bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting Autophagy Potentiates Imatinib-Induced Cell Death in Philadelphia Positive Cells Including Primary CML Stem Cells.
Authors: Bellodi Cristian , Lidonnici Maria Rosa , Hamilton Ashley , Helgason Gudmundur V , Soliera Angela R , Ronchetti Mattia , Galavotti Sara , Young Kenneth W , Selmi Tommaso , Van Etten Richard A. , Donato Nicholas , Hunter Ann , Dinsdale David , Tirro’ Elena , Vigneri Paolo , Nicotera Pierluigi , Dyer Martin J.S. , Holyoake Tessa , Calabretta Bruno , Salomoni Paolo 
Published: April 2020

Abstract
Imatinib mesylate (IM), a potent ATP-competitive inhibitor of the BCR/ABL tyrosine kinase, has become standard therapy for patients with chronic myelogenous leukemia (CML). However, the main limitations of IM- and second generation tyrosine kinase inhibitor (TKI)-based therapy are the insurgence of resistance in patients and the intrinsic refractoriness of primitive Philadelphia-positive stem cells. Therefore, there is the need to develop new therapeutic approaches that, in combination with TKI, might be more effective in targeting the stem cell population and preventing the outgrowth of TKI-resistant CML cells. TKI-induced elimination of BCR/ABL-dependent intracellular signals is known to trigger apoptosis, but it is unclear whether this also activates additional cell death and/or survival pathways. We show that IM treatment induces autophagy in CML blast crisis cell lines, CML primary cells and p210BCR/ABL-expressing 32Dcl3 (32D) myeloid precursor cells, but not in 32D cells expressing v-Src or the IM-resistant T315I p210BCR/ABL mutant. IM-induced autophagy does not involve c-Abl, as it is also observed in cells co-expressing p210BCR/ABL and the IM-resistant T315I c-Abl mutant. Induction of autophagy is associated with endoplasmic reticulum-stress and is suppressed by depletion of intracellular calcium. By contrast, ectopic Bcl-2 expression does not block IM-induced autophagy. Suppression of autophagy by pharmacological inhibitors or siRNA-mediated knockdown of essential autophagy genes enhances cell death induced by IM in cell lines and primary CML cells, demonstrating that induction of autophagy has a pro-survival effect. Critically, the combination of TKI with autophagy inhibitors results in near complete elimination of phenotypically (CD34+38−) and functionally (colony forming cells) defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKI in the treatment of CML.
Bcr-Abl Promotes CML Cell Proliferation through the Suppression of PHLPP Expression.
Authors: Hirano Isao , Nakamura Satoki , Yokota Daisuke , Ono Takaaki , Shigeno Kazuyuki , Fujisawa Shinya , Shinjo Kaori , Ohnishi Kazunori 
Published: April 2020

Abstract
[Background] The proto-oncogene Akt/PKB is activated in many human cancers including leukemias, and regulates the cell survival and apoptosis. Akt/PKB is activated by growth factors or the phosphatidylinositol-3,4,5-triphosphate via PI3K, phosphorylates many substrates for cell survival or proliferation. The tumor suppressor PTEN prevents Akt/ PKB phosphorylation and activation. On the other hand, the PH domain leucine-rich repeat protein phosphatase (PHLPP) dephosphorylates Akt/PKB, induces apoptosis, and suppresses tumor growth. It has been reported that the expression levels of PHLPP are significantly reduced in several colon cancer and glioblastoma cell lines, and Akt/PKB phosphorylation is elevated. However, its function is little known in CML. We found that the expression of PHLPP gene and protein was suppressed in CML cells. In this study, we have investigated the expression and the function of the PHLPP in CML cells.
