bluuebunny/crossref_metadata_2025 huggingface dataset:
Caspase Activated Rock-1 Allows Erythroblasts Enucleation Independently of Cytokine Signaling
Authors: Courtois Genevieve , Vandekerckhove Julie , Chang Yunhua , Ribeil jean-Antoine , Kersual Joelle , Debili Najet , Vainchenker William , Varet Bruno R. , Hermine Olivier
Published: April 2020

Stem Cell Factor (SCF) and Erythopoietin (Epo) are strictly required for preventing apoptosis and stimulating proliferation, allowing differentiation of erythroid precursors from CFU-E until the polychromatophilic stage. In contrast, terminal maturation generating erythrocytes occurs independently of cytokine signaling by a mechanism poorly understood. Terminal differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size and nucleus with chromatin condensation and disappearance of organelles that require transient caspase activation. These events are followed by nuclei extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Two serine/threonine protein kinases, Rock-1 and Rock-2, the main Rho effectors, are key regulators of the actomyosin-based contractility. To determine the role of the Rho/Rock pathway in the terminal stage of erythropoiesis, we used an in vitro model of erythroid differentiation based on human hematopoietic cells derived from cord blood. We showed that in early step, SCF stimulates Rho/Rock pathway that is involved in erythroblasts proliferation and cytokinesis until the basophilic stage. Thereafter, Rock-I is activated independently of Rho signaling by caspase-3-mediated cleavage. To investigate the role of Rock during erythroid terminal differentiation, we used a Rock inhibitor (Y-27632). At day 8 of the CD36+ control culture, 55% of cells exhibited a mature phenotype (30% polychromatic, and 25% orthochromatic erythroblasts). By contrast, in the Y-27632 treated culture, there were no more than 23% mature cells (22% polychromatic and 1% orthochromatic erythroblasts) and 47% exhibited apoptotic features. Most of these apoptotic cells exhibited mature morphology suggesting that apoptosis occurred during the late step of maturation at the time of caspase activation. The percentage of enucleated cells was low in these culture conditions, thus, to determine whether or not Rock was also involved in the enucleation process, we used a coculture on MS5 stromal layer system (Giarratana, Nat Biotechnol 2005). At day 18, the rate of enucleated cells was 30% in the control culture whereas it was two-fold decreased in the Y-27632 treated culture and many of mature cells were instead dying by apoptosis. This finding suggested that Rock inhibition could be also involved in the enucleation process. By using shRNA technique, we demonstrated that Rock-1 is involved in terminal maturation and enucleation through phosphorylation of the light chain of Myosin II (MLC2). Therefore, in this differentiation system, caspase activation allows final maturation independently of cytokine signaling through kinase cleavage and activation.
The Unfolded Protein Response (UPR) Is Activated in Human Acute Myeloid Leukemia (AML) and Suppresses Translation of the CCAAT/Enhancer Binding Protein-Alpha (CEBPA) by Induction of the RNA-Binding Protein Calreticulin
Authors: Schardt Julian , Eyholzer Marianne , Mueller Beatrice U , Pabst Thomas
Published: April 2020

Deregulation of the myeloid key transcription factor CCAAT/enhancer binding protein alpha (CEBPA) is a common event in AML patients. We previously reported that the RNA-binding protein calreticulin efficiently blocks CEBPA translation and is specifically induced in core binding factor (CBF) leukemias. In addition, calreticulin is a crucial component of the unfolded protein response (UPR) which is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). In vitro studies suggested that the UPR is activated in some solid cancers and thereby involved in tumor development. The role of the UPR during leukemogenesis has not been addressed so far. Here, we investigated the induction of the spliced variant of the X-box binding protein 1 (XBP1s) as a marker for activated ER stress and determined the expression of key mediators of the UPR such as calreticulin and the 78-kDa glucose-regulated protein (GRP78) in leukemic cells from 92 consecutive AML patients. Increased expression of the XBP1 spliced variant was detected in 16 of 92 AML patients. Consistently, this group also had increased mRNA and protein levels of calreticulin and GRP78. In patients expressing the XBP1 spliced variant, CEBPA protein was hardly detectable in contrast to AML patients not expressing the XBP1 spliced variant. Moreover, treatment of myeloid leukemic cells with compounds activating the UPR – such as thapsigargin - confirmed rapid induction of XBP1s, GRP78 and calreticulin in myeloid cells whereas CEBPA protein levels decreased. In addition, conditional expression of calreticulin in U937 cells suppressed CEBPA protein. At the molecular level, we identified two functional ER stress response elements (ERSE) in the calreticulin promoter, and both elements were found to be necessary for full induction of calreticulin following ER stress. The presence of the tripartite nuclear transcription factor Y (NFY) and activating transcription factor 6 (ATF6), as well as an intact binding site for the YY1 transcription factor (YY1) within these ERSE motifs appeared to be crucial for mediating sensitivity to ER stress. Finally, chromatin-immunoprecipitation assays indicated that binding of NFY and YY1 to the ERSE motifs is induced after induction of ER stress in vivo. Therefore, we conclude that the UPR is activated in a subgroup of AML patients. Activation of the UPR involves induction of calreticulin expression by the ATF6 pathway and ultimately leads to suppressed CEBPA translation, thus contributing to the block in myeloid differentiation in these leukemias.
MMP-14 Mediates Migration of Acute Myelogenous Leukemia Cells
Authors: Shirvaikar Neeta , Jalili Ali , Mirza Imran , Ilnitsky Sara , Korol Chris , Larratt Loree M. , Turner A. Robert , Janowska-Wieczorek Anna
Published: April 2020

