bluuebunny/crossref_metadata_2025_split huggingface dataset:
Triptolide Cooperates with Cytarabine To Induce Apoptosis in Acute Myeloid Leukemia.
Authors: Pigneux Arnaud , Uhalde Maïalen , Mahon François X. , Lacombe Francis , Praloran Vincent , Milpied Noel-Jean , Belloc Francis 
Published: October 2019

Abstract
Triptolide, a diterpene triepoxide isolated from the Chinese herb Tripterygium wilfordii Hook.f, has shown antitumor activity in a broad range of solid tumors and on leukemic cells in vitro. Here, we examined its pro-apoptotic effects on THP1 myeloid cell line and primay AML blasts in association with cytarabine. THP1 cells were treated with increasing concentrations of Ara-C in the presence or not of Triptolide (5 ng/ml). Apoptosis was measured by flow cytometry at the mitochondrial level by using the ΔΨm sensitive probe DiOC6(3). This low dose of Triptolide did not induce any apoptosis in 24 hrs. However, it significantly increased the apoptosis induced by Ara-C in the range 0.1–2 μg/ml (p<0.001) (figure 1). At 48 hrs, this cooperative effect was observed for lower doses of Ara-C (0.05–0.1 μg/ml)(figure 2).
Fig. 1 Fig. 1. Fig. 2 Fig. 2.
When measuring the IκB content by western blot, although triptolide alone was found able to decrease IκB (p<0.05), there was no effect on the IκB content of Ara-C-treated cells. Similarly, no effect of the Ara-C/Triptolide association was found in the DNA binding activity of NFκB. The DNA synthesis activity was measured by flow cytometry using the BrdUrd method and a dose dependent DNA synthesis inhibition was found with Triptolide from 2 to 20 ng/ml. Moreover, the association of 5 ng/ml Triptolide with 0.05 or 0.1 μg/ml of Ara-C showed an increase in the DNA synthesis inhibition as compared with both drugs separately. However, while Ara-C induced an accumulation of cells in the S compartment of the cell cycle, the cell cycle distribution was not significantly altered by Triptolide alone, suggesting that this drug induced also a slowing down during all the cell cycle. The association Triptolide/Ara-C was also investigated in vitro on primary blast cells from 26 AML patients. The blood or bone marrow mononuclear cells were cultured for 48 to 72 hrs in the presence of a cytokine cocktail (Stem α.4B) to recruit the cells into the cell cycle. The samples were then treated with 7.5 ng/ml Triptolide, 0.05 or 0.1 ng/ml Ara-C or both drugs in combination for 24 and 48 hrs. Apoptosis was measured by flow cytometry, using FITC-Annexin V. The blast cells were discriminated on the basis of their low CD45 expression. The combination of both drugs yielded a percentage of apoptotic cells that was significantly higher than each drug administered separately (p<0.05). These results show that low concentrations of Triptolide which were not shown to induce apoptosis by themselves were able to potentiate the pro-apoptotic and anti-proliferative effects of Ara-C. The potentiation by triptolide of the antileukemic activity of cytarabine in vitro warrants further clinical investigations for the treatment of AML patients, specially in elderly patients in which low-dose cytarabine treatment could be improved by an association with Triptolide.
Rational for Specific Inhibition of Both PI3K/AKT and mTORC1 Activities in Acute Myelogenous Leukaemia.
Authors: Tamburini Jerome , Chapuis Nicolas , Bardet Valerie , Park Sophie , Verdier Frederique , Dreyfus Francois , Lacombe Catherine , Bouscary Didier 
Published: October 2019

Abstract
We previously reported that constitutive activation of PI3K/AKT activity in primary blasts from patients with AML is related to the expression of the p110d isoform of class IA PI3K which is activated by an upstream, actually unknown kinase (Sujobert P, Blood, 2005, 106, 1063). Activation of PI3K activity might make AML cells susceptible to mTORC1 inhibition, which was recently shown to be activated in AML cells. mTORC1 inhibition by Rapamycin or RAD001 may control both proliferation and survival of leukemic cells. The mechanisms leading to mTORC1 activation in AML are actually unknown
In this report, we studied the interactions between PI3K and mTORC1 pathways in primary blast cells from the bone marrow of patients with primary AML at diagnosis. We observed that specific inhibition of mTORC1 activity with RAD001 upregulates PI3K activity, as evidenced by an increased phosphorylation of AKT on Ser 473. In 8 patients, the mean increase of AKT phosphorylation in the presence of RAD001 was 200% (130%–500%). Such results were observed in most patients with constitutive activation of PI3K, but very rarely in patients without constitutive PI3K activity.
We then studied the activation of the mTORC1 complex when a full inhibition of PI3K activity with IC87114, a specific inhibitor of p110d isoform, was performed. mTORC1 activity, as assessed by phosphorylation of downstream targets, p70S6K, S6 ribosomal protein and 4EBP1 was not inhibited by IC87114 (10μM) in PI3K+ patients, whereas RAD001 (10nM) completely or near completely inhibited all phosphorylation events. Similar results were detected in the MV4-11 cell line. Moreover, we detected constitutive activation of mTORC1 in several PI3K- patients. These results clearly indicate that mTORC1 activation is no related to PI3K activity. In accordance with that, we showed that simultaneous inhibition of PI3K and mTORC1, with IC87114 and RAD001 respectively, additively inhibited the proliferation of blast cells and the clonogenicity of leukemic progenitor cells. Our results provide evidence that RAD001 relieve mTORC1 mediated feedback inhibition on AKT activation, as recently described in multiple myeloma and some cancer cell lines. We also demonstrate that mTORC1 activation occurs independently of PI3K activity. We are currently studying the upstream mechanisms resulting in mTORC1 activation. Finally, functional studies support a clear strategy for combining an mTORC1 inhibitor with an inhibitor of PI3K.
SHP-1 (PTPN6) Gene Supression by FLT3/ITD Defines an Aggressive Subgroup of Myeloid Malignancies.
Authors: Mena-Duran Armando V. , Pajuelo Juan C. , Cervera-Zamora Jose , Garcia-Casado Zaida , Valencia Ana , Barragan Eva , Benlloch Luis , Mustelin Tomas , Sanz Guillermo , Sanz Miguel A. 
Published: October 2019

Abstract
Recently, Chen et al (J Biol Chem.2005;280:5361–9) showed SHP-1 (PTPN6) gene supression by FLT3/ITD in TF-1 cell lines and in primary cells from AML patients. To address the prognostic impact that both together FLT3/ITD and SHP-1 gene suppression may have in myeloid malignancies patients, we have studied 85 patients diagnosed with de novo AML and MDS. SHP-1 gene expression was quantified by real time PCR and compared with normal CD34+ cells from healthy donors. FLT3/ITD was detected by conventional agarose gel electrophoresis after PCR. FLT3/D835 was detected by melting curve methodology. FLT3 mutations were correlated with higher WBC and blasts count at diagnosis (p=0.001). FLT3/ITD had a negative impact on DFS compared with wt or D835 mutations (log-rank p=0.054). Median levels for SHP-1 were lower in leukemic blasts than in normal peripheral blood CD34+ cells (p=0.001). SHP-1 lowest expression was found in those patients where FLT3 mutations were present (p=0.001). SHP-1 lowest expression was associated with higher blasts count in BM at diagnosis (p=0.017).

Having both Flt3 mutations and lowest expression for SHP-1 were strongly associated with higher WBC (> 20 x109/L) (p=0.005). OS univariate analysis yielded a worse prognosis for patients having both lower SHP-1 expression and harboring an Flt3 mutation (p=0.021).
Figure Figure
In summary, SHP-1 (PTPN6) gene supression by FLT3 constitutively activating mutations confers a bad prognosis for myeloid malignancies patients and is associated with hyperleukocytosis at diagnosis.
Differences in the Localization of Receptor Tyrosine Kinase FLT3 in Human Hematopoietic Stem and Progenitor Cells and Leukemic Blasts.
Authors: Koch Sina , Ryser Martin , Bornhaeuser Martin , Ehninger Gerhard , Thiede Christian 
Published: October 2019

Abstract
Activation of type III receptor tyrosine kinase FLT3 by its ligand controls cell proliferation and differentiation in normal hematopoiesis. Constitutively activating mutations of FLT3 are common abnormalities in acute myeloid leukemia. Internal tandem duplication (ITD) mutations of the FLT3 receptor are found in approximately one third of patients with AML and normal karyotype and have been shown to be associated with inferior response towards chemotherapy. One possible way how mutant FLT3 receptors may be mediating its pathological effects is by altering the normal localization of the receptor protein. To determine the intracellular localization of wild type and mutant FLT3 receptors, confocal laser scanning microscopy was performed in cultured leukemic cell lines (THP-1, EOL-1, MV4-11) and primary hematopoietic stem cells (HSC) from healthy donors. HSC (CD34+) cells were prepared from human bone marrow and leukapheresis products of healthy donors by MACS technique using CD34 and CD135 (FLT3) coupled microbeads. Additionally, different leukemic cell lines were transduced with retroviral vectors encoding FLT3wt or FLT3-ITD receptors.
In normal HSC, only 4–13% are expressing FLT3, and unexpectedly FLT3 staining was observed both in the plasma membrane and evenly distributed in the cytoplasm. The intracellular FLT3wt was not localized to the endoplasmic reticulum (ER), but part of it co-localized with the Trans-Golgi-network (TGN) and early endosomes.
In leukemic cell lines endogenously expressing either wt or mutant FLT3, wtFLT3 localized to the plasma membrane and uniformly to the cytoplasm whereas mutant FLT3 strongly accumulated in a perinuclear region but did not localize to the plasma membrane. Stable transfection of leukemic cell lines with wt or mutant FLT3 confirmed this localization pattern. In leukemic cell lines neither FLT3wt nor FLT3-ITD accumulated in the ER or in the Golgi apparatus, but instead FLT3wt and FLT3-ITD seem to reside in the TGN and in early endosomes as shown by immunofluorescence costaining with the organelle markers.
These data indicate that although perinuclear accumulation of mutant FLT3 is observed in all cells analyzed, wtFLT3 is also found intracellularly. FLT3-ITD might reside in a different state concerning glycosylation and/or phosphorylation (Schmidt-Arras et al., Mol Cell Biol, 2005) and therefore it may activate alternative or additional signaling cascades by having access to different or additional substrates. Further studies will clarify, whether intracellularly located wtFLT3 in healthy HSC and FLT3wt-expressing leukemic cells is also involved in FLT3 signaling.
Donor CD8+ T Cells Mediate GVL without GVHD in Recipients Conditioned with Anti-CD3 mAb.
Authors: Zhang Chunyan , Lou Jingwei , Li Naninong , Todorov Ivan , Lin Chia-Lei , Cao Yu-An , Contag Christopher H. , Kandeel Fouad , Forman Stephen , Zeng Defu 
Published: October 2019

Abstract
Donor CD8+ T cells play a critical role in mediating graft versus leukemia (GVL), but also induce graft versus host disease (GVHD) in recipients conditioned with total body irradiation (TBI). Here, we report that injections of donor C57BL/6 (H-2b) or FVB/N (H-2q) CD8+ T with bone marrow cells induced chimerism and eliminated BCL1 leukemia/lymphoma cells without GVHD in anti-CD3-conditioned BALB/c (H-2d) recipients. In contrast, the same dose of donor CD8+ T and marrow cells induced lethal GVHD in TBI-conditioned recipients. In addition, the anti-CD3-conditioned long-term complete chimeras without prior exposure to host-type BCL1 cells also eliminated the tumors when being challenged with BCL1 cells 120 days after HCT. This is in contrast to the report that long-term complete chimeras induced with delayed donor lymphocyte infusion lost GVL activity. Using in vivo and ex vivo bioluminescent imaging, we observed that donor CD8+ T cells expanded rapidly and infiltrated GVHD target tissues in TBI-conditioned recipients, but donor CD8+ T cell expansion in anti-CD3-conditioned recipients was confined to lympho-hematological tissues. This confinement was associated with lack of up-regulated expression of α4β7 integrin and chemokine receptors (i.e. CXCR3) on donor CD8+ T cells. In addition, host-reactive donor CD8+ T cells in anti-CD3-conditioned recipients were only partially deleted, and the residual cells were rendered heterogeneous: some unresponsive/anergic, some Tc2, some Foxp3+ suppressive cells, and some effector cells. The whole population of residual donor CD8+ T cells from anti-CD3-conditioned recipients mediated GVL without GVHD in TBI-conditioned secondary recipients. These results indicate that anti-CD3-conditioning separates GVL from GVHD via confining donor CD8+ T cell expansion to host lympho-hematological tissues as well as tolerization of the residual donor CD8+ T cells, in which the residual host-reactive effector cells mediate persistent GVL, and the regulatory CD8+ T cells prevent them from damaging host tissues.
Ape-1/ref-1 Mediated Redox Regulation of Retinoic Acid Receptor Transcription Function in Myeloid Leukemia Cell Differentiation and Apoptosis.
Authors: Robertson Kent A. , Colvin Edwin S. , Luo Meihua , Reed April , Kelley Mark R. 
Published: October 2019

Abstract
Ape1/ref-1is a multifunctional base excision DNA repair protein that is involved in the repair of abasic sites in DNA. However, it also has a distinct role in the redox regulation of a variety of cellular proteins, such as Fos, Jun, p53, NFkB, PAX, HIF-1a, HLF, and others. Ape-1/ref-1 maintains these proteins in a reduced state thereby facilitating their DNA binding and transcriptional activation capability. HL-60 cells are known to respond to retinoic acid (RA) with terminal granulocytic differentiation and apoptosis, which is mediated through the RA receptors. Previous experiments suggested that Ape1/ref-1 expression is related to apoptosis. To further define this relationship, we used retroviral gene transduction to over-express HA-tagged Ape1/ref-1 in HL-60 myeloid leukemia cells. We observed that the RA-induced growth inhibition, apoptosis, and differentiation of HL-60 cells over-expressing Ape1/ref-1 was significantly enhanced compared to wild type HL-60 cells. To further understand the mechanism of this effect we performedgel shift experiments in vitro with Ape1/ref-1, retinoic acid receptor alpha (RAR-α), and a retinoic acid response element (RARE) under varying redox conditions andco-transfection experiments in CV-1 cells with Ape1/ref-1 and RAR-α using an RARE linked to a luciferase reporter.
Results:gel shift experiments demonstrate a redox dependent binding of RXR-α and RAR-α to their RARE which is mediated by Ape1/ref-1;western blot analysis of transfected CV-1 cells revealed proper expression of each transfected construct including RAR-α, RXR-α and Ape1/ref-1; andexamination of RA-treated CV-1 cells for RARE-linked luciferase expression demonstrated Ape1/ref-1 enhancement of RAR activated transcription of the luciferase reporter.
In conclusion, our data supports the contention that Ape1/ref-1 expression may be important for enhancing RA-induced myeloid differentiation and programmed cell death through a redox based mechanism in transcription of target genes.
BCR-ABL Activates IGF-1 Expression and Signaling in Chronic Myelogenous Leukemia Blast Crisis Cell Lines.
Authors: Pastural Elodie , Lakshmikuttyamma Ashakumary , Takahashi Naoto , Pearson Derek , Sheridan David , DeCoteau John F. , Ronald Geyer C. 
Published: October 2019

Abstract
CML blast crisis is characterized by the continued presence of the Philadelphia chromosome, which expresses the P210 BCR-ABL fusion protein, and the acquisition of additional molecular and chromosomal alterations. Evolution from CML chronic phase to blast crisis is associated with loss of heterozygosity at chromosome region 1p36, which contains the putative tumor suppressor RIZ1. We found that RIZ1 expression decreases during progression from CML chronic phase to myeloid blast crisis and that forced RIZ1 expression in CML blast crisis (CML-BC) cell lines decreases proliferation, increases apoptosis, and enhances differentiation. Furthermore, we found that RIZ1 represses IGF-1 autocrine production and blocks the activation of the IGF-1 receptor, AKT, and ERK signaling pathways in CML-BC cell lines thereby implicating RIZ1 control of the IGF-1 pathway in the regulation of these phenotypes. As BCR-ABL induces a growth factor independent phenotype due to the activation of autocrine growth factor production, we analyzed IGF-1 expression in CML-BC cell lines following exposure to imatinib. We found that imatinib treatment reduced IGF-1 mRNA and extracellular IGF-1 suggesting that RIZ1 may suppress blast crisis by counteracting the ability of BCR-ABL activate RIZ1 regulated genes. We characterized the signaling pathways used by BCR-ABL to activate IGF-1 expression and found that the HCK inhibitor PP2 and STAT5b shRNA reduced IGF-1 expression whereas the JAK2 inhibitor AG490 increased IGF-1 expression. These results suggest that BCR-ABL activates HCK, which in turn activates STAT5b and induces IGF-1 expression. To confirm the importance of BCR-ABL induced IGF-1 signaling to CML-BC cell line viability, proliferation and apoptosis, we characterized these cellular phenotypes in the presence of IGF-1 receptor blocking antibody and the IGF-1 receptor tyrosine kinase inhibitor AG1024. Blockage of autocrine IGF-1 signaling in CML-BC cell lines using an anti-IGF-1 receptor blocking antibody reduced cell viability and decreased proliferation. AG1024 treatment of CML-BC cell lines decreased proliferation and induced apoptosis. Together these results demonstrate that RIZ1 counteracts the ability of BCR-ABL to induce IGF-1 signaling in CML-BC cell lines, which influences cell proliferation, apoptosis, and differentiation. Our findings highlight RIZ-1 and IGF-1 signaling pathways as potential therapeutic targets for treating CML blast crisis.
The G Protein-Coupled Receptor cysLT1 Is Expressed in Acute Myeloid Leukemia Mediating Cell Migration and Survival.
Authors: Rether Christina , Hengstebeck Sandra , Xue Xingkui , Boehmler Andreas , Kanz Lothar , Seitz Gabriele , Mohle Robert 
Published: October 2019

Abstract
Previous studies have shown that the G protein-coupled receptor (GPCR) cysLT1, which recognizes inflammatory lipid mediators (cysteinyl-leukotrienes), is expressed in immature hematopoietic and tumor (eg. colon cancer) cells. In the present study, we analyzed leukemic blasts from 19 patients with newly diagnosed acute myeloid leukemia for analysis of expression and function of cysLT1. By RT-PCR, cysLT1 mRNA was found in all primary AML cells. Quantitative TaqMan PCR demonstrated high levels particularly in acute myeloblastic leukemia without maturation (FAB M1). There was also a positive correlation between the cysLT1 mRNA level and the expression of CD34 and CD-117 (c-kit), indicating that high cysLT1 expression corresponds to an AML phenotype resembling normal hematopoietic progenitor cells. CysLT1 was functionally active in AML blasts, as demonstrated by intracellular calcium fluxes and actin polymerization induced by the ligand LTD4. Similar to mRNA expression, strongest responses were seen in AML FAB M1. As LTD4 can be produced in the bone marrow by stromal cells and may contribute to bone marrow infiltration of AML, chemotaxis was analyzed. Surprisingly, already low concentrations (10nM) induced significant chemotaxis of AML blasts, while higher concentrations (up to 1 uM) were less effective in a dose-dependent manner. Incubation of myeloid leukemic cell lines (eg. KG1a) and primary AML blasts with the specific cysLT1 antagonist MK571 resulted in significantly reduced viability after 48 h in a dose-dependent manner (10nM-10uM), suggesting also an autocrine function of cysLT1 ligands. To explore signal transduction pathways involved in leukemic cell proliferation and chemotaxis, we found that in AML cell lines, LTD4 induced phosphorylation of Erk/MAP kinase, wich is related to proliferation, and Pyk2, which represents a focal adhesion kinase-like signaling molecule that links GPCRs with cell migration, while the Akt pathway was not involved. We conclude that cysLT1 is consistently expressed in acute myeloid leukemia, and mediates both chemotactic and proliferative responses. Therefore, cysLT1 antagonists, which are already used in the treatment of allergy, may improve the effectiveness of antileukemic therapy.
The Predictive Value of a “Positive” ASH Abstract in AML Therapeutics.
Authors: Estey Elihu H. , Bedikian Slyva H. , Witter Dianne C. , Pierce Sherry A. , Giles Francis J. 
Published: October 2019

Abstract
ASH abstracts describing outcomes of trials of new therapies (Rxs) are presumably read in anticipation that positive results, although necessarily preliminary, will herald therapeutic advances. Here we examine the predictive value of a “positive” (pos) ASH abstract. We reviewed abstracts, presented in oral or poster sessions from 1993–2001, dealing with early phase trials of new drugs, used alone or together with more standard Rx, in adult AML (APL excepted). A research nurse (RN), a medical editor (ME), and E. Estey (EE) reviewed the abstracts independently, with the latter’s decision final. There was 80–85% concordance among the 3 reviewers, with EE tending to view fewer abstracts as pos. 63/91 abstracts (69%) were judged pos from a therapeutic standpoint (based typically on conclusions that the rx was “active” or “effective”), 14 were considered “negative”, and 14 inconclusive. The 63 pos abstracts involved 37 separate drugs, i.e. several drugs were the subject of > 1 pos abstract, including gemtuzumab, and topotecan combinations (5 abstracts each) and PSC and IL-2 (without histamine) (4 abstracts each). A “pub med” search indicated that 45/63 (71%) pos abstracts, covering 27 of the 37 separate drugs, subsequently appeared in peer-reviewed journals, with half the unpublished abstracts appearing at the 2000 or 2001 ASH meetings. The pos conclusion was unaltered in 38 of the 45 and was changed to negative in 5 and to inconclusive in 2. Three of the 37 pos drugs have either migrated into clinical practice (gemtuzumab) or been pos in a randomized trial (IL-2 + histamine, cyclosporine + infusional daunorubicin + ara-C). Four of the pos drugs (PSC833 + chemotherapy. HuM195, fludarabine + ara-C, troxacitabine + ara-C) have appeared, in some pt subsets, negative in randomized trials. However, 81% (30/37) of the drugs reported pos in ASH abstracts remain, at least 5 years from date of ASH publication, either outside the scope of clinical practice or unevaluated in randomized trials. We suggest that the reasons for this phenomenon be explored.