bluuebunny/crossref_metadata_2025 huggingface dataset:
Thalidomide Derivative CC-4047 Inhibits Osteoclast Formation by down Regulation of PU.1.
Authors: Lentzsch Suzanne , Anderson Gulsum , Kurihara Noriyoshi , Honjo Tadashi , Anderson Judith , Mapara Markus Y. , Stirling David , Roodman David
Published: April 2020

CC-4047 (Actimid) is an immunomodulatory analog of thalidomide that has stronger anti-myeloma and anti-angiogenic activity than thalidomide, but its effects on human osteoclast lineage are unknown. Early osteoclast progenitors are of hematopoietic origin and progressively differentiate into mature bone resorbing multinucleated osteoclasts. We investigated the effects of CC-4047 and thalidomide on human osteoclastogenesis, using in vitro receptor activator of NFκ-B ligand/M-CSF stimulated culture system of bone marrow cells. Three weeks of treatment of primary bone marrow cultures with 100 μM CC-4047 decreased osteoclast formation accompanied by complete inhibition of bone resorption. Interestingly, osteoclast formation was also inhibited when cultures were treated with CC-4047 only for the first week (90% inhibition). In contrast, inhibitory effect was greatly diminished when the drug was given for only the last week (25% inhibition), indicating that inhibition of osteoclast formation is an early event. The inhibitory effect of CC-4047 on osteoclastogenesis was not induced by cell death, but by a shift of lineage commitment to granulocyte-CFU at the expense of GM-CFU that are osteoclast progenitors. Further studies revealed that this shift is mediated through down regulation of the transcription factor PU.1, which is critical for early osteoclast formation. In contrast to CC-4047, thalidomide was a significantly less potent inhibitor of osteoclast formation and bone resorption. These results provide the first evidence that CC-4047 blocks osteoclast differentiation at the early phase of osteoclastogenesis. Therefore, CC-4047 might be a valuable drug targeting both the tumor and osteoclastic activity in patients with multiple myeloma and potentially other diseases associated with the development of osteolytic lesions.
HoxA10 Represses Transcription of Multiple Genes Encoding Respiratory Burst Oxidase Proteins in Undifferentiated Myeloid Cells.
Authors: Lindsey Stephan , Lu YuFeng , Eklund Elizabeth A.
Published: April 2020

HoxA10 is a homeodomain (HD) transcription factor which is maximally expressed in committed myeloid progenitors and is overexpressed in acute myeloid leukemia (AML). Consistent with this, forced overexpression of HoxA10 in murine bone marrow leads to AML. Our studies identify a mechanism by which HoxA10-overexpression blocks differentiation, contributing to leukemogenesis. Previously, we found that HoxA10 represses transcription of the CYBB gene. This gene encodes the gp91phox component of the phagocyte respiratory burst oxidase and is actively transcribed in myeloid cells differentiated beyond the promyelocyte stage. CYBB transcription continues until cell death and is further increased in mature phagocytes by inflammatory mediators such as IFNg and LPS. In undifferentiated myeloid cells, HoxA10 represses CYBB transcription via a proximal promoter cis element with homology to the consensus sequence for HoxA10 DNA-binding as a heterodimer with Pbx1 (also a HD protein). However, we found that HoxA10 repression of CYBB transcription is Pbx1-independent and HDAC2-dependant. During cytokine induced differentiation, phosphorylation of two HoxA10 HD tyrosine residues decreases DNA-binding affinity and transcriptional repression. In the current studies, we investigate the impact of HoxA10 on transcription of the NCF2 gene. This gene, which encodes the p67phox phagocyte oxidase component, is transcribed concurrently with the CYBB gene during myelopoiesis and the inflammatory response. The NCF2 promoter also includes a sequence homologous to the HoxA10/Pbx1 DNA-binding consensus. In the current studies, we determine that HoxA10 functionally represses NCF2 transcription in undifferentiated myeloid cells in a Pbx1-independent, HDAC2-dependant manner. Additionally, we determine that NCF2-transcriptional repression by HoxA10 is abolished by phosphorylation of HD tyrosine residues in response to differentiating cytokines. In these studies, we also investigate the impact of IFNg-treatment of monocytes on HoxA10 phosphorylation and gp91phox and p67phox expression. We find that treatment of monocytes with this inflammatory mediator results in hyper tyrosine phosphorylation of HoxA10, which is necessary for IFNg-induced gp91phox and p67phox expression in these cells. These experiments suggest that HoxA10 contributes to differentiation block in AML by repressing transcription of genes that confer the mature myeloid phenotype. HoxA10 tyrosine phosphorylation abrogates transcriptional repression and may provide a molecular target for differentiation induction in AML. Additionally, HoxA10 hyper phosphorylation during the inflammatory response further increases oxidase gene expression. This represents the first demonstration that functions of HoxA10 contribute to gene regulation in mature phagocytes, contributing to the system of host defense.
Lineage Instruction by Oncogenic Tyrosine Kinase Fusions.
Authors: Shigematsu Hirokazu , Mizuno Shin-ichi , Iwasaki Hiromi , Arinobu Yojiro , Ozawa Hidetoshi , Sternberg David W. , Huntly Brian , Gilliland D. Gary , Akashi Koichi
Published: April 2020

