bluuebunny/crossref_metadata_2025 huggingface dataset:
NIPA Targets Nuclear Cyclin B1 in a Cell-Cycle Dependent Manner.
Authors: Bassermann Florian C. , von Klitzing Christine , Kluempen Silvia , Bai Ren-Yuan , Ouyang Tao , Morris Stephan W. , Peschel Christian , Duyster Justus
Published: October 2019

Ubiquitin-mediated destruction of regulatory proteins marks the vital means of controlling cell cycle progresssion. The E3 ubiquitin-ligases are prominent in this process, as they allow the transfer of ubiquitin to the target protein and mediate substrate binding specificity. Recently, a new class of E3 ligases referred to as SCF complexes has been identified that consists of four subunits:SKP1, Cul1, Roc1 and an F-box protein, the latter of which determines substrate specifity.

We previously reported the cloning of NIPA (nuclear interaction partner of ALK) in complex with constitutively-active oncogenic fusions of ALK, which contribute to the development of certain lymphomas and sarcomas. Subsequently we characterized NIPA as a human F-box protein that determines a novel SCF complex (SCFNIPA) whose cell cycle regulated activity is restricted to interphase to allow for substrate expression at G2/M and mitosis. Phosphorylation of NIPA in late S-phase was found to be the underlying mechanism of SCFNIPA inactivation.

We have now identified the key mitotic regulator cyclin B1 to serve as the relevant substrate of the SCFNIPA complex. This targeting process is restricted to interphase and directed towards the nuclear pool of cyclin B1. Inactivation of NIPA by siRNAs results in nuclear accumulation of cyclin B1 in interphase and an elevation of cells in S-phase and mitosis. In contrast, expression of a phosphorylation deficient NIPA mutant that retains constitutive SCFNIPA activity throughout the cell cycle arrests cells at early prophase thus delaying mitotic entry. Both effects are likely attributable to either cyclin B1 accumulation in the case of NIPA inactivation by siRNA or untimely cyclin B degradation at G2/M upon expression of the constitutively active SCFNIPA complex. Cyclin B1 is physiologically kept cytoplasmic during interphase and premature nuclear accumulation has been associated with untimely mitotic entry, loss of checkpoint control and genomic instability. Our data provides a mechanism to inhibit premature nuclear accumulation of cyclin B1 in the mammalian cell cycle. NIPAs association with NPM-ALK of ALCL has been shown to be associated with NIPA phosphorylation and thus to the inactivation of the SCFNIPA complex. The mechanism described above may therefore provide a framework for understanding how this oncogene interferes with the physiologic regulation of cyclin B - a potential mechanism by which NPM-ALK transforms hematopoietic cells.
Adenoviral Mediated TGF-β1inhibition in a Murine Model of Myelofibrosis Can Prevent and Cure Bone Marrow Fibrosis Development.
Authors: Gastinne Thomas , Wagner-Ballon Orianne , Tulliez Micheline , Chagraoui Hedia , Vainchenker William , Giraudier Stephane
Published: October 2019

Myelofibrosis is a clinical feature of several hematopoietic disorders and is most prominent in idiopathic myelofibrosis. It is characterized by excessive deposits of extracellular matrix proteins which occur as a marrow microenvironment reactive response to cytokines released from the clonal malignant myeloproliferation. The observation that mice exposed to high systemic levels of thrombopoietin (TPO) invariably develop a myelofibrosis within a period of 4 to 8 weeks has allowed the demonstration of the crucial role of TGF-b1 released by hematopoietic cells in the promotion of myelofibrosis (Chagraoui et al Blood, 2002 ; 100 :3495). The aim of this study was to investigate whether TGF-b1 inhibition could directly inhibit the fibrosis development in a curative approach of this murine model.

An adenovirus encoding for TGF-b1 soluble receptor was constructed. SCID mice were infected with this adenovirus to measure the TGF-b1 inhibitory effect. We measure TGF-b1-Rs (TGF-b1 soluble receptor) concentration and TGF-b1 levels in 5 groups of mice injected with 0- 6.5 106- 6.5 107- 6.5 108-and 6.5 109 pfu/mice. We demonstrated that 6.5 108 pfu/mice was able to completely inhibit 10 fold increase of circulating TGF-b1 and that 1 microgram of soluble receptor was able to inhibit 1 ng of TGF-b1.

All subsequent experiments were performed by injecting 1 109 pfu/mice either shortly after transplantation (preventive) or after 30 days (curative). Briefly, 46 mice (2 individuals experiments n= 16 and n= 30) were transplanted with 5-FU treated syngeneic bone marrow cells transduced with a retrovirus encoding for murine TPO. Retroviral transduction efficiency varied from 67% to 87%. All mice developed a myeloproliferative syndrome characterized by an increase in platelet count, a leukocytosis, an increase in progenitor cells in the blood circulation and a 1000-fold increase in TPO level. TGF-b Rs (13 to 32 micrograms/ml) was detected in the blood of all mice injected with the TGF-b1-Rs Adeno. A dramatic decrease in TGF-b1 level was noticed in all these treated mice in comparison to the control groups. Histological analysis demonstrated that the two approaches (curative or preventive) were identical to abolish fibrosis development in bone marrow as well as in the spleen. However, all mice which received the TGF-b1-RS adenovirus developed a hepatitis leading to death 6 weeks to 3 months after infections.