Matrix metalloproteinase (MMP)-14 expression correlates with progression and metastasis of multiple tumor cell types and is a major mediator of cell migration and invasion. MMP-14 possesses a trans-membrane domain that tethers the enzyme to the plasma membrane and not only activates proMMP-2 but also degrades extracellular matrix (ECM) by pericellular proteolysis and cleaves several non-ECM molecules, including adhesion molecules and chemokines. To assess the role of MMP-14 in leukemic dissemination we evaluated its expression in myeloid cell lines and primary acute myelogenous leukemic (AML) samples. Using RT-PCR, flow cytometry and Western blotting we found that MMP-14 is highly expressed in

leukemic myeloid cell lines THP-1, U937, HEL and K562; and primary samples from 37 out of 40 patients (pts) diagnosed with AML (WHO classification, AML with recurrent cytogenetic translocations: 9 pts; AML with multilineage dysplasia: 4 pts; AML not otherwise categorized: 27 pts).

Moreover, primary AML blasts secreted the 72 kDa proenzyme form of MMP-2 into media (zymography) which became activated after co-culture of AML blasts with bone marrow (BM) stromal cells. This activation was inhibited by the potent MMP-14 inhibitor epigallocatechin-3-gallate (EGCG). We also found that

migration of primary AML cells (trans-Matrigel invasion assay) was inhibited by EGCG; and silencing of MMP-14 by transfecting THP-1 cells and AML blasts with MMP-14 siRNA oligonucleotides resulted in reduced trans-Matrigel migration of these cells.

Given that AML blasts constitutively secrete TNF-α, we confirmed that

AML blasts express mRNA for TNF-α as well as its receptors TNFR1 and TNFR2; and TNF-α levels are elevated in the plasma of AML patients.

However, we demonstrated that recombinant human (rh) TNF-α further upregulated MMP-14 expression in AML blasts (RT-PCR, Western blot) and increased its incorporation into membrane lipid rafts (confocal microscopy). Moreover, rh TNF-α- stimulated AML blasts

were found to be more potent activators of pro-MMP-2 than unstimulated cells (zymography), indicating that activation may occur via upregulation of MMP-14; had significantly higher migration which was inhibited by EGCG and also by the TNF-α receptor inhibitor Enbrel; and rh TNF-α had no effect on the migration of MMP-14 siRNA-silenced AML cells.

Other factors tested, for example, SDF-1, IL-1 and TGF-β, had no effect on MMP-14 expression in AML cells (flow cytometry). In conclusion, we report here for the first time that MMP-14 is expressed in AML blasts and is modulated by TNF-α. We suggest that, by pericellular degradation of ECM and activation of latent MMP-2 secreted by AML blasts, MMP-14 may contribute to the highly proteolytic BM microenvironment in AML and the invasive phenotype of this malignancy.
Single Cell Molecular Genotyping Reveals Significant Molecular Heterogeneity at Diagnosis and Emergence of Clonal Dominance at Relapse
Authors: Zeng Rong , Meshinchi Soheil
Published: April 2020

Acute myeloid leukemia is a heterogeneous disease with significant variation in morphologic, cytogenetic, and molecular characteristics. We used single cell molecular genotyping to investigate the extent of molecular heterogeneity in individual patients with FLT3/ITD, WT1 and c-KIT mutations. Diagnostic leukemic samples from patients with FLT3/ITD (N=10) with varying FLT3/ITD allelic ratios (proportion of mutant to wild type product, ITD-AR) were flow sorted to immunophenotypic homogeneity (CD45dim/CD34+/CD33+) and individual leukemic blasts were isolated and directly genotyped. Single cell signal recovery as evident by the detection of FLT3 signal ranged from 39% to 88% (median 58%). Single cells from FLT3/ITD-positive samples demonstrated significant molecular heterogeneity with the presence of 3 distinct FLT3 genotypes: heterozygous FLT3/ITD, homozygous FLT3/ITD and wild type FLT3 (FLT3/WT). Proportion of cells with homozygous FLT3/ITD varied from 3% to 80% and directly correlated with the diagnostic ITD-AR, where those with high allelic ratios had a higher proportion of homozygous ITD cells and in those with the highest ITD-AR, with demonstrated 13q uniparental disomy (13q UPD), cells with homozygous ITD were the dominant clone. We further evaluated relapse specimens from 3 patients with FLT3/ITD, where cells with homozygous ITD was a minor clone at diagnosis (15%–30%). Single cell FLT3 genotyping of these specimens revealed significant expansion and clonal dominance of the homozygous ITD at relapse, where >85% of leukemic cells harbored homozygous ITD (and presumed 13q UPD). We demonstrated similar molecular heterogeneity in patients with WT1 and c-KIT mutations, with detection of homozygous mutations in 20–60% of the cells tested. Single cell genotyoing demonstrates significant molecular heterogeneity in AML that may not be evident by conventional methods. This data provides direct evidence that cells with homozygous FLT3/ITD and associated 13q UPD may be present as a minor population at the time of diagnosis and emerge as the dominant clone at relapse. Such a molecular heterogeneity and emergence of clonal dominance by an individual clone may provide insight into mechanism of disease resistance. Extent of molecular heterogeneity may also be important in our interpretation of data obtained from biologic specimens.
Poly ADP Ribose Polymerase (PARP) Inhibitors Induce Apoptosis Alone or Synergistically with Histone Deacetylase Inhibitors in Primary Acute Myeloid Leukemic Patient Cells
Authors: Gaymes Terry J , Shall Sydney , Farzaneh Farzin , Mufti Ghulam J
Published: April 2020

It has been previously reported that cells defective for double strand break (dsb) DNA repair can be selectively targeted for apoptosis through abrogation of their PARP activity. It has also been reported that leukemic cells have inherent defects in dsb DNA repair. Exploitation of DNA repair defects using PARP inhibitors (PI) thus represents a specific and less toxic form of therapy for a number of hematological malignancies. In order to test the efficacy of PI therapy we analysed primary cells from acute myeloid leukemia (AML) patients and the potential for combination therapy with inhibitors of DNA methyltransferase (DNMTi) or histone deacetylase inhibitors (HDACi). We report that from a panel of 12 AML patients, 2 AML patient cells demonstrated abnormal cell cycle profiles and apoptosis in response to the PI, KU-0058948 (1μM). In contrast, normal control cells displayed standard cell cycle profiles and no apoptosis in response to PI. Clonogenic cytotoxicity assays also showed that these PI sensitive AML patient cells exhibited between 45–55% cell survival compared with 100% cell survival in control cells (p<0.05) in response to PI. The homologous recombination (HR) DNA repair component, rad51 forms foci in response to DNA damage. In HR compromised cells, rad51 foci fail to form. In response to PI, immunofluorescent studies show that the 2 PI sensitive AML patients cells demonstrated severely reduced rad51 foci formation (<1%) compared to PI insensitive and normal control cells (15%). This confirmed that PI targets the HR deficiencies in PI sensitive cells. Histone H2AX, phosphorylated in response to DSB had greatly increased foci formation in PI sensitive cells compared to PI insensitive control cells as a result of unrepaired DNA damage (32.3 vs 19.3%)(p<0.05). We next explored the use of PI in combination with DNMTi or HDACi. Whilst KU-0058948 offered only additive effects on DNMTi cytotoxicity, a non-cytotoxic concentration of KU-0058948 (10nM) behaved synergistically with HDACi potentiating the cytotoxic effect of MS275 by 45% compared to MS275 alone (p<0.05). Furthermore, non-cytotoxic doses of MS275 (50nM) potentiated the cytotoxic effects of KU-0058948 in PI sensitive cells (25–30%) compared to KU-0058948 alone (P<0.05). In conclusion, we have shown that primary AML cells are sensitive to the cytotoxic actions of PI. We have also showed that PI acts synergistically in combination with HDACi. PARP inhibitors can potentially exploit dsb repair defects in leukemic cells paving the way for a targeted therapy for leukemia.
Phase I/II Study of Tipifarnib and Bortezomib in the Treatment of Poor Risk Adult Acute Myeloid Leukemia
Authors: Paolini Stefania , Ottaviani Emanuela , Lama Barbara , Salmi Federica , Clissa Cristina , Curti Antonio , Finelli Carlo , Giannoulia Panagiota , Iacobucci Ilaria , Laterza Claudio , Papayannidis Cristina , Parisi Sarah , Piccaluga Pier Paolo , Vianelli Nicola , Saglio Giuseppe , Fanin Renato , Morabito Fortunato , Zaccaria Alfonso , Lauria Francesco , Amadori Dino , Baccarani Michele , Martinelli Giovanni
Published: April 2020

Background. Outcome of elderly acute myeloid leukemia (AML) patients is dismal. Tipifarnib (Zarnestra, Z) and Bortezomib (Velcade, V) are new, targeted-treatments that might improve current results. In particular, Z is a farnesyltransferase inhibitor effective in AML, allowing complete remission (CR) rates ranging from 8 to 22%. Notably, the response rate appeared superior among patients with higher RASGRP1/APTX gene expression ratio. V is a proteasome inhibitor, possibly effective in AML as for phase I studies. Interestingly, Z and V appeared synergistic in AML cell lines.

Aim. We designed a phase I/II study aiming to assess the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of V in association with standard doses of Z and the efficacy and toxicity of the combination, in AML patients >18 years, unfit for conventional therapy, or >60 years, in relapse. Furthermore, we aimed to assess the correlation between RASGRP1/APTX ratio and treatment response.

Methods. V was administered as weekly infusion for three consecutive weeks (days 1, 8, 15), starting from 0.7 mg/m2 and increasing by 0.3 mg/m2 until the DLT was reached. Z was administered at the daily dose of 600 mg BID for 21 consecutive days. Response was assessed at the end of each cycle (28 days). Patients’ withdrawn was planned in case of progression or stable disease after six cycles. Real-time quantitative PCR (q-PCR) was used for RASGRP1/APTX genes evaluation.

Results. 45 patients were enrolled: 12 in the phase I and 33 in the phase II. 11/12 patients were evaluable in the phase I. Five patients received V at the dose of 0.7 mg/m2 without reporting DLT. Conversely, DLT was reached at the dose of 1.0 mg/m2 due to grade III SNC toxicity, recorded in 1/6 patients.

22/45 patients were evaluable for treatment response; transient reduction of peripheral blasts (> 50% to baseline) was observed in 20/22 cases. 2 patients achieved CR and 1 obtained a partial response (PR). 1 patient had a haematological improvement (HI), 12/22 had a stable disease (SD) and 5/22 showed progressive disease (PD). Interestingly, the 4 responders (CR+PR+HI) had a significant higher RASGRP1/APTX ratio respect to non responder (p=0.017).

Conclusion. We conclude that the MTD of V in association with Z is 1.0 mg/m2. The association seemed to be safe, with response rate (18%) similar to what reported for Z alone. Finally, though in few cases, RASGPR1/APTX ratio was confirmed to be associated to treatment response.
Effects of Recipients’ Body Weight and Total Nucleated Cell Dose on Outcomes of Unrelated Bone Marrow Transplantation: Analysis of Clinical Data of 3,114 Leukemia Patients from the Database of the Japan Marrow Donor Program
Authors: Yamashita Takuya , Akiyama Hideki , Ohashi Kazuteru , Onizuka Makoto , Mori Shin-ichiro , Sao Hiroshi , Miyamura Koichi , Sakamaki Hisashi
Published: April 2020

In allogeneic bone marrow transplantation (BMT) from unrelated donor of Japan Marrow Donor Program (JMDP), the standard bone marrow nucleated cell dose for transplantation is regulated as 3.0×10E8 total nucleated cells (TNC)/recipient’s body weight (RBW) (kg). RBW are also used to estimate the target harvest volume of bone marrow from the donor. So RBW determine the dose of harvested TNC, and these factors will influence the outcomes of BMT. We retrospectively analyzed the clinical data of 3,114 adult transplant patients (median age 33, range 16–70) from extracted the database of JMDP. The diagnosis of these patients were AML (n=1257), ALL (n=927) and CML (n=930). Five years overall survival (OAS) of all cases was estimated as 47.6%. Possible factors that could influence on survival and hematological recovery were analyzed by using proportional hazards analysis. In univariate analysis, TNC>=3.0×10E8/RBW was one of the significant factors to improve OAS (p=0.022). TNC/recipients’ ideal body weight (IBW) whose body mass index (BMI) was over 22 could be also a significant favorable factor for OAS (p=0.014). In multivariate analysis, variables correlated with improving OAS were TNC>=2.0×10E8/IBW (p=0.011), using tacrolimus for GVHD prophylaxis (p=0.011), younger recipient’s age (p=0.013). TNC>=2.0×10E8/IBW were also powerful variables correlated with the recovery of neutrophils, reticulocytes and platelets. These findings suggest that TNC/IBW can substitute for TNC/RBW to evaluate infused cell dose. And in our data set, use of IBW to estimate the harvest volume from the donor brought 10.7% reduction of target bone marrow volume compared with use of RBW. JMDP sets up the upper limit of harvested bone marrow volume based on the donor’s BW, but heavyweight recipients often require larger volume of bone marrow from the donors. Appropriate evaluation of TNC for transplantation and recipients’ BW by using BMI may be one of the useful methods to improve outcomes of unrelated BMT and avoid excess risk and stress of unrelated donors.
Antibody Development in Pediatric Sickle Cell Patients Undergoing Erythrocytapheresis: A 10 Year Review
Authors: Lockwood William B , Godfrey Gwendolyn J , Bertolone Salvatore , Raj Ashok
Published: April 2020

Background: The formation of alloantibodies and/or autoantibodies in multitransfused pediatric sickle cell disease patients undergoing monthly erythrocytapheresis (RBCX) during a 10-year period was evaluated utilizing a retrospective patient chart review.

MATERIALS AND METHODS: Clinical charts and transfusion services serological computer records of 32 patients were reviewed under an IRB approved protocol. All patients were seen in the Pediatric Sickle Cell Clinic, Kosair Children’s Hospital (KCH) between 1998 and 2008. Included in the data review were patient gender, ABO/Rh blood type, interval between RBCX start date and last RBCX, number of RBC components transfused, identity of existing antibody(ies) prior and subsequent to RBCX, and time interval for development of new antibody. All patients received C, E, and K antigen negative RBCs based on their phenotype at the start of their RBCX.

RESULTS: Thirty-two patients received between 8 to 555 RBC transfusions (Table 1). An allo- or autoantibody developed in 5/15 patients (33.3%) receiving C-, E-, K- RBCX; 3/10 patients (33.3%) receiving E-, K- RBCX; 2/4 patients (50%) receiving C-K- RBCX. An autoantibody developed in 3/32 patients (9.4%) as did a low frequency antibody (Low) not otherwise identified. One patient each developed an anti-Kpa and anti-M. In the two patients receiving K- RBCX, no new antibody was identified. Three patients developed anti-E and one patient developed anti-K after receiving partially matched RBCs on an emergency basis.

Table 1. Antibody (Ab) development following erythrocyapheresis (RBCX) of patients with sickle cell disease (n=32)

Transfused Phenotypes Number Patients New Ab Ab ID #RBC to New Ab (mean±SD) Second Ab Ab ID #RBC to Second Ab (mean±SD) *NA=Not applicable C- E- K- 15 5 E, K, M, Auto, Low 34±67 0 NA* NA C- K- 4 2 Auto, Low 114±43 1 Low 203±0 E- K- 11 3 E, Kpa, Auto 22±17 1 Auto 69±0 K- 2 0 NA NA 0 NA NA

Conclusions: Performing RBCX using C, E, K antigen phenotypically matched RBCs prevented development of new alloantibodies in 75% of multitransfused patients. Development of autoantibody or antibody to a low frequency RBC antigen can still occur in any patient receiving RBCX. In this series, all patients, including those who developed RBC antibodies, were able to continue long term RBCX without additional complications. Routine use of C-, E-, K- RBCs is recommended in RBCX to prevent formation of clinically significant antibodies.
Lenalidomide and Rituximab Are a Promising Combination in Vitro, in Vivo Preclinically and in a Phase I/II Clinical Trial in Relapsed/Refractory Mantle Cell Lymphoma
Authors: Wang Michael , Zhang Liang , Fayad Luis , Hagemeister Fredrick , Neelapu Sattva , Samaniego Felipe , Pro Barbara , Younes Anas , Miller Scott , Knight Robert D. , Zeldis Jerome B. , Yi Qing , Romaguera Jorge
Published: April 2020

Relapsed/refractory mantle cell lymphoma (MCL) is difficult to treat. Rituximab (R) targets CD20 antigen on the surface of MCL cells while lenalidomide (Len) may target the microenvironment of MCL cells and enhance the antibody-dependent cellular cytotoxicity (ADCC) activity of R. To test this hypothesis, we initiated preclinical studies and a phase I/II clinical trial. In the preclinical study we found that Len and R induced growth inhibition and apoptosis of both cultured and fresh primary MCL cells. Len enhanced R-induced apoptosis via upregulating phosphorylation of c-Jun N-terminal protein kinases (JNK), Bcl-2, Bad; increasing release of cytochrome-c; enhancing activation of caspase-3, -8, -9 and cleavage of PARP. Daily treatment with Len increased NK cells by 10 times in SCID mice. The combination of Len and R decreased tumor burden and prolonged survival of MCL-bearing SCID mice. In the phase I/II clinical trial, Eligible patients (pts) with MCL had 1–4 lines of prior therapies. Treatment consisted of Len given orally daily on days 1–21 of a 28-day cycle and R 375 mg/m2 by IV infusion weekly for 4 weeks only during the first cycle with the first dose on Day 1 in Cycle 1. A standard 3+3 dose escalation was used to determine MTD with Len doses at 10 mg, 15 mg, 20 mg, and 25 mg. Detailed toxicity profile in phase I was reported previously (Wang et al, ASH 2007). Two DLT s occurred at 25 mg including 1 grade 3 hypercalcemia and 1 grade 4 non-neutropenic fever during the first cycle. Six patients from phase 1 were at 20 mg dosage level. One patient from phase 1 was initially at 25 mg dosage level and was subsequently reduced to 20 mg dosage level due to DLT. Eight patients have been enrolled in the phase II trial at MTD. In the 14 patients evaluated at 20 mg dosage level in phase II, median age was 68 (51–77); median prior therapies were 2 (1–4); median cycles received to date were 4 (range 2–26). Grade 3/4 hematologic toxic events included neutropenia (35), febrile neutropenia (2), and thrombocytopenia (11). There was no grade 3–4 anemia. Grade 3 non-hematologic toxic events included fatigue (2) and myalgia (1). Fourteen pts at MTD (20 mg) including 7 in phase I plus 7 in phase II were evaluable for response. Eight out of 14 pts achieved responses including 4 CRs, 4 PR s, 2 SD and 4 PD s.

Conclusions: Lenalidomide in combination with rituximab provided a synergistically therapeutic effect on mantle cell lymphoma cells by enhancement of apoptosis and R-dependent NK cell-mediated cytotoxicity preclinically. Lenalidomide plus rituximab showed early evidence of response with a very favorable toxicity profile in a phase I/II clinical trial. Updated information will be presented at the conference.
A FVIII C1 Domain Loop Containing Phe2093 Contributes Affinity to the Binding of Recombinant FVIII C1C2 Proteins to Activated Platelets
Authors: Hsu Ting-Chang , Pratt Kathleen P. , Thompson Arthur R.
Published: April 2020

Factor VIII (FVIII) circulates bound to von Willebrand factor, and upon proteolytic activation it dissociates and attaches to activated membranes, e.g. at the platelet surface, that expose negatively-charged phosphatidylserine. This membrane association is mediated entirely by the FVIIIa light chain (A3-C1-C2), and the C2 domain is known to be a primary contributor to the membrane affinity. We previously demonstrated that the C1 domain, which is homologous to C2, also contributes to the affinity for activated platelets, (Hsu et al., Blood111, 200–208, 2008). Our earlier work showed that the platelet affinity of recombinant C1C2, as well as the total number of binding sites per platelet, were significantly higher than those measured for recombinant C2. Thus C1 either interacts with the platelet surface directly, or it stabilizes a conformation of C2 that promotes membrane binding. In this study, the affinities for activated platelets of a series of mutant C1C2 proteins were evaluated to determine residues involved in FVIII binding to platelets. C1C2 and C2 proteins were generated with a free cysteine residue substituted for the wild-type serine at position 2296 in C2 (S2296C does not disrupt the protein structure or affect membrane binding), to which a sulfyhydryl-linked fluorescein probe was attached covalently (C1C2* and C2*). Single or double alanine substitutions were introduced at the two beta-hairpin turns in C2 that mediate membrane binding (M2199A, F2200A, L2251A, L2252A), and at C1 residue F2093, which aligns with C2 residue L2252. Washed platelets were activated with SFLLRN-amide, incubated for 1 hr with the wild-type or mutant proteins, and analyzed by flow cytometry on a FACSCaliber. The relative binding affinities were estimated by using multiple protein concentrations and comparing the fluorescent signals to the values for saturation binding of C1C2* and C2*. Alanine substitutions at all of these positions resulted in decreased binding of C1C2*, comparable to the difference in affinity of C2* versus C1C2* (figure 1A). C1C2-F2093C was then generated, and the introduced sulfhydryl was blocked by adding free cysteine, thus introducing the bulky, charged cystine residue at this putative membrane-binding site (C1C2-F2093C-C). Binding assays utilizing detection by monoclonal antibody ESH8, which does not interfere with membrane attachment, followed by a PE-labeled secondary antibody showed that C1C2-F2093C-C had markedly reduced binding to activated platelets compared to C1C2 (figure 1B). These results are consistent with the hypothesis that the C1 domain contacts the membrane directly when FVIIIa becomes attached to the activated platelet surface and indicate that F2093 contributes significantly to this interaction.

Figure Figure
Antisense Oligonucletide Mediated Depletion of Factor VII Provides Protection from Ferric Chloride Induced Thrombosis without Increased Bleeding Risk in Mice
Authors: Crosby Jeff , Zhao Chenguang , Gao Dacao , Siwkowski Andy , Zhang Hong , Monia Brett P.
Published: April 2020

The Tissue Factor-Factor VIIa complex is the initiating protease in the extrinsic pathway of the coagulation cascade. Knockout of Factor VII in mice results in embryonic lethality. However, only humans with severe Factor VII deficiency exhibit any coagulopathy. In order to determine the potential of Factor VII as an anticoagulant target and the utility of antisense oligonucleotides (ASOs) as Factor VII inhibitors, ASOs were designed to promote RNaseH mediated degradation of Factor VII RNA and evaluated in mice. Factor VII is predominantly produced in the liver, a tissue that is tractable for antisense mediated target depletion. Following three weeks of treatment, the Factor VII ASO was well tolerated and supported significant reduction of Factor VII RNA and protein in mouse liver (ED50 of 3.5 mg/kg for RNA and 2.8 mg/kg for protein). Greater than 95% reduction in Factor VII RNA levels was observed at a dose of 25 mg/kg, administered twice weekly. Following three weeks exposure to 100 mg/kg/wk, prothrombin times (PT) from platelet poor mouse plasma was more than doubled, compared to untreated animals, while activated partial thromboplastin times (aPTT) was only increased 16%. Antithrombotic activity of Factor VII ASO was determined following ferric chloride injury to the inferior vena cava. ED50 for inhibition of thrombosis was 24 mg/kg/wk. The effects of Factor VII depletion on hemostatic control were also determined using a mouse tail nick model. No increase in blood loss was detected, compared to untreated animals, up to a weekly exposure of 80 mg/kg, the highest dose utilized in the study. Compared to warfarin, ASO mediated depletion of Factor VII promoted equivalent protection from thrombosis following ferric chloride injury while maintaining better hemostatic control. These findings support the development of an ASO drug targeting Factor VII as an anticoagulant.
Endothelial and Hematopoietic Tissue Factor Are Unnecessary in Two Mouse Models of Hemostasis and Thrombosis
Authors: David Tovo H , Sulciner David , Cornelissen Ivo , Coughlin Shaun R
Published: April 2020

Endothelial and hematopoietic tissue factor are unnecessary in two mouse models of hemostasis and thrombosis

