bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Glutaminase Activity Of L-Asparaginase Is Not Required For Anticancer Activity Against Asns-Negative Cell Lines
Authors: Lorenzi Philip L , Chan Wai Kin , Anishkin Andriy , Purwaha Preeti , Rogers David M , Sukharev Sergei , Rempe Susan B , Weinstein John N 
Published: October 2019

Abstract
L-Asparaginase (L-ASP) is a key component of acute lymphoblastic leukemia therapy. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. In the present study, we tested the hypothesis that L-ASP glutaminase activity is required for anticancer activity. We first used molecular dynamics simulations of the clinically used E. coli L-ASP enzyme to guide engineering of mutants that lack glutaminase activity. Dynamic mapping of enzyme-substrate contacts identified the backbone amine of residue Q59 as having frequent contact with glutamine but not asparagine substrate. That difference identified Q59 as a promising mutagenesis target for modifying substrate selectivity. Saturation mutagenesis and screening of the resulting Q59 mutants identified Q59L as retaining asparaginase activity yet exhibiting undetectable glutaminase activity. Using Q59L to test the glutaminase-anticancer hypothesis, we observed no anticancer activity by Q59L against cell lines that do express asparagine synthetase (ASNS), including six leukemia lines—CCRF-CEM, SR, MOLT-4, K562, NALM-6, and REH—and two ovarian cancer lines—OVCAR-8 and SK-OV-3. Wild-type (WT) L-ASP, on the other hand, effected a dose-response in all of those cell lines, suggesting that glutaminase activity is required to kill cancer cells that express ASNS. Unexpectedly, Q59L exhibited potent anticancer activity against cell lines that do not express detectable ASNS, including the leukemia cell lines Sup-B15 and RS4;11 and ASNS siRNA-treated OVCAR-8 cells. We conclude that the glutaminase activity of L-ASP is not necessary for anticancer activity against cell types that do not express ASNS. Since Q59L is expected to exhibit reduced toxicity relative to wild-type L-ASP because of its reduced glutaminase activity, these findings provide rationale for clinical assessment of Q59L L-ASP for the treatment of ASNS-deficient cancers.

Disclosures:
Lorenzi: ERYtech Pharma: Consultancy, Membership on an entity’s Board of Directors or advisory committees, US 7985548, US 7985548 Patents & Royalties. Off Label Use: L-asparaginase is an enzyme-drug approved for treatment of acute lymphoblastic leukemia.
Differential Response Of Leukemic Blasts To Dexamethasone and Prednisone Occurs At Sub-Saturating Doses
Authors: Pufall Miles A. , Yang Hongxing , Shelton Dawne N. 
Published: October 2019

Abstract
Dexamethasone (dex) and prednisone (pred) are highly related glucocorticoids that are used in the treatment of leukemias and lymphomas. Recently it was shown that dex produces better outcomes in children with high-risk B cell precursor acute lymphoblastic leukemia than pred. Although this result was consistent with the accepted potencies of the two drugs, the reason for this difference is not well understood. Glucocorticoids work primarily by binding and activating the glucocorticoid receptor, which, when bound, associates with genomic response elements to orchestrate gene expression programs. We hypothesized that dex regulates key genes that drive apoptosis in BCP-ALL more strongly. To test this we used microarrays to measure the differential expression of genes in response to saturating doses of dex and pred in three cell lines. Dex and pred induced very similar patterns of gene expression in each cell type, with an overlap of about 60%. Surprisingly, when we compared the 40% of genes that were regulated by dex or pred alone in each cell line, there were no commonly regulated genes among all cell lines. Further, for the 150 genes commonly regulated by both dex and pred in all three cell lines, there were no differences in the magnitude of response, suggested that the difference might lie at a lower, more functional doses. We tested this using RNA sequencing at five concentrations of both dex and pred in a primary patient sample. Consistent with their potency, the average regulated gene required eight times more pred than dex. Interestingly, when we performed unsupervised clustering of regulated genes by concentration, we found more modest differences in the concentration required for the onset of regulation, and that pred selectively suppressed expression of a block of genes at lower concentrations. These data suggest that rather than specific genes being regulated by dex, or simply a more potent response of the same genes, that the difference in apoptosis induction may be due to surprisingly different responses at sub-saturating concentrations.

Disclosures:
No relevant conflicts of interest to declare.
GLI1 Inhibitor GANT61 Deregulates stat3 Phosphorylation In Chronic Lymphocytic Leukemia Cells
Authors: Wang Cheng , MM Kang Lu , Wang Xin 
Published: October 2019

Abstract

Background
Hedgehog(Hh) family has come to be recognized as key fundamental mediators of many carcinomas, but conflicting results exist about its role in chronic lymphocytic leukemia (CLL). Here we examined the effect of GLI inhibitor GANT61 to investigate the role of the Hh-signaling pathway in CLL.

Methods and Results
We conduct real-time PCR for Hedgehog family members on isolated mononuclear cells from peripheral blood (n=35) and bone marrow cells(n=6) to evaluate the presence of the Hh-signaling pathway in CLL. There's no significant difference between peripheral blood and bone marrow cells in levels of Hh members. Profiling of cognate Hh pathway members revealed reduced expression of three key Hh signaling effectors, Patched, Smoothened (SMOH) and GLI, in peripheral blood mononuclear cells (PBMC), whereas transcription levels of other investigated members(SHH, IHH, DHH, GLI2, GLI3 etc.) resembled normal B-lymphocyte levels. However, we found a great heterogeneity for the expression levels of the Hh family with a subset of about 25% of CLL PBMC samples showing high transcript levels ( 1.5-fold than the median) for GLI1 and SMO. There is a direct positive correlation between GLI1 expression and SMO expression. We performed western-blot in CLL PBMC samples and found a positive correlation between phosphorylation of stat3 and GLI1 (figure 1). We examined the activity of GANT61 on viability of cell lines and primary CLL cells (N=3) in vitro by CCK8. GANT61 reduced the cell viability to 65% ± 14% after 24 hours of culture at concentration of 20uM (mean +/¨C SD, P < 0.05). We found that the capacity of GANT61 to inhibit CLL cell viability was associated with stat3 phosphorylation, which is time and dose dependent (figure 2).

Conclusion
These results suggest that in CLL Hh pathway is closely related to stat3 pathway. Moreover, these studies reveal a potential mechanism for the anti-leukemia activity of GANT61 which might inhibit viability of CLL cells by deregulating stat3 phosphorylation.

Disclosures:
No relevant conflicts of interest to declare.
Comparison Of Clinical Implications Of p16 Deletion In B-Lineage Acute Lymphoblastic Leukemia
Authors: Xu Na , Xiao Xiaozhen , Zhou Xuan , Gao Guanlun , Xiao Yajuan , Du Qingfeng , Xiaoli Liu 
Published: October 2019

Abstract

Objective
This paper probes into the impact of P16 gene deletion on the prognosis of children and adults B-ALL.

Method
With the adoption of the Flow Cytometry, G-banding Technique Karyotype Analysis and Fluorescence in situ hybridization (I-FISH) technology, this paper will retrospectively analyze the clinical data of the immunological and cytogenetic detection to the total 124 initial onset B-ALL patients, who were treated in our hospital from January 2006 to December 2008, including children (73 cases, median age 6 years old) and adults (51 cases, median age 29 years).

Result
With the adoption of the I-FISH technology, the result data of the survey are exemplified as follows: in the group of 73 cases of child B-ALL patients, the incidence of P16 gene homozygous deletion, loss of heterozygosity and non-deletion are 13.7% (7 cases), 5.9% (3 cases), 80.4% (41 cases); in the group of 51 cases of adults, they are 13.7% (7 cases), 5.9% (3 cases), 80.4% (41 cases). In this data,recurrence rate in patients with p16 gene deletion is 43.5% (10 cases) in group of children, which is lower than that 90.0% (9 cases) in group of adults. The difference is statistically significant (P = 0.013 ). Overall Survival (OS) is compared in patients of children and adults with and without p16 gene deletion. The result shows that children aged from 1 to 14 with p16 gene deletion have lower Overall Survival (OS) than that without p16 gene deletion (P = 0.013). And it is the same in adult patients older than or are 15 years old (P = 0.020). The long-term disease-free survival DFS is no significant difference (P = 0.154) between that with and without p16 gene deletion in child group. On the contrary, the P16 gene deletion DFS is significantly lower than those without it in the adult group (P <0.001).

Conclusion
p16 gene deletion in B-ALL patients of children and adults is equally poor prognostic factors. Consequently, it is significant for the assessing prognosis and guiding clinical treatment by making a definite status of the p16 gene deletion.

Disclosures:
No relevant conflicts of interest to declare.
Monoclonal Igκ and Igλ Antibodies Minimize Cross-Reactivity With NK Cells In a Multicolor Immunophenotyping Panel
Authors: Deal Emily M. , Inokuma Margaret S. , Yang Xiaoyun , Shye Shou , Franzblau Manuela , Gladding Daiva A. , Crespo Oliver , Wai Hobert 
Published: October 2019

Abstract
Immunophenotyping provides a valuable aid to leukemia and lymphoma diagnosis and classification. Comprehensive initial screening panels must include markers that identify all major cell populations, such as T cells, B cells, and NK cells. It also is useful to further phenotype the subsets of the major populations within the same tube. This can be achieved by using a single fluorochrome for two markers that are specific for subsets of different cell types (for example, CD8 FITC for NK cells and Igλ FITC for B cells). Such a cocktail minimizes the amount of patient sample required for classification while simultaneously decreasing workflow, reagent consumption, and time.
The current study examines the interaction between Igλ FITC, Igκ PE, CD8 FITC, and CD56 PE surface staining. We identified that Igλ and Igκ polyclonal antibodies cause an artificial dragup of the CD8 FITC NK population in a fraction of healthy donors. This dragup results in the majority of NK cells appearing to be CD8+, which is consistent with the phenotype of chronic lymphoproliferative disorders of NK cells [Am J Clin Pathol 127:881; Br J Haematol 120:1026].
Since this artificial dragup may indicate an inappropriate result for the patient specimen, we assessed the effects of different Igκ and Igλ antibodies on this stain. The highly conserved nature of Igλ and Igκ across species caused us to evaluate polyclonal antibodies column-adsorbed against cross-reactive antibodies in addition to total polyclonal and monoclonal antibodies. We conclude that monoclonal Igκ and Igλ antibodies minimize the phenotypic effects on the NK population when using the same fluorochromes, while providing sufficient separation to discriminate Igκ+ and Igλ+ B cells. These studies decrease the risk of errors in disease classification in assays that simultaneously evaluate subsets of multiple cell types.

Disclosures:
Deal: BD Biosciences: Employment. Inokuma:BD Biosciences: Employment. Yang:BD Biosciences: Employment. Shye:BD Biosciences: Employment. Franzblau:BD Biosciences: Employment. Gladding:BD Biosciences: Employment. Crespo:BD Biosciences: Employment. Wai:BD Biosciences: Employment.
Increased ADAM28 Levels In Plasma and Bone Marrow Cells of Patients With Adult Acute Leukemia
Authors: Zhang Xiaohui , Wang Qianming , Hanby Hayley A , Zhou Shiyuan , Zhou Yi , Wang Min , Zhao Jingzhong , Huang Xiao-Jun , Zheng Long 
Published: October 2019

Abstract
ADAM28, a member of the ADAM (A Disintegrin And Metalloproteinase) family, cleaves various substrates including von Willebrand factor. Two isoforms of ADAM28: the membrane associated (ADAM28m) and the secreted (ADAM28s) were identified. Both were overexpressed in solid tumors including breast carcinoma, non-small cell lung cancer, and bladder transitional cell carcinoma. Overexpression of ADAM28 has been shown to promote cell growth, invasion, and metastasis of solid tumors in humans. However, little is known about expression and potential role of ADAM28 in hematological malignancies. In this study, we determined ADAM28 protein and/or mRNA expression in plasma and/or bone marrow cells of patients with various acute and chronic leukemia. An informed consent was obtained from all participants. Plasma and bone marrow aspirates were obtained from a total of 65 patients including 24 patients with acute lymphoblastic leukemia (ALL), 23 patients acute myelocytic leukemia (AML), and 18 patients with other leukemia. Twenty one healthy volunteers were also included in the study (Table 1). By flow cytometry, we showed that mean fluorescent index ratios that are specific for ADAM28m were significantly <>
<>increased in the bone marrow malignant cells in patients with ALL (2.9±0.13) and AML (2.1±0.17) compared with those in the control (0.4±0.06). The p values were <0.01 and <0.05, respectively. In addition, plasma levels of ADAM28s antigen assessed by a specific sandwich ELISA were significantly higher in patients with ALL (386.1±44.6 pg/ml) (p<0.01) and AML (291.3±24.0 pg/ml) (p<0.05) than those in the control (120.7±12.1 pg/ml). Furthermore, the mRNA levels encoding ADAM28m (1.7±0.0) and ADAM28s (2.9±0.1) in the bone marrow cells were significantly higher than those in the control (p< 0.05) (Table 1). Interestingly, cellular ADAM28 overexpression was inversely associated with cellular apoptosis in ALL patients. In four ALL patients with an early relapse, ADAM28 levels (both protein and mRNA) in bone marrow cells were significantly higher than those in patients with complete remission. In conclusion, our data indicate for the first time that a measurement of plasma and bone marrow ADAM28 levels may help identify patients with high risk of early relapse. Whether a treatment that targets ADAM28 has an effect in adult acute leukemia remains to be determined.

