bluuebunny/crossref_metadata_2025 huggingface dataset:
The Wilms’ Tumor Suppressor Wt1 Activates Transcription of the Erythropoietin-Receptor in Hematopoietic Progenitor Cells.
Authors: Dame Christof , Kirschner Karin M. , Hagen Patricia , Hussels Christiane S. , Scholz Holger
Published: October 2019

The Wilms’ tumor suppressor, Wt1, has been implicated in the stage-specific quiescence and differentiation of hematopoietic progenitor cells. Since Wt1 deficiency compromises the proliferation and differentiation of erythroid blast-forming units (BFU-E) and colony-forming units (CFU-E), we analyzed the potential role of the transcriptionally active Wt1 isoform, Wt1(-KTS), in regulating the expression of the erythropoietin receptor (EpoR). CD117-positive hematopoietic progenitor cells isolated from the liver of Wt1-deficient murine embryos (Wt1−/−) at 11.5 dpc exhibited a 10-fold lower proliferative responsiveness to recombinant erythropoietin than CD117-positive cells from wild-type embryos (Wt1+/+), or embryos with heterozygous Wt1-deletion (Wt1+/−). Benzidine staining revealed a reduced fraction of hemoglobin-containing cells in Wt1-deficient cells in comparison to Wt1+/+ cells after 48 hrs of culture of CD117-positive cells in the presence of 6 U rhEpo/ml medium. Consistently, expression of the EpoR was significantly reduced in hematopoietic progenitor cells that lack Wt1 compared to Wt1+/+ cells. The decrease of EpoR mRNA was not due to a general down-regulation of gene expression in Wt1−/− CD117-positive cells, since levels of c-kit and c-mpl expression were normal. In the heart of wild-type and Wt1−/− embryos, which was analyzed as a non-hematopoietic tissue expressing both EpoR und Wt1, no difference in Epo-R expression was found. Transient transfection of Wt1(-KTS) into human hepatoma-derived HepG2 cells increased EpoR transcripts approximately 2-fold. A luciferase reporter plasmid carrying 309 bp of the proximal human EpoR promoter was stimulated 8-fold by co-transfection with Wt1(-KTS). The responsible cis-element in the EpoR promoter was identified by mutation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. In summary, we identified the EpoR gene to be the first downstream target of Wt1 within the hematopoietic cytokine receptor superfamily. Our data indicate that activation of the EpoR gene by Wt1 is a critical mechanism in normal murine hematopoiesis. However, our data do not support a role for Wt1 in EpoR expression in non-erythoid tissues, such as the heart.
Influence of Mannose Binding Lectin (MBL) Gene Polymorphisms on Infectious Complications during Treatment for Childhood Malignancy.
Authors: Dommett Rachel M. , Bajaj-Elliott Mona , Chisholm Julia , Klein Nigel
Published: October 2019

Infection remains a major cause of morbidity and hospitalisation in children receiving chemotherapy. At present clinical parameters provide a guide to risk of severe infection but it has become increasingly apparent that, among patients with the same diagnosis and treatment regimen, not all suffer equally from infectious complications. Mannose binding lectin is a pattern recognition molecule of the innate immune system which, upon binding to a wide range of microorganisms, activates the lectin pathway of complement. Polymorphisms in the MBL2 gene result in low levels of MBL protein and are frequently associated with increased susceptibility to infection. Studies investigating the role of MBL in defence against infection following chemotherapy have reported conflicting findings to date. MBL replacement therapy is a potential treatment option in the future and we consider it imperative that we clarify the role of MBL in this clinical setting. Clinical data from episodes of febrile neutropenia (FN) in children aged 0−16 receiving chemotherapy for childhood cancer was recorded prospectively from April 2004−March 2005, including clinical and microbiological evidence of infection, antibiotic days and duration of admission. MBL genotyping was performed using a reverse hybridisation technique and results were analysed against FN outcome. 269 children were recruited into the study. A total of 513 episodes of FN were captured over the study period from 211 patients. 58 patients had no recorded episodes of FN. There was no association between age, sex, ethnicity or diagnosis and MBL genotype. 75% of subjects had a haematological malignancy and of these 84% had acute lymphoblastic leukaemia (ALL). Overall, patients with MBL2 polymorphisms experienced more episodes of FN than wildtype individuals (median 2 and 1, respectively, p=0.074). Analysis of episodes with documented clinical/microbiological infection revealed that the proportion of patients with ≥ 3 episodes was 14.6% in those with polymorphisms and 8% in wildtype, p=0.045. This trend was also true for the supgroup of patients with ALL. The duration of inpatient stay for FN, used as a surrogate measure of severity, was influenced by MBL genotype in some groups of patients. Longer inpatient stays and antibiotic days were most apparent in the MBL deficient patients with high risk diagnoses e.g. AML and B NHL who spent up to 4.5 days longer/per episode in hospital than high risk wildtype patients. These results suggest that MBL deficiency influences both susceptibility to FN and outcome of FN episodes in this study cohort. The effect of MBL deficiency differs between diagnostic groups and may be most important in those patients who are at higher risk of severe FN by virtue of their underlying diagnosis and treatment regime.
The Wilms’ Tumor Gene WT1 Is Over-Expressed in Immature Leukemia Cells but Not Necessary for Leukemia Development in Mouse Leukemia Models.
Authors: Hosen Naoki , Sugiyama Haruo , Weissman Irving L.
Published: October 2019

The Wilms tumor gene WT1, which is over-expressed in almost all leukemia, is one of the most promising targets for immunotherapy. To clarify which cells express WT1, we generated a knock-in reporter GFP mice (WT1GFP/+) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term hematopoietic stem cells (HSCs) and very few (<1%) of multipotent progenitor cells. WT1 was expressed in 17.4% of megakaryocyte-erythrocyte progenitor cells and 4.1% of common myeloid progenitor cells, while the frequencies of WT1 expressing cells in mature granulocyte or lymphocyte were very low (~1%). In contrast, in murine leukemias induced by TEL/PDGFβR + AML1/ETO or BCR/ABL, WT1 was expressed in 40.5% or 38.9% of c-kit+lin-Sca-1+ (KLS) leukemia cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSC). Furthermore, WT1 was not expressed fetal liver HSC population, mobilized HSCs by cyclophosphamide and granulocyte-colony stimulating factor, or HSCs stimulated with SCF, Flt3L, and IL3 in-vitro. These results suggest that WT1 expression in is not a surrogate marker of cell proliferation. To examine the function of WT1 in leukemogenesis, we used recombinant lentivirus to overexpress WT1. HSCs transduced with either empty or WT1-expressing lentivirus were transplanted into lethally irradiated recipients. The FACS analysis of BM cells at 4 months post transplant showed very low chimerism of WT1-expressing cells compared to control GFP-expressing cells. Moreover, differentiation block of WT1-expressing cells was not observed. These results indicate that WT1 does not induce proliferation or block differentiation in-vivo. Next, we tested whether leukemias could be induced from the WT1-deficient fetal liver hematopoietic cells. All of three mice transplanted with E13.5 WT1-defeicient fetal liver cells transduced with TEL/PDGFβR+AML1/ETO developed leukemia. Similarly, 3 out of 4 mice transplanted with p210BCR/ABL-transduced WT1-deficient fetal liver cells developed leukemia. These results indicate that WT1 is not necessary for leukemia development in two leukemia models examined. Collectively, WT1 is expressed on immature leukemic cells but not normal HSCs, highlighting its potential as a specific target for therapy. However, the exact function of WT1 in leukemogenesis remains unclear from these studies.
Activation Mechanisms of STAT5 by Oncogenic Flt3-ITD.
Authors: Choudhary Chunaram , Brandts Christian , Schwable Joachim , Tickenbrock Lara , Sargin Bulent , Ueker Andrea , Bohmer Frank D. , Berdel Wolfgang E. , Müller-Tidow Carsten , Serve Hubert
Published: October 2019

Mutations in the receptor tyrosine kinase Flt3 represent one of the most common known genetic lesions in AML. Internal tandem duplication (ITD) mutations clustered in the juxtamembrane domain are the most frequent and best characterized mutations found in Flt3. We and others have previously reported that oncogenic activation of Flt3 by ITD mutations activate aberrant signaling including activation of STAT5, up-regulation of STAT targets like-Pim and SOCS proteins and repression of the myeloid transcription factors Pu.1 and c/EBP-alpha (Mizuki et al., Blood96: 3907–14, 2000; Mizuki et al., Blood101: 3164–73, 2003). Earlier studies have observed activation of Src and Jak kinases by Flt3-ITD but the mechanisms of STAT5 activation by Flt3-ITD and the role of Src or Jak kinases in this process remains unclear. Using the specific small molecule inhibitors PP-1 and PP-2, we analyzed the role of Src kinases in Flt3-ITD mediated STAT5 activation. Treatment of Flt3-ITD expressing 32D cells with PP-1 and 2 had only marginal effect on Flt3-ITD induced STAT5 activation, which was due to a proportional inhibition of Flt3 kinase by these inhibitors. Similar results were obtained with AG490, reportedly a specific inhibitor of Jak2. However, use of cell a line deficient for all Src family kinases revealed a Src-independent activation of STAT5 by Flt3-ITD. Also, using cell lines deficient for Jak2 or Tyk2 we show that Flt3-ITD induced STAT5 activation independent of Jak2 or Tyk2 kinases. To rule out a possible redundant role of different Jak kinases in Flt3-ITD mediated STAT5 activation, we used SOCS1, a target of STAT5 and a potent molecular inhibitor of all Jak kinases. Surprisingly, retroviral transduction of 32D cells with SOCS1 inhibited IL-3 but not Flt3-ITD mediated proliferation of these cells. Similar results were obtained with SOCS3, another inhibitor of Jak kinases. Detailed signaling analyses of stable 32D cells co-expressing Flt3-ITD and SOCS1 revealed that activation of the STAT5 and up-regulation of STAT5 target gene Pim-2 by Flt3-ITD is completely resistant to SOCS1. In contrast, expression of SOCS1 severely inhibited IL-3 mediated STAT5 activation. Furthermore, using purified recombinant STAT5 and Flt3 kinase in in-vitro kinase assays, we show that STAT5 is indeed a direct target of Flt3. Taken together, our data show that Flt3-ITD directly activates STAT5 independent of Src or Jak kinases, providing the mechanistic basis of Flt3-ITD induced STAT5 activation. Furthermore, our finding that Flt3-ITD up-regulates SOCS proteins but activate STAT5 in a SOCS1 resistant manner may have important implications for Flt3-ITD-mediated modification of cytokine signaling.
Characterization of a Novel R-RAS Mutation Cloned from Primary AML Cells.
Authors: Reuter Christoph W.M. , Aydilek Erdem , Onono Fredrick O. , Morgan Michael A. , Henningfeld Kristine A. , Ganser Arnold
Published: October 2019

Mammalian RAS genes encode a family of small GTPases, which are involved in signal transduction pathways resulting in cell growth and differentiation. Many human cancers, including hematologic malignancies, have been found to harbor activating H-, K- and N-RAS mutations. We report here the discovery and characterization of a novel, activating R-RAS mutation cloned from primary AML cells. Total RNA was purified from AML blasts, reverse transcribed into cDNA and R-RAS-specific PCR products were sequenced on an ABI310 automated DNA sequencer. A mutation in codon 84 of R-RAS (Thr to Ala, T84A) was detected in a female AML patient with normal karyotype. The R-RAS T84A mutant was cloned into a hygromycin B-resistant pCGN vector and used to stably transfect BA/F3 cells, an IL-3-dependent murine pro B-cell line. The well-described R-RAS Q87L activating mutant was employed as a positive control for R-RAS-induced transformation of BA/F3 cells as well as for activation of signal transduction pathways. The R-RAS T84A mutant protected BA/F3 cells from the apoptotic effects of IL-3 starvation, albeit to a lesser extent than the R-RAS Q87L mutant. Interestingly, these two R-RAS mutants activate distinct signal transduction pathways. For example, the R-RAS Q87L mutant preferentially caused activation of the RAS-to-MAPK signal cascade as evidenced by Western blots of affinity precipitations with the GST-conjugated c-Raf-1 RAS binding domain, which is designed to bind GTP-loaded (activated) RAS proteins. The R-RAS T84A mutant activated the PI-3K pathway as demonstrated by Western blots of affinity precipitations with the GST-coupled RAS binding domain of the p110-alpha subunit of PI-3K. Additionally, the R-RAS T84A mutant induced a stronger phosphorylation of the PI-3K down-stream target Akt upon IL-3 stimulation (0.1 and 10 ng/mL, 10 minutes) of BA/F3 cells cultured overnight without IL-3. In conclusion, a novel R-RAS mutation was found in primary AML blasts and the mutant protein activates anti-apoptotic signaling through the PI-3K/Akt pathway.
Oncogenic CBL Mutants Have Transforming Potential in Cells Expressing the FLT3 Tyrosine Kinase.
Authors: Reindl Carola , Vempati Sridhar , Quentmeier Hilmar , Mellert Gudrun , Hiddemann Wolfgang , Spiekermann Karsten
Published: October 2019

CBL is a E3-ubiquitin ligase, that negatively regulates many receptor tyrosine kinases (RTKs). Translocations (t(4;11); t(11;14) involving CBL in humans and two oncogenic CBL deletion mutants, CBL-70Z and v-CBL, inducing B-cell lymphomas in mice, were described. The RTK Fms-like tyrosine kinase 3 (FLT3) is expressed by the leukemic cells of 70–90% of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) and is highly implicated in the malignant transformation by harbouring activating mutations, aberrant autocrine stimulation or overexpression.

We hypothesized that CBL could play a role in regulating FLT3 activity and if mutated, could cause aberrant FLT3 signaling in hematopoietic cells. To analyze if CBL mutants could lead to transformation of FLT3-expressing cells we stably transduced Ba/F3 cells with CBL-WT, CBL-70Z or v-CBL alone or together with FLT3-WT. Coexpression of FLT3-WT and a CBL mutant, CBL-70Z or v-CBL, but not CBL-WT or expression of a CBL-construct alone, conferred IL-3 independent growth to Ba/F3 cells. In presence of FLT3 ligand FLT3-WT/CBLmutant-expressing cells showed hyperproliferation. To determine whether the proliferative advantage of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells is mediated by FLT3 we cultivated the cells in presence of selective FLT3 inhibitors, SU5614 and PKC412. Both inhibitors were able to abrogate the IL-3 independent and FL-stimulated growth of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells. The FLT3-WT receptors and the downstream signaling pathway, STAT5, were constitutively active in FLT3-WT/CBLmutant-expressing cells in contrast to control cells. To clarify the mechanism of transformation we performed internalization assays. The rate and speed of internalization of the FLT3-WT receptors were not altered in FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells compared to FLT3-WT cells. However, we could show that CBL-WT promotes FLT3 degradation. To investigate if CBL mutations play a role in acute leukemias we screened the cDNA of 62 AML and 6 ALL cell lines and found a CBL deletion mutant in MOLM-13 and MOLM-14 cells lacking exon 8, which comprises parts of the linker and RING finger domain. Hence, this mutation targets the same functionally important regions of the CBL protein than the CBL-70Z deletion.

In summary, CBL-70Z and v-CBL, but not CBL-WT, confer a transforming potential to cells expressing FLT3. The pro-proliferative phenotype of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells is mediated by an increase in FLT3 tyrosine kinase activity. The CBL-70Z and v-CBL mutations were hypothesized to act in a dominant negative manner abrogating the negativ-regulatory function of CBL-WT. We identified a CBL deletion mutant in the MOLM-13 and MOLM-14 cells, that is reminiscent of CBL-70Z, indicating that CBL mutations might play a role in the pathogenesis of acute leukemias.
Towards a Pediatric Approach in Adults with Acute Lymphoblastic Leukemia (ALL): The GRAALL-2003 Study.
Authors: Huguet Françoise , Raffoux Emmanuel , Thomas Xavier , Leguay Thibaut , Chevallier Patrice , Escoffre Martine , Reman Oumedaly , Caillot Denis , Vey Norbert , Boissel Nicolas , Hunault Mathilde , Buzyn Agnes , Chalandon Yves , Delannoy Andre , Vernant Jean-Paul , Lheritier Veronique , Bene Marie-Christine , Macintyre Elizabeth , Ifrah Norbert , Dombret Herve
Published: October 2019

Introduction: In a recent French FRALLE versus LALA comparison, we have demonstrated a large benefit in outcome when adolescents and young adults were treated in a pediatric rather than an adult ALL protocol (Boissel JCO 2003). Similar provocative results have been observed in three other pediatric versus adult ALL studies (Stock ASH 2000, de Bont Leukemia 2004, Testi ASH 2004). One explanation may be the larger amounts of steroids, vincristine (VCR), and L-asparaginase (L-aspa) administered in pediatric patients. The GRAALL-2003 study was thus designed to offer a pediatric approach in adults with Ph-negative ALL until 60 years of age.

Methods: Treatment included a 5-drug induction, high dose-intensity consolidation blocks, delayed intensification, and 2-year maintenance. The comparison with the former LALA-94 protocol showed a 8.6-fold, 3.7-fold, and 16-fold increase in cumulative doses of prednisone, VCR, and L-aspa, respectively. One difference with childhood ALL therapy remained indication of allogeneic SCT in first CR which was offered to all patients with high-risk factor and a donor. In addition, induction was reinforced with a hyper-cyclophosphamide sequence (HyperC) in case of poor early response (cortico- and/or chemoresistant ALL). In the present report, 212 GRAALL-2003 patients with Philadelphia-negative ALL aged 15–55 years with a median follow-up of 18 months were compared to 712 patients previously treated in the LALA-94 trial.

Results: Cohorts were comparable in terms of prognostic factors. CR rate was significantly higher in GRAALL patients (93 vs 88%, P=0.02) due to a reduction in resistant disease (0.5 vs 8%, P<0.001) with comparable induction death (6 vs 5%, P=0.37) rate. When compared to the LALA-94 study, 2-year EFS and overall survival were markedly improved (56 vs 41% and 66 vs 54%; P=0.0002 and 0.02, respectively). Similar differences were observed after censoring SCT patients at transplant time, underlining the role played by chemotherapy modification. Because of a worse tolerance to induction and post-remission therapy, the benefit associated with the GRAALL approach decreased with advanced age, becoming not statistically significant over 40 years of age. Finally, outcome improvement was higher in T- than in B-lineage ALL (2-year overall survival, 75 vs 51% and 61 vs 56%; P=0.016 and 0.25, respectively). Among patients with T-ALL, poor early responders (allocated to HyperC induction) tended to have a better EFS than good early responders, suggesting a major role of HyperC in this ALL subset.

Conclusion: In conclusion, a pediatric approach benefits to younger adults with ALL.
Gene Expression Patterns Identify Patients with Non-Small Cell Lung Cancer (NSCLC) and Ovarian Cancer Who Are at Increased Risk for Venous Thromboembolism (VTE).
Authors: Ramiah Veshana S. , Potti Anil , Peterson Rebecca , Harpole David , Berchuck Andrew , Ortel Thomas L.
Published: October 2019

Background: VTE is the leading cause of death in patients with cancer. The 1-year survival rate in patients diagnosed with cancer at time of VTE is 12% compared to 36% in cancer patients without thrombosis. Cancer patients who develop VTE have higher mortality during hospitalization and during surgery. VTE in cancer patients portends a poorer prognosis and may indicate a more aggressive phenotype. There are, as yet, no clinical or laboratory parameters that have clinical utility in identifying this important group of patients with cancer who are at risk for developing VTE.

Methods: We explored whether gene expression profiling could define phenotype-specific metagenes (aggregate patterns of gene expression) that distinguish cancer patients with and without VTE. The medical history of 95 patients with NSCLC and 37 patients with ovarian cancer was reviewed to identify patients with VTE after the initial diagnosis of cancer but not within 6 weeks of surgery. Separate sets of controls with NSCLC and ovarian cancer, respectively, were identified from the same groups, matched by age, gender and clinical stage, but without VTE for at least 2 years following the diagnosis of cancer. RNA was extracted and gene array data obtained using Affymetrix U133 GeneChips. Gene expression data was analyzed using binary regression methodologies.

Results: 13/95 (13.5%) patients with NSCLC and 6/37 (16%) with ovarian cancer had VTE and met inclusion criteria. Using the metagene approach, a discriminator gene set (n=50) that differentiated patients with NSCLC and VTE from patients with NSCLC without VTE was identified. A separate discriminator gene set was identified for the ovarian cancer group. A leave-one-out cross validation performed to validate the reliability of the discriminator metagene set was 85% accurate in identifying patients with NSCLC and VTE. Similar analysis for the ovarian cancer patients was limited by the small number of patients identified. Significant biological differences were seen between the comparison groups in the NSCLC subset, including genes such as P53, VEGFC, E2F4, TFPI and EPHB2. Expression differences in the ovarian subset similarly included P53, but also included genes not seen in the NSCLC group, such as H-ras, Tissue factor and Factor X.

Conclusions: Our data suggests that a genomic approach can identify patients with cancer at risk for VTE. In addition, these results also suggest that different tumor types might possess unique expression signatures associated with increased thrombotic risk.
Platelet PF-4 Is an Early Marker of Tumor Angiogenesis.
Authors: Klement Giannoula L. , Cervi David , Yip Tai-Tung , Folkman Judah , Italiano Joseph
Published: October 2019

Early tumor detection and treatment is one of the most important determinants of cancer survival. We reported previously, that a ‘platelet angiogenesis proteome’ can detect clinically occult tumors in mice. We now present evidence that changes in platelet content of platelet factor-4 (PF-4) can be a consistent predictor of tumor growth across a spectrum of cancers. The differentially expressed protein was initially identified as a ~8200Da band by SELDI-ToF analysis. The protein band was isolated and positively identified by peptide sequencing and anti-PF-4 SELDI MS as PF-4. The platelet-associated PF-4 appeared to be up-regulated in early growth of human liposarcoma, mammary adenocarcinoma, osteosarcoma and down-regulated in a RIP-Tag model of pancreatic islet cell carcinoma. A 120 day follow-up study of liposarcoma revealed ≥2-fold increases of platelet-associated PF-4 at 19, 30 and 120 days. Concomitantly, only a ~0.5–1 fold decrease in PF-4 in plasma was observed. There were minimal changes in the plasma of animals bearing other human tumor xenografts. We conclude that platelet-associated PF-4, but not its plasma counterpart, may be a promising marker of early tumor growth or tumor recurrence.
Nitric Oxide and Asymmetric Dimethlyarginine Levels in Malignancy Associated Thrombosis and Their Modulaiton by Anticoagulants.
Authors: Hoppensteadt Debra , Fareed Daniel , Iqbal Omer , Lale Ajit , Fareed Jawed
Published: October 2019

Nitric oxide (NO) represents one of the major endothelial derived vasoactive mediators. Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NO synthase which inhibits NO production at Pathophysiologic levels. While the association of ADMA in cardiovascular diseases is known, no information on the ADMA levels is available in cancer patients. To test the hypothesis that endothelial dysfunction in cancer patients may result in increased ADMA levels, plasma samples were analyzed from an open label, multidose, active comparator designed study in which all patients (n=110) were initially treated with low molecular weight heparin, enoxaparin (E) at 1–1.5 mg/kg sc for 5 days. These patients were further subdivided. One group continued to receive E whereas the other group received warfarin (W) for up to 12 weeks. Baseline blood samples (BL), 5 days post E (IPE) and 4–6 week samples from the E and W were analyzed for ADMA and NO levels (Cardiovasics, Palo Alto, CA; R&D Systems, Minneapolis, MN, respectively). In comparison to the aged matched controls both the ADMA and NO levels were markedly elevated in cancer patients. The E treated group showed a marked decrease in the ADMA levels which persisted throughout the treatment period. However in the W converted group the ADMA levels rebounded to an increased level indicating that E differentially regulated ADMA in these patients. The down regulation pattern of NO was similar for both enoxaparin and warfarin. These results suggest that patients with cancer and thrombosis exhibit simultaneous up regulation of ADMA and NO. While enoxaparin and warfarin show a differential regulation of ADMA both result in down regulation of NO. The fact that E regulates ADMA is highly suggestive of its regulatory role in iNOX which may be involved in the inflammatory response in cancer patients. Furthermore, these studies underscore the anti-inflammatory mechanisms by which low molecular weight heparins such as E, mediate their therapeutic effects in cancer asscoiated thrombosis.

Group Nitric Oxide (uM) ADMA (uM) Aged matched controls 18±5 1.51±0.52 BL 63±18 5.34±1.5 IPE 22±9 2.81±0.9 4–6 weeks enoxaparin 20±8 2.45±0.9 4–6 weeks warfarin 25±8 3.67±1.1
MicroRNA Signatures Associated with Cytogenetics and Outcome in Acute Myeloid Leukemia.
Authors: Garzon Ramiro , Volinia Stefano , Liu Chang G. , Pichiorri Flavia , Palumbo Tiziana , Fabbri Muller , Cimmino Amelia , Alder Hansjuerg , Flomenberg Neal , Coombes Kevin R. , Calin George A. , Andreeff Michael , Croce Carlo M.
Published: October 2019

MicroRNAs are small non-coding RNAs of 19–25 nucleotides in length that are negative regulators of gene expression. Findings over the last few years indicate that microRNAs are involved in fundamental cellular process, including development and hematopoietic differentiation. Acute myeloid leukemia (AML) is a heterogeneous disorder that is characterized by proliferation of immature cells. Although there are well defined molecular subtypes of AML, the pathogenesis in the majority of cases is largely unknown. Focusing on known genes will not likely suffice to uncover the nature of the AML. The integration of a whole genome approach including non-coding RNA gene products may lead to an improve understanding of the biology of AML.

Methods: To determine whether microRNAs are associated with known cytogenetic abnormalities and biological features in AML, we evaluated the microRNA expression profiles of 176 samples of adult AML with intermediate and poor risk cytogenetics and 10 CD34+ cells from healthy donors using a microarrays platform. After normalization, data were analyzed using significance analysis of microarrays and prediction analysis of microarrays software. An independent set of 28 patients with AML was used to validate the signatures using quantitative real time PCR. Treatment response was evaluated in 29 newly AML diagnosed patients 4 to 6 weeks after induction chemotherapy with idarubicin and cytarabine by bone marrow examination. Complete remission was defined as less than 5% blasts in the bone marrow. Otherwise it was categorized as resistant disease.

Results: We found several microRNAs differentially expressed between CD34+ cells and all the AML samples. A subset of these microRNAs reflects the differentiation stage of the leukemias and correlate with the French-American-British classification of AML. Likewise, microRNAs are closely associated with the prevalent cytogenetic abnormalities. A common signature including the over expressed miR-20; miR-17, miR-25 and miR-191 are associated with short overall survival, while miR-29b is found down-regulated in patients with resistant disease. Furthermore, we proved experimentally that miR-29b regulates negatively MCL-1, a critical apoptosis regulator, which has been found up-regulated and associated with relapse and chemotherapy resistance in leukemia.

Conclusions: MicroRNAs expression in AML is closely associated with differentiation stage, morphology and cytogenetics. A subset of MicroRNAs is correlated with survival and treatment response.
Requirement of Transferrin for Iron-Induced Hepcidin Expression in the Zebrafish.
Authors: Fraenkel Paula G. , Dooley Kim A. , Zon Leonard I.
Published: October 2019

The zebrafish mutant gavi was identified in a large-scale chemical mutagenesis screen for embryonic anemia. Two alleles were discovered: gaviHE067 and gaviIT029, both of which have a homozygous recessive pattern of inheritance. The gavi mutants developed severe hypochromic anemia at 48 hours post-fertilization and died by 14 days post-fertilization. To determine the cause of the hypochromic anemia, low resolution genetic mapping was performed which revealed transferrin as a candidate gene. Sequencing of transferrin cDNA from the mutants confirmed that each allele of gavi harbors a mutation that results in aberrant splicing, while quantitative realtime RT-PCR demonstrated virtually absent transferrin expression in the mutants. Our previous work demonstrated that expression of hepcidin, an iron regulatory gene, is upregulated 18 hours following iron dextran injection of the zebrafish embryo. This effect was not impaired by a defect in ferroportin1 (Fraenkelet al., (2005) J Clin Invest115:1532–1541), an iron exporter and ligand for hepcidin. To evaluate the effects of iron loading on transferrin-deficient mutants, gaviIT029 homozygotes at 48 hours post-fertilization were either anesthetized and injected with iron dextran, or anesthetized but not injected. Transferrin expression was assayed 18 hours post-treatment. gaviIT029 homozygote embryos exhibited low levels of hepcidin expression that failed to increase following iron injection. These findings provide the first evidence to support the hypothesis that transferrin is required for the induction of hepcidin expression in response to iron loading in vivo.
Investigation of the Iron Responsive Elements of the Hepcidin Promoter by In Vivo Imaging of Mice.
Authors: Flanagan Jonathan , Truksa Jaroslav , Peng Hongfan , Lee Pauline , Beutler Ernest
Published: October 2019

Hepcidin is an acute-phase response antimicrobial peptide that has emerged as a central regulator of iron absorption. It performs this function by modulating iron transfer by both the intestinal epithelial cells and macrophages. Low or high hepcidin levels result in increased or decreased iron absorption respectively. Hepcidin is mainly liver-derived and regulated at least, in part, transcriptionally. Hypoxia, erythroid demand, iron content and inflammation have all been shown to influence hepcidin mRNA expression in intact animals. Regulation of hepcidin by cytokines and by hypoxia is readily demonstrated in primary hepatocytes or in hepatocyte lines, but incubating hepatocytes or mixtures of hepatocytes and macrophages or marrow cells with iron does not increase transcription of hepcidin. Thus, how iron excess stimulates hepcidin production in hepatocytes remains a mystery. To provide a better understanding of hepcidin gene expression in response to these regulatory stimuli, we took advantage of the fact that 2 hepcidin genes exist in mice. Hepcidin 1 is homologous to human hepcidin while hepcidin 2 is the result of a tandem duplication of murine hepcidin 1. In mice hepcidin 1 mRNA expression responds to iron and to inflammation, but hepcidin 2 responds only to iron. We established a whole animal in vivo bioluminescence imaging assay to measure the activity of hepcidin promoter constructs in the animals’ liver after transfecting hydrodynamically hepcidin promoter/luciferase constructs into mice. Murine hepcidin 1, murine hepcidin 2 and human hepcidin promoters were all shown to have basal luciferase activity. Luciferase expression was lower in HFE knockout mice than in wild type controls. When transfected mice were stimulated with iron or endotoxin, the murine hepcidin 1 and human hepcidin constructs were found to respond to both of these stimuli. The murine hepcidin 2 transfected mice showed a weak response to iron stimulation while there was no stimulation by endotoxin. These findings validate, in an in vivo system, what is known about these promoters from other studies. Subsequent experiments have focused on making shorter hepcidin promoter constructs to identify the key regions of the hepcidin promoter involved in iron induction of hepcidin expression. This strategy will allow us to map the iron response site of the hepcidin promoter and this should aid in identifying transcription factors and upstream stimuli involved in regulating expression of hepcidin by iron.
HAMP and HVJ as Candidate Modifier Genes in Type1 Hereditary Hemochromatosis with High Iron Burden and in Porphyria Cutanea Tarda (PCT).
Authors: Ajioka Richard S. , Phillips John D. , Weiss Robert B. , Dunn Diane M. , Kushner James P.
Published: October 2019

Homozygosity for the HFE C282Y mutation accounts for approximately 90 percent of HFE-associated (type 1) hereditary hemochromatosis. The clinical phenotype in C282Y homozygotes, however, ranges from simply an elevated percent saturation of transferrin to organ damage due to iron overload. Modifier genes have been proposed to explain this phenotypic variability. Hepatic siderosis is a nearly constant finding in patients with PCT. Approximately 20 percent of patients with PCT are homozygotes for the C282Y HFE mutation but the cause of hepatic iron loading in the remaining 80 percent is not known. Approximately one-third of patients with PCT are heterozygotes for mutations of the uroporphyrinogen decarboxylase (URO-D) gene (familial PCT) but pedigree studies indicate that clinical expression occurs only in those URO-D heterozygotes who develop hepatic siderosis. Most patients with PCT have no URO-D mutations (sporadic PCT) but virtually all sporadic cases have hepatic iron overload. Two genes known to affect iron homeostasis are hepcidin (HAMP) and hemojuvelin (HJV). Heterozygosity for HAMP and HJV mutations have been associated with marked iron overload in a small number of patients with type 1 hemochromatosis (Blood.2004; 103:2835–40; Blood Cells Mol Dis.2004; 33:338–43). We asked if mutations of HAMP or HJV could account for hepatic iron overload in highly penetrant C282Y homozygotes and in PCT patients with or without HFE mutations. We sequenced the HAMP and HJV genes in 96 hemochromatosis patients with grade 3–4 (scale 0–4) hepatic parenchymal cell stainable iron (HPCSI) and 96 PCT patients with variable degrees of hepatic siderosis. Ninety-four percent (90) of the hemochromatosis patients were C282Y homozygotes, 4.2 percent (4) were C282Y heterozygotes and 2.1 percent (2) were wild type 282 homozygotes. No exonic changes or splice site mutations were detected in either the HAMP or HJV genes. Eighty-three of the 96 PCT patients were genotyped at the HFE locus. Twenty-five percent (21) were C282Y homozygotes, 23 percent (19) were C282Y heterozygotes and 52 percent (43) were wild type 282 homozygotes. No exonic changes or splice site mutations were detected in the HJV gene of patients with PCT but two PCT patients were found to be heterozygotes for HAMP mutations. The first had the previously identified 212G→A transition leading to a G71D substitution. The second had a 248A→C transversion corresponding to K83R in the peptide. Both of these PCT patients were HFE 282 wild type homozygotes but both had grade 4 HPCSI. These data indicate that heterozygosity for mutations of HAMP or HJV rarely modifies the iron loading phenotype in either type 1 hemochromatosis or PCT. Other modifier loci must exist.
Role of HFE in Hepatic Iron Overload: Novel Findings Demonstrate Direct Effects of Mutant C282Y on Hepcidin and Calreticulin mRNA Expression.
Authors: Pinto Jorge P. , Ramos Pedro , de Almeida Sergio , Oliveira Susana , Breda Laura , Michalak Marek , Rivella Stefano , de Sousa Maria
Published: October 2019

Studies done in non-hepatic cell lines, focusing on the interaction between HFE with TFR1 and β-2M proved insufficient to explain the discrepancies found in the clinical penetrance of hemochromatosis in subjects carrying the C282Y mutation. Our first goal was to investigate the role of HFE wild type (wt) and mutant proteins (C282Y and H63D) in a human hepatic cell line, focusing on the cellular localization and interaction of HFE with the expression of other iron related proteins. HFE mutant C282Y was found to be retained in the endoplasmic reticulum (ER). Thus, in addition, we investigated the effect of HFE wt and mutant proteins on Calreticulin, which is a chaperon protein that responds to ER stress and has a protective effect on oxidative damage in some cell lines. Here we report setting up a stable transfection of wt- and mutant-HFE in a hepatic cell line (HepG2) and examine the intracellular distribution of wt- and HFE mutants, their effect on iron intake independently of TFR1 and on the expression of other iron and ER stress response genes, namely Hepcidin and Calreticulin. In addition, we validated some of the novel effects of HFE on Calreticulin using peripheral blood mononuclear cells from HFE patients. The localization of the HFE variants was analyzed using KDEL and Golgin-97 as ER and the Golgi complex markers, respectively. HFE C282Y shows a high degree of overlap with the ER markers, confirming a retention of this variant in this organelle. Over-expression of the HFE wt impaired the intake of 55Fe relatively to transfected control cells (P<0.008) independently of TFR1, as demonstrated by RNAi silencing. Hamp RNA expression was decreased in cells over expressing C282Y in comparison to HFE wt cells (P<0.011). Finally over-expression of HFE wt decreases Calreticulin mRNA, whereas the C282Y had an opposite effect, compared to the control cell line. A similar result was observed in peripheral blood mononuclear cells (PMBC) of C282Y homozygous HFE patients, compared to wild type blood donors (P<0.006). Interestingly, this data suggest that synthesis of the HFE mutant C282Y triggers a protective effect on oxidative damage mediated by Calreticulin. In fact, HepG2 cells over-expressing C282Y showed lower levels of ROS than HFE wt (P<0.004). This observation might contribute to explain some of the discrepancies seen in the clinical penetrance of the disease in C282Y carrying subjects. The direct effect of the mutant HFE C282Y on mRNA expression of hepcidin also demonstrated here for the first time corroborates and provides a molecular basis for earlier reports of low hepcidin levels in HH patients and in Hfe-KO mice.
Novel Rearrangements and Mutations in the Alpha-Globin Gene Cluster Detected by a New Alpha-Globin Dosage Assay, Cation Exchange HPLC and Comprehensive DNA Sequencing.
Authors: Hantash Feras M. , Gallivan Monica V. , Mario Mikula , Kelsey Starn , Wang Sheng-Biao , Sun Weimin , Strom Charles M.
Published: October 2019

The alpha globin gene cluster contains two highly homologous alpha globin genes, HBA1 and HBA2, that code for identical proteins. Mutations in the alpha globin gene cluster are predominantly large deletions causing the loss of either one copy of the alpha-globin gene (e.g. -α3.7 and -α4.2 deletions) or both copies (e.g. --THAI, --FIL or --MED). A few large deletions encompassing the regulatory HS-40 region have also been described in alpha-thalassaemia patients. Point mutations and small base pair insertions or deletions have also been detected in HBA1 and HBA2 genes. Seven common large deletions in the alpha globin gene cluster are detected by a gapped-PCR assay. These common mutations and some other types of rearrangements can be detected by Southern blot, a laborious and time consuming method. However, these methods may not accurately identify the total number of copies of alpha globin like genes. We designed a single-tube alpha globin gene dosage assay (αGDA) using semi-quantitative fluorescent PCR (SQF PCR) for detecting the total number of alpha globin genes. Primers that amplify specific fragments from HBA1 and HBA2 genes, a fragment between the alpha globin pseudogenes, and three fragments flanking and including the HS-40 regulatory region were included in a single PCR reaction together with primers that amplify fragments from 3 different normalization genes. Using the αGDA, we were able to detect in patient samples varying copy numbers of alpha globin genes and to identify the nature of DNA rearrangements between HBA1 and HBA2. We also identified novel alpha globin conversion events that were verified by DNA sequencing. We also designed a complimentary comprehensive DNA sequencing assay to detect point mutations and small base pair insertions or deletions in the HBA1 and HBA2 genes. Using this method, and in combination with cation exchange HPLC and agarose gel electrophoresis, novel mutations in alpha globins were identified and submitted to the globin gene server, including Hb Linwood (α2 40 Lys>Gln), Hb Creve Coeur (α2 24 Tyr>Asp), and Hb Westborough (α-3.7 130 Ala>Val). The simplicity of αGDA will allow the replacement of the laborious Southern blot analysis to detect large deletions in the alpha globin gene cluster and to provide accurate information of total a-globin gene dosage, while the DNA sequencing assay will allow the detection of known and novel variants.
Towards a New Therapy for Hemophilia A: Evaluation of Multiple Approaches To Prolong the In Vivo Efficacy of Recombinant Factor VIII.
Authors: Murphy John E. , Pan Clark , Mei Baisong , Strauss Jonathan , Tjandra Hendri , Tang Liang , Esmon Pamela , Newgren James , Jesmok Gary , Landskroner Kyle , Parmathi Mohan , Radtke Klaus-Peter , Severs Joanne , Teare John , Jiang Haiyan , Fournel Michael
Published: October 2019

Prophylactic therapy with rFVIII has been shown to have a significant positive impact on the treatment of Hemophilia A. One of the impediments to effective prophylaxis is the requirement for frequent injections necessitated by the 12–14 hour circulating half-life of FVIII. We have evaluated a number of approaches to modify FVIII to reduce the need for frequent injections. In one approach, the active form of FVIII was stabilized by addition of a disulfide linkage between the A2 and A3 domains. As previously reported1, these molecules exhibited prolonged FVIII activity in a number of in vitro assays following activation. In vivo characterization of these molecules is in progress. Other approaches focused on increasing the circulating half-life of FVIII. In an attempt to reduce FVIII clearance by the liver receptor LRP, site-directed mutations in the reported LRP binding region of the FVIII A2 domain were generated. Twenty mutants, with single amino acid changes, were analyzed in mouse and rabbit recovery studies and no significant increase in recovery was observed. Since the point mutants may not have covered a large enough surface of the LRP binding domains, polyethlylene glygol (PEG) modification was used to disrupt a larger surface area. Previously, attachment of PEG moieties to FVIII had been shown to lead to an increase in plasma half-life in animal models; however, standard pegylation chemistries result in significant product heterogeneity and thus may not be suitable for commercial production. We have developed a novel method, based on protein engineering, to introduce PEG to specific cysteine residues on the surface of FVIII. Using this method, different molecular weight PEGs have been conjugated to sites in the A1, A2 or A3 domains of FVIII. Analysis of the pegylated proteins confirms that the attachment of PEG is highly specific to the engineered site. The pegylated products retain activity based on the two stage chromogenic assay, but exhibit reduced activity when analyzed by the one stage coagulation assay. Pharmacokinetic studies, performed in mouse and rabbit, show that pegylation increases half-life in a manner that is proportional to PEG molecular weight. Using a number of injury models in hemophilic mice, the pegylated molecules have been shown to be efficacious in stopping bleeds. The prospects for using site-specific pegylation of FVIII to produce a therapeutic for treatment of hemophilia A will be discussed.
Identifying Novel Blood Proteins and Protein Isoforms Using Tandem Mass Spectra.
Authors: Fermin Damian , Allen Baxter B. , Blackwell Thomas W. , Menon Rajasree , Adamski Marcin , States David J.
Published: October 2019

Blood is a complex fluid that samples all tissues in the human body. Despite complete sequence determination of the human genome, defining genes and gene products remains a challenge. Here, we apply tandem mass spectroscopy as new source of unbiased data to interrogate genomic sequence and identify novel protein coding sequences. A six-frame translation of the Human genome was used as the query database to search for novel blood proteins in the data from the HUPO PPP. Significance is assessed using a Poisson statistical model incorporating the length of the matching sequence and the frequency of spectrum matches observed in searching the database [Nat Biotech 2006 24(3):333–8]. Matches are binned by X!Tandem hyperscore, and statistics for each score class are considered independently. The overall probability that the matches to an ORF occurred at random is calculated as the product of the probability that the matches in each score category occurred at random. The expected number of random matches, E, is calculated as the product of the probability that an ORF match occurred at random multiplied by the number of ORFs searched. The confidence in an ORF identification is 1/(1+E). An open reading frame is considered significant if confidence is greater than 95%. Expanding recently published work [Genome Biol2006; 7(4):R35], we have identified 837 significant open reading frames coding for 18852 peptides falling within 914 exons of 413 genes. Out of 8856 candidate ORFs outside the boundaries of known genes, 3246 of them achieved a confidence >= 0.95. We also required the XG ORFs to be supported by at least 3 distinct ESTs. Twenty four of the XG ORFs were found to have a significant alignment to the mouse genome. Of these, 13 of the alignments encompassed a coding region for one of the diagnostic peptides associated with the ORF. Gene models for the XG ORFS were derived from the GENSCAN prediction made for their coding regions. This analysis suggests that alternative splicing of blood protein genes is common and that much remains to be learned about the protein constituents of blood.