In conclusion our approach support the major role of TGF-b1 in myelofibrosis development as previously demonstrated by our group using TGF-b1 KO mice. Moreover, the present results suggest that TGF-b1 inhibition could be a relevant approach to treat patients presenting a TGF-b1 mediated bone marrow fibrosis.
In Vitro Analysis of Myeloid Proliferation Caused by Oncogenic Kras.
Authors: Braun Benjamin S. , Gavin Joehleen , Tuveson David A. , Jacks Tyler E.
Published: October 2019

Somatic mutations in NRAS and KRAS2 are found in approximately one third of cases of myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD) or acute myeloid leukemia (AML). Cases without RAS mutations frequently demonstrate genetic lesions which result in activation of Ras, such as FLT3 mutations, Bcr-Abl expression, or inactivation of the NF1 tumor suppressor. We have exploited a conditional oncogenic Kras allele that was generated by homologous recombination to directly address the effects of expressing a mutant K-RasG12D protein from the endogenous promoter in primary hematopoietic cells. We crossed this mutation into the Mx1-Cre strain, and induced Cre recombinase expression by administering polyinosinic-polycytidilic acid. These mice uniformly and rapidly develop a progressive MPD with leukocytosis and massive splenomegaly, and succumb after a median of 84 days. In vitro, myeloid cells expressing an oncogenic allele of Kras from the endogenous promoter demonstrate a dramatic hyperproliferative phenotype and hypersensitivity to hematopoietic growth factors. We have further characterized these findings, and have found that, despite the rapid accumulation of mature myeloid cells in vivo and in vitro, proliferation occurs mainly in immature, Mac1-negative progenitors. Therefore, myelopoiesis in Mx1-Cre, KrasG12D mice is analagous to, but more productive than that in normal mice. Kinetic data is most consistent with a major effect of K-rasG12D on cell cycle activity, with minimal effects on apoptosis. This system provides a novel and tractable platform for determining how oncogenic RAS alters myeloid homeostasis, for investigating how oncogenic RAS influences signaling networks in primary cells, and for preclinical testing of targeted therapeutics in primary cells.
HMGA2, a Member of the High Mobility Group Gene Family, Is Expressed at the Protein Level in Peripheral Blood CD34+ Cells of Patients with Idiopathic Myelofibrosis and Polycythemia Vera.
Authors: Huang Stephen , Xu Minjiang , Bruno Edward , Barosi Giovanni , Prchal Josef , Hoffman Ronald
Published: October 2019

Idiopathic myelofibrosis (IM) is a chronic myeloproliferative disorder associated with increased numbers of CD34+ cells circulating in the peripheral blood (PB). To characterize these cells, we transplanted CD34+ or CD34− cells from either G-CSF mobilized PB or IM PB into NOD/SCID mice to test their hematopoietic stem cell function. IM CD34+ cells, but not CD34−, cells engrafted in NOD/SCID mice, demonstrating that IM PB CD34+ cells contain true bone marrow repopulating cells. Furthermore, the differentiation program of IM CD34+ cells was quite different than that of CD34+ cells isolated from normal donors receiving G-CSF. G-CSF mobilized PB CD34+ cells generated predominantly CD19+ cells (B-lymphocytes), while IM PB CD34+ cells generated predominantly myeloid cells as well as larger numbers of CD41+ cells (megakaryocytes), but few CD19+ cells. The molecular basis for this stem cell defect in IM remains poorly defined. We hypothesized that the High Mobility Group Gene, HMGA2, might play a role in the biogenesis of IM. HMGA2 is a nuclear protein, normally expressed only during embryonic and fetal development, which acts as an architectural transcription factor important in the growth and differentiation of cells of mesenchymal origin. It has been reported that this gene has a direct effect on chromatin configuration by promoting DNA relaxation and that it may control the transcriptional activities of several genes. Rearrangement of the HMGA2 gene has frequently been detected in human benign tumors of mesenchymal origin including lipomas and sarcomas. In addition, the gene has been shown to be overexpressed in squamous cell carcinomas of the oral cavity. Previous work has suggested that HMGA2 is overexpressed in the PB mononuclear cells of patients with IM at the mRNA level (Andrieux, J. et al, Genes, Chromosomes & Cancer, 39: 82, 2004). Since IM and polycythemia vera (PV) are hematological malignancies that originate at the level of the hematopoietic stem cell, we examined the expression of HMGA2 in CD34+ and CD34− cell fractions isolated from the PB of patients with IM and PV as well as G-CSF mobilized normal donors. Using Western Blotting, we were unable to detect HMGA2 protein in either G-CSF mobilized CD34+ or CD34− cells. By contrast, HMGA2 was clearly expressed in the CD34+ cell fraction, but not the CD34− fraction isolated from IM and PV patients. These data indicate that increased HMGA2 protein levels are unique to IM and PV CD34+ cells and that the aberrant expression of this gene in the CD34+ progenitor cells may contribute to the stem cell defect in these myeloproliferative disorders.
Analysis of the Fate and Function of Antigen-Specific CD8 T Cells during B Cell Lymphoma Progression.
Authors: Brayer Jason , Cheng Fengdong , Horna Pedro , Suarez Ildefonso , Wang Hongwei , Sotomayor Eduardo M.
Published: October 2019

There is now overwhelming evidence that tumor-induced antigen (Ag)-specific T cell tolerance represents a critical problem in tumor immunology. Early studies of CD8 T cell tolerance equated peripheral tolerance with either ignorance or clonal deletion, although more recent evidence has suggested that this may be only partly accurate. While murine modeling outwardly supports the contention that high-affinity tumor-specific CD8 T cell responses are centrally deleted, cognate CD8 T cells displaying an Ag-experienced phenotype can nonetheless be detected in regional draining lymph nodes (dLN) or in non-lymphoid sites where the Ag is present. However, these CD8 T cells are typically deficient in one or more effector functions, including cytokine production, cytotoxicity, or proliferative capacity. To better define the state of Ag-specific CD8 T cell responsiveness in the face of progressive tumor, we adoptively transferred hemagglutinin (HA) Ag-specific Clone 4 (CLN4) CD8 T cells into animals bearing a genetically modified B cell lymphoma expressing HA as a model tumor antigen (A20HA). Analysis of the fate and function of these transferred antigen-specific CD8 T cells revealed that they encountered antigen in vivo, were capable of mounting an initial response to A20HA but this response was not sustained. Indeed, while a prominent CTL activation was observed in the spleen and draining lymph nodes of tumor bearing mice within 14 days of T cell transfer, responses (HA-specific proliferation, IFN-γ production and cytotoxicity) began to wane by day 21 after T cell transfer, and in particular their ability to produce IFN-γ. A similar pattern of transient activation followed by loss of CD8 T cell function has been also observed in an in vivo model of high-dose peptide induced antigen-specific CD8 T cell tolerance. Given our recent demonstration that the disruption of Stat3 signaling in APCs overcomes CD4 T cell tolerance we determined next whether Stat3 deficient APCs may be inherently better at cross-presenting tumor-Ags and elicit therefore a more productive and sustained CD8 T cell response. In an in vitro system in which tumor cells expressing a model tumor antigen (EL4mOVA) were cultured with APCs genetically devoid of Stat3 signaling and anti-OVA CD8 T-cells (OT-I), we found that these T cells displayed an enhanced function relative to antigen-specific CD8 T-cells that encountered antigen on APCs with an intact Stat3 signaling. Currently, we are investigating whether CD8 T-cell tolerance to tumor antigens occurred -or not- in tumor bearing mice with a genetic disruption of Stat3 signaling in APCs. Furthermore, given the emerging role of other members of the STAT family in regulation of APC function, we are exploring whether targeted disruption of Stat1, 4 and 6 can alter the ability of the CD8 T-cell to sustain a protective response or, more importantly to recover function once tolerance is induced.
Enhanced Platelet Aggregation and Thrombogenic Tendency in Adiponectin-Deficient Mice.
Authors: Kato Hisashi , Kashiwagi Hirokazu , Shiraga Masamichi , Honda Shigenori , Miyata Shigeki , Yamamoto Junichiro , Kurata Yoshiyuki , Funahashi Tohru , Shimomura Iichiro , Tomiyama Yoshiaki , Kanakura Yuzuru
Published: October 2019

Adiponectin is a 30 kDa protein secreted specifically from adipocytes and structurally composed of two distinct domains, C-terminal collagen-like domain and N-terminal complement C1q-like globular domain. Adiponectin is abundantly present in plasma at high concentration ranging from 2 to 30 μg/ml. The plasma levels of adiponectin decreased in patients with obesity and diabetes. Recently it has been demonstrated that adiponectin has an anti-atherogenic activity. Hypoadiponectinemia is an independent risk factor for coronary artery disease in men. However, the role of adiponectin in hemostasis and thrombosis still remains obscure. In this study, we examined its role in hemostasis and thrombosis using adiponectin-deficient (APN-KO) mice (Nat. Med. 2002 Maeda et al.). APN-KO mice were fed by normal chaw and studied at 8–12 weeks old. There were no differences in platelet counts, PT, APTT and plasma fibrinogen levels between APN-KO and Wild-Type mice. Neither Wild-Type nor APN-KO mice showed detectable atherosclerotic lesion in carotid artery as well as whole aorta. We examined tail-bleeding times as a measure of primary hemostasis. The tail bleeding time was 96.9 ± 34.9 seconds in APN-KO mice, which was shorter than that in wild type mice (130.9 ± 52.1 seconds, n=30, p<0.05). We next studied thrombus formation in mice carotid artery using He-Ne laser induced in vivo thrombus formation model. Thrombus formation was induced by the interaction of irradiated He-Ne laser with evans blue dye injected into blood flow. The thrombus volumes formed during 10 minutes were significantly larger in APN-KO mice (6.74 ± 2.87 x 107 arbitrary units for wild-type v.s. 13.4 ± 4.25 x 107 arbitrary units for APN-KO mice, n=10, p<0.01). Adenovirus-mediated supplement of adiponectin compensated for the thrombotic tendency in APN-KO mice. In order to clarify the effects of adiponectin on platelet functon, we performed ex vivo experiments. In platelet aggregation studies under stirring conditions using platelet-rich plasma, platelet aggregation induced by low concentrations of agonists (ADP 2.5μM, collagen 2.5μg/ml, PAR4 peptide 75μM) was enhanced in APN-KO mice. Again the adenovirus-mediated supplement of adiponectin compensated for the enhancement of platelet aggregation. We next studied the thrombus formation on collagen coated surface under flow conditions. The thrombus formation was enhanced in APN-KO mice under shear rate at 250s−1. Our data provide a first evidence that adiponectin plays a role in hemostasis and thrombosis as a negative modulator of platelet function.
Multiple Hepatic Clearance Receptors Facilitate Elimination of Secretory Mucins that Can Enter the Bloodstream in Pathological States and Generate Platelet-Rich Microthrombi.
Authors: Wahrenbrock Mark G. , Varki Ajit
Published: October 2019

Epithelial cells that line hollow organs typically secrete mucins and their proteolytic fragments vectorially, into the lumen of the organ. Fragments of these large, heterogeneously O-glycosylated molecules can enter the blood stream during injury, inflammation or malignancy involving such organs. We have previously shown that intravenous injection of human carcinoma mucin fragments into mice triggered a “Trousseau”-like syndrome consisting of extensive formation of platelet- and leukocyte-rich microthrombi that were thrombin-independent, but L-and P-selectin-dependent and heparin-sensitive (Wahrenbrock et al., 2003, J Clin Invest, 112, 853–62). Thus, we hypothesized that mechanisms should exist to clear such mucin fragments when they enter the bloodstream in various pathological states. Indeed, intravenously injected radiolabeled mucin fragments had a circulating half-life of ~1 minute in mice, due to rapid and specific clearance by liver reticuloendothelial cells. Competition of uptake by inhibitors of known glycan-recognizing hepatic clearance receptors showed that several overlapping and non-overlapping systems work in concert to remove mucin fragments from the circulation. The systems involved include the hepatic asialoglycoprotein receptor, the macrophage Gal/GalNAc and Mannose receptors, and the endothelial S4GGnM and hyaluronan receptors. Studies of genetically deficient mice, and competition studies amongst various differently glycosylated mucins confirm this result. Thus, multiple hepatic clearance receptors collaborate to varying degrees to ensure elimination of the many polydisperse and differentially glycosylated forms of secretory mucins that might enter the blood circulation from various hollow organs, thus limiting their ability to cause pathology involving disseminated platelet and leukocyte aggregation. This process could also potentially modulate circulating levels of mucin epitopes that are sometimes used as diagnostic criteria in cancer (e.g., CA125, CA19-9 etc.). Furthermore, it can explain why mucin-type clustered O-glycosylation is very uncommon on circulating plasma proteins.
Sequential Treatment with Reduced Intensity Allogeneic Stem Cell Transplantation and Imatinib for Chronic Myelogenous Leukemia (CML.
Authors: Champlin Richard , Ghosh Shubhra , McCormick Gloria , Andersson Borje , de Lima Marcos , Anderlini Paolo , Qazilbash Muzaffar , Giralt Sergio , Cortes Jorge
Published: October 2019

