bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fap1 Controls Beta-Catenin Activity in a Gsk3-Beta Dependent Manner in Chronic Myeloid Leukemia.
Authors: Huang Weiqi , Bei Ling , Eklund Elizabeth A. 
Published: October 2019

Abstract
Abstract 2404
Fap1 (Fas-associated phosphatase 1) interacts with the C-terminus of multiple proteins via a PDZ-type protein-protein interaction domain. Interaction of Fap1 with Fas results in Fas-de-phosphorylation and inhibits Fas-induced apoptosis. In previous studies, we determined that PTPN13, the gene encoding Fap1, is a target gene for the interferon consensus sequence binding protein (referred to as Icsbp or Irf8). Icsbp functions as a leukemia suppressor in chronic myeloid leukemia (CML), and we previously demonstrated Icsbp/Fap1-dependent apoptosis resistance in cells expressing the CML-associated oncoprotein, Bcr-abl. Another known interaction partner for Fap1 is the adenomatous polyposis coli protein (Apc). However, the functional significance of this interaction has not been previously explored. In the current stuides, we investigate whether interaction between Fap1 and Apc impacts the pathogenesis of CML. Apc is involved in assembly of a multi-protein complex that includes Apc, Axin, Gsk3-beta and beta-catenin. Association of these proteins results in serine/threonine phosphorylation (pS/T) of beta-catenin by Gsk3-beta. pS/T beta-catenin is released from the complex and degraded by the proteasome. Previous studies demonstrated that pS/T of beta-catenin, and consequent beta-catenin degradation, is impaired in CML, but the mechanism for this impairment is unknown in most cases. We find that Icsbp-dependent increase in Fap1 results in increased interaction between Fap1 and Apc in Bcr-abl+ myeloid progenitor cells. We also find that increased Fap1/Apc interaction is associated with inactivation (de-phosphorylation) of Gsk3-beta and a decrease in inhibitory pS/T of βcatenin. We find that this results in increased beta-catenin protein and activity. We also find that Fap1 co-immuno-precipitates with Apc, Axin, Gsk3-beta and beta-catenin. This result suggests the possibility that Gsk3-beta is a substrate for Fap1 protein tyrosine phosphatase activity. Consistent with this hypothesis, we find that Fap1 is able to dephosphorylate Gsk3-beta in vitro. We tested the functional significance of this observation in experiments using an activated form of Gsk3-beta that cannot be dephosphorylated at a key residue (Y216D-Gsk3-beta). We find that expression of this Gsk3-beta mutant partly reverses the effect of Bcr-abl and Fap1 on beta-catenin protein stability and activity. Increased beta-catenin activity is a characteristic of leukemia stem cells (LSC) in CML, and increasing beta-catenin activity in the bone marrow is associated with poor prognosis in CML. Therefore, these studies identify a novel pathway that regulates events significant to CML pathogenesis. These studies also identify a role for Apc in the pathogenesis of leukemia, and another mechanism for the leukemia suppressor effect of Icsbp.

Disclosures:
No relevant conflicts of interest to declare.
Sleeping Beauty Transposon Insertional Mutagenesis Identifies Drivers of Erythroid Leukemia in Mice.
Authors: Loeb Keith R. , Hughes Bridget T , Fissel Brian M. , Choi Yongiae E. , Pogosov Galina , Knoblaugh Sue E , Wood Brent L. , Clurman Bruce E. 
Published: October 2019

Abstract
Abstract 2407

Background and Methods:
Cyclin E-Cdk2 is a cell cycle regulator that promotes the G1-S transition by phosphorylating proteins involved in cell division and genome duplication. Cyclin E activity is frequently increased in many cancers, usually as a result of deregulation of mechanisms that control cyclin E protein levels and activity. Cyclin E T74A/T393A mice, in which cyclin E has been rendered resistant to degradation by the Fbw7 ubiquitin ligase by ablating two phosphorylation sites, exhibit enhanced epithelial proliferation and erythroid hyperplasia with inefficient erythropoiesis and macrocytic anemia. However, despite this widespread hyperproliferation, cyclin E T74A/T393A mice do not develop spontaneous tumors. To accelerate tumorigenesis and identify cooperating oncogenes in cyclin E T74A/T393A mice that complement the block in erythroid differentiation, we performed an insertional mutagenesis screen using the Sleeping Beauty (SB) transposon system.