Disclosures:
No relevant conflicts of interest to declare.
Genomic Analysis Of Notch Mutations In a Case Of Alagille Syndrome With Acute Lymphoblastic Leukemia
Authors: Bernardi Simona , Tononi Paola , Vargas Sergio Marin , Bassi Giulio , Ambrosetti Achille , Meneghini Vittorio , Scaffidi Luigi , Martinelli Giovanni , Iacobucci Ilaria , Pizzolo Giovanni , Delledonne Massimo , Krampera Mauro 
Published: October 2019

Abstract
Alagille syndrome (ALGS), or arteriohepatic dysplasia, is a congenital multisystem disease due to Notch signalling pathway mutations, most commonly affecting JAG1 (ALGS type 1), and more rarely NOTCH2 (ALGS type 2), leading to hepatic, lung, renal and ocular dysfunction (chronic cholestasis, peripheral pulmonary artery stenosis, dysplastic kidneys pigmentary retinopathy), and skeletal abnormalies (minor vertebral segmentation, characteristic facies, posterior embryotoxon/anterior segment defects). ALGS is an autosomal dominant disease, but it is characterized also by variable penetrance and clinical expression and somatic/germline mosaicism.
A 20-year-old man with ALGS was admitted to the University Hospital of Verona because of pancytopenia. Following analyses led to the diagnosis of Philadelphia chromosome/bcr-abl-negative, CD10-positive, B-lineage acute lymphoblastic leukemia (common B-ALL).
In order to identify the genetic components involved in this complex phenotype, we sequenced the exome of a bone marrow sample collected from the patient. By genome interpretation with Knome pipeline applied to the reference genome UCSC hg19, we found missense variants both in NOTCH2 (E38K) and JAG1 (P871R) genes that are mainly involved in the syndrome, although their effect on protein function was predicted not to be deleterious. However, we detected putative damaging mutations in genes such as PAX5 (R38H) and NOTCH1 (K1821N) which might be strongly related to the observed disease. In fact, PAX5 is a member of PAX protein family of transcription factors implicated into regulation of early development, that binds NOTCH2 and likely altering its functionality. On the other hand, NOTCH1 is involved in cell growth and proliferation and thus the predicted alteration of function of the corresponding protein may have an important role in neoplastic transformation.

Disclosures:
No relevant conflicts of interest to declare.
Body Mass Index and Uncontrolled DM Impact On Acute Myeloid Leukemia (AML) Outcomes
Authors: Abdelfatah Mohamed , Anand Ankit , Kanaan Zeyad , Al-Ameri Ali , Haller Nairmeen 
Published: October 2019

Abstract

Background
The incidence of obesity is increasing worldwide and is associated with numerous adverse health outcomes. Obesity was associated with more toxicity and worse prognosis in pediatric AML patients. On the other hand diabetic patients diagnosed with AML have multiple complications related to infection organ failure and chemotherapy side effects, Previous epidemiological studies in children have shown significant correlations in incidence between AML and DM in children.

Aim
To study Body-Mass Index (BMI) and uncontrolled DM impact on clinical outcome of adult AML pts.

Methods
A total of 187 adult pts with AML between 2002-2011 were enrolled. Retrospective data included demographics, labs, cytogenetics and outcome. LeukemiaNET Standardization (LNS), complete remission (CR), overall survival (OS) and relapse free survival (RFS) were obtained (Dohner E, Blood 2010). BMI of <18.5 defined as underweight, 25-30 was defined as overweight, while >30 as obesity. On the other hand Fasting blood glucose and Hemoglobin A1c were measured, DM severity was classified according to guidelines, as non diabetics (A1c)<5.7 and normal FBG<100, high risk for DM with (A1c) from (5.7 - 6.4) and uncontrolled DM (A1c) >6.5. Fischer's and Wilcoxon tests were used for comparatives between groups, cox proportional hazards and logistic regression for associations for OS/RFS and CR, Kaplan-Meier test for OS and RFS estimates via JMP software V9.0. IRB approval was obtained according to Helsinki declaration.

Results
The median age was 70 years, with 98 (52%) were men. 45 pts (24%) had complex cytogenetics, 28 pts (15%) had AML progressed from MDS. Median BMI was 28.7 (range 14.8-51.4). 10 (5%) were underweight, 34 (18%) had normal BMI, 59 (32%) were overweight, and 84 (45%) were obese. 149 pts (80%) were not Diabetic, 12 pts (6%) had high risk for DM, 26 pts (14%) uncontrolled DM. Overall Median OS was 23 weeks; 46 weeks in normal BMI, 30 weeks in overweight, 14 weeks in obese, 15 weeks in underweight, A according to DM subgroups; 24 weeks non-diabetics, 15 weeks had high risk for DM, 37 had uncontrolled DM. At diagnosis, BMI or Hemoglobin A1c classes were not associated with age, sex, white blood count (WBC), platelets, blasts, ECOG status, LNS; nor treatment toxicities. Even though median survival in weeks was lower in obese and underweight pts, Median OS and RFS were not different in univariate or across BMI subgroups (p=0.32 and 0.22) or Hemoglobin A1c subgroups (p=0.19 and 0.71)

Conclusions
BMI and uncontrolled DM did not show statistically significant correlation with LNS, relapse rates, OS or RFS.

Disclosures:
No relevant conflicts of interest to declare.
Recurrent STAT6 Mutations In Follicular Lymphoma
Authors: Malek Sami , Kaminski Mark S. , Li Hongxiu , Ouillette Peter , Jones Sian , Fox Heather , Jacobi Kathryn , Saiya-Cork Kamlai , Bixby Dale , Lebovic Daniel , Shedden Kerby , Sabel Michael , Marentette Lawrence , Cimmino Vincent , Chang Alfred , Yildiz Mehmet 
Published: October 2019

Abstract

Introduction
Follicular lymphoma (FL) constitutes the second most common non-Hodgkin’s lymphoma in the Western world. FL carries characteristic recurrent structural genomic aberrations. However despite recent advances, knowledge regarding the coding genome in FL is still evolving and is currently incomplete.

Methods
To further our understanding of the genetic basis of follicular lymphoma (FL), we used solution exon capture of sheared and processed genomic DNA isolated from FACS-sorted lymphomatous B-cells and paired CD3+ T-cells isolated from twenty three cases of FL and one case of DLBCL (which was transformed from prior FL), followed by paired-end (96-101 base pair read length per side) massively parallel sequencing. The sequence data were characterized by a mean depth of coverage of 41, and 90% of bases in the target region were covered by at least 10 reads. Bioinformatics pipelines developed by our bioinformatics core served as the primary data source to nominate candidate mutated genes in downstream data analysis.

Results
The bioinformatics pipeline nominated 711 distinct candidate mutations in 24 FL cases. Sanger sequence validation confirmed 39 recurrently (≥ 2 FL cases) mutated genes. Genes with confirmed mutations in ≥ 2 FL cases in the discovery cohort were subsequently selectively expanded into a combined FL validation cohort of 114 cases. In addition to frequent mutations in MLL2, CREBBP, BCL2, TNFRSF14, EZH2, OCT2, ARID1A, IRF8 and MEF2B, we here report novel mutations in STAT6 in FL. STAT6 mutations were identified in 11% (12/114) of FL and predominantly affected the DNA binding domain (DBD; comprising STAT6 amino acids 268-430). Two FL cases each carried two distinct STAT6 mutations, presumably targeting both alleles. Of interest, the majority of FL-associated STAT6 mutations affected a single amino acid codon (codon 419), resulting in the STAT6 mutants p.419D>D/G or p.419D>D/H. These FL-associated STAT6 mutations are distinct from mutations previously described in primary mediastinal B-cell lymphoma (PMBCL).
Given the involvement of STAT6 in signal transduction pathways activated by multiple cell surface receptors, as well as the recently described involvement of STAT6 in antiviral innate immunity (involving an interaction between the STAT6 DBD and the protein STING), we are currently exploring functional consequences of the novel STAT6 mutations in FL and cell line models.

Conclusion
We report identification of somatic mutations in STAT6 in 11% of FL. These mutations predominantly affected the STAT6 DNA binding domain. We identify a novel STAT6 mutation hotspot in STAT6 codon 419 (p.419D>D/G or p.419D>D/H).

Disclosures:
Lebovic: Genentech: Speakers Bureau; Allos/Spectrum: Speakers Bureau; Celgene: Speakers Bureau; Onyx: Speakers Bureau.