Chronic myelogenous leukemia is uniquely sensitive to the graft-vs-malignancy effects. Donor lymphocyte infusion (DLI) can induce a high rate of durable remissions in patients relapsing after BMT. Imatinib mesylate may also induce remission in patients with recurrent disease post BMT. This suggests a strategy of sequential therapy using a reduced intensity allogeneic transplant to achieve initial cytoreduction with post transplant imatinib and DLI for recurrent disease as an approach to reduce the risks associated with allogeneic transplantation. We also assessed whether the addition of imatinib to the preparative regimen could be done without increased toxicity to take advantage of the synergistic effects of imatinib with cytotoxic chemotherapy. Patients received a reduced intensity regimen of imatinib (800 mg/d), busulfan ( 8 mg/m2 IV), fludarabine (160 mg/m2), Thymoglobulin (5 mg/kg) and allogeneic stem cell transplantation. Patients receive tacrolimus and mini-methotrexate for GVHD prophylaxis. Those who had molecular evidence of disease at 3 months receive imatinib and those with residual disease 3 months later receive DLI.

14 patients with Ph+ CML have been entered and have follow-up greater than 100 days. 8 had a matched sibling, 5 an unrelated donor and 1 a one antigen mismatched related donor. 4 received marrow and 10 blood progenitor cell transplants. All had received prior imatinib and all except 2 had failed to achieve a major cytogenetic response. 7 were in first chronic or accelerated phase and 7 were in > 2nd chronic phase after prior accelerated phase or blast crisis. All had hematologic control while receiving imatinib-based therapy. Median age was 48 (range 23–70) years.

All patients achieved engraftment and none have died from treatment related causes. 3 developed grade 1–2 acute GVHD and 4 have developed chronic GVHD. 13 patients achieved cytogenetic complete remission, one patient failed to respond and was taken off study. 3 had cytogenetic progression with one having fatal recurrence of lymphoid blast crisis. 9 patients who were pcr positive for bcr-abl rearrangement at 3 months received imatinib; 7 have had ongoing disease reduction by quantitative pcr, 1 currently has no response and 1 patient had progressive disease and was taken off study. None have received DLI. 13 patients remain alive with 11 in cytogenetic remission.

In summary, addition of imatinib to the preparative regimen was not associated with increased toxicity. This strategy allows for allogeneic transplantation using a reduced intensity regimen with capture of persistent or recurrent disease by post transplant imatinib and/or DLI. The low rate of severe morbidity and treatment related mortality makes this approach attractive for CML patients failing to have an optimal initial response to imatinib based therapy.
The Activation of Stat3 by the Stk Receptor Tyrosine Kinase Requires Src Kinases and Gab2, and Is Essential for Friend Virus Induced, Epo-Independent Growth of Primary Erythroid Cells.
Authors: Ni Shuang , Heal Hami , Xu Jie , Paulson Robert , Correll Pamela H.
Published: October 2019

Friend virus-induced erythroleukemia is a model for the study of the multi-stage nature of carcinogenesis. The early stage of the disease is characterized by a polyclonal expansion of infected erythroid precursor cells, and the later stage is marked by the emergence of fully transformed cells in the spleen, blood, bone marrow and liver, leading to the development of erythroleukemia. Previous findings have demonstrated that a naturally occurring, N-terminally truncated form of the stk receptor tyrosine kinase (Sf-stk) provides signals necessary for the polycolonal expansion of infected cells. Signal transducer and activator of transcription (Stat) proteins play an important role in normal and malignant hematopoiesis. Moreover it has been shown that growth factors can induce Stat3 in a Src family kinase (SFK)-dependent manner. Here we show that dominant negative Stat3 and c-Src significantly inhibit the BFU-e and CFU-e colony number induced by friend virus. In addition, the non-specific SFK inhibitor PP1 also abrogated Epo-independent colony formation. Moreover we found that the inhibition of SFKs by PP1 suppresses Stat3 tyrosine phosphorylation activated by Sf-stk. To elucidate the signaling mechanism by which Sf-stk activates Stat3, we mutated the docking site tyrosines to phenylalanine. Our results indicated that the mutation of Y436F eliminates Sf-stk-induced Stat3 tyrosine phosphorylation. Studies from our lab have further demonstrated that tyrosine 436 of Sf-stk is essential for the cytokine-independent growth of primary erythroblasts induced by Friend virus, by providing a docking site for Grb2, which recruits Gab2 to induce erythroleukemia. To further determine the relationship between Gab2 and Stat3, we generated fusion proteins, in which Sf-stk lacking the docking site tyrosines is fused to Gab2 or Gab1. The Sf-stk/Gab2, but not Sf-stk/Gab1, fusion protein supported Epo-independent colony formation and induced Stat3 tyrosine phosphorylation. In conclusion, these results suggest that SFKs, Gab2 and Stat3 are all important in Friend virus induced erythroleukemia mediated by Sf-stk, and that Gab2 and SFKs lie upstream of Stat3 in this signaling pathway.
Flt3 Internal Tandem Duplications Cooperate with Wnt Signaling in Leukemic Signal Transduction.
Authors: Tickenbrock Lara , Schwable Joachim , Wiedehage Markus , Steffen Bjoern , Choudhary Chunaram , Berdel Wolfgang E. , Muller-Tidow Carsten , Serve Hubert
Published: October 2019

Flt3 internal tandem duplications cooperate with Wnt signaling in leukemic signal transduction

Lara Tickenbrock, Joachim Schwable, Markus Wiedehage, Bjoern Steffen, Chunaram Choudhary, Wolfgang E. Berdel, Carsten Muller-Tidow and Hubert Serve

Department of Medicine, Hematology and Oncology, University of Muenster, D-48149 Muenster, Germany

The type III receptor tyrosine kinase (RTK) Flt3 plays an important role in survival and proliferation of hematopoietic progenitor cells. Somatic mutations of Flt3 consisting of internal tandem duplications (ITD) occur in 25% of patients with acute myeloid leukemia (AML), and are associated with a poor prognosis. They result in Flt3-ligand (FL) independent kinase activation of Flt3. The Wnt signal transduction pathway has recently been implicated to be an important regulator of early hematopoietic stem cell fate decisions. Also, we previously showed that activation of Wnt-dependent Tcf transcriptional activity is induced by the leukemia-associated fusion proteins PML-RAR? and AML1-ETO. Here, we show that Flt3-ITD mutations enhance basal and Wnt3a-stimulated Tcf activity in myeloid cells. Microarray experiments analyzing Flt3-ITD target genes in 32D cells revealed up to 8-fold induction of Frizzled-4, a cell surface receptor for Wnt proteins, by Flt3-ITD mutations. This could be verified by real-time RT-PCR and Western Blot analyses. In functional analyses, we explored the synergism of Flt3-ITD and the activation of Wnt signaling. Flt3-ITD mutations induced the accumulation of ?-Catenin and TCF-dependent transcriptional activity. Also, the presence of Flt3-ITD highly sensitized cells for ?-Catenin-induction by the Wnt3a. Wnt3a incubation enhanced 32D cell proliferation in the presence of activated Flt3 receptors. More importantly, Flt3-ITD-mediated clonogenic growth highly depended on the activity of Tcf transcription factors, since transient transfection of Flt3-ITD cells with dominant negative TCF4 almost abolished 32D cell colony growth. Our results indicate that Flt3-ITD mutations mediate their leukemogenic effects in part through the activation of Wnt-dependent signaling pathways, possibly defining this signal system as a converging point of leukemogenic events elicited by Flt3-mutations and leukemia-associated fusion proteins.
Impact of Cytogenetics on Outcome of Mismatched Unrelated Donor Hematopoietic Stem Cell Transplantation for Adults with AML.
Authors: Tallman Martin , Dewald Gordon , Lazarus Hillard , Gandham Sharavi , Nelson Gene , Logan Brent , Rowe Jacob , Litzow Mark , Keating Armand , Mehta Jayesh , Wetzler Meir , Warren Tanya , Weisdorf Daniel
Published: October 2019

Matched unrelated donor hematopoietic stem cell transplantation (MUD HSCT) is a potentially curative treatment for patients with acute myeloid leukemia (AML). The graft-versus-leukemia (GVL) effect may be potent enough to overcome the otherwise poor prognosis associated with AML though its efficacy for high risk cytogenetic subgroups is uncertain. To test this hypothesis, we analyzed outcomes by cytogenetic risk group in 324 patients in first complete remission (CR1), and 440 in CR2 undergoing NMDP-facilitated MUD HSCT between 1988–2002. Using the SWOG/ECOG classification of cytogenetic risk groups (Slovak et al. Blood, 2000) cytogenetics were classified as favorable in 14% of patients, intermediate in 71% and unfavorable in 16%. 56% of the patients were male and 42% were > 35 years at HSCT. 76% of patients and donors were matched at HLA-A, -B and -DRB1, 17% were mismatched at one or more loci and 7% were potentially matched (serologically matched at HLA-A and -B and potentially allele matched at -DR).

Disease Status N Kaplan-Meier Estimate for Survival at 5 years Kaplan-Meier Estimate for Disease-Free Survival at 5 years Cumulative Incidence for 100 Day Transplant-Related Mortality Cumulative Incidence for Relapse at 5 years * p-value indeterminate; ** p=0.01 CR1 324 32 ± 6% 32 ± 5% 32 ± 5% 18 ± 4%     Intermediate 227 33 ± 7% 32 ± 7% 31 ± 6% 16 ± 5%*     Unfavorable 85 31 ± 11% 31 ± 10% 29 ± 10% 23 ± 9%* CR2 440 36 ± 5% 35 ± 5% 25 ± 4% 16 ± 3%     Favorable 93 46 ± 10% 44 ± 10% 25 ± 9% 10 ± 6%**     Intermediate 313 33 ± 6% 32 ± 5% 27 ± 5% 16 ± 4%**     Unfavorable 34 37 ± 17% 38 ± 16% 15 ± 12% 32 ± 15%**

