bluuebunny/crossref_metadata_2025_split huggingface dataset:
Syk Is a New Target for AML Differentiation.
Authors: Hahn Cynthia K. , Ross Kenneth N. , Kakoza Rose M. , Carr Steven A. , Du Jinyan , Ong Shao-En , Golub Todd R. , Stegmaier Kimberly 
Published: April 2020

Abstract
One of the challenges of phenotype-based small molecule screening has been the difficulty of protein target identification for newly discovered compounds. For example, we previously performed a gene expression-based small molecule library screen, which identified gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, as an inducer of acute myeloid leukemia (AML) differentiation in cell lines and primary patient cells. Neither EGFR transcript nor protein is expressed in the tested AML cell lines, thus precluding inhibition of this kinase as the mechanism of AML differentiation. However, because multiple EGFR inhibitors induce this phenotype, we hypothesize that a shared off-target kinase is the target in AML differentiation. In order to identify candidate gefitinib targets of AML differentiation, we utilized a proteomics method: peptide immunoprecipitation-HPLC-mass spectrometry. This approach enriches for phospho-tyrosine peptides with immunoprecipitation (IP) after enzyme digestion. In contrast to IP at the protein level, the final mixture following peptide-IP contains primarily phospho-tyrosine-containing peptides. We treated the AML cell line HL-60 with gefitinib versus vehicle and then identified peptide sites with loss of phosphorylation with gefitinib treatment. Spleen tyrosine kinase (Syk) was identified as one of the only kinases with loss of phosphorylation post treatment. Syk is a nonreceptor tyrosine kinase, important in normal B-cell differentiation, and implicated in malignancies such as myelodysplastic syndrome and lymphoma. We first confirmed with IP western immunoblotting the inhibition of Syk phosphorylation with gefitinib treatment in HL-60 cells. We next confirmed with both pharmacological inhibition and genetic loss of Syk the induction of differentiation in the AML cell lines HL-60 and U937. Numerous determinants of myeloid maturation were tested: a complex differentiation gene expression signature, cellular morphology, cell surface proteins CD11b and CD14 expression, and nitro-blue tetrazolium (NBT) reduction, a functional assay for myeloid differentiation. Two reported pharmacological inhibitors of Syk scored positive on all measurements of differentiation. Furthermore, the shRNA construct inducing the most complete loss of Syk also scored the highest on all measurements of differentiation. The most convincing data that Syk is the true target of these inhibitors would be the identification of an inhibitor resistant Syk mutant, analogous to BCR-ABL or c-Kit mutants that confer imatinib resistance. To this end, a Syk random mutagenesis screen is ongoing. In summary, these data identify Syk as a strong candidate target of gefitinib, demonstrate that inhibition of Syk can induce AML differentiation, and identify Syk as a potential target for AML differentiation therapy.
Low Erythrocytic Glutathione Peroxidase-1 Activity Correlates with Hemolytic Rate in Patients with Sickle Cell Disease and Is Elevated on Hydroxyurea.
Authors: Kato Gregory J. , Cho Chun Seok , Yang Seung Ha , Bae Sung Won , Lee Jong Seo , Gladwin Mark T. , Rhee Sue Goo 
Published: April 2020

Abstract
Background: Glutathione peroxidase-1 (GPX1) is the most significant catalytic antioxidant in the red cell. Congenital deficiency of GPX1 has been reported in association with hemolytic anemia due to oxidant stress. Hemolytic rate has been implicated in the development of sickle vasculopathy.
Methods: Red cell pellets were collected from 32 patients with sickle cell disease and 17 healthy African American control subjects, and were stored frozen until assayed. A novel microplate immunoaffinity capture assay was used that measures both immunoreactive GPX1 protein and enzyme activity.
Results: Patients with SCD had higher levels of red cell GPX1 activity than controls (2.42 ± 0.12 vs. 1.86 ± 0.15 units/mg red cell protein (mean ± SEM), p=0.006). GPX1 activity correlated with low reticulocyte count (r=−0.50, p=0.004)(Fig. 1A), low indirect bilirubin (r=–0.45, p=0.01) and high fetal hemoglobin expression (r=0.39, p=0.03). GPX1 activity in SCD patients on hydroxyurea was much higher than those not on hydroxyurea (2.75 ± 0.12, n=21 vs. 1.79 ± 0.13, n=11, p&lt;0.001)(Fig. 1B), suggesting that hydroxyurea might induce GPX1 expression. In the patients off hydroxyurea, low GPX1 activity closely correlated with high serum lactate dehydrogenase level, a marker of high hemolytic rate (r=–0.85, p=0.002), suggesting that low GPX1 levels might contribute to rapid hemolytic rate. In a multivariate model, GPX1 was independently associated with reticulocyte count even when adjusted for fetal hemoglobin level.
Conclusions: In addition to fetal hemoglobin, hydroxyurea may also induce expression of GPX1, a potent antioxidant that might have a role in decreasing hemolysis associated with the robust oxidant stress of SCD. This finding is consistent with previously published evidence of GPX1 induction by hydroxyurea in cancer cell lines, with increased antioxidant function. Additional studies are needed to confirm this phenomenon in a larger cohort of patients with SCD and to further evaluate the combined effect of fetal hemoglobin co-induction, but this finding suggests a potential mechanism by which hydroxyurea may produce clinical benefit in SCD even in the absence of significant fetal hemoglobin induction.
Figure Figure
Hypermethylation of DNA and Increased Expression of DNMT1 May Represent a Protective Mechanism from Oxidative Stress and DNA Damage in MDS.
Authors: Jankowska Anna M. , Gondek Lukasz P. , Szpurka Hadrian , Nearman Zachary P. , Tiu Ramon , Maciejewski Jaroslaw P. 
Published: April 2020

Abstract
Epigenetic changes including altered cytosine methylation have been hypothesized to play a pathogenic role in myelodysplastic syndrome (MDS) and other myeloid malignancies. While the specific functional consequences of CpG island methylation depend on the pattern of silenced promoters, the triggers for changes in the methylation program are unknown. DNA damage is likely to interfere with faithful heritability of cytosine methylation patterns during cell division. For example, formation of 8-oxoguanine (8-OG) in CpG islands may result in hypomethylation through inhibition of methylation-sensitive human maintenance methyltransferase DNMT1. Alternatively, hypermethylation may be a part of cellular response to oxidative damage e.g., through stimulation of DNMT1 activity. When we investigated expression of various DNA repair genes in highly purified CD34+ progenitor and stem cells of patients with MDS (19 RARS/RA, 16RAEB1/2/sAML, 2 CMML) using expression arrays, we found a significant and concordant up-regulation of N-methylpurine DNA glycosylase MPG; this finding was confirmed by TaqMan PCR in 21/37 MDS patients tested. Over-expression of MPG may be in response to increased DNA alkylation, but MPG has been described to co-localize in methylated CpG islands and could reflect their increased methylation. We have stipulated that up-regulation of MPG is a consequence of ongoing DNA damage and chose to study other parameters that could support this theory. For example, incorporation of 8-oxoguanine can be measured by flow cytometry and when we used this method, we determined that an increased oxidative DNA load/stress was found in 67% of all MDS cases studied (n=24) and correlated with advanced stage of MDS (p&lt;.006), cytopenia (e.g., neutropenia p&lt;.002) and a greater propensity for AML transformation (p&lt;.02). We also studied expression of DNA glycosylase/AP lyase OGG1 with the capacity to excise oxidized guanine from DNA and found up-regulation of OGG1 mRNA levels in 11/37 patients. To investigate whether oxidative/alkylating DNA damage is indeed associated with alteration of methylation we have measured 2 parameters:
global methylation using an ELISA-based colorimetric assay specific for methylcytosine and expression of DNMT1 using TaqMan PCR.
We determined that patients with over-expression of OGG1 were characterized by lower expression of DNMT1 (&gt;2x control levels) and inversely correlated with global methylation levels. In contrast, those patients with over-expression of DNMT1, a corresponding increase in global methylation was observed. In sum, our results are consistent with the inverse relationship between hypermethylation and oxidative stress or alternatively that hypermethylation may be a protective response to an ongoing DNA damage in a subset of MDS patients.
CliniMACS® Cell Enrichment Using NKp46 — A Large Scale, Single-Step NK Cell Isolation Method.
Authors: Bondzio Ilka , Schmitz Jurgen , Huppert Volker 
Published: April 2020