Results:
Expression of SB by the inducible Mx-Cre caused rapid leukemiagenesis within 9–15 weeks post poly (I/C) injection in both control and cyclin E T74AT393A mice. The majority of tumors were T-cell lymphomas/leukemias and erythroid leukemias. Surprisingly, both wild type and cyclin E T74AT393A mice developed erythroid leukemias at similar frequencies. The erythroleukemia were composed of homogeneous populations of immature proerythroblasts akin to pure erythroleukemia in humans. Immunophenotypic profiling showed dim CD45 with variable CD117 (c-kit) and CD71 (transferrin receptor) expression. Preliminary sequencing of 29 erythroid leukemias identified 27 common insertion sites (CIS), including several genes associated with AML (Flt3, Erg and Ets1) as well as erythropoiesis (Fli-1 and Epo), highlighting the specificity of the SB-induced mutations for myeloid and erythroid leukemias. Preliminary analyses have also revealed preferential gCISs in erythroid leukemias isolated from cyclin E T74AT393A mice.

Conclusion:
We conclude that the Mx-Cre SB mutagenesis system provides a unique and rapid murine model of pure erythroid leukemia. We have identified a set of genes that promote erythroleukemia in mice may represent potential therapeutic targets for human erythroid and myeloid leukemias.

Disclosures:
No relevant conflicts of interest to declare.
High Musashi-2 Expression Is an Unfavorable Prognostic Factor in Adult B-Cell Acute Lymphoblastic Leukemia.
Authors: Mu Qitian , Wang Yungui , Chen Bing , Qian Wenbin , Meng Haitao , Tong Hongyan , Chen Feifei , Ma Qiuling , Ni Wangmao , Chen Sai-Juan , Jin Jie 
Published: October 2019

Abstract
Abstract 2508

Background:
Musashi-2 (MSI2) can inhibit translation of the mRNA encoding NUMB, the NOTCH signaling inhibitor, and play a vital role in the maintenance of hematopoietic stem cells. Musashi-1 (MSI1), another isoform of Musashi family, positively regulates NOTCH1 expression. Recent studies have demonstrated that MSI2 expression was up-regulated during chronic myelogenous leukemia (CML) progression and its high expression was associated with poor outcome of CML and acute myeloid leukemia (AML). However, the prognostic significance of MSI2 expression and the relation between MSI2 and NOTCH1 expressions in acute lymphoblastic leukemia (ALL) remain unknown.

Methods:
In this study, real-time PCR was used to measured expression levels of MSI2 and NOTCH1 signaling related genes in 116 B-ALL and 24 T-ALL patients, in whom more than 70% leukemic cells were morphologically detected in bone morrow (BM) at diagnosis. Clinical and Molecular data in relation with MSI2 expression level were analyzed.

Results:
In our B-ALL cohort, although MSI2 expression was not associated with gender, age, white blood cell (WBC), t(9;22)/BCR-ABL and IK6 variant of IKZF1, patients with high MSI2 expression had inferior overall survival (OS) (P=0.007) and event free survival (EFS) (P=0.002) than patients with low MSI2 expression. Multivariate analysis showed that high MSI2 expression was an independently prognosic factor in OS (P=0.001, HR=2.641, 95%CI, 1.494–4.668), EFS (P<0.001, HR=2.562, 95%CI, 1.513–4.218) and RFS (P=0.043, HR=2.057, 95%CI, 1.023–4.137). Moreover, MSI2 expression level had a positive correlation with that of NOTCH1 (P<0.001), but not c-MYC (P=0.432) or HES1 (P=0.509). Similarly, NOTCH1 expression level in patients with high MSI2 expression (0.98, range 0.02 to 13.58) was significantly higher than that in patients with low MSI2 expression (0.42, range 0.01 to 3.55, P=0.001), but c-MYC and HES1 expression levels between patients with high MSI2 expression and low MSI2 expression showed no significant differences (both P>0.05).

Conclusion:
Our data suggests MSI2 high expression can indicate poor prognosis in adult B-ALL accompanying with elevated NOTCH1 expression but not activating NOTCH1 downstream pathway.

Disclosures:
No relevant conflicts of interest to declare.
Genome-Wide Genotype-Based Risk Model for Survival in Acute Myeloid Leukemia Patients with Normal Karyotype.
Authors: Kim Hyeoung-Joon , Choi Hangseok , Kim Yeo-Kyeoung , Sohn Sang Kyun , Moon Joon Ho , Kim TaeHyung , Zhang Zhaolei , Kim Dennis Dong Hwan 
Published: October 2019

Abstract
Abstract 2526

Introduction:
Single nucleotide polymorphism (SNP) is an inter-individual genetic variation which could explain inter-individual differences of response/survival to chemotherapy. The present study was attempted to build up risk model of survival for acute myeloid leukemia (AML) patients with normal karyotype (AML-NK).

Methods and materials:
A total of 247 patients with AML-NK was included into the study. Genome-wide SNP array (Affymetrix SNP-array 6.0) was performed in the discovery set (n=118), and genotypes were analyzed for overall survival (OS). After identifying significant SNPs for OS in single SNP analyses, risk model was constructed. Replication was performed in an independent validation cohort (n=129).

Results:
Out of 632,957 autosomal SNPs meeting genotype data filtration criteria, a total of 82 SNPs were selected and passed into the next step of validation in an independent cohort. In the risk model generation step, finally 4 SNPs (rs2826063, rs12791420, rs11623492 and rs2575369) were meeting stringent criteria for SNP selection as follows: 1) p-value < 0.10 from Cox proportional hazards regression model in adjustment with age and WBC counts at diagnosis; 2) minor allele frequency > 0.05; 3) call rate > 95.0%; 4) high linkage disequilibrium r2 < 0.8. These 4 SNPs were introduced into the risk model, and patients was grouped into 2 groups according to the number of deleterious variables including 4 SNPs and 2 clinical variables (i.e. age and WBC counts at presentation): risk score 0–2 as a low risk (n=80) and 3–6 as a high risk (n=38). The risk model could stratify the patients according to their OS in discovery (p=1.053656•10−4) and in validation set (p=5.38206•10−3). The risk model showed a higher AUC than those being incorporated only clinical or only 4 SNPs, suggesting improved prognostic stratification power of combined model.

Conclusion:
Genome-wide SNP based risk model obtained from 247 patients with AML-NK can identify high risk group of patients with poor survival using genome wide SNP data. (Clinicaltrials.govIdentifier:NCT01066338)

Disclosures:
No relevant conflicts of interest to declare.
Induction of NK Cell Reactivity in Breast Cancer by Fc-Engineered NKG2D-Ig Fusion Proteins
Authors: Raab Stefanie , Steinbacher Julia , Grosse-Hovest Ludger , Schmiedel Benjamin J , Steinle Alexander , Kanz Lothar , Salih Helmut R 
Published: October 2019

Abstract
Abstract 253
NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. Their capability to mediate Fc-receptor dependent effector functions like antibody dependent cellular cytotoxicity (ADCC) largely contributes to the clinical success of anti-tumor antibodies like Herceptin (Trastuzumab®), which is approved for treatment of breast cancer displaying HER2/neu-overexpression. Notably, only about 20% of breast cancer patients show overexpression of HER2/neu. Moreover, this antigen is also expressed on healthy cells, and application of Herceptin is associated with side effects. In contrast, ligands of the activating immunoreceptor NKG2D (NKG2DL) are widely expressed on malignant cells, but generally absent on healthy tissues. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as tumor-antigens for Fc-optimized NKG2D-Ig fusion proteins targeting breast cancer cells for NK cell ADCC and IFN-γ production. NKG2D-Ig fusion proteins with distinct modifications in their Fc portion were generated by amino acid exchange as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT) or Herceptin, our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcγRIIIa receptor (CD16), respectively. This resulted in lacking (NKG2D-Fc-KO) or highly enhanced (NKG2D-Fc-ADCC) NK cell activation. In cultures of NK cells and breast cancer cells, NKG2D-Fc-KO significantly reduced NK cell reactivity due to blockade of NKG2DL-mediated activating signals, while NKG2D-Fc-WT substantially enhanced NK reactivity by induction of ADCC and cytokine production. Notably, the effect of our NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT and, in case of HER2/neu low targets, also that of Herceptin, resulting in potently enhanced NK anti-tumor reactivity. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing cancer cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to its highly increased affinity to CD16 NKG2D-Fc-ADCC potently enhances NK cell reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, NKG2D-Fc-ADCC may thus constitute an attractive means for immunotherapy, especially of HER2/neu-low or -negative breast cancer.

Disclosures:
No relevant conflicts of interest to declare.
Human Natural Killer (NK) Cells: Differential Expression of Phosphatase and Tensin Homologue Deleted On Chromosome Ten (PTEN) During NK Cell Development Regulates Its Cytolytic Activity Against Leukemic Target Cells
Authors: Briercheck Edward L. , Trotta Rossana , Cole Jordan P. , Cole Tyler D. , Hartlage Alex S. , Martin Chelsea K. , Leone Gustavo , Caligiuri Michael A. 
Published: October 2019

Abstract
Abstract 254
Human NK cells are CD56(+)CD3(-) large granular lymphocytes characterized by the ability to directly kill virally infected or malignantly transformed cells. Five stages of human NK cell development can be identified in secondary lymphoid tissue. Stage 4 NK cells are immature CD56bright and have poor cytolytic activity against sensitive leukemic target cells at rest while stage 5 NK cells are mature CD56dim and have potent cytolytic activity against the same leukemic cells at rest. Both stage 4 CD56bright and stage 5 CD56dim NK cells can be found circulating in blood. We sought to determine the mechanism responsible for this different cytolytic activity by exploring changes in gene expression between CD56bright NK cells and CD56dim NK cells. We first observed that CD56bright NK cells expressed ∼5 fold greater amounts of PTEN protein over CD56dim NK cells by western blot (n=5, p < 0.04). Given that PTEN is a lipid phosphatase that opposes the PI3K/AKT pathway, we hypothesized that it may negatively regulate NK cell cytolytic activity. In order to test this, we used lentiviral infection to overexpress PTEN in the human NK cell line NK-92. Relative to cells infected with an empty lentiviral vector, NK-92 cells with overexpression of PTEN showed decreased cytotoxicity against sensitive leukemic target cells by at least 25% at all effecter:target ratios (n=4, p < 0.02). Next, we infected primary human NK cells with the same vector and showed an approximately 30% reduction in degranulation of cytolytic mediators as determined by CD107a mean fluorescent intensity (M.F.I.) when cultured with sensitive leukemic target cells (n=4, p<.08). Consistent with this, murine NK cells were isolated from FVB mice with a heterozygous germline deficiency in Pten (Pten+/−) and showed at least a 25% increase in cytotoxicity against sensitive lymphoma target cells (n=4, p<.0001). At the molecular level, when NK-92 cells overexpressing PTEN were bound to their sensitive leukemic target cells, they showed decreased activation along the AKT and ERK1/2 signaling pathways, which are known as positive regulators of NK cell cytotoxicity. Collectively, we identify a functional role for the tumor suppressor PTEN in normal human NK cell development. We demonstrate differential expression of PTEN in immature and mature human NK cells, show that it is a negative regulator of NK cell cytolytic activity, and suggest that this differential expression may in fact account for the difference in baseline cytolytic activity found in the CD56bright and CD56dim human NK cell subsets, possibly via the AKT and ERK1/2 pathways.

Disclosures:
No relevant conflicts of interest to declare.
Testing in Vitro Sensitivity of Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia.
Authors: Groth-Pedersen Line , Valentin Rebecca , Schmiegelow Kjeld 
Published: October 2019