These data suggest that with the exception of the 5-year relapse rate, results of cytogenetics have little apparent influence on the outcome for patients undergoing MUD HSCT for AML in CR1. In CR2, results in patients with favorable cytogenetics appear to be better than those with intermediate or unfavorable cytogenetics, but are not statistically significantly different. Effective GVL and protection against relapse is observed, even in high risk cytogenetic subgroups. In this retrospective study, other prognostic factors may influence the outcome, but overall survival for patients with unfavorable cytogenetics appears at least as good as previously reported for matched sibling HSCT.
An In-Frame Deletion of Six Amino Acids and a Point Mutation in the Disintegrin Domain of ADAMTS-13 Associates with a Case of Congenital Thrombotic Thromocytopenic Purpura.
Authors: Tao Zhenyin , Nolasco Leticia , Aubrey Bernardo , Rice Lawrence , Moake Joel F. , Dong Jing-Fei
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) is characterized by severe thrombocytopenia, hemolytic anemia, and diffuse and non-focal neurological findings. Microthrombi found in these patients are predominantly composed of platelets and von Willebrand factor (VWF). Recent studies suggest that the systemic thrombosis in TTP is mostly due to the congenital or acquired deficiency of the VWF-cleaving metalloprotease ADAMTS-13, which cleaves the ultra-large and hyperreactive VWF to smaller and less active form found in plasma. Most congenital cases of TTP have so far been identified in children. Here, we report a 60-year-old Caucasian man with a history of chronic relapsing TTP over thirty years, requiring plasma transfusion every 24 days in recent years. Repeat assays showed no ADAMTS13 activity in patient’s plasma under both static and flow conditions without inhibitors being detected. We therefore examined the possible genetic defects in the ADAMTS-13 gene of this patient. Genomic DNA was extracted from patient’s white blood cells and exons of ADAMTS-13 gene were amplified by polymerase chain reaction. The amplified DNA fragments were then screened for mutations by direct DNA sequencing. We identified a deletion of 18 base pairs from G1095 to G1112 (GTGCTCCAAGGGTCGCTG) in the exon10 of ADAMTS-13 gene, resulting in a deletion of six amino acids (C366 to C371) in the disintegrin domain of the metalloprotease. A point mutation (W365C) occurred immediately before the deletion due to a nucleotide realignment. The patient is heterozygote for the deletion. This is the first report of a deletion mutant (without frame shift and truncations) in the disintegrin region that has previously been demonstrated as critical for the ADAMTS-13 function by in vitro mutagenesis, epitope mapping of autoantibody to the metalloprotease in patients with adult acquired TTP, and identification of natural occurring mutations in patients with congenital TTP. Our ongoing studies are to determine the impact of these two mutations on the synthesis, release, and cleavage of ADAMTS-13.
Postremission Therapy in Adult Acute Myeloid Leukemia (AML): A Randomized Comparison of Intensified Consolidation Therapy without Maintenance Therapy Against Conventional Consolidation with Maintenance Therapy -JALSG AML-97 Trial-.
Authors: Miyawaki Shuichi , Sakamaki Hisashi , Ohtake Shigeki , Yagasaki Fumiharu , Mitani Kinuko , Matsuda Shin , Kishimoto Yuji , Miyazaki Yasushi , Asou Norio , Ogawa Yoshiaki , Honda Sumihisa , Ohno Ryuzo
Published: October 2019

Between 1997 and 2001, JALSG conducted a randomized study to assess the optimal post remission therapy for adult AML in the first CR. The JALSG AML97 enrolled 809 previously untreated AML patients (pts) aged 15–64 yrs. Induction therapy consisted of cytarabine (100mg/m2 day1–7) and idarubicin (IDR 12mg/m2 day1– 3). If the patients did not achieve remission after the first induction therapy, the same therapy was given once more. Pts were categorized into good, intermediate or poor risk groups by risk factors based on the previous JALSG AML studies. All CR pts were randomized to receive either the intensified short course post remission regimen (arm A) or the conventional JALSG’s post remission regimen for AML including maintenance therapy (arm B). Arm A: 1) AraC 200mg/m2 day1–5+ Mitoxantrone (MTZ) 7mg/m2 day1–3, 2) AraC 200mg/m2 day1–5+Daunorubicin (DNR) 50mg/m2 day1–3, 3) AraC 200mg/ m2 day1–5+ Aclacinomycin (ACR) 20mg/m2 day1–5, 4) AraC 200mg/m2 day1–5+ Etoposide (ETP) 100mg/m2 day1–5 + Vincristine (VCR) 0.8mg/m2 day 8 + Vindesine (VDS) 2 mg/m2 day10. Arm B: 1) AraC 200mg/m2 day1–5 + MTZ 7mg/m2 day1–3, 2) Behenoyl AraC (BHAC) 200mg/m2 day1–7 + ETP 100mg/m2 day1–5 + DNR 50mg/m2 day1–3 + 6 mercptopurine (6MP) day1–7, 3) BHAC 200mg/m2 day1–7 + ACR 14mg/m2 day1–7, and then 6 courses maintenance therapy: 1) BHAC 170mg/m2 day1–5 + DNR 50mg/m2 day1,4+6MP day1–7, 2) BHAC 170mg/m2 day1–5 + MTZ 5mg/m2 day1–3, 3) BHAC 170mg/m2day1–5 + ETP 80mg/m2 day1,5,7 + VDS 2mg/m2 day1,8, 4) BHAC 170mg/m2 day1–5 + ACR 14mg/m2 day1–4 + 6MP day1–7, 5) BHAC 170mg/m2 day1–5 + DNR 50mg/m2 day1–4 + 6MP day1–7, 6) BHAC 170mg/m2 day1–5 + ETP 80mg/m2 day1,5,7 + VDS 2mg/m2 day1,8.

Result: Of the 809 pts registered, 789 pts (median age: 45 years) were evaluable. 621 pts (78.7%) achieved CR after one or two courses of induction therapy. The 5-year OS rate of arm A was 45.6% and of arm B 53.2% (p=0.3259). The 5-year DFS rate of CR patients was 34.8% in arm A and 28.9% in arm B (p=0.4978). Among the good risk group, the 5-year OS rate of arm A was 62.1% and of arm B 70.2% (p=0.5068), and the 5-year DFS rate of arm A was 53.4% and of arm B 42.0% (p=0.3719). Among the intermediate risk group, the 5-year OS rate of arm A was 35.6% and of arm B 45.5% (p=0.4776), and the 5-year DFS rate of arm A was 26.0% and of arm B 26.1% (p=0.9653). Among the poor risk group, the 5-year OS rate of arm A was 29.7% and of arm B was 33.4% (p=0.6523), and the 5-year DFS rate of arm A was 20.4% and of arm B was 13.5% (p=0.6339). In conclusion: JALSG’s conventional post remission therapy consisting of 3 courses of consolidation and 6 courses of maintenance therapy could be replaced by a shorter duration of intensified consolidation therapy.
Low Dose Ara-C Versus Hydroxyurea with or without Retinoid in Older Patients Not Considered Fit for Intensive Chemotherapy: The UK NCRI AML14 Trial.
Authors: Burnett Alan K. , Milligan Donald , Prentice Archie G. , Goldstone Anthony H. , McMullin Mary F. , Wheatley Keith
Published: October 2019

