bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevated FoxM1 Expression Promotes Cell Cycle Progression by Induction of KIS Expression and Contributes to the Development of Leukemia Cells.
Authors: Keiji Okinaka , Nakamura Satoki , Hirano Isao , Ono Takaaki , Fujisawa Shinya , Shigeno Kazuyuki , Shinjo Kaori , Ohnishi Kazunori
Published: November 2019

[Background] FoxM1, a member of the Fox transcription factor family, plays an important cell cycle regulator of both the transition from G1 to S phase and progression to mitosis. FoxM1 expression was also found to be up-regulated in some solid tumors (basal cell carcinomas, hepatocellular carcinoma, and primary breast cancer). These results suggested that FoxM1 plays a role in the oncogenesis of malignancies. However, it is unknown whether FoxM1 expression contributes to the development or progression of leukemia cells. Therefore, we investigated how FoxM1 regulated the cell cycle of leukemia cells and the expression analysis of the FoxM1 gene in patients with acute leukemias.

[Methods] The cells used in this study were human acute leukemia cell lines, U937 and YRK2 cells. Primary acute myeloblastic (25 AML (4 M1, 11 M2, 6 M4, 4 M5)) cells were obtained from the peripheral blood. Human normal mononuclear cells (MNCs) were isolated from peripheral blood (PB) of healthy volunteers after obtaining informed consents. For analysis of proliferation and mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, Aurora kinase B, and KIS) in leukemia cells, MTT assays and western blot were performed in all cell lines, which untransfected or transfected with siRNA FoxM1, respectively. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells untransfected or transfected with siRNAFoxM1 by PI staining. For analysis of FoxM1 mRNA, quantitative RT-PCR was performed in all cell lines and clinical samples.

[Results] In all leukemia cell lines, the expression of FoxM1B mRNA were significantly higher than normal MNCs. When transfected with the siRNA FoxM1 in leukemia cells, suppression of FoxM1 caused a mean 71% (range 62 to 80%) reduction in S phase cells and a mean 4.4-fold (range 3.2 to 5.6-fold) increase in G2/M phase cells compared to controls. MTT assay demonstrated that the proliferation of the siRNA FoxM1 transfected cells was inhibited compared to the untransfected cells. Moreover, FoxM1 knockdown by siRNA in leukemia cells reduced protein and mRNA expression of Aurora kinase B, Survivin, Cyclin D1, Skp2 and Cdc25B, while increased protein expression of p21and p27. In the clinical samples obtained from patients with acute leukemias, the FoxM1B gene was overexpressed in 22/25 (88%). The relative folds of FoxM1B gene expression were for AML: 2.83 compared to normal MNCs.

[Conclusions] In this study, we report in the first time that FoxM1 is overexpressed in myeloid leukemia cells. These results demonstrated that expression of FoxM1 is an essential transcription factor for growth of leukemia cells, and regulate expression of the mitotic regulators. Moreover, we showed that FoxM1 induced the expression of KIS protein. Therefore, FoxM1 might be one of moleculer targets of therapy for acute leukemias.
A Phase I Trial of AT9283, a Multitargeted Kinase Inhibitor, in Patients with Refractory Hematological Malignancies.
Authors: Ravandi Farhad , Foran James , Verstovsek Srdan , Cortes Jorge , Wierda William , Boone Patricia , Borthakur Gautam , Sweeney Trish , Kantarjian Hagop
Published: November 2019

AT9283 is a small molecule inhibitor of aurora kinases A and B, JAK2, and JAK3 (IC50 <5nM in each case). AT9283 was administered as a 72-hour continuous intravenous infusion to patients with refractory hematological malignancies at 6 different dose levels: 3, 6, 12, 24 and 48 mg/m2 daily x 3. Inclusion/exclusion criteria, and definitions of DLT and MTD were standard. A 3+3 dose escalation phase I design was implemented. To date, 20 patients have been treated: 3 at 3, 3 at 6, 7 at 12, 3 at 24 and 4 at 48 mg/m2/daily x 3. Seven were females; median age was 58 years (range 22 to 86); 19 patients had previously treated acute myeloid leukemia (AML); 15 were in second or subsequent salvage; abnormal cytogenetics were present in 15. Two patients had undergone prior matched unrelated allografts. The median number of prior chemotherapy regimens was 2 (range 1 to 5). Pharmacokinetic data is available for patients treated at early dose levels only. Median systemic clearance varied up to two-fold between patients with some suggestion of a non-linear increase in exposure at higher dose levels. At the completion of the infusion, the plasma concentration of AT9283 decreased rapidly exhibiting a multiphasic elimination and a terminal half-life of 6 to 13 hours. At doses of 3 to 48 mg/m2/D x 3, no MTD has been defined; 2 patients treated at 12 mg/m2/D x 3 developed tumor lysis syndrome which resolved spontaneously in one case and required hemodialysis in the second. This dose level was expanded to treat 6 patients. Patients received a median of two cycles of treatment (range 1 to 3). So far, 6 of 20 patients (30%) exhibited evidence of anti-leukemia activity, with significant reductions in bone marrow blasts (≥ 50%) observed at all dose levels: In summary, AT9283 shows good early results and continues to accrue patients to define the DLT and MTD with the 72-hour infusion schedule. In vitro studies suggest that prolonging the duration of infusion will increase the drug’s biological effect. This will be explored following the identification of the MTD for a 72-hour infusion.
A Phase I/II Dose Escalation Trial of ONCOHIST® (Recombinant Human Histone H1.3) in Patients with Relapsed or Refractory Acute Myeloid Leukemia.
Authors: Zeppezauer Michael , Formicka-Zeppezauer Grazina , Gross Peter , Joernvall Hans , Schubert Joerg , Pfreundschuh Michael
Published: November 2019

Background: Histone H1.3 suppresses tumor growth of leukemic cells in vitro, ex vivo and in animal models. To evaluate toxicity and efficacy of ONCOHIST, recombinant human histone H1.3 (rhH1.3) in patients suffering from acute myelogenous leukemia, we initiated a phase-I/II dose escalation study.

Methods: This was an open-label unicentric phase I/II study. Nine patients suffering from relapsed or refractory acute myeloid leukemia (median age: 69 years, range 49 to 83 years) with a life expectancy of at least one month who were unable or unwilling to receive curative chemotherapy without major organ dysfunction were eligible for the trial. Exclusion criteria were presence of HIV, HBV, or HBC infection, heparin treatment during the 2 weeks before enrolment, and medical conditions known to potentially interfere with rhH1.3 treatment such as rheumatoid arthritis or SLE, as well as circulating anti-histone H1.3 antibodies. One treatment course consisted of 9 applications in 3 weeks. 3 patients were treated at each dose level. Starting dose level was 245 mg/m2 and in the absence of dose-limiting toxicities, the dose was increased to the next higher dose level (392 and 628 mg/m2 rhH1.3) in the third week of the treatment course. Primary endpoints of the study was the definition of the maximal tolerated dose and dose-limiting toxicities of the study drug.

Results: To date, 9 patients have been treated and all patients are evaluable for toxicity and efficacy. No side-effects were observed except for one atrial fibrillation under infusion of rhH1.3, which was considered not to be related to the study drug. All patients completed one course of therapy (9 applications), and one responding patient received a second course without side effects. No dose-limiting toxicities were observed and the maximal tolerated dose has not been reached at 628 mg/m2. With respect to efficacy, the formal criteria of response were not met in any of the 9 patients. However, two patients had a temporary increase of their platelet counts while on therapy, and in one patient platelet counts raised from 22 000/mm3 to 100 000/mm3, remaining stable for 3 months. Two patients achieved a reduction of blasts upon treatment.

Conclusion: rhH1.3 is well tolerated at the doses treated so far. While the achieved serum levels are still below the growth-inhibiting concentrations in vitro, first clinical effects have been observed. Additional patients and higher dose levels are needed to delineate the efficacy and toxicity profile of rhH1.3 for the treatment of acute myelogenous leukemias.
RGD-Substituted High Molecular Weight Hyper-Branched Polyglycerols (HPG) Are Effective Platelet Inhibitors.
Authors: Zhang Jerry G. , Krajden Oren , Kainthan Rajesh K. , Kizhakkedathu Jayachandran N. , Gyongyossy-Issa Maria I.C.
Published: November 2019

Peptides containing Arg-Gly-Asp (RGD) sequences are known to bind to integrins which mediate cell adhesion and therefore have been utilized in applications such as antithrombotics and tissue engineering. Although RGD and related peptides show promise, their unfavourable pharmacokinetic profiles and susceptibility to in vivo proteolysis hinder their clinical usefulness. Peptide-polymer conjugates can address one of these challenges by extending the peptide’s residence in plasma. Hyperbranched polyglycerols (HPGs) are biocompatible polyether polyols with very long plasma circulation half-lives (t1/2 ∼ 60h) and as such are ideally suited to be carriers in such conjugates. HPGs of three different molecular weights were conjugated with RGD peptides at various substitution levels. Some of the terminal hydroxyl groups of polyglycerols were converted to vinyl sulfone groups which were subsequently utilized to couple cysteine terminated RGD peptides. The following conjugates were made: 515 kDa with substitution levels of 100:1, 500:1 and 1000:1; 100 kDa with substitution levels of 100:1 and 1000:1; and 3 kDa with substitution levels of 1:1, and 10:1. RGD-coupled HPG inhibited fibrinogen binding to platelet glycoprotein IIb-IIIa as detected by flow cytometry using anti-fibrinogen. Compared to free RGD (Ic50 = 5 x 10-5 M), inhibition of fibrinogen binding increased with increasing HPG molecular weight and increasing RGD-substitution for the high MW conjugates: Ic50 for the 515 kDa conjugates were 6.3 x 10−8 M for the 100:1 substitution, 4.7 x 10-8 M for the 500:1 substitution, and 4.1 x 10−8 M for 1000:1 substitution; Ic50 for the 100 kDa conjugates were 7.0 x 10−7 M for the 100:1 substitution and 1.2 x 10−7 M for the 1000:1 substitution. Ic50 for the 3 kDa conjugates were 2 x 10−5 M for the 1:1 substitution, 2 x 10−4 M for 10:1 substitution. Similarly, platelet function, as demonstrated by MnCl2-initated aggregation was inhibited in a dose- and molecular weight-dependent manner by HPG-RGD conjugates. Platelet aggregate formation and aggregate size were confirmed by microscopy. However, unsubstituted HPG had no effect on platelet fibrinogen binding and neither conjugated nor unconjugated HPG increased platelet CD62 surface expression. Trypsin digestion (but not SBTI-treated trypsin) removed the inhibitory activity of the conjugates and thus confirmed that both fibrinogen binding and aggregation-inhibition were dependent on the RGD peptide sequence. Such multi-determinant peptide-HPG conjugates represent a new approach to the development of antithrombotic drugs.
Rivaroxaban Has Protective Effects in a Model of Disseminated Intravascular Coagulation (DIC) in Rats.
Authors: Perzborn Elisabeth , Hirth-Dietrich Claudia , Fischer Elke , Groth Martin , Hartmann Elke , Sperlich-Wulf Kerstin
Published: November 2019

Introduction: DIC is a complication that occurs during serious infection with Gram-negative bacteria. Endotoxin is a component of the bacterial cell wall that elicits a cytokine-mediated cascade of tissue factor-dependent hypercoagulable reactions. The resulting hypercoagulable state may be inhibited by potent anticoagulation. Rivaroxaban is an oral, direct Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. The aim of this study was to examine the effects of rivaroxaban in a rat model of endotoxin-induced DIC.

Methods: Rivaroxaban (0.1–10 mg/kg p.o.) or vehicle control (PEG400/H2O 60/40%, 5 mL/kg p.o.) were administered 30 minutes before endotoxin injection (lipopolysaccharide O55 B5 [LPS], 250 μg/kg i.v.) to conscious rats. Blood samples were withdrawn from anesthetized animals by heart puncture 4 hours after LPS injection and fibrinogen, platelet count, thrombin-antithrombin (TAT) complex levels and IL-6 were measured.

Results: The induction of DIC was indicated in the placebo + LPS group vs vehicle control by decreased fibrinogen (2.12±0.08 vs 2.69±0.10 g/L) and platelet count (571±28 vs 904±30×109/L) an increase in TAT (75±9 vs 2±1 μg/L) and IL-6 (8.6±1.23 μg/L vs 0.028±0.020 μg/L). Pretreatment with rivaroxaban (0.1–10 mg/kg p.o.) dose-dependently ameliorated the effects of LPS on fibrinogen, platelets and TAT. Rivaroxaban 10 mg/kg p.o. normalized fibrinogen (2.58±0.07 g/L) and TAT (5.6±1.2 μg/L) and increased platelet count (703±19×109/L) (Table). Rivaroxaban also slightly reduced the plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) (4.24±0.67 μg/L).

Conclusions: These results show that direct, selective inhibition of FXa by rivaroxaban effectively normalized the hypercoagulable reactions of endotoxemia with a slight modulating effect on the generation of pro-inflammatory active cytokines, such as IL-6, in the rat DIC model. Further research into the use of rivaroxaban in the management of DIC is therefore warranted.

Effects of rivaroxaban in an endotoxin-induced DIC model in rats 4 hours after LPS injection. Results show mean ± SEM. Vehicle control Placebo + LPS Rivaroxaban 1 mg/kg + LPS Rivaroxaban 10 mg/kg + LPS DIC, disseminated intravascular coagulation; IL-6, interleukin-6; LPS, lipopolysaccharide O55 B5; SEM, standard error of the mean; TAT, thrombin-antithrombin ## p<0.01 vs sham, ### p<0.001 vs sham, *p<0.05 vs LPS/placebo, ** p<0.01 vs LPS/placebo, *** p<0.001 vs LPS/placebo Fibrinogen (g/L) 2.69±0.10 n=5 2.12±0.08## n=14 2.41±0.09 n=14 2.58± 0.07** n=7 Platelets (×109/l) 904±30 n=5 571±28### n=8 655±23 n=6 703±19* n=7 TAT (μg/L) 1.8±0.8 n=5 75.2±9.4### n=14 65.3±8.0 n=14 5.6±1.2*** n=7 IL-6 (μg/ml) 0.028±0.020 n=5 8.60±1.23### n=6 5.01±0.47 n=6 4.24±0.67 n=7
A Study of Bedside Visual Art To Decrease Anxiety and Improve Pain and Mood in Cancer Patients: The Mayo Clinic Experience.
Authors: Wolanskyj Alexandra P. , Curry Elizabeth A. , Ehlers Shawna L. , Eagle Debra M. , Larson Dirk R. , Colby Colin L. , Bauer Brent A. , Scanlon Paul D.
Published: November 2019

Background: The treatment of cancer-related symptoms continues to represent a major challenge for health care providers. Traditional approaches often require pharmacologic interventions which have potential side effects. A wide variety of approaches using complementary and alternative medicine (including music therapy) have demonstrated some early benefit. The role of a visual art intervention on pain, anxiety and mood has not been studied to date.

Patients and Methods: Our patient population consisted of 21 Hematology inpatients at the Mayo Clinic (19 women, 2 men with a median age of 53.4 years, [range19 to 73]), with the following hematological disorders: Acute Leukemia (n = 6); Lymphoma (n = 5); Multiple Myeloma (n = 5); and a variety of other malignant conditions (n =5). Patients and their families were invited to participate actively or passively (i.e. observe) in a 1 to1 visual art intervention, conducted by a trained artist educator using different forms of art media. Baseline assessments of pain, anxiety and mood were conducted just prior to the intervention using Visual Analog scales (VAS), the Spielberger State Trait Anxiety Inventory and the Positive and Negative Affect Schedule (PANAS) respectively. The same scales were administered immediately following completion of the art intervention.

Results: Twenty of 21 patients completed the intervention, and all 20 patients actively participated. A significant improvement in positive mood scores (4.6 [95%CI: (1.7, 7.5)] or 14.6% change, p=0.003), decrease in negative mood scores (−3.4 [95%CI: (−0.7, −6.1)] or 18% change, p=0.016) and in anxiety scores (−9.2 [95%CI: (−14.2, −4.1)] or 21.6% change, p=0.001) were observed. Among the 14 patients who reported some pain (VAS>0) prior to the intervention, the mean VAS pain score improved significantly (−1.4 [95%CI: (−2.4, −0.3)] or 35.1% change, p=0.017). Universally all patients commented that they would participate in art-based interventions in the future

Conclusions Bedside visual art interventions can decrease pain and anxiety, and improve mood in hospitalized hematological patients with cancer diagnoses. These intriguing findings warrant future placebo-controlled studies, and suggest that these alternative measures should be considered as an adjunct to conventional therapies in patients with cancer-related pain, and symptoms of anxiety and decreased mood.
The MLL PHD Fingers of MLL Block MLL Fusion Protein Mediated Transformation.
Authors: Muntean Andrew G. , Hess Jay L.
Published: November 2019

Mixed Lineage Leukemia (MLL) is a histone H3K4 methyltransferase that is rearranged in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). MLL is required for the maintenance of Hox gene expression. Deregulation of Hox genes by MLL fusion proteins, which fuse MLL in frame to one of over 50 different translocation partners, is critical for transformation. In these translocations, the DNMT homology (CXXC) domain is always included, but the set of adjacent plant homeodomains (PHD), which includes four PHD fingers and a bromodomain, is invariably excluded. PHD fingers have recently been described to bind tri-methylated histone H3K4 and others report PHD domains binding transcriptional co-repressors, such as Mi-2a of the NuRD complex. However, the role of the PHD fingers in MLL is not well understood. To determine the function of the PHD fingers in MLL, we performed bone marrow transduction and colony assays with the MLL fusion protein MLL-AF9, engineered to contain the PHD domain region (MLL-PHD-AF9). These experiments showed that inclusion of the PHD fingers inhibited immortalization as shown by the absence of compact colonies in methylcellulose replating assays and inhibition of proliferation in liquid cultures. Initial experiments with PHD finger deletions to map the inhibiting activity suggest inclusion of any PHD fingers beyond the first PHD finger, results in inhibition of transformation. To monitor the transcriptional activity of the retrovirally infected bone marrow cells, total RNA was isolated from cells harvested after the second replating, when significant differences were seen in colony morphology and size. Consistent with the transformation inhibition, Hoxa9 gene expression was found to be significantly repressed with respect to expression detected in transformed MLL-AF9 cells as determined by qPCR. To confirm this effect is directly due to the MLL fusion proteins, we performed luciferase assays with an MLL responsive myc E-box luciferase construct in MLL −/− MEFs. We found a specific and robust activation of the reporter in the presence of MLL-AF9, which was severely compromised by the inclusion of the PHD fingers. Together, these results suggest the PHD fingers act as transcriptional repressors that inhibit transformation. Our results provide an explanation for the finding that translocations including the coding region for C terminal PHD fingers do not occur in human leukemias and suggest that this region is also involved in the regulation of wild type MLL. We are currently studying the mechanisms of transcriptional repression mediated by the PHD fingers by isolating interacting proteins and assessing their effect on transcription and transformation.
Prognostic Impact of Chromosomal Abnormalities in Elderly Patients with Multiple Myeloma Treated with High-Dose Melphalan (MEL140) and Autologous Stem Cell Transplantation.
Authors: Liebisch Peter , Straka Christian , Wimmer Astrid , Wendl Christiane , Hinke Axel , Ibach Stefan , Döhner Hartmut
Published: November 2019

Background: Although chromosomal aberrations (CA) have emerged as important outcome predictors in multiple myeloma (MM), the prognostic significance of many recurring genomic changes is still unknown. Moreover, there is only scarce data on the implication of chromosomal abnormalities in elderly patients (pts.) receiving high-dose chemotherapy (HD-CTX) followed by autologous stem cell transplantation (ASCT). Aims: To evaluate the prognostic relevance of recurring chromosomal aberrations as detected by cIg-FISH for elderly pts. with MM receiving HD-CTX and ASCT. Materials: Between 05/2001 and 08/2006, 549 pts. 60–70 yrs. of age with newly diagnosed symptomatic MM entered the DSMM II trial of the Deutsche Studiengruppe Multiples Myelom to receive two cycles of HD-CTX (melphalan 140 mg/sqm) followed by ASCT after 3–4 cycles of dexamethason-based induction chemotherapy (IC; arm A1) or no IC (arm A2). cIg-FISH and DNA probes mapping to chromosome bands 1q21.2, 9q34, 11q25, 13q14, 14q32, 17p13, and 22q11 were applied to all pts. from whom sufficient bone marrow specimen were obtained. 152 pts. with a median age of 65 yrs. for that information on all genomic loci was available were included in the present analysis. The clinical database was last updated in 04/2007.

Results: The median follow-up of time in the present series was 83 (1–264) weeks. Univariate analysis showed significantly shorter EFS in 14 out of 152 pts. (9%) with chromosome 17p13 deletion (17p−; 46 vs. 99 weeks, p<0.0001) and 18 out of 152 pts. (12%) with translocation t(4;14) (57 vs. 99 weeks, p=0.001). Clinical and laboratory variables as well as all other CA had no significant impact on EFS. Multivariate analysis revealed 17p− and t(4;14) as independent predictors of inferior EFS (p=0.0005 and p=0.01, respectively). OS was significantly shorter in pts. with 17p− (99 vs. 219 weeks, p=0.001) and in 17 out of 152 pts. (11%) with deletion of chromosome band 22q11 (127 weeks vs. not reached, p=0.002). There was a trend towards a shorter OS in pts. with t(4;14).

Conclusions: Our data suggest that HD-CTX and ASCT does not provide long-term disease control in elderly pts. with t(4;14) or 17p−. Therefore, HD-CTX and ASCT cannot be recommended for these high-risk pts. (overall ∼20% of cases). 13q− and +1q21.2 were not prognostic markers in the present study. Collection of clinical data and molecular cytogenetic analysis of further cases is ongoing.
Genome-Wide Analysis of 30 Burkitt Lymphomas Using High-Density SNP Arrays Reveals Frequent Cryptic Alterations Including Amplification of the microRNA 17-92 Locus.
Authors: Havelange Violaine , Théate Ivan , Callet-Bauchu Evelyne , Mugneret Francine , Michaux Lucienne , Barin Carole , Dastugue Nicole , Lippert Eric , Saussoy Pascale , Penther Dominique , Vikkula Miikka , Poirel Hélène A.
Published: November 2019

Burkitt lymphomas/leukemias (BL) are highly aggressive mature B-cell malignancies characterized by a constant MYC rearrangement. MYC deregulation is an oncogenic event known to be necessary but not sufficient in BL pathogenesis. Additional (epi)genetic alterations should cooperate, such as inactivation of the p14arf-MDM2-TP53 pathway which is frequently detected. We recently demonstrated the independent negative prognostic impact of specific chromosomal alterations, del(13q) and +7q, in childhood BL. We also showed by Multi-FISH that 13q abnormalities are more heterogeneous than expected and that13q gains are underestimated by conventional cytogenetics (CC). DNA from 30 BL (15 adults/15 children) was analyzed with 50K Xba single nucleotide polymorphisms (SNP) arrays (Affymetrix), in order to characterize additional allelic imbalances and losses of heterozygosity. 47 gains and 30 losses were found in 22 BL; 57 chromosomal imbalances were cryptic (not detected by CC). A recurring 13q amplification was detected in 6 BL. The minimal amplified region (MAR) of 4.35 Mb includes 3 candidate genes: C13orf25, GPC5 and GPC6. One C13orf25 transcripts contains a miRNA 17–92 cluster. The amplicon was unmasked with BAC probes in 2/8 other screened BL with 13q abnormalities. This amplification was associated with a terminal deletion (minimal size: 5Mb) in half cases. A recurring 11q abnormality was also assessed by FISH with a MAR of 3.2 Mb containing candidate genes such as CBL, BLR1. Acquired partial uniparental disomies (pUPD) are characterized by loss of heterozygosity without chromosomal deletion. 73 pUPD (>5 Mb) were found in 15 BL; 14 were telomeric and probably resulted from one mitotic recombination. The pathogenic role of pUPD remains unclear. Some of them may be polymorphisms also detected in healthy population. Other may reflect genomic instability but in our series, no higher incidence of pUPD was detected in BL with a complex karyotype. Thirdly, non random pUPD may render the neoplastic cell homozygous for a preexisting mutation leading to the activation of an oncogene or inactivation of a tumor suppressor gene (TSG). Four telomeric pUPD on 1p, 9p, 17p and 17q were found in at least 2 BL. We confirmed homozygous mutations of 2 TSG (TP53 and CDKN2A) located on 17p and 9p pUPD, respectively. These pUPD may be another way of p14arf-MDM2-TP53 inactivation. Moreover, these results suggest an involvement of the miR 17–92 cluster known to interact with the MYC oncogene in BL pathogenesis. The expression pattern of the different mature miR 17–92 in BL with and without 13q amplification will be presented at the meeting.
BCR/ABL and Other Oncogenic Fusion Tyrosine Kinases (FTKs) Stimulate Single Strand Annealing (SSA), the Rare and Extremely Unfaithful DNA Double-Strand Break Repair Pathway To Facilitate Genomic Instability in Hematologic Malignancies.
Authors: Cramer Kimberly , Penserga Emir T. , Reddy Akhil , Wosen Jonathan , Skorski Tomasz
Published: November 2019

Chromosomal translocations are responsible for the appearance of oncogenes encoding BCR/ABL-related fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFR and NPM/ALK, which induce hematological malignancies such as chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelomonocytic leukemia (CMML), and anaplastic large cell lymphoma (ALCL). We showed that cells transformed by BCR/ABL and other FTKs generate higher levels of reactive oxygen species (ROS) than normal counterparts. Enhanced ROS production in malignant cells causes extensive oxidative DNA damage resulting in elevated numbers of DNA double strand breaks (DSBs) measured by immunofluorescence detecting histone H2AX phosphorylated on serine 139 (γ-H2AX) and localized near DSB sites. Since DSBs may be lethal for cells, they are usually repaired by homologous recombination and non-homologous end-joining. Occasionally, a third mechanism called single-strand annealing (SSA) may be involved, too. During SSA an exonuclease degrades one strand at the DNA ends until homology is revealed (for example between two Alu sequences) allowing the broken ends to anneal and the intervening sequence to be clipped off. Therefore, SSA is extremely unfaithful, resulting in a deletion of sequences between the repeats. Because ∼50% of the mammalian genome consists of repeat sequences, SSA is potentially an important pathway of mutagenesis. For example, intrachromatid SSA events can give rise to interstitial deletions leading to a loss of a tumor suppressor gene. To determine the activity of SSA, a well-defined genetic model designed to exclusively measure SSA was applied. The model consists of a reporter cassette containing two fragments of the gene encoding green fluorescent protein (GFP) with a unique I-SceI restriction site introduced into one fragment. Expression of I-SceI restrictase will cause a DSB, which if repaired by SSA will result in recovery of a functional GFP gene generating a GFP+ cell. A single copy of the SSA reporter cassette was integrated in the genomic DNA of parentals and FTK-transformed cells. Cells were transfected with an expression vector encoding I-SceI restrictase and red fluorescent protein with mitochondrial localization (RFP-Mito) to control transfection efficiency. The number of double-positive (GFP+/RFP-Mito+) cells was scored in the population of RFP-Mito+ cells. FTKs induced an approximately 3–30 fold increase of the frequency of SSA events. Therefore, elevated levels of ROS-induced DSBs combined with increased frequency of a relatively rare but extremely unfaithful SSA repair may contribute to genetic instability in hematologic malignancies induced by FTKs.
Foxo1 Expressed in Osteoblasts Promotes the Leukemogenic Properties of β-Catenin By Activating Notch Signaling
Authors: Kode Aruna , Mosialou Ioanna , Manavalan Sanil J , Feldstein Julie Teruya , Bhagat Govind , Berman Ellin , Kousteni Stavroula
Published: November 2019

Osteoblasts affect self-renewal and expansion of hematopoietic stem cells (HSCs) and homing of healthy hematopoietic and tumor cells into the bone marrow. Moreover, in the mouse, constitutive activation of b-catenin (Ctnnb1CAosb mice) in osteoblasts is sufficient to alter the differentiation potential of myeloid and lymphoid progenitors and to trigger the development of acute myeloid leukemia (AML). Because the same genetic event is associated with AML development in humans we sought to better understand its molecular bases. For that purpose we examined in vivo whether FoxO1, a transcription factor known to interact with β-catenin, affects its AML inducing properties. Deleting one allele of FoxO1 mice from the osteoblasts of leukemic Ctnnb1CAosb mice prevented the appearance of anemia, peripheral monocytosis, neutrophilia and lymphocytopenia that are otherwise observed in Ctnnb1CAosb mice. Similarly, FoxO1 haploinsufficiency in osteoblasts of Ctnnb1CAosb mice prevented the shift in the differentiation of HSCs to the myeloid lineage and the increase in the LSK+/CD150+/CD48- subset of long term repopulating HSC progenitors (LT-HSCs). Histological analysis showed that myeloid and megakaryocyte dysplasias observed in Ctnnb1CAosb mice and associated with their AML phenotype were also rescued in Ctnnb1CAosb;FoxO1osb+/-animals. As a result FoxO1 haploinsufficiency in osteoblasts prevented the early lethality of Ctnnb1CAosb mice since Ctnnb1CAosb;FoxO1osb+/-mice lived and were healthy for at least one year, the entire time that they were observed. At the molecular level FoxO1 interacts with b-catenin in osteoblasts to induce expression of the Notch ligand Jagged-1 which initiates the dysmyelopoiesis leading to AML. ChiP/Re-ChiP assays and transient transfection experiments showed that this interaction requires the formation of a b-catenin/Foxo1 complex at two distinct locations in the regions of the Jagged-1 promoter. It also leads to subsequent activation of Notch signaling in LT- HSC progenitors. These events induce the leukemogenic transformation of HSCs. These findings identify FoxO1 through its expression in osteoblasts as a factor affecting hematopoiesis and provide a molecular mechanism whereby the FoxO1/ activated b-catenin interaction results in AML. They further support the notion that targeting the bone marrow niche may be a new strategy to treat leukemia and raise the prospect that FoxO1 oncogenic properties may occur in other tissues.

Disclosures

No relevant conflicts of interest to declare.
Generation and Characterization of FVIII-Specific CAR-Transduced Regulatory T Cells
Authors: Naumann Anja , Kim Yongchan , Königs Christoph , Scott David W
Published: November 2019

The development of inhibitory antibodies (inhibitors) against FVIII is the most critical complication in the treatment of hemophilia A patients as hemostasis can no longer be reestablished by FVIII replacement therapy. Immune tolerance induction by frequent FVIII infusions is demanding, costly and not successful in all treated patients leading to an urgent need for the development of new therapeutic approaches for the prevention or treatment of FVIII inhibitors. Regulatory T cells (Tregs) are important for the maintenance of tolerance and have a high therapeutic potential in the context of autoimmune or inflammatory immune disorders. As Tregs are polyclonal, treatment with Treg pools comprises the risk of a general immunosuppression. Thus, the establishment of antigen-specific Tregs could be of great benefit for a broad range of patients, including inhibitor positive hemophilia A patients. To create such specific Tregs, a FVIII-specific scFv isolated out of a synthetic phage display library was used to generate a second generation chimeric antigen receptor (CAR).

To verify the specificity of the CAR for FVIII and the functionality of the recombined cytoplasmic domain (CD28 and CD3zeta), naïve CD4 T cells were retrovirally transduced with the generated CAR construct and a proliferation assay was conducted in the presence of plate-bound or soluble FVIII, as well as soluble FVIII presented by autologous irradiated PBMCs. Proliferation of transduced cells was more effective when FVIII was presented plate-bound or by PBMCs. In a therapeutically-relevant setting, this would be very promising, as transduced T cells should not be activated by soluble FVIII in the bloodstream but rather by FVIII presented on antigen-presenting cells in lymphatic organs.

Next, functionality of the CAR construct in Tregs was addressed. Transduced Tregs showed extracellular expression of the scFv and could be stimulated MHC-independently with FVIII. Such stimulated cells showed increased expression of Treg activation markers LAP and GARP. Thus, by using the generated CAR for transduction of Tregs, it was possible to create FVIII-specific Tregs that can be stimulated MHC-independently, opening new possibilities for therapeutic approaches in hemophilia A patients with FVIII inhibitors.

Disclosures

No relevant conflicts of interest to declare.
Engineered Antigen-Specific Human Regulatory T Cells Suppress Both FVIII-Specific T- and B-Cell Responses
Authors: Kim Yong Chan , Zhang Aihong , Pratt Kathleen P. , Shevach Ethan , Scott David William
Published: November 2019

Expansion of human regulatory T cells (Tregs) for clinical applications offers great promise for the treatment of undesirable immune responses in autoimmunity, transplantation, allergy and anti-drug antibody responses, including inhibitor responses in hemophilia A patients. However, polyclonal Tregs are “nonspecific” and therefore their therapeutic application could potentially cause global immunosuppression. To avoid this undesirable outcome, the generation of antigen-specific Tregs would represent a significant advance. In this study, we demonstrate the production and properties of engineered antigen-specific Tregs created by transduction of a recombinant T-cell receptor into expanded human FoxP3+ Tregs. The DNA sequence of this T cell receptor was obtained from a FVIII-specific T cell clone that had been expanded from blood donated by a hemophilia A subject. The resulting engineered FVIII-specific Tregs efficiently suppressed both proliferation and cytokine production of FVIII-specific CD4 T effector cells. Moreover, studies with an HLA-transgenic, FVIII-deficient mouse model demonstrated that antibody production from FVIII-primed spleen cells in vitro was profoundly inhibited in the presence of these FVIII-specific Tregs, suggesting that this novel approach has translational potential for the prevention and/or reversal of inhibitor responses in hemophilia A patients. Furthermore, these FVIII-specific Tregs successfully cross-suppressed unrelated, bystander T effector cells (specific for a myelin peptide) only in the presence of FVIII and the unrelated, stimulatory myelin peptide. These data strongly imply that such FVIII-specific Tregs would be effective at suppressing multiple effector cells, e.g., CD4 T effector cells specific for other FVIII epitopes besides that recognized by the Tregs expressing the engineered TCR, in the local milieu when FVIII is present. Thus, we propose that FVIII-specific Tregs have potential as a therapeutic tool to induce tolerance to FVIII, by preventing or reversing inhibitor formation in hemophilia A patients.

Supported by NIH RO1-HL061883 (DWS), funding from Bayer and CSL Behring (KPP) and intramural support from NIAID (EMS). We thank the Puget Sound Blood Center for enrolling hemophilia A patients and we thank all blood donors.

Disclosures

Kim: Henry Jackson Foundation: YCK and DWS have a provisional patent filing via the Henry Jackson Foundation on Design and use of specific regulatory T cells to induce immune tolerance Patents & Royalties. Scott:Henry Jackson Foundation: YCK and DWS have a provisional patent filing via the Henry Jackson Foundation on Design and use of specific regulatory T cells to induce immune tolerance Patents & Royalties.
Cytotoxic T-Cell Responses (CTL) Against Several Leukemia-Associated-Antigens (LAA) Related to the Detected Cytokine Profile in the Course of Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) and Donor Lymphocyte Infusion (DLI) in Patients with Different Hematological Diseases
Authors: Hofmann Susanne , Götz Marlies , Herbst Cornelia , Schneider Vanessa , Wiesneth Markus , Bunjes Donald , Döhner Hartmut , Greiner Jochen
Published: November 2019

T-cell responses play a central role in maintaining remission and prolonging overall-survival in patients with hematologic malignancies after allo-HSCT and delayed DLI. The role of LAA and mHag has to be elucidated although there is evidence that the graft-versus-leukemia-effect observed after DLI is based on CTL-mediated immunity which is reactive against mHag and LAA.

In this study, we analysed peripheral blood and serum samples of 10 patients with AML, T-NHL, CML and Multiple Myeloma in the course of allo-HSCT and before and after DLI for specific CTL responses against several LAA and established a cytokine profile. Epitopes derived from PRAME, NPM1mut, RHAMM, WT-1 and other LAA were tested. Immune reactions of CTL were measured in ELISpot assays (INF-gamma and granzyme B). In addition tetramer assays were performed. The corresponding cytokine profile of several cytokines was analysed for further interpretation of the clinical data.

In all patients we could detect a CTL response against at least one of the tested LAA in the course of DLI. Two patients were AML patients with NPM1-mutation (NPM1mut) who received preemptive DLI in molecular relapse and showed a polyspecific CTL-response against several epitopes including those derived from the mutated region of NPM1. The frequency of CTL-responses changed during the course. All of the analysed cytokines IL18, CD40L, IL1ra, IL6, IL7, IL10, IL15, IL17, IL18, IL2, IFNg and TGFß were detected in at least one of the samples of the patients, but the pattern differed dependent on the time point after allo-HSCT and DLI, immune-suppression, immune-reconstitution, GvHD and disease status. Some patients showed an increase in cytokine levels in the course of therapy, e.g. in one patient with AML with NPM1mut who received preemptive DLI in molecular relapse we detected significant higher levels of several cytokines after DLI. The patient was in molecular remission again and had developed GvHD.

Taken together, we detected CTL responses against several LAA after allo-HSCT with an increase of polyspecific T cell responses after DLI in some tested patients. These might support the role of CTL against LAA in the control of disease. Especially patients with AML with NPM1mut seem to be a very interesting population with T-cell activation after DLI. Moreover, cytokine profiles might be important and need to be correlated to the cellular responses as well as the clinical data, to further characterize the mechanisms of DLI.

Disclosures

No relevant conflicts of interest to declare.
Longer Procedure Duration and Large Volume Harvests Reduce the Likelihood of Achieving the Requested Cell Dose from Unrelated Bone Marrow Donors
Authors: Anthias Chloe , Arkwright Rebecca , Billen Annelies , Madrigal J. Alejandro , Shaw Bronwen E.
Published: November 2019

Introduction

The factors influencing the quality of bone marrow (BM) harvests and total nucleated cell count (TNC) achieved are poorly understood. As the number of BM harvest procedures falls (due to an increasing proportion of harvest requests for peripheral blood stem cells), concerns are raised around maintaining expertise in this area. We investigate, in four Anthony Nolan collection centers, the procedure- and donor -related factors influencing the quality of harvests, and likelihood of achieving the requested TNC.

Materials & Method

110 consecutive BM harvests of healthy unrelated donors (UD)s were performed according to standard Anthony Nolan protocols. Harvest quality was defined as TNC/ml volume collected. Univariate analyses of factors influencing the TNC and harvest quality were performed using Chi squared or Fisher’s test where appropriate.

Results

110 BM harvests were performed in 80 male and 30 female donors. The median donor age was 28 years (range 18-56 years).

The median TNC requested was 4x108/kg, and median harvested TNC 4.2x108/kg recipient weight, with the requested TNC dose achieved in 50% of harvests. Higher volume harvests (greater than the median volume 1200 mls) were significantly less likely to achieve TNC> 4x108/kg recipient weight (38% vs 70% p=0.001), as were harvests with a procedure time greater than 30 minutes (p=0.037). We investigated donor factors influencing the harvest yield and found that donor/recipient weight discrepancy significantly impacted the TNC achieved: with only 18% of BM harvests from donors who weighed less than their recipient achieving TNC >4x108/kg compared to 62% harvests from donors who were heavier than their recipient (p=0.001). No other donor factor had a significant influence on the TNC.

The median TNC/ml collected was 18.2x10^6/ml and harvests in donors>40 years were more likely to be of lower quality (p=0.036). No other donor factors affected harvest quality, however a significant variation between the 4 collection centers was observed: both the harvest quality (p=0.014) and volume collected (p=0.035) varied significantly between centers.

Conclusions

Donor weight is a strong determinant of bone marrow harvest yield; we recommend that when multiple matched UDs are available, a donor with high body weight should be selected for BM harvest. Procedure factors including large volume harvests and long procedure times reduce the likelihood of achieving the requested TNC, and significant variation was seen between centers. Given the declining number of requests for BM harvests, it is crucial these are performed by the most experienced centers and that systems exist to ensure operators retain their expertise.

Disclosures

No relevant conflicts of interest to declare.
An SCF-FBXW7 Ubiquitin Ligase Mediated Feedback Loop Facilitates GATA Factor Switching and Reinforces Commitment to Terminal Erythroid Maturation
Authors: Ghamari Alireza , Jaensch Elizabeth , Giadone Richard , Akie Thomas E , Xu Jian , Bauer Daniel , Orkin Stuart H. , Cantor Alan B.
Published: November 2019

The GATA family transcription factors GATA2 and GATA1 play reciprocal roles during terminal erythroid and megakaryocytic maturation. GATA2 is expressed early in hematopoiesis and is required for early progenitor cell proliferation and survival. It must be down regulated in order for terminal maturation to occur. In contrast, GATA1 increases in expression during erythropoiesis and megakaryopoiesis, and is required for terminal maturation of these lineages. During this process, GATA1 displaces GATA2 at a large number of chromatin loci, typically leading to repression of early progenitor genes and activation of terminal maturation genes. This “GATA factor switch” is facilitated by the considerably shorter half-life of GATA2 (~30 min) compared to GATA1 (>4-6 hours). GATA2’s short half-life is mediated by ubiquitin-proteosomal degradation mechanisms. Yet the ubiquitin complex(es) responsible for GATA2-specific clearance has not been identified. In this study, we show that the Skip1-Cullin-Fbox (SCF) substrate recognition factor Fbxw7 is involved in GATA2 ubiquitin-mediated degradation. Proteomic and co-immunoprecipitation experiments show physical association of Fbxw7 with GATA2, but not GATA1. CRISPR/Cas9 deletion of Fbxw7 in G1ER4 cells results in elevated GATA2 steady-state protein levels, a prolonged GATA2 half-life, delayed GATA2 clearance upon estradiol induction, and impaired erythroid terminal maturation. Importantly, Fbxw7 mRNA and protein levels normally increase during terminal erythroid maturation and this occurs in a GATA1 dependent manner. We identified a key cis-regulatory element upstream of the Fbxw7 gene that is occupied by GATA2 during early erythropoiesis, but becomes bound by GATA1/TAL1 during late erythroid maturation in induced G1-ER4 cells and human CD34+ in vitro differentiated erythroblasts. This is accompanied by the acquisition of active enhancer histone marks at this site. Deletion of this regulatory element in G1ER4 cells blocks the GATA1 dependent increase in Fbxw7 mRNA transcript levels during erythroid cell maturation. Lastly, we identified a family with congenital hypoplastic anemia (lacking mutations in all known Diamond Blackfan Anemia genes) who harbor a germline Fbxw7missense mutation. Collectively, these findings identify GATA2 as a novel Fbxw7 substrate and uncover an ubiquitin-mediated post-transcriptional GATA factor feedback loop that reinforces GATA factor switching and commitment to terminal erythroid maturation.

Disclosures

Cantor: Amgen: Membership on an entity's Board of Directors or advisory committees.