Tissue Factor (TF), in combination with Factor VII, activates the extrinsic coagulation pathway leading to thrombin generation and clot formation. TF is expressed by epithelial, smooth muscle and perivascular cells; in addition endothelial and hematopoietic cells can express TF, especially in response to inflammatory stimuli. Despite extensive studies the exact cellular source of TF during clot initiation and propagation has not yet been clearly identified, and the importance of endothelial or hematopoietic TF in hemostasis and thrombosis is unknown. To address the latter, we measured bleeding time and arterial thrombus formation in TF flox/flox: Tie-2p/e Cre conditional mice, in which endothelial and circulating TF expression is absent. We observed that TF deficiency in the endothelial and hematopoietic compartment was not associated with defective hemostasis. In a FeCl3- induced carotid injury model, in contrast to PAR4 null mice, clotting in TF conditional mice did not significantly differ from that of wild type littermates. This study suggests that TF derived from endothelial or blood cells is not necessary for hemostasis and thrombosis in the models studied.

Figure Figure
Procoagulant Activity and Phosphatidylserine of Amniotic Fluid Cells
Authors: Zhou Jin , Liu Shuchuan , Ma Ming , Hou Jinxiao , Yu Hongjuan , Lu Chengfang , Gilbert Gary , Shi Jialan
Published: April 2020

Amniotic fluid (AF) may induce disseminated intravascular coagulation when it enters maternal circulation by breaching the placental-maternal circulation barrier. The precise mechanism of the procoagulant activity of AF is unclear, we speculate that AF cells have procoagulant activity due to the externalization of phosphatidylserine (PS). The present study aims to demonstrate that, in addition to tissue factor (TF), the PS that is externalized on AF cells is important for the procoagulant activity of AF. Ten AF samples from parturient women were analyzed and normal platelets, neutrophils, and lymphocytes were harvested as controls. Lactadherin, a glycoprotein, binds to membranes containing PS, inhibits prothrombinase activity, factor Xase activity, and tissue factor-factor VIIa activity by blocking PS-containing membrane binding. Thus lactadherin was utilized as a PS probe for flow cytometry and confocal microscopy to enable comparison of PS distribution with TF and intrinsic factor Xase complex formation. Procoagulant activity of AF cells was first measured in plasma with AF cells serving as thromboplastin. Activity of AF cells supporting intrinsic and extrinsic factor Xase complexes was measured in purified systems. Lactadherin, as an agent to block exposed PS, inhibited 85% of intrinsic and extrinsic factor Xase activity. Competition binding studies indicated that lactadherin competed for 55% of factor VIII binding sites. However, binding of factor VIII was completely inhibited by PS-containing vesicles and by mAb that recognize the factor VIII C2 domain indicating that all fVIII binding was mediated by the membrane-binding motif or an overlapping epitope. Confocal microscopy identified patches and a rim-pattern indicating a diffuse PS exposure. Lactadherin binding sites and TF distributed to discreet, but overlapping regions of the cells. These results indicate that PS exposure parallels procoagulant activity on AF cells and is required for at least 85% of intrinsic and extrinsic factor Xase activities. However, the topographical pattern of PS exposure differs from the pattern of TF and the pattern of binding site distribution for intrinsic factor Xase complexes. Thus, the results imply that intrinsic factor Xase and extrinsic factor Xase activity are localized to small cell regions where PS exposure coincides with TF and intrinsic factor Xase binding sites, respectively.
Raf-1 Is Over Expressed in Chronic Lymphocytic Leukemia and Promotes Cell Survival by Phosphorylation of ERK and BAD
Authors: Wang Miao , Kluin P.M. , Rosati Stefano , Luinge Marjan , Daenen Simon M. G. J. , Visser Lydia
Published: April 2020

Introduction: The serine threonine kinase Raf-1 plays a protective role in many cell types, but its expression and function in CLL cells has not been studied in detail. In the present study, we analyzed Raf-1 expression and tested the hypothesis that Raf-1 is critical for CLL cell survival.

Materials and Methods: By using qRT-PCR and western blot, we compared the expression of Raf-1 of mRNA and protein levels in purified B cells from 45 CLL cases and CD38 negative B cells from 5 reactive tonsils. By western blot, we analyzed the activity of phospho-Raf-1 (ser338) and its downstream targets (phospho-ERK1/2, phospho-BAD) in 23 CLL cases and 4 CLL cell lines (JVM-3, MEC-1, MEC-2 and MO1043) before and after IgM stimulation. By immunoprecipitation, we analyzed if Raf-1 co-localizes with Bcl-2. We correlated the change in phosphorylation status (Raf-1, ERK and BAD) in response to IgM stimulation with the ZAP-70/SYK mRNA ratio, which was detected by qRT-PCR in purified B cells from CLL cases. After using specific inhibitors, including the Raf-1 inhibitor GW5074, the Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137, we investigated apoptosis by Annexin V flowcytometry in CLL cases and CLL cell lines as well as cell cycle changes in the 4 cell lines by flowcytometry.