There is no adequate treatment for older patients with AML who are not considered fit for intensive chemotherapy who comprise a significant proportion of the AML population. As part of the ongoing NCRI (formerly MRC) AML14 Trial in patients over 60 years patients were randomised to Low Dose Ara-C (20mg bd for 10 days every 4–6 weeks) versus Hydroxycarbamide (Hydroxyurea). Because of preclinical studies showing sensitization to Ara-C, patients were in addition randomised to receive All-transretinoic acid or not (45mgs/m2 for 60 days).

Two hundred and four patients entered; 129 had a WHO score of <2; 155 patients were >65 yrs; 108 had do novo, 53 secondary disease and 28 had high risk MDS (blasts >10%). One hundred and ninety-nine patients entered the HU vs LD-Ara-C randomisation and 204 entered the ATRA randomisation. The arms were balanced with respect to age; sex; disease type; WBC and performance score.

Complete remission was seen in 1 of 92 (1%) patients in the HU arm and 15 of 94 (17%) in the LD Ara-C arm (P=0003). Overall survival was considerably improved (hazard ratio 0.61, 95% Cl 0.45 to 0.82, p=0.001). This improvement in outcome was not obtained at the expense of less well tolerated treatment: toxicity and supportive care requirements were similar between the two groups. However, there were no significant differences in outcome between patients given ATRA or not overall or within the treatment arms, although numbers were too small to rule out moderate benefits or disbenefits of treatment (hazard ratio for OS 0.97, 95% Cl 0.73 to 1.28, p=0.8).

We conclude that LD-Ara-C in the schedule chosen could be adopted as a standard of care for older patients not fit for intensive chemotherapy but the outcome remains poor. Future strategies could combine Low Dose Ara-C with novel agents. This trial received a research grant from the UK Leukaemia Research Fund.
Hospital Resource Utilization in Acute Invasive Aspergillosis (AIA) Patients (Pts) Treated with Voriconazole (VOR) or Amphotericin B (CAB).
Authors: Wingard John R. , Herbrecht Raoul , Marciniak Anne , Roberts Craig S.
Published: October 2019

Background: Treatment of AIA typically involves prolonged hospitalization and substantial use of intensive care (ICU) resources. This study compared consumption of hospital resources by AIA pts receiving initial treatment with either VOR or CAB.

Methods: Data were collected prospectively as part of a clinical trial comparing VOR to CAB in AIA of immunocompromised pts, where VOR showed a significant improvement in success and a relative survival benefit (Herbrecht R et al, NEJM2002; 347: 408–15). Pts in the modified-intent-to-treat population with completed resource utilization data were included. Total days of hospitalization, days of ICU, and hospitalization-free survival days during 12 weeks following initiation of therapy were compared. The impact of survival on resource consumption was explored.

Results: There were 143 and 131 pts in the VOR and CAB arms, respectively, with resource utilization data. Resource utilization data are presented in the Table.

Resource Utilization by Treatment Arm

VOR Mean (SD) CAB Mean (SD) Difference (95% CI) Total Hospital Days 27.8 (22.7) 27.7 (20.8) 0.1 (−5.1, 5.3) Total Hospital Days (survivors) 30.2 (24.2) 33.3 (22.2) −3.1 (−10.3, 4.0) ICU Days 5.6 (10.7) 8.1 (14.4) −2.5 (−5.5, 0.5) ICU Days (survivors) 4.0 (10.9) 8.1 (14.6) −4.1 (−7.9, −0.2) Hospitalization-free survival (days) 40.3 (29.8) 28.4 (28.5) 11.9 (4.9, 18.8)

Conclusion: VOR pts had significantly longer hospitalization-free survival and had a trend toward fewer ICU days than CAB pts. Improved survival with less hospital resource utilization and lower total drug acquisition costs (Lewis JS et al, ICAAC 2003, A1359) support the economic benefit of using VOR for primary therapy for AIA.
High Dose Therapy with Autologous Blood Stem Cell Transplantation (ASCT) for Follicular Lymphoma: Long-Term Outcome According to Histologic Grade.
Authors: Pohlman Brad , Jin Tony , Summers Kristie , Kuczkowski Elizabeth , Kalaycio Matt , Sobecks Ronald , Andresen Steven , Bernhard Laura , Cherni Kelly , Baker Janet , Bates Jennifer , Bolwell Brian J.
Published: October 2019

Follicular lymphoma is generally an indolent disease with a relatively long natural history requiring multiple therapies over many years. The optimal combination and sequence of these therapies continue to evolve. Despite substantial supporting evidence (including a recently published, randomized study), the role of high dose therapy with ASCT in follicular lymphoma has been questioned. Therefore, we reviewed the Cleveland Clinic experience to determine the long-term outcome of follicular lymphoma patients according to histologic grade. Between June 1991 and June 2004, 105 patients with relapsed, grade 1–3, follicular lymphoma (without histologic transformation) received high dose CBV (n=9) or BuCyVP (n=96) and ASCT at the Cleveland Clinic. The median follow-up among survivors is 4.4 (0.1–11.4) years.

Table of patient, disease, ASCT characteristics, and outcome

Variable Grade 1 (n=45) Grade 2 (n=36) Grade 3 (n=24) p-value Age: median (range) 49(35–62) 51(33–58) 53(42–64) 0.042 Male sex: N (%) 23(51) 20(56) 12(50) 0.89 Years from diagnosis to ASCT: median (range) 3.0(0.4–15.7) 3.0(0.9–17.6) 2.3(0.6–15.2) 0.34 Prior # chemotherapy regimens: 2–3/>4, N (%) 36(80)/9(20) 32(89)/4(11) 19(79)/5(21) 0.49 Disease status at ASCT: CR/PR, N (%) 6(13)/33(73) 10(28)/22(61) 6(25)/15(63) 0.58 Bone marrow status at ASCT: +/−, N (%) 8(23)/27(77) 4(13)/28(88) 4(18)/18(82) 0.54 Prior radiation therapy: N (%) 14(31) 11(31) 5(21) 0.63 LDH > normal at ASCT: N (%) 28(62) 27(77) 13(54) 0.16 Tumor bulk >10 cm at ASCT: N (%) 8(18) 6(17) 5(21) 0.92 Disease progression: N (%) 20(44) 13(36) 11(46) – Death: N (%) 17(38) 11(31) 10(42) –

Kaplan-Meier freedom from progression and overall survival according to histologic grade are shown:

Figure Figure

By Cox proportional univariate analysis, male sex, ≥4 prior chemotherapy regimens, and elevated LDH predicted a higher risk of progression while prior radiation therapy and bone marrow involvement predicted a higher risk of death. By Cox proportional multivariate analysis, male sex and elevated LDH predicted a higher risk of progression while prior radiation therapy and tumor bulk predicted a higher risk of death. In conclusion, approximately half of all patients that receive high dose therapy and ASCT for relapsed follicular lymphoma of any histologic grade enjoy long-term remissions and survival. ASCT remains one of the most beneficial treatment options for many patients with relapsed follicular lymphoma.
Tandem Autologous Stem Cell Transplantation Following High Dose Chemotherapy for Transformed Low Grade and Refractory High Grade Lymphoma.
Authors: Dubruille Viviane , Morineau Nadine , Moreau Philippe , Guillaume Thierry , Mahe Beatrice , Chevalier Patrice , Luc Harouseau Jean , Noel Milpied
Published: October 2019

The results of autologous stem cell transplantation following high dose chemotherapy for transformed low grade, high grade refractory or relapsing early (<1 year) lymphoma are disappointing. We describe a tandem intensive chemotherapy followed by autologous stem cell transplantation in 37 patients between April 1996 and February 2004. The median age was 49 yo (33–63yo); 19 patients had a transformed low-grade lymphoma, 7 patients a high grade early (<1year) relapse and 11 patients had induction failure. Pre transplant therapy consisted of 2 to 4 courses of polychemotherapy with anthracycline or with cisplatine. The first intensive chemotherapy program consisted of mitoxantrone 45mg/m² at day 1 and cytarabine 1000mg/m² at days 1,2,3,4 followed by autologous stem cell transplantation at day 5. The second intensive treatment consisted of fractioned total body irradiation (12 Gy) and cyclophosphamide 60mg/m² for two days followed by autologous stem cell transplantation in 35 patients and of BEAM (carmustine 300mg/m² at day 1, cytarabine 200mg/m²/12hours at days 2,3,4,5; etoposide 400mg/m² at days 2,3,4,5; melphalan 140mg/m² at day 6) followed by autologous stem cell transplantation in 2 patients who previously received radiotherapy. The regimen was well tolerated with no treatment related death. With a median follow-up of 34.4 months from day 0 of the first intensive treatment, 22 patients are alive in complete remission. The 5y Kaplan Meyer probability of survival is 59%; 15 patients relapsed after a median of 10.6 months (1.9–30.1) and died. The 5y OS in each category is: transformed low grade lymphoma: 68%; high grade early relapse: 29%; induction failure: 64%. This tandem intensive chemotherapy is feasible, well tolerated and effective for transformed low grade lymphoma and induction failure but seems to be disappointing for high grade in early relapse and new therapeutic approaches need to be explored for this category.
Low Incidence of Complete Remission but Comparable Event-Free Survival(EFS) and Overall Survival (OS) with a History of Prior Monoclonal Gammopathy of Undetermined Significance (MGUS) or Smoldering Myeloma (SMM) among Patients Receiving Total Therapy 2 (TT 2).
Authors: Fassas Athanasios , Barlogie Bart , Rasmussen Erik , Milner Teresa , Terry Julian , Zangari Maurizio , Talamo Giampaolo , Lee Choon-Kee , Thertulien Raymond , Crowley John , Tricot Guido
Published: October 2019

Symptomatic/progressive MM sometimes develops on the background of MGUS or progresses through a smoldering MM phase (SMM); other patients may have initially a solitary plasmacytoma of bone (SPC). This study examines the survival implications of pre-existing conditions such as MGUS (n=22), SMM (n=22) or SPC (n=20) among 668 enrolled in TT 2. TT 2 consists of intensive remission induction, melphalan-based tandem autotransplants; consolidation chemotherapy and interferon maintenance. 2 yr estimates of CR/near-CR (only IFE-positive) were similar in de novo MM (44%/67%) and in those with preceding SPC (46%/67%); both were higher than in case of preceeding SMM (24%/38%) or MGUS (17%/49%) (p=.009/p=.007). According to multivariate analysis, the time to CR was shorter in the presence of LDH > 190 U/L (HR 1.33, p=.003) and IgA isotype (HR 1.3, p=.08) but longer in the case of preceeding MGUS or SMM (HR 0.44, p=.02). Kaplan Meier plots of EFS and OS were similar for de novo MM and patients with the 3 precursor conditions. We conclude: (1) the low incidence of stringently defined CR among patients with preceeding MGUS or SMM does not negatively impact EFS or OS after initiation of therapy for symptomatic/progressive MM; (2) this lower incidence of CR may reflect the re-establishment of a stable precursor state due to the high level of drug resistance of these low proliferative activity conditions; (3) patients with de novo MM not achieving CR but having a prolonged EFS/OS may be those with a clinically unrecognized precursor condition; (4) measurements other than M protein-reduction must be identified to better assess minimal residual disease.

Figure Figure