bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction and Regulation of HSC with Osteopontin Is Mediated through α4β1 and α9β1Integrins, Which Are Differentially Expressed in the HSC Pool.
Authors: Haylock David N. , Whitty Genevieve A. , Williams Brenda , Storan Melonie J. , Nilsson Susie K.
Published: October 2019

Osteoblasts are a key cellular component of the hemopoietic stem cell (HSC) niche and directly regulate the HSC pool. Molecules synthesised by osteoblasts both promote or inhibit HSC proliferation. Osteopontin (Opn) is an osteoblast produced, RGD containing protein with roles in cell adhesion and migration. Until recently, the role of Opn in hemopoiesis was seen as restricted to the regulation of bone turnover. However, from analysis of hemopoiesis in the Opn null mouse, we have demonstrated that Opn plays a critical role in regulating the HSC pool. Furthermore Opn is critical in trans-marrow migration and lodgement of HSC within the BM after transplantation. When added to in vitro HSC cultures, exogenous thrombin-cleaved Opn also inhibits cell proliferation and potently suppresses HSC differentiation. We have now demonstrated that this interaction occurs in an RGD-independent manner via the cryptic SVVYGLR epitope revealed on the N-terminal fragment of Opn following thrombin cleavage. This epitope has previously been shown to bind to α4β1 and α9β1. HSC are known to express α4β1, but we have now shown that within the HSC pool this occurs in a differential manner, mimicking that of CD38, with more committed CD34+CD38+ cord blood progenitors having the highest levels of expression. In addition, we have shown the previously unrecognised characteristic of human marrow and cord blood HSC, the expression of α9β1, which also occurs in a differential manner, but mimicking CD34. Expression of α9β1 is highest on cord blood CD34+CD38− cells, a population highly enriched for HSC. Using the synthetic SVVYGLR peptide in culture, we re-capitulated the thrombin-cleaved Opn induced suppression of HSC differentiation in a dose dependent manner. Antibody blocking experiments demonstrated that binding to this peptide was occurring through both α4β1 and α9β1. In contrast, suppression of HSC proliferation and differentiation did not occur through the upstream alternate α4β1 binding site. Furthermore, we have now demonstrated endogenous binding of Opn to α4β1 and α9β1 to cord blood HSC in vivo. Together, these data provide strong evidence that Opn is an important component of the HSC niche which acts as a physiological negative regulator. Furthermore, our studies identify the previously unrecognised characteristic of HSC, the expression of α9β1, which together with α4β1 provides two receptors on HSC with differing expression signatures and potentially a mechanism for fine tunning the physiological effects of Opn.
Synergistic Activity of the Aurora Kinase Inhibitor MK-0457 (VX-680) with Idarubicin, Ara-C, and Inhibitors of BCR-ABL.
Authors: Hoover Russell R. , Harding Matthew W.
Published: October 2019

MK-0457 (VX-680) is a reversible small molecule kinase inhibitor that targets Aurora A, B, and C with Ki values of 0.7, 18, and 4.6 nM, respectively. MK-0457 also inhibits Flt3 (Ki = 30 nM), and both the wild type and the T315I mutant of BCR-ABL (Ki = 30 and 40 nM, respectively). Clinical trials are ongoing in patients with solid tumors and hematological malignancies. Recent data show that MK-0457 is active in patients against subtypes of AML, BCR-ABL T315I mutant CML, and Philadelphia positive (Ph+) ALL. To support multi-agent clinical trials, the activity of MK-0457 in combination with idarubicin, Ara-C, and BCR-ABL inhibitors was investigated. The viability of a panel of AML, ALL, and CML cell lines was assessed following single agent and either simultaneous or sequential combinations of agents. Combination effects were evaluated using the Bliss Independence Model. MK-0457 as a single agent markedly inhibited leukemia cell viability (at 72 hrs) with an IC50 range of 20–300 nM for MV4-11, Molt-4, Molm-13, K562, LAMA-84, MEG-01, and KU812F cells. Additionally, MK-0457 inhibited the viability of BaF3 cells transformed by wild type, T315I, or Y253F mutants of BCR-ABL with similar IC50s (approximately 300 nM). The sequential combination of MK-0457 followed by either idarubicin or Ara-C showed greater synergy than simultaneous combinations in a cell line dependent manner. MK-0457 displayed strong synergy in simultaneous combination with Gleevec (imatinib mesylate) in a panel of human CML-derived cell lines and BaF3 cells expressing wild type BCR-ABL. MK-0457 enhanced the Gleevec-mediated cell death of K562 leukemia cells as evidenced by increased caspase activity, PARP cleavage, and induction of the sub-G1 population. At concentrations where synergy was observed by cell viability analysis, the MK-0457/Gleevec combination resulted predominantly in aneuploidy and G2/M arrest, consistent with inhibition of Aurora kinases by MK-0457. These results support the clinical evaluation of MK-0457 combined with idarubicin and Ara-C in AML and with BCR-ABL inhibitors in CML and Ph+ ALL.
NPM-ALK Fusion Tyrosine Kinase of Anaplastic Large Cell Lymphoma Exerts Its Transforming Potential by Increasing Translation of JUNB through mTOR and S6K1.
Authors: Staber Philipp B. , Vesely Paul , Ott Rene , Haq Naznin , Linkesch Werner , Schauer Silvia , Hrzenjak Andelko , Kenner Lukas , Dirks Wilhelm , Kadin Marshall E. , Sternberg David W. , Hoefler Gerald
Published: October 2019

The nucleophosmin (NPM) - anaplastic lymphoma kinase (ALK) fusion protein, which is the product of the balanced chromosomal rearrangement t(2;5)(p23;q35), occurs in about 50% of nodal anaplastic large cell lymphoma (ALCL). Expression of this fusion kinase results in neoplastic transformation by modulating multiple intracellular signaling molecules, such as the PI3-kinase and the ERK1/2 kinases.

Here we show high activation of JunB in various human ALCL cell lines. Moreover, using human embryonic kidney (HEK293) and murine hematopoietic Ba/F3 cells ectopically expressing NPM-ALK, we demonstrate that NPM-ALK is the trigger for JunB activation.

Knock down of JUNB in NPM-ALK expressing cells using RNA interference results in upregulation of p16INK4a and downregulation of Cyclin D1, causing G1/S cell cycle arrest. Thus, JunB plays a critical oncogenic role in NPM-ALK transformed cells.

Using the PI3-kinase inhibitor LY294002 and the MEK-inhibitor UO126, we demonstrate that NPM-ALK-induced JunB activity as well as cellular proliferation are dependent on both PI3-kinase and MEK-ERK signaling. Moreover, we illustrate that NPM-ALK and subsequently PI3-kinase activate AKT and mTOR/S6K1 which are implicated in the translational control of specific mRNA molecules containing a polypyrimidine motif (see pathway diagram). Since JUNB mRNA harbors such a motif we confirmed that JUNB mRNA is translated in large polysomes using ribosomal gradient preparations. Selective block of mTOR with the immunosuppressant rapamycin decreases proliferation and reduces protein but not mRNA levels of JunB in human and murine NPM-ALK positive cell lines. A similar effect is seen when inhibiting the upstream activator of mTOR, PI3-kinase. In contrast, a significant decrease of JUNB mRNA levels is shown in cells treated by the MEK-inhibitor UO126.

An analogous effect of NPM-ALK is observed in ALCL patient samples. Hyperphosphorylation of S6K1 at Thr389 indicating activated mTOR/S6K1 is seen in 9/10 NPM-ALK positive ALCL cases in contrast to only 1/15 of NPM-ALK negative ALCL samples (P < 0,001). Hence, these findings suggest that the signaling cascade NPM-ALK→PI3K→AKT→mTOR/S6K1 is crucial to induce JUNB translation in human NPM-ALK positive ALCL.

In conclusion, we reveal that JunB acts as an oncogene in NPM-ALK positive neoplastic cells and therefore represents a valid therapeutic target. Our data indicate a novel mechanism for regulation of AP-1 activity in general and suggest a new therapeutic approach to specifically modulate translation of an oncogene.

Figure Figure
Biochemical and Functional Analysis of Germline KRAS Mutations That Cause Disorders of the Noonan Syndrome Spectrum.
Authors: Schubbert Suzanne , Sovik Oddmund , Bollag Gideon , Nguyen Hoa , Rowe Sara L. , Kratz Christian , Zenker Martin M. , Niemeyer Charlotte Marie , Molven Anders , Shannon Kevin
Published: October 2019

Noonan syndrome is characterized by short stature, facial dysmorphism, and cardiac defects. We and our colleagues discovered novel de novo germline KRAS mutations that introduce V14I, T58I, or D153V amino acid substitutions in individuals with NS and a P34R alteration in an individual with cardio-facio-cutaneous (CFC) syndrome, which has overlapping phenotypic features with NS. Recombinant V14I and T58I K-Ras proteins display defective intrinsic GTP hydrolysis and impaired responsiveness to the GTPase activating proteins (GAPs) p120 GAP and neurofibromin. We also found that V14I and T58I K-Ras render primary hematopoietic progenitors hypersensitive to growth factors and deregulate signal transduction in a cell lineage specific manner (Nature Genetics38, 331, 2006). We recently began interrogating the P34R and D153V K-Ras mutant proteins and a novel CFC-associated F156L K-Ras mutant protein. Both P34R and D153V K-Ras display normal levels of intrinsic GTP hydrolysis. In contrast, F156L K-Ras displays defective intrinsic GTP hydrolysis that resembles oncogenic G12D K-Ras. P34R K-Ras is completely resistant to both GAPs, which is intriguing as these data suggest that the ability of GAPs to down-regulate Ras-GTP levels is dispensible for development. In contrast, D153V K-Ras is responsive to both GAPs. We expressed mutant Ras proteins in COS-7 monkey kidney cells to investigate activation of Ras and downstream effectors. Cells expressing P34R, D153V, F156L, and G12D K-Ras demonstrate elevated levels of Ras-GTP and phospho-MEK in basal and serum-starved conditions. We conclude that germline KRAS mutations that cause human disease encode proteins with distinct mechanisms of biochemical activation. Further investigation of this allele series will provide insights about the relative importance of the intrinsic Ras GTPase, GAPs, and other biochemical mechanisms in controlling the growth of different cell lineages.
Regulation of NOTCH2 Signaling in B-CLL: Involvement of PKC-δ.
Authors: Hubmann Rainer , Duechler Markus , Hilgarth Martin , Schnabl Susanne , Demirtas Dita , Schwarzmeier Josef D. , Jaeger Ulrich , Shehata Medhat
Published: October 2019

We have previously shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002 May 15;99(10):3742–7). NOTCH2 plays a determining role in the development/homeostasis of CD5+ B1 B-cells and of the related marginal zone (MZ) B2 B-cells, suggesting a potential role for NOTCH2 in B-CLL leukemogenesis. Using electrophoretic mobility shift assays (EMSA) we demonstrate that freshly isolated B-CLL patient samples (n=30) express an activated form of nuclear NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 surface expression, cytogenetics).

Although the majority of cultured B-CLL samples lose their N2IC activity within one day, DNA-bound N2IC complexes could be maintained by low concentrations of the PKC-stimulating phorbol esther PMA (1ng/ml). This was accompanied by the upregulation of CD23. The effect of PMA on N2IC activation and CD23 expression was abrogated by the PKC-δ inhibitor Rottlerin. Since wild type NOTCH2 signaling is regulated through binding to its ligand followed by γ-secretase mediated cleavage and release of the intracellular domain (N2IC), we induced NOTCH2 signaling by PMA in the presence or absence of the γ-secretase inhibitors (GSI) DAPT and compound E. Results demonstrated that the PMA-induced NOTCH2 activity is resistant to GSI treatment in 24 out of 30 B-CLL cases (80%). This suggests that the leukemic cells from the majority of B-CLL patients express an activated form of N2IC which is independent from γ-secretase cleavage and, thus, do not reqire NOTCH2 ligands for signaling.

In conclusion, the results suggest that PKC-δ is involved in the activation/nuclear translocation of N2IC in B-CLL cells. The data may also suggest that deregulated NOTCH2 signaling is an early step in the development of B-CLL and might be critically involved in the aberrant response of the malignant clone to cell fate modulating stimuli acting through PKC.
Lymphoid Microenvironment Inhibits Apoptosis in B-CLL Cells: Involvement of PI3-K/AKT Pathway and PTEN.
Authors: Shehata Medhat , Schnabl Susanne , Demirtas Dita , Schwarzmeier Josef D. , Hilgarth Martin , Duechler Markus , Gaiger Alexander , Hubmann Rainer , Jaeger Ulrich
Published: October 2019

The activation of anti-apoptotic mechanisms in the leukemic B cells in chronic lymphocytic leukemia (B-CLL) through the interaction with their microenvironment may lead to prolonged survival and the accumulation of the malignant clone. The aim of this study is to elucidate the influence of the lymphoid microenvironment in the activation of the potent anti-apoptotic PI3-K/Akt pathway and to investigate the pattern of expression of the tumor suppressor PTEN in B-CLL. Stromal fibroblasts of bone marrow (BMF), spleen (SF) and lymph gland (LGF) were used as an in vitro model for lymphoid microenvironment. Pharmacological inhibitors and siRNAs against PI3-K and Akt were applied to explore the anti-apoptotic effect of this pathway in B-CLL. The results showed that co-cultivation of B-CLL cells with human BMF, LGF, and SF significantly inhibited apoptosis and prolonged survival of the leukemic cells in comparison to suspension cultures. To explore the involvement of PI3-K/Akt pathway in the anti-apoptotic effect of stromal fibroblasts, co-cultures were performed in presence of PI3-K inhibitors (wortmannin or LY294002) or siRNAs against PI3-K and Akt1. These inhibitors significantly reduced the supportive effect of stromal fibroblasts and induced apoptosis in B-CLL cells. The leukemic cells were more sensitive to PI3-K inhibition than T cells, monocytes and stromal fibroblasts. Induction of apoptosis was associated with a significant decrease in the intracellular levels of PIP3, PI3-K, PDK1, Akt1, NF-kappa B, IKK, and dephosphorylation (activation) of PTEN. Since PTEN activity, as a negative regulator for PI3-K signalling, is controlled by its phosphorylation at the tail domain, we studied the pattern of PTEN protein expression in B-CLL. Western blotting demonstrated that the total PTEN in PBMC of B-CLL patients (n=40) is comparable to healthy individuals (n=8). However, using phosphospecific anti-PTEN antibody demonstrated that samples of B-CLL patients highly express phosphorylated (inactive) forms of PTEN in comparison to healthy persons. In conclusion, the results demonstrate that PI3-K/Akt pathway is involved in inhibition of apoptosis of B-CLL cells and suggest that interaction of the leukemic cells with lymphoid microenvironment may lead to the activation of this pathway. The data also suggest that targeting this pathway represents a feasible therapeutic approach in B-CLL.
Oncogenic NRAS, KRAS, and HRAS Induce Distinct Hematological Diseases in Mice.
Authors: Parikh Chaitali , Subrahmanyam Ramesh , Ren Ruibao
Published: October 2019

RAS family proteins (NRAS, HRAS and KRAS 4B/4A) are small GTPases that play a central role in transducing signals that regulate cell proliferation, survival and differentiation. The RAS proteins interact with a common set of activators and effectors and, therefore, share many biochemical and biological functions. However, the RAS proteins associate with different microdomains of the plasma membrane as well as other internal cell membranes and are capable of generating distinct signal outputs. Mutations that result in constitutive activation of RAS proteins are associated with approximately 30% of all human cancers, including 20–30% acute myeloid leukemia (AML) and 50–70% chronic myelomonocytic leukemia (CMML). Different RAS oncogenes are preferentially associated with different types of human cancer. In myeloid malignancies, NRAS mutations occur (in approximately 70% of cases), more frequently than KRAS mutations, while HRAS mutations are rare. The mechanism underlying the different frequencies of RAS isoforms mutated in myeloid leukemia is not known. One possibility is that oncogenic RAS proteins have different leukemogenic potentials. To test this possibility, we compared the ability of the three oncogenic RAS proteins to induce leukemias using the same animal model. We have shown previously that oncogenic NRAS rapidly and efficiently induces CMML- or AML-like disease in an improved mouse bone marrow transduction and transplantation model. We found here that in the same model system, oncogenic KRAS invariably induces a CMML-like disease that is similar to what has been shown in a mouse conditional knock in model for oncogenic KRAS. Surprisingly, all mice receiving oncogenic HRAS infected bone marrow cells develop an AML-like disease. The HRAS mice have the shortest disease latency, followed by NRAS and then KRAS. HRAS induced disease also appears to be more aggressive than N or KRAS and is usually accompanied by massive pulmonary infiltration and hemorrhages. These studies demonstrate that all three RAS oncogenes have the potential to induce myeloid leukemias, yet have distinct leukemogenic potentials. The underlying mechanism of the discrepancy between the frequency of HRAS mutation in human myeloid leukemia and its leukemogenic potential in mice is not clear, but the models established here provide a system for further studying the molecular mechanisms in the pathogenesis of myeloid malignancies and for testing targeted therapies.
High-Throughput Genetic Profiling of Acute Myeloid Leukemias: Novel Mutations Detected in Signal Transduction Pathway Genes by a Strategy Utilizing Endonuclease-Based Heteroduplex Scanning and DNA Sequencing.
Authors: Lilleberg Stan L. , Hempel Jill , Durocher Jeff , Foster Damien , Hartman Christopher , Stoddard Jason , Walters Brooke , Nickerson Michael L. , Koul Manika
Published: October 2019

The Raf/MEK/ERK, Wnt/beta-catenin, JAK/STAT and PI3K/Akt signal transduction pathways have key roles in regulation of cell cycle progression and apoptosis, and are current focal points of therapeutic development and intervention strategies for hematopoietic neoplasias. Although mutations in several pathway-associated genes known to contribute to the malignant phenotype have been described, the mutation status of genes encoding components of these pathways as a whole remains to be determined. This has yet to be achieved in even a single type of cancer. In this study, we applied a novel mutation scanning strategy (HTMS) that utilizes SURVEYOR Endouclease heteroduplex cleavage analysis of patient derived PCR products, along with selective fluorescent DNA sequencing in a combinatorial fashion to achieve high-throughput, accuracy, and sensitivity. Comprehensive screening of the protein coding portions of 33 signal transduction pathway genes was performed on PCR products from 192 AML samples and 48 controls. Target genes included receptor kinases (FLT3, KIT, CSF1R, FGFR3, NOTCH1), cytoplasmic kinases (ABL1, SRC, PIK3CA, JAK2), GTPases (KRAS, NRAS, HRAS), transcription factors (MYC, GATA1, CEBPA) and tumor suppressors (PTEN, P53). The Raf/MEK/ERK (genes: BRAF, ARAF, CRAF, PTPN11, MEK1/2, ERK1/2) and Wnt/beta-catenin (genes: APC, CTNNB1, CDH1, AXIN1, AXIN2, GSK3B, PP2A) pathways were emphasized. Using this high-throughput mutation scanning (HTMS) methodology, over 10 million base pairs from 192 cancer genomes and 48 control genomes was analyzed for somatic alterations and inherited polymorphisms in these genes. In the AML sample set, we identified 393 somatic mutations (2.0 mutations/AML), of which 54 (13.7%) have not been previously reported. These novel variants included point mutations in key functional domains of FLT3, KIT, NRAS, BRAF, ARAF, PTPN11, ABL1, FGFR3, MYC, NOTCH1, APC, CTNNB1, and GSK3B. At least one mutation was found in 100% of the AML samples. The relevance of these somatic mutations to the AML pathogenesis awaits detailed functional studies. The approach demonstrated in this study represents an effective in-depth mining strategy for high-throughput mutation analysis, compared to the standard re-sequencing approach for profiling of complex genetic diseases like cancer. The HTMS approach alleviates the current bottleneck of rigorous, manual sequence analysis that is required to identify somatic mutations. By integrating mutational profiling with comparative genomic hybridization, high-density SNP screening, and RNA profiling, many genetic changes relevant to cancer diagnosis, treatment and patient management, will surely be discovered, along with new targets for therapeutic intervention.
RAX Activates Tumor Suppressor p53.
Authors: Pan Yu , Bennett Richard L. , May W. Stratford
Published: October 2019

Recently, our laboratory identified RAX as a unique cellular activator for the interferon-induced double-stranded RNA-dependent protein kinase, PKR. Cellular stresses such as IL-3 withdrawal from factor dependent hematopoietic cells, inflammatory cytokine and chemotherapy treatment or viral infection promote RAX phosphorylation with activation of PKR leading to inhibition of new protein synthesis. In addition, PKR can also regulate the transcription factors p53, STAT1 and NF-kB. Now we report a novel activity for RAX in regulating the expression and activity of the tumor suppressor, p53. Results indicate that increased RAX expression in p53 expressing cells, such as HEK293, mouse embryo fibroblast (MEF) or human osteosarcoma U2OS promotes an increase in the steady-state level of endogenous p53 and its activation of the p53 target gene, p21. Furthermore, when RAX is expressed in U2OS cells or coexpressed with p53 in p53 null H1299 cells, enhanced, dose-dependent p53 transcriptional activity is observed as assessed using a p53 luciferase reporter. Consistent with these findings, flow cytometry demonstrates that either RAX or PKR can stimulate p53-dependent G1 arrest that precedes apoptosis. Since p53 is a major ‘sensor’ for DNA damage leading to cell cycle arrest and apoptosis, we also examined whether RAX enhances p53’s response to DNA damage. Following gamma irradiation or Cisplatin treatment of p53 expressing cells, expression of exogenous RAX augments while ‘knock down’ of endogenous RAX by siRNA or expression of the dominant-negative, RAX (S18A) mutant, potently blocks any increase in p53 and p21 expression compared to vector-only control cells. While we find no evidence that RAX directly interacts with p53, results indicate that RAX promotes p53 SUMOylation but does not affect p53 ubiquitination or interaction with MDM2. We now propose that RAX augments the p53 stress-dependent cellular response potentially by a PKR-dependent mechanism involving SUMOylation. Since p53 is only mutated in apparently 15% of hematologic malignancies versus more than 50% in solid tumors, it is envisioned that a more detailed understanding of the mechanism by which RAX activates p53 may serve as a basis for novel antileukemic strategies.
Outcome of Infants Less Than One Year of Age with Acute Lymphoblastic Leukemia Treated with the Interfant-99 Protocol.
Authors: Pieters Rob , Schrappe M. , de Lorenzo P. , Hann I. , Vora A. , deRossi G. , Biondi A. , Felice M. , Hovi L. , Mechinaud F. , Szczepanski T. , Ferster A. , Janka G. , Rubnitz J. , Silverman L. , Stary J. , Campbell M. , Mann G. , Suppiah R. , Valsecchi M.
Published: October 2019

Introduction

Acute lymphoblastic leukemia (ALL) in infants under 1 year of age is rare and has a poor outcome compared to ALL in older children. It is characterized by a high expression of MLL gene rearrangements, high tumor load and myeloid features. In 1999, a large collaborative international study, Interfant-99, was initiated with the aims to determine: (1) the outcome of a new treatment protocol including ALL and AML elements; (2) in a randomised way the value of a late intensification course with high dose araC and methotrexate; (3) which clinical and biological factors have independent prognostic value within infant ALL. Event-free survival (EFS) and overall survival (OS) were primary endpoints and analysed on an intention to treat basis.

Results

17 study groups representing >20 countries enrolled 482 patients leading to by far the largest trial ever reported in infant ALL. 79% of cases had an MLL rearrangement. Of these, 53% was t(4;11), 20% t(11;19), 11% t(9;11) and 16% had other MLL partner fusion genes.

Death in induction rate was 3.8% and 2.3% did not achieve CR at the end of induction, so CR rate was 94%. Death rate in CCR was 5.2%. Relapses occurred in 36% of cases and were mainly isolated bone marrow relapse. Median time from 1st CR to relapse was 8 months. The overall 4-year EFS is 47% and OS is 55%. This is at least comparable to the best historical controls and better than most of those of the participating study groups. Especially outcome of high-risk patients, defined by poor response to one week prednisone, had improved. The late intensification course with HD-araC and HD-MTX did not improve outcome.

Cox model showed that MLL rearrangement and age <6 months were strong independent prognostic factors for poor outcome. WBC > 300x10e9/L and poor prednisone response were also of independent prognostic value. The outcome was not depending on the type of MLL rearrangement.

Conclusions

Results of this first international treatment protocol for infant ALL are very satisfactory. Early bone marrow relapse remains the major cause of treatment failure indicating that early treatment intensification is necessary. The large international collaboration has enabled the start of studies to improve outcome for infant ALL. The new Interfant-06 study will stratify the patients based upon MLL status, age and WBC and will study the value of the use of two early "AML" courses in a randomised way.
Mutations of the Catalytic Subunit of Class IA PI3K Are Constitutively Active and Confer Factor-Independent Growth to Early Hematopoietic Cells.
Authors: Horn Stefan , McCubrey James , Stocking Carol , Truemper Lorenz , Basecke Jorg
Published: October 2019

Background: Activating mutations of the catalytic subunit of class IA phosphoinositide 3-kinase alpha (PIK3CA) are clustered in small hot-spot regions of the PIK3CA gene, including exon 9 within the helical domain and exon 11 within the kinase domain. They have been linked to several human neoplasias, including colorectal, breast and hepatocellular cancers. In acute leukemias, PIK3CA mutations have not been investigated in larger chorts and so far only been observed in a few patients. Since the PI3K/Akt/GSK3beta pathway is an important signaling cascade of receptor tyrosine kinases (e.g. Flt3R, Kit) which are frequently activated in acute leukemias we investigated the functional activity of PIK3CA mutants.

Materials and methods: We transfected early hematopoietic cells (Ba/F3 cell line) with PI3KCA exon 9 and 11 mutations and investigated the cells in an in vitro factor-independent growth assay and pharmacologic inhibition experiments.

Results: We demonstrate that mutations in the helical or kinase domain of PIK3CA lead to the constitutive activation of PI3Kalpha in Ba/F3 cells, inducing factor-independent growth of the IL3-dependent cells. The frequency of IL3-independent Ba/F3 cells after tranfection with exon 9 and 11 PIK3CA mutants was equivalent to the frequency confered by PIK3CA mutants containing the membrane localization signal of either src or ras. Proliferation and survival of the cells were inhibited by the PI3K inhibitors LY294002 and Quercitin or an inhibitor of the PI3K downstream target Akt. Inhibition occurred in a dose- and time-dependent manner and could be reverted by addition of IL-3. One of the major targets of PI3K/Akt signaling is GSK3beta which becomes inactivated after Akt-mediated phosphorylation. By using a GSK3beta-specific inhibitor or LiCl we could show that the inactivation of GSK3beta alone did not result in factor-independent growth of Ba/F3 cells. However, GSK3beta inhibition led to a delay in the induction of cell death after IL3-withdrawal.

Conclusion: Activating mutations of PIK3CA, associated with several human neoplasias and acute leukemia are functionally active in hematopoietic cells, confer factor independency and respond to PI3K/Akt inhibition.
Dominant-Negative Effect of NPM-RAR on NPM Mediated Regulation of p53.
Authors: Redner Robert L. , Swaney Erin M. , Rush Elizabeth A.
Published: October 2019

We have been studying the variant Acute Promyelocytic Leukemia (APL) translocations with a view towards identifying common molecular pathways underlying APL. The t(5;17) variant of APL fuses the N-terminal 117 amino acids of nucleophosmin (NPM) with the C-terminal 402 amino acids of the retinoic acid receptor alpha (RAR). NPM plays a role in several important pathways within cells beyond its originally described function in the transport of ribonucleoproteins. It serves a chaperone function in transport of proteins such as p53, Tat, Rb, YY1, IRF-1, and Arf. NPM binds to and becomes a target of CDK2/cyclin E to play a role in regulation of centrosomal duplication. The importance of NPM in myeloid leukemias has been highlighted by the recent finding that mutation of the NPM C-terminus is the most common genetic event in non-APL acute myeloid leukemia. We have previously shown that the RAR portion of NPM-RAR acts as a dominant-negative towards wild-type RAR. We now investigate the hypothesis that NPM-RAR serves as a dominant negative for NPM and partially contributes to the leukemic phenotype through disruption of NPM-dependent cellular functions. We have focused our studies on the effects of NPM-RAR on p53. It has previously been shown that NPM directly binds to p53 to increase p53 stability and p53-dependent transcription. The p53-interaction domain maps to a region of NPM that is not contained in NPM-RAR. We hypothesized that NPM-RAR might bind directly with NPM, disrupt NPM association with p53, and thereby indirectly affect p53 activity. We first used pull-down assays of Maltose Binding Protein (MBP)-NPM or MBP-NPM-RAR fusions incubated with 35S-NPM to determine that the N-terminal domain of NPM-RAR is sufficient to interact with NPM. To investigate whether NPM-RAR alters NPM interaction with p53, we transfected COS cells with expression vectors encoding NPM, p53 and either NPM-RAR or control plasmid (pBluescript). Complexes were precipitated with an anti-NPM antibody (that recognizes epitopes not contained in NPM-RAR) and immunoblotted with an anti-p53 antibody. We found a decrease in the p53 signal captured by NPM in the lysate expressing NPM-RAR, compared to the control lysate. To further investigate the effects of NPM-RAR on p53, we made use of a transcriptional assay utilizing a reporter gene under control of a p53 response element. We co-transfected COS cells with a mix containing NPM-RAR, p53, and NPM expression plasmids, along with a p53-luciferase reporter (containing a 20 base pair p53 response element derived from the p21 promoter), and a ß-gal transfection control plasmid, or a similar mix in which NPM-RAR was replaced by an equivalent amount of carrier DNA (pBluescript). We found that the co-expression of NPM-RAR led to a decrease in the luciferase activity. These results support our hypothesis that NPM-RAR binding to NPM interferes with NPM’s ability to regulate p53.
Human Platelet Tissue Factor Is Localized in Lipid Rafts and Is Associated with GPIb-IX-V Complex in the Membrane: Association between Adhesive and Procoagulant Activities of Platelets.
Authors: Panes Olga , Matus Valeria , Sáez Claudia G. , Gárate Alan , Pereira Jaime , Mezzano Diego
Published: October 2019

The binding of platelet membrane GPIb-IX-V to extracellular matrix proteins and von Willebrand factor initiates the adhesion, spreading and activation of platelets. On the other hand, tissue factor (TF) triggers the blood coagulation by binding FVII/VIIa. In resting platelets, TF is stored mainly as a non active (“encrypted”) form, possibly through a dimer- or heterodimer complex. The encryption seems not to affect the TF capacity to bind FVII and VIIa, in such form that the membrane-based complex would be “primed” to trigger the clot formation upon platelet activation. Accordingly, the platelet adhesion and activation would be tightly interwoven with thrombin generation. Given the described localization of GPIbα in platelet lipid rafts, which appears to modulate its activity, we investigated whether TF is also present in these cholesterol-rich domains of the platelet membrane. Continuous sucrose gradients generated by ultracentrifugation at 180.000g x 18h at 4°C were used to obtain cholesterol-rich fractions from platelet lysates (1% Triton X-100 at 4°C). Western blots of each gradient fraction using a polyclonal anti-TF antibody revealed a protein of ~60kDa only in the fractions rich in cholesterol (using flotillin-1 as a marker). TF-dependent procoagulant activity (PCA), assessed by FXa generation was measured in resting and 5μM TRAP-stimulated, leukocyte-free platelets. In both cases, PCA was reduced after membrane cholesterol depletion with methyl-β-cyclo-dextrin. We also found that PCA was significantly enhanced in washed platelets treated with VWF and ristocetin, suggesting some form of functional or structural relationship between GPIb-IX-V complex and TF. GPIb-IX was immunoprecipitated (IP) from human platelet membranes by anti-GPIbα MoAbs (AP-1 or SZ2), in both resting and stimulated platelets (TRAP or VWF-ristocetin). The IP product was revealed with MoAb anti-TF (American Diagnostica, clone 4509) or polyclonal anti-TF, detecting a protein of ~47kDa. When we used MoAb anti-TF for the IP and MoAb anti-GPIX (BL-H6, anti-CD42a) to reveal the western blot, two distinct bands of ~22 and ~47kDa were disclosed, probably corresponding to GPIX and a heterodimer of GPIX and TF, respectively. These observations indicate that, at least a fraction of the platelet TF is localized in lipid rafts and that TF is also in close association with the GPIb-IX-V complex. This co-localization probably has important functional implications as demonstrated by the defective thrombin generation in patients with Bernard-Soulier syndrome. Moreover, this functional association may be relevant in both normal hemostasis and thrombus formation and growth.
Role of Gender in the Clinical Presentation of a First Deep Venous Thrombosis (DVT) in Outpatients.
Authors: Andreou Roseann , Koru-Sengul Tulay , Linkins Lori-Ann , Bates Shannon M. , Ginsberg Jeffrey S. , Kearon Clive
Published: October 2019

BACKGROUND: As clinical assessment of pretest probability (PTP) is now considered the first step in the diagnostic evaluation of DVT it is important to know if the clinical features of DVT are the same in men and women.

OBJECTIVES: We sought to answer the following questions: 1) Is the prevalence of DVT the same in men and women who are referred for diagnostic testing? 2) Are the clinical features of DVT the same in men and women? 3) Does the Wells’ clinical prediction model for first DVT perform similarly in men and women?

METHODS: We performed a retrospective analysis of individual patient data from three studies that evaluated diagnostic testing in outpatients with a suspected first episode of DVT. Associations were assessed using univariable and multivariable analyses.

RESULTS: A total of 1838 patients were eligible for final analyses, of whom 1132 (62%) were women and 706 (38%) were men. DVT was objectively diagnosed in 208 patients (11%). QUESTION 1: The prevalence of DVT was higher in men (14%) than in women (9%) (p=0.001). In a multivariable analysis that included eleven other clinical variables male gender was independently associated with a higher prevalence of DVT (OR 1.5, 95% CI 1.1–2.1). QUESTION 2: Of the confirmed episodes of DVT, “swelling of the entire leg” was observed more often in women (41%) than in men (16%) (p<0.001). This difference was partly explained by more extensive thrombosis in women than in men; the common femoral vein was involved on ultrasound in 48% of women and 22% of men (p< 0.001). The frequency of other clinical features was similar in men and women. QUESTION 3: The prevalence of DVT was higher in men than in women who were categorized by the Wells model as having a low PTP (6.9% vs 3.6%; p=0.025) or a moderate PTP (16.9% vs. 8.7%; p<0.001), but similar in the high PTP category (40% vs 44%; p=0.6). Five clinical features were more strongly associated with presence of DVT in women than in men: “swelling of the entire leg” (OR 7.7 vs. 1.6; p<0.001), “difference in calf circumference >3cm” (OR 8.2 vs. 2.9; p<0.001), “pitting edema” (OR 4.4 vs. 1.8; p=0.004), “age > 60 yrs” (OR 1.8 vs. 0.9; p=0.02) and “bedridden for greater than 3 days” (OR 3.7 vs. 1.4; p=0.05).

CONCLUSIONS: Among outpatients referred with a suspected first episode of DVT, the prevalence of DVT is higher in men than in women but DVT is more extensive in women. Entire leg swelling is a more common and important sign of DVT in women than in men and the Wells prediction rule behaves differently in men than in women.
Risk Stratification by MLL Gene Status and Outcome of Acute Lymphoblastic Leukemia in Infants: A Report from the Japan Infant Leukemia Study Group.
Authors: Tomizawa Daisuke , Koh Katsuyoshi , Nagayama Jun , Sato Takashi , Kinukawa Naoko , Isoyama Keiichi , Kosaka Yoshiyuki , Oda Takanori , Oda Megumi , Hayashi Yasuhide , Horibe Keizo , Mizutani Shuki , Ishii Eiichi
Published: October 2019

Background: Acute lymphoblastic leukemia (ALL) in infants represent 2.5% to 5% of all childhood ALL, and has an inferior therapeutic outcome compared to that of older children. Among several risk factors, rearrangement in MLL gene is most closely associated with this poor outcome. This is the first series of cooperative group trials stratifying ALL in infants with MLL gene status.

Procedure: Infants with ALL, age less than 12 months, were registered on two consecutive multi-center trials designated MLL96 and MLL98. All the patients were tested for MLL gene rearrangements by Southern Blot analysis and/or fluorescence in situ hybridization. According to the MLL gene status, patients with rearranged MLL gene were allocated to intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT), and those with germline MLL were treated with standard chemotherapy alone (total treatment duration was 83–85 weeks). In MLL98 study, treatment intensification and early use of HSCT were introduced compared to MLL96 study.

Results: A total of 101 infants were enrolled between December 1995 and December 2002. Seventy-nine had rearranged MLL and 22 had germline MLL. Event-free survival (EFS) of all 101 infants with ALL was 52.0% at 5 years; 5-year EFS for MLL rearranged cases was 39.7%, and for MLL germline cases was 95.5% (p<0.001). Among the MLL rearranged patients, 3-year EFS improved from 34.0% in MLL96 trial (N=41) to 43.6% in MLL98 trial (N=38). Although their were no secondary malignancies among the whole enrolled patients, physical growth impairments was observed in MLL rearranged patients, while no significant late effects were observed in MLL germline patients.

Conclusions: Risk stratification with MLL gene status was successful for infants with ALL. Excellent results were obtained for the germline MLL group using chemotherapy alone, and early introduction of allogeneic HSCT in first remission seemed effective for the MLL rearranged group. However, use of HSCT with less toxic conditioning regimens or development of effective chemotherapy including molecular target therapy are needed for MLL rearranged patients in order to avoid severe late effects for infants.
Risk of Venous Thromboembolism Is Not Increased by Elevated TAFI Levels.
Authors: Folkeringa Nienke , Brouwer Jan Leendert P. , Veeger Nic J.G.M , van der Meer Jan
Published: October 2019

TAFI (Thrombin-activatable fibrinolysis inhibitor) is a suppressor of fibrinolysis and high plasma levels of TAFI are assumed to induce venous thromboembolism (VTE). A few studies published on this subject identified TAFI as a mild risk factor for VTE. The risk may be increased if high TAFI levels are combined with other trombophilic defects. We performed a retrospective study to asses the absolute risk of VTE associated with elevated levels of TAFI and the contribution of concomitant thrombophilic defects. Subjects were recruited from a previous large family cohort study, which was designed to estimate the risk of VTE in first degree relatives of probands with documented VTE and a hereditary deficiency of antithrombin, protein C or protein S. Probands were excluded from analysis. Blood samples of relatives were tested on deficiencies of antithrombin, protein C and protein S, prothrombin G20210A, factor FV Leiden, hyperhomocysteinemia and increased levels of factor VIII:C, IX:C and XI:C. TAFI activity was additional measured by chromogenic assay on plasma samples stored at minus 80 degrees Celsius.

We analysed 350 of 457 relatives, who were tested on TAFI. TAFI levels observed in women were lower than in men (median 97 versus 106 U/dL) and increased with age (median from 95 U/dL at age 15–30 yr to 107 U/dL at age>60 yr). Relatives with TAFI levels above the 90th percentile (>128 U/dL) had the same absolute risk of VTE as relatives with TAFI levels below the 90th percentile. Annual incidences (AI) were 0.74 versus 0.72 % per year: relative risk (RR) 1.0; 95% CI.0.51–2.10 (Table).

Concomitant thrombophilic defects were demonstrated in 82% of the relatives. These defects were equally distributed among relatives with TAFI levels of >90th versus <90th percentile. Relatives with TAFI levels above the 90th percentile and concomitance of at least one other thrombophilic defect were not at increased risk of VTE compared to relatives with lower TAFI levels and concomitant thrombophilic defects (RR 0.83; 95% CI 0.41–1.68).

Our data do not support that eleveated TAFI levels are a risk factor for VTE. The high absolute risk of VTE among relatives is mainly due to the presence of other thrombophilic defects rather than elevated TAFI levels, as interactions between TAFI and other thrombophilic defects were not demonstrated.

VTE (n) Observation years AI (95% CI) RR (95% CI) TAFI > 90th percentile 9 1209 0.74 (0.3–1.4) 1.0 (0.51–2.10) Concomitance 9 1147 0.78 (0.4–14.9) 0.83 (0.41–1.68) No concomitance 0 62 0 TAFI < 90th percentile 53 7364 0.72 (0.5–0.9) Ref Concomitance 53 5724 0.93 (0.7–1.2) Ref No concomitance 0 1426 0
Women with the Prothrombophilic HPA-1b Genotype of Platelet Integrin αIIbβ3 Are at Increased Risk of Intrauterine Growth Restriction.
Authors: Gerhardt Andrea , Howe Nadja , Kruessel Jan S. , Bender Hans G. , Scharf Rudiger E. , Zotz Rainer B.
Published: October 2019

Background: Disturbances in the placental vascular development with endothelial inflammation and thrombotic occlusion of the placental vasculature are associated with fetal intrauterine growth restriction (IGR). As shown in previous studies, the HPA-1b allele of the b subunit of the essential platelet integrin αIIbβ3, also known as glycoprotein IIb–IIIa, can be associated with increased platelet thrombogenicity, leading to arterial vascular occlusion also triggered by inflammatory endothelial alterations. We performed a case-control study to assess HPA-1b as risk determinant for fetal IGR.

Materials and Methods: Fifty two women with fetal IGR (defined by birth weight below the 5th- and 10th percentile for gestational age and sex) were evaluated. Women with other reasons of IGR (history of venous thrombosis, fetal loss, and preeclampsia) were excluded. The fetuses were born alive after the 24th week of gestation. As control subjects, 307 normal women with at least one previous pregnancy and no previous pregnancy complication were included.

Results: There was a significant risk association of the HPA-1b/1b genotype (birth weight below 5% percentile: odds ratio (OR) 6.3 (95% confidence interval (CI) 1.4–21); birth weight below 10% percentile: OR 4.6 (95% CI 1.4–15). OR for the HPA-1b/1b genotype (birth weight below 5% percentile) in subgroups with increased levels of fibrinogen (above median > 284mg/dl) were 17.7 (95% CI 2.9–106) and increased levels of FVIII:C (above median > 147%) were 22.1 (95% CI 3.8–127). No significant difference between the genotype distribution was found in patients with low levels of fibrinogen and/or FVIII:C. Similar results were found for women with IGR with a birth weight below the 10% percentile. There was no significant risk association for factor V Leiden G1691A, prothrombin G20210A mutation, and MTHFR 677TT.

Conclusions: The platelet receptor genotype HPA-1b/1b is significantly associated with IGR. As possible pathological mechanism, an increased HPA-1b-associated thrombogenicity of platelets leading to IGR via interaction with an inflammatory vascular process and/or via increased levels of FVIII:C or plasma fibrinogen have to be considered. Pharmacological control of the observed platelet thrombogenicity in patients at risk may be of clinical relevance. In consequence, it will be of importance to examine whether this critical subgroup of patients can benefit from prevention with specific antiplatelet agents.
Calcein and the Labile Iron Pool.
Authors: Soe-Lin Shan , Buss Joan L. , Tang Evelyn , Ponka Prem
Published: October 2019

The labile iron pool is a putative cytosolic compartment of loosely bound, redox-active, chelator-accessible iron. Iron contained within this pool is thought to influence the activity of iron regulatory proteins (IRPs), which bind to iron response elements (IRE) during low iron conditions; this association blocks the translation of ferritin mRNA, and stabilizes transferrin receptor mRNA. High levels of labile iron have been shown to promote oxidative stress. As this pool has such profound effects upon cellular iron homeostasis, there has been great interest in the development of methods to measure labile iron. Calcein, a fluorescent iron chelator, has been widely used to monitor the labile iron pool. When the non-fluorescent acetoxymethyl ester moiety (calcein-AM), enters cells, it is immediately cleaved by cytosolic esterases to its cell-impermeable, fluorescent calcein form. Iron binding to calcein quenches its fluorescence, which can subsequently be recovered following the loss of its iron to a stronger chelator. The difference in fluorescence between the bound and unbound calcein forms is thought to be proportional to the labile iron pool itself. While this method has been commonly exploited, it is unknown whether calcein may over-estimate the size of the labile pool by stripping iron from sources where it may be loosely bound, or by intercepting iron during its passage from one compartment to another. Although it is believed that calcein exerts very little direct influence on cellular iron homeostasis and acts only as a passive sensor of labile iron, some recent evidence from our lab indicates that this may not be the case. We have observed that incubation with calcein results in the activation of IRP-2 and stabilization of HIF-1α, a potent physiological regulator governing the expression of genes involved in oxygen sensing and iron metabolism. Furthermore, we have found that the size of the labile iron pool as measured by calcein was proportional to the amount of calcein loaded in HeLa and K562 cell lines. These findings suggest that calcein may be able to perturb cellular iron homeostasis, and may not accurately reflect the size of the labile iron pool. While calcein may still be used for comparative purposes under identically controlled conditions, its usefulness as a quantifying agent should be regarded with caution.