Oncogenic tyrosine kinase fusions play a key role in transforming hematopoietic stem and progenitor cells. Bcr/Abl (B/A) and TEL/PDGFβR (T/P) tyrosine kinase fusions were isolated from chronic myeloid leukemia and chronic myelomonocytic leukemia respectively. The constitutive activation of these fusions mainly promotes cell growth of target cells inducing myeloproliferative disease (MPD), which could be a precondition for transformation into acute leukemia. We found that these kinase fusions not only stimulate autonomous cell growth, but also specify cells’ myeloid fate by their own myeloid lineage instructive signals. We retrovirally transduced either the B/A or the T/P gene into purified murine hematopoietic progenitor and stem cells. Transducion of B/A or T/P into hematopoietic stem cells (HSCs) and common myeloid progenitors (CMPs) resulted in formation of spontaneous myeloid colony in cytokine- independent manners. Interestingly, B/A-transduced HSCs (B/A+ HSCs) spontaneously formed granulocyte/monocyte (GM) and megakaryocyte/erythroid (MegE) colonies, while T/P-transduced HSCs (T/P+ HSCs) only generated GM colonies. T/P+ HSCs did not generate MegE-related colonies even in the presence of erythropoietin or thrombopoietin, suggesting that T/P transduces GM-restricted permissive signals. Both B/A+ HSCs and T/P+ HSCs displayed markedly reduced potential for B cells in vitro and vivo. We then transduced B/A or T/P into purified common lymphoid progenitors (CLPs) to test their effect on lymphoid commitment. In a limiting dilution assay, 1 in 7 control-GFP transduced CLPs generated B cell progeny in vitro, while only 1 in 150 B/A+ CLP and 1 in 500 T/P+ CLPs differentiated into B cells. Instead, surprisingly, the majority of B/A+ and T/P+ CLPs differentiated into granulocytes and monocytes in vitro and in vivo. These results indicate that B/A and T/P can specify the myeloid fate counteracting the lymphoid differentiation. We then tried to locate the responsible signaling domain for growth-stimulating or instructive signals by utilizing a series of tyrosine-to-phenylalanine mutants of T/P at the major autophosphorylation sites of PDGFβR. The substitution of only two juxtamembrane tyrosine residues resulted in impairment of cytokine-independent cell growth of HSCs. In contrast, CLPs transduced with mutant T/P substituted at all eight major tyrosine residues were still reprogrammed to differentiate into the myeloid lineage. These data collectively suggest that oncogenic tyrosine kinase fusions can specify the leukemic phenotype by their own instructive signals, which use independent signaling domain for growth promoting ones.
Dynamics of Calcium Mobilization in Platelets during Thrombus Formation in a Living Mouse.
Authors: Dubois Christophe , Panicot-Dubois Laurence , Furie Barbara C. , Furie Bruce
Published: April 2020

Platelet accumulation at sites of vascular injury arrests bleeding but also plays a critical role in the pathogenesis of thrombosis, leading to ischemia in myocardial infarction or stroke. Intracellular calcium mobilization in platelets is a critical step in the activation of platelets and formation of the platelet thrombus. Here we show the relationship of the dynamics of intracellular calcium mobilization with platelet accumulation into the developing thrombus in a living mouse. Following injection of 100 x 106 fura-2 loaded platelets into a living mouse we used high speed intravital multi-channel digital fluorescence microscopy to monitor calcium status in circulating and thrombus-bound platelets during thrombus development. One population of platelets binds transiently to the developing thrombus but does not mobilize calcium. The mean duration of platelet-thrombus interaction for these platelets is 11 sec. Another population of platelets undergoes calcium mobilization after binding to the developing thrombus. The time interval from attachment to calcium mobilization for individual platelets varied from 1.0 to 12 sec, with a median of 3.5 sec. More than 90% of platelets that undergo calcium mobilization do so with in 5 sec of attachment. The calcium mobilization in the thrombus bound platelets is reversible. About two thirds of the platelets return rapidly to the basal Ca2+ state while the remaining thrombus bound platelets maintain an elevated Ca2+ level for an extended period. The mean duration of platelet-thrombus interaction is 35 sec with a range of 1.5 sec to 284 sec (median duration 39.5 sec) as calculated from multiple independent observations of single platelets. In each platelet studied, only one calcium peak is detected per platelet. There is a close correlation between the duration of calcium mobilization in an individual platelet and the time that the platelet remains attached to the developing thrombus, suggesting a relationship of calcium-dependent events and platelet-thrombus affinity. A population of platelets binds to the thrombus, mobilizes calcium and remains associated with the thrombus. Using widefield deconvolution techniques to obtain planar images and increased numbers of dye-loaded platelets, individual platelets could be observed undergoing sustained calcium elevation within the thrombus. As the platelet thrombus reaches maximal size at about 120 sec, calcium mobilization continues in the stable core of the thrombus for several minutes, then decreases. These studies describe thrombus formation in a living animal under conditions in which the endothelium and vessel wall, blood cells and plasma components, and flowing blood are preserved in the absence of anticoagulants. Our results indicate that stable platelet thrombus formation is dependent upon durable calcium mobilization, and that intracellular calcium regulates thrombus development and maturation in vivo.
The Role of the cGMP-Dependent Protein Kinase II in Platelet Activation and In Vivo Thrombosis.
Authors: Li Zhenyu , Zhang Guoying , Yin Hong , Feil Robert , Hofmann Franz , Du Xiaoping
Published: April 2020

