bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient MGMTP140K-Mediated In Vivo Selection and Chemoprotection of Long-Term Repopulating Cells in Nonhuman Primates Following Reduced Intensity Conditioning.
Authors: Beard Brian C , Trobridge Grant D , McCune Jeannine S , Kiem Hans-Peter
Published: October 2019

Abstract 3572

Poster Board III-509

Strategies using gene-modified hematopoietic stem cells to treat various severe hematopoietic diseases, including but not limited to hemoglobinopathies, will likely require high levels of gene marking. Here we have established efficient and stable in vivo selection in nonhuman primates using methylguanine methyltransferase (MGMTP140K). In the macaque (Macaca nemestrina) we were able to increase pre-chemotherapy lentiviral gene marking levels of 11.3% in granulocytes and 15.3% in lymphocytes to a post-chemotherapy gene marking level of 76.9% in granulocytes and 49.0% in lymphocytes. Furthermore, stable increases in gene marking were also observed in red blood cells (RBCs) and platelets (PLTs) with a pre-chemotherapy gene marking level of 5.6% and 6.7%, respectively, and a post-chemotherapy gene marking level of 15.2% and 64.0%, respectively. Importantly, the chemotherapy regimen was well tolerated, and engraftment was polyclonal as determined by analyzing long-term repopulating clones by LAM-PCR. In order to minimize extra-hematopoietic toxicity we have began to test a more clinically applicable conditioning regimen in the macaque model. This reduced intensity conditioning regimen should allow treatment of patients with severe hematopoietic or infectious diseases, who may not tolerate a high dose conditioning regimen. We tested targeted busulfan for conditioning to provide sufficient myelosuppression and to facilitate engraftment of chemoprotected hematopoietic stem cells while minimizing extra-hematopoietic toxicity. Following conditioning with busulfan (4 mg/kg/day for 2 days) and infusion of gene modified cells (∼1.7 × 107 CD34-selected cells/kg), there was moderate cytopenia with ANC <500/mL for 7 days and thrombocytopenia with a nadir of 18,000/mL. Following stable hematopoietic recovery, we observed gene marking, determined by RT-PCR, in total white blood cells as a provirus copy number of 0.04 (∼4% gene marking) that, following a single cycle of O6BG (x2) and BCNU, rose to 0.16 (∼16% gene marking). Currently, gene marking has been stable for more than 9 months following chemotherapy. The treatment was well tolerated with only transient elevated liver enzymes following O6BG/BCNU treatment and no additional extra-hematopoietic toxicity has been observed. Clonality studies before and after in vivo selection is underway using a combination of LAM-PCR and a modified whole genome pyrosequencing approach. In summary, we have attained efficient and stable in vivo selection of long-term repopulating cells in nonhuman primates, and have extended this approach to use a reduced intensity conditioning regimen that should be well tolerated in patients with many hematopoietic diseases.

Disclosures:

No relevant conflicts of interest to declare.
The Transient Neutropenia Following G-CSF Administration Is Associated with An Accumulation of Neutrophils in the Pulmonary and Splenic Vasculature.
Authors: Dejesus Catherine E. , Egen Jackson G. , Alvarez Xavier , Metzger Mark E. , Combs Christian A. , Malide Daniela A. , Yu Zu Xi , Donahue Robert E.
Published: October 2019

Abstract 3586

Poster Board III-523

Shortly following G-CSF administration there is a dramatic yet transient disappearance of neutrophils from the circulation. To evaluate this disappearance, C57BL/6 LysM-EGFP knock-in male mice between 6-12 weeks of age were used. The LysM promoter/enhancer is specifically expressed in neutrophils and monocytes permitting evaluation of tissue distribution using EGFP marker localization by confocal microscopy. The ratio of EGFP+ leukocytes to background cellularity was determined in fixed volumes of tissue collected from the lung, liver, spleen, and kidney of mice either receiving saline (n=4) or at 15 minutes (n=6), 30 minutes (n=6), or 240 minutes (n=4) following 50ug/kg SQ rmG-CSF administration. No changes in ratio were observed in triplicate images of the liver or kidney of mice following G-CSF administration; however, there was a significant increase in EGFP+ leukocytes in the spleen and lung shortly following G-CSF administration. It increased from a control value of 6.3(2.6)% (n=12) to 15.9 (6.8)% (n=18) at 30 minutes in the lung and from 1.6 (0.7)% (n=12) to 8.3 (4.2)% (n=18) at 30 minutes in the spleen. While the value for the pulmonary images was less at 240 minutes (9.2 (5.1)%) the level of EGFP+ cells in the spleen continued to rise (12.3 (7.2)%). The increases were in neutrophils based on nuclear morphology. Thus the transient loss of neutrophils from the circulation following G-CSF administration appeared to be due to an accumulation of neutrophils within the pulmonary and splenic tissues. To confirm this observation, tissues were collected from three rhesus macaques 30 minutes following 10ug/kg SQ G-CSF. These tissues were compared to those of a control. Neutrophils were labeled with CD18 and a secondary antibody conjugated to FITC for quantification by confocal microscopy. As before, ratios of fluorescence were determined. The mean threshold intensity for CD18+ cells for the lung lobes for those animals receiving G-CSF (3.8 (3.0)%, n=54) was greater than control specimen lungs (1.5(1.1)%, n=18) and slightly increased for the spleen( from 4.1(3.7)% to 8.5 (6.3)%, n=9). Detailed confocal microscopy using a myeloid/histiocyte antigen clone Mac387 which labels neutrophils and monocytes, an endothelial marker anti-caveolin-1-Cy3 antibody, and a nuclear stain (To-Pro3) showed (see below, stained green, red, and blue, respectively) that the neutrophils were elongated, appeared activated, and were tightly adhered to the endothelium of the pulmonary vasculature. These results suggest that the transient neutropenia following G-CSF administration is associated with an accumulation of neutrophils within the pulmonary and splenic tissues.