Abstract
Natural Killer (NK) cells show potent anti-leukemic activity in vitro and thus NK cells isolated using the CliniMACS® Cell Separation System are currently under clinical evaluation as part of a treatment for acute myeloid leukemia (AML). (Uharek L et al., 2003, Gentilini C et al., 2005; Koehl U et al., 2004, 2005; Passweg, JR et al., 2005, 2006; Miller J et al., 2005). Currently, the processing of highly purified, clinical-grade NK cells requires a two-step method using the CliniMACS Instrument: Depletion of CD3+ cells followed by enrichment of CD56+ cells (Iyengar R et al., 2003). This procedure shows excellent performance (reviewed in Passweg et al., 2005) with yields of 30–60%, purities of &gt;85%, &gt; 99.97%/3.5 log T cell depletion but is time consuming. Simpler one-step procedures have thus been tried to reduce the processing time (CD3 depletion, CD3/CD19 depletion, CD6 depletion, CD56 enrichment) but result in a more heterogeneous final cell product that usually contains less than 60% NK cells. NKp46 (CD335) has been demonstrated to be primarily expressed by NK cells and may thus be a suitable selection marker for one-step NK cell enrichment. We evaluated an NKp46 enrichment strategy using the autoMACS™ Pro Separator, NKp46-Biotin, and Anti-Biotin MicroBeads. We could enrich CD56+CD3- cells from peripheral blood mononuclear cells (PBMCs) to a purity of 90% with a yield of 40–60% and a &gt;99.92%/3.1 log depletion of T cells (n = 2). These results encouraged the evaluation of this procedure on a clinical scale by using the CliniMACS Cell Separation System. Using the CliniMACS Plus Instrument, NK cells could be enriched to 73–87% purity (n = 3) from Buffy Coats derived from whole blood. T cells were efficiently depleted (&gt;99.92%/3.0 log) to such a degree that NKp46-selected NK cell products may fulfil the release criteria for NK cell donor lymphocyte infusions (NK-DLI) in the context of haploidentical stem cell transplantation. While CD56+ CD3- NK cells are not activated by antibody binding during cell separation, magnetic labelling using anti-NKp46 antibodies may trigger NK cell activation. NKp46-mediated NK cell activation may be synergistically enhanced by the use of IL-2. Functional studies of NK cells isolated by different methods will be required to further evaluate their feasibility for use in NK-DLIs.
Phase II Trial of the mTOR Inhibitor RAD001 in Relapsed and/or Refractory Waldenstrom Macroglobulinemia: The Dana Farber Cancer Institute Experience.
Authors: Ghobrial Irene M. , Chuma Stacey , Sam Amy , Leduc Renee , Nelson Marybeth , O’Connor Kelly , Harris Brianna , Soumerai Jacob , Warren Diane , Jacobsen Eric , DeAngelo Daniel , Anderson Kenneth C. , Richardson Paul G. , Witzig Thomas E. , Treon Steven P. 
Published: April 2020

Abstract
INTRODUCTION: Previous studies have demonstrated the clinical activity of the mTOR inhibitor RAD001 in low- grade lymphomas. Our preclinical studies demonstrated activity of mTOR inhibitors in Waldenstrom Macroglobulinemia (WM) cell lines and patient samples. This phase II study aimed to determine safety and activity of the oral mTOR inhibitor RAD001 (Novartis Pharmaceutical, MA) in patients with relapsed or refractory WM.
METHODS: Patients who had at least one previous therapy for WM, and who had symptomatic relapsed or refractory disease were eligible. NCI CTCAE v3.0 was used for toxicity assessment. All patients received daily RAD001 at 10 mg. A cycle was considered 28 days. Patients were allowed to stay on therapy until progression of disease or excessive toxicity. This study was conducted in a collaborative effort between Dana Farber Cancer Institute (DFCI) and Mayo Clinic College of Medicine. Here, we report the data on the patients accrued at DFCI.
RESULTS: 19 pts (15 men and 4 women) have been treated to date. All patients had symptomatic disease and required therapy. The median number of lines of prior treatment was 3 (range 1 – 5) including included rituximab, nucleoside analogues (fludarabine or 2-CDA), combination chemotherapy (e.g. CHOP, CVP), chloramucil, and bortezomib. The median IgM at baseline was 3330 mg/dL (range 1010– 7410). The median follow on RAD001 was 8 months (range 3 – 22 months). Eighteen pts are currently evaluable for response. Best responses to RAD001 after 2 cycles using IgM monoclonal protein were as follows: partial remission in 8 (44%), minimal response in 5 (28%). Progressive disease occurred in 4 (22%) and stable disease occurred in 1 (6%). The overall response rate (PR+MR) was 72%. The median duration of response has not been reached (3–22+ months). Patients tolerated therapy well without significant toxicities. Grade 3 and 4 toxicities included grade 4 thrombocytopenia in 1 patient, grade 3 pneumonia in 1 patient, grade 3 hyperglycemia in 1 patient and grade 3 mucositis in 1 patient. Other adverse events of grade 2 or lower included nail cracking, mucositis, diarrhea, and fatigue. Attributable toxicities otherwise proved manageable with appropriate supportive care, and RAD001 was generally well tolerated. One patient enrolled on the study withdrew consent and changed to hospice care within 3 weeks of therapy, and passed away due to disease progression.
CONCLUSIONS: The use of the oral RAD001 single agent RAD001 in patients with relapsed or refractory WM was welltolerated and demonstrated significant activity achieving an overall response rate in 72% of patients. Future studies of combination of this agent with rituximab and bortezomib are currently being planned.
Activity of the Multi-Targeted Kinase Inhibitor, AT9283 on Imatinib-Resistant CML Models.
Authors: Tanaka Ruriko , Squires Matthew S , Kimura Shinya , Yokota Asumi , Mallett Kirsty , Smyth Tomoko , Thompson Neil T , Nagao Rina , Yamauchi Takahiro , Ushiki Takashi , Lyons John F , Maekawa Taira 
Published: April 2020

Abstract
CML is caused by a consistent genetic abnormality, termed the Philadelphia chromosome, that results from a reciprocal (9;22) translocation leading to the expression of the BCR-ABL fusion protein. Although treatment has been revolutionized by the introduction of tyrosine kinase inhibitors which target Abl activity, reactivation of Abl signaling via several different point mutations remains problematic. In particular the mutation of Threonine 315 to Isoleucine (T315I) confers resistance to all existing therapies with tyrosine kinase inhibitors in the clinical settings. We describe the in vitro and in vivo effects of AT9283, a potent inhibitor of several protein kinases, including Abl kinase (wild type BCR-ABL and several of the drug resistant mutant variants that have arisen in clinical practice e.g. T315I), JAK2, JAK3 and Aurora kinases A and B, on imatinib-resistant CML cells including those harboring BCR-ABL (T315I). AT9283 has potent anti-proliferative activity in a panel of BaF3 and human cell lines expressing the BCR-ABL or its mutant forms. In BaF3 BCR-ABL wild-type and T315I mutant cells and K562 CML cells we observed inhibition of substrates of both BCR-ABL (STAT5) and Aurora B (Histone H3) at concentrations &gt;300nM and &lt;100nM, respectively, suggesting that AT9283 is capable of inhibiting Aurora and BCR-ABL simultaneously in these cell lines. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3, human CML cell lines or primary CML patient samples expressing the BCR-ABL or its mutant forms. Specifically AT9283 prolonged the survival of mice engrafted intravenously with either BaF3 BCR-ABL T315I, or E255K cells when administered intraperitoneally twice daily at doses of either 6.25 or 10mg/kg or once daily at 15mg/kg when administered 5 days in every week repeated twice. Maximal survival advantage was conferred at either 10mg/kg twice daily or 15mg/kg once a day. Similar data were obtained in an intravenous model using primary CML cells taken from a patient harbouring the BCR-ABL E255K mutation. We also present data from ongoing studies showing increased survival rates in these in vivo model systems following multiple cycles of AT9283 administered on the 15mg/kg once daily schedule. These data together support further clinical investigation of AT9283 in patients with treatment resistant CML.
Pre- vs. Post-National Coverage Determination (NCD) Blood Utilization and Hemoglobin Values among Medicare Patients Treated with Erythropoietic-Stimulating Agents (ESAs) for Chemotherapy-Induced Anemia (CIA)
Authors: Burton Tanya , Larholt Kay , Apgar Elizabeth , Pashos Chris , Bookhart Brahim , Corral Mitra , Piech Catherine Tak , Mckenzie R. Scott 
Published: April 2020