Results: In comparison to normal resting (CD38 negative) B cells, there was a strongoverexpression of Raf-1 in CLL cells, both at at the mRNA and protein level. Using qRT-PCR there was an almost linear correlation between Raf-1 and Bcl-2 expression. Moreover, the phosphorylation status of Raf-1 and ERK in response to IgM stimulation strongly correlated with the ZAP-70/SYK mRNA ratio. Using immunoprecipitation and confocal miscroscopy we found colocalization of Raf-1 with Bcl-2, which might account for the observed constitutive activation of BAD in CLL cells. The Raf-1 inhibitor GW5074, Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137 all led to p-Raf-1 inhibition as well as downregulation of p-ERK and p-BAD. Additionally, all three inhibitors downregulated cyclin D3 and cyclin E, which are important for G0/G1 transition. We also found that GW5074 induced apoptosis in CLL cell lines and primary cells of CLL cases.

Conclusion/Discussion: In conclusion, our study identifies Raf-1 as a critical anti-apoptotic and cell cycle regulating kinase in CLL cells.
Deregulation of MicroRNA Underlies Severe Congenital Neutropenia Pathogenesis
Authors: Velu ChinavenMeni S. , Baktula Avinash M , Bourdeau Tristan , Grimes H. Leighton
Published: April 2020

The zinc finger protein Growth factor independent-1 (Gfi1) is a transcriptional repressor that regulates hematopoietic stem cell (HSC) maintenance and granulocytic lineage differentiation. Humans with severe congenital neutropenia (SCN) display mutations in GFI1 (encoding GFI1N382S) which generate dominant negative acting proteins. GFI1N382S proteins sequester limiting cofactors to deregulate a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs and suggest that that transcriptional control of microRNA genes is critical for GFI1N382S-associated SCN phenotypes. First, the expression of Gfi1 and miR21 and miR-196 is reciprocal: 1) in wild type and Gfi1−/− marrow cells, 2) during normal differentiation from common myeloid progenitors (CMP) to granulocyte monocyte progenitors (GMP), 3) during treatment-induced differentiation of human myeloid leukemia cell lines, and 4) upon conditional deletion of Gfi1 in primary sorted murine CMP and GMP. Biochemical analyses reveal that miR21 and miR-196 are direct transcriptional targets of Gfi1. Subsequently, forced expression of wild type Gfi1 rescues expression of microRNA in Gfi1−/− Lin- bone marrow cells, while forced expression of Gfi1N382S in wild type Lin- bone marrow cells significantly deregulates miR-21 and miR-196 expression. Similarly, we demonstrate elevated miR21 and miR196b levels in CD34+ cells from a GFI1N382S SCN patient. Flow cytometric analysis and colony assays reveal that the overexpression or knockdown of either miR induces changes in myeloid development, but that co-expression of both miR (as seen in Gfi1−/− mice and GFI1N382S SCN patients) completely blocks G-CSF-induced granulopoiesis. These data provide a molecular understanding of SCN disease pathogenesis.
Chronic Lymphocytic Leukemia Cells Promote Increased BAFF Expression in Monocytes Partially through TNF-Alpha Leading to Increased CLL Cell Survival
Authors: Messmer Davorka , Chang Hsu-Hsiang , Kipps Thomas J.
Published: April 2020

The tumor microenvironment plays a critical role in growth, metastasis, and survival of tumors. Chronic lymphocytic leukemia (CLL) B cells of many patients can rapidly undergo apoptosis in vitro unless co-cultured with accessory cells, which elaborate factors that promote CLL-cell survival. A major type of such accessory cells is nurse-like cells (NLC), which prior studies demonstrated could differentiate from CD14+ blood mononuclear cells when exposed to CLL B cells in vitro, and presumably in vivo. In the absence of CLL cells, blood mononuclear CD14+ cells typically differentiate into macrophages that have a distinctive morphology from that of NLC. We found that, compared with macrophages, NLCs consistently expressed significantly higher levels of B-cell activating factor (BAFF) (n=11), a tumor-necrosis-factor (TNF)-related ligand that promotes survival of B cells, including CLL B cells. To investigate the influence of CLL cells on the differentiation of NLC in vitro, purified CLL B cells or B cells of healthy donors were cultured for 24h to generate conditioned media. When CD14+ blood mononuclear cells of healthy donors were cultured for 7 days in media conditioned by CLL cells, but not in non-conditioned media or in media conditioned by normal B cells, the cells assumed the morphology of NLC, acquired high-level expression of BAFF, and developed an enhanced capacity to support CLL cell survival in vitro. Similar activity was also found in the sera of CLL patients, but not in the sera of healthy control donors. The capacity of either CLL-conditioned media (n=4) or CLL-patient sera (n=9) to induce NLC differentiation leading to increased expression of BAFF was significantly reduced by a decoy receptor for TNF-a, suggesting that TNF-a contributes to the differentiation of NLC in vitro. However, TNF-a alone was not sufficient to induce high-level expression of BAFF on blood mononuclear CD14+ cells, suggesting that additional factors are involved. The results show a previously unrecognized symbiosis between CLL cells and accessory cells in their microenvironment. Because the survival of neoplastic B cells in vivo potentially depends upon NLC found in the lymphoid tissues of patients with CLL, identification of the factor(s) involved in inducing their differentiation could provide a novel target for therapy of patients with this disease.
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

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.

Figure Figure Figure Figure
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

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.