[Methods] The cells used in this study were human CML cell lines, K562 and Meg01 cells. Primary CML (CP; n=10) cells were obtained from the peripheral blood or bone marrow. Human normal mononuclear cells (MNCs) were isolated from peripheral blood or bone marrow of healthy volunteers after obtaining informed consents. For analysis of PHLPP mRNA expression, quantitative RTPCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the levels of Akt phosphorylation in CML cells, MTT assays and western blot were performed in all cell lines transfected with Bcr-Abl or PHLPP siRNA, respectively. For colony analysis, the colonies of CFU-GEMM, CFUGM, and BFU-E were counted in CML stem/progenitor cells transfected with PHLPP siRNA or treated with Abl kinase inhibitors.
[Results] In CML cell lines, the expressions of PHLPP mRNA and protein were significantly increased more than normal MNCs by treatment with Abl kinase inhibitors or transfection with Bcr-Abl siRNA. On the other hands, in CML cells transfected with the PHLPP siRNA, it is shown that the phosphorylation levels of Akt were markedly increased compared to the untransfected cells. In CML stem/progenitor cells obtained from patients with CML, the expression of PHLPP gene was suppressed in 10/10 (100 %) of CML. This higher expression was not associated to the counts of blasts of PB (P &lt; 0.001). The transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of PHLPP mRNA and protein, and decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusions] Our results suggest that the Bcr- Abl suppressed the expression of PHLPP mRNA and protein, and the phosphorylation of Akt in CML cells to induce the proliferation of CML cells. Moreover, induction of PHLPP expression might regulate the proliferation of CML stem/progenitor cells.
Long-Term Follow-up Results of IFM9903 and IFM9904 Trials Comparing Non Myeloablative Allotransplantation with Autologous Transplantation in High-Risk De Novo Multiple Myeloma.
Authors: Moreau Philippe , Garban Frederic , Attal Michel , Michallet Mauricette , Marit Gerald , Hulin Cyrille , Benboubker Lotfi , Doyen Chantal , Mohty Mohamad , Yakoub-Agha Ibrahim , Leyvraz Serge , Casassus Philippe , Avet-Loiseau Hervé , Garderet Laurent , Mathiot Claire , Harousseau Jean-Luc 
Published: April 2020

Abstract
The IFM99-03 and IFM99-04 trials were conducted from April 2000 to August 2004. Patients younger than 66 years with high-risk (b2microglobulin &gt; 3 and chromosome 13 deletion by FISH analysis at diagnosis) de novo multiple myeloma (MM) were included and prospectively treated. In both protocols, induction regimen consisted of VAD (4 courses) followed by melphalan 200 mg/m2 (HDM200) plus autologous peripheral blood stem cell transplantation (ASCT). When a HLA-sibling donor was available, ASCT was followed by reduced-intensity conditioning regimen (RIC) allograft (fludarabine, antithymocyte globulin and low dose busulfan): IFM9903 protocol (Garban et al, Blood2006;107:3474–3480). When no donor was available, patients were randomised to receive a second ASCT with HDM220 +/− anti-IL6 monoclonal antibody (BE-8, 250 mg total dose, Diaclone Besançon, France): IFM99-04 protocol (Moreau et al, Blood2006;107:397–403).284 patients met eligibility criteria and received at least one course of VAD. 65 had an available HLA-identical sibling donor and were included in the IFM99-03 trial, and 219 were included in the IFM 99-04 trial. Patients were older in the tandem ASCT trial (median age, 58 vs 54 years; P = .006) and the b2-microglobulin level was also higher in the latter group (median, 4.9 mg/L vs 4.1 mg/L; P = .049). At the reference date of July 1st, 2008, on an intent-to-treat basis, considering the entire population of 284 patients, with a median follow-up of 56 months, the EFS did not significantly differ from tandem ASCT to single autograft followed by allo-RIC (median 22 vs 19 months, P = 0.58). Nevertheless, there was a trend for a superior OS in the double ASCT trial (median 48 vs 34 months, P = 0.07). When considering the comparison of the results of the 166 patients /219 who completed the whole tandem ASCT protocol with those of the 46 patients /65 who underwent the entire auto/allo-RIC program, no difference was observed regarding EFS (median 25 vs 21 months, P = 0.88), but there was again a trend for a superior OS in favour of double ASCT (median OS, 57 vs 41 months, P = 0.08), due to a longer survival after relapse in the tandem ASCT arm. These long-term results indicate that, in a subgroup of high-risk patients with de novo MM, a tandem autologous transplant procedure is at least equivalent or even superior to a combination of autologous followed by RIC allogeneic stem cell transplantation.