Disclosures:

No relevant conflicts of interest to declare.
The Role of Beta 2 Integrins in Macrophage Migration During Resolution of Inflammation.
Authors: Kushchayeva Yevgeniya , Mishchuk Darya , Ugarova Tatiana
Published: October 2019

Abstract 3600

Poster Board III-537

The mobilization of blood monocytes and their differentiation into macrophages during the immune-inflammatory response helps to prepare the tissue for resolution. During the resolution phase of inflammation macrophages do not die locally: some cells emigrate by draining lymphatics whereas some remain at the site of inflammation. The major myelo-monocytic integrin αMβ2 (Mac-1, CD11b/CD18), together with two related integrins αDβ2 (CD11d/CD18) and αXβ2 (CD11c/CD18), mediate critical adhesive reactions of monocyte/macrophages. However, the roles of these adhesion receptors in control of macrophage retention at sites of inflammation and their emigration to lymph nodes are unclear. Using a mouse model of sterile peritonitis induced by thioglycollate injection, we examined the dynamics of macrophage β2 integrins during the resolution phase of inflammation. Macrophages were defined by FACS analyses as a population of cells expressing αMβ2high, αDβ2+ and CD115+. The initial population of resident β2, positive for βDβ2 and negative for αXβ2. The thioglycollate-challenged mice showed a ∼4-fold increase in macrophages on day 3 followed by a progressive decrease to normal resident cell numbers by day 13. Expression of αMβ2 on macrophages on day 3 decreased by 2.5-fold as a result of dilution of the initial population of αMβ2high resident macrophages by infiltrating blood monocytes expressing αMβ2low. However, after day 3, the density of αMβ2 on macrophages gradually increased and by day 13 returned to the high levels characteristic of resident macrophages. By contrast, expression of αDβ2 and αXβ2 on inflammatory macrophages increased by 2-fold by day 6-9 compared to that on resident macrophages and then returned to the resident levels by day 3. Thus, although the number of macrophages decreased from day 3 to day 9 by several fold, the population of macrophages which remained in the peritoneum was enriched in cells expressing the high levels of αMβ2 and αDα2. Tracking migration of fluorescently labeled peritoneal cells demonstrated that a population of macrophages which leaves the inflamed peritoneum and enters lymph nodes consists of cells expressing low levels of αMβ2 and αDβ2. These data suggested that upregulation of β2 integrins, especially αMβ2, may be responsible for the retention of macrophages in the peritoneum. Indeed, the rate of macrophage emigration from the peritoneum in the αMβ2-deficient mice was significantly higher than that in wild-type mice. The results indicate that macrophage emigration from the inflamed site is controlled by the level of integrin αMβ2 and αDβ2 with low expressors being migratory and high expressors remaining in the peritoneum. The data also highlight the importance of integrins αDβ2 and αXβ2 as specific markers of inflammatory macrophages.

Disclosures:

No relevant conflicts of interest to declare.
Rescue of Erythropoietin-Deficient Mice From Anemia by Complementation with BAC Transgene.
Authors: Yamazaki Shun , Suzuki Norio , Obara Naoshi , Pan Xiaoqing , Hirano Ikuo , Jishage Kou-ichi , Honda Kurisu , Minegishi Naoko , Yamamoto Masayuki
Published: October 2019

