apheresis CS-3000 machine later 30 minutes. GVHD prophylaxis consisted of cyclosporine A, Mycophenolate mofetil(MMF) and CD25 antibodies.

Results All eleven patients passed smoothly the hematopoietic suppression stage and white blood cells and plate count was recovery normal in 11–15 days and 15–21 days after transplant. Eight patients get full donor chimerism(FDC) and three patients get mixed chimerism (MC) in which two patients converted to FDC in 28 days and 1 patient still remained of the MC after three months. In two patients the bone marrow derived MSCs cotained 5% donor MSCs in MSCs cultrued by STR-PCR on day +30, +60 and +90d after MSC infusion. Among the all of eleven patients, the incidence of aGVHD(grade I-II=4, and grade III=1) was 45.5%(n=5), the incidence of cGVHD was 27.3%(n=3) and leukaemia relapsed rate is 9.1%(n=1). Follow-up 3 to 18 months, survive rate of patients is 72.7%(n=8) and the following is continous.

Conclusions We conclude that co-transplantation haploiddentical MSCs and PBSCT with reduced toxicity condition is safe and effective. MSCs may play an very important rules in improving the donor engraftment and reduce the GVHD in haploidentical transplant.
Unique Reversible but Serious Adverse Events in Very Young Infants Undergoing Unrelated Donor Umbilical Cord Blood Transplantation for Non-Malignant Conditions: Increased Autoimmune Cytopenias and Hypertrophic Cardiomyopathy.
Authors: Lakshminarayanan Sonali , Szabolcs Paul , Parikh Suhag , Wood Susan J. , Allison June , Kurtzberg Joanne
Published: October 2019

Between September 1998 and June 2004, 15 young infants, <3 months of age, 60% males, 14 Caucasian, 1 African American, underwent unrelated donor umbilical cord blood transplantation for treatment of non-malignant conditions [early infantile Krabbe disease (n = 9), MLD (n = 1), MPS I (n = 1), MPS II (n = 2), Tay Sachs Disease (N = 1) or Beta Thalassemia major (n = 1)]. All infants were prepared for transplant with oral busulfan 20mg/m2/dose (targeting a steady state of 600–900ng/ml), cyclophosphamide 50mg/kg/dose daily x 4, and equine ATG 30mg/kg/dose daily x 3. Cyclosporine and solumedrol were used for prophylaxis against GvHD. All infants engrafted with donor cells, although 1 patient with MPS II never achieved >50% donor chimerism. Two patients died 3 and 5 years post transplant from respiratory failure (1 patient with MPS II and incomplete chimerism) and progressive Tay Sachs Disease, respectively. Grafts were mismatched at 1 (n = 8) or 2 (n = 7) HLA loci and 8/15 donor/recipient pairs were ABO mismatched. Nine patients developed mild to moderate acute GvHD. Within 6–10 months of transplant, six of the 15 patients developed autoimmune cytopenias (autoimmune hemolytic anemia +/− ITP), diagnosed clinically by Coombs positivity and antiplatelet antibodies respectively. They required therapy with steroids, imuran, dacluzimab, rituximab, and in one patient, splenectomy. There was no correlation with ABO or HLA matching or GvHD and the incidence of autoimmune disease. All patients recovered within 3–24 months. Eight of the 15 newborns developed concentric cardiomyopathy while on solumedrol and either cyclosporine or FK506 for GvHD treatment and/or prophylaxis. One patient required intubation and prolonged mechanical ventilation. The others remained relatively asymptomatic. Over time and with weaning of steroid therapy, their left ventricular hypertrophy resolved and heart function returned to normal. Both autoimmune cytopenias and hypertrophic cardiomyopathy occur in higher frequency in very young infants undergoing unrelated donor umbilical cord blood transplantation. The etiology of the autoimmune disease is not currently known while the cardiac disease is a result of therapies used to treat and prophylax against GvHD.
Long Term Survival Analysis after High-Dose Melphalan Chemotherapy Followed by Autologous Stem Cell Transplantation for Treatment of AL Amyloidosis: A Single Centre Experience.
Authors: Perz Jolanta B. , Rahemtulla Amin , Giles Chrissy M. , Szydlo Richard , Apperley Jane F.
Published: October 2019

Introduction: AL amyloidosis (AL) is a clonal plasma cell disease with a median survival of 10–14 months without therapy. Phase II studies report remission rates in up to 50% of patients (pts) after high-dose melphalan (HDM) and autologous stem cell transplantation (ASCT). We retrospectively analysed AL pts who underwent HDM and ASCT at the Hammersmith Hospital in London since 1996.

Patients and methods: 19 pts (12 m, 7 f), median age 51 y (40 – 63 y) with lambda (14) or kappa (5) AL (2 pts with multiple myeloma stage IA) had the following leading organ involvements: 10 kidney, 3 cardiac, 3 liver or gut, 3 other. 12 pts had more than 2 involved organs. According to risk criteria for HDM (Comenzo and Gertz, 2002) 12 pts were high, 2 pts intermediate and 5 pts low risk. Stem cells were mobilised with Cyclophosphamide (4 g/m2) in 3 pts, Etoposide (1.6 g/m2) in 1 patient, G-CSF alone in 14 pts, 1 bone marrow harvest was performed. Melphalan dose was: 8 pts 200 mg/m2, 5 pts 140 mg/m2, 6 pts 100 mg/m2. The median number of infused stem cells was 3.45 x 106 CD 34+ /kg bw (1.96 – 11.3 x 106 CD 34+ /kg bw).

Results: Treatment related mortality (TRM) was 26%. In 14 patients who survived longer than 100 days from ASCT 6 pts achieved a haematological remission (5 a complete remission, 1 a partial remission) but in 8 patients no remission was achieved: 2 pts received a second ASCT, 2 pts further chemotherapy, 2 pts required haemodialysis. The mean survival from HDM and ASCT is 58.1 months (SD 9.64 months, 95% CI 39.21 – 76.99). The 1 - year and 2 - years overall survival from HDM and ASCT was 73% and 62%, respectively. The overall survival dropped to 42% after 6 years and the main cause of death was progressive or relapsed AL.

Conclusions: HDM and ASCT is feasible for patients with AL, however, TRM was high at 26% in a group of maily high risk patients. 62% of patients survived longer than 2 years, but disease relapse and deaths from relapsed or progressive disease occurred after longer than 2 years from HDM. Thus, new treatment strategies have to be investigated to treat disease progress and relapse in order to improve the long-term survival after HDM and ASCT in patients with AL.
Truncated Human EpoR Causes Polycythemia in Fetal Erythropoiesis through Stat5 Hyperactivation.
Authors: Yoon Donghoon , Watowich Stephanie S. , Prchal Josef T.
Published: October 2019

Gain-of-function mutations causing truncations of the cytoplasmic domain of the human erythropoietin receptor (EPOR) result in a dominantly inherited disorder-primary familiar congenital polycythemia. The consequence of EpoR truncation is polycythemia and in vitro hypersensitivity of erythroid precursors to erythropoietin (Epo). We have replaced the murine EpoR gene with a wild-type human EPOR (whEPOR) gene or a mutant human EPOR gene (mthEPOR) that we initially identified in a family with polycythemia. This mutation produces a truncated EpoR mutant with deleted cytoplasmic C-terminal domain after the first tyrosine residue of the intracellular domain. We reported that the mice containing mthEPOR allele have polycythemia and the adult whEPOR mice have anemia. To study the effects of these genotypes on fetal erythropoiesis, we examined fetuses E14.5 homozygotes wild type mouse EpoR, and human EPOR alleles. Hematocrit results show: MthEPOR fetuses were polycythemic compared to mouse EpoR (mthEPOR vs mouse EpoR; 34.58 ± 1.24 % vs 27.49 ± 0.90 %, P<0.001 by t-test), and whEPOR were anemic (whEPOR vs mouse EpoR; 18.61 ± 0.79 % vs 27.49 ± 0.89 %, P<0.001 by t-test). To study the pathophysiology of these phenotypes, we analyzed the erythroid differentiation using differential expression of CD71 and TER119 surface antigens by FACS in the fetal liver cells (FLs). FLs from mthEPOR homozygote had increased population on early and late basophilic erythroblast stage compared to mouse EpoR, but the populations of differentiated erythroid progenitors from mthEPOR homozygote were significantly lower than mouse EpoR and whEPOR homozygotes. We proposed that the erythroid progenitors from mthEPOR exit fetal liver to fetal blood circulation prematurely. The peripheral blood from mthEPOR and mouse EpoR s was analyzed by morphology and FACS. We show that the majority of population in peripheral blood from mthEPOR homozygote were earlier erythroid progenitors (high CD71/low TER119 population in mthEPOR 47.53 %, vs 13.14 % mouse EpoR; high CD71/high TER119 population from mthEPOR 11.49 % vs 65.00 % mouse EpoR). To evaluate the Epo signal transduction we tested the kinetics and dose responses of Epo at Stat5 phosphorylation level. The mthEPOR expressing FLs showed a markedly increased and sustained Epo sensitivity compared to mouse EpoR expressing FLs. In contrast, in whEPOR expressing FLs, Stat5 phosphorylation was significantly lower even at the highest concentration of Epo. We conclude that mthEPOR causes fetal polycythemia and is associated with premature exit of early erythroid progenitors from fetal liver to fetal circulation. In contrast whEPOR is associated with more severe anemia than we observed in adult erythropoiesis. The degree of Epo induced Stat5 activation plays an important role in generation of anemic and polycythemic phenotypes.
siRNA Versus Antisense Locked Nucleic Acids: Stay Single!.
Authors: Kearney Phil , Westergaard Majken , Hansen Henrik F. , Staarup Ellen M. , Koch Troels , Ørum Henrik , Hansen Jens Bo
Published: October 2019

Much discussion has centred around the utility and benefits of siRNA in both target validation and as a therapeutic option. This has been driven by significant publications including that of Soutcheck et al (Nature432, 173–177 2004), which demonstrated liver targeting as well as in vivo efficacy when siRNA against ApoB was tethered to a cholesterol moiety.

Santaris Pharma has developed a third generation nucleic acid chemistry referred to as locked nucleic acid (LNA) which delivers unmatched affinity and stabiliy benefits, largely overcoming the drawbacks associated with traditional antisense molecules. We therefore sought to compare this chemistry with targets which siRNA has been successfully used in in vivo/in vitro settings. The same motif used in the Soutcheck study was targeted with a LNA molecule, and the free siRNA activity was compared to the cholesterol linked and free LNA molecules in their ability ot down regulate ApoB expression. LNA (SPC3197) inhibited ApoB expression by 90% while at an equimolar concentration siRNA was ineffective in the liver and jejunum. Cholesterol linked siRNA was only effective in the jejunum (c50% reduction in mRNA) Fig1. Only the LNA mediated inhibition of ApoB expression was paralleled by a drop in serum cholesterol in the host animal.

In a second model siRNA molecules targeting Hif-1a mRNA (Yu et al Lab Invest84, 553–561 2004) were compared to our lead LNA molecule targeting Hif-1a, SPC2968. Interestingly in in vitro analyses these 2 molecules were equally effective. However in a murine model the increased half life of the LNA molecules translated to a potent inhibition of Hif-1a as measured by QPCR. This effect was noted in jejunum and liver, and persisted for at least 4 days. Hif-1a inhibition mediated by siRNA was not seen in any tissue analysed (Fig 2).

Finally a 3rd molecule targeting Bcl-2 has entered clinical Phase 1 trials, and data will be presented documenting its improved affinity and stabitily in relation to competitor molecules such as Genasense.

Figure Figure
Survival Impact of Time Repeated bcl2/IgH Rearrangement Measurements in Follicular Lymphoma Patients Treated with Front-Line Autotransplantation in the GELF-94 Trial by the GELA.
Authors: Florence Broussais , Nicolas Mounier , Marie-Hélène Delfau , Jean-Marie Cayuela , Elisabeth MacIntyre , Frederic Davi , Catherine Sebban , Coralie Belanger , Nicole Brousse , Pauline Brice , Corinne Haioun , Andre Tilly , Pierre Feugier , Andre Bosly , Bertrand Coiffier , Gilles Salles
Published: October 2019

In the autotranplantation framework, an undetectable MRD before ASCT seems to protect for disease relapse and to increase survival. Using data from the GELF94 trial, which randomized consolidation treatment after achieving clinical response between front line ASCT and chemotherapy, we aimed to assess the impact of MRD on survival controlling for consolidation treatment and time repeated measurement. Of 401 patients included from 07/94 to 03/01, 209 received 12 cycles of CHVP (cyclophosphamide, doxorubicin, teniposide, and prednisone) plus interferon alfa in 18 months (CHVP-I arm) and 192 received 4 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) then high-dose therapy with total body irradiation and ASCT (CHOP-HDT arm). Bcl2/IgH rearrangement was prospectively assayed by PCR in bone marrow (BM) and in blood at diagnosis and repeated every 6 months during the first year then annually. Time repeated measurement was stopped at clinical relapse or instigation of a new treatment. At diagnosis, 225 patients had material available for bcl2/IgH rearrangement analysis: BM for 182 (45 %) patients and blood for 199 (50%). In the latter, 105 of the 199 patients (53%) were found to have a bcl2/IgH rearrangement in the MBR breakpoint, whereas bcl2/IgH rearrangement in mcr was observed in 5 patients (3%) and no rearrangement at MBR or mcr in the remaining 94 patients (44%). No differences were found according to bcl2/IgH rearrangement in terms of response rate (82% for bc2- vs 80% for bcl2+) and.5 yrs survival (85% for bcl2- and 79% for bcl2+). Time repeated bcl2/IgH rearrangement measurements were available for 142 patients (ASCT n=75, chemo n=67): in BM for 79 patients and in blood for 85 patients. The clinical characteristics were well balanced between patients with/without time repeated measurement. By time dependant Cox’s model, the significant prognostic factors for survival were age below 40 yrs (RR= 21, p=0,005), complete clinical response (RR=5, p=0.02) and bcl2/IgH rearrangement negativity (RR=4, p=0.03). There was no treatment impact. These findings confirm the importance of molecular response in addition to the clinical response as a critical factor for prognosis. The results of this study suggest that in FL patients, the eradication of cells bearing the Bcl2/IgH rearrangement is highly desirable early in the treatment and should be maintained as long as possible.
Recurrent De Novo Mutations of EPOR Gene in Primary Congenital Polycythemia.
Authors: Mojica-Henshaw Mariluz P. , Laverdiere Caroline , Prchal Jaroslav F. , Prchal Josef T.
Published: October 2019

Primary familial and congenital polycythemia (PFCP) is a rare inherited disorder presenting with elevated red blood cell mass, elevated hemoglobin concentration and low levels of erythropoietin. Ten mutations in the erythropoietin receptor (EPOR) gene to date have been associated with PFCP. All of these mutations result in deletion of 59 to 82 amino acids from the carboxy terminal of EpoR which has been shown to contain a negative regulatory domain. Here, we describe a 2-year old boy of French-Canadian descent presenting with polycythemia and splenomegaly. Sequencing of the EPOR gene showed the proband to be heterozygous for a G to A transition in nucleotide 6002 (G6002A). The mutation generates a stop codon instead of tryptophan at amino acid 439, leading to a truncated EpoR. The association of the G6002A mutation in the EPOR gene with PFCP has been previously described in a large Finnish family (dela Chapelle et al., Proc Natl Acad Sci USA1993; 90: 4495) and in a 16-year old boy of English descent (Percy et al., Br J Hematol1998; 100:407). The G6002A mutation in both cases was considered to have arisen independently based on differences in a microsatellite polymorphism in the 5′UT of EPOR and the absence of the mutation in the immediate family of the English boy. We studied our proband’s parents for the G6002A EPOR mutation and did not find it. Their parentage was confirmed using 24 different microsatellite markers. This indicates that the G6002A mutation in the proband arose de novo. Since the mutation arose de novo, in vitro methycellulose cultures of erythroid progenitors isolated from peripheral blood of the proband were grown in the presence of increasing concentrations of Epo to rule out genetic mosaicism. The erythroid progenitors showed hypersensitivity to Epo as is characteristic of PFCP. However, we did not find evidence supportive of genetic mosaicism as all 70 BFU-E colonies analyzed were heterozygous for the G6002A mutation. Previously, two other polycythemia-associated EPOR mutations, 5974insG (Sokol et al., Blood1995; 86:15) and 5959G>T (Kralovics et al., Am J Hematol2001; 68:115) were shown to have arisen de novo. This case is thus the fourth instance out of 13 reported cases of polycythemia-associated EPOR mutations that has arisen de novo. Because of the rarity of polycythemia-associated EPOR mutations, their frequent de novo occurrence suggests that these mutations do not have a selective advantage but are detrimental. Their possible association with increased risk of thromboembolic and atherosclerotic disease due to chronically augmented Epo signaling is being explored by ongoing clinical studies.
MicroRNA Expression and Regulation of Hematopoiesis in CD34+ Cells: A Bioinformatic Circuit Diagram of the Hematopoietic Differentiation Control.
Authors: Georgantas Robert W. , Hildreth Richard , Alder Jonathan , Croce Carlo M. , Calin George A. , Civin Curt I.
Published: October 2019