Abstract
Abstract 2543
Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in children and adolescents. Although improved risk grouping, anti-cancer treatment, and supportive care have resulted in survival rates above 80 %, 15–20 % of patients experience a relapse, which is associated with an inferior prognosis. Relapse is caused by persistence of minimal residual disease (MRD) primarily in the bone marrow during chemotherapy, and methods for identification of drugs that are capable of eliminating the MRD cell population is necessary, but not currently available. In vitro sensitivity testing has been carried out at the time of diagnosis and shows predictive value of treatment outcome and correlation to MRD levels. However, the in vitro sensitivity profiles have not been applicable for treatment stratification, since the vast majority of leukemic cells present at diagnosis are rather chemosensitive, as opposed to the MRD population. Thus, the more sensitive clones mask the more resistant ones. We hypothesized that in vitro sensitivity testing of the more resistant MRD cells remaining in bone marrow after the induction therapy could help to stratify patients to individualized chemotherapy.
The greatest challenge in carrying out in vitro sensitivity testing on MRD cell populations is the low number of leukemic cells available, i.e. 0.1–5%. Traditionally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flourometric microculture cytotoxicity assays have been used for in vitro sensitivity testing. They, however, need several million cells for in vitro sensitivity testing of 3 drugs. We aimed at developing a cell death assay applicable on small ALL cell populations. We show that a flow cytometry based assay using annexin V (reflecting exposure of phophatidylserine on the outer cell membrane during early apoptosis) and 7-Aminoactinomycin D (reflecting cell membrane rupture during late apoptosis) staining is a reliable method for evaluating early and late stages of cell death in small ALL cell populations. Both in ALL cell lines REH (B-cell precursor (BCP) ALL, t(12;21), Jurkat (t-ALL), RS4;11 (BCP ALL, t(4;11)) and Nalm-6 (BCP ALL, t(5;12)) and in primary ALL cell samples the results are highly reproducible and show the same relative sensitivity profile as when a large number of cells are used. This cell death assay only demands a total of 20.000 cells to determine the in vitro sensitivity for 3 drugs run in triplicates at 4 concentrations. This assay is thus applicable for sorted MRD populations accounting for 0.1% of the mononuclear cells in the bone marrow.

Disclosures:
No relevant conflicts of interest to declare.
Induction of NK Cell Reactivity Against AML Cells by Fc-Engineered RANK-Ig Fusion Proteins.
Authors: Nuebling Tina , Schmiedel Benjamin J , Azuma Miyuki , Schneider Pascal , Grosse-Hovest Ludger , Kanz Lothar , Salih Helmut R 
Published: October 2019

Abstract
Abstract 2625
NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. A clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of anti-tumor antibodies. In acute myeloid leukemia (AML) NK cells largely contribute to the therapeutic efficacy allogenic stem cell transplantation (SCT). Recently we demonstrated that AML cells functionally express the TNF family member RANK ligand (RANKL) which impairs NK cell anti-leukemia reactivity (Schmiedel et al., ASH annual meeting 2011). Here we developed a strategy to combine blocking of the NK inhibitory effects of RANKL with targeting of AML cells for NK cell ADCC. To this end we generated fusion proteins consisting of the extracellular domain of RANK and a human IgG1 Fc part that was modified by amino acid exchange. Compared to wild type RANK-Fc fusion protein (RANK-Fc-WT), our mutant RANK-Fc-ADCC (S239D/I332E) displayed highly enhanced affinity to FcγRIIIa (CD16) on NK cells. Primary AML cells expressed substantial levels of RANKL in 53 of 78 (68%) investigated patient cases, and our RANK-Ig fusion proteins bound to AML cells in a target antigen-specific manner. Treatment with both RANK-Fc-WT and RANK-Fc-ADCC clearly reduced the release of RANKL-induced immunomodulatory factors like TNF, IL-6, IL-8 and IL-10 by AML cells. When the effects of the fusion proteins on NK cell ADCC were studied we found that treatment with RANK-Fc-WT only slightly enhanced NK cell reactivity against RANKL-positive patient AML cells. However, RANK-Fc-ADCC potently induced NK cell ADCC and cytokine production in response to AML targets in a target antigen-dependent manner due to the functional properties of its engineered Fc moiety. Taken together, our Fc-engineered RANK-Fc-ADCC fusion protein may serve to modulate the cytokine milieu involved in AML pathophysiology and target RANKL-expressing leukemia cells for NK anti-tumor reactivity. Thus, RANK-Fc-ADCC constitutes an attractive immunotherapeutic means for the treatment of AML, e.g. for elimination of minimal residual disease after conventional therapy including SCT.

Disclosures:
No relevant conflicts of interest to declare.