Abstract 3614

Poster Board III-550

To maintain tissue oxygen homeostasis, erythropoietin (Epo) regulates the erythropoiesis. During embryonic development Epo is produced in the liver, but after birth and throughout adulthood Epo is produced and secreted from the kidney and support definitive erythropoiesis in the bone marrow. Anemia or hypoxic stress provokes transcription of the Epo gene and the mechanisms how the Epo gene expression is activated in a tissue-specific and hypoxia-inducible manner are one of the research topics of the molecular biology or gene regulation study. Since Epo-null mice die in utero of definitive erythropoiesis failure in the fetal liver, physiological significance of the Epo production for the adult erythropoiesis remains to be clarified. We have found that transgenic reporter gene expression driven by a BAC clone containing 180-kb mouse Epo gene locus recapitulates the Epo gene expression in the liver and kidney. To examine whether the Epo gene transcriptional activity within the 180-kb BAC is sufficient to recapitulate physiological Epo production, we attempted to rescue Epo-null mice from embryonic lethality with a BAC transgene harboring a floxed allele of the Epo gene. We found that transgene-derived Epo recovered the erythropoiesis in the fetal liver of Epo-null mice and Epo-null mice with the BAC transgene were born showing a Mendelian ratio. The rescued mice showed no hematological abnormality and were fertile. In the rescued mice bleeding anemia transiently increased plasma Epo level and after recovery from anemia plasma Epo level declined similarly to wild-type mice. In an RT-PCR analysis Epo production in the liver and kidney of the rescued mice was practically equivalent to that in the wild-type counterpart, while other tissues produced only marginal amount of Epo. These results thus demonstrate that the BAC transgene contains the Epo gene regulatory domain region sufficient to fully recapitulate the endogenous Epo gene expression and to compensate for the Epo-deficiency. We also found that the Epo gene deletion from the BAC transgene by general deletor-Cre transgene provoked the Epo-null phenotype, demonstrating that conditional inactivation of the Epo gene is attained in the BAC-rescued mice. The latter system will be a precious tool to investigate adult stage-specific roles of Epo.

Disclosures:

Yamamoto: Chugai Pharmaceutical, Co., Ltd. : Research Funding.
Single Cell Phospho-Flow Analysis of Cytokine Stimulation in Human Hematopoietic Progenitors Reveals That G-CSF Acts Directly On Human Hematopoietic Stem Cells.
Authors: Gibbs Kenneth , Gilbert Penney , Weissman Irving L. , Blau Helen M. , Nolan Garry P. , Majeti Ravindra
Published: October 2019

Abstract 3617

Poster Board III-553

Hematopoietic stem and progenitor cells (HSPC) give rise to all blood cell lineages for the lifetime of an individual. However, their low frequency has precluded the use of traditional biochemical methods to study their signaling biology. We developed a phospho-specific flow cytometry approach that allows for the simultaneous interrogation of multiple elements of signaling networks in primary human HSPC at the single cell level without prospective purification by cell sorting. We found that HSPC contain biochemically distinct signaling subpopulations, and that, although considered quiescent, HSC from cord blood and adult bone marrow respond directly to several cytokine stimuli through multiple signaling nodes including JAK/Stat, Erk, and Akt. Surprisingly, we found that a subset of HSC respond rapidly and directly to in vitro G-CSF stimulation through Stat3 signaling. The addition of G-CSF to purified cultures of HSC significantly increased the percentage of cells that had entered the cell cycle. Moreover, when G-CSF was added to cultures of purified human HSC spatially segregated in hydrogel microwell arrays, we observed a significant increase in the frequency of HSC proliferation. These results demonstrate that contrary to the accepted paradigm, G-CSF acts directly on human HSC. Cytometric single cell characterization of the molecular behavior of HSPC in a wide variety of hematopoietic malignancies can now be applied to study the signaling behavior of other primary and rare stem cell populations.

Disclosures:

Weissman: U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties; Stem Cells Inc.: Cofounder and Director; Cellerant Inc.: Cofounder; Amgen: Equity Ownership. Nolan:BD Biosciences: Consultancy.
STAT3 Controls Neutrophil Progenitor Growth and Differentiation During Emergency Granulopoiesis.
Authors: Zhang Huiyuan , Nguyen-Jackson Hoainam , Panopoulos Athanasia , Li Haiyan S , Murray Peter J , Watowich Stephanie
Published: October 2019

Abstract 3619

Poster Board III-555

Granulocyte colony-stimulating factor (G-CSF) controls neutrophil production in the bone marrow under steady state conditions and during demand-driven hematopoiesis occurring in response to infection. STAT3 is a principal signaling molecule activated by the G-CSF receptor (G-CSFR). We previously reported that STAT3 has an important role in demand-driven granulopoiesis, although its cellular and molecular mechanisms have been unclear. To address this, we investigated STAT3 function in emergency granulopoiesis stimulated by G-CSF administration or infection with Listeria monocytogenes, which is restrained by the G-CSF response pathway in vivo. Our results show that STAT3-deficiency renders hematopoietic stem cells and myeloid progenitors refractory to the proliferation-inducing effects of G-CSF or Listeria monocytogenes infection. STAT3-deficient myeloid progenitors have a cell autonomous defect in G-CSF-responsive cell cycle progression and undergo delayed granulocyte maturation relative to wild type cells. To define STAT3 target pathways in granulocytic progenitors, we investigated the expression of CCAAT enhancer binding protein (C/EBP) beta, a transcription factor that is necessary for G-CSF-driven emergency granulopoiesis. We found that STAT3 directly regulates G-CSF-responsive C/EBPbeta expression by binding to Cebpb promoter. Moreover, we show that STAT3 and C/EBPbeta co-regulate c-Myc during emergency granulopoiesis. These results place STAT3 as a crucial G-CSF-responsive signal transducer during demand-driven granulopoiesis, through its regulation of critical transcription factors in developing granulocytes.

Disclosures:

Zhang: Amgen: Research Funding. Nguyen-Jackson:Amgen: Research Funding. Watowich:Amgen, Inc: Research Funding.
Mesenchymal Stem Cells Convert Human Macrophages to a Novel Type of Alternatively Activated Macrophages.
Authors: Kim Jaehyup , Hematti Peiman
Published: October 2019

Abstract 3632

Poster Board III-568

Mesenchymal stem cells (MSCs) are capable of modulating the immune system through interaction with a wide range of immune cells. This study investigates the hypothesis that interaction of MSCs with macrophages could play a significant role in their anti-inflammatory/immune modulatory effects. All studies were approved by IRB of University of Wisconsin School of Medicine and Public Health. MSCs were culture expanded from discarded bone marrow filters after bone marrow harvest from normal healthy sibling HLA-matched donors. We used passages -35 for our experiments. Ex vivo culture expanded MSCs were characterized by their cell surface phenotype (positive for MSC markers: CD29, CD44, CD73, CD90, and CD105; and negative for hematopoietic markers: CD31, CD34, and CD45), and their differentiation potential into bone, fat and cartilage. Monocytes were isolated from peripheral blood mononuclear cell fraction of healthy donors using CD14+ Miltenyi magnetic bead cell separation method. We cultured human CD14+ monocytes for seven days without any added cytokines to generate macrophages, and then co-cultured them for three more days with culture-expanded MSCs. We used cell surface antigen expression and intracellular cytokine expression patterns to study the immunophenotype of macrophages at the end of this co-culture period, and phagocytic assays to investigate their functional activity in vitro. Macrophages co-cultured with MSCs consistently showed high level expression of CD206, a marker of alternatively activated macrophages, in addition to being positive fro CD14 marker. Using CD1a and CD209 staining we did not detect presence of any dendritic cells either at the end of seven days culture of monocyte-derived macrophages or at the end of co-culture period. Furthermore, macrophages that were co-cultured with MSCs expressed high levels of IL-10 and low levels of IL-12, as determined by intracellular staining, typical of alternatively activated macrophages. However, macrophages co-cultured with MSCs also expressed high levels of IL-6 and low levels of TNF-α, compared to controls. Functionally, macrophages co-cultured with MSCs showed a higher level of phagocytic activity using Alexa 488-conjugated E. coli phagocytic assay. In summary we describe a novel type of human macrophage generated in vitro after co-culture with MSCs that assume an immunophenotype defined as IL-10 high, IL-12 low, IL-6 high and TNF-α low secreting cells. These MSC-educated macrophages may be a unique and novel type of alternatively activated macrophages with potentially significant role in tissue repair.

Disclosures:

No relevant conflicts of interest to declare.
A Role for the Transcriptional Coactivator PRC1 in Mitochondrial Biogenesis During Erythroid Development.
Authors: Xu Xiuling , Friedman Jeff S.
Published: October 2019

Abstract 3642

Poster Board III-578

Red cell development requires unparalleled mitochondrial metabolic and biosynthetic capacities to support hemoglobin formation. In addition to meeting the energetic requirements of the erythroblast and reticulocyte, mitochondria are the site of multiple steps in heme biosynthesis and are essential for iron utilization. As such, perturbation of mitochondrial function in hematopoietic cells appears to differentially affect red cell development. For instance, we show that the drug chloramphenicol (an inhibitor of mitochondrial protein synthesis) preferentially inhibits erythroid colony formation with relatively little effect on other types of myeloid progenitors. Similarly, we show that knockout of the mitochondrial superoxide dismutase (Sod2) preferentially inhibits BFU-E, CFU-E and GEMM colonies. Clinically, several mutations in mitochondrial DNA or in nuclear encoded mitochondria-localized proteins preferentially disrupt erythroid development resulting in sideroblastic anemia. In order to better understand regulation of mitochondrial activity during red cell development/differentiation, we have investigated expression of transcription factors and coactivators implicated as regulators of mitochondrial biogenesis in other tissues including PGC-1 α and β, ERR proteins, NRF 1 and NRF 2 in normal and Sod2 deficient murine erythroblasts. Microarray and qPCR data suggest that neither of the classic transcriptional coactivators PGC-1 α or β are expressed at appreciable levels in erythroblasts. However, a PGC-1 related coactivator, PRC1 is detectable at both the transcript and protein level in developing erythroid cells. In the Sod2 deficiency model, one of the most prominent transcriptional changes we have observed in erythroblasts is a broad down regulation of expression of nuclear encoded mitochondria localized protein transcripts including multiple components of the electron transport chain, TCA cycle enzymes and mitochondrial ribosomal proteins. We show that PRC1 expression is down regulated in Sod2 deficient cells—suggesting that this coactivator plays a key role in mitochondrial biogenesis during erythroid development. Further, using the MEL cell line, we show that PRC1 expression is turned on following induction of differentiation with DMSO. These results identify PRC1 as a novel, and perhaps exclusive regulator of mitochondrial biogenesis during erythroid development. A better understanding of regulation of PRC1 expression and identification of transcriptional targets of PRC1 during erythroid development will improve our understanding of how perturbations in mitochondrial physiology affect the red cell compartment.

Disclosures:

No relevant conflicts of interest to declare.
Epigenetic Control of C/EBPa by Noncoding RNAs.
Authors: Di Ruscio Annalisa , Ebralidze Alexander K , D'Alò Francesco , Voso Maria Teresa , Leone Giuseppe , Tenen Daniel G.
Published: October 2019

Abstract 3644

Poster Board III-580

Little is currently known about the role of noncoding RNA transcripts (ncRNA) in gene regulation; although most, and perhaps all, gene loci express such transcripts. Our previous results with the PU.1 gene locus showed a shared transcription factor complex and chromatin configuration requirements for biogenesis of both messenger and ncRNAs. These ncRNAs were localized within the nuclear and cytoplasmic compartments. Disrupting ncRNAs in the cytoplasmic cellular fraction results in increased PU.1 mRNA and protein. Recently, we have focused on the C/EBPa gene locus and observed extensive noncoding transcription. The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo. In this study, we characterize ncRNAs derived from the C/EBPa locus and demonstrate their functional role in regulation of C/EBPa gene expression. First, northern blot analysis and RT PCR determined a predominantly nuclear localization of the C/EBPa ncRNAs. Second, strand-specific quantitative RT PCR demonstrated a concordant expression of coding and noncoding C/EBPa transcripts. Next, we investigated the results of ablation of ncRNAs using a lentiviral vector containing ncRNA-targeting shRNAs on the expression of the C/EBPa gene. We have observed that reduced levels of ncRNAs leads to a significant downregulation of the expression of coding messenger RNA. These data strongly suggest that C/EBPa ncRNAs play an important role in maintaining optimal expression of the C/EBPa gene at different stages of hematopoiesis and makes targeting noncoding transcripts a novel and attractive tool in correcting aberrant gene expression levels.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Mechanisms of the Pre-TCR Alpha Chain (pTa) Enhancer Transcriptional Regulation by Notch1.
Authors: Tremblay Cedric Stevens , Tremblay Mathieu , Herblot Sabine , Hoang Trang
Published: October 2019

Abstract 3649

Poster Board III-585

The Notch1 pathway and pre-TCR signalling are critical regulators of thymocyte development that have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Although the pre-TCR is required for Notch-dependent T-ALL, the role of pre-TCR alpha (pTa) as a Notch1 target remains controversial. Previous work in our laboratory has shown that the pTa gene is a target gene of the E2A-HEB transcription factors and that the SCL and LMO1 or LMO2 oncogenic transcription factors inhibits E2A activity and pTa expression, thereby causing thymocyte differentiation arrest during the pre-leukemic stage. SCL and LMO1/2 are implicated in 25% of T-ALL cases and induce aggressive T-ALL in transgenic (tg) mice when inappropriately expressed in the thymus. To define the mechanism regulating pTa expression by Notch1 and SCL, we used pTa regulatory sequences as a tool. To this end, we have performed transcriptional assays by co-delivering a luciferase reporter gene construct driven by the 200 bp pTa enhancer sequence with or without SCL or Notch1 intracellular domain (N1ICD), the functional fragment of the Notch1 receptor. Sequence analysis of the pTa enhancer revealed the presence of four E boxes that are potential binding sites for E2A-HEB and a CSL consensus sequence that can recruit the CSL/RBP-Jk transcription factor. Transcriptional assays in double negative thymomas indicated that the integrity of E box 2 and 3 (that recruit E2A-HEB) are essential for pTa enhancer activity in DN thymocytes, confirming the importance of E2A-HEB for pTa expression in these cells. In contrast, the CSL consensus sequence is important but not essential for pTa enhancer activity in these cells, consistent with previous results based on a conditional Notch1 knock out. In the presence of N1ICD, pTa enhancer sequences are strongly activated, either in fibroblasts or in double negative T cell lines in a dose dependent manner. This activation requires the integrity of the CSL consensus sequence but not of the E boxes. Moreover, we found that N1ICD strongly overrides the inhibitory activity of SCL on the pTa enhancer. Inhibition by SCL requires the integrity of E2 and E3 but not the CSL, confirming that SCL inhibits E2A-HEB activity, without affecting N1ICD. Together, our observations indicate that the regulation of pTa enhancer activity by Notch1 and E2A/HEB occur though independent mechanisms.

Disclosures:

No relevant conflicts of interest to declare.
PU.1 - c-Jun Interactions Are Crucial for PU.1 Function in Myeloid Development.
Authors: Bartholdy Boris , Yamamoto Yukiya , Evans Erica , Crispino John , Tenen Daniel G.
Published: October 2019

Abstract 3651

Poster Board III-587

The Ets transcription factor PU.1 is a master regulator absolutely required for the differentiation of monocytes, macrophages, and B cells in the fetal liver and in the adult bone marrow. PU.1 drives hematopoietic differentiation partly through direct protein-protein interactions with other transcription factors, such as the AP-1 transcription factor c-Jun. We have shown that c-Jun can be recruited to promoters which do not include AP-1 binding sites, such as the MCSFR promoter, and act as a PU.1-dependent co-activator. To address the functional importance of this interaction, we identified and studied PU.1 point mutants that lost the capability to physically interact with c-Jun while retaining normal DNA binding affinity. These mutants failed to efficiently transactivate a PU.1 target reporter, and, more importantly, were unable to induce monocyte/macrophage differentiation of the PU.1-deficient immature myeloid 503 cell line.

Subsequently, we have generated knock-in mouse models harboring these single point mutations by means of homologous recombination. The mutant mice phenotypically resemble PU.1-deficient mice, have an early block in hematopoiesis, and die perinatally. We show that the mutant PU.1 mRNA and protein is expressed in long-term and short-term hematopoietic stem cells, but that the maturation into lymphoid primed multipotent progenitor (LMPP) and later progenitor populations is severely blocked, leading to an almost complete loss of mature B, T and myeloid cells. Collectively, our data strongly suggest that the PU.1-c-Jun interaction is crucial for normal PU.1 function in vivo during murine fetal hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Diagnostic Value and Limitations of FDG-PET for the Staging of Childhood Lymphomas.
Authors: Kabickova Edita , Votrubova Jana , Sumerauer David , Mejstrikova Ester , Hrusak Ondrej , Kyncl Martin , Kodet Roman , Stary Jan , Belohlavek Otakar
Published: October 2019

Abstract 3658

Poster Board III-594

Introduction

Positron emission tomography with fluorine-18-fluorodeoxyglucose (FDG-PET) has the potential to detect malignant cells by their increased glycolysis. PET can detect early changes, before they are apparent on anatomic imaging. Whole-body PET can also detect lesions at unexpected sites. However, physiologic FDG uptake in non-malignant conditions limits the specificity of PET. PET is predominantly used in adult lymphoma patients, limited number of studies were published dealing with PET and PET/CT in childhood lymphomas. The aim of our study was to assess the usefulness of PET for initial staging of pediatric lymphomas and to evaluate benign pathologic causes of FDG uptake.

Patients and methods

Over a period of 5 years 86 children and adolescents with lymphoma (58 with Hodgkin's lymphoma, 28 with non-Hodgkin's lymphoma) had complete staging work-up including FDG-PET or PET/CT and were included into this prospective study. Patients were aged 4-19 years, 57 (66%) were boys. No patient had CNS lymphoma infiltration. PET findings were correlated with conventional staging methods (CSM) including CT, ultrasound, and bone marrow examination. Discordant findings were verified by MRI and follow-up radiographic studies including PET.

Results

PET revealed 40 additional lymphoma manifestations in 34% (26/86) of studies and correctly upstaged 15% (13/86) of patients. Only 2% (2/86) of children were not accurately staged by PET, when PET failed to visualize diffuse bone marrow infiltration (extent of 15% cells) in 1 patient, and missed small pulmonary metastases (≤6 mm) in 1 child. Compared with CSM, PET had significantly higher sensitivity (97% vs. 83%), specificity (100% vs. 89%), and significantly higher accuracy (98% vs. 84%). Physiologic thymic FDG uptake was observed in 15% (13) of patients; diffuse increased FDG bone marrow activity had 22% (19) of children, and intense FDG activity in fatty tissue occurred only in 8% (7) of patients.

Conclusions

PET imaging was highly sensitive in detecting all subtypes of pediatric lymphomas and has potential to accurately define the disease stage except the lungs, where CT has excellent sensitivity. Understanding of the physiologic FDG biodistribution and benign pathologic causes of FDG uptake is essential for accurate scan interpretation.

Supported by grants: IGA NS/9997-4, NS/10480-3, MSM 0021620813 and MZO FNM 2005

Disclosures:

No relevant conflicts of interest to declare.
B Cell Depletion Reduces the Number and Function of T- Regulatory Cells and Enhances the Anti Tumor response
Authors: Tadmor Tamar , Zhang Yu , Dunn Robert , Shin Seung-uon , Cho Hyung-Mee , Rosenblatt Joseph
Published: October 2019

Abstract 3668

Poster Board III-604

Increasing evidence suggests that B lymphocytes play a central role in inhibiting the immune response against certain tumors, but the underlying mechanisms by which B cells facilitate tumor growth are still poorly understood.

In this study, we investigated how the presence or absence of B cells affects expansion and function of T- regulatory cells (‘Tregs’) in a murine tumor model (EMT-6). We compared tumor growth, and the number and function of T- regs cells in wild type immune competent mice ( ICM), B cell deficient mice ( BCDM) and /or in BALB-C mice following B- cell depletion induced by injection of anti murine CD20 antibodies (mCD20 Ab, 18B12, mouse IgG1,k, Biogen-IDEC) Mice were either tumor-naïve or implanted with EMT6 mammary adenocarcinoma cells.

Absence of B cells as in BCDM completely inhibited tumor growth in the majority of mice, while B cell depletion in normal mice substantially slowed the growth of EMT-6 tumors compared to wild type mice (ICM). Substantial T regs expansion, as defined by CD4+/CD25+/FOXP3+ cells, was evident on day 26 post tumor inoculation in EMT-6 tumor bearing ICM in comparison to the non- tumor bearing mice ( 15.2 +/− 1.2. % and 11.9 +/− 1.1% respectively), isolated from spleen as compared to naïve or tumor bearing BCDM (10.1+/− 0.2% and 10.8+/− 1.2%) The percentage and absolute number of T-regs in the spleen, tumor draining lymph nodes and tumor bed were significantly reduced in the BCDM and/or B cell depleted ICM compared to tumor bearing ICM (10%+/−0.8, 13.9+/− 1.23% and 17+/− 1.3% respectively p<0.01. data from single cell suspensions isolated from spleens on day 20 post tumor inoculation). Similar effects of B cell depletion on the numbers of T-regs were observed in the setting of pre-established EMT6 mammary tumors. In contrast to tumor bearing mice, differences in T-reg number and function were minimal in tumor free B cell deficient or in B cell depleted naïve mice compared to ICM.

T-reg function, measured by suppression assay and proliferation assays, was also markedly reduced in tumor bearing BCDM compared to ICM. Combining B cell and T-reg depletion using i.p. injection of anti CD 25 antibody (PC61 or PBS) resulted in similar rates of tumor regression in B cell depleted mice as were seen in BCDM suggesting that the combination of B cells depletion and further depletion of Tregs augmented anti-tumor response.

In conclusion, our studies indicate that B cell depletion may play a useful role in augmenting the T cell anti-tumor response, in part due to their effects on T-regulatory cell biology.

Disclosures:

Dunn: Biogen IDEC: Employment.
Involvement of Fatty Acid Synthase in Azacytidine-Induced Cytotoxicity in Waldenstrom's Macroglobulinemia.
Authors: Xu Lian , Patterson Christopher J , Sun Jenny , Yang Guang , Zhou Yangsheng , Hunter Zachary , Hatjiharissi Evdoxia , Ciccarelli Bryan , Gong Ping , Liu Xia , Tseng Hsiuyi , Ioakimidis Thea , Manning Robert , Hanzis Christina , Cao Yang , Lewicki Megan , Treon Steven
Published: October 2019

Abstract 3731

Poster Board III-667

Azacytidine is a potent demethylation agent and has been used for the treatment of myelodysplastic syndrome. Despite a role in reactivating epigenetically silenced tumor-suppressor genes, the mechanisms of action for azacytidine are not well understood. The relationships between the reactivation of tumor-suppressor genes and the significance for clinical responses also remain to be established. Azacytidine can incorporate into both RNA and DNA strands and may therefore act through multiple pathways. We therefore investigated the therapeutic potential of azacytidine in Waldenstrom's Macroglobulinemia (WM), and characterized certain molecular changes associated with azacytidine. We found that azacytidine exhibited significant dose-dependent cytotoxicity against WM cell lines, and primary WM cells. Treatment of BCWM.1 WM cells with 2 uM of azacytidine rapidly induced cell cycle arrest at G1, and also induced apoptosis at 48 hours. Cleavage of caspase 3, 7, 8, 9 and PARP-1 suggested an involvement of mitochondrial and death receptor pathways in azacytidine-induced apoptosis. The BH3 proteins Puma and Bim, and the pro-apoptotic protein Bax were up-regulated while the anti-apoptotic proteins Bcl-2 and Bcl-xl remained unchanged after treatment with azacytidine. In addition, azacytidine did not induce Akt and NFκB pathways. To further elucidate molecular changes associated with azacytidine treatment, we performed genome-wide microarray expression in BCWM.1 WM cells treated with azacytidine. We observed that the gene encoding fatty acid synthase (FASN) was among the most down-regulated of targets. FASN has been linked to tumor cell proliferation, and its expression was observed by us to be upregulated by microarray studies in WM patients, and further validated as a highly over-expressed target by quantitative RT-PCR analysis in WM patients versus healthy donors. Moreover, by these studies we show that knockdown of FASN by small interfering RNA resulted in growth arrest, and also induced apoptosis in BCWM.1 WM cells. Cerulenin, a specific inhibitor of FASN derived from a natural product, induced strong cytotoxicity against BCWM.1 and primary WM cells at 20 uM in 24 hours. In addition, a synergistic induction of apoptosis in BCWM.1 WM cells was observed in the presence of both azacytidine and cerulenin. The results of this study support an important role for FASN in azacytidine-induced cytotoxicity in WM cells, and highlight a novel mechanistic pathway for this agent, as well as a novel target for drug therapy in WM. A clinical study investigating the activity of azacytidine in WM is planned.

Disclosures:

No relevant conflicts of interest to declare.
Phase II Trial of 90y-Ibritumomab Tiuxetan Treatment as Consolidation After 6th R-CHOP Chemotherapy in Patients with Limited-Stage, Bulky Diffuse Large B Cell Lymphoma.
Authors: Yang Deok-Hwan , Kim Won Seog , Suh Cheolwon , Kwak Jae-Yong , Jung Joo-Sep , Kim Sung Hyun , Kim Jin Seok , Lee Je-Jung , Kim Hyeoung-Joon
Published: October 2019

Abstract 3751

Poster Board III-687

Limited-stage non-Hodgkin's lymphomas with bulky mass are known to have a poor prognosis as like to those with advanced disease. There are very limited data to compare the results of chemotherapy alone with those of combined modality treatment. However, defining local failure at bulky sites as one component of relapse, radioimmuotherapy (RIT) appears to reduce failure rates at sites of bulky disease. We assessed the clinical efficacy and toxicity of 6th R-CHOP chemotherapy and followed by ibritumomab tuixetan (Zevalin¢ç) consolidation in patients with bulky diffuse large B cell lymphoma (DLBCL).

Patients and Methods

This prospective, multi-center phase II clinical trial included the patients following as: i) patients with newly diagnosed CD20+ DLBCL ii) patients with stage I/II and bulky disease defined by maximum diameter greater than 10 cm or any mediastinal mass exceeding 1/3 the maximum trans-thoracic diameter (more than 8 cm) iii) patients that achieved a complete (CR) or partial response (PR) after 6th R-CHOP chemotherapy iv) aged over 18 years and ECOG performance status 0-2. Conventional, single dose of ibritumomab tuixetan (0.4 mCi/kg) was administered after 6th R-CHOP. Clinical response was evaluated after completion of 3rd and 6th R-CHOP and at 8 and 20 weeks after RIT, and assessed according to revised International Workshop Criteria.

Results

Twenty patients were treated with ibritumomab tuixetan as consolidation. Median age was 45.5 years and median size of bulky mass was 10.0 × 6.6 at diagnosis. 5 patients (35%) presented with high-intermediate or high risk by IPI and 4 patients underwent debulking operation. After a median of 17.5 months follow-up, the overall response rate was 95%, with a 80% CR and 15% PR. Two patients with PR after R-CHOP chemotherapy improved to CR after consolidation. However, four patients (20%) experienced a relapse with one of them progressed within 1 month after RIT. 2-year probability of progression-free survival (PFS) was 78.9 ±9.1%. The adverse events were mainly hematologic toxicities (more than grade3); neutropenia (60%) and thrombocytopenia (35%). Only one patient experienced a febrile neutropenia.

Conclusions

Consolidation treatment with Ibritumomab tuixetan resulted in a favorable response with tolerable toxicity in patients with bulky DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Direct Inhibition of Fas Activation by CD74.
Authors: Berkova Zuzana , Garcia Michael , Wang Shu , Wise Jillian Frances , Tao Rong-Hua , Daniluk Urszula , Samaniego Felipe
Published: October 2019

Abstract 3770

Poster Board III-706

Hematopoietic cancers are commonly resistant to Fas-mediated apoptosis that contributes to their resistance to chemotherapy and reversing Fas resistance has become a primary interest in treating chemotherapy-resistant hematopoietic cancers. Wide expression of Fas receptor yet the limited extent of Fas-mediated apoptosis in tissues suggest a strict regulation of Fas signaling. A recently identified tissue-specific modulator of Fas-mediated apoptosis is hepatocyte growth factor (HGF) receptor HGFR/Met. HGFR/Met binds Fas and inhibits Fas signaling at an immediate early step. However, upon binding of ligand HGF to HGFR/Met, Fas is released and Fas signaling is restored. We hypothesized that hematopoietic cancers may express similar inhibitors of Fas, that can be targeted to resensitize cancer cells to Fas and thus also to therapy. We screen lymphoma cells for for such inhibitors. B cell lymphoma-derived BJAB cells and primary patient samples were incubated with Fas agonistic antibody. Upon removal of antibody excess, cell lysates were immuno-depleted of CH-11 antibody-bound and thus activated Fas. The remaining supernatant was then precipitated a second time with anti-Fas antibody B-10 that binds to the intracellular region of Fas. Protein bands present in B-10 precipitates and absent in CH-11 precipitates were excised from silver-stained gel and analyzed by mass-spectroscopy. CD74, also termed the invariant chain of MHC II and known to mediate pro-survival signaling as a receptor for macrophage migration inhibitory factor (MIF), was found to exclusively associate with activation-resistant Fas. Intriguingly, CD74 is known to be overexpressed in 80% to 95% of hematopoietic cancers and also in some solid tumors.

Using RNA interference we showed that CD74 negative cells are more sensitive to Fas-mediated apoptosis and that CD74 overexpression confers resistance to Fas both in vitro and in mouse tissues. Incubation of cells with peptides or anti-CD74 antibody LL1 disrupts the CD74-Fas inhibitory complex and sensitizes CD74-positive cells to Fas-mediated apoptosis. Analysis at the molecular level revealed that CD74 interferes with the immediate early steps in Fas signaling – binding of agonistic CH-11 antibody and subsequent clustering of Fas receptor.

We anticipate that specific disruption of the CD74-Fas interaction by CD74-derived peptides or anti-CD74 antibodies will sensitize cancer cells to Fas-mediated apoptosis. This approach represents a novel strategy for modulation of cell surface death receptors to produce effective treatments for CD74-positive cancers.

Disclosures:

No relevant conflicts of interest to declