bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Analysis of Cyclin D1 in Mantle Cell Lymphoma (MCL) by Specific Lentiviral shRNA Mediated Knockdown.
Authors: Klier Margit , Anastasov Natasa , Angermeier Daniela , Raffeld Mark , Fend Falko , Quintanilla-Martinez Leticia 
Published: April 2020

Abstract
Introduction: Cyclin D1 overexpression is the hallmark of MCL. However, the importance of cyclin D1 for the maintenance of MCL still remains to be defined. Therefore, the aim of this study is to elucidate the role of cyclin D1 overexpression using the siRNA technology in well-characterized MCL cell lines, as a model system.
Material and Methods: A highly efficient cyclin D1-shRNA (96% knockdown) was identified using a lacZ-cyclin D1 fusion gene reporter system in HEK-293T cells. This shRNA was cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Seven MCL cell lines were analyzed (Granta 519, Jeko-1, Rec-1, Z-138, UPN-1, Hbl-2 and JVM-2), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantitate the knockdown effect. The effect of cyclin D1 knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis.
Results: The infection rates varied among the different MCL cell lines. Rec-1 and Hbl-2 showed low infection rates (50%) even at high MOI’s (multiplicity of infection), whereas UPN-1 and JVM-2 had moderate infection rates (80%). Jeko-1, Granta 519 and Z-138 showed high infection rates (almost 100% of the cells). Despite the good tranfection rate, the downregulation of cyclin D1, as measured by Western Blot and qRT-PCR, was about 80% in Granta 519, and 65% in Jeko-1 and Z-138. No IFN response, as secondary effect was identified. Interestingly, no apoptosis was observed, and there was only a moderate retardation of growth (60% of control cells) with 10% shift from the S phase to G1 phase of the cell cycle when compared to the controls, suggesting that other cell cycle proteins might compensate, at least partially, for the loss of cyclin D1. Accordingly, cyclin D2 showed upregulation in Western blot analysis and qRT-PCR, whereas the phosphorylation status of retinoblastoma protein on Ser780 was reduced and the expression of the CDK inhibitor p27Kip1 increased. No changes were observed in the expression of cyclin D3, Cyclin E, CDK4 and CDK2.
Conclusions: In this study, a system that enables the specific downregulation of cyclin D1 in MCL cell lines was established. Surprisingly, the downregulation of cyclin D1 in MCL cell lines resulted in only a moderate inhibition on cell growth with no apoptosis. The reasons for this might be 1) that the upregulation of cyclin D2 compensates for cyclin D1 downregulation, and/or 2) that the chromosomal translocation leading to cyclin D1 overexpression is an initiating event in MCL lymphomagenesis followed by secondary genetic events at later stages of the disease, which make cyclin D1 dispensable. This finding has important implications for MCL therapy, as strategies targeting only cyclin D1 might be hampered by the redundancy of the system, resulting in a low probability of treatment response.
Synergistic Induction of Apoptosis by Simultaneous Disruption of the Bcl-2 and mTOR/Akt Pathways in Acute Myeloid Leukemia.
Authors: Zeng Zhihong , Samudio Ismael , Andreeff Michael , Konopleva Marina 
Published: April 2020

Abstract
Activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway provides survival signals for leukemic cells and blockade of this pathway may facilitate cell death. Akt/mTOR activation is a frequent event in AML that translates into significantly shorter survival of AML patients (S.M. Kornblau, ASH 2007). Bcl-2 family proteins are key regulators of apoptosis that are known to promote tumorigenesis and chemoresistance. We have recently reported that ABT-737, a small molecule BH3 mimetic, effectively kills AML progenitor cells via the disruption of Bcl-2:Bax, but its activity is largely diminished in cells overexpressing Mcl-1 (M. Konopleva, Cancer Cell 2006). We have now investigated anti-leukemia efficacy of concomitant blockade of mTOR/Akt, known to regulate Mcl-1 levels, and of Bcl-2 signaling. Combined use of rapamycin derivative temsirolimus and ABT-737 induced striking apoptotic responses in AML cell lines OCI-AML3 and MOLM13 leukemic cells. Mechanistically, temsirolimus downregulated expression of the anti-apoptotic protein Mcl-1 and induced expression levels of the proapoptotic BH3-only protein Noxa which specifically neutralizes Mcl-1. These effects were seen upon prolonged temsirolimus treatment that inhibited mTORC2 complex (Zeng, Blood 2007) and downregulated Akt phosphorylation. In primary AML samples co-cultured with bone marrow-derived stromal cells, ABT-737 at 25nM induced apoptotic cell death in CD34+ AML progenitor cells from 13 of the 16 samples tested. Temsirolimus/ABT-737 combination markedly enhanced apoptosis induction in 8/9 primary samples that were sensitive to temsirolimus alone (specific apoptosis, temsirolimus 15±5%; ABT-737, 28±5.9%; temsirolimus+ABT, 45±5.9%, p&lt;0.001) and in 3/7 samples resistant to temsirolimus (p=0.7). Further, combined blockade of mTOR/AKT and Bcl-2 pathway effectively induced apoptosis in 3 AML samples resistant to ABT-737 alone. Taken together, these results indicate that temsirolimus can directly alter the Bcl-2 apoptotic rheostat via induction of Noxa and decreased Mcl-1 expression sensitizing leukemia cells to apoptosis induced by BH3 mimetics. The combined use of agents interfering with AKT/mTOR and Bcl-2 pathways may be a highly effective anti-leukemia strategy that induces apoptosis in AML progenitor cells in the context of bone marrow microenvironment.
