bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Two Novel Chemokines (CCL2 and CXCL2) in B-Chronic Lymphocytic Leukaemia (B-CLL) and Prolonged Survival of Primary B-CLL Cells in Vitro
Authors: Gill Devinder , Burgess Melinda , Knop Louise , Mollee Peter , McMillan Nigel 
Published: April 2020

Abstract
Aim.
Investigate the in vitro culture conditions that would allow the long-term culture of primary CLL cells and Identify cytokines responsible for B-CLL survival in vitro
Method. Blood and/or bone marrow was collected, after informed consent, from patients with CLL. Diagnosis of CLL was made according to NCI criteria. Mononuclear cells were cultured at various cell densities in RPMI 1640 medium + 10% heat inactivated FCS. Human cytokine array was performed at baseline day 0 and on supernatant of B-CLL cells which had been cultured in complete media for 7 days using ChemiArray system (Human Cytokine Antibody Array III, Chemicon)
Results. Samples were cultured from 19 CLL patients (9 females, 10 males with median age of 60 years; range 44–90). Patients were predominately untreated, early stage with only 3 patients at Binet stage C or Rai stage 4. The overall trend was that 80% of the total cell population died rapidly leaving a resident population of CD19+CD5+ cells (Fig 1). By increasing the initial seeding cell density to high levels (≥5×107/ml) CLL cells can be cultured out to approximately 90 days without additional stromal cells support from another source. Decreasing the seeding cell densities resulted in reduced cell survival. The surviving cells belonged to the malignant clone and were all CD5+/19+, EBV negative and mostly quiescent, with only 3.5% of the CLL B cells actively dividing. Certain patients’ cells exhibited much better in vitro survival than others but no correlation was found with any clinical parameters including clinical stage (Rai or Binet), lymphocyte doubling time, time to treatment, CD38 positivity, IgVH mutation status or LDH levels. Two novel soluble factors, the chemokines CCL2 (MCP-1) and CXCL2 (GROb), were identified in the culture media which appear to enhance survival of CLL B cells. Addition of exogenous CCL2 and CXCL2 resulted in improved in vitro survival of CLL B cells, while blocking these growth factors with specific antibodies resulted in decreased survival (Fig 2). By immunohistochemistry and intracytoplasmic flow cytometry, the CCL2 chemokine appears to be predominately secreted by the stromal cells.
Conclusion. This study demonstrates the prolonged survival of B-CLL in vitro without additional stromal cell support. Furthermore, novel cytokines CCL2 and CXCL2 appear to prolong the survival of B-CLL cells in vitro. This culture system and these chemokines may allow us to gain insight into factors modulating B-CLL survival and potentially could lead to targeted therapy as well as serve as an appropriate model to test new therapies.
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Disruption of the Bcr-Abl/Jak2/HSP90 Network Complex Leading to Induction of Apoptosis in Drug-Resistant CML Cells by a New Compound WP1193
Authors: Samanta Ajoy K. , Chakraborty Sandip N. , Samanta Susmita , Wang Yan , Sun Xiaoping , Schlette E. , Priebe Waldemar , Arlinghaus Ralph B. 
Published: April 2020

Abstract
In chronic myelogenous leukemia (CML), treatment of early stage disease has been significantly improved with the discovery of imatinib mesylate (IM, Gleevec), but with the progression of the disease, drug resistance develops which is due to mutation of the ATP binding site domain within the Bcr-Abl tyrosine kinase, the activation of Lyn kinase (a Src-related tyrosine kinase) and other mutations that produce a more aggressive disease. Thus, patients relapse and the disease progresses to blast crisis (BC) stage where survival is limited to only several months. No suitable drug treatment for BC is available, and the mechanism of drug resistance for BC is unclear. A new synthesized small molecule, WP1193, a caffeic acid derivative, induced apoptosis in all types of drug-resistant CML cells including IM-resistant T315I BCR-ABL+ cells, IM-resistant CML cells with up-regulated Lyn tyrosine kinase (K562-R) and cells from blast crisis CML patients. Treatment of BCR-ABL+ cells with WP1193 inhibited Janus kinase activities, leading to strong reduction of Bcr-Abl; this treatment also reduced STAT3 and pTyr 705 STAT3 protein levels. CML cell lines contain a large signaling network complex composed of Bcr-Abl, Jak2, HSP90 and its client proteins. HSP90 is a molecular chaperone that plays an important role in conformational maturation and stabilization of client proteins (Bcr- Abl, Jak2, STAT3, etc.), which are physically associated in the signaling network complex. This network complex (estimated to be between 2–5 million daltons) has been characterized by gel filtration column chromatography. This complex is disassembled by treatment of Bcr-Abl+ cells with WP1193 (10 μM for 3–6 h). Treatment of the cells with WP1193 also inhibited binding of STAT3 to its consensus DNA sequence and reduced levels of HSP90α transcripts, HSP90α protein and its client proteins, which include Bcr-Abl, Jak2 and Akt. WP1193 also reduced solid tumors in mice inoculated with IM-resistant K562-R cells, and inhibited leukemic effects caused by T315I BCR-ABL+ cells. These results indicate that disruption of the Bcr-Abl/Jak2/HSP90 network complex by WP1193 overcomes drug resistance in advanced stage CML cells. Since disruption of this network complex causes apoptosis induction in all IM-resistant and late stage CML patient cells tested so far, and since no significant toxicity was observed in short-term mouse experiments, WP1193 may have medical utility for the treatment of IM-resistant forms of CML and late stage CML where other drugs fail. One novel feature of WP1193 is its ability to reduce the activity of several important components of the network, namely Bcr-Abl, Jak2 and STAT3 activities and reduction of HSP90α, which then causes a high level of apoptosis in IM-resistant cells and late-stage CML cells.
Disturbed NK Cell Compartment in CML Patients with Anti-Leukemia Immune Responses Under Treatment with Imatinib Mesylate
Authors: Chen Christiane I , Maecker Holden T , Falkow Rhoda , Lee Peter P. 
Published: April 2020

Abstract
Imatinib mesylate has revolutionized the treatment of CML and, as an oral agent with few side effects, became the first-line therapy for patients with CML. However, patients who discontinue the drug invariably relapse. Currently, the only curative treatment for CML is allogeneic stem cell transplantation (ABMT). A major mechanism of ABMT is immunological as evidenced by the poor clinical outcome with T cell-depleted ABMT. To determine anti-leukemia immune responses in patients in remission on imatinib, blood and bone marrow samples before (12 samples) and serially after treatment (59 samples, range 10 months – 35 months) were collected and analyzed. The NK cell (CD56+CD3−) compartment in the 12 CML patients were significantly decreased before treatment (median 3,4 % vs 10 %, p&lt;0.01) and remained low in patients in remission on imatinib (5 % vs 10 %, p&lt; 0.01) compared to 32 age matched healthy donors. Within the CD8+ T cell compartment, naïve T cells (CD45+CD27+) were significantly higher before treatment (55.5 % vs 37.7 %, p=0.04) and decreased slowly to normal levels after successful treatment (41.5 % vs 37.7 %, p=0.28), whereas memory (CD45−CD27+) T cells presented the opposite way. They were significantly lower before treatment (21.7 % vs 31.1 %, p=0.03) and increased slowly to normal levels after successful treatment (27.5 % vs 31.1 %, p=0.39). However, the percentage of T cells (CD4+, CD8+, CD4+CD25+ regulatory T cells) and B cells were not significantly different. These results correlated (p&lt;0.01) with T cell responses to autologous leukemia cells at the same time points, analyzed by IFN-g ELISPOT assay (median 36 SFCs vs 5 SFCs, p&lt;0.01), and were confirmed by cytokine flow cytometry (IFN-g 3.6 % vs 0.29 % and TNF-a 6.6 vs 0.1 %). These results suggest that a significant portion of CML patients in remission with imatinib develop an anti-leukemia T cell response, whereas the NK cell compartment is disturbed. Mechanisms by which imatinib treatment leads to anti-leukemia immune responses, and the molecular targets to which these cells are directed, will be further investigated. This knowledge will be useful in the development of immunotherapy strategies against CML and may, in combination with imatinib, lead to eradication of residual leukemia cells for a durable cure.
Degrasyn (a novel tyrophostin) Impacts BCR-ABL Protein Level and Is Cytotoxic to Chronic Myeloid Leukemia Early Progenitors
Authors: Peterson Luke F , Sun Hanshi , Bornmann William , Peng Zhenghong , Maxwell David , Donato Nicholas , Talpaz Moshe 
Published: April 2020

Abstract
Despite the potent anti-proliferative and apoptotic effects of tyrosine kinase inhibitors (imatinib, dasatinib) on mature CML cells these compounds have limited activity on Chronic Myeloid Leukemia (CML) early progenitors (Lin-/CD38−/CD34+). Mechanisms of resistance to these agents in the early progenitor population are poorly understood but are distinct from BCR-ABL mutation induced resistance. Degrasyn (WP1130) was previously shown by our laboratory to inhibit CML cell growth and colony formation. Further structure-activity relationship studies led to the development of a more potent compound, CP2005 (CP). CP displays enhanced activity in suppressing growth and activating apoptosis in various CML and ALL cell lines (IC50 ~1 microM). Here we show that total CD34+ from CML patients are more effectively growth inhibited by CP2005 compared to WP1130. An anti-leukemic effect was also noted in primitive progenitors and was associated with the induction of apoptosis. BaF3 cells expressing an EGFP-tagged BCR-ABL protein treated with CP or WP1130 show that both facilitate translocation of BCR-ABL from the cytoplasm to a detergent insoluble compartment which subsequently leads to degradation of BCR-ABL. We observed that the Akt/mTOR pathway is activated by CP which could be partially suppressed by mTOR or PI3K/Akt inhibition (rapamycin and wortmannin, respectively). CP-induced BCR-ABL translocation was associated with a reduction in the level of tyrosine-phosphorylated Stat3 and Stat5. However, although there is activation of survival signals associated with Akt-signaling 2 microM CP is able to promote significant apoptosis (67%) as a single agent in CML early progenitors. Although imatinib was minimally effective in reducing the early progenitor cell survival, co-incubation of imatinib with CP2005 resulted in near complete induction of apoptosis in early progenitor cells (~ 93%), suggesting that the combined effects of both CP and imatinib on BCR-ABL protein degradation and kinase inhibition markedly impair the survival of CML early progenitors. The combination of BCR-ABL degradation and kinase inhibition may be effective in engaging pro-apoptotic signaling in CML early progenitor cells.
Study of Expression of Ku70 and NHEJ in CML Cells
Authors: Fu Chengcheng , Chen Peishuai , Depei Wu , Chen Zixing 
Published: April 2020

Abstract
Objective FThe DNA double strand breaks (DSB) in mammalian cells are predominantly repaired by a process called non-homologous DNA end joining (NHEJ). Ku70 played a pivotal role in NHEJ pathway. As a common hematological malignant disease with unique chromosomal translocation t (9;22), chronic myeloid leukemia (CML) can be considered as a paradigm for neoplasias that evolve through a multi-step process. As we reported before, protein Ku70 expressed significantly higher in CML than in normal BM cells. Meanwhile, its expression level in blast phase was markedly higher than that in chronic phase. The present study furthermore aims to investigate the expression of the gene Ku70 in CML cells at different clinical stage and reveal the correlation among the expression of the gene Ku70, the protein Ku70 and BCR-ABL in cells of CML. The NHEJ efficiency to repair DSB in CML was also investigated.
Methods: Bone marrow cells were collected from 24 cases of normal adults and 27 cases of de novo diagnosed CML patients. 15 CML patients were in chronic phase and 12 in blast phase. The expression of gene Ku70 was detected by RT-PCR and RQ-RT-PCR. The fusion gene BCR-ABL was detected by TaqMan probe Real Time PCR, using ABL as the internal control. Nucleic extracted proteins were used to determine NHEJ efficiency by an in vitro end-ligation system.
Results: The mean level of NHEJ activity of normal BM cells and CML cells were 18.6±13.1% vs 24.8±14.9%, .024. The gene Ku 70 expression in normal BM cells and CML cells were 31.08±8.41 vs 544.63±1185.71 copies/10,000 β-Actin copies, P=0.039. gene Ku 70 expressed significantly higher in blast phase (1103.31±1645.62 copies/10,000 β-Actin copies, P&lt;0.01). There were positive correlations of BCR-ABL when compared with the expression of the gene Ku70 (r=0.573 P=0.002) and with the protein Ku70 (r=0.705 P&lt;0.001) in CML cells. There was also significantly correlations between the expression of the gene Ku70 and the protein Ku70 (r=0. 808, P&lt;0.001).
