bluuebunny/crossref_metadata_2025 huggingface dataset:
The Parthenolide Derivative LC-1 Is An Effective Single Agent and Is Highly Synergistic with Existing Therapies in Multiple Myeloma.
Authors: Walsby Elisabeth J , Hewamana Saman , Burnett Alan , Knapper Steven , Fegan Chris , Pepper Chris
Published: April 2020

Multiple myeloma (MM) remains incurable with conventional therapeutic agents and has a median survival of only 3–5 years. Therefore, there is clearly a need for novel treatment strategies that can change the natural pathology of this condition. The nuclear factor κB (NF-κB) family of transcription factors is constitutively activated in MM cell lines and the majority of MM patients. Since NF-κB has known oncogenic activity in a number of human malignancies, targeted inhibition of this family of proteins may be useful in the treatment of MM. We and others have recently shown that the parthenolide derivative LC-1 has activity in acute myeloid leukaemia (AML) and chronic lymphocytic leukaemia (CLL) cells. Unusually, it induces apoptosis via the activation of both the intrinsic and extrinsic pathways and apoptosis is preceded by marked inhibition of NF-κB. Importantly, LC-1 is more potent against primary AML blasts and CLL lymphocytes than normal bone marrow progenitors and normal B-cells and T-cells. In this study we set out to evaluate LC-1 in MM cell lines and plasma cells derived from MM patients. LC-1 was cytotoxic to MM cell lines H929, U266 and JJN3 and induced apoptosis in a dosedependent manner resulting in an overall LD50 of 3.6mM (±1.8) after 48 hours in culture. Primary myeloma plasma cells, identified by CD38 and CD138 positivity, had a mean LD50 for LC-1 of 5.4mM (±1.6) after 48 hours of in vitro culture. Normal bone marrow cells were significantly less sensitive to the effects of LC-1 under the same conditions (P = 0.0007). Treatment of MM cell lines with LC-1 resulted in a decrease in the nuclear localization of NF-κB, as evidenced by a dose-dependent decrease in the DNA binding capacity of the NF-κB subunit RelA after 4 hours of treatment. To demonstrate whether synergy exists between LC-1 and existing MM therapies, the H929 cell line was treated for 48 hours with LC-1 and doxorubicin (32:1), melphalan (1:1) or bortezimib (1:500) and the combination indices (CI) calculated using the median effect method. A combination index of less than 1 denotes synergy. LC-1 did not show synergy with doxorubicin (CI >1) but was synergistic with melphalan and bortezimib (CI values of 0.53 and 0.59 respectively). Taken together our data clearly demonstrate that LC-1 has activity in MM cell lines and primary MM cells. Its ability to inhibit the nuclear localization of NF-κB is important to its cytotoxic effects. Furthermore, it may also provide an explanation for the synergy demonstrated with melphalan and bortezimib. These results provide a rationale for exploring the potential of LC-1 in clinical studies.
Therapeutic Antimyeloma Effect of Dendritic-Tumor Cells Hybrids Transduced with Adenovirus Encoding CD40L.
Authors: Alvarez Eva , Moga Esther , Sierra Jorge , Briones Javier
Published: April 2020

Dendritic cells (DCs) are the main antigen presenting cells and play a pivotal role in the stimulation of T-cell immune responses. DCs cultured in the presence of a single tumor antigen can elicit an immune response against tumor cells expressing that antigen. However, simultaneous use of several tumor antigens may be advantageous since polyclonal activation of T cells against different tumor antigens may be a better approach to eradicate tumor cells. In this sense, fusions of dendritic and tumor cells (FCs) show a broad spectrum of tumor antigens, both known and unidentified, to be presented by class I and II MHC. Although prophylactic vaccines were successful in murine models, the results in the therapeutic setting have been unsatisfactory. We hypothesised that enhancing costimulation of FCs would help to break tumor tolerance once the tumor is established. To this purpose, we transduced FCs with a recombinant adenovirus encoding CD40L (AdvCD40L or AdvGFP as control) and we studied the therapeutic antitumoral effect of the administration of FC-CD40L in a murine model of myeloma. DCs obtained from day 7-bone marrow cultures of Balb/c mice were fused with tumor cells, a syngeneic murine myeloma cell line (4TOO). FCs hybrids were generated with PEG and selected after culturing in HAT medium plus GM-CSF for 7 days. FC were quantified by determining the percentage of cells that coexpress specific DC (CD11c) and tumor markers (CD138). Mean fusion efficiency was 30% (20–40%) and FCs expressed moderate levels of CD80, CD83, CD86, CD54, CD40 and MHC II and did not express CD40L. FC-CD40L showed a significant increase of expression of costimulatory molecules (CD80, CD86, CD54, and MHC II) compared to FC-GFP (p=0.011). Moreover, in a syngeneic mixed lymphocyte reaction, FC-CD40L induced a two-fold higher T-cell proliferation than FC-GFP or FC alone. In addition, FC-CD40L had improved migration to lymphoid tissues, preferentially to spleen, in comparison with FC-GFP (2.8% versus 1.6%). The antitumor effect of FC-CD40L was analyzed in vivo. Mice (n=10 per group) were injected i.v. with 2.5×105 tumor cells and treated with irradiated FC, FC-GFP or FC-CD40L (1×106 cells each) on days 2, 6 and 10 after tumor challenge. 40% of mice treated with FC-CD40L had long-term survival (>120 days). In contrast, all of mice treated with FC or FC-GFP died between days 25 and 35 (p=0.012). In parallel, treatment with mixed cells (not fused DC+ tumor cells), mix transduced with AdvGFP, or mix transduced with AdvCD40L did not provide any significant antitumor effect. We conclude that FCs transduced with AdvCD40L better stimulate in vitro and in vivo immune responses than FC alone and may provide a new strategy for treating patients with multiple myeloma or lymphoma.
Short-Term Culture of Umbilical Cord Blood-Derived Stem Cells Enhances the Engraftment into NOD/SCID Mice through the Increased Expression of CXCR4.
Authors: Ohno Norioki , Kajiume Teruyuki , Sera Yoshihiko , Sato Takashi , Kobayashi Masao
Published: April 2020

Human umbilical cord blood (CB) is rich source of hematopoietic stem cells (HSCs) and provides an attractive alternative to bone marrow or mobilized peripheral blood transplantation. However, major disadvantage of CB transplantation is the relatively low number of HSCs in each CB unit, which severely limits its usefulness in clinical transplantation. A part of CD34+ cells expresses cell surface CXCR4, which has been found to be critical for bone marrow engraftment by human hematopoietic stem cells. In the present study, we examined the effect of short-term culture on CXCR4 expression and engraftment in CB-derived CD34+ cells transplanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. CB-derived CD34+-enriched samples were divided into three aliquots and cultured. The first aliquot was incubated for 2 h in RPMI-1640 medium alone, whereas the second was incubated for 2h in RPMI-1640 medium containing the specific CXCR4 antagonist, AMD3100. As a control, the third sample was neither treated nor incubated. Surface CXCR4 expression on CD34+ cells significantly increased after incubating the cells in medium alone for 2 h (7.7 to 19.8%); this increase was blocked by the addition of AMD3100. No difference in CXCR4 mRNA expression was noted after incubating CD34+ cells for 2 h. The short-term incubation seems to influence the translocation of CXCR4 from the interior of the cell to the cell surface. CD34+ cells cultured for 2 h showed significantly increased transmigrational activity toward SDF-1 and homing activity in NOD/SCID mice. However, those were lower in non-cultured cells and cells treated with AMD3100. Next, we assessed the engraftment of cells cultured into NOD/SCID mice. Engraftment analysis was performed 8 weeks after transplantation. The presence of human cells was detected by the cell surface expression of CD45, CD34, CD33, and CD19. The CD34+ cells cultured demonstrated significantly increased engraftment of human cells in the bone marrow of NOD/SCID mice compared with that of CD34+ cells without culture or with culture in the presence of AMD3100 (p < 0.01). The recipients exhibited CD19+ B-lymphoid cells, CD33+ myeloid cells, and CD34+ immature cells, but neither CD3+ T cells nor CD45+ cells in bone marrow. Finally, we performed a secondary transplant of engrafted cells from previously transplanted mice to confirm long-term engraftment and self-renewal of cultured CB cells. The successful engraftment was observed in mice injected with primary cultured cells, demonstrating that these cultured cells are self-renewing and capable of long-term engraftment. These observations suggest that the increase of the cell surface expression of CXCR4 on CD34 cells resulted in the retention of human umbilical cord blood cells within the bone marrow through the engraftment of homing activity.
Absolute Lymphocyte Count at the Time of Relapse Predicts Survival in Patients with Diffuse Large B-Cell Lymphoma.
Authors: Porrata Luis F. , Ristow Kay , Haberman Thomas M , Witzig Thomas E , Inwards David James , Ansell Stephen M , Johnston Patrick B , Micallef Ivana N. , Colgan Joseph P , White William L , Markovic Svetomir N
Published: April 2020

The International Prognostic Index at the time of relapse (IPI-R) has been reported to predict clinical outcome in patients with relapsed diffuse large B-cell lymphoma (DLBCL) undergoing autologous stem cell transplantation (ASCT) [Jabbour et al, Leukemia & Lymphoma2005;46(6):861–867; Lerner et al, Biology of Blood and Marrow Transplant2006;13:486–492; and Costa et al, Bone Marrow Transplant2008;41:715–720]. The absolute lymphocyte count (ALC) has been reported as a prognostic factor for survival at diagnosis, during standard chemotherapy and at day 15 post-ASCT for multiple hematological malignancies; however, no reports have addressed whether ALC at the time of relapse (ALC-R) predicts survival. Thus, we assessed the prognostic significance of ALC-R in patients with relapsed DLBCL. To be included in the study, patients were required to have been diagnosed with relapsed DLBCL, have ALC values available from the time of relapse, and to be followed at Mayo Clinic, Rochester. From Feb 2,1987 until March 6, 2006, 121 DLBCL patients qualified for the study. The overall survival (OS) was measured from the time of relapse to the date of death or last follow-up and progression-free survival (PFS) was defined as the time from relapse to the time of progression, relapse, death, or last follow-up. The median age at relapse was 68 years (range:25–88 years) and the median ALC-R was 1.21 x 109/L (range: 0.33–5.99 x 109/L). The value of ALC-R ≥ 1.0 x 109/L was used for the analysis based on previous publications. The groups (ALC-R ≥ 1 or < 1 x 109/L) were balanced for the IPI-R (p=0.1), as well as for undergoing ASCT (p=0.4). Superior OS was observed in patients with an ALC-R ≥ 1.0 x 109/L (N = 76) [median OS: 27.5 months, 3 years OS rates of 42%] compared with patient with an ALC-R < 1.0 x 109/L (N =45) [median OS: 9.4 months, 3 years OS rates of 13%] (p <0.0001). Superior PFS was also observed in patients with an ALC-R ≥ 1.0 x 109/L (N = 76) [median PFS: 14.8 months, 3 years PFS rates of 30%] compared with patient with an ALC-R < 1.0 x 109/L (N =45) [median PFS: 6.2 months, 3 years PFS rates of 11%] (p <0.001). ALC-R was an independent prognostic factor for OS [RR = 0.551, p < 0.009] and PFS [RR = 0.674, p < 0.02] in the multivariate analysis when compared with the IPI-R and whether patients underwent ASCT or not. Our study supports the hypothesis that ALC at relapse predicts clinical outcome in DLBCL and suggests that patient host immunity is an important variable predicting survival in relapsed DLBCL.
Chromosomal Instability Causes Genetic and Clonal Heterogeneity in Myeloproliferative Neoplasms and Is Not Restricted to JAK2-V617F Positive Cells
Authors: Jäger Roland , Olcaydu Damla , Berg Tiina , Gisslinger Bettina , Gisslinger Heinz , Kralovics Robert
Published: April 2020

A number of oncogenic mutations have been identified in myeloproliferative neoplasms (MPN) in the past few years. Among these JAK2-V617F is most frequent, followed by mutations of the thrombopoietin receptor MPL and JAK2 exon 12. In addition, cytogenetic lesions occur frequently in MPN detected either at diagnosis or later in the course of the disease. To explore the genetic aberrations present in MPN patients, we performed microarray genotyping using Affymetrix SNP 6.0 arrays in a series of 71 MPN patients with variable presence of JAK2 and MPL mutations. More than half of the analyzed patients exhibited loss of heterozygosity (LOH) in at least one chromosomal region. Complex karyotypes with two and more regions with LOH were detected in 18 patients. Uniparental disomy (UPD) on chromosomes 9p, 1p, 11q, 14q and 17q represented the largest proportion of LOH detected followed by deletions on chromosome 13q, 20q, and 12p. All patients with UPD on chromosome 1p were homozygous for the MPL-W515L mutation. We observed frequent aberrations of chromosome 7 including monosomy, deletions on 7p and 7q, and UPD of 7q. Using microsatellite PCR, we validated the microarray findings and further determined the frequency of these aberrations in a total of 367 MPN patients. Multiple occurrences of individual chromosomal lesions allowed us to define the minimal genomic regions involved in deletions or UPDs. The sizes of the common deleted regions (CDRs) were variable ranging from 9 mega base pairs (Mb) to 0.5 Mb. The CDR on chromosome 7p included only the IKZF1 and FIGNL1 genes previously shown to associate with leukemic transformation. To determine the clonal composition of the hematopoietic progenitor pool of patients with complex karyotypes we genotyped individual BFU-E and CFU-GM colonies in a series of 27 patients. We observed a remarkable clonal heterogeneity at the progenitor cell level. Using four clonal markers we defined 9 different types of clonal structures. In a set of patients, JAK2-V617F or MPL-W515L mutations occurred before the acquisition of chromosomal deletions. Other patients acquired deletions before the acquisition of JAK2-V617F. In summary, our results show that somatic mutations in MPN are not acquired in a predetermined order as seen in other malignancies, but occur randomly. The chromosomal instability in MPN is not caused by JAK2-V617F exclusively, since many patients show aberrations outside of the JAK2-V617F positive clone. Heterogeneity of somatic mutations in MPN leads to high clonal variability within the progenitor pool potentially affecting therapeutic outcome. Thus, targeting JAK2-V617F alone may not lead to restoration of polyclonal hematopoiesis. An individualized therapeutic approach and/or combination therapy might be necessary to achieve clonal remission in MPN.
Specific Inhibition of Syk Is Sufficient to Disrupt Proliferation and Survival of Non-Hodgkin’s Lymphoma Cell Lines without Concomitant Inhibition of JAK.
Authors: Coffey Greg , Luan Peng , Delaney Suzanne , Pandey Anjali , Jia Zhaozhong , Xu Qing , Bauer Shawn , Song Yonghong , Conley Pamela B. , Phillips David , Sinha Uma
Published: April 2020

Recent studies indicate that dual inhibition of the spleen tyrosine kinase (Syk) and Janus kinases (JAK) by agents such as R788 (tamatinib fosdium) are clinically efficacious in patients with non-Hodgkin’s lymphoma. Since Syk has been implicated in B cell activation, proliferation, and survival, the present study was designed to determine whether the specific inhibition of Syk alone would suffice to disrupt proliferation and survival of lymphoma cells. To test this, a series of compounds were synthesized and screened to identify those that specifically inhibited Syk in a panel of in vitro multi-kinase assays at 300nM. Of these, three were selected for this study; compounds P459-72, P505-15, and P420-89. All three compounds inhibited purified Syk with IC50’s in the 6-43nM range. P459-72 was highly Syk specific. P505-15 also inhibited purified Lyn with an IC50 of 199nM. P420-89 inhibited multiple kinases in the BCR signaling pathway, in addition to JAK1, 2, and 3; IC50’s of 0.63-6.2nM. Assays using the non-Hodgkin’s lymphoma B cell lines Ramos, SUDHL-4 and -6 showed that each compound also inhibited BCR-induced Syk auto-phosphorylation and BLNK phosphorylation (IC50’s in the 100–400nM range) as well as subsequent Ca2+ flux and ERK phosphorylation (IC50’s in the 50–156nM range). In contrast, the activity of the Src family member Lyn, upstream of Syk in the BCR signaling pathway and responsible for Syk tyrosine phosphorylation at amino acid position 352, was not affected. Cellular proliferation was also attenuated in these lymphomas (IC50’s in the 1–5μM range), and all three compounds induced apoptosis to various extents between 1 and 3μM. Interestingly, while P505-15 did inhibit purified Lyn with an IC50 of 199nM, this did not translate into inhibition of Lyn activity upon BCR cross-linking in cells at concentrations where Syk activity was inhibited. A structurally similar compound with a greater than one hundred fold lower Syk inhibitory activity (P528-85) had no effect on the proliferation and survival of these B cell lines. Finally, the cellular effects of the Syk inhibitors required an active BCR signaling pathway, as the B cell line Toledo which lacks BCR expression was insensitive to these compounds; proliferation was inhibited with IC50’s in the 9-38μM range with no induction of apoptosis below 10μM, the highest concentration tested. Taken together, these data suggest that the specific inhibition of Syk, without concomitant inhibition of JAK, may be sufficient for the treatment of non-Hodgkin’s lymphoma.
A Molecular Network Comprising MicroRNA-223, E2F1 and C/Ebpα in Granulopoiesis and in Acute Myeloid Leukemia.
Authors: Pulikkan John Anto , Dengler Viola , Peerzada Abdul , Bohlander Stefan , Tenen Daniel G. , Behre Gerhard
Published: April 2020

MicroRNAs play crucial roles in gene expression programmes and have been demonstrated to have major influence in various biological processes. Recent findings suggest aberrant regulation of microRNAs is a hall mark of many cancers including leukemia. MicroRNA-223 (miR-223) is regulated by the transcription factor CCAAT enhancer binding protein α (C/EBPα) and is upregulated during granulopoiesis. miR-223 mutant mice display defects in granulopoiesis pointing out the importance of miR-223 during granulopoiesis. Recent studies suggest that loss of function or expression of C/ EBPα is a major step in the development of acute myeloid leukemia (AML). Using an inducible cell line model, we show that C/EBPα upregulates microRNA-223 expression during granulopoiesis. Based on these findings, we hypothesized that miR-223 could be downregulated in human AML. Here we report that miR-223 is downregulated in different subtypes of AML as analysed by quantitative Real-Time RT-PCR. We investigated what are the critical targets of miR-223 during granulopoiesis. Computational analysis suggests that E2F1, the transcription factor that promotes cell cycle progression which is inhibited by C/EBPα during granulopoiesis, could be a putative target of miR-223. By luciferase assay using 3’UTR of E2F1, we show that E2F1 is a potential target of miR-223. miR-223 downregulates E2F1 by translational repression as revealed by reduction in E2F1 protein level. Silencing of miR-223 leads to upregulation of E2F1 protein level as analyzed by Western blot analysis. Proliferation assays as well as cell cycle analysis demonstrate that miR-223 blocks cell cycle progression in myeloid cells. Interestingly, sequence analysis of miR-223 promoter revealed putative E2F1 binding sites. We demonstrate that E2F1 inhibits the microRNA-223 promoter activity through its transactivation domain as shown by promoter assay. Furthermore, overexpression of E2F1 down regulates the expression of miR-223, suggesting E2F1 acting as a transcriptional repressor of the miR-223 gene. Meanwhile, C/EBPα transactivates miR-223 promoter activity. We also report that E2F1 is able to block granulocytic differentiation. Recent studies demonstrate that disruption of E2F1 inhibition by C/EBPα leads to leukemia, pointing out the significance of E2F1 inhibition in the development of AML. Our data support a circuitry comprising miR-223, C/EBPα and E2F1 as major components of the granulocyte differentiation programme, which is deregulated in AML. Manipulation of miR-223 could be therapeutically relevant in AML subtypes in which E2F1 inhibition is deregulated.
Cytoskeletons of X-Linked GATA-1, G208S Macrothrombocytes Are Deficient in Talin.
Authors: White James G. , Burris Steven M. , Thomas Angela
Published: April 2020

Germline mutations in the X-linked transcription factor, GATA-1, cause several hematopoietic disorders resulting in anemia and/or macrothrombocytopenia. Features of the platelet ultrastructural pathology, including hypo- and agranular megathrombocytes (Mtc) in all mutations, tubular membrane sheets often in parallel association in megakaryocytes and Mtc of patients with the V205M and G208S variations, platelets sequestered in Mtc and platelets in platelets in Mtc (as many as five in one Mtc) in the V205M, G208S and R216Q disorders, together with platelets attached to platelets attached to platelets forming large Mtc in the V205M and G208S mutations have helped to explain how Mtc are formed and why they fail to separate from proplatelets of megakaryocytes and enter the circulation as single cells. However, the findings do not explain why patients with GATA-1 mutations often have serious bleeding problems. Major receptors for hemostasis and thrombosis are present on GATA-1 Mtc, although, in some cases, slightly reduced in frequency. Yet, while the cells bind to and spread fully on glass and plastic, they adhere poorly to vWF or collagen coated glass slides under flow conditions, aggregate less well than normal platelets when stirred with collagen or ristocetin in an aggregometer and fail to retract clots. The findings suggest that internal activation mechanisms for the surface receptors are defective. The present study has solubilized GATA-1 Mtc from two males with the G208S mutation and normal platelets in sodium dodecyl sulfate/ EDTA in the presence of protease inhibitors and analyzed their cytoskeletal proteins on reduced, one dimensional polyacrylamide gels using a Mini-Slab Gel Apparatus. Gels were electrophoresed by the method of Laemmli. Proteins were stained with colloidal Coomassie G250 blue. GATA-1 Mtc cytoskeletal proteins were the same as those from the normal subjects, except for the absence of Talin. Western blot analysis to prove the missing protein is Talin was carried out. Normal platelet and GATA-1 Mtc cytoskeletal proteins were transferred to nitrocellulose sheets. Identification and localization of the missing protein was accomplished using a mouse monoclonal antibody against human Talin and a secondary anti-mouse IgG peroxidase conjugate. The Western blot analysis confirmed that the missing Mtc protein is Talin. Recent studies have shown that binding of the 235–245 kD actin-binding protein Talin to the β subunit cytoplasmic tails of integrins is the final step of integrin activation. The absence of Talin in GATA-1 Mtc may result in failure of full integrin activation accounting for functional failure in hemostasis.
A Novel Histidine Tyrosine Phosphatase, TULA-2, Associates with Syk and Negatively Regulates GPVI Signaling in Platelets.
Authors: Thomas Dafydd H , Dangelmaier Carol , Jin Jianguo , Tsygankov Alexander , Kunapuli Satya P. , Daniel James
Published: April 2020

Glycoprotein VI (GPVI) is the primary platelet receptor for collagen signaling. Following damage to the vascular endothelium, the GPVI receptor interacts with the exposed sub-endothelial collagen. This interaction initiates a signaling cascade involving phosphorylation of the dual ITAM motif of the FcRγ chain by Fyn and Lyn, followed by the recruitment, phosphorylation and activation of Syk. This leads to the eventual activation of PLCγ2 and the release of calcium from intracellular stores to cause platelet activation. While a lot is known about the activation processes involved in GPVI signaling less is known about its negative regulation. The T-cell ubiquitin ligand (TULA) family of proteins has been implicated in the negative regulation protein tyrosine kinase (PTK)-dependent signaling pathways. More recently, it has been shown the TULA family member, TULA-2, exhibits phosphatase activity towards PTKs, including Syk, and this activity is responsible for the negative regulation of T-cell receptor signaling (Mikhailik et. al. 2007, Agrawal et. al. 2008). Thus, we investigated the role of TULA-2 in the negative regulation of the GPVI signaling cascade. We show that TULA-2 is expressed in both human and murine platelets. Deletion of TULA-2 in murine platelets manifests itself functionally as enhanced aggregation in response to the GPVI agonist convulxin as well as enhanced dense granule secretion when compared to wild type platelets. No difference was witnessed in response to the PAR4 agonist AYPGKF. TULA-2-deficient platelets also exhibit sustained hyperphosphorylation of Syk at tyrosines 525 and 526 as well as hyperphosphorylation of PLCγ2 at tyrosines 753 and 759, indicative of enhanced kinase and phospholipase activity respectively. GST-pulldown experiments suggest that Syk and TULA- 2 are able to associate in resting and convulxin stimulated platelets and in-vitro phosphatase assays demonstrate that TULA-2 can dephosphorylate Syk at tyrosines 525 and 526. Taken together, these data suggest that TULA-2 is a negative regulator of GPVI signaling and this regulation is mediated by an association of TULA-2 with Syk, allowing the dephosphorylation of Syk at catalytically important tyrosine residues.
Functional Genomics of the Platelet ADP and Collagen Signalling Pathways.
Authors: Garner Stephen F , Jones Chris I , Stephens Jonathan , Bray Sarah L , Angenent Will , Burns Philippa , Farndale Richard W , Rice Catherine , Dudbridge Frank , Watkins Nick , de Bono Bernard , Deloukas Panos , Goodall Alison H , Ouwehand Willem
Published: April 2020

The extent of platelet response to agonists varies between individuals and is to a large extent genetically controlled. Here we present the results of a systems biology study designed to define the association between sequence variation and platelet functional phenotype. The exons and flanking intronic regions of 98 genes, with known function in platelets, were sequenced in 48 Caucasoid CEPH DNA reference samples, identifying 1949 SNPs, approximately half of which were novel. From this enriched data set 1340 SNPs, comprising tagging and non-synonymous validated SNPs, were mounted on an Illumina Golden Gate array and typed in 500 DNA samples from a cohort of healthy subjects whose platelet functional phenotype was measured by flow cytometry, using fibrinogen binding and P-selectin expression in response to pathway-specific ADP (10−7M), or CRP-XL (0.1μg/ mL) stimulation. Thirty-six SNPs in 16 genes showed an association (p<0.005) with one or more platelet functional trait. To explore the mechanism by which sequence variation alters the platelet response, further tests were performed on platelets from individuals recalled on the basis of genotype for the SNPs with the lowest p-value for association in two of the genes, ITPR1 and VAV3. A SNP within the 3’UTR located in a putative transcription factor binding site of the VAV3 gene, for which the minor allele was associated with increased P-selectin expression following ADP stimulation (p=0.004), was associated with a higher VAV3 protein expression in subjects with the minor allele (p=0.03). Conversely, the minor allele of the lead SNP in ITPR1, associated with increased fibrinogen binding and P-selectin expression in response to ADP but not CRP-XL (p=0.003, >0.2, respectively), did not show significant effect on the level of ITPR1 protein, but there was increased mobilisation of [Ca2+]i in response to ADP (but not CRP-XL) with the minor allele (p=0.004). By applying systems biology to platelets we have identified SNPs in 16 genes which together account for up to 60% of the genetic variation underlying platelet response to ADP and CRP-XL. These data provide novel insights into key “hubs” in well-studied platelet signalling networks where sequence variation modifies the response phenotype.
Increased Nucleosomal DNA Fragmentation in Leukocytes of Thalassemia Patients.
Authors: Walter Patrick B , Porter John B , Evans Patricia , Kwiatkowski Janet , Neufeld Ellis J. , Coates Thomas D. , Giardina Patricia , Grady Robert jW , Vichinsky Elliott , Olivieri Nancy F. , Trachtenberg Felicia , Alberti Daniele , Fung Ellen , Ames Bruce , Lui Annie , Harmatz Paul
Published: April 2020

Iron overload has been shown to induce DNA damage and may be implicated in leukocyte apoptosis. Previously we have shown that indirect markers of apoptosis such as caspase activity and Bax levels were higher in leukocytes of thalassemia patients. We have now assessed whether nucleosomal DNA fragmentation, a hallmark of apoptosis, is higher in leukocytes of iron-overloaded thalassemia patients compared with control subjects.

Methods: Thalassemia Clinical Research Network patients participating in the Novartis CICL670A0107 trial (a randomized comparison of deferasirox, an oral iron chelator, vs. DFO) were eligible and 44 (25 male, mean age: 21.8 ± 11.1 yrs) were enrolled in the study. Fasting blood samples were obtained

after a 5-day washout of DFO prior to commencing treatment with study drug, and 24 hours post-chelator at 1, 6, and 12 months on study.

Thirty healthy controls matched for age, sex, race and antioxidant usage (15 male, mean age 24.5 ± 9 yrs) also supplied a blood sample. Plasma, serum and cells were separated by centrifugation. The classic marker for apoptosis, nucleosomal DNA fragmentation was determined by a high throughput ELISA assay that used monoclonal antibodies directed against single- and double-stranded DNA bound to histones (H1, H2A, H2B, H3, and H4) and thus specifically detected mono- and oligonucleosomes (measured in ng/μg leukocyte protein). Detection of free mono- or oligonucleosomes is a direct measurement of apoptotic DNA fragmentation. DNA fragmentation results were log-transformed prior to analysis and means are reported.

Results: At baseline, thalassemia patients had elevated nucelosomes compared to the controls (351.5 vs. 74.9 ng/μg leukocyte protein, p = 0.001). This high level of leukocyte DNA fragmentation was unchanged throughout the study. Furthermore, nucleosomal DNA fragmentation was positively correlated with CRP (r = 0.3, p = 0.01), WBC (r = 0.2, p = 0.05) and ANC (r = 0.2, p = 0.04), suggesting apoptosis is present in increased inflammatory states.

Conclusions: This finding demonstrates that nucleosomal DNA fragmentation is higher in thalassemia compared to control patients and thus provides further evidence that there is increased apoptosis in circulating leukocytes of thalassemia patients.
Is It Possible to Stop Imatinib in Patients with Chronic Myeloid Leukemia? An Update from a French Pilot Study and First Results from the Multicentre « Stop Imatinib » (STIM) Study
Authors: Mahon Francois-Xavier , Huguet Francoise , Guilhot Francois , Legros Laurence , Nicolini Franck E , Charbonnier Aude , Guerci Agnes , Rea Delphine , Varet Bruno R. , Gardembas Martine , Guilhot Joelle , Etienne Gabriel , Milpied Noel-Jean , Aton Emilie , Reiffers Josy , Rousselot Philippe
Published: April 2020

Imatinib (IM) has greatly improved survival rates in chronic myeloid leukemia (CML). However, all patients (pts) must continue treatment for an unknown period of time. A pilot study of the first pts who discontinued IM therapy was previously reported (Rousselot et al. Blood. Blood2007;109:58–60). The new, multicentre « Stop Imatinib » (STIM) study was started in July 2007. The aim of this study is to evaluate in a larger cohort the persistence of complete molecular remission (CMR) after stopping IM, and to determine the factors that could influence the persistence of CMR. The criteria for inclusion were IM treatment for at least 3 years and sustained CMR. Sustained CMR was defined as BCR-ABL/ABL levels below a detection threshold corresponding to a 5-log reduction (undetectable signal using RQ-PCR) for at least 2 years. Molecular relapse, defined as RQ-PCR positivity, was taken into account if confirmed in two successive assessments. In cases of molecular relapse, pts were re-treated with IM at 400 mg dailyIn the pilot study, 7 out of 15 pts relapsed within 6 months, but CMR was re-attained in all cases after IM was re-started. The other 8 pts (4 male, 4 female) are still in CMR, with a median follow up of 37 months (range 26–49 months) after IM discontinuation. All pts were pre-treated with interferon-alpha (IFN) and most responded to IFN before IM treatment. The STIM study included 50 pts from 18 centres (20 male, 30 female), with a median age of 62 years (range 32–81 years). Of these, 25 pts had received no pre-treatment with IFN. By July 2008, 34 pts had a follow up ≥ 6 months. Eighteen pts relapsed within the first 6 months: 3 pts in month 2 (M2), 8 pts in M3, 4 pts in M4, and 3 pts in M5. One patient relapsed after more than 6 months (M8). Among the 19 pts who relapsed, 11 were not IFN pre-treated and 8 were IFN pre-treated (relapse rate 44% vs 32%). Ten IFN pre-treated pts with follow up ≥ 6 months have not relapsed (M12 in 2 pts, M10 in 5 pts, M8 in 1 pt, M7 in 2 pts), and 5 pts with follow up ≥ 6 months who were not IFN pre-treated have not relapsed (M12 in 1 pt, M10 in 1 pt), M8 in 1 pt, M6 in 2 pts).

These studies confirm that CMR can be sustained after discontinuation of IM, particularly in pts pre-treated with IFN with a long follow-up (pilot study). Among pts in the STIM study who were not pre-treated with IFN, more than half have not relapsed, and 20% have reached a follow-up ≥ 6 months and not relapsed. Updated data will be presented but we conclude that it is possible to stop treatment in pts with sustained CMR, even in those treated with IM as a single agent.
Iron-Overload Induces Apoptosis in Cardiomyocytes and Hepatocytes Via Mitochondrial/Caspase-3 Pathways.
Authors: Yang Mo , Chan Shing , Cheung Yiu Fai , Ha Shau Yin , Chan Godfrey ChiFung
Published: April 2020

Cardiomyopathy and liver damage due to iron-overload are the major complications in patients with beta-thalassaemia major. Iron-overload may induce apoptosis in cardiomyocytes and hepatic cells, and that TPO may exert protective effect on apoptosis of cardiomyocytes (Circulation, 2006). In this study, we demonstrated firstly that iron induced apoptosis in cardiomyocytes. Using H9C2 cells, we have shown that iron reduced cell viability in a dose-dependent manner (0.003–3 mM) (n=6). By annexin V and PI staining, apoptotic cells were found to be significantly increased after iron treatment (0.3 mM, 72 hrs) (n=6). The expression of active caspase-3 was significantly increased in iron-treated cells. Furthermore, iron treatment increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential (n=6). Secondly, we found that TPO exerted cardio-protective effect on iron-induced apoptosis. H9C2 cells were cultured in the presence of iron (0.3 mM) with or without TPO (5, 10, 20, 50, 100 ng/mL, 72 hrs). The cell viability was significantly increased with the treatment of TPO at 50 ng/mL and 100 ng/mL (n=4). Dot-plot analysis of annexin V/PI staining demonstrated that TPO (50 ng/mL) significantly reduced the population of apoptotic cells (n=6). Incubation with TPO also decreased the iron-induced caspase-3 expression (n=6). Flow cytometric dot-plot analysis of H9C2 cells also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO group (n=6). The population of phospho-Akt and Erk1/2 were also significantly increased after treatment by TPO (P<0.05, n=4). Human liver cell line MIHA was also used as a cell model. We showed that iron-overload reduced cell viability in a dose-dependent manner (0.0375–0.6 mM) (n=7). By annexin V and PI staining, apoptotic cells were found to be significantly increased after iron treatment (0.15–0.6 mM) for 72 hrs (n=7). The expression of active caspase-3 was also significantly increased in iron-treated cells (n=5). We also found that TPO exerted proliferation effect on MIHA cell by activation of phospho-Akt. However, MIHA cells were cultured in the presence of iron (0.3 mM) with TPO (50 ng/mL, 72 hrs). The cell viability was not significantly increased with the treatment of TPO (n=5). Dot-plot analysis of annexin V/PI staining did not demonstrated that TPO reduced the population of apoptotic cells induced by iron-overload (n=5). Also, incubation with TPO did not decrease the iron-induced caspase-3 expression in these cells (n=5). Our findings suggest that iron-overload induces apoptosis in cardiomyocytes and hepatocytes via mitochondrial/caspase-3 pathways and that TPO might exert a protective effect on iron-overload induced apoptosis via the activation of Akt and Erk1/2 pathways in cardiomyocytes.
Growth Differentiation Factor 15 (GDF15) and Erythropoietin (EPO) Levels in Beta Talassemia Major Patients.
Authors: Salussoglia Ilaria , Volpe Gisella , Fracchia Silvia , Roggero Simona , Longo Filomena , Piga Antonio
Published: April 2020

Background: The serum level of GDF15 has been recently indicated as a possible marker of erythropoiesis (Tanno et al., Nature 2007) suggesting a role of its over-expression in contributing to iron overload in thalassemia syndromes by inhibiting hepcidin expression. The aim of present study has been to evaluate GDF15 serum levels in a homogeneous series of thalassemia patients and the relationship with transfusional parameters and iron status markers.

Methods: A group of consecutive patients with beta thalassemia major followed at our institution were included in the study. All patients were on regular transfusion and iron chelation treatment. Quantification of GDF15 on serum samples was performed with DuoSet ELISA for human GDF15 (R&D Systems) following the manufacturer’s protocol (Tanno et al., Nature 2007). Each patient had also a blood test for haemoglobin (Hb), serum iron, ferritin, transferrin, transferrin saturation and EPO levels. Liver Iron Concentration by SQUID and cardiac iron by MRI T2* have been assessed. The mean hemoglobin levels of the previous year (pre-transfusional, post-transfusional and mean) have been calculated for each individual. The presence of mild thalassemic mutations was used to classify mild or severe genotype. Clinical status has been assessed on the presence/absence of main complications (heart disease, liver disease, diabetes, hypothyroidism). Statistical analysis was performed using the software Statistica (StatSoft).

Results: One hundred-forty patients (73 male, 67 females) were studied. The mean age was 27.9 ± 9.0 years (range: 3.5–42). One hundred (71%) were splenectomised. Betathalassemia major patients had elevated GDF15 serum levels (mean 6892 ± 6894 pg/mL; range 720–52521) in comparison with healthy volunteers (273 ± 104 pg/mL; range 129–401). GDF 15 levels were strongly related to EPO levels (r=0,81; p<0,001). GDF15 levels were not related with age, gender, spleen, clinical status and iron markers. Patients with a severe genotype had higher GDF15 levels than mild genotype patients. GDF15 levels had a negative correlation with Hbs (p<0,05 for actual Hb and pre-transfusional Hb; p<0,001 for post-transfusional Hb and mean Hb). In thalassemia major patients with a severe genotype, GDF15 levels within thrice the normal range have been observed only in patients with pre-transfusional Hb above 9,6, post-transfusional Hb above 12,5 and a mean Hb above 11,3.

Conclusions: In beta thalassemia major patients on regular transfusion and iron chelation, serum GDF15 levels are high, inversely related to the haemoglobin levels maintained. Further studies of this marker may lead to a rethinking of the optimal transfusion therapy in these conditions.
Thrombin Inhibits NF-κb and RhoA Pathways in Cytokine-Stimulated Vascular Endothelial Cells When EPCR Is Occupied by Protein C.
Authors: Rezaie Alireza R. , Bae Jong-Sup
Published: April 2020

We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by protein C switches the PAR-1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells. To determine whether the occupancy of EPCR by protein C renders thrombin a protective enzyme, thus up-regulating the expression of signaling molecules in the antiinflammatory pathways, we investigated the effects of thrombin and thrombin receptor agonist peptides (TRAP) on TNF-a-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A by monitoring the expression of cell surface adhesion molecules (VCAM- 1, ICAM-1 and E-selectin), adhesion of neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factorkB (NF-kB) pathway. Analysis of the results indicates that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither thrombin nor TRAP influenced the proinflammatory effects of TNF-a in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin and TRAP, thus leading to inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNFa-stimulated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-kB pathways in response to TNF-a in cells pretreated with protein C-S195A. Based on these results we conclude that when EPCR is bound by its natural ligand protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses in vascular endothelial cells.
Endothelial Dysfunction and Paradoxical Resistance to Thrombosis in a Transgenic Mouse Model of Severe Hyperhomocysteinemia.
Authors: Dayal Sanjana , Jensen Melissa J , Faraci Frank M , Kruger Warren D , Lentz Steven R.
Published: April 2020

Hyperhomocysteinemia induced by low folate or high methionine diets produces endothelial dysfunction and accelerated thrombosis in mice, but it is not known if these vascular effects are influenced by homocysteine-independent effects of dietary perturbation. To determine the vascular phenotype of severe hyperhomocysteinemia in the absence of dietary intervention, we utilized a recently developed transgenic mouse model of cystathionine β-synthase (CBS) deficiency. In this model, the early lethality of homozygous Cbs-deficient mice (Cbs-/-) is rescued by a zinc-inducible human CBSI278T (hCBS-I278T) transgene under the control of a metallotheinein promoter. The I278T mutation is a common CBS mutation found in human patients with homocystinuria. We bred Cbs+/− male to Cbs+/− hCBS-I278T female mice. The mothers were given drinking water supplemented with 25 mmol/L zinc sulfate. Pups were weaned at 4–5 weeks of age and did not receive supplemental zinc after weaning. At 20 weeks of age, plasma total homocysteine was significantly higher in Cbs-/- hCBS-I278T mice (350±17 μmol/L) compared with Cbs+/+ hCBS-I278T (8.0±0.6 μmol/L) or wild-type Cbs+/+ (7.2±0.8 μmol/L) littermates (P<0.001). Maximal dilatation of pial arterioles to the endotheliumdependent dilator, acetylcholine, was decreased significantly in Cbs-/- CBS-I278T mice (10±2 %) compared with Cbs+/+ hCBS-I278T (25±2 %) mice (P<0.001). Dilatation responses to the endothelium-independent vasodilator, nitroprusside, were similar in both groups. Surprisingly, the time to stable occlusion after photochemical injury (rose bengal and green laser) of the carotid artery was significantly delayed in Cbs-/- hCBSI278T mice (71±10 minutes) compared with Cbs+/+ hCBS-I278T (30±10 minutes) or Cbs+/+ mice (33±8 minutes) (P<0.05). The tail bleeding time was comparable in all 3 groups. We conclude that severe hyperhomocysteinemia due to genetic deficiency of Cbs produces endothelial dysfunction but not accelerated thrombosis in the absence of dietary intervention in mice.
The Multikinase Inhibitor Sorafenib Inhibts Proliferation in B- and T-Lymphoblastic Cell Lines Via the PI3K/Akt Pathway.
Authors: Schult Catrin , Dahlhaus Meike , Boldt Sonja , Glass Änne , Ruck Sabine , Sawitzky Mandy , Lange Sandra , Amoroso Francesca , Freund Mathias , Neri Luca Maria , Junghanss Christian
Published: April 2020

Background: The phosphatidylinositol 3 Kinase (PI3K)/Akt signalling pathway is involved in the regulation of proliferation, apoptosis and angiogenesis. Dysregulation of PI3K/Akt has been described for several solid and hematological tumors. Its role in acute lymphoblastic leukemogenesis (ALL) is unkown. Here, we investigated whether the inhibition of PI3K/Akt kinases influences apoptosis, necrosis and cell proliferation.

Patients and Methods: ALL and high grade NHL cell lines with different cytogenetics and phenotypes were used (SEM, RS4;11, REH, Jurkat, DOGKIT). Phosphorylationstatus of Akt (Ser473, Thr308) and FOXO3A (Thr32) were determined by western blot (WB). Cells were incubated for 96h with LY29004 (5μM, 12.5μM, 25μM; PI3K inhibitor) or sorafenib (0.73μM, 7.3μM). In order to detect synergistic effects with other cytotoxic drugs cells were treated with combinations of sorafenib and doxorubicine, cytosin-arabinoside, and mTOR inhibitor (RAD001), respecitvely. Cell number, apoptosis, necrosis, protein phosphorylation and metabolic activity were determined at 4h, 24h, 48h, 72h, and 96h by microscopy, flow cytometry, WB and WST-1 testing. Whole genome Affymetrix gene expression arrays (U133 Plus 2.0) were performed in order to detect differential gene expression compared to controls treated with DMSO (dimethylsulfoxid) only.

Results: In all ALL cell lines phosphorylated Akt (pAKT) was detected. Levels of pAkt and pFOXO3A differed between cell lines significantly, with Jurkat and SEM demonstrating high activation levels. PI3K inhibition by LY29004 led to decreased proliferation in SEM cells with only slight increases in apoptosis and moderate increase in necrosis rates (20%). Sorafenib inhibited the proliferation of SEM, Jurkat and RS4;11 significantly, with most pronounced effects at 96h. Maximal apoptosis and necrosis rates increased and ranged from 11 to 39% and 53 to 84%, respectively. Metabolic activity decreased significantly already after 24h. As early as 0.5h after treatment complete disappearance (SEM, RS4;11) or marked decrease (Jurkat) in levels of pAkt and pFOXO occurred. Combination of sorafenib with conventional cytotoxic drugs failed to demonstrate synergistic effects. Sorafenib treatment induced differential gene expression in several genes e.g. genes involved in apoptosis such as BIMBAM, caspases and CDKs.

Conclusions: Inhibition of the PI3K/Akt pathway seems to be a potential therapeutic target in ALL and high grade lymphoma cells. Sorafenib as a multikinase inhibitor approved for clincial application in solid tumors displayes significant antileukemic activity in vitro and might be a potential drug for a targed therapy approach in ALL.