Use of a Potassium Channel Blocker to Inhibit Effector-Memory T Cells Isolated from Recipients of Allogeneic BMSCT.
Authors: Cameron Cheryl , Woodliff Jeffrey , McOlash Laura , Hari Parameswaran , Bredeson Christopher , Margolis David , Truitt Robert L. 
Published: April 2020

Abstract
Following allogeneic blood or marrow stem cell transplantation (BMSCT), donor T cells are persistently exposed to host alloantigens and develop into effector-memory (TEM) cells. CD4+ T-EM cells are thought to play a role in perpetuating chronic GVHD (cGVHD) (K. Yamashita et al., BLOOD103:3986, 2004). In contrast to central memory (T-CM) and naïve CD4+ T cells, human CD4+ T-EM cells over-express the voltagegated potassium channel Kv1.3 when activated. As a result, Kv1.3 has emerged as an attractive pharmacological target for the selective suppression of T-EM cells, especially in the setting of autoimmune disease (C. Beeton et al., Mol Pharm67:1369, 2005). We hypothesized that over-expression of Kv1.3 by T-EM cells obtained from allogeneic BMSCT patients with cGVHD would make them preferential targets for Kv1.3-specific potassium channel blockers. To our knowledge, no one has examined the effect of Kv1.3 blockers on T-EM cells from patients with cGVHD. In this study, we isolated T-EM cells as well as central-memory (T-CM) and naïve T cells from fresh (n = 5) and frozen (n = 4) peripheral blood (PB) of patients who had undergone allogeneic BMSCT more than 6 months earlier as well as from normal volunteers by fluorescence-activated cell sorting (FACS). All BMSCT patients had some clinical evidence of cGVHD. T-EM cells were identified by their characteristic immunophenotype: CD4+ CD45RO+/RA- CCR7- CD62L Low. The percentage of CD4+ T-EM cells in 9 patients with cGVHD was 45 ± 21% (range 19%–70%) as compared to approximately 11% in healthy controls. Sorted T-EM, T-CM and naïve CD4+ T cells were polyclonally activated ex vivo using Dynal beads coupled with anti-CD3/CD28 monoclonal antibodies. The proportion of cells secreting gamma interferon (γIFN) was quantified using EliSpot assays to assess functional activity. We first compared the kinetics of γIFN secretion by activated T-EM cells to that of T-CM and naïve T-cells. Only T-EM cells secreted γIFN within 24 hrs of activation; T-CM cells secreted γIFN ≥ 48 hrs after activation, and naïve T cells required ≥ 96 hrs. ShK peptide, a highly specific inhibitor of Kv1.3 channels derived from the sea anemone Stichodactyla helianthus, was added at the initiation of 24-hr cultures. Dose response assays confirmed that T-EM cells in both BMSCT patients and normals were preferential targets of the Kv1.3-specific peptide. On average, T-EM cells were inhibited 26%, 55%, and 69% with ShK concentrations of 1, 10 and 100 nM, respectively. With 100nM ShK, T-EM cells sorted from the peripheral blood of BMSCT patients were inhibited an average of 44 ± 21% (range 17–67%). Our data, using polyclonally activated T-EM cells, support further testing of Kv1.3 blockers against host-alloantigen-specific T-EM cells obtained from BMSCT recipients and as a potential therapeutic agent for cGVHD.