Abstract
Background: In July 2007, the Centers for Medicare and Medicaid Services issued ESA coverage limitations for cancer patients with CIA through a national coverage determination (NCD). Clinical outcomes based on ESA dosing described in the NCD have not been reported in prospective observational or clinical trial data. To understand hematologic outcomes in the Medicare population treated in pre- and post-NCD time period, an analysis of hematologic outcomes from the DOSE (Dosing and Outcomes Study of Erythropoiesis-Stimulating Therapies) registry, an ongoing prospective observational study, was conducted.
Methods: ESA-treated cancer patients with CIA were selected based on Medicare primary coverage, available baseline hemoglobin (Hb) value, and receipt of at least two ESA administrations. Data were categorized into two timeframes based on date of initial ESA administration: pre-NCD (4/06-4/07) and post-NCD (10/07-5/08). Baseline demographics, Hb values during ESA treatment, and blood utilization patterns were analyzed.
Results: 288 patients were identified (Pre-NCD – 230; Post-NCD – 58) from 41 sites. Patient groups were similar with respect to age, gender, weight, and tumor type. ESA treatment duration was significantly greater in the pre-NCD group (70 days vs. 54 days, p = 0.0011). As shown in the table, differences with regard to blood utilization and Hb values were observed between the Pre-NCD and Post-NCD populations. A significantly greater proportion of patients required a blood transfusion and the number of units administered per study patient was significantly higher in the Post-NCD group. Hb levels were significantly lower at all time points of observation in the Post-NCD group.
Pre-NCD Post-NCD p-value Transfusion Outcomes Proportion of Patients Transfused 18.3% 32.8% p = 0.0157 Mean No. of Units per Study Patient 0.5 1.1 p = 0.0089 Hematologic Outcomes: Mean Hb (SD) Baseline 10.6 (0.8) 9.6 (0.5) &lt; 0.0001 Week 4 11.1 (1.3) 9.9 (1.1) &lt; 0.0001 Week 8 11.2 (1.3) 10.4 (1.3) 0.013 Week 12 11.1 (1.3) 9.8 (1.2) 0.0002 Week 16 11.0 (1.1) 9.7 (0.2) 0.018
Conclusion: Greater blood utilization and lower Hb values were observed in Medicare CIA patients treated with ESAs during the Post-NCD time period compared to the Pre- NCD time period. The impact of the NCD on patient outcomes is important to providers and hospital systems and warrants further research.
Wnt/β-catenin and Pten/Akt Signaling Interaction Drives Hematopoietic Stem Cell Self-Renewal and Leukemogenesis.
Authors: Perry John M. , Grindley Justin C. , He Xi , Li Linheng 
Published: April 2020

Abstract
Self-renewal is the central property of stem cells but remains inadequately defined. Separately, the Pten/Akt and Wnt/β-catenin signaling pathways have been implicated in regulating hematopoietic stem cells (HSCs) and in leukemia development. Using an HSC-specific conditional mutant model, we studied the effects of Pten deletion combined with constitutively active β-catenin. HSC-specific deletion of Pten leads to a relatively moderate HSC expansion in spleen with increased myeloid differentiation. In contrast, HSC-specific activation of β-catenin results in functional failure by differentiation blockage. However, unlike single mutants, double mutant mice exhibit a novel phenotype including dramatic expansion in HSCs without extensive differentiation both in vitro and in vivo. Furthermore, β-catenin deficiency prevents the relatively moderate expansion of HSCs in Pten mutants. Our data reveal that these two pathways interact to coordinately drive HSC proliferation while inhibiting both differentiation and apoptosis. Together, the Pten/Akt and Wnt/β-catenin pathways cooperate to drive self-renewal, with uncontrolled self-renewal leading to leukemia development.
Specific Induction of Granzyme B in Human B Cells by Viral Antigens.
Authors: Hagn Magdalena , Maier Julia , Syrovets Tatjana , Simmet Thomas , Jahrsdoerfer Bernd 
Published: April 2020

Abstract
B cells are not currently known to produce granzyme B (GrB) in (patho-) physiological settings. We recently reported that B-chronic lymphocytic leukemia cells and normal B cells treated with interleukin-21 (IL-21) and anti-B cell receptor antibodies (anti-BCR) produce functional GrB. Here we demonstrate for the first time that viral antigens can also induce specific peripheral B lymphocytes to produce and secrete substantial amounts of active GrB. Using FACS, ELISpot, immunofluoresence and western blot we show that B cells from subjects recently vaccinated against tick-borne encephalitis virus (TBEV) but not unvaccinated subjects respond to viral TBEV antigens with GrB secretion in a dosedependent manner. This response is direct and occurs only in the presence of co-activation with certain IL-2 family cytokines such as IL-21. Similar results were found with other viruses including hepatitis B and rabies. GrB production in B cells required activation of JAK1 and STAT3 and inhibition of JAK1 by pyridone 6 completely abrogated GrB induction by viral antigens or anti-BCR. Our findings suggest GrB secretion by B cells may be part of a novel, anti-viral immune response mechanism. Further studies will elucidate whether or not granzyme B-secreting B cells can act as cytotoxic cells towards virus-infected cells.