Phase I Study of S-trans, Trans-farnesylthiosalicylic Acid (FTS), a Novel Oral Ras Inhibitor, in Patients with Refractory Hematologic Malignancies.
Authors: Borthakur Gautam , Ravandi Farhad , O’Brien Susan , Beran Miloslav , Williams Betsy , Goldsweig Howard , Giles Francis 
Published: April 2020

Abstract
Background: Ras activation (mutational or non-mutational) is a key pathway for survival and proliferative advantage of leukemic cells. Farnesyl transferase inhibitors (FTI) that are thought to alter lipid modification of Ras and thereby alter its membrane targeting, are in clinical trial in hematologic malignancies but their target may not be specific for Ras. Toxicities of FTIs include diarrhea, skin rash and hepatic toxicity. FTS is an oral Ras inhibitor that causes dislocation of Ras from its membrane location by competing directly with farnesylated Ras to bind to its putative membrane binding proteins. FTS does not inhibit farnesyl transferase enzyme.
Methods: We report on an ongoing phase I study of FTS in patients with relapsed/refractory hematologic malignancies in which FTS is administered orally twice daily on days 1–21 of a 28 day cycle in a “3+3” dose escalation design.
Results: To date, 18 patients (pts) have been enrolled, with 17 pts treated (1 cycle=2, 2 cycles=8, 4 cycles=2, 5 cycles=1, 6 cycles=2, 8 cycles=1 and 9 cycles=1). Four pts were enrolled at dose level (DL) of 100 mg, 3 pts at each DL of 200, 400, 600 and 800 mg and 2 pts at DL of 900 mg. One pt enrolled at 100 mg DL was never treated. Median age was 72 years (range, 35 to 85 years), median ECOG status 1 (range 0–2) and median no. of prior therapies 2 (range, 0–7). Diagnoses included: Acute myelogenous leukemia (AML) = 9 pts, myelodysplastic syndrome (MDS) = 5 pts, chronic myelomonocytic leukemia (CMML) = 2 pts, chronic myelogenous leukemia (CML) = 1 pt. and Myeloproliferative Disorder = 1 pt. Preliminary analysis of pharmacokinetic data indicates that both Cmax and AUC increase with dose, but less than dose proportionately. Detailed PK data will be available for presentation at the American Society of Hematology 2007 Annual Meeting. Of 17 pts evaluable for response, 7 pts (41%) (4 MDS, 1 CMML, 1 CML, 1 AML) had hematological improvement (HI) (1 in three lineages, 6 in one lineage) without any complete remission. These responses lasted for a median of 9 weeks (range, 4 to 26 weeks). Currently, 2 patients are continuing on study with improvement in cytopenia and stable disease for over 20 weeks each. Grade 1–2 diarrhea has been the only non-hematologic toxicity seen, observed in 14 of 17 (82%) pts evaluable for toxicity and is completely correctable with oral antidiarrheals.
Conclusion: FTS shows promising activity in refractory hematologic malignancies and to date has minimal toxicity.
Expressing N-RasG12D from the Endogenous Promoter Induces Myeloproliferative Disease (MPD) and Cooperates with Retroviral Insertional Mutagenesis (RIM) To Generate Acute Myeloid Leukemia (AML).
Authors: Li Qing , Haigis Kevin , McDaniel Andrew , Huang Benjamin , Miller Michelle , Shieh Angell , Kogan Scott , Wolff Lynda , Jacks Tyler , Shannon Kevin 
Published: April 2020

Abstract
Somatic NRAS and KRAS mutations are common in myeloid malignancies, particularly juvenile and adult chronic myelomonocytic leukemia (JMML and CMML) and AML. Use of the interferon-inducible Mx1-Cre transgenic strain to activate oncogenic KrasG12D expression from the endogenous Kras promoter induces an aggressive MPD with 100% penetrance that is fatal by ∼100 days of age. These Mx1-Cre, KrasG12D mice do not develop AML, which suggests that additional mutations are required. To identify genes that might cooperate with KrasG12D in leukemogenesis, we performed RIM with MOL4070LTR virus. Mx1-Cre, KrasG12D pups were injected with this virus at birth and with pIpC at 3 weeks of age to induce Cre recombinase expression. Approximately 12% of these mice developed either AML or a transplantable MPD-like leukemia. In parallel studies, we intercrossed Mx1-Cre mice with a novel strain of loxP-STOP-loxP (LSL) NrasG12D “knock in” mice to investigate the effects of expressing NrasG12D in the same experimental system. Although Mx1-Cre, NrasG12D mice develop MPD, this disease is remarkably less aggressive with a much later onset of disease and longer survival compared to Kras mutant mice. Consistent with the attenuated disease phenotype, myeloid progenitors from Mx1-Cre, NrasG12D mice form fewer cytokine independent colonies in methylcellulose cultures and are less hypersensitive to growth factors than KrasG12D progenitors. Importantly, 90% of Mx1-Cre, NrasG12D mice that were injected with MOL4070LTR at birth and with pIpC at 3 weeks old developed AML or MPD-like leukemia, most of which demonstrate myelomonocytic features that are reminiscent of human M4 AML. We are cloning retroviral integrations from these leukemias and have identified Evi1 and other common insertions. Although both NRAS and KRAS are commonly mutated in myeloid malignancies, our data indicate that expressing these oncoproteins at endogenous levels in primary stem/progenitor cells results in distinct phenotypes. We recently showed that fetal liver cells infected with MSCV viruses expressing NrasG12D, Kras4aG12D, or Kras4bG12D demonstrate similar patterns of cytokine-independent colony formation and growth factor hypersensitivity. These data raise the possibility that variable levels of expression of Ras isoforms in different hematopoietic compartments may underlie some of our results. These models provide tractable systems for identifying cooperating genes in leukemogenesis and platforms for testing and designing new therapeutic agents.
Outcomes for Patients Who Fail High Dose Chemoradiotherapy and Autologous Stem Cell Rescue for Relapsed and Primary Refractory Hodgkin Lymphoma.
Authors: Moskowitz Alison , Yahalom Joachim , Kewalramani Tarun , Tejerina Manfred , Zelenetz Andrew D. , Moskowitz Craig H. 
Published: April 2020

Abstract
Background: Limited data exists regarding the outcome for patients (pts) with relapsed and primary refractory Hodgkin Lymphoma (HL) who fail high dose chemoradiotherapy (HDT) and autologous stem cell rescue (ASCR). The aim of this study is to determine survival time and evaluate the role of second transplantation in pts who have HL-related ASCR failure.
Results: At Memorial Sloan Kettering Cancer Center, from 1995–2005, two hundred and twelve pts with relapsed or primary refractory HL underwent HDT/ASCR after responding to ICE-based salvage chemotherapy on one of four consecutive clinical trials. Eighty-one patients failed HDT, including 10 treatment related mortality (4 deaths within 100 days from ASCR, 5 deaths due to secondary malignancies, and 1 death, 10 years after ASCR due to cardiomyopathy); leaving 71 (33%) pts with biopsy-confirmed progression; median time to progression was 5.7 months. There was 43 males (60%), median age at the time of ASCR was 29 (range 17–65). At a median follow-up of 2 years for surviving patients, the median overall survival from ASCR failure was 25 months. Only seventeen of the 71 pts are currently alive (24%), nine (12.6%) of whom are in remission. Remission duration from ASCR of &lt; 3 months predicted for OS; 1 of 17 pts who relapsed within 3 months are currently alive vs 16 of 54 pts with remission duration of &gt; 3 months; median OS are 13.5 and 27.3 months respectively (p=0.02). Treatment regimens following ASCR failure varied and included gemcitabine-based chemotherapy, MOPP, investigational therapies, radiotherapy and second transplantation. The impact of the various treatment regimens on OS following HL-related ASCR failure was difficult to assess due to the heterogeneity of regimens administered. Treatment with a second transplant had a favorable impact on OS, however only two of 17 pts with remission duration &lt; 3 months were able to receive a second transplant because of rapid disease progression. Twenty-three patients underwent a second transplant of which seven are failure free (1/4 autologous, 6/19 allogeneic). The median OS was 40 months for patients transplanted vs. 19.3 months (p=0.05) for patients ineligible for a second transplant.
Conclusion: Patients who fail HDT/ASCR due to HL progression have a median OS of two years. Relapse within 3 months of ASCR is associated with a poor OS and investigational or novel therapy should be explored in this cohort of pts. An allogeneic transplant following HL-related ASCR failure is associated with a 30% chance of long term survival and should be strongly considered in pts whose remission duration from their initial ASCR is &gt;3 months.
FANCD2 Purification and Identification of a Functionally Important Phosphorylation Event at Serine 331.
Authors: Zhi Gang , Tomashevski Andrei , Ward Michael , Newcomb William , Brown Jay , Semmes Oliver J. , Kupfer Gary M. 
Published: April 2020

Abstract
Fanconi anemia (FA) is an inherited hematological disorder characterized by birth defects, bone marrow failure, and cancer susceptibility. FA cells are characterized by spontaneous chromosomal instability and hypersensitivity to cross linking reagents such as mitomycin C (MMC). Cloned FA genes cooperate in a signaling pathway activated by DNA damage during replication, and monoubiquitination of FANCD2 is the critical downstream event. The check point kinase ATR is required for the efficient monoubiquitination of FANCD2. Inducible ATR-dependent phosphorylation of threonine 691 and serine 717 are reported to enhance the monoubiquitination of FANCD2 and resistance to MMC. In the present study, we have devised means to purify the FANCD2 protein into multiple subcomplexes. In doing so, we have identified at least 3 distinct subcomplexes: a pure multimeric precursor form comprised of FANCD2 polypeptides, a 1 MD form that is chromatin bound, and a 2 MD form that is in the nucleosol. Electron microscopic analysis of the precursor form reveals homogenous appearing complexes. Mass spectroscopic analysis of the 1MD FANCD2 complex has identified the FANCI protein, among others, which confirms recent reports of FANCD2-FANCI interaction. Further mass spectroscopic analysis of FANCD2 has identified a novel phosphorylation site on FANCD2, serine 331, which is further confirmed by immunoblot using phosphospecific antibody against phospho-serine 331. This phosphorylation site is conserved in other species, including mouse, rat, xenopus, and tetraodon nigroviridis. Single amino acid mutation by alanine at this site results in a complete loss of monoubquitination of FANCD2 and resistance to MMC, suggesting that the phosphorylation of S331 is a functionally significant event. Moreover, this site fits the consensus sequence for a CHK1 substrate, which in turn is a substrate of target for CHK1, a substrate of ATR, which is also required for monoubiquitination of FANCD2. These findings further support the correlation between phosphorylation and monoubiquitination of FANCD2 in the DNA damage response with the intervention of CHK1 as a mediator of ATR and suggest dynamic regulation of FANCD2 subcomplexes within the FA pathway.
Targeted Disruption of GAS6-Mertk Pathway Leads to Defects in Physiological Clearance of Expelled Nuclei from Erythroblasts by Bone Marrow Macrophages.
Authors: Kadi Linda , Burnier Laurent , Sugamele Rocco , Carmeliet Peter , Lemke Greg , Earp H. Shelton , Matsushima Glenn K. , Schapira Marc , Angelillo-Scherrer Anne 
Published: April 2020

Abstract
Late in erythropoiesis, nuclei are expelled from erythroblasts and engulfed by macrophages located in the blood island. Expelled nuclei expose phosphatidylserine (PS) on their surface, which is used as a signal for their engulfment by macrophages. PS opsonins milk-fat-globule EGF8 (MFG-E8) and Growth arrest-specific gene 6 product (GAS6) together with their respective receptors avb5 (and avb3) and Axl/Mertk/Tyro3, are involved in the phagocytosis of apoptotic cells. Because fetal liver and bone marrow macrophages do not express MFG-E8, GAS6-Mertk pathway might constitute a major pathway for the engulfment of nuclei expelled from erythroblasts. To test this hypothesis, we isolated nuclei from late-stage erythroblasts from the spleens of phlebotomized mice (500 μl), and tested the capacity of bone marrow-derived macrophages (BMDM) from mice deficient either in GAS6 (GAS6−/−), Axl (Axl−/−), Mertk (mertkkd) or Tyro3 (Tyro3−/−) to internalize these nuclei. Spleen erythroblasts were isolated 4 days after phlebotomy and cultured for 5 hours. Nuclei were obtained after disconnection from reticulocytes by mechanical shaking. Released nuclei were then identified by flow cytometry according to their size and their positive staining for the erythroid lineage marker Ter119 and Annexin V for PS labelling. Purity of the preparation was double-checked by morphological examination of cytospin preparations. Primary BMDM isolated from wild-type (WT) controls and GAS6−/−, Axl−/−, Mertkkd, Tyro3−/−, Axl/Tyro3−/−, Axl−/−/Mertkkd mice were incubated with nuclear preparation for 3 hours. BMDM were then washed to remove un-engulfed nuclei, analyzed in bright field and stained with May-Grünwald-Giemsa. Phagocytosis was determined by counting the number of BMDM with ingested nuclei and the phagocytosis index indicated the number of engulfed nuclei per macrophage. We found that GAS6−/− BMDM cleared 30% less nuclei than WT BMDM (p&lt;0.01). We observed a slight decrease of internalization capacity for Axl−/− BMDM, whereas Tyro3−/− BMDM engulfed the nuclei as efficiently as WT BMDM. In contrast, Mertk deficiency nearly abolished the nuclei phagocytosis (p&lt;0.001). Axl/Tyro3−/− and Axl−/−/Mertkkd BMDM were tested in comparison with WT BMDM and single knockouts, and did not show any cumulative effects when compared to single knockouts. Thus, Mertk was critical for the phagocytosis of nuclei from erythroblasts whereas the role of Axl and Tyro3 appeared to be negligible. In conclusion, we have shown that GAS6 and its receptor Mertk were involved in late erythropoiesis when nuclei are expelled from the erythroblasts and engulfed by BMDM in the blood island. Indeed, GAS6 binding to nuclei exposing PS on their surface might form a bridge between PS and Mertk receptor on BMDM, allowing their efficient clearance.
A Case of HPP with a Novel Combination of α and β Spectrin Mutations.
Authors: Natarajan Kavita , Kutlar Ferdane , Li Tao , Gallagher Patrick , Kutlar Abdullah 
Published: April 2020

Abstract
Hereditary pyropoikilocytosis (HPP) is a red cell membrane disorder classified under the broad umbrella of hereditary elliptocytosis (HE). It is usually the most severe form of HE and is inherited in an autosomal recessive manner. HE results from alterations in RBC membrane cytoskeleton proteins: spectrin, ankyrin, pallidin, band 4.1, and band 3. Quantitative or qualitative deficiencies result in varying clinical syndromes. In general, mutations in the α gene are closer to the amino terminal, and those in the β spectrin gene are nearer the carboxyl terminal and affect the formation of tetramers. We describe a patient associated with a novel mutation in the β and α spectrin genes. A 19 year old black female with a lifelong history of anemia and jaundice that worsened since age 15 presented with history of early satiety of a few months duration. Neither parent had a history of a blood disorder; a maternal uncle had history of splenectomy in his thirties. Her pre-splenectomy labs showed a Hb of 4.8g/dl with a MCV of 62, RDW of 38.8, and chemistries consistent with non-immune hemolytic anemia. Imaging and clinical exam revealed a spleen of 19cms and bizarre red cell morphology including RBC fragmentation and microcytosis. Post splenectomy the patient is entirely asymptomatic with a Hb of 13.2 with pathologic exam of the spleen showing benign, enlarged, red pulp consistent with hypersplenism. Genomic DNA was extracted from peripheral blood. Sequencing of the β-spectrin exon 30 was undertaken at the MCG Sickle Cell Center that revealed a GCT → CCT mutation (Ala → Pro) in codon 2053. This mutation has been previously reported in the homozygous state in a young boy from Mali with HPP (β spectrin Kayes). Since we could not explain the relatively severe phenotype in this patient with heterozygous β spectrin Kayes, further molecular analyses was performed at Yale. This revealed a mutation in the α-spectrin exon 5; L207P, α spectrin St.Louis. This is the 2nd reported case of HPP due to compound heterozygosity for an α and β spectrin mutation and the first combination of α spectrin St. Louis and β spectrin Kayes.
African American female, 19 years old Diagnoses: “Pyropoikilocytosis” Sequence data: Spectrin bate-Kayes African American female, 19 years old Diagnoses: “Pyropoikilocytosis” Sequence data: Spectrin bate-Kayes