Although it was previously believed that the intracellular secondary messenger cGMP inhibits platelets, we have recently shown that cGMP-dependent protein kinase I (PKG I) in fact plays a stimulatory role in platelet activation. However, there are apparent differences between the PKG inhibitors and PKG I knockout in their effects on platelet activation. PKG inhibitors are more potent in inhibiting platelet activation than PKG I knockout. More importantly, although platelet secretion and aggregation induced by collagen were inhibited by PKG inhibitors, they are not significantly affected in PKG I knockout platelets. There are two types of PKG, PKG I and PKG II. PKG II has not been previously described in platelets. Here we show that PKG II mRNA is expressed in platelets using RT-PCR with primers specific for a C-terminal fragment of human PKG II cDNA. We further cloned the complete cDNA of human PKG II by RT-PCR using the purified human platelet mRNA as a template. Furthermore, PKG II from platelet lysates was pulled down by cGMP conjugated agarose beads and detected by western blot using a polyclonal antibody against PKG II. These data indicate that PKG II is expressed in platelets. To investigate the role of PKG II in platelet activation, washed wild type or PKG II knockout (PKG II−/−) mouse platelets in tyrode’s solution were exposed to platelet agonists. Platelet aggregation and ATP secretion induced by low concentrations of collagen were significantly reduced in PKG II deficient mice, indicating that PKG II plays important roles in collagen-induced platelet activation. PKG II−/− platelets also showed reduced aggregation and secretion to low dose of a thromboxane A2 (TXA2) analog, U46619. However, low dose thrombin-induced platelet activation was not negatively affected in PKG II−/− platelets, but was inhibited in PKG I−/− platelets. To evaluate the in vivo role of PKG II, we compared in vivo thrombus formation of wild type and PKG II knockout mice using the FeCl3-injured carotid artery thrombosis model. The time to the formation of stable thrombus in PKG II−/− mice (median, 420.0 seconds, n=15) is significantly prolonged compared to wild type mice (median, 321.0 seconds, n=15) (p=0.031). Tail-bleed time analysis also indicated a remarkably prolonged bleeding time in PKG II−/− mice (the median bleeding time was 73.50 seconds (n=18) in wild type mice, 454.50 seconds (n=20) in PKG II knockout mice) (p=0.0008). Thus, PKG II plays an important role in promoting platelet activation, thrombosis and hemostasis. PKG I and PKG II have differential roles in platelet activation induced by different platelet agonists.
FcRγ-Chain Signals in GPVI-Deficient Platelets.
Authors: Liu Junling , Ware Jerry , Jackson Carl W. , Gartner T. Kent
Published: April 2020

The receptor glycoprotein VI (GPVI) is required for hemostasis and collagen-induced platelet aggregation. GPVI and FcRγ-chain form a noncovalent signaling complex. Genetic ablation of murine FcRγ-chain prevents expression of GPVI, and immunodepletion of GPVI removes FcRγ chain from platelet membranes. The GPVI/FcRγ-chain relationship has confounded elucidation of the individual role(s) of GPVI and FcRγ-chain in signal transduction. For example, botrocetin (bt)/von Willebrand factor (vWf) and γ-thrombin induced aggregation of washed platelets each appear to be FcRγ-chain-dependent. However, since FcRγ-chain−/− platelets also lack GPVI, the independent role(s), if any, of these signaling molecules in this aggregation could not be established. But, genetic ablation of murine GPVI does not exclude expression of the FcRγ-chain. Therefore, based on analyses of genetically deficient GPVI platelets that express FcRγ-chain, we demonstrate here that bt/vWf and thrombin-induced aggregation of washed platelets are each dependent on FcRγ-chain thereby demonstrating that FcRγ-chain can function in the absence of GPVI. Presumably, another receptor or receptors is/are able to activate FcRγ-chain in GPVI −/− and GPVI +/+ platelets. GPIb may not directly activate FcRγ-chain because GPIb elicited activation of αIIbβ3 is not FcRγ-chain dependent. The integrin αIIbβ3 appears to be common to the signaling described here because both the non-agglutination elicited bt/vWf-induced, and the γ-thrombin-induced TxA2 production and ATP secretion were β3-dependent. Therefore, αIIbβ3 may have activated this FcRγ-chain-dependent signaling. This hypothesis was tested by investigating the role of FcRγ-chain in αIIbβ3-mediated outside-in signaling induced by a palmitoylated derivative of the peptide KVGFFKR, a peptide which directly activates αIIbβ3 causing β3-dependent signaling that elicits TxA2 production, ATP secretion and platelet aggregation. As with γ-thrombin-treated FcRγ-chain−/− platelets, pKVGFFKR-treated FcRγ-chain−/− platelets did not produce TxA2, secrete ATP or aggregate. But, pKVGFFKR-treated GPVI−/− platelets produced the wild type level of TxA2, secreted the wild type level of ATP, and aggregated normally demonstrating that pKVGFFKR-induced αIIbβ3-mediated outside-in signaling is FcRγ-chain dependent. These results support the hypothesis that activation of FcRγ-chain is αIIbβ3-dependent in the signaling described here. The results of ELISA studies support this conclusion.
Reduced Intensity Conditioning (RIC) Allogeneic Stem Cell Transplantation (allo-SCT) for Patients with Acute Lymphoblastic Leukemia (ALL): A Survey from the European Group for Blood and Marrow Transplantation (EBMT).
Authors: Mohty Mohamad , Labopin Myriam , Boiron Jean-Michel , Theorin Niklas , Fauser Axel , Rambaldi Alessandro , Boogaerts Marc , Slavin Shimon , Majolino Ignazio , Blaise Didier , Rocha Vanderson
Published: April 2020

The role of RIC allo-SCT for adult patients with ALL is still under considerable debate. While the use of such so-called nonmyeloabaltive or RIC regimens has emerged as an attractive modality to decrease transplant-related toxicity, it is still unclear whether such regimens are associated with a graft-vs.-leukemia (GVL) effect in adult ALL. This report describes the results of 91 adult ALL patients (62 M and 29 F) treated in different centers affiliated with the EBMT. Median age was 40 y. (range, 17–66). Twenty nine (32%) patients were transplanted in first complete remission (CR), 28 (31%) beyond first CR, and 34 (37%) in advanced disease. In this cohort, 39 patients (51%) had a Ph+ or t (4;11) ALL. The majority of patients received a PBSC graft (78%) from an HLA-matched sibling donor (n=62; 68%). The RIC regimen included Fludarabine (80%) associated to low dose TBI in 22 patients (24%), and/or low dose Bussulfan (<8mg/kg). ATG was used in 29 patients (33%), mainly in unrelated HSCT. With a median follow-up of 34 months (range, 7–76), the incidences of grade II-IV and grade III-IV acute GVHD were 35% and 13% respectively. At 2 years, chronic GVHD occurred in 49% of patients. Cumulative incidence of transplant-related mortality at 3 years (TRM; 11 fatal infections, 5 refractory GVHD, 6 other) was 24% (15% in patients transplanted in first CR). Cumulative incidence of relapse at 3 years was 58%+/−5 in this high risk group. Three years leukemia-free survival (LFS) was 18% in the whole study group, with this being significantly higher (34%) in patients transplanted in first CR. Patients transplanted in advanced refractory disease had the worst LFS (5%). In multivariate analysis, disease status at time of allo-SCT (first CR vs. other) was the strongest factor associated with an improved LFS (P=0.04; RR=2.1; 95%CI, 1.02–4.20).

The results of this retrospective registry based study suggest that RIC allo-SCT for adult ALL may represent a valid therapeutic option when a conventional standard conditioning is not possible. Of note, results obtained in patients transplanted in first CR (LFS and TRM) compare favorably with previous results achieved after standard myeloablative allo-SCT, warranting further investigations in this subgroup. Also, better understanding of the putative GVL effect in adult ALL, in addition to designing maintenance strategies (imatinib in Ph+ ALL for eg.) after RIC allo-SCT, may further improve the outcome of adult ALL.
Contrasting Effects of Post-Transplant Lymphopenia on Proliferation and Degranulation in Antigen-Specific CD8 Memory T Cells.
Authors: Shatry Alwi M. , Levy Robert B.
Published: April 2020

Lymphopenia following bone marrow transplant (BMT) can induce expansion of donor T cells which help contribute to reconstitution in recipients. We are interested in elucidating the responses of transplanted CD8 T memory (TM) to lymphopenic signals in the recipient splenic and marrow environments. To compare proliferative responses of marrow and spleen populating antigen-specific CD8 TM to lymphopenic signals, purified (95% pure) CFSE labeled B6 CD8 TM were co-transplanted with syngeneic or allogeneic TCD-BM to ablatively conditioned (9.0 Gy) syngeneic B6 recipients. Antigen-specific CD8 TM were obtianed following priming B6 mice against BALB.B antigens. An H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to identify immunodominant H60-specific CD8+ cells which expressed a central memory (TM) phenotype (CD44+, Ly6C+). Three days post-transplant of these CD8 TM, recipient spleen and marrow cells were analyzed for CFSE dilution in gated H60+CD8+ T cells. Transferred H60+ CD8+ cells in the marrow compartment consistently showed greater (5 – 6 cell divisions) proliferative activity compared to those populating the spleen (1 – 2 divisions) in recipients of syngeneic (i.e. homeostatic) as well as allogeneic TCD-BM. Thus, the marrow compartment apparently provided greater support for the early homeostatic proliferation of these MiHA-specific CD8 TM. Furthermore, the presence of antigen in B6 recipients of allogeneic BM did not significantly enhance the proliferation of transferred CD8+ TM, indicating that homeostatic signals induced greater overall proliferation than antigen activation in the simultaneous presence of both signals early post-transplant. To examine for non-proliferative functional activity in transplanted antigen-specific CD8 TM, degranulation was assessed by surface expression of LAMP-1 (CD8 107a) which was analyzed 3 days after the transfer of purified splenic CD8 T cells from B6BALB.B into lethally irradiated (9.0 Gy) syngeneic B6 mice. Analysis of recipient spleeen and marrow showed that transferred antigen-specific in these tissues markedly upregulated surface LAMP-1 expression. Interestingly, there was greater frequency (~90%) of CD8+ TM in the spleen expressing LAMP-1 than in the bone marrow (56%). To determine if this degranulation response was limited to transplanted TM, a homogeneous population of naive T cells (TN) from OT-1 RAG−/− TCR tg mice was prepared and transplanted in an identical manner. LAMP-1 expression was marginally detectable in these cells. In total, these results suggest that transplanted CD8 TM and TN respond differently in the homeostatic environment post-transplant. Moreover the observations may suggest that CD8 TM can exhibit effector-like activity in the lymphopenic milieu immediately post-transplant. These findings have implications for the relative contribution of hematopoietic compartments during immune reconstitution after HCT.
The Menin Tumor Suppressor Protein Is an Essential Oncogenic Cofactor for MLL-Associated Leukemogenesis.
Authors: Yokoyama Akihiko , Somervaille Tim , Cleary Michael L.
Published: April 2020

The Mixed Lineage Leukemia (MLL) protein is a histone methyltransferase that is mutated in clinically and biologically distinctive subsets of acute leukemia. MLL normally associates with a cohort of highly conserved cofactors to form a macromolecular complex that includes menin, a product of the MEN1 tumor suppressor gene, which is mutated in heritable and sporadic endocrine tumors. We demonstrate here that oncogenic MLL fusion proteins retain an ability to stably associate with menin through a high-affinity, amino-terminal, conserved binding motif and that this interaction is required for the initiation of MLL-mediated leukemogenesis. Furthermore, menin is essential for maintenance of MLL-associated but not other oncogene induced myeloid transformation. Acute genetic ablation of menin reverses aberrant Hox gene expression mediated by MLL-menin promoter-associated complexes, and specifically abrogates the differentiation arrest and oncogenic properties of MLL-transformed leukemic blasts. These results demonstrate that a human oncoprotein is critically dependent on direct physical interaction with a tumor suppressor protein for its oncogenic activity, validate a potential target for molecular therapy, and suggest central roles for menin in altered epigenetic functions underlying the pathogenesis of hematopoietic cancers.
Identification of Novel Signalling Pathways in Myeloblasts from Patients with Acute Myeloid Leukemia Using a Phosphoproteomic Strategy.
Authors: Craddock Charles , Tauro Sudhir , Griffiths Michael , Cook Mark , Singh Harprit , Wakelam Michael
Published: April 2020

Acquired somatic mutations which result in constitutive activation of signal transduction pathways are increasingly recognised in acute myeloid leukemia (AML). Reasoning that since protein tyrosine phosphorylation is an important mechanism mediating the transduction of proliferative and survival signals we have utilised a proteomic strategy to identify dysregulated phosphoproteins in myeloblasts from patients with AML. Using an anti-phosphotyrosine antibody we have immunoprecipitated proteins from AML blasts, separated proteins by SDS PAGE and identified proteins within distinct bands by mass spectrometry. Results in primary AML blasts have been compared with CD34+ hemopoietic progenitors harvested from GCSF mobilised normal donors. The methodology was validated using vanadate-stimulated HL60 cells. 10 patients with a median age of 51 (range 16–90 yrs) were studied. One patient had favorable risk cytogenetics, three adverse risk cytogenetics and six a normal karyotype. Mutations in the flt-3 tyrosine kinase were present in four patients. Blasts from every patient demonstrated phosphorylation of MAP kinase (MAPK) implying activation of the ras-MAPK cascade. In contrast CD34+ cells from three normal donors demonstrated weak or no MAPK phosphorylation. Since each AML sample demonstrated MAPK phosphorylation irrespective of flt-3 status we next examined their phosphotyrosine-proteome. This identified a number of phosphorylated and non-phosphorylated proteins in signalling complexes in anti-phosphotyrosine immunoprecipitates. Notable examples amongst these were receptors (Ephrin type-A3, interleukin-13, GPCR43), signalling intermediates (TAPP1, RASA1, GDI-2, LARG, SGEF, cortactin, Cd-2 associated protein) and transcription factors (ELK-1, HFK-1). These proteins identify three functionally distinct groups in AML blasts which were not detected in normal CD34+ progenitors: (i) intermediates of a tyrosine kinase mediated ras-MAPK signalling cascade (ii) intermediates in PtdIns-3-kinase mediated signalling presumably suppressing apoptosis and (iii) regulators of the actin cytoskeleton and thus cell movement. Surprisingly the strategy also identified tyrosine phosphorylation of the 5-HT3A receptor and the MRCP1-ABC transporter in a number of patients suggesting a role for these proteins in the pathogenesis of certain subtypes of AML. The phosphorylation of a number of these identified proteins was confirmed immunochemically. Thus adoption of a phosphoproteomic methodology has identified novel phosphoproteins in AML which require further validation. Our data also demonstrate a common intracellular signalling pathway (ras-MAPK) in distinct AML patients regulated by differing primary events. These observations provide information of value in the rational development of targeted therapies in AML.
Assessment of PML-RARa Real-Time Quantitative PCR for Definition of Molecular Relapse in APL Patients (Results of the APL Study Group).
Authors: Cassinat Bruno , De Botton Stephane , Ades Lionel , Raffoux Emmanuel , Escoffre-Barbe Martine , Baruchel Andre , Miclea Jean-Michel , Reman Oumedaly , Gardembas Martine , Brault Philippe , Milpied Noel , Legrand Olivier , Dombret Herve , Fenaux Pierre , Chomienne Christine
Published: April 2020

Although patients with Acute Promyelocytic Leukaemia (APL or AML M3) and the classical t(15;17) translocation can now benefit from efficient treatments, some 10 to 20% of patients still relapse at 5 years. In order to improve the clinical outcome of APL patients, the early detection of disease reactivation should allow treatment at molecular relapse rather than at complete haematological relapse. To this end accurately defining molecular relapse is compulsory. Recent studies suggest that RQ-PCR methods applied to the detection of minimal residual disease (MRD) could provide such criteria. Our laboratory has established a RQ-PCR method (Cassinat et al., 2001) using Taqman technology. Standard curves are established and the PML-RARa copy number is normalized according to the expression of the housekeeping gene PBGD. A total of 260 APL patients (APL study Group) have been followed from 2000 to 2004 with a total of 970 samples analyzed. Median follow up for all these patients was 24 mths (range: 1–83 mths) with a median number of samples per patient of 6 (range: 1–28). During this period, 38 patients presented at least one positive result after a median negative period of 13 mths and 13 of them relapsed (median follow-up of 17 mths, range from 3 to 43mths). To evaluate the value of this re-positivity, we tried to determine whether a predictive threshold of PML-RARa copy as assessed by RQ-PCR method could allow detecting patients with molecular relapse. At the time of the first positive result, patients were in hematological CR. Patients were grouped according to the normalized PML-RARa copy number and the risk of relapse was studied in each group. No relapse (n= 0/17; 0%) was noted in patients with less than 10 copies (group A). 38% relapses (n= 5/13) in patients with 10 to 102 copies (group B) and 100% relapses (n=8/8) in patients with more than 102 normalized copies (group C). Negative RQ-PCR was reached again in all patients that did not relapse. Interestingly in groups B and C, interval to hematological relapse appeared to be related to the number of PML-RARa copies at first positivity (median interval to relapse was 6 months in group B and 2 months in group C). For 3 positive patients of group B, 3 consecutive samples were analysed until relapse: copy numbers increased exponentially. From these results we conclude that RQ-PCR is a powerful tool to define the molecular relapse in APL patients as it is able to accurately identify two subgroups of patients with either a 100% or no risk of relapse wilts median follow up of 24 mths. These patients with true molecular relapse should probably receive salvage therapy without waiting for the hematological relapse.
Primary CNS Lymphoma - High-Dose-Chemotherapy with Autologous PBSCT and Hyperfractionated Radiotherapy as First-Line-Therapy - Results of a Multicenter Phase II Study.
Authors: Illerhaus Gerald , Marks Reinhard , Derigs Guenther , Peschel Christan , Frickhofen Norbert , Guttenberger Roland , Kubin Thomas , Ostertag Christoph B. , Finke Juergen
Published: April 2020