MicroRNAs (miRs) are a recently realized class of epigenetic elements which block translation of mRNA to protein. MicroRNAs have been shown to control cellular metabolism, apoptosis, differentiation and development in numerous organisms including drosophila, rat, mouse, and humans. Recently, miRs have been implicated in the control of hematopoiesis. Importantly, both aberrant expression and deletion of miRs are have been associated with the development of various cancers. In a previous study, we determined the gene expression profiles of HSC-enriched, HPC-enriched, and total CD34+ cells from human PBSC, BM, and CB. One rather surprising finding from this study was that virtually all of “hematopoietic important” genes were expressed at virtually identical levels within all populations examined. One of our hypotheses to explain this phenomena was that miRs may control differentiation by controlling protein expression from these “hematopoietic” RNAs. To examine the possible role of miRs in normal hematopoiesis and their relation to the HSPC transcriptome, we used mir-miroarrays to determine the miR expression profile of primary normal human mobilized blood and bone marrow CD34+ hematopoietic stem-progenitor cells (HSPCs). We have combined this miR data with (1) our extensive mRNA expression data obtained previously for CD34+ HSPCs, CD34+/CD38−/Lin- stem cell-enriched, CD34+/CD38+/Lin+ progenitor-enriched populations, and total CD34+ HSPC (Georgantas, Cancer Research 64:4434) and (2) miR target predictions from various published algorithms. Combining these datasets into one integrated database allowed us to bioinformaticly examine the global interaction of HSPC mRNAs and miRs during hematopoiesis. The 3′UTR sequences from many of these “hematopoietic” mRNA were cloned behind a luciferase reporter. K562 cells were transfected with these luc-3′UTR constructs, confirmating that expression of many important hematopoietic proteins are controlled by miRs. Based on our bioinformatic and protein expression studies, we present a global in silico model by which microRNAs control and direct hematopoietic differentiation. Actual in vitro and in vivo studies addressing the action of specific miRs in hematopoietic differentiation are presented in separate abstracts.
Structural Basis and Cellular Consequences of Potent Inhibition of the Aurora Kinases, Wild Type Abl Kinase and a T315I Multi-Drug Resistant Mutant Form of Abl Kinase by MK-0457 (VX-680).
Authors: Pollard John R. , Charlton Peter A. , Cheetham Graham M.T. , Falcon Susanna C. , Golec Julian M.C. , Griffiths Matthew R. , Weber Peter
Published: October 2019

MK-0457 (VX-680) is a potent small molecule inhibitor of the Aurora-family of protein kinases that inhibits cell proliferation through aberrant mitosis and failed cytokinesis. Affected cells are subsequently deleted by apoptosis. Tumor regression and increased survival have been reported in multiple in-vivo models and in January 2005 MK-0457 entered Phase I clinical trials as the first in class Aurora inhibitor. Although MK-0457 demonstrates significant selectivity over the vast majority of kinases tested, it has been shown to potently inhibit Flt-3 and Abl, both of which are targets for anti-cancer therapies. Most notably MK-0457 potently inhibits a series of mutant Abl kinases including the multi-drug resistant T315I form that is commonly expressed in Gleevec refractory CML and ALL. We report here the first MK-0457 / Aurora co-complex crystal structure and show that it adopts a closed inactive conformation. By comparison with other reported kinase structures we provide a structural basis for the cross-reactivity with wild type and drug resistant mutants of the Abl kinase. We also show that MK-0457 blocks both Aurora and Abl cellular kinase activity and displays potent cytotoxic activity against a range of leukemic cell lines, including Gleevec resistant cells that are reliant on mutant Abl for growth and survival. Similar observations have recently been made in the clinic, where new data show that MK-0457 has activity in patients with BCR-ABL T315I mutant CML and Philadelphia positive ALL.
Tal1 and a DNA Binding Mutant (Tal1R188G;R189G) Cooperate with LMO2 To Induce Leukemia in Mice.
Authors: Draheim Kyle M. , Erdkamp Kimberly , Arous Eward , Calvo Jennifer A. , Kelliher Michelle A.
Published: October 2019

LMO2 is a member of the “LIM only” protein family and required for primitive erythropoiesis and adult vasculogenesis and angiogenesis. In erythroid cells, LMO2 interacts with TAL1 and E47 and LDB1 and GATA-1, thereby forming a transcriptional complex whose target genes include EKLF, CKIT, and p4.2 (protein 4.2). LMO2 was first implicated in leukemogenesis when it was identified in chromosomal translocations t(11;14)(p13;q11) and t(7;11)(q35;p13) found in T cell acute lymphoblastic leukemia (T-ALL) patients. Ectopic expression of LMO2 in mice recapitulates the human disease, albeit at low penetrance and following a long latency. LMO2 has been shown to synergize with TAL1, yet the mechanism of oncogene cooperativity is unknown. Two models have been proposed: the first suggests that LMO2 and TAL1 synergize by forming an active transcriptional complex that induces expression of target genes such as retinaldehyde dehydrogenase 2 (RALDH2) and TALLA1 (a surface marker of T-ALL). The second model proposes that LMO2 and TAL1 sequester the E47/HEB heterodimer, resulting in inhibition of E47/HEB-mediated transcription. To distinguish between these models, we mated our Tal1 transgenic mice and our DNA binding mutant of Tal1(R188G:R189G) with Lmo2 transgenic mice. As expected, transgenic expression of Lmo2 induced disease in 23% of mice after 302 days. Similar to published studies, Tal1 and Lmo2 expression dramatically inhibited thymocyte development and induced T cell leukemia in 100% of the mice with a mean latency of 108 days. To test whether the DNA binding properties of tal1 were required to cooperate with LMO2, we mated mice expressing a DNA binding mutant of Tal1(R188G;R189G) with Lmo2 transgenic mice and found that tumors were induced with similar kinetics; 100% of mice developed disease with an average latency of 107 days. These data suggest LMO2 does not require the DNA-binding properties of Tal1 to induce leukemia in mice and support the model that LMO2 contributes to leukemia through E47/HEB sequestration and inhibition.
Myc Promotes Tumorigenesis by Supressing Nfkb2.
Authors: Keller Ulrich , Huber Juergen , Nilsson Jonas , Hall Mark , Peschel Christian , Cleveland John L.
Published: October 2019

Rel/NF-kappaB transcription factors are mediators of immune responses, cell survival, and transformation, and are frequently deregulated in cancer. The NF-kappaB2 subunit is associated with chromosomal translocations or deletions in lymphoid malignancies, and deletion of the COOH-terminal ankyrin domain of NF-kappaB2 results in increased lymphocyte proliferation. Here, we report that activation of the Myc oncogene leads to suppression of Nfkb2 expression in early passage mouse embryonic fibroblasts and primary bone marrow-derived B cells. Accordingly, transgenic expression of c-Myc in the Eμ-Myc model of human Burkitt lymphoma results in reduced nfkb2 transcript and NF-kappaB2 p100 and p52 protein levels in pre-cancerous B cells. Nfkb2 expression is further reduced in the majority of Eμ-Myc lymphomas and in human Burkitt lymphoma. Nfkb2 suppression by Myc occurs at least in part by transcriptional repression as shown by promoter studies. To evaluate the relevance of Myc-mediated suppression of Nfkb2 for tumorigenesis, consequences of complete Nfkb2 loss were evaluated in vivo. In pre-cancerous B cells of Myc-transgenic mice, loss of Nfkb2 affects Myc-induced apoptosis while B cell proliferation is unaffected. Deletion of Nfkb2 results in an acceleration of lymphoma development in Eμ-Myc transgenic mice. Therefore, Myc-induced Nfkb2 suppression promotes lymphomagenesis.
Application of High Throughput Screening To Identify Novel Small Molecule Inhibitors of αIIbβ3-Mediated Platelet Adhesion to Fibrinogen.
Authors: Blue Robert , Jiroušková Markéta , Karan Charles , Coller Barry S.
Published: October 2019

Two separate conformational changes have been proposed to accompany activation of platelet αIIbβ3: 1) leg separation leading to extension of the head region composed of the αIIb propeller and β3 βA (I-like) domains, and 2) a swing-out motion at the junction of the β3 βA (I-like) and hybrid domains. Small molecule inhibitors of αIIbβ3 competitively block the RGD ligand binding site and variably induce conformational changes in αIIbβ3 as judged by the binding of ligand-induced binding site (LIBS)-specific monoclonal antibodies. In an attempt to identify molecules that may inhibit αIIbβ3 activation without initiating the conformational changes associated with ligand binding, we screened 33,264 compounds from four different chemical libraries (Prestwick, Chembridge, Cerep and ChemDiv) for their ability to inhibit the adhesion of washed platelets in HEPES-modified Tyrode’s buffer with 1 mM Ca2+/0.5 mM Mg2+ to immobilized fibrinogen adsorbed from a 50 μg/ml solution. When tested at a final concentration of 16 μM, a total of 102 compounds (0.31%) demonstrated greater than 50% inhibition of platelet adhesion, and two of these (Figure 1) demonstrated >30% inhibition of the initial wave of ADP-induced aggregation of platelets in citrated platelet-rich plasma. IC50s for inhibition of ADP (5 μM)-induced platelet aggregation for compounds 1 and 2 were 13 ± 4.5 and 17 ± 5 μM (n=3), respectively. Compounds 1 and 2 also inhibited fibrinogen binding to platelets induced by the activating LIBS antibody AP5 with IC50s of 27 and 30 μM, and 20 and 27 μM, respectively, in two experiments. Since AP5 binds to and directly activates αIIbβ3, it is likely that the compounds’ inhibitory effects are due to direct binding to αIIbβ3 rather than inhibition of signal transduction. In two separate experiments, compound 1 at 15 - 20 μM produced variable increases in the binding of LIBS mAbs AP5, PMI-1 or LIBS1 to unactivated and ADP-activated platelets, whereas tirofiban (20 μM) consistently increased the binding of each mAb. Compound 2 did not increase the binding of any of the mAbs. Neither compound contains a negatively charged carboxyl group, which mediates the interaction of the Asp group in RGD ligands with the β3 MIDAS metal ion, but compound 1 has a carbonyl group that may potentially interact with the MIDAS metal ion. Compound 1 resembles 1,2-fused pyrimidine derivatives that have previously been demonstrated to inhibit platelet aggregation (Roma et al., Bioorg. Med. Chem. 2003, 11, 123). We conclude that high throughput screening of molecular libraries can identify novel compounds that inhibit αIIbβ3 and that one of them appears to inhibit αIIbβ3 without inducing conformational changes in the receptor.

Figure Figure
Functional Characterization of the Bcl-2 Splice Variant Beta.
Authors: Schinkothe Timo , Leistert Conrad , Staib Peter
Published: October 2019

Bcl-2 family proteins are decisive regulators of the intrinsic apoptosis mechanisms. The best known antiapoptotic protein is Bcl-2, which functions via the interaction with proapoptotic proteins due to its association to mitochondrial membranes. Besides the widely described splice variant Bcl-2alpha (26kDa), another 22kDa splice variant Bcl-2beta exists. Bcl-2beta is described as a cytosolic and functionless protein, because of its transmembrane domain deficit.

In this study, we demonstrated for the first time that Bcl-2beta has a function and might act in the linkage between extrinsic and intrinsic pathway of apoptosis and may also play a role in hematopoietic malignancy. By performing binding studies using immuno-coprecipitation (IP) we were able to demonstrate that both splice variants bind to Bad and Bax. Furthermore, Bcl-2beta but not Bcl-2alpha is able to bind to the proapoptotic protein Bid which is localized in the cytoplasma. Bid is known as a linker which transmits an extrinsic apoptotic signal into the intrinsic pathway. Additionally, an overexpression of Bcl-2beta results in an increased chemoresistance to different cytotoxic drugs in-vitro. This effect was stronger than it was seen with the overexpression of Bcl-2alpha. We were also able to show an expression of Bcl-2beta in PBMC in 6 of 12 samples of patients with B-CLL. Bcl-2alpha was expressed in all cases. Using confocal laser scanning microscopy, we also analyzed the sub-cellular localisation of both splice variants.

Conclusion: Various models attempt to describe the mechanisms of Bcl-2 family proteins. It seems that Bcl-2beta is a protein which can function in the cytosole as well as membrane associated in an antiapoptotic manner. Its specific interaction with Bid suggests that Bcl-2beta is the cytosolic counterpart of the Bid induced pathway. The intracellular transformation from Bid to tBid, which links the extrinsic to the intrinsic apoptotic pathway, as well as the proapoptotic property of tBid could be inhibited by Bcl-2beta.
MDS1-EVI1 and EVI1 Overexpression Results in Changes in the Behavior of Murine Hematopoietic Cells.
Authors: Metais Jean-Yves , Wieser Rotraud , Dunbar Cynthia E.
Published: October 2019

We have found non-random patterns of retroviral integration in long-term hematopoietic repopulating cells in the rhesus macaque, with frequent integration events of MLV vectors into the MDS1-EVI1 gene complex. These findings, along with reports regarding frequent integration events in the same gene complex in patients with chronic granulomatous disease receiving MLV-transduced hematopoietic cells in a clinical trial and the ability of MLV vectors activating expression of this gene via integration to immortalize primary murine bone marrow cells, suggests these gene products could have important roles in normal and leukemic hematopoiesis. Expression from this gene complex can result in translation of at least three distinct proteins: MDS1, EVI1, and MDS1-EVI1. EVI1 has been the most studied protein of this locus. Its overexpression, as a consequence of chromosomal rearrangement or viral integration, is associated with leukemia. MDS1-EVI1 contains a PR domain that is lacking in EVI1 and is thought to possibly be antagonistic to EVI1, however the location of the integrations in our prior rhesus studies would indicate that overexpression of either gene product could be immortalizing. Both proteins share the same expression profile in normal tissues as well as most reports of myeloid leukemias. To investigate the impact of the three gene products on hematopoietic cells, we cloned murine mds1, evi1, and mds1-evi1 into the pMIEV-GFP retroviral vector and produced ecotropic vector particles. These were used to transduce the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these various gene products. Gene expression analysis using Afflymetrix arrays demonstrated that both EVI1 and MDS1-EVI1 expression produced dramatic changes in gene expression profiles of these cells, compared to MDS1 and control vector. For instance, EVI1 transduced cells overexpressed oncogenes such as small G proteins belonging to the RAS family. There was modulation of genes implied in hematopoiesis, apoptosis, TGF beta signaling, and cell cycle. To assess changes in cell cycling of transduced BaF3 cells we used a flow cytometric assay, which unraveled an arrest in G1 phase only when EVI1 was overexpressed. These changes were concomitant to an increased metabolic activity as measured by an MTT assay. Further studies of these different pathways have to be performed in order to confirm the results obtained by the DNA chips analysis. Primary murine bone marrow cells could be immortalized after transduction by both EVI1 and MDS1-EVI1 vectors, compared to MDS1 and control vectors. Mice have been transplanted with primary bone marrow cells transduced with all vectors, and are being followed for hematopoietic changes or leukemia. In conclusion, both MDS1-EVI1 and EVI1 overexpression appear to result in marked changes in the behavior of primitive hematopoietic cells.