bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment with Imatinib in Very Elderly (> 75 Years) CML Patients.
Authors: Ata Roberto Latagli , Breccia Massimo , Sica Simona , Spadea Antonio , D’Addosio Ada , Carmosino Ida , De Matteis Silvia , Stefanizzi Caterina , Volpicelli Paola , Napoleone Laura , Vozella Federico , Petti Maria Concetta , Alimena Giuliana
Published: April 2020

Median age of patients with CML is about 65 years, according to SEER 2000–2005 data. However, the treatment with Imatinib in elderly has been seldom specifically addressed: in particular, there is a lack of data regarding very elderly CML patients. To highlight peculiar aspects of toxicity and efficacy of Imatinib in this subset which accounts for at least 10–15% of all CML cases, we revised retrospectively 28 CML patients treated with Imatinib when aged > 75 years from 4 haematological Institution in Rome; there were 12 males and 16 females, median age at Imatinib was 78.7 years (IR 75.8 – 82.9), Sokal Risk at diagnosis was intermediate in 20 patients, high in 6 and not valuable in 2. One or more concomitant diseases requiring specific treatments were present in 26/28 patients (92.8%), with 15 patients (53.5%) assuming 3 or more concomitant drugs. Nine patients (32.1%) have been pretreated ≥ 6 months with HU before starting Imatinib; on the whole, median time from diagnosis to Imatinib was 1.8 months (IR 0.7 – 25.1). Starting dose of Imatinib was 400 mg/day in 23 patients (82.1%) and 300 mg/day in 5 patients (17.9%); overall, 15 patients (53.5%) (14/23 at 400 mg starting dose and 1/5 at 300 mg starting dose) needed a dose reduction and 6 (21.4 %) discontinued Imatinib for toxicity (early toxicity in 4 and late toxicity in 2). Excluding the 4 patients who discontinued Imatinib due to early toxicity, maximum tolerated daily dose during treatment was 400 mg in 8 patients, 300 mg in 13 patients and 200 mg in 3 patients. According to CTC-AE, grade 3 – 4 haematological and extra-haematological toxicities were observed in 7 (25%) and 12 (42.8%) patients, respectively; 2 patients (7.1%) presented a pleural effusion during Imatinib treatment. All patients were fully evaluable for response to Imatinib, with a median treatment period of 30.6 months (IR 15.7 – 49.2); six patients (21.4%) failed any response, (including 4 patients who discontinued Imatinib due to early toxicity) and 22 (78.6%) achieved a complete haematological response (CHR). Among these 22 patients in CHR, 4 refused any other karyotipic or molecular evaluation (3 are still alive in CHR, 1 died in CHR from unrelated cause after 44,5 months) and 17/18 (60.7% of all 28 patients) achieved a cytogenetic response (CyR), major in 1 patient and complete in 16 patients. In addition, 7 patients (25% of all 28 patients) achieved a complete molecular remission, with an undetectable BCR/ABL hybrid gene at qualitative nested PCR. In conclusion, Imatinib at reduced daily dose of 300 mg seems a relatively safe and quite effective treatment for very elderly CML patients; from our data, no upper age limit should be given for TKinhibitors treatment but also very elderly (and with concomitant severe diseases) patients should have this chance of cure.
DNA-Based Monitoring of Minimal Residual Disease(MRD) in Chronic Myeloid Leukemia(CML).
Authors: Morley Alexander A. , Bartley Paul , Ross David M , Latham Sue , Martin-Harris Henry , Budgen Brad , Wilczek Vicki , Branford Susan , Hughes Timothy
Published: April 2020

Aim: To develop and evaluate a DNA-based method for monitoring of MRD in CML

Background: At present, monitoring of MRD in CML uses RNA and reverse transcription (RT-PCR). This leads to a number of disadvantages, including the potential for RNA degradation, requirement for reverse transcription, difficulty in standardisation and only an indirect relationship between assay result and cell number.

Methods: A highly multiplexed, short-range PCR using six BCR primers and a pool of 282 ABL primers was used to amplify across the BCR-ABL translocation breakpoint and was followed by 2–3 rounds of “bottleneck PCR”, a technique recently-developed in our laboratory, which adjusts primer concentration so as to minimise non-specific amplification and facilitate highly multiplexed PCRs. The breakpoint was isolated and sequenced, patient-specific primers were synthesised, and MRD was quantified using 10 μg of DNA and a 3 round nested quantitative PCR incorporating a Taqman probe in the third round. Samples from patients on treatment were divided and assayed both by this technique and by RT-PCR. 10 μg of DNA from a normal individual was used in each assay as a control for non-specificity.

Results: The BCR-ABL breakpoint was successfully isolated and sequenced in 28 of the 29 patients studied and MRD was assayed in 38 samples from 24 patients. Follow-up samples from 4 patients were unavailable MRD was detected and measured by both methods in 22 samples, detected and measured only by DNA-PCR in 10 samples and not detected by either method in 6 samples. The limit of detection of RT-PCR was in accord with previous results which indicated it to be a mean decrease of 4.5-logs below baseline. The mean limit of detection of DNA-PCR was an MRD of 7.2 x 10−7. Assay precision was determined by performing independent replicate assays on different days and by different individuals on the 32 samples with MRD detectable by DNA-PCR. The median SD of a single assay was 0.15 log units with the range being 0.00 – 0.64 log units.

Figure Figure

Conclusions

Monitoring of MRD by DNA-PCR is feasible in the great majority of patients with CML. In terms of possible clinical benefits, DNA-PCR is more sensitive than RT-PCR, and provides a direct measure of leukemic cell number in the individual patient. Use of DNA rather than RNA simplifies specimen collection and transport. In terms of laboratory benefits, DNA-PCR obviates reverse transcription and inter-laboratory standardisation. The principal disadvantage of the method is the initial cost, although this may be able to be amortised over several assays if monitoring is ongoing.
Monitoring of Adenovirus Load in Stool by Real-Time PCR Permits Early Detection of Impending Invasive Infection in Patients after Allogeneic Stem Cell Transplantation
Authors: Lion Thomas , Kosulin Karin , Landlinger Christine , Rauch Margit , Jugovic Dragana , Pötschger Ulrike , Lawitschka Anita , Peters Christina , Fritsch Gerhard , Matthes-Martin Susanne
Published: April 2020

Disseminated adenovirus (AdV) infection is associated with high mortality in allogeneic stem cell transplant recipients. We have demonstrated that molecular detection of AdV in peripheral blood (PB) precedes the onset of life-threatening virus disease (Lion et al., Blood 102:1114–20, 2003). In most instances, detection of AdV in stool preceded the onset of viremia, thus raising the possibility that intestinal infections represent a common source of virus dissemination. To address this question, we have investigated 170 consecutive allogeneic transplantations in 138 pediatric patients transplanted at our center. Patients were monitored for the presence and the load of AdV in stool and in PB by a real-time PCR approach covering the entire spectrum of known human AdV serotypes. Fifty one patients (37%) tested positive in serial stool samples, revealing adenoviruses of nearly all subgenera, with strong predominance of serotypes 2 and 1 of species C. The overall risk of developing viremia in patients displaying or lacking intestinal AdV infection was 31% and 0%, respectively (p<0.0001). Among patients who had maximum AdV levels in stool ≤1×10E6 copies/g (n=29) neither experienced viremia during the post-transplant course, while the incidence of viremia in individuals with peak virus levels in stool above this threshold (n=22) was 73% (p<0.0001). In contrast to earlier observations, the recovery of T-cells including the CD4 and CD8 subsets showed no statistically significant correlation with high AdV loads in stool, occurrence of viremia or disseminated disease (p-values for CD4 and CD8 cells: 0.5833 and 0.6313). The recovery of NK cells showed a borderline-significant correlation with the above parameters (p=0.0442). Our observations indicate that serial measurement of AdV levels in stool specimens by RQ-PCR provides a parameter permitting early diagnosis of impending invasive infection. The median time span between detection of AdV load in stool above 1×10E6 copies/g and first observation of viremia was 10 days (range 0–191). Quantitative monitoring of AdV loads in stool could therefore provide a rationale for early onset of antiviral treatment in attempts to prevent progression to life-threatening systemic infection.
Induction of Hyperproliferative Fetal Megakaryopoiesis by An N-Terminally Truncated GATA1 Mutant.
Authors: Shimizu Ritsuko , Kobayashi Eri , Engel James , Yamamoto Masayuki
Published: April 2020

Two types of GATA1-related leukemias have been noticed. One is the erythroid leukemia develops in mice as a consequence of deficient expression of GATA1, while the other is acute megakaryoblastic leukemia (AMKL) that arises in Down syndrome (DS) children as a consequence of somatic N-terminally truncation (ΔNT) mutation of GATA1 (reviewed in Shimizu et al, Nat Rev Cancer 2008). We hypothesized that modeling of the human mutation in mice might provide novel insights into human TMD and DS-AMKL, since the GATA1 N-terminal (NT) function is apparently important in these disorders. A previous report showed that immature megakaryocytic progenitors in the yolk sac and early fetal liver of knock-in mice that expressed a mutant GATA1 protein lacking amino acids 3 to 63 (and not 1–83, as in the DS-associated AKML patients) developed a hyperproliferative phenotype, but also that these aberrant cells disappeared from fetal blood by E14.5. The same phenotype was detected in conditional knockout mice in which Gata1 exon 2 (which encodes the wild-type start codon) was deleted. Since this phenotype was substantially different from human TMD, the contribution of the GATA1 NT 83 amino acids to human TMD pathogenesis remained obscure. Therefore, we adopted a transgenic complementation/rescue approach to more directly analyze NT domain function. We generated mice that were rescued from GATA1-deficient lethality by transgenic expression of a GATA1 mutant lacking the N-terminal 83 amino acids, a protein that is identical to the mutant GATA1 factor encountered in human TMD and DS-AKML cases. We discovered that expression of this shortened GATA1 protein in mice phenocopies human TMD in DS children preceding AMKL; immature megakaryocytes accumulate massively in the perinatal livers of mutant mice. However, unlike the usual disease progression in humans, these mouse mutants never develop leukemia. These results unequivocally revealed that expression of the short form of GATA1 is sufficient to induce hyperproliferative fetal megakaryopoiesis, but additional mutation(s) to the GATA1/Gata1 N-terminal deletion is necessary to transform those hyperproliferative megakaryocytic cells into overt leukemic cells in both mouse and human. Thus, this model significantly clarifies our understanding of the distinct molecular mechanisms by which the GATA1 protein can contribute to the multi-step leukemogenesis.
NK-Like Homeodomain Proteins Regulate NOTCH3-Signaling in T-Cells.
Authors: Nagel Stefan , Venturini Letizia , Przybylski Grzegorz K. , Grabarczyk Piotr , Meyer Corinna , Kaufmann Maren , Battmer Karin , Schmidt Christian A. , Drexler Hans G. , Scherr Michaela , MacLeod Roderick A.F.
Published: April 2020

Three NK-like (NKL) homeobox genes, TLX1/HOX11, TLX3/HOX11L2 and NKX2- 5/CSX, have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Here we screened further NKL genes in 24 T-ALL cell lines by RT-PCR and identified common expression of MSX2, highlighting this homeobox gene as a potential physiological family member in T-cells. Subsequent quantification of MSX2 confirmed expression in primary hematopoietic cells demonstrating higher levels in CD34+ stem cells when compared to peripheral blood cells or mature CD3+ T-cells. Analysis of core thymic factors in T-ALL cell lines, including IL7, BMP4, TGFbeta, NOTCH and T-cell receptor signaling, suggests their involvement in MSX2 regulation during T-cell differentiation. Chromosomal and genomic analysis of the MSX2 locus (at 5q35) uncovered deletion in t(5;14)(q35;q32) positive T-ALL cell lines associated with low expression levels of MSX2 and ectopic activation of TLX3 or NKX2-5, respectively. For functional analysis we lentivirally transduced T-ALL cells for overexpression of either MSX2 or oncogenic TLX1 and NKX2-5. These cells displayed transcriptional activation of NOTCH3-signaling, as indicated by expression array profiling and real-time PCR analysis of NOTCH3, HES1 and HEY1. The sensitivities to gamma-secretase inhibitor analyzed by MTT-assay of cells overexpressing MSX2, TLX1 or NKX2-5, respectively, were consistently decreased. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with repressor proteins of the NOTCH-pathway, SPEN/MINT and TLE1/GRG1, as shown by co-immunoprecipitation, probably representing one mechanism of (de)regulation. Elevated expression of NOTCH3 and HEY1 mRNA was detected in TLX1/3 positive T-ALL patients, confirming data obtained from cell lines. In conclusion, we have defined expression patterns, regulation and targets of MSX2 in hematopoietic cells, to reveal a novel modulatory activity in T-cell differentiation operating via NOTCH-signaling, and in leukemogenesis when replaced or supplemented by oncogenic NKL homeodomain proteins.
Results of the First International Round Robin for the Quantification of Urinary and Plasma Hepcidin: Need for Standardization
Authors: Kroot Joyce JC , Kemna Erwin HJM , Bansal Sukhvinder , Busbridge Mark , Campostrini Natascia , Girelli Domenico , Hider Robert C , Koliaraki Vasiliki , Mamalaki Avgi , Olbina Gordana , Tomosugi Naohisa , Tselepis Chris , Ward Douglas G , Ganz Tomas , Hendriks Jan CM , Swinkels Dorine W.
Published: April 2020

The hepatic peptide hormone hepcidin plays a central role in body iron metabolism. Quantification of hepcidin concentrations in urine and plasma holds promise as a biomarker for diagnosis and monitoring of disorders of iron metabolism. To date, various mass spectrometry (MS, time of flight and liquid chromatography-MS/MS) as well as immunochemical (IC, competitive radio-immunoassay and ELISA) and functional methods exploiting an internal or external standard, have been developed to measure hepcidin levels in both plasma and urine. Only a few of them have been published. The hepcidin levels quantified by these methods are likely to differ substantially in analytical characteristics. Therefore, comparative studies are imperative for progress in the field by allowing meaningful exchange of hepcidin results derived from various laboratories. In this study we compared the analytical characteristics of several available urinary and plasma hepcidin assays as a first step towards standardization of hepcidin quantification methods. Six and 8 laboratories participated in an international inter-laboratory evaluation of hepcidin levels from a common human urinary (n=8) and plasma (n=7) sample set, respectively, exploiting MS (n=5) and IC (n=3) based methods. Participating laboratories were requested to perform 3 hepcidin measurements for each sample on 4 different days, making a total of 96 urine and 84 plasma measurements per laboratory. The methods differed widely in mean urine and plasma hepcidin level, i.e. range 2.9–427.1 nmol/mmol creatinine and 11.4–124.6 nM, respectively. Coefficient of variation (CV)-ranges for the between-sample and withinsample variation were 126.1–191.5 % and 10.8–33.8%, respectively, for urine and 40.4–96.6 % and 5.5–31.0%, respectively, for plasma. The ratio between-sample CV/within-sample CV varies between 15.3 and 4.1 for urine and 15.1 and 2.4 for plasma. Hepcidin results of most methods mutually correlated (plasma: R range 0.82–1.00; urine: R range 0.73–0.99). We conclude that for the various methods

absolute hepcidin values differ widely, but generally correlate and the ratio between-sample and within sample CV vary greatly, suggesting relevant differences in ability to detect small differences in hepcidin concentrations.

To allow future comparison, exchangeability and transferability of hepcidin data, we recommend

publication of all hepcidin results according to STARD (www.stard-statement.org) to ensure complete reporting of analytical characteristics and to develop initiatives for standardization of assays.
Mir-34a as Part of the Chemotherapy Resistance Network in Chronic Lymphocytic Leukemia.
Authors: Zenz Thorsten , Mohr Julia , Eldering Eric , Kater Arnon P , Bühler Andreas , Winkler Dirk , Kienle Dirk , Dürig Jan , Mertens Daniel , Döhner Hartmut , Stilgenbauer Stephan
Published: April 2020

The central role of 17p (TP53) deletion and 11q (ATM) deletion in identifying patients with CLL who respond sub-optimally to chemotherapy suggests a prominent role of the DNA damage pathway in CLL. MiR34s have been shown to be direct p53 targets and mediate some of the p53 dependent effects.

To assess the role of miR-34s in a large cohort of CLL patients (n=60), we studied miR34a and miR34b/c levels together with a detailed assessment of the DNA damage response. While no expression of miR-34b/c could be detected, we found diverse expression levels of miR34a. MiR34a levels in CLL are up-regulated after DNA damage in the presence of functional p53. In contrast, cases with 17p deletion showed low levels of miR-34a and failure to up-regulate miR-34a after irradiation (median basal expression 2.5 (1.0–15.78) vs. 32.8 (0.89–337.8) in cases without 17p deletion (p<0.0001); after irradiation (median 5.8 (1.38–12.9) vs. 121.8 (1.79–608.9) (p<0.0001)). Since we also observed heterogeneous expression in cases without 17p deletion we investigated causes for impaired miR-34a expression in the absence of 17p- and found a strong correlation of impaired miR-34a levels with impaired DNA damage response, TP53 mutations (without 17p deletion) and F-refractory disease (in the absence of 17p deletion). Importantly, decreased miR-34a expression was associated with resistance to apoptosis and the expression levels differentiated subgroups of CLL with different rates of apoptosis after irradiation better than induction of p21/p53 or cytogenetics. In order to assess the relation of miR-34a to other known targets of p53 we compared the miR-34a expression to mRNA levels of p21, Bax and Puma. Up-regulation of miR-34a after irradiation was associated with induction of Bax and p21 but not with PUMA.

Low expression of miR-34a is associated with p53 inactivation but also chemotherapy-refractory disease, impaired DNA damage response and apoptosis, irrespective of 17p deletion/TP53 mutation. The demonstration of decreased miR-34a basal levels in cases with TP53 mutation could be interpreted as an oncogenic function of mutated TP53, where the mutated protein acts to decrease miR-34a levels and thus leads to reduced apoptosis. The mechanism underlying miR-34a regulation and function warrants further study.
Mice Lacking Wasp-Interacting Protein Evolve Anti-Platelet IgAs That Impair Platelet Responses Mediated by the Collagen Receptor GPVI.
Authors: Falet Hervé , Marchetti Michael P. , Simon Delphine , Geha Raif S. , Hartwig John H.
Published: April 2020

Wiskott-Aldrich Syndrome (WAS) is a hematopoietic disorder characterized by immune deficiency, eczema and severe thrombocytopenia with small platelets. However, the role of the WAS protein (WASp), mutated or absent in WAS, is unclear in platelets because platelets that lack WASp function normally. WASp constitutively associates with WIP in resting and activated platelets. We investigated the role of the platelet WIP-WASp complex by using mice that lack either WIP or WASp. WIP knockout (KO) platelets lack WASp and WIP expression is reduced in WASp KO platelets, indicating a requirement for both proteins for each other’s integrity. WIP KO, but not WASp KO mice evolve platelet-associated antibodies of the IgA class, as evidenced by immunoblot analysis and flow cytometry. We present evidence that platelet-associated IgAs specifically target the collagen receptor GPVI in WIP KO mice:

protein tyrosine phosphorylation, including that of phospholipase C-γ2, and calcium mobilization are impaired in IgA-presenting WIP KO platelets stimulated through GPVI, resulting in α-granule secretion, integrin αIIbβ3 activation and actin assembly defects; binding of the anti-GPVI antibody JAQ1 to normally expressed GPVI is reduced in IgA-presenting WIP KO platelets; JAQ1 induces the irreversible loss of GPVI from circulating platelets in WT mice, but not in WIP KO mice that bear anti-platelet IgAs.

Together, the data indicate that platelet-associated IgAs negatively modulate GPVI signaling in WIP KO mice.
The Effect and Underlying Mechanism of Melatonin on Platelet Formation and Survival in a Thrombocytopenic Model.
Authors: Yang Mo , Zhou Min , Ye Jie Yu , Cheung Yiu Fai , Chan Shing , Ha Shau Yin , Chan Godfrey Chifung
Published: April 2020

Thrombocytopenia is a major treatment-related problem for cancer patients on chemo- or radiotherapy. In clinical studies, melatonin has been shown to be able to lower the frequency of thrombocytopenia in these cancer patients. However, the actual effects and underlying mechanism of melatonin on megakaryocytopoiesis, and platelet formation/survival have not been formally investigated. Our previous studies have demonstrated that serotonin can stimulate hematopoiesis and megakaryocytopoiesis (Yang et al, Stem Cells, 2007). Melatonin is an acetylated product of serotonin. To delineate the effect of melatonin on platelet production, we hypothesize that melatonin has therapeutic effects on thrombocytopenia by promoting megakaryocytopoiesis and enhancing the recovery of platelets via activation of Akt/Erk signaling. The effect of melatonin on thrombopoiesis was investigated in an irradiated mouse model (4 Gy). Eighteen mice were randomly divided into three groups (6 in each). Group 1 (normal control, N) received no irradiation or melatonin; Group 2 (model control, C) and Group 3 (melatonin, M) received 4 Gy total body irradiation. After irradiation, melatonin (10mg/kg.d) was injected intraperitoneally into Group M for consecutively 21 days. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from the three groups on day 0, 7, 14, and 21.Our results showed that melatonin enhanced the recovery of platelets and WBC counts. We also investigated the in-vitro effect of melatonin on CFU-MK formation at different doses (0–-500 nM). The results showed that melatonin significantly promoted CFU-MK formation at a dose dependant manner. The maximal concentration was at 200 nM (P<0.01, n=6). The size of CFU-MK with melatonin treatment was much larger and each MK cell was more mature. Melatonin also stimulated the formation of bone marrow CFU-F. Using megakaryocytic cell CHRF-281, we further found that melatonin exerted protective effect on serum-free induced apoptosis of CHRF cells. The cell viability was significantly increased with the treatment of melatonin. Flow cytometric dot-plot analysis demonstrated that the population of apoptotic cells (Annexin V/PI staining) was significantly decreased with melatonin treatment (200nmol/L) for 72 hrs (n=4). It also showed trends of amelioration of caspase-3 expression and the proportion of cells containing JC-1 monomers (indicating a trend in the drop of mitochondrial membrane potential). The population of phospho-Akt and Erk1/2 were significantly increased after treatment by melatonin. Our results showed that melatonin had a promoting effect on platelet formation and survival in a thrombocytopenic model. The underlying mechanisms may be involved in directly stimulating megakaryocytopoiesis and having anti-apoptotic effect in megakaryocytes via the activation of Akt/Erk signaling.
Integrin-Independent Role of CalDAG-GEFI in Neutrophil Chemotaxis.
Authors: Carbo Carla , Goerge Tobias , Hattori Hidenori , Duerschmied Daniel , Cifuni Stephen M. , Luo Hongbo R. , Wagner Denisa D.
Published: April 2020

Neutrophil chemotaxis and transmigration towards a source of inflammation are two crucial processes for host defense against infection that rely on integrin function. Recently, integrin-independent migration of dendritic cells to the lymph node has been brought to light, although neutrophil migration in the presence of EDTA was reported many years ago. Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), is a small signaling protein that plays a key role in the activation of beta-1, beta-2, and beta-3 integrins in platelets and neutrophils by activating the small GTPase Rap1. We explored the role of CalDAG-GEFI in integrin-independent chemotaxis in neutrophils. Here we report that CalDAG-GEFI−/− neutrophils have impaired chemotaxis that is independent of integrin function. In a chemotaxis transwell assay towards LTB4 and in the presence of 10mM EDTA, CalDAG-GEFI−/− neutrophils had a 50% reduction in transmigration over 60 minutes compared to wild-type (WT) neutrophils (p<0.05). In separate experiments we confirmed that the transwell assay is independent of integrins using either CD18−/− neutrophils or WT neutrophils plus a blocking anti-CD18 monoclonal antibody. We previously showed that LTB4 signaling upstream of CalDAG-GEFI was not affected in CalDAG-GEFI−/− neutrophils, as assessed by intracellular calcium flux measurements. Using videomicroscopy to visualize the live migrating neutrophils in a horizontal plate in the presence of 10mM EDTA, we found that the reason CalDAG-GEFI−/− neutrophils fail to reach the chemotactic stimulus (10 pg/mL LTB4) is because they have a significantly reduced migration speed compared to WT neutrophils (16 um/sec vs. 23 um/sec, p<0.05), and also because they have an abnormal chemotactic directionality, with a directionality index (the distance between the start and finish points of a migrating neutrophil/total distance covered by the migrating neutrophil) of 0.84 vs 0.94 in WT neutrophils, p<0.05. We investigated whether the observed differences in chemotaxis between CalDAG-GEFI−/− and WT neutrophils could be explained by differences in F-actin polymerization. Using fluorescence microscopy, we found that the percentage of CalDAG-GEFI−/− neutrophils with F-actin pseudopodia after LTB4 stimulation was significantly lower compared to WT neutrophils (22% vs. 56.7%, p<0.05), suggesting that CalDAG-GEFI−/− neutrophils have a defect in F-actin polymerization. Overall, our studies suggest that CalDAG-GEFI plays a role in the mechanisms that regulate both the migration speed and direction of neutrophils during chemotaxis, independent of its established role in integrin activation.
Comprehensiveness of Quality of Life Instruments in Capturing Concerns Related to Chemotherapy-Induced Neutropenia
Authors: Nina Lathi A , Isogai Pierre , Mittmann Nicole , DeAngelis Carlo , Cheung Matthew , Sandra Knowles , Piliotis Eugenia , Shear Neil , Walker Scott
Published: April 2020

Neutropenia is a serious hematologic consequence of cancer chemotherapy that can lead to further complications such as febrile neutropenia (FN). FN is potentially life threatening and often requires hospitalization. Few studies have evaluated the impact of neutropenia on quality of life (QoL). This study quantified QoL using two nonneutropenia-specific instruments, the EQ-5D questionnaire, a generic tool used to measure health-related QoL, and the Functional Assessment of Cancer Therapy - General (FACT-G) questionnaire, and a neutropenia-specific instrument, the Functional Assessment of Cancer Therapy - Neutropenia (FACT-N) questionnaire. The FACT-G is a 27-item questionnaire that examines QoL in patients with cancer using four subscales. A neutropenia-specific subscale (NSS) has been developed for use with the FACT-G; this combined questionnaire is the FACT-N. Data were collected from patients, who provided informed consent, and who were admitted to Sunnybrook Health Sciences Centre, Toronto, Canada, for the treatment of chemotherapy-induced FN. Linear regression models were fitted to examine the relationship of scores from the neutropenia-specific instrument with those obtained from the other instruments. Two models were fitted using the NSS as the response variable. Predictors for the regression models were the FACT-G scores for each of the subscales (physical, emotional, social and functional wellbeing) and the five domains of the EQ-5D (mobility, self-care, usual activity, pain/discomfort and anxiety/depression) along with the visual analog scale (VAS) component of this tool. The physical and emotional wellbeing subscales of the FACT-G had a strong relationship to the NSS (p < 0.05); the social and functional well-being subscales had a much weaker relationship (p > 0.5). For the EQ-5D, the pain/discomfort domain had the strongest relationship to the NSS (p=0.18); the remaining domains, with or without the VAS, all demonstrated a weaker relationship (p > 0.5). Model fit was assessed by the adjusted R2 statistic; it was 0.54 when FACT-G subscales were used as the predictors compared to −0.04 for the EQ-5D domains indicating that the FACT-G was a better predictor of neutropenia-related concerns. Neutropenia concerns appear to be more closely related to cancer specific QoL compared to general quality of life as demonstrated by the stronger relationship of the NSS to the FACT-G than to the EQ-5D. This may be due to the comprehensiveness of the FACT-G questionnaire where a possible score anywhere from 0 to 24 or 28 can be obtained in each of the subscales, compared to three-point descriptive system for each of the domains of the EQ-5D.
Enforced Nr4a2 expression Drives HSCs into Quiescence.
Authors: Sirin Olga , Conneely Orla M , Goodell Margaret A.
Published: April 2020

Hematopoietic stem cells (HSCs) are used as a paradigm for understanding somatic stem cell biology, yet the factors that regulate their self-renewal and differentiation are poorly understood. Our lab has recently discovered that within the hematopoietic lineage, Nr4a2 expression is restricted to the stem and progenitor cell compartment. Furthermore, we have observed a dramatic decrease in its expression when the bone marrow cells are exposed to 5-fluorouracil which forces quiescent HSCs into cycle. We therefore hypothesize that Nr4a2 may be involved in maintaining stem cell quiescence.

To better understand the role of Nr4a2 in hematopoiesis, we have performed gain-of-function studies in which Nr4a2 is overexpressed in HSCs. Bone marrow transplantation (BMT) experiments revealed that Nr4a2 overexpressing cells contributed significantly less to the peripheral blood compared to the control (1% vs 35%, see fig). This suggests that enforced Nr4a2 expression negatively affects HSC regeneration potential. We also found that Nr4a2 overexpression has a negative effect on the overall proliferation capacity with an in vitro methocult assay. Together, these results support our hypothesis that the high level expression of Nr4a2 regulates HSC quiescence by inhibiting HSC self-renewal and/or differentiation.

Cell cycle analysis demonstrates that enforced expression of Nr4a2 causes an increase in the number of cells in quiescence. These data are supported further by analysis at a molecular level revealing that overexpression of Nr4a2 increases expression of cell cycle inhibitors such as p18 and p19, both of which are important for Go to G1 entry. By uncovering the role of Nr4a2 in hematopoiesis we aim to achieve not only a better understanding of the regulation of stem cell self-renewal and differentiation but also of the pathways involved in HSC cell cycle regulation.

Figure Figure
C/Ebpβ Inhibits Apoptosis in Hematopoietic Cells Via Interaction with NF-κb p50.
Authors: McFall Rebecca , Paz-Priel Ido , Friedman Alan D.
Published: April 2020

The CCAAT/enhancer binding protein (C/EBP) family of proteins bind DNA via their C-terminal basic region – leucine zipper (BR-LZ or bZIP) domains and activate transcription via N-terminal trans-activation domains. We previously demonstrated that C/EBPα inhibits apoptosis when the immature hematopoietic Ba/F3 cell line is withdrawn from IL-3 and that a C/EBPα BR mutant that does not bind NF-κB p50 but retains the ability to bind NF-κB p65 is inactive. We have now sought to extend these findings to C/EBPβ. We first created two point mutations in C/EBPβ, R240G and ΔK253. The C/EBPβ(R240G) is analogous to the C/EBPα(R300G) BR variant that does not bind NF-κB p50 or inhibit apoptosis, and C/EBPβ(ΔK253) is analogous to the C/EBPα(ΔK313) LZ variant that does not bind DNA but retains the ability to interact with NF-κB p50 and inhibit apoptosis. In co-ip experiments using transiently transfected 293T extracts, both C/EBPβ and C/EBPβ(ΔK253) interacted with NF-κB p50, whereas C/EBPβ(R240G) did not, as predicted. In contrast, all three isoforms bound NF-κB p65. And in gel shift assay, C/EBPβ bound a consensus C/EBP site, whereas neither C/EBPβ(R240G) nor C/EBPβ(ΔK253) bound DNA, again as expected. Ba/F3 cell lines were then generated stably expressing C/EBPβ-ER, C/EBP(R240G)-ER, or C/EBPβ(ΔK253)-ER proteins, via transduction with pBabePuro retroviral vectors and puromycin selection of pooled populations. Western blot analysis confirmed similar expression of each transgene. Ba/F3 lines were removed from IL-3 and cultured with or without estradiol, to activate the ER fusion proteins. Remarkably, both CEBPβ-ER and C/EBPβ(ΔK253)-ER prolonged survival of the cells during the first 48 hrs after IL-3 withdrawal, similar to the activity of C/EBPα-ER, whereas C/EBPβ(R240G)-ER was ineffective. Current efforts focus on identifying genetic targets of C/EBPβ:NF-κB p50 cooperation relevant to the observed inhibition of apoptosis, on assessing activities of C/EBPβ and it variants in normal hematopoietic cells isolated from adult marrow, and on determining the activities of the long and short (LAP and LIP) C/EBPβ translational isoforms. Overall, the finding that C/EBPβ inhibits apoptosis in cooperation with NF-κB p50 is significant due to the expression of both C/EBPβ and activated NF-κB in several human lymphoma subsets and in solid tumors such as breast cancer. Targeting this interaction with small molecules may provide a novel therapeutic approach for these malignancies.
JunB Limits Hematopoietic Stem Cell (HSC) Functions as a Protective Mechanism against Initiation of Myeloid Malignancy
Authors: Santaguida Marianne T , Schepers Koen , King Bryan C , Passegue Emmanuelle
Published: April 2020