Primary NHL of the CNS (PCNSL) carries a poor prognosis despite initial response to steroids and radiotherapy (RT). Addition of methotrexate (MTX) to RT has improved the prognosis of patients (pts) with PCNSL, but a significant proportion of patients are still not cured. To improve relapse free survival and to reduce neurotoxicity we initiated a multicenter phase II study with early dose intensified chemotherapy (CT) and PBSCT followed by hyperfractionated whole-brain radiation (WBRT) for pts aged under 65yrs. The use of high-dose (HD) lipophilic blood-brain-barrier-penetrating agents (BCNU, Thiotepa) in addition to maximum doses of water-soluble agents (MTX, AraC) is a novel approach in the treatment of PCNSL. To reduce the risk of delayed neurotoxicity, intrathecal chemotherapy was completely avoided and CT was administered before RT. Induction treatment included 3 repetitive cycles of HD-MTX (8g/m2). AraC (2x 3g/m2) and thiotepa (40mg/m2) followed by rG-CSF were used for stem-cell-mobilisation. The conditioning regimen included BCNU (400mg/m2) and thiotepa (2x5mg/kgBW) prior PBSCT. Additional hyperfractionated WBRT (45 Gy, 2x1Gy/d) was administered as consolidation. From 1999 to 2003 thirty pts (age under 65y) have been enrolled in the study (median age 54, range 30–64y). 21 of 30 pts responded to HD-MTX (6 CR, 15 PR). 22/30 pts received high dose CT and autologous PBSCT according to the protocol. Four pts refractory to MTX proceeded to RT directly and 1 patient died from PD subsequently. One patient died because of treatment-related liver toxicity after High-Dose MTX. Beside cytopenia no severe acute toxicities [WHO Grade 3 or 4] were observed after high-dose chemotherapy. After intention-to-treat analysis the response-rate was 89%. All patients that completed the protocol obtained CR (22/22). Three of these pts died, two died during follow-up due to relapse after 1 or 5 years respectively, the third patient died after 25 mo due to progressive heart failure. With a median follow-up of 42 months (range 3–84 mo) the overall survival of all pts included and pts that fulfilled the protocol is 71,3% and 86,5%, respectively. We conclude that sequential systemic application of high-dose differential acting cytostatic agents with consolidating hyperfractionated radiotherapy is very effective and well tolerable. In a new multicenter phase II study, pts will be treated with more intensive high dose CT and PBSCT omitting consolidating radiotherapy.
The Factor XI Receptor on Collagen Activated Platelets Is gC1qR/P33.
Authors: Baglia Frank A. , Bradford Harlan R. , Colman Robert W. , Peerschke Ellinor I.B. , Ghebrehiwet Berhane , Walsh Peter N.
Published: April 2020

Factor XI (FXI) binds to high affinity sites on the surface of thrombin-stimulated platelets where it is efficiently activated by thrombin. We have previously shown that FXI binding to thrombin-activated platelets is mediated by residues in the FXI Apple 3 (A3) domain interacting with leucine-rich repeat sequences within the NH2-terminal globular domain of glycoprotein (GP) Ibα (J. Biol. Chem.277:1662–1668, 2002; J. Biol. Chem.279:45470–45476, 2004; J. Biol. Chem.279:49323–49329, 2004). The cell surface receptor protein gC1qR/P33, expressed on collagen-stimulated platelets, binds FXII and high molecular weight kininogen (HK). We now provide evidence that the FXI binding site on collagen-activated platelets is gC1qR/P33: 1) two anti-gC1qR/P33 mAbs, 60.11 and 74.5.2, inhibited FXI binding to collagen-stimulated platelets whereas SZ-2 (a monoclonal antibody directed against GPIbα) does not; 2) by surface plasmon resonance, FXI was bound specifically to gC1qR/P33 in a Zn2+-dependent fashion (Kdapp ~5 nM); 3) soluble gC1qR/P33 inhibited the binding of FXI to collagen-stimulated platelets with an IC50 ~10 nM. Optimal binding of FXI to collagen-activated platelets (~1,500 binding sites per platelet; Kd ~10 nM) was achieved in the absence of HK. A synthetic peptide comprising residues Asn 235-Arg 266 within the FXI A3 domain inhibited FXI binding to collagen-activated platelets with a Ki ~10 nM which is identical to the Kd for FXI binding to platelets, whereas A1, A2 and A4 peptides had no effect. We then investigated whether gC1qR/P33 could promote activation of FXI by thrombin or FXIIa. In the presence of Zn2+ and Ca2+ ions, and the absence of HK, FXI was activated by thrombin in the presence of gC1qR/P33 at rates comparable to those seen in the presence of thrombin-activated platelets, whereas rates of FXI activation by FXIIa in the presence of gC1qR/P33 were 8-fold lower. Thus, thrombin is the preferred activator of FXI bound to gC1qR/P33. We conclude that whereas FXI binds to GPIbα on thrombin-activated platelets, it binds to gC1qR/P33 on collagen-activated platelets. Finally, we show here that both collagen and vonWillebrand factor can inhibit FXI binding to GPIbα, suggesting that on collagen-activated platelets gC1qR serves as an alternative FXI receptor that promotes FXI activation by thrombin.
Primary Acute Myeloid Leukemic Blasts Display Constitutive Serine 585 Phosphorylation within the 14-3-3 Binding Motif of the GM-CSF/IL-3 Receptor Required for Survival Signalling.
Authors: Thomas Daniel , Powell Jason , Lee Szu-Hee , Lopez Angel F. , Mark Guthridge
Published: April 2020

Acute myeloid leukemia (AML) is characterised by defects in the regulation of hematopoietic cell survival, differentiation and proliferation. IL-3 and GM-CSF exert potent survival and proliferative effects on a range of myeloid progenitor cells. Previously we have identified a novel cytoplasmic motif of the human GM-CSF/IL-3 receptor beta chain responsible for coordinating survival and proliferative responses in primary hematopoietic cells. The motif contains a serine residue (585) that is phosphorylated in response to cytokine and couples to the PI3K/AKT pathway via the 14-3-3 adaptor protein and is required for cell survival (Guthridge et al, Molecular Cell6:99, 2000;Blood103:820, 2004). An adjacent tyrosine residue (577) within the motif is phosphorylated via Jak2 and associates with proliferative function. We investigated the phosphorylation status of the beta chain motif in response to GM-CSF stimulation in blasts cells from 12 patients with AML, and in blood monocytes from 6 healthy volunteers. Leukemic samples consisted of >99% blasts after buffy coat separation according to cytospin morphology. All leukemic blasts and blood monocyte samples expressed alpha and beta chain by flow cytometry. The phosphorylation status of residues 577 and 585 were examined utilising beta chain phospho-specific antibodies, after stimulation of AML blasts or blood monocytes with GM-CSF and beta chain immunoprecipitation. Human monocyte preparations all showed inducible serine 585 phosphorylation and tyrosine 577 phosphorylation. In contrast AML blasts displayed strong constitutive serine 585 phosphorylation in 10 out of 12 patients. No discernible tyrosine 577 phosphorylation in the absence of cytokine was observed for the AML samples. Beta chain phosphorylation pattern was independent of FAB classification. Inhibition of Jak2 did not prevent serine 585 phosphorylation of the receptor. Constitutive Akt activation was observed in AML blasts. The data provides evidence for dysregulation of a novel GM-CSF/IL-3 receptor mediated survival pathway involving serine/threonine kinases, 14-3-3 and Akt that is active in primary AML blasts in the majority of patients. This data would suggest that dysregulated activation of serine/threonine kinases and/or phosphatases at the receptor level may contribute to leukemogenesis.
Validation of the Prognostic Significance of the Disease Specific Gene CLLU1 in Chronic Lymphocytic Leukemia.
Authors: Josefsson Par , Jurlander Jesper , Geisler Christian H. , Pedersen Lone B. , Bach Lone , Andersen Mette K. , Leffers Henrik , Buhl Anne Mette
Published: April 2020

We have recently identified and cloned a novel disease specific gene CLLU1, with a strong prognostic significance in a small test cohort of CLL patients (ASH 2004, #770). To validate this finding in a larger series, 176 newly diagnosed, previously untreated CLL patients referred to Rigshospitalet, Copenhagen in 1991–1999, were investigated. Clinical characteristics of the population were: 116 stage A patients (66 %), median age 69.9 years, 98 males (56%), median follow up 59 months. The end points for the statistical analysis were overall survival and time to initiation of treatment. At time of analysis 91 (52%) patients had died and 104 (59%) had commenced treatment. Frozen patient samples, taken at time of diagnosis, were investigated for CLLU1 expression, as well as for mutational status, CD38 expression, ZAP-70 expression and cytogenetic aberrations. CLLU1 expression was assayed by QRT-PCR using the cDNA1 splice variant, and expressed as fold upregulation above the CLLU1 level in normal B-cells. CD38 and ZAP-70 analysis was performed by flow cytometry, using a 20 % cut-off level. Cytogenetic analysis was performed by FISH. The previously reported (ASH 2004, #770) median upregulation of CLLU1 in CLL cells was accurately reproduced and confirmed (25.5-fold (N = 176) vs. 27.7-fold (N = 59) in test population). Also in accordance with the pilot study, segregation of the patients based on the median CLLU1 expression level divided the population in two groups with significantly different times to initiation of treatment and borderline significant difference in overall survival. However, further analysis found an optimal cut-off level at 40-fold upregulation of CLLU1 expression; use of this cut-off demonstrated a highly significant difference in overall survival for patients with upregulated expression of CLLU1 (p = 0.0023). The median overall survival was 60.2 months for patients with CLLU1 expression above 40-fold compared to 100.8 months for the group with lower expression level. Likewise the time to initiation of first treatment was significantly shorter in the high-level expression group (p = 0.0018, median time to first treatment 9.3 months vs. 54.9 months). Furthermore, CLLU1 expression was upregulated in all poor prognostic subgroups investigated: Binet stage B or C, IgVH unmutated, unfavorable cytogenetics, high CD38 expression and high ZAP-70 expression. Our new study confirms that CLLU1 is a strong prognostic factor in CLL, comparable to other established prognostic markers. The exclusive upregulation of CLLU1 expression in CLL suggests a putative role for CLLU1 in the pathogenesis of CLL, and makes this gene a strong candidate for future gene-targeted treatment strategies.

Figure Figure
17p Deletion Predicts for Inferior Overall Survival after Fludarabine - Based First Line Therapy in Chronic Lymphocytic Leukemia: First Analysis of Genetics in the CLL4 Trial of the GCLLSG.
Authors: Stilgenbauer Stephan , Kröber Alexander , Busch Raymonde , Eichhorst Barabara , Kienle Dirk , Winkler Dirk , Hopfinger Georg , Lichter Peter , Emmerich Bertold , Hallek Michael , Döhner Hartmut ,   The GCLLSG
Published: April 2020

The CLL4 trial evaluated first line treatment with fludarabine (F) or F + cyclophosphamide (FC) among 375 CLL patients up to 65 years enrolled between 1999 and 2003. FC resulted in a higher overall response rate (94% vs. 83%; P=0.001), longer median progression free survival (PFS) (48 vs. 20 months (m); P=0.001), but no difference in overall survival (OS) (Eichhorst et al., ASH 2003 and submitted). At enrollment, genomic aberrations and the VH mutation status have been analyzed for 307 and 280 cases thus far. The most frequent genomic aberrations were 13q-: 50.3%, 13q- single: 34.1%, 11q-: 20.3%, +12q: 13.7%, 6q-: 8.7%, 14q-: 6.1%, and 17p-: 4.9%. VH was mutated in 33.9% and unmutated in 66.1% of cases. The aberrations 13q- and 13q- single were more frequently observed in the VH mutated subgroup (45.4% vs. 32.7% and 35.5% vs. 18.8%, respectively), while 11q- (26.9% vs. 12.2%) and 6q- (14.2 vs. 1.7%) were more common in the VH unmutated subgroup. Clinical outcome was evaluated in subgroups defined by genomic aberrations and VH status for both treatment arms combined. In univariate analyses, significant associations were found for the following parameters: the overall response rate was significantly lower in the subgroup with 17p- (53.8% vs. 89.6%, P=0.001), the median PFS was significantly shorter in the subgroups with 11q- (17.4 vs. 26.8 m, P=0.044), 14q- (14.5 vs. 24.5 m, P=0.018), and 17p- (11.0 vs. 24.1 m, P=0.002), and the median OS was significantly shorter in the subgroup with 17p- (15.9 m vs. not reached, 75% survival at 43.8 m, P<0.001, Figure 1). Multivariate analysis was performed including the treatment arms, specific genomic aberrations, and the VH mutation status as possible prognostic factors: the parameters with significant adverse impact were F monotherapy (HR 1.70, 95%CI 1.12-2.58, P=0.013) and 17p- (HR 3.67, 95%CI 1.66-8.14, P=0.001) regarding PFS, but only 17p- (HR 7.32, 95%CI 2.44-21.90, P<0.001) regarding OS. In conclusion, this first analysis of the prospective CLL4 trial shows that 17p- CLL is characterized by a short OS after F-based first line therapy. In future trials, alternative primary treatment strategies of proven efficacy in 17p- CLL such as alemtuzumab should be considered for these patients.

Figure Figure
Early Results from LRF CLL4: A UK Multicenter Randomized Trial.
Authors: Catovsky Daniel , Richards Susan , Hillmen Peter
Published: April 2020

On behalf of the UK NCRI CLL Trials Group and the Adult Leukaemia Working Party. The CLL4 trial opened in 2/99 and closed in 10/04. 783 previously untreated patients were randomized and 6 were excluded. 12% of patients were entered from outside the UK (Argentina, Ireland, Italy, New Zealand and Russia). Median follow-up is 21 months. The male:female ratio was 2.8:1 and the distribution by Binet stages was A progressive 25%, B 45% and C 30%. One third of cases were aged <60 years, one third 70 years or over. Patients were randomized between chlorambucil (10mg/m2 x 7 days)(n=387) and fludarabine alone (FDR)(n=194) or in combination with cyclophosphamide (FC) (n=196). FDR and FC were given intravenously (IV) in the first 2 years (FDR 25mg/m2 x 5 days and FDR 25mg/m2 plus cyclophosphamide 250mg/m2 for 3 days). Early in 2001 oral FDR became available and the protocol was modified to allow its use: FDR 40mg/m2 x 5 days and FDR 24mg/m2 plus cyclo 150mg/m2, both for 5 days. Differences between the IV and oral formulations will be presented elsewhere in this meeting (Hillmen et al). Responses were assessed by bone marrow biopsies. Responses to treatment are summarized in the table below in 661 patients with available data. Within each age group treatment affected response (CR+nodPR v PR/NR/PD: p=0.0003 for <60 years, p<0.0002 for 60–69 years and p=0.002 for ≥70 years) with highest responses with FC and lowest with chlorambucil. The toxicities profile showed more neutropenia with FC (55%) than FDR (40%) and chlorambucil (29%) and more hospitalisations with FC and FDR. However, and as reported in ASH 2004, there were more hemolytic anemias after chlorambucil (13%) than with FDR (10%) and FC (4%). There was more nausea/vomiting and alopecia with FC than with the other regimens. Progression free survival (PFS) up to 10/04 showed fewer events with FC (O/E 0.5) than FDR (O/E 1.1) and chlorambucil (O/E 1.3); FDR+FC v Chl p<0.00005; FC v FDR p<0.0005. PFS at 3 years is chlorambucil 23%, FDR 31% and FC 62%. Analysis of survival thus far showed no differences between the three regimens. We conclude that the combination of FC is associated with a greater quality of responses, more myelotoxicity and less hemolytic anemia than FDR and chlorambucil and it could become the new standard regimen for CLL upon which future regimes will build. CLL4 has generated a wealth of prognostic information: FISH, VH mutations, expression of CD38 and ZAP-70 which will allow a better characterization of risk groups and their response to first-line therapy. Some of these analyses will be presented in this meeting (Oscier et al).

Chlorambucil FDR FC * within the first year and not assessable for response No. patients 309 176 176 Complete Response (CR) 8% 15% 37.5% Nodular partial response (NPR) 18.5% 27% 22% Partial response (PR) 42.5% 35% 31% No response/progressive disease (NR/PD) 28% 18.5% 6.5% Died early* 3% 4.5% 3%