bluuebunny/crossref_metadata_2025_split huggingface dataset:
Increased PKR Expression Promotes MDS Evolution to Acute Leukemia in the NHD13 Mouse Model
Authors: Byrne Michael T. , Bennett Richard L , Cheng Xiaodong , Carter Christopher M , May W. Stratford 
Published: October 2019

Abstract
Protein kinase R (PKR) is an interferon-inducible serine-threonine kinase that is activated in response to inflammatory cytokines and cellular stressors. When over-expressed in murine hematopoietic tissues, PKR results in peripheral blood (PB) and bone marrow (BM) features consistent with myelodysplastic syndrome (MDS). We hypothesized that PKR can cooperate with MDS driver mutations to promote MDS and acute leukemia. To test this, we crossed the Nup98-HoxD13 (NHD13) transgenic model of MDS with our TgPKR and PKR-KO mice to generate NHD13-TgPKR and NHD13-KO mice. These mice were aged alongside NHD13 and wild type (WT) controls. Using RT-PCR, we evaluated PKR expression in NHD13 mice. 9/13 NHD13 mice developed acute leukemia with leukocytosis and/or increased BM blasts whereas 4/13 had MDS only without PB or BM evidence of acute leukemia. Non-leukemic NHD13 mice had PKR expression levels below WT controls (RQ: 0.412 to 0.744). Alternatively, PKR expression in the leukemic group was significantly higher than WT controls and non-leukemic mice (RQ: 3.3 vs. 1.0 and 0.5, respectively) and was associated with shortened survival (mean: 304 vs. 345 days; p < 0.05). We then compared NHD13-TgPKR vs. NHD13-KO mice and discovered significantly lower white blood cells (WBC) counts in the NHD13-TgPKR group (2.7 vs. 5.6 x 106 cells/ml, p <0.05; n=8/group) with significant differences in lymphocytes and neutrophils. With aging, NHD13-KO mice lived significantly longer than NHD13 mice (352.1 vs. 314.3 days, p <0.05) and NHD13-TgPKR mice (273.9 days, p <0.05). There were minor differences in the frequency of acute leukemia between the groups (NHD13-TgPKR: 58.3% vs. NHD13-KO: 41.6%). Next, we evaluated the frequency of somatic mutations in PB reticulocytes using the PIG-A assay that we have previously validated for measuring genomic instability of BM derived cells. We discovered an increased frequency of somatic mutations, indicative of greater genomic instability of the MDS HSPC, in 9-month-old NHD13-TgPKR (3.64%) vs. WT (<0.01%), NHD13 (0.26%), and NHD13-KO (0.18%) mice (p <0.05). Finally, the pharmacologic inhibition of PKR in NHD13 mice improved the WBC count (1.6 vs. 3.6 x 106 cells/ml, p <0.05) and significantly reduced genomic instability (0.11 vs. 0.39%, p <0.05). Collectively, these data indicate that increased PKR expression is associated with the development of acute leukemia and shortened survival in the NHD13 mouse model. Further, NHD13-TgPKR mice, when compared to NHD13-KO mice, have a more aggressive MDS phenotype with deeper PB cytopenias, increased BM blasts, and shortened survival resulting from increased genomic instability. These data indicate that PKR can cooperate with MDS driver mutations and suggests that PKR may be a promising target for future MDS therapies, particularly in patients with high-level PKR expression.

Disclosures
No relevant conflicts of interest to declare.
Mirs-138 and -424 Control Palmitoylation-Dependent CD95-Mediated Cell Death By Targeting Acyl Protein Thioesterases 1 and 2 in Chronic Lymphocytic Leukemia
Authors: Berg Valeska , Rusch Marion , Vartak Nachiket , Juengst Christian , Schauss Astrid , Waldmann Herbert , Hedberg Christian , Pallasch Christian P. , Bastiaens Philippe , Hallek Michael , Wendtner Clemens-Martin , Frenzel Lukas P. 
Published: October 2019

Abstract
Introduction: Resistance towards CD95-mediated apoptosis is a hallmark of many different malignancies, like it is known from primary chronic lymphocytic leukemia (CLL) cells. Moreover, apoptosis mediated through CD95 is an essential mechanism to eliminate e.g. auto-reactive or virally infected cells. However, its mode of action is still not fully understood. Recently, it could be shown that palmitoylation of CD95 can influence its signaling properties. Nevertheless, the role and regulation of palmitoylated CD95 still needs to be determined.
Methods and results: Previously, we could show that miR-138 and -424 are down-regulated in CLL cells. By applying luciferase reporter assays, mutations of the binding sites qRT-PCR and immunoblots after transfection of both miRs, we identified two new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby are significantly over-expressed in CLL cells. Interestingly, our data reveal that expression of APTs is already controlled by miRs on mRNA level. This way APT1 is regulated by miR-138 and expression of APT2 is controlled by miR-424. So far, APTs are the only enzymes known to promote de-palmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared to normal B cells as we determined by click-chemistry, which is a non-radioactive method to determine palmitoylated proteins. Importantly, via acyl-biotin exchange assays with subsequent immunoprecipitation of CD95 and fluorescence lifetime imaging microscopy (FLIM) to Foerster resonance energy transfer (FRET) in living cells we identified APTs to directly interact with CD95 to promote de-palmitoylation, thus impairing apoptosis mediated through CD95. As proof of concept APTs were inhibited specifically by siRNAs, miRs-138/-424 or our pharmacological inhibitor Palmostatin B. Thereby we could restore CD95-mediated apoptosis in CLL cells and other cancers, pointing to a central regulatory role of APTs in CD95 apoptosis.
Conclusion: The identification of the de-palmitoylation reaction of CD95 by APTs as a miRNA target provides a novel molecular mechanism how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel post-translational modification in CLL. In light of global palmitoylome studies, which show that potentially palmitoylated proteins are involved in all central cellular processes, such as protein transport, survival, migration, apoptosis and B-cell receptor signaling, this emphasizes the importance of palmitoylation and might put it on par with modifications like phosphorylation.

Disclosures
No relevant conflicts of interest to declare.
A Novel in Vitro Three Organ System Assay Identifies a Small Molecule Ubiquitin Analog with in Vivo Activity Against Myeloma
Authors: Vatolin Sergei , Phillips James G , Govindgari Shravya , Grabowski Dale , Parker Yvonne , Lindner Daniel , Cotta Claudiu , Reu Frederic J. 
Published: October 2019

Abstract
Taking a mechanistically unbiased approach to the discovery of new myeloma drugs we developed a three organ system assay that selected anti-myeloma compounds from a primary 30,000 small molecule ATP-based screen in one sandwich setup for tolerability by normal bone marrow, stability towards liver enzymes, and activity in the context of cell barriers, bone marrow stromal support, and short, kidney-clearance-like exposure (fig.1). The most promising compound (CCF642) emerging from this test had IC50s around 500 nM in all 8 MM cell lines tested including a proteasome inhibitor refractory line while the IC50 was not reached in 5 healthy bone marrow samples at doses up to 6750 nM. Similar sensitivity as in MM was seen in 7 lymphoma cell lines. Investigation of its mechanism of action revealed that CCF642 immediately stimulates protein ubiquitination which is followed by degradation of major myeloma survival factors (c-MYC, IRF4, NF-κB) and apoptosis, while normal bone marrow cells increase ubiquitination to a much lesser degree and only transiently without undergoing cell death. Experiments with an active biotinylated analog showed that it preferentially enters myeloma cells with cytoplasmic accumulation and over 10-fold lower entry into normal bone marrow. Immunoblots revealed that it becomes covalently attached to proteins in a time- and distribution pattern that parallels ubiquitination responses. In vitro, it bound to ubiquitin activating enzyme UBA1 at the active site cysteine in a similarly stable and reversible way as ubiquitin. Selective alkylation of cysteine sulfhydryl groups inhibited binding of biotinylated CCF642 to UBA1, while addition of ATP and ubiquitin led to its dissociation from UBA1. CCF642 may therefore at least in part act as a small molecule ubiquitin analog. Accordingly CCF642 binding to target proteins of UBA1 containing HeLa fraction II was increased by addition of ATP and blocked in the presence of EDTA. Injected twice a week IP at 30mg/kg it suppressed systemically engrafted luciferase expressing 5TGM1 mouse myeloma cells in syngeneic mice comparable to bortezomib given at the MTD for this mouse strain (0.5mg/kg SC twice a week) with equal prolongation of survival. Results support use of our sandwich assay to select myeloma drug candidates for in vivo testing and reveal a new UBA1 interacting small molecule ubiquitination enhancer with promise for clinical translation.
Figure 1: Sandwich Three Organ System Assay Figure 1:. Sandwich Three Organ System Assay

Disclosures
No relevant conflicts of interest to declare.
A Novel ICAM-1/CD54 Antibody Identified By Phage Display with Potent Pre-Clinical Anti-Myeloma Activity
Authors: Klausz Katja , Burger Renate , Kellner Christian , Peipp Matthias , Gramatzki Martin 
Published: October 2019

Abstract
Introduction: Despite novel agents in multiple myeloma (MM) a substantial number of patients is still in need of better disease control. Monoclonal antibodies provide therapeutic options in other tumor entities but in MM no antibody has been approved yet. Two antibodies currently evaluated namely Daratumumab (CD38) and Elotuzumab (CD319), are ahead and achieved phase 3 clinical trials, but other antigens on myeloma cells could also be promising targets. Here, a cell-based phage display screening strategy is presented that identified a novel intercellular adhesion molecule-1 (ICAM-1)/CD54 antibody suitable for myeloma immunotherapy.
Methods: Synthetic human single-chain fragment variable (scFv) phage display libraries were pre-absorbed with granulocytes and T lymphocytes of healthy individuals and subsequently panned with plasma cell lines INA-6 and JK-6L. Phage binding characteristics were evaluated by cellular ELISA and flow cytometry. The coding sequence of the leading candidate MSH-TP15 was used to generate scFv-Fc fusion proteins and fully human IgG1 antibodies with wild type or modified Fc domains for functional in vitro and in vivo testing.
Results: Panning of human phage display libraries with a newly established subtractive cellular screening approach resulted in phages with preferential binding to MM cells. Based on its reactivity with MM cell lines and patient-derived CD138+ malignant plasma cells from bone marrow, pleural effusion and peripheral blood, the leading candidate MSH-TP15 was selected. Notably, MSH-TP15 showed no or only marginal binding to B and T lymphocytes, NK cells, monocytes and granulocytes of healthy individuals and MM patients. Recent studies identified ICAM-1/CD54 as the antigen recognized by MSH-TP15. The antibody alone was not capable to significantly inhibit growth of plasma cell lines in vitro, however induced antibody-dependent cell-mediated cytotoxicity (ADCC), an important killing mechanism of many therapeutic antibodies. An Fc-engineered antibody variant of MSH-TP15 triggered dose-dependent ADCC against INA-6, L363, RPMI-8226, U266 and MM1.S plasma cell lines and freshly isolated tumor cells using blood mononuclear cells and NK cells of healthy donors as effector cells. In contrast, complement-dependent cytotoxicity was not induced. Importantly, in vivo studies revealed that MSH-TP15 completely prevented tumor engraftment in the INA-6 myeloma xenograft model.
Conclusions: The cell-based screening of phage display libraries led to the identification of a novel ICAM-1/CD54 antibody that is effective in antibody-mediated cellular cytotoxicity of MM cells in vitro and completely prevented myeloma tumor engraftment in vivo. The relevant mechanisms of action of MSH-TP15 will be defined with different antibody variants and in vivo models to further evaluate its potential for an antibody-based treatment approach of MM.

Disclosures
No relevant conflicts of interest to declare.
Novel Series of ALK2 Inhibitors That Alter Hepcidin Expression As Potential Therapeutics for Anemia of Chronic Disease
Authors: Kim Wontak , Maughan Kyle S. , Soh Katherine K. , Bearss Jeremiah J. , Bahr Brigham L. , Bearss David J. , Warner Steven L. 
Published: October 2019

Abstract
Anemia of chronic disease (ACD) is an inflammatory cytokine driven disease characterized by hypoferremia despite adequate iron stores. This is largely due to hepcidin, a master regulator of iron homeostasis, which blocks enterocytes from absorbing iron and preventing iron release from macrophages by binding to ferroportin. It is known that bone morphogenetic proteins (BMP) up-regulate hepcidin by activating the SMAD signaling pathway through the activin-like kinase receptor 2 (ALK2). Therefore, ALK2 has emerged as a potential therapeutic target to modulate hepcidin levels and treat ACD.
We have developed a novel series of small molecule ALK2 inhibitors with promising activity in preclinical models of ACD. Using well-established cell-based and animal models of hepcidin signaling and anemia, we optimized and validated the activity of the most promising preclinical lead candidates. These compounds demonstrate significant activity in downregulating hepcidin expression in BMP-induced cell culture studies at concentrations of 100 nM or lower. Importantly, this hepcidin lowering activity was observed at concentrations that exhibited no cytotoxicity suggesting the compounds have a clean selectivity profile. The compounds also demonstrated remarkable activity in animal models of anemia, including an acute model induced by the administration of turpentine oil and a more chronic model induced by tumor formation and growth. Treatment with the lead candidates completely reversed the induction of hepcidin expression in these models and also decreased the symptoms of anemia as measured by serum iron and red blood cell levels. From these data, we have nominated a candidate to advance into IND-enabling studies that has favorable drug-like properties. We anticipate a clinical development strategy that focuses on anemia of cancer with subsequent expansion into anemia associated more broadly with other inflammatory and chronic diseases

Disclosures
Kim: Tolero Pharmaceuticals: Employment. Maughan:Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Bahr:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.
An Inflammatory Pathway Mediating Rapid Hepcidin-Independent Hypoferremia
Authors: Muckenthaler Martina U. , Guida Claudia , Altamura Sandro , Klein Felix A. , Galy Bruno , Boutros Michael , Ulmer Arthur J. , Hentze Matthias W. 
Published: October 2019

Abstract
Hypoferremia represents an innate immune response to infection and inflammation sequestering iron from pathogens. The iron-hormone hepcidin is induced by such stimuli, causing degradation of the iron exporter ferroportin (Fpn) and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here we report the discovery of a fast, hepcidin-independent hypoferremia pathway. Stimulation of the toll-like receptor (TLR) 2 and TLR6 triggers profound decreases in Fpn mRNA and protein expression in bone marrow-derived macrophages, liver and spleen of mice without changing hepcidin expression. Furthermore, C326S Fpn knock-in mice with a disrupted hepcidin/Fpn regulatory circuitry respond to injection of the TLR2/6 ligand FSL1 by Fpn down regulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia, uncovering a rapid and potent inflammatory response pathway.

Disclosures
No relevant conflicts of interest to declare.
Predisposition to Burkitt Lymphoma in Williams-Beuren Syndrome
Authors: Guenat David , Rizzari Carmelo , Lundin Catarina , Borg Christophe , Soulier Jean , Rohrlich Pierre-Simon 
Published: October 2019

Abstract
Williams-Beuren Syndrome (WBS) is a multisystem disorder caused by a 1.5 Mb hemizygous deletion at chromosome 7q11.23. Here, we report two novel cases of Burkitt lymphoma in WBS children. With the addition of these latest two case reports the number of pediatric cancer reported in WBS has reached 11 cases. Strikingly, 8 (73%) of them were Burkitt lymphoma. In addition, a sporadic Burkitt lymphoma in a third child without WBS but carrying a somatic deletion of the WBS critical region was investigated. Array-based Comparative Genomic Hybridization and Next Generation Sequencing were used to analyze the DNA isolated from normal tissues and lymphoma cells of the 3 patients. Microsatellite instability was also investigated. The constitutional hemizygous deletion observed in the normal DNA of the 2 WBS patients corresponded to the typical WBS critical region and there was no evidence of homozygous deletion or mutation at 7q11.23 in the tumor DNA. Remarkably, the somatic deletion observed in the sporadic NHL was similar to the one observed in WBS patients. Microsatellites were stable in the 3 patients. A number of genes mapping to the WBS critical region are involved in DNA repair (PMS2L, BAZ1B, RFC2, GTF2I family genes). Haploinsufficiency of one or of a combination of these genes could be involved in the predisposition to Burkitt lymphoma in patients with WBS. More generally, this observation illustrates the putative role of genes located at 7q11.23 in lymphomagenesis, thus pointing to potentially new targets for anti-cancer therapies.

Disclosures
No relevant conflicts of interest to declare.
PARP Inhibitor Selectively Induces Cell Death in E2A-Hlf Positive Leukemia
Authors: Takagi Masatoshi , Piao Jiuhua , Kamiya Takahiro , Masutani Mitsuko , Mizutani Shuki 
Published: October 2019

Abstract
Background: Defects in homologous recombination repair (HRR) have long been known to contribute to genomic instability leading to tumor development. Poly (ADP-ribose) polymerase (PARP) exerts various cell biological effects, such as maintenance of genomic stability, energy metabolism and cell death. PARP is indispensable in DNA repair machinery, especially in base excision repair (BER). PARP inhibition convert DNA double strand breaks from DNA single strand breaks induced by alkylating agents. These DNA double strand breaks can be repaired by HRR. Therefore, PARP inhibitor induces synthetic lethality in HRR defective cancer cells. Such lethality was successfully shown in BRCA1 or 2 mutated breast cancers. However, only a limited study has been performed other than breast cancers. Some tumors including hematological malignancies are defective in HRR function leading to a possibility to be sensitized to PARP inhibitor.
Methods: Sensitivity to PARP inhibitor was screened using 28 leukemia cell lines. HRR activity was measured by DR-GFP HRR assay. Expression of proteins involves HRR was evaluated by cDNA microarray analysis and western blotting.
Results: E2A-HLF positive leukemia showed susceptibility to PARP inhibitor. This experiment suggests that expression of E2A-HLF chimeric messenger RNA sensitize the leukemic cell to PARP inhibitor. To elucidate whether E2A-HLF genuinely sensitize the cell for PARP inhibition, E2A-HLF was transduced into PARP inhibitor resistant Burkitt cell line, Daudi, using retrovirus. Compared with mock infected Daudi cell, E2A-HLF expressed Daudi cell showed increased sensitivity to PARP inhibitor. This experiment suggests that E2A-HLF expressed cell is defective HRR pathway. To test this hypothesis, HRR assay using DR-GFP construct was employed. HRR between the two nonfunctional GFP genes to generate a functional GFP gene can be triggered by transient transfection of the I-SceI expression vector, which introduces a DNA double-strand break (DSB) in one of the two GFP genes. HRR proficiency can be determined by the number of cells expressing the GFP protein. DR-GFP HRR assay exhibited defect of HRR function in E2A-HLF expressed cell. Interestingly, expression of BRCA1 was decreased in E2A-HLF transfected cell, which presumably link with decreased HRR activity.
Conclusions: Increased sensitivity to PARP inhibitor in E2A-HLF positive leukemia was caused by decreased HRR activity by E2A-HLF expression. PARP inhibitor will be a novel therapeutic approach for refractory leukemia, especially with E2A-HLF translocation. While PARP inhibitor monotherapy is an attractive proposition for treating such as HRR defective E2A-HLF expressed leukemia, combination of HRR inhibitor will be a universal strategy for various types of leukemia.

Disclosures
No relevant conflicts of interest to declare.
Egr-1, a Stress Response Transcription Factor and Myeloid Differentiation Primary Response Gene, Behaves As Tumor Suppressor in CML
Authors: Maifrede Silvia , Liebermann Dan , Hoffman Barbara 
Published: October 2019

The transcription factor early growth response 1 (Egr-1) gene was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There’s evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with therapy derived MDS and AML; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). BCR-ABL driven leukemia (Chronic Myelogenous Leukemia [CML]) was chosen as a model system to investigate the role of Egr-1 as a tumor suppressor for leukemia. CML is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. To assess the effect of Egr-1 on BCR-ABL driven leukemia, lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-GFP retrovirus (bone marrow transplantation {BMT}). It was observed that loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Analysis of hematopoietic organs is consistent with loss of Egr-1 accelerating development of CML but not altering the nature of the disease. Cells expressing BCR-ABL that are null for Egr-1 exhibit increased survival and proliferation, which can account for some of the above observations. Substantial evidence is available that loss of Egr1 increases the leukemia initiating cell population, which can account for the more rapid development of disease. An increased population of lineage negative BM cells was observed in Egr-1-/- BCR-ABL recipient mice compared to animals transplanted with WT BCR-ABL BM. Serial BMT has shown that Egr-1-/- BCR-ABL BM has an increased leukemic burden compared to the WT counterpart. Furthermore, data from serial colony transfer experiments confirmed that loss of Egr-1 increases leukemia initiating cells. Preliminary studies on the effect of gain of Egr-1 function are consistent with the tumor suppressor behavior of Egr-1. These data as well as analysis of human CML samples will be presented. Further investigation could result in novel targets for diagnosis, prognosis, and targeted therapeutics, including strategies for activating Egr-1 expression that can be used to treat CML, as well as other leukemic diseases.

Disclosures
No relevant conflicts of interest to declare.