The JunB/AP-1 transcription factor plays a crucial role in controlling homeostasis of the hematopoietic stem cell (HSC) compartment and functions as a tumor suppressor in mice. We have previously demonstrated that mice lacking JunB accurately recapitulate important clinical aspects of human myeloid malignancies, including chronic myelogenous leukemia(CML). Using this model, we have shown that leukemia-initiating stem cells (LSCs) can arise from the hematopoietic stem cell (HSC) compartment. Now, we have identified the mechanisms by which JunB regulates HSC functions and discovered how inactivation of this key transcription factor leads to HSC transformation into LSCs that overproduce myeloid progenitors and drive myeloproliferative disease (MPD) development in vivo. We found that JunB normally controls the proliferation of the long-term HSCs (LT-HSCs) by regulating the expression levels of key cell cycle regulators, including the CKI p57 and the polycomb group gene Bmi1, and limits the rate at which LT-HSCs produce myeloid progenitors by maintaining appropriate responsiveness to both Notch and TGF-β signaling pathways. These functions place JunB at the center of a complex network of intrinsic and extrinsic regulations that ensure coordinate regulation of HSC proliferation and differentiation, independently of LT-HSC maintenance and self-renewal activity. These results provide a mechanism for disease initiation in a broad range of human myeloid malignancies, including those in which JunB inactivation has already been reported. Moreover, they expand our understanding of the normal biology of the HSC compartment and provide a mechanism to explain how mutations that increase the proliferation of a single LT-HSC can become dominant and lead to the expansion of an aberrant clone within the otherwise normal HSC compartment. Understanding clonal dominance of LT-HSCs is clearly at the root of designing new therapeutic treatments for a broad spectrum of hematological malignancies.
Human Hematopoietic Stem Cells and Leukemic Cells Form Cadherin-Catenin Based Junctional Complexes with Mesenchymal Stromal Cells
Authors: Wuchter Patrick , Saffrich Rainer , Wagner Wolfgang , Wein Frederik , Schubert Mario Stephan , Eckstein Volker , Ho Anthony D.
Published: April 2020

The interaction between human hematopoietic stem cells (HSC) and their niche plays a key role in regulating maintenance of “stemness” and differentiation. We have demonstrated that a feeder layer of human mesenchymal stromal cells (MSC) can serve as a surrogate model for the niche for human HSC. We could also show, MSC are intimately connected to one another by a novel kind of adhering junction, consisting of villiformto-vermiform cell projections (processus adhaerentes). With this background, we have analyzed the intercellular junctional complexes between HSC and MSC. In comparison, we also studied the cell-cell contacts between leukemia cells (LC) and MSC.

MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were isolated from umbilical cord blood. Leukemia cells that were CD34+ were obtained from bone marrow aspirates from patients suffering from acute myeloid leukemia at the time point of initial diagnosis. After 24–48 hours of co-cultivation, we stained the cellular contacts with a panel of antibodies specific for various components of tight, gap and adherens junctions. Using advanced confocal laser scanning microscopy in combination with deconvolution and volume rendering software, we were able to produce 3D-images of intercellular junctions between HSC/MSC as well as between LC/MSC. To examine the specific function of N-cadherin, we analyzed the effect of siRNA knock down of N-cadherin in MSC upon co-cultures of HSC and MSC.

Intercellular connections between HSC and MSC are mainly characterized by podia formation of the HSC linking to the adjacent MSC. At the intimate contact zone to the MSC, we have identified the cytoplasmic plaque proteins alpha- and beta-catenin, co-localized with the transmembrane glycoprotein N-cadherin. Additionally, we compared these findings with a similar setting consisting of human LC co-cultured with feeder-layer of MSC. Our results demonstrated that in comparison to HSC, the proportion of leukemia cells adherent to the feeder-layer is significantly lower and podia formation is less frequent (ratio 1:3). However, the mechanism of adhesion through cadherin-catenin-complex has remained the same. At a functional level, we found that siRNA knock down of N-cadherin in MSC resulted in decreased adhesion of HSC to MSC and in a reduction of cell divisions of HSC. These results confirm that direct cellular contact via N-cadherin-based junctions is essential for homing and adhesion of HSC to the cellular niche and subsequently for the regulation of self-renewal versus differentiation in HSC.
SCL and Associated Proteins Distinguish Active from Repressive GATA Transcription Factor Complexes.
Authors: Deng Wulan , Tripic Tamara , Cheng Yong , Zhang Ying , Vakoc Christopher , Hardison Ross , Blobel Gerd
Published: April 2020

GATA-1 controls hematopoietic development by activating and repressing gene transcription, yet the in vivo mechanisms that specify these opposite activities are unknown. By examining the composition of GATA-1 associated protein complexes in a conditional erythroid rescue system, we detected the SCL/TAL1, LMO2, Ldb1, E2A complex in vivo at all positively acting GATA-1-bound elements examined. ChIP-on-chip tiling arrays for SCL and Ldb1 revealed that chromatin occupancy of SCL and Ldb1 are extremely tightly correlated. Importantly, they showed a strong tendency for co-occupancy with GATA-1. In deed, all regions occupied by GATA-1 that showed enhancer activity were occupied by SCL and Ldb1, independent of the proximity of E-box sequences. Similarly, the SCL complex is present at all activating GATA elements in megakaryocytes and mast cells. In striking contrast, at sites where GATA-1 functions as a repressor, the SCL complex is depleted. A DNA-binding defective form of SCL maintains association with a subset of active GATA elements indicating that GATA-1 is a key determinant for SCL recruitment. Knockdown of LMO2 selectively impairs activation but not repression by GATA-1. ETO-2, an SCL-associated protein with the potential for transcription repression is also absent from GATA-1-repressed genes but, unlike SCL, fails to accumulate at GATA-1 activated genes. Together, these studies identify the SCL complex as a critical and consistent determinant of positive GATA-1 activity in multiple GATA-1-regulated hematopoietic cell lineages.
Bidirectional Control of Transcription Factor GATA2 and Cyclin/Cdks in Hematopoietic Cells.
Authors: Minegishi Naoko , Harigae Hideo , Yamamoto Masayuki
Published: April 2020

GATA2 is a transcription factor indispensable for development and maintenance of hematopoietic stem cells. Hematopoietic stem cells in G0 phase express GATA2 (Suzuki N, PNAS 103: 2202 2006), but proliferating hematopoietic progenitor cells, as well as proliferating cells in several other tissues, also express this factor. Gata2 knockout experiments indicate that GATA2 regulates cell proliferation; however, precise mechanisms have not been elucidated. Previously, we found oscillatory expressions of GATA2 during the cell cycle. In G1/S and G2/M phases, Cdk4 and Cdk2, forming complexes with several cyclins, phosphorylate GATA2 and induce GATA2 degradation (Koga S, Blood109: 4200, 2007). In this study, we demonstrate the bidirectional control between GATA2 and Cyclin/Cdk systems. To identify GATA2 target genes regulating cell proliferation, we searched for conserved GATA factor-binding sequences in evolutionarily conserved regions (ECRs, http://ecrbrowser.dcode.org), and found conserved GATA sequences in ECRs in intron 3 and intron 4 of the CyclinD2 (Ccnd2) gene, and in intron 3 and 3′UTR of the CyclinE1 (Ccne1) gene. In ChIP experiments using synchronized Ba/F3 and P815 cells, GATA2 binding to these regions was high in late G1 phase, and then rapidly declined in early S phase. One hour treatment using a Cdk1/2 inhibitor, roscovitine, cancelled this regression in early S phase, implicating Cdk2 as a negative regulator of GATA2 binding to target genes. Significant GATA2 binding to Ccnd2 and Ccne1 genes was also detected in embryonic day 13.5 fetal liver cells. Exogenous GATA2 expression augmented transcriptional activity of these binding regions in luciferase assay. Induction of GATA2 siRNA repressed Ccnd2 and Ccne1 mRNA expression. Furthermore, Ccnd2 and Ccne1 mRNA expression, as well as Gata2 mRNA expression, was significantly reduced in placental and aortic regions of embryonic day 10.0 conceptihar boring insertions of green fluorescent protein cDNA at translation start sites of bothGata2 alleles (Minegishi N, Blood102: 896, 2003). These results indicate that Ccnd2 andCcne1 genes are direct targets of GATA2. GATA2 binds to Ccnd2 and Ccne1 genes and activates their transcription in the G1 phase of proliferating cells. In G1/S transition, Cdk2activity, probably enhanced by CyclinE1 expression, attenuate GATA2 binding to Ccnd2and Ccne1 genes. In this fashion, bidirectional control of GATA2 and G1 cyclins may contribute to the transient expression of CyclinD2 and CyclinE1 in G1 phase, and to the acceleration of S-phase entry. In addition, it appears plausible that lack of Cdk activities causes differences in GATA2 functions in hematopoietic stem cells in G0 phase.
The G Protein-Coupled Receptor cysLT1 Mediates Gi Protein-Dependent Chemotaxis and Adhesion of CD34+ Cells in Vitro Similar to CXCR4, but Not Progenitor Mobilization in Vivo.
Authors: Drost Adriana , Jaggy Lena , Kanz Lothar , Möhle Robert
Published: April 2020

CysLT1, which is expressed in several cell types during inflammation and allergy, and the homing related chemokine receptor CXCR4 both belong to the family of G protein-coupled receptors (GPCR). Similar to CXCR4, the cysteinyl-leukotriene receptor cysLT1 is strongly expressed in CD34+ cell lines and CD34+ hematopoietic progenitor cells (HPC). We therefore compared the effects mediated by cysLT1 on HPC to those observed after activation of CXCR4. The most potent cysLT1 ligand LTD4 induced chemotaxis and adhesion of CD34+ HPC to endothelial cells, immobilized VCAM-1 and fibronectin, which was comparable to effects induced by the CXCR4 ligand SDF-1. CysLT1- and CXCR4-mediated effects were inhibited by pertussis toxin (PTX), suggesting that both GPCRs employ the same Gi-protein-dependent signaling pathways in CD34+ HPC. This is supported by identical time courses of intracellular calcium fluxes and actin polymerization induced by LTD4 and SDF-1, as measured by time-dependent flow cytometry. Given the striking similarities of cysLT1- and CXCR4-mediated effects in vitro, one might expect also overlapping functions in vivo. We therefore investigated whether blocking of cysLT1 is associated with HPC mobilization. As cysLT1 antagonists are established for therapy and prophylaxis in patients with allergic and exercise-induced asthma, circulating CD34+ progenitors were enumerated after initiation of a treatment with the cysLT1 antagonist montelukast, used as asthma prophylaxis in otherwise healthy subjects. The number of CD34+ cells or white blood counts did not differ significantly from the baseline value 2, 4, 8, 12, and 24 hours after administration of 10 mg montelukast, in contrast to previous studies analyzing stem cell mobilization induced by CXCR4 antagonists. This corresponds with our in vitro findings that LTD4 is produced by bone marrow endothelium and stromal cells only when deprived of hematopoietic cells, in contrast to the constitutive production of SDF-1. We conclude that cysLT1 is not involved in bone marrow retention of HPC during steady-state hematopoiesis, but may modulate HPC homing when its ligands are produced either locally (i.e. bone marrow aplasia) or systemically (i.e. inflammation).