Macrophage Infiltration of Skin Lesions Correlates to Prognosis of Gvhd; A Clue to Refractory Gvhd
Authors: Nishiwaki Satoshi , Terakura Seitaro , Goto Tatsunori , Seto Aika , Watanabe Keisuke , Imahashi Nobuhiko , Tsukamoto Shokichi , Shimba Makoto , Ozawa Yukiyasu , Miyamura Koichi , Ito Masafumi 
Published: April 2020

Abstract
The pathophysiology of acute graft-versus-host disease (aGVHD) is a complex process. Although it is considered an alloimmune attack on host tissues mounted mainly by donor CD8-positive T cells, skin infiltration of T cells is not always the case in biopsy specimens of skin aGVHD, suggesting the involvement of other cellular components. In detailed observation, CD163-positive macrophages which have abilities of antigen presentation and releasing cytokines were frequently observed. We retrospectively reviewed 66 biopsy specimens of skin lesions clinically thought to be aGVHD performed within 100 days after allogeneic stem cell transplantation, and analyzed the relationship between types of infiltrating cells and clinical outcomes. Paraffin-section immunohistochemical analysis was performed using a monoclonal antibody against CD8 and CD163. Counting the total number of CD8-positive T cells and CD163-positive macrophages in 4 fields with 200-fold magnification, median number of CD-8 positive T cells was 78.5 (range; 2–305), and that of CD163-positive macrophages was 149 (range; 38–372). Infiltration of over 100 cells of CD8-positive T cells (CD8ov100) correlated with HLA class I mismatch and grade III–IV a GVHD, while infiltration of over 200 cells of CD163-positive macrophages (CD163ov200) correlated with grade III–IV aGVHD and refractory aGVHD. In logistic analyses, CD163ov200 (Odds 6.29 (95%CI 1.57–25.0); p=0.009) was identified as the only negative predictors of aGHVD outcome. Overall survival of patients with CD163ov200 was significantly lower than that of those with infiltration of under 200 cells of CD163-positive macrophages (63.0% vs. 40.6% at 1 year, respectively; p=0.02). In 23 patients treated with steroid for aGVHD, CD163ov200 was the only significant predictor of refractory aGVHD outcome (Odds 10.9 (95%CI 1.37–83.3); p=0.02), and overall survival of patients with CD163ov200 was significantly lower than that of those with infiltration of under 200 cells of CD163-positive macrophages (63.5% vs. 12.5% at 1 year, respectively; p=0.0004). These results indicate that CD163-positive macrophages may provide a clue for the pathophysiology of refractory GVHD and new treatment strategies.
Recombinant PDGF-BB Enhances Platelet Recovery in Mice with Radiation Induced Thromobocytopenia.
Authors: Ye Jie Yu , Chan Godfrey ChiFung , Chan Shing , Luo Jue Cong , Yang Mo 
Published: April 2020

Abstract
Platelet-derived growth factor (PDGF) involves in the regulation of hematopoietic stem cells. Imatinib mesylate (Gleevec), a PDGF receptor inhibitor, induces thrombocytopenia in 50% of patients with chronic myeloid leukaemia (CML). Our previous studies showed that PDGF enhances megakaryocytopoiesis in vitro via its receptors directly. However, the in vivo effect of recombinant PDGF in thrombocytopenic models has not been reported. In this study, we investigated the in vivo effect of PDGF as well as thrombopoietin (TPO) on hematopoietic stem cells and platelet production in a radiation treated mouse model. We also explored its potential molecular mechanisms on thrombopoiesis. A radiation induced myelosuppression with thrombocytopenic model was established using 4-Gy-irradiated mice. PDGF-BB (1 mg/kg/day) and TPO (1 mg/kg/day) were injected separately after radiotherapy into the mice. Peripheral blood