Conclusions: The gene and protein of Ku70 were expressed significantly higher in CML than in normal BM cells with NHEJ activity was also enhanced in CML cells. Meanwhile, Ku70 expression level in acute phase was markedly higher than that in chronic phase. There were significantly correlations among the expression of fusion gene BCR-ABL, the gene Ku70 and the protein Ku70 in cells of CML This Study illustrates DNA instability in CML cells which was mainly damaged by DSB, and this kind of DNA damage was repaired by NHEJ pathway predominantly. NHEJ pathway plays an important role in disease progression of CML.
Interim Positron Emission Tomography (PET) in Diffuse Large B-Cell Lymphoma: Independent Expert Nuclear Medicine Evaluation of ECOG 3404
Authors: Horning Sandra J. , Juweid Malik E. , Schoder Heiko , Wiseman Gregory , McMillan Alex , Swinnen Lode J. , Advani Ranjana , Gascoyne Randy D. , Quon Andrew 
Published: April 2020

Abstract
Background: Positive interim PET scans have been associated with inferior outcomes in DLBCL treated with chemotherapy, alone or with rituximab. In the ECOG 3404 study for bulky and advanced DLBCL, PET scans at baseline and after 3 R-CHOP are centrally reviewed by a single reader; those with positive scans cross-over to R-ICE after 4 R-CHOP, whereas those with negative scans continue on R-CHOP. The primary endpoint of E3404 is progression-free survival. To determine the reproducibility of interim PET scan interpretation, we convened an expert panel.
Methods: Three external nuclear medicine physicians visually scored baseline and interim PET scans independently and blinded to other clinical information or outcome. ECOG study criteria were binary (0,1) based on residual disease in initially involved sites with uptake greater than the liver. London criteria were on a scale of 0–5, where 4–5 was positive, based on increased uptake relative to the liver. Overall scores and agreement among experts were evaluated for both criteria, with application of the kappa statistic to correct for chance.
Results: Using the ECOG criteria, external reviewers were in complete agreement in 68% of 38 interim scans and completely agreed with the central review in the same 68% cases. Agreement among the experts was 71% employing the London criteria in these cases. The range of PET+ interim scans by reviewer was 16.8% to 34.2% (p=NS) by both ECOG and London criteria. The kappa statistic for overall pairwise correlation between readers was 0.445 (0.396–0.533) using ECOG and 0.502 (0.396–0.630) using London criteria – indicating moderate consistency. Areas of disagreement often, but not exclusively, related to bone disease, the shape and focality of residual uptake, splenic disease, and rare scans without CT-fusion.
Conclusions: These data show that visual criteria, either ECOG or London in this series, for interim PET results are moderately reproducible among individual nuclear medicine experts. Our finding of variability among experts indicates the need for caution in interpreting interim PET results in studies and in practice. Review of all E3404 cases is planned after accrual is completed (projected 12/08). Other ongoing studies evaluating interim PET after 1–3 cycles of therapy, the application of quantitative criteria, and consensus panels may provide further valuable information.
DNA-Dependent Protein Kinase Is a Therapeutic Target in Poor Prognosis B-Cell Chronic Lymphocytic Leukemia
Authors: Willmore Elaine , Elliott Sarah L. , Crawford Clark J. , Summerfield Geoffrey , Mainou-Fowler Tryfonia , Jackson Graham , Evans Paul , Newton Paula , Gowans Duncan , Durkacz Barbara W. 
Published: April 2020

AbstractPoor prognosis B-cell chronic lymphocytic leukemia (CLL) is characterised by del(17p), del(11q) and unmutated IgVH genes. Mutational inactivation of p53 and ataxia telangiectasia-mutated kinase (ATM) are more frequent in these patients and confer drug-resistance. Over-expression of DNA-dependent protein kinase (DNA-PK), the enzyme that mediates DNA double strand break (DSB) repair via non homologous end joining (NHEJ), also correlates with chemo-resistance. Thus, alterations in DNA damage signalling pathways are associated with poor risk CLL. We have shown that DNA-PK is a new therapeutic target in CLL1, and are evaluating the efficacy of novel small molecule inhibitors of DNA-PK in ex vivo studies using leukemic lymphocytes from a well-characterised cohort of CLL patients (n=85). We hypothesised that targeting DNA-PK would inhibit NHEJ and thus sensitise CLL cells to drug-induced DNA damage.NU7441 and KU-0060648 are potent small molecule inhibitors of DNA-PK, developed in collaboration with KuDOS Pharmaceuticals (Cambridge, UK). Lymphocytes were treated with fludarabine, chlorambucil, and Topoisomerase II poisons (mitoxantrone, etoposide, doxorubicin) in the presence or absence of NU7441 (1 μM) or KU-0060648 (0.2 mM). There was a concentration-dependent decrease in viability in response to single agent treatment (XTT/apoptosis assays) that was potentiated in the presence of a DNA-PK inhibitor. For example, 14/18 cases tested with mitoxantrone (currently in clinical trials) were sensitised by NU7441. Measurement of γH2AX foci formation (a surrogate marker for DSB) after Mitoxantrone treatment showed foci formation within 3 hr (n=4), which was maximally potentiated at 24hr following co-incubation with NU7441, implicating DNA-PK as a mediator of DSB repair following drug treatment. Stratification by karyotypic status demonstrated striking results. Although del(17p) cases were more resistant to mitoxantrone (mean LC50 1.2 mM ± 0.2) compared to del(13q) cases (mean LC50 0.4 mM ± 0.03), they had the greatest sensitization (7–13 fold) to Mitoxantrone by NU7441 (p=0.0006), indicating the particular effectiveness of this combination in del(17p) cases. Consistent with this observation, DNA-PK expression (Western blot and activity assays) was highest in del(17p) cases, confirming the utility of this novel drug combination. Whereas Topoisomerase IIα expression was negligible (Western blotting), Topoisomerase IIβ expression varied 3-fold. RT PCR analyses are underway to further study expression of DNA-PK and Topoisomerase II in this cohort. Taken together, these data show that use of a DNA-PK inhibitor increases the therapeutic index of drugs currently used to treat CLL and identify a targeted and novel approach for poor prognosis disease.
The Role of Heme Oxygenase 1 in Erythroid Differentiation.
Authors: Santos Daniel Garcia dos , Schranzhofer Matthias , Chies Jose Artur Bogo , Ponka Prem 
Published: April 2020

Abstract
Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. However, if left unguarded, non-protein-bound heme promotes free radical formation, resulting in cell damage and tissue injury. The highest amounts of organismal heme (75–80%) are present in circulating red blood cells (RBC) whose precursors synthesize heme with rates that are at least 1 order of magnitude higher than those in the liver (on the per cell basis), which is the second most active heme producer in the body. The only physiological mechanism of heme degradation is performed by heme oxygenases (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme and are, therefore, involved in the control of cellular heme levels. Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO-1 plays any role in erythroid cell development under physiological or pathophysiological conditions. In this study we have shown that HO1 protein is expressed in uninduced murine erythroleukemic (MEL) cells and that its levels, somewhat surprisingly, do not decrease during DMSO-induced erythroid differentiation. Moreover, we demonstrated that heme significantly induces HO1 in both uninduced and induced MEL cells. Additionally, we investigated the effect of overexpressed HO1 on heme and iron metabolism in stably transfected MEL cells (MEL-HO1) and their non-transfected counterparts. Compared to wild type cells, DMSO-treated MEL-HO1 cells displayed a reduction in heme stability (measured by the incorporation of 59Fe into heme) in addition to impairment of erythroid differentiation. Moreover, although wild type and transfected cells expressed similar levels of transferrin receptors in the uninduced state, MEL-HO1 cells, as compared to wild type MEL cells, showed only a small increase in transferrin receptors upon treatment with DMSO. Finally, we measured apoptosis using annexin-V and observed an increase in the number of apoptotic cells in HO1 transfectants, but not in wild type MEL cells. These results suggest that an as yet unknown mechanism exists to protect heme against endogenous HO1 action during physiological erythroid differentiation. In addition, our results showing that high levels of HO1 in erythroid cells cause heme catabolism and a defect in erythroid differentiation raise the possibility that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias.