bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Wnt Pathway Signaling by Thalidomide and Revlimid: Studies in a Drosophila Model System.
Authors: Stewart A. Keith , Zhu Yuan Xiao , Yahyapour Maryan , Manoukian Armen , Scanga Sam E.
Published: October 2019

High throughput sequencing, gene expression profiling and protein biochemistry in myeloma have all consistently revealed elevated expression of wnt signaling pathways in malignant plasma cells. Indeed, downregulation of the Wnt pathway in myeloma cells has recently been shown to inhibit myeloma cellular proliferation. Preliminary pharmacogenomic studies have also suggested that hyperactivation of the wnt signaling antagonist DKK-1 is associated with response to the immunomodulators thalidomide and revlimid. The mechanism of action for these therapeutically active drugs is however by no means clear as multiple biologic consequences of treatment have been proposed. We report here use of a drosophila model to examine wnt signaling inhibition by these pharmaceuticals. We employed a unique drosophila larval imaginal disc culture system in which wnt pathway activity is monitored through control of LacZ expression by the distalless promoter. In this system 10uM of both thalidomide and revlimid reproducibly inhibit lacZ expression when compared with vehicle controls. Western blots of larva confirmed downregulation of expression of armadillo (the drosophila b-catenin homologue) by both drugs but particularly revlimid. Lithium Chloride is an inhibitor of the drosphila GSK3b homologue shaggy and thus mimics wnt signaling by stabilizing b-catenin. The effect of Lithium could not be overcome by thalidomide or revlimid indicating that the action of these drugs is upstream of shaggy (or GSK3). Next we employed a fly transgenic for wingless which is embryonic lethal. By adding either drug to larval culture medium the lethality of wingless expression was reversed. Indeed drosophila embryos fed thalidomide exhibited developmental plate abnormalities. We next sought evidence that similar effects were evident in revlimid treated human myeloma. As previously reported most myeloma cell lines studied expressed b-catenin and this protein was downregulated by revlimid treatment of human myeloma cell lines co-incident with inhibition of growth as measured by MTT assay. We sought, but failed to find evidence of up-regulation of the wnt signaling pathway antagonist DKK-1 using an ELISA assay on pre and post treatment serum samples in patients responding to thalidomide.The implications of wnt signaling inhibition as a primary or secondary readout of therapeutic efficiency in MM may be of substantial importance in subsequent design of drug therapies or combination therapies.
Cladribine Alone or in Combination with Cyclophosphamide or Cyclophosphamide and Mitoxantrone as First Line Treatment in Chronic Lymphocytic Leukemia: An Early Report of Prospective Randomized Study.
Authors: Robak Tadeusz , Blonski Jerzy Z. , Gora-Tybor Joanna , Kasznicki Marek , Dwilewicz-Trojaczek Jadwiga , Tomaszewska Agnieszka , Konopka Lech , Ceglarek Bernadeta , Kloczko Janusz , Sokolowski Jaroslaw , Dmoszynska Anna , Kowal Malgorzata , Zawilska Krystyna , Stella-Holowiecka Beata , Sulek Kazimierz , Kuliczkowski Kazimierz , Warzocha Krzysztof , Calbecka Malgorzata , Skotnicki Aleksander
Published: October 2019

The aim of the study was to determine the efficacy and toxicity of cladribine alone (2-CdA) and in combination with cyclophosphamide - CY (CC) or CY and mitoxantrone - MIT (CMC) in previously untreated patients with progressive or symptomatic chronic lymphocytic leukemia in a randomized, multicenter study. 2-CdA was given at a dose of 0.12 mg/kg/d in 2 h I.V. infusion for 5 consecutive days in monotherapy and for 3 days in combination. In CC and CMC programmes cyclophosphamide was administered at a dose of 650 mg/m2 I.V. on day 1 and additionally mitoxantrone 10 mg/m2 I.V. on day 1 in CMC. Courses were repeated at 28 day intervals or longer if myelosuppression and/or infection developed, for a maximum of 6 courses. The response criteria were those recommended by NCI sponsored Working Group. Minimal residual disease (MRD) were evaluated by flow cytometry if CR was achieved.

In conclusion, the results of our study indicate that 2-CdA combined with CY or CY+MIT as first line therapy give higher CR rate than 2-CdA alone. However, CMC is more toxic than 2-CdA or CC. We recommend CC combination for further studies.

Treatment 2-CdA CC CMC p value Entered pts 167 169 163 Evaluated pts 143 152 139 CR 37 (25.9%) 43 (28.3%) 55 (39.6%) 0.03 OR 106 (74.1%) 125 (82.2%) 110 (79.1%) 0.2 MRD 15 (46.9%) 22 (82.2%) 25 (56.8%) 0.7 Median OR duration (years) 1.67 1.81 1.43 0.1 Relapse 48 (45.3%) 45 (29.6%) 43 (30.9%) 0.2 AIHA 10 (7.0%) 10 (6.6%) 5 (3.6%) 0.4 Thrombocytopenia grIII/IV 25 (17.5%) 25 (16.4%) 32 (23.0%) 0.3 Neutropenia grIII/IV 27 (18.9%) 43 (28.3%) 52 (37.4%) <0.001 Infections 39 (27.3%) 47 (30.9%) 53 (38.1%) 0.07 Died 39 (27.3%) 30 (19.7%) 37 (26.6%) 0.4
FLT3 ITD and FLT3 Tyrosine Kinase Domain Mutants Induce Different Phenotypes in a Murine Bone Marrow Transplant Model.
Authors: Grundler Rebekka , Miething Cornelius , Thiede Christian , Peschel Christian , Duyster Justus
Published: October 2019

Activating mutations of FLT3 are frequent in patients with acute myeloid leukemia (AML). Two distinct types of FLT3 mutations are most common: Internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence in approximately 30% of patients with AML and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3 ITD mutation seem to have a significantly worse prognosis, whereas the impact of TKD mutations on clinical outcome has not yet been determined. Recently, point mutations within the activation loop of FLT3 were also found in a significant percentage of infant and childhood acute lymhoblastic leukemia (ALL).

Previous studies demonstrate that mice receiving transplants of bone marrow retrovirally infected with FLT3 ITD develop a myeloproliferative disease. The effect of FLT3 TKD mutations in vivo has not yet been investigated.

To examine the transforming properties of FLT3 TKD mutants in primary hematopoietic cells, we used a bone marrow transplant model (BMT). Therefore we transduced bone marrow with retrovirus expressing either FLT3 D835Y or FLT3 I836M+R and transplanted it to lethally irradiated syngeneic recipient mice. As control we also transplanted mice with FLT3 WT and ITD infected bone marrow, respectively. We found that mice transplanted with FLT3 ITD developed a myeloproliferative disorder in mice, as previously described. In contrast, mice transplanted with FLT3 TKD mutants developed a lymphoid disease with distinct hematologic manifestation.

Most recipients of FLT3 TKD transduced bone marrow developed T lymphoma syndrome, characterized by massive enlargement of thymus and lymph nodes. Some mice developed a B lymphoid leukemia with splenomegaly and enlarged lymph nodes. Interestingly, the disease latency of 53 to 183 days (median 102 days) of FLT3 TKD mutants contrasted with FLT3 ITD mice, which succumbed myeloproliferative disease within 53 to 70 days (median 58 days).

The lymphoid manifestation and longer latency of FLT3 TKD in a murine BMT model together with the absent influence of FLT3 TKD mutations on clinical outcome of AML patients suggest differences in cell signaling between FLT3 TKD mutants and FLT3 ITD. The TKD mutants seem to require lymphoid cell context for full malignant transformation, whereas FLT3 ITDs transform myeloid cells.
Pro-Apoptotic Synergistic Interactions between ERK1/2 and Bcl-2 Inhibitors in Acute Myeloid Leukemia Cells.
Authors: Ricciardi Maria Rosaria , Konopleva Marina , Ruvolo Peter P. , McQueen Teresa , Milella Michele , Andreeff Michael
Published: October 2019

We previously reported that activation of the Ras/MEK/ERK signaling pathway alone or in combination with high levels of Bcl-2 confers poor prognosis in patients with acute myeloid leukemias (AML). In this study, we investigated whether the simultaneous disruption of these two pathways by pharmacological inhibition of ERK1/2 and Bcl-2 utilizing novel small molecule inhibitors would induce pro-apoptotic activity in myeloid leukemic cells. For inhibition of ERK1/2 signaling, we utilized the MEK inhibitor CI-1040 (Pfizer Global Research and Development). Functional Bcl-2 inhibition was achieved by the novel BH3 peptide binding domain inhibitor A438744.7 (Abbott Laboratories). A dose-dependent increase in apoptosis as assessed by an increase in phosphatidylserine externalization was observed in OCI-AML3 cells after 24h incubation with CI-1040 or A438744.7 (both in a range between 0.25 and 2 μM). When these agents were used in combination at a fixed 1:1 ratio, a dramatic enhancement of cell killing was observed especially at lower concentrations (70.63%±9.95 Annexin V+ cells at 0.25 μM of each compound). Isobologram analysis (Chou and Talalay method) revealed a Combination Index (CI) < 1 (CI=0.114 and 0.436 at 0.25 μM and 2 μM, respectively) suggesting the strongly synergistic nature of these interactions. In addition, combined treatment with CI-1040 and A438744.7 resulted in a substantial increase of mitochondrial damage and caspase cleavage. Pre-incubation (1-hour) with a pan-caspase inhibitor (IDN-1965) was able to completely abrogate sensitivity to the inhibitors. We further demonstrated that bcl-2 overexpression prevented induction of apoptosis by low doses of both, MEK and BH3 inhibitors, whereas enforced Bcl-XL expression essentially abrogated the lethal effects of A438744.7, but not of CI-1040. Finally, to evaluate whether the combined strategy of ERK1/2 and Bcl-2 inhibition would be potentially applicable to leukemia patients, primary cells were isolated from AML samples and exposed to CI-1040 and A438744.7, as single agents or in combination at a 1:1 ratio for up to 96 hours. In each sample, drugs administrated individually were minimally toxic. Co-administration of CI-1040 and BH3 inhibitors displayed strong synergistic lethal effects toward primary cells from AML patients (CI=0.45±0.42 at 0.25 μM and CI=0.20±0.22 at 0.5 μM after 48h incubation). In conclusion, simultaneous exposure to nanomolar concentrations of MEK and bcl-2 inhibitors induced mitochondrial dysfunction, caspases activation and striking synergistic pro-apoptotic activity in myeloid leukemic cells. Similar synergistic interactions occured in primary AML samples. Together, these data strongly suggest that therapeutic strategies combining MEK and Bcl-2 inhibitors warrant further examination in AML.
Highly Synergistic Interaction between Farnesyltransferase Inhibitors and the Chk1 Inhibitor UCN-01 to Induce Apoptosis in Human Leukemia Cells through Interruption of Both Akt and MEK/ERK Pathways and Activation of SEK1/JNK.
Authors: Dai Yun , Rahmani Mohamed , Pei Xin-Yan , Khanna Payal , Dent Paul , Grant Steven
Published: October 2019

Ras mutations, which result in constitutive Ras activation, occur frequently in human malignancies, including leukemia. This finding has prompted the development of farnesyltransferase inhibitors (FTIs), which interfere with Ras farnesylation and membrane translocation necessary for Ras function. However, FTIs alone have not yet fulfilled their clinical potential, raising the possibility that their role may lie in combination with other agents. The present studies examined interactions between the farnesyltransferase inhibitor L744832 and the Chk1 inhibitor UCN-01 in human leukemia cells. Combined exposure of U937 cells to sub-toxic concentrations of UCN-01 (100nM) and the FTI L744352 (10μM) resulted in a dramatic and highly synergistic increase in mitochondrial dysfunction, apoptosis, and loss of clonogenicity. Similar interactions were noted in other leukemia cells (HL-60, Raji, Jurkat) and primary AML blasts, and with other farnesyltransferase inhibitors (e.g., FTI-277). These events were accompanied by cleavage of the anti-apoptotic proteins Bcl-2, XIAP, and Mcl-1. Co-administration of L744832 blocked UCN-01-mediated phosphorylation of MEK/ERK, leading to down-regulation of phospho-CREB and -p90RSK, and activation of p34cdc2 and SEK/JNK. Combined treatment also resulted in pronounced reductions in levels of phospho-Akt, -GSK, -p70S6K, -mTOR, -FKHR, -caspase-9, and -Bad. Ectopic expression of Bcl-2 or Bcl-xL but not dominant-negative caspase-8 blocked UCN-01/L744832-mediated mitochondrial dysfunction and apoptosis, but did not prevent activation of p34cdc2 and JNK, or inactivation of MEK/ERK and Akt. Enforced expression of myristolated Akt but not constitutively-active MEK significantly attenuated UCN-01/L744832-induced apoptosis. However, dual transfection with constitutively active Akt and MEK resulted in further protection from UCN-01/L744832-mediated lethality. Finally, down-regulation of JNK1 by siRNA significantly reduced the lethality of the UCN-01/L744832 regimen. Together, these findings suggest that farnesyltransferase inhibitors interrupt the cytoprotective Akt and MAPK pathways while reciprocally activating SAPK/JNK in leukemia cells exposed to UCN-01, and in so doing, dramatically increase mitochondria-dependent apoptosis. They also raise the possibility that combined treatment with FTIs and UCN-01 may represent a novel therapeutic strategy in leukemia.
The Role of the Cytoplasmic Domain of the FcγRI α Chain in FcγRI Signaling.
Authors: Hunter Sharon , Huang Zhen-Yu , Chien Paul , Kim Moo-Kyung , Worth Randall G. , Daniels Anthony B. , Kim-Han Tai-Hee , Indik Zena K. , Schreiber Alan D.
Published: October 2019

The high affinity Fcγreceptor FcγRI, consists of a ligand binding α chain and an associated γ chain subunit. Signaling by FcγRI is thought to be mediated through the cytoplasmic domain (CYT) of the γ chain which contains the ITAM sequence essential for many Ig receptor signaling events. For example, we have demonstrated that the γ chain is required for FcγRI mediated phagocytosis. The FcγRI α chain CYT lacks known signaling motifs and consists of 64 amino acids whose function has not yet been defined. Evidence is beginning to accumulate, however, for the participation of the FcγRIα CYT in FcγRI mediated signaling events. For example, we previously demonstrated that the FcγRIα CYT contributes to modulation of FcγRI mediated cytokine release (eg IL-6) and calcium flux. We have now observed that in the absence of γchain expression, FcγRIα efficiently induces the internalization of aggregated IgG complexes in transfected COS-1 cells. Taken together, the data suggest that the FcγRIα CYT contributes to signaling by FcγRI. We, therefore, utilized yeast two-hybrid screening to identify effector molecules that bind to the FcγRIα CYT. Of especial interest among the identified binding molecules is β arrestin-2, recently defined as an adaptor molecule linking receptors such as the LDL and type III TGF-β receptors to the endocytic pathway. Also of interest is Snapin, an adaptor molecule involved in membrane trafficking. These studies should help to define sequences in the cytoplasmic domain of FcγRIα that mediate and/or modulate important FcγR functions.
Proviral Tagging Combined with Human Microarray Data Reveals Genes and Pathways That Cooperate in Disease Induction.
Authors: Dave Utpal P. , Akagi Keiko , Dupuy Adam , Suzuki Takeshi , Jenkins Nancy A. , Copeland Neal G.
Published: October 2019

Retroviruses induce hematopoietic cancer by integrating into the genome and insertionally mutating cancer causing genes and provide powerful molecular tags for cancer gene identification. Retroviruses induce cancer following several rounds of insertional mutagenesis and clonal selection; as a result, each tumor contains multiple retroviral integrations that mark the location of cooperating cancer genes. Some retroviral integrations occur early in tumor formation and initiate tumorigenesis while others occur late and promote tumor progression. Using ligation-mediated PCR (LM-PCR), a new technique that permits the automated cloning and sequencing of thousands of retroviral integration sites per day, we are now able to rapidly identify the many initiation and progression events that cooperate to produce a mouse tumor. We have used LM-PCR to identify the myriad of genes that cooperate with activating mutations at [itallic]Lmo2 in leukemia induction. We compare our mouse results with the published microarray data for 19 human tumors that overexpress LMO2 and demonstrate a remarkable correlation between the genes causally associated with murine Lmo2 leukemias and the genes overexpressed in human LMO2 leukemias. Our studies highlight the power of LM-PCR for sampling tumor microheterogeneity and provide a powerful new means for interpreting and annotating human microarray data.
Effect of Factor XI or Factor IX Deficiency on an Arterial Occlusion Model.
Authors: Gailani David , Cheng Qiufang , Xu Lin , Ogletree Martin , Wang Xinkang
Published: October 2019

Factor XI (fXI) and factor IX (fIX) are zymogens of plasma proteases that are required for normal formation and maintenance of a blood clot. Recent work has implicated these proteins in the pathogenesis of vascular thrombosis. Epidemiologic studies indicate that high levels (top 10% of normal distribution) of fXI or fIX are independent risk factors for venous thromboembolism, increasing risk ~2-fold. Recently, it was shown that fXI deficiency protects mice from carotid artery occlusion in a ferric chloride (FeCl3 ) injury model. FeCl3-induced thrombus formation involves thrombin generation, in addition to platelet activation and von Willebrand factor. We used a modified version of the FeCl3 model to study the antithrombotic effects of complete fXI or fIX deficiency. In wild type C57Bl/6 mice, carotid artery flow measured by Doppler flow probe is completely blocked within 10 minutes of applying 3.5% FeCl3 to the vessel. 3.0% FeCl3 induced occlusion in some (5 of 8) mice by 30 minutes, while no animal treated with 2.5% FeCl3 experienced occlusion. FXI and fIX deficient mice were fully protected from occlusion induced by 3.5% or 5% FeCl3. Some fXI (4 of 8) and fIX (4 of 6) deficient animals developed occlusion with 7.5% FeCl3, while occlusion occurred in all mice at 10% FeCl3. To put the effect of fXI or fIX deficiency on this model into perspective, it requires a very high dose of heparin (1000 U/kg) to produce similar protection. With 5% FeCl3, heparin at 200 U/kg only protects 50% of wild type mice from occlusion, despite prolonging the activated partial thromboplastin time beyond the upper limit of the assay (> 500 secs). High dose aspirin (100 mg/kg) did not prevent occlusion induced by 5% FeCl3, despite producing a nearly complete block of arachidonic acid-induced platelet aggregation in vitro. While fXI and fIX deficiency affect the FeCl3 model similarly, they have significantly different impacts on a tail bleeding time (TBT) assay. FXI deficient and wild type mice have similar mean TBTs (265 ± 68 and 287 ± 92 secs, respectively), while fIX deficiency causes prolonged bleeding (1561 ± 125 secs, p < 0.01). In comparison, heparin (200 units/kg) causes the TBT to exceed the upper limit of the assay (1800 seconds), while aspirin (30 mg/kg) modestly increases the TBT (~2.2-fold). The data indicate that fXI and fIX are involved in thrombus formation in the FeCl3 model, and support a growing body of evidence that thrombin formation through the fIX/fXI axis contributes to thrombotic disease. Given the mild bleeding diathesis associated with fXI deficiency, inhibition of fXI may be a useful component of therapy for treating or preventing thrombus formation, and would be associated with a relatively low risk of bleeding.
The Three-Dimensional Structure of the cGMP-Dependent Protein Kinase I - α Leucine Zipper Domain and Its Interaction with the Myosin Binding Subunit.
Authors: Zhou Guo-Ping , Surks Howard K. , Schnell Jason R. , Chou James J. , Mendelsohn Michael E. , Rigby Alan C.
Published: October 2019

Vascular smooth muscle contractile state is tightly coupled to myosin light chain phosphorylation. Myosin light chain phosphorylation is regulated by the counter regulatory activities of myosin light chain kinase and myosin phosphatase. Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase. This in turn functions to dephosphorylate myosin light chains. Mechanistically this involves a protein-protein interaction mediated by a leucine zipper (LZ) domain that is localized within the N-terminus of cGK1α and the C-terminus of myosin-binding subunit (MBS). To better understand the importance of this interaction in regulating smooth muscle vascular tone we have determined the three-dimensional structure of the LZ domain of cGK1α, (cGKIα1–59) using triple resonance, multidimensional nuclear magnetic resonance (NMR) spectroscopy. We have expressed the LZ domain (cGKIα1–59) in E. coli under conditions facilitating the uniform incorporation of 13C/15N and 2H isotopic labels and purified this expressed protein. Circular dichroism data supported that cGK1α is comprised of an α-helix, while our NMR data identified that this helix commences at residue Ala9 and extends through to residue Leu47 of the LZ domain. 15N backbone longitudinal and transverse relaxation rates are best fit to a rotational diffusion anisotropy model that is consistent with a coiled-coil dimmer structure, not a monomeric helix. This suggests that cGKIα1–59 is comprised of a coiled-coil conformation over this same region. A time course investigation carried out with the LZ domain and a homobifunctional amine reactive crosslinker [dithio-bis(succinimidyl propionate)] (DSP) supported the presence of a homodimer. Furthermore, 15N heteronuclear single quantum correlation (HSQC) chemical shift mapping data has been used to identify residues that are critical for interacting with the C-terminal region of the myosin binding subunit (MBS). These data have allowed us to identify those residues necessary for cGK1α dimerization, while simultaneously permitting a better understanding of the critical determinants of cGK1α and MBS that are involved in regulating the contractile state of smooth muscles within blood vessels. The spatial and temporal contractile state of these blood vessels is important in the physiologic control of blood vessel tone and plays a role in the pathogenesis of hypertension and atherosclerosis a leading cause of death in the US and worldwide.
Proteomics of Membrane from Human Sickle and Normal Fractioned Red Cells Identifies Different Expression of Stress-Response Proteins.
Authors: Turrini Franco , Carta Franco , Biondani Andrea , Roncada Paola , Cappellini Maria D. , Corrocher Roberto , De Franceschi Lucia
Published: October 2019

Sickle cell disease (SCD) is characterized by the presence of sickle hemoglobin (HbS), which has the unique property of polymerizing when deoxygenated and thereby triggering erythrocyte sickling and dehydration. Repeated HbS polymerization is associated with generation of dense, distorted red cells characterized by an extensive membrane oxidative damage and abnormal activation of membrane cation transport pathways. The recently developed proteomic technologies represent a useful tool for identification of new functional pathways in sickle red cells. We studied the membrane proteoma of sickle red cells from homozygous patients (SS), not carrying alpha-thalassemia, not undergoing hydroxyurea treatment and not transfused at least three months before the study. The buffy-coat was removed and the red cells were separated into two fractions corresponding to the reticulocytes enriched fraction and to the dense red cell fraction (n= 8). Following osmotic lysis, the red cell ghost proteins were separated by one- dimensional or by two-dimensional gel electrophoresis (1-DE, 2-DE). Using the ImageMaster 2D Platinum (5.0 Amersham-Biosciences) software the 2-DE gels from control and sickle cells were compared, allowing us to identify spots differently expressed in sickle cells compared to normal cells. The selected spots were excised and trypsinized before mass spectrometric analysis and compared with the SWISS-PROT and NCBI non-redundant databases. In sickle reticulocytes enriched fraction compared to the control one, we selectively identified the following cystokeleton proteins: four spots for beta actin, two spots for myosin light chain and a train of four proteins identified as protein 4.1, one of which was phosphorylated on Tyr13, a protein similar to flotillin 2, carbonic anhydrase I and the following stress-response proteins: heat shock protein 70 (HSP70) protein 9B, HSPC108 and peroxiredoxin II (PrxII), which was phosphorylated in Thr142. In dense sickle red cell fraction compared to normal one we selectively identified a train of two spots for 4.1R, band 3 and aldolase and two spots identified as HSP70 protein 5.

The identified proteins were then confirmed by western-blotting analysis.

These data suggest that in sickle red cells HSP70, HSP108 function may be required by the extensive assistance in the refolding of misfolded proteins most likely related to abnormal sickle red cell oxidative environment as also supported by the presence of phosphorylated peroxiredoxin II, which has been recently proposed as regulator of redox-sensitive signaling in other cell types.
PDE-4 Inhibitor Rolipram Prevents Hypoxia Induced Pulmonary Hypertension in Transgenic Sickle Cell Sad Mice.
Authors: De Franceschi Lucia , Malpeli Giorgio , Janin Anne , Scarpa Aldo , Leboeuf Christhophe , Platt Orah , Leboulch Philippe , Payen Emmanuel , Beuzard Yves , Brugnara Carlo
Published: October 2019

Pulmonary hypertension (PH) is one of the major causes of morbidity and mortality for young-adult patients with sickle cell disease (SCD). Little is known about the role of vaso-active agents, pro-inflammatory and pro-fibrotic mediators in the genesis of PH in SCD.

We developed a model for studying PH in transgenic sickle cell SAD mice. Wild-type (WT) and SAD mice aged between 4 and 6 months were divided in groups of 7 animals each. One group from each strain served as control under normoxia, while a second group from each strain was exposed to 7 days hypoxia (8% oxygen). The following parameters were evaluated in normoxic and hypoxic groups: lung histopathology, complete blood count, reticulocytes, bronchoalveolar lavage (BAL) total leokocyte and neutrophil count, BAL levels of IL-6, IL-10, IL-1β and TNF-α. The expression of nine genes was examined by quantitative RT-PCR. These included endothelin-1 (ET-1), endothelin-1 receptor (ET-1R), cycloxygenase-2 (COX2), NF-kBp65, transforming-growth-factor-1β (TGF-1β), metalloproteinase-2 and 9 (MMP-2, MMP-9), IL-10 and IL-1β.

Two of the seven SAD mice exposed to hypoxia died at day fourth. The remaining five mice were sacrified at day seven and showed: a) increased pulmonary arterial wall thickness and α-actin staining, suggesting a vascular remodeling; b) lung injury compatible with PH in SCD; c) increased BAL total leukocyte and neutrophil count; d) increased BAL IL1β, IL10, IL6 and TNF-α; e) increased peripheral neutrophil and reticulocyte counts, hematocrit and hemoglobin levels. In lungs of hypoxic SAD mice, ET-1, ET-1R, TGF-1β, MMP-2, MMP-9, IL-1β and IL-10 gene expression were induced, with no significant changes in NF-Kbp65 gene expression. These data were consistent with a model of PH in SCD.

We then assessed the effect of the PDE-4 inhibitor ROLIPRAM on the PH in seven hypoxic SAD mice. ROLIPRAM was administrated at the dosage of 30 mg/Kg/day by gavage. No deaths were observed in this group. ROLIPRAM a) protected SAD mice from hypoxia induced lung injury; b) decreased BAL total leukocyte and neutrophil count; c) decreased BAL IL-1β, IL-6 and IL-10 levels; d) reduced peripheral neutrophil count; d) normalized and/or modulated the expression of the hypoxia-induced genes. These data suggest that the PDE-4 inhibitor ROLIPRAM has anti-inflammatory properties and could have beneficial effects on the hypoxia-induced airway-remodeling and possibly PH in sickle cell disease.
Arginine Supplementation of Sickle Transgenic Mice Leads to a Rapid Decrease in MCHC and Percent Dense Cells and a Slow Increase When Supplementation Is Discontinued.
Authors: Fabry Mary E. , Etzion Zipora , Bookchin Robert M. , Suzuka Sandra M. , Nagel Ronald L.
Published: October 2019

Nitric oxide (NO) is produced from arginine by nitric oxide synthase (NOS) and is essential to the maintenance of vascular tone. Plasma arginine levels are reduced in sickle cell anemia patients, and we have previously reported that S+S− Antilles mice have lower plasma arginine than control mice (C57BL). Long term arginine supplementation (5% arginine in chow) restored plasma arginine levels in S+S− Antilles mice to that found in C57BL mice, and, in S+S− Antilles mice, MCHC and the percent high density red cells were reduced. Our observation that Ca++-activated K+ channel [K(Ca) channel or Gardos channel] activity is reduced in supplemented vs non-supplemented S+S− Antilles mice can account for reduced red cell density and dense cell formation (Blood99: 1103, 2002). The time course of arginine induced changes in red cell indices in sickle transgenic mice has not yet been studied. Using the Advia 120 Hematology System, we found that, within 7 days after the onset of arginine supplementation, MCHC* (CHCM in the Advia system) fell from 33.4±0.5 g/dL to 31.0±0.2 (mean ±SD, P<0.000002, N=6) and the percent high density red cells (MCHC>37 g/dL) decreased from 10.5±4.3 % to 4.5±1.6 (P<0.01, N=6). MCV rose from 43.6±0.8 fL to 46.4±0.5 (P<0.00003, N=6). Reticulocyte counts and mean corpuscular hemoglobin did not vary significantly. Although plasma arginine levels increase within a few hours of oral administration and persist for only a few hours, no change in red cell indices was observed 7 days after discontinuing arginine supplementation, and the indices slowly returned to baseline levels after 40 days. In summary, MCHC* and the percent high density RBCs decrease significantly within 7 days of the onset of arginine supplementation. When arginine supplementation is withdrawn, the return to baseline values is much slower and required more than 40 days to complete. These observations have important implications for arginine supplementation in sickle cell anemia patients. In particular, the persistence of decreased MCHC* and reduced percent high density RBCs for an extended period of time after supplementation was withdrawn parallels our previously reported observation of persistent improvement of perfusion in sickle transgenic mice after supplementation was withdrawn and suggests that daily administration of arginine may not be necessary once an induction period has passed.
Drug Selection Enrichment of Type II Pneumocytes Following Transplant of P140K MGMT Lentiviral Transduced Hematopoietic Stem Cells.
Authors: Reese Jane S. , Roth Justin C. , Gerson Stanton L.
Published: October 2019

Recent reports have shown that bone marrow derived cells can differentiate into epithelial cells of the lung. Data indicate that this event occurs in the absence of cell fusion, however the numbers of donor-derived cells are present at a low frequency. In this study, we investigated whether hematopoietic donor-derived Type II pneumocytes could be enriched by lentiviral or retroviral expression of P140K MGMT. MGMT encodes O6 alkylguanine DNA alkyltransferase (AGT), a major drug resistance protein for DNA damage induced by BCNU. This gene transfer approach provides a selective advantage to wild type donor cells by transduction with a mutant human AGT (P140K MFG-MGMT) which confers resistance to BG and BCNU. P140K transduced whole bone marrow or Sca+kit+lin- cells were transplanted into lethally irradiated wild type recipients followed by two or three doses of 30 mg/kg BG and 7.5–10 mg/kg BCNU. After drug selection, human AGT expressing murine cells comprised up to 99% of bone marrow in mice harvested 5–7 months after transplant (n=8). These mice also showed enrichment for the total number of donor derived alveolar cells after drug treatment from 25% (n= 2 untreated mice) to 46% ± 9.7 (n=4 mice; p=.0001). The high percentage of donor cells was attributed to the hematopoietic cells present in the lung, however there were also donor-derived cells with epithelial morphology. Similar bone marrow plasticity studies have elicited controversy, largely due to the technical limitations of in situ detection of organ specific donor cells. To overcome the inability to accurately detect single cells in lung tissue, we used dispase mediated cell dissociation followed by analysis of single cell suspensions by fluorescent microscopy. Type II pneumocytes were detected using a Pro-surfactant protein C antibody that is specific for alveolar Type II cells. Donor P140K transduced cells were co-detected using an antibody to AGT. Using this method, 10–12% of total cells isolated from the lung were Type II pneumocytes and 1.5–8% of these cells co-expressed AGT in mice treated with BG and BCNU (n=3). This indicated drug selection mediated expansion of transplanted marrow-derived cells with characteristics of lung epithelial Type II pneumocytes. These numbers represent an increase compared to untreated mice in which we were unable to detect donor-derived Type II cells (n=2) and published results indicating 0.2–0.6% of total lung cells are bone marrow-derived epithelial cells. This study indicates that MGMT mediated drug selection of hematopoietic cells, using lentiviral MGMT gene transfer, enhances their capacity for organ specific selection and repopulation of lung alveolar epithelial cells. Drug selection gene transfer may augment transdifferentiation and have therapeutic implications for pulmonary disorders.
Both the Activity and Stability of the Transcriptional Repressor PLZF Are Modified by the Class III Histone Deacetylase SIRT1.
Authors: McConnell Melanie-Jane , Langley Emma , Martinez-Martinez Yolanda , Kouzarides Tony , Licht Jonathan D.
Published: October 2019

The transcription factor PLZF is expressed in hematopoietic development and rearranged in t(11;17) acute promyelocytic leukemia (APL). PLZF expression is high in the quiescent progenitor CD34+ cell, and declines during differentiation along myeloid and erythroid lineages. PLZF encodes a BTB-Zinc finger transcriptional repressor that inhibits the cell cycle through inhibition of targets genes such as cyclin A and c-myc through the recruitment of histone deacetylase complexes. However, PLZF itself is regulated by acetylation. In a separate study we demonstrate that acetylation of PLZF by p300/CBP enhances the transcriptional repression activity of PLZF. Through a yeast two-hybrid study we found that PLZF associates with the protein deacetylase SIRT1. SIRT1 is a member of the sirtuin family of class III histone deacetylases. In model organisms such as yeast, worms and flies, sirtuins play a common role in lifespan extension. The interaction between PLZF and SIRT1 was confirmed by co-precipitation of endogenous proteins and localized to the zinc fingers of PLZF, the region targeted for acetylation by p300/CBP. Acetylation of PLZF mediated by p300/CBP was reversed by SIRT1. Furthermore while acetylation of PLZF enhances its ability to repress transcription, co-expression of SIRT1 decreased PLZF transcriptional repression activity, consistent with loss of acetylation. Conversely, inhibition of SIRT1 activity with nicotinamide enhanced both PLZF acetylation and transcriptional repression of PLZF on its endogenous target gene c-myc. Further, increasing PLZF acetylation by inhibition of SIRT1 was associated with decreased PLZF stability. Acetyl-PLZF levels were stabilized by inhibition of the proteosomal degradation machinery, together implying that PLZF acetylation and activation results in increased protein turnover. These data point to the increasing complexity of the role of acetylation in transcriptional regulation and stand in contrast with data for the related Bcl6 repressor where acetylation of the protein inhibits repression. These data also indicate a novel function for sirtuins in regulation of transcriptional repression.
Transplantation of Human Aldehyde Dehydrogenase Expressing Cells Leads to Widespread Tissue Distribution of Donor Cells in the Novel NOD/SCID/MPSVII Xenotransplantation Model.
Authors: Hess David A. , Craft Timothy P. , Wirthlin Louisa , Herrbrich Phillip E. , Hofling A. Alex , Sands Mark S. , Nolta Jan A.
Published: October 2019

Beta-glucuronidase (GUSB) is a lysosomal enzyme expressed in virtually all human and murine cell types. Transplantation of GUSB-expressing human hematopoietic stem cells (HSC) into the recently developed GUSB-deficient NOD/SCID/MPSVII mouse allows for the sensitive histochemical detection of xenotransplanted cells in hematopoietic and non-hematopoietic tissues. Most notably, human cells expressing GUSB can be detected without reliance on the expression of human cell surface markers. In addition, we have recently characterized a novel population of reconstituting HSC from human umbilical cord blood (UCB) by lineage depletion (Lin−) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. We have also demonstrated the robust hematopoietic engraftment of ALDHhiLin− cells in the NOD/SCID model. Because donor cell surface protein expression may be altered in non-hematopoietic tissues due to cell fusion or other mechanisms, we have used the NOD/SCID/MPSVII model to study the tissue distribution of ALDHhiLin− or ALDHloLin− cells in multiple organs. Tail vein injection of 2x105–4x105 ALDHhiLin− cells into sub-lethally irradiated (300cGy) NOD/SCID/MPSVII mice (n=3) demonstrated high levels of hematopoietic (human CD45+ cells) engraftment in the BM (70.5±15.1%), spleen (7.0±2.8%) and peripheral blood (17.0±10.7%). In contrast, injection of 2x105–106 ALDHloLin− cells (n=3) did produce significant human cell engraftment in these hematopoietic tissues, with only 1 of 3 mice engrafting at 0.3% CD45+ cells in the murine BM. By using GUSB-specific histochemical staining, significant human engraftment was detected, with single cell sensitivity, in hematopoietic (BM and spleen) and non-hematopoietic (liver, pancreas, and lung) tissues of mice transplanted with ALDHhiLin− cells. GUSB activity was colocalized with CD45 immunohistochemical staining in the BM, liver, and pancreas of mice transplanted with ALDHhiLin− cells. Human cells were not detected in the muscle or brain of these engrafted mice. In contrast, mice transplanted with equal doses of ALDHloLin− cells showed no GUSB expression in the BM, spleen, liver, pancreas, lung, muscle, and brain. We are currently developing a flow cytometric assay to enumerate the frequency of GUSB expressing cells in the tissues of the transplanted mice. These data indicate that ALDHhiLin− UCB cells demonstrate previously uncharacterized engraftment in the liver, pancreas, and lung of NOD/SCID MPSVII mice. This novel model provides a unique opportunity to visualize donor human cells after xenotransplantation without reliance upon the expression of cell surface proteins, in situ hybridization, or cell labeling using viral transduction or membrane dyes.
Pulmonary Hypertension: A Common Complication in Thalassemia.
Authors: Vichinsky Elliott P. , Morris Claudia R. , Hagar Ward , Gardner Jennifer , Singer Sylvia Titi , Foote Drucilla , Quirolo Keith C.
Published: October 2019

Pulmonary hypertension (PHT) is a complication that is associated with high mortality rate. It is increasingly recognized in thalassemia intermedia (TI) as a leading factor in heart failure and death. Undetected PHT has been reported in 60–75% of patients. Data on PHT and thalassemia major is limited. However, these patients have many risk factors for PHT, including splenectomy, red cell phosphatidylserine exposure, coagulation abnormalities and iron overload. Since chronic hemolysis continues despite transfusion, these patients are also at risk for hemolysis-associated PHT. This is a recently described syndrome where free hemoglobin scavenges nitrous oxide and catalyzes the formation of reactive oxygen species. The purpose of this study is to determine the prevalence of PHT in transfused thalassemia patients and its risk factors. We compared the echocardiogram of 28 patients with their transfusion history, iron stores, chest x-ray, age, hemoglobin level, splenectomy status and cardiac function.

The prevalence of PHT in this population was 57%. Sixteen of the 28 patients had a tricuspid regurgitant jet velocity ≥2.5 m/s, indicating PHT. 5 patients had a jet velocity ≥ 2.9 m/s suggestive of moderate PHT. Patients with PHT were more likely to be older (29±10 vs. 24±7 years without PHT, r=0.52, p=0.01) and male (56%). 41% of the female patients undergoing echocardiogram exam had PHT, while 82% of the men screened had PHT (p=0.03). PHT was also inversely related to ferritin level (r=−0.46, p=0.02). A history of an abnormal CXR occurred more frequently in the PHT group (38% vs. 8%, p=0.08), but did not reach statistical significance. However, there was no difference in hemoglobin level, creatinine, splenectomy rate (63% vs. 58%) or abnormal cardiac function between patients with PHT vs. those without PHT.

Two of the 16 patients with PHT have initiated therapy with hypertransfusion or sildenafil. An intermittently transfused 25-year-old female patient lowered the tricuspid jet velocity from 3.2 to 2.4 m/s following hypertransfusion program. The second patient, a 44-year-old chronically transfused female, lowered her tricuspid jet velocity from 3 to 2.3 m/s within one month of sildenafil therapy.

In conclusion, PHT is a common complication in transfused thalassemia patients that is under recognized and under treated. In contrast to other complications, adequate chelation and low iron stores are not protective. Since secondary PHT is associated with a high mortality and morbidity, annual screening with echocardiogram for all thalassemia patients is recommended. Early identification of PHT and its risk factors may prevent the irreversible cardiomyopathy that may develop. Prospective studies evaluating therapies, including hypertransfusion, sildenafil and arginine are needed.
Non-Invasive Monitoring of Hepatic Iron Concentration during Oral Chelation in Patients with Non-Regularly Transfused β-Thalassemia/Hb E Disease.
Authors: Pootrakul Pensri , Chua-anusorn Wanida , Fleming Adam , Clark Paul , Sirankapracha Pornpan , Kachintorn Udom , Chanyawattiwongse Somsak , Metarugcheep Pichest , St. Pierre Tim
Published: October 2019

Current non-invasive measurement techniques of hepatic iron concentration (HIC) include magnetic susceptometry (SQUID) and the magnetic resonance imaging (MRI) methods utilising T2 and T2*. HIC can be quantified through image measurement of the proton transverse relaxation rate (R2) (St. Pierre et al Blood 2004). The potential for using the St Pierre method to monitor changes in HIC of patients with β-thalassemia/Hb E undergoing iron chelation therapy was investigated.

Seventeen non-tansfusion dependant β-thal/Hb E patients who had not previously undergone chelation were studied. Subjects were chelated with the oral iron chelator Deferiprone (DFP) and had their HIC measured using both the R2-MRI and biopsy non-heme iron techniques pre and post treatment. Ferritin levels were also assayed for comparison. The subjects ages ranged from of 13 to 53 years (mean 31.6, SD 11.5). DFP was administered at a low dose of 50 mg/kg/Day with divided doses 2 –3 times daily. The periods of drug exposure ranged between 53 and 77 weeks (mean 64.1, SD 8.4).

HIC by R2-MRI and tissue iron chemical analysis, and serum ferritin

Measurement R2-HIC (mg/g DW) Biopsy-HIC (mg/g DW) Serum Ferritin (ng/ml) Mean ± SD Range Mean ± SD Range G.Mean ± SE Range Initial 17.8 ± 6.6 5.7 – 29.7 18.3 ± 9.0 6.0 – 40.1 2526 ± 432 842 – 6072 Final 8.4 ± 7.9 1.0 – 23.9 7.4 ± 7.3 0.2 – 25.5 416 ± 236 113 – 4030

The results show a significant decrease of HIC after long term administration of DFP with MRI and biopsy (p= .0004 and p<0.0001 respectively). Spearman rank correlations of R2-HIC with the liver non-heme Fe and serum ferritin measures gave positive values of 0.866 (p < 0.0001) and 0.768 (p < 0.0001) respectively. The mean reduction of R2-HIC, biopsy-HIC and serum ferritin were 53%, 69%, and 74%, respectively. A decrease of 20.5% (SD ±14.9%) in the standard deviation of the R2 distribution was observed suggesting a decrease in iron heterogeneity accompanied the mean HIC decrease. The results suggest that R2 MRI has the potential to be used as a clinical monitoring tool in chelation therapy.
A Novel Murine Model for Human Waldenstrom’s Macroglobulinemia.
Authors: Tassone Pierfrancesco , Neri Paola , Kutok Jeffery , Tournilhac Olivier , Santos Daniel Ditzel , Hatjiharissi Evdoxia , Venuta Salvatore , Anderson Kenneth C. , Treon Steven , Munshi Nikhil C.
Published: October 2019

The pre-clinical studies of investigational agents in Waldenstrom’s Macroglobulinemia (WM) have been limited by the lack of an in vivo animal model, which is representative of the disease in patients. We and others have previously demonstrated growth of primary myeloma cells in immunodeficient mice bearing a human bone marrow (huBM) implant (SCID-hu mice). We therefore evaluated the use of this model for propagation of WM tumor cells. Four weeks following implantation of human fetal bone chip in SCID mice, mononuclear cells obtained from BM aspirates of consenting WM patients were directly inoculated into the bone chip. Mice were then evaluated every 2 weeks for the presence of human immunoglobulins. Seven of 13 patient samples engrafted in mice 4 to 28 weeks after WM cell inoculation as confirmed by detection of circulating human IgM and/or human k or l chain of the same type as that from the patient from whom the BM aspirate was obtained. The levels of human paraproteins, which were detected in murine sera, showed a time-dependent increase. Importantly, immunohistochemical analysis of human bone chips retrieved from the mice showed diffuse infiltration of CD20+, IgM+ and/or k+ or l+ lymphoplasmacytic cells as well as mast cells, which, we recently demonstrated, provide growth and survival signals for WM cells (JCO 2004 22:571S). Consistent with our previous observations with the MM SCID-hu mouse model, hereto we did not observe infiltration of murine tissues by WM cells confirming the essential nature of the human BM microenvironment in supporting WM cell expansion. These studies therefore demonstrate the utility of the SCID-hu mouse model for the study of WM, and provide a pre-clinical model for the investigation of novel agents for the treatment of WM.