platelets, WBC and RBC from PDGF-BB, TPO and control groups were collected and analyzed on day 0, 7, 14 and 21 respectively. The mice were sacrificed on day 21 and their bone marrows were harvested for CFU assays and histology analysis. PDGF-BB, like TPO, showed a significant promoting effect in platelet production (n=5 p= 0.0078) as well as CFU-MK formation (n=5 p=0.0019) compared with the untreated control group. Histology images also indicated that PDGF-BB increased the number of the megakaryocytic cells and its progenitors in bone marrow of irradiated mice. In order to find out the underlying mechanism, we further studied the in vitro effect of PDGF using megakaryocytic cell line M-07e. Our results demonstrated that PDGF alone (100–200ng/ml) significantly activated the PI-3K/AKT signaling pathway (n=9 p=0.014). PDGF plus PI-3K inhibitor (wortmannin, 100nM) was shown to attenuate the expression of AKT (n=9, p=0.0112). Consistently, imatinib mesylate (Gleevec, 1uM) negatively interfered the expression of AKT when combined with PDGF-BB ((n=4, p= 0.0288). PDGF-BB was found to have a similar anti-apoptotic effect as TPO on M07e cells as showed by Annexin V (n=8, p=0.0281), Mitochondrial Membrane Potential (JC-1) (n=7, p=0.0042), and Caspase-3 (n=5, p=0.0080) assays respectively. In summary, our findings showed that PDGF-BB had an in vivo thromobopoietic effect on the radiation induced myelosuppressive mice models. This effect was likely mediated via PDGF receptor with subsequent activation of PI-3K/AKT pathway, which leads to anti-apoptotic effect on megakaryocytes.
A Myelodysplastic Syndrome Associated with a Clonal GATA1 Mutation.
Authors: Freson Kathleen , De Waele Liesbeth , Thys Chantal , Wittevrongel Christine , Vandenberghe Peter , De Vos Rita , Vyas Paresh , Van Geet Christel 
Published: April 2020

Abstract
Germline exon 4 mutations in the N-terminal zinc finger (N-finger) of transcription factor GATA1 cause a variable degree of X-linked dyserythropoiesis with macrothrombocytopenia. Congenital neutropenia and anemia are described in patients with an exon 2 germline splicing mutation resulting in exclusive expression of the short isoform of GATA1 (GATA1s). Similar but acquired somatic GATA1 exon 2 splicing mutations occur in children with Down Syndrome (DS) and congenital transient myeloproliferative syndrome (TMD) or acute megakaryoblastic leukemia (AMKL). The present study describes a somatic exon 4 GATA1 mutation in a 19-year-old boy with myelodysplastic syndrome (MDS) who has a lifelong history of anemia, neutropenia, severe bone pains and hepatosplenomegaly. Bone marrow biopsy demonstrates bilineage dysplasia with deficient granulopoiesis and dysmegakaryopoiesis. The patient has a normal platelet count but an increased Ivy bleeding time and a prolongation of the PFA100 occlusion time. Platelet ATP secretion and platelet aggregation are decreased. The a to g substitution is present in GATA1 mRNA from platelets and bone marrow mononuclear cells. The mutation is also detected in genomic DNA from purified common myeloid progenitors, but not in common lymphoid progenitors, peripheral blood lymphocytes and skin fibroblasts. This GATA1- E200G mutation is located in a highly conserved residue at the beginning of the N-finger, but does not perturb DNA binding to GATA sites nor reduces its interaction with FOG1. In contrast, the mutation is located at the exonic splice site and is associated with alternative splicing and reduced GATA1 protein levels. In conclusion, this study expands the GATA1 phenotype-genotype spectrum with the development of MDS and platelet dysfunction due to a somatic GATA1 splice mutation.
M-CSF Inhibition Selectively Targets Tumor Angiogenesis and Lymphangiogenesis
Authors: Kubota Yoshiaki , Suda Toshio 
Published: April 2020

Abstract
Anti-angiogenic therapy in cancer is desired to be selective for tumor angiogenesis. The best validated approaches for limiting tumor angiogenesis involve blockade of the VEGF pathway. However, recent studies show that VEGF blockade damages healthy vessels resulting in toxic side effects (Maharaj et al. J. Exp. Med. 2008) and that interrupting the VEGF blockade induces rapid vascular regrowth in tumors (Mancuso et al. J. Clin. Invest. 2006). Therefore, other targets are currently being explored. Macrophage-colony stimulating factor (M-CSF), a cytokine required for differentiation of monocyte-lineage cells including macrophages promotes formation of a high-density vessel network in tumors suggesting therapeutic potential of M-CSF inhibition (Lin et al. J. Exp. Med. 2001). Moreover, M-CSF is abundantly expressed in highly-metastatic cancers such as breast cancer and osteosarcoma, suggesting high dependency on macrophages. However, the physiological role of M-CSF in vascular and lymphatic development as well as the precise mechanisms underlying anti-angiogenic effects of M-CSF inhibition is not clarified. Here, utilizing osteopetrotic (op/op) mice, we show that M-CSF deficiency leads to lack of both LYVE-1+ and LYVE1− macrophages, resulting in defects not only in vascular but also lymphatic development. To clarify the anti-cancer effects of M-CSF inhibition, we utilized a novel mouse model of osteosarcoma, in which mice were transplanted with a newly established osteosarcoma cell line, AX, which were developed from c-Myc-overexpressing Ink4a/ARF−/− bone marrow-derived stromal cells. In this model, systemic treatment with small molecule c-fms inhibitors (Ki20227) effectively suppressed tumor angiogenesis and lymphangiogenesis disorganizing extracellular matrices. Consequently, c-fms inhibitors almost completely suppressed tumor metastasis, and protected mice from cancer death. Different from VEGF blockade, interruption of M-CSF inhibition did not promote rapid tumor regrowth. Continuous M-CSF inhibition did not affect healthy vascular and lymphatic systems outside the tumor. These results suggest M-CSF-targeted therapy is an ideal therapeutic strategy against cancer alternative to VEGF blockade.
The Second Signal in Antigen-Presenting Cells: Complementary JAK1/JAK2 and CD40 Signalling Mediate Pro-Inflammatory Activation.
Authors: Conzelmann Michael , Rodionova Elena , Hess Michael , Zota Annika , Giese Thomas , Wagner Andreas , Breit Stephen , Hecker Markus , Ho Anthony D. , Dreger Peter , Luft Thomas 
Published: April 2020

Abstract
CD40Ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. CD40L induces pro-inflammatory activation of CD40 expressing antigen-presenting cells (APC) such as DCs, monocytes, B-cells and endothelial cells; however, CD40 activation alone is insufficient to induce interleukin (IL)-12p70 secretion. Using quantitative Western blotting and siRNA inhibition of protein expression we demonstrate that cytokine-induced Janus kinase (JAK)1 and/or JAK2 activation mediates a complementary second signal for the production of IL- 12p70, whereas tyrosine kinase (Tyk)2 activation has inhibitory effects. Furthermore, JAK1 activation reciprocally reduced IL-10 production. The mechanism of JAK modulation of CD40 signals in dendritic cells (DCs) involved transcriptional regulation. The Th1 and Th2 cytokines interferon (IFN)-γ and IL-4 both enhance IL-12p70 via activation of the same JAKs. However due to a weaker JAK activity, IL-4 signalling has to persist much longer than IFN-γ signalling in order to induce similar amounts of IL-12p70. This different dependence on signalling persistence may give rise to the distinct biological characteristics of Th1/Th2 cytokines and may explain the pleiotropic in vivo and in vitro effects of IL-4. Complementary CD40 and JAK activity is essential for IL-12p70 secretion by all human APC studied. This strict requirement of two complementary signals opens a new way of interfering with CD40L induced APC activation indirectly by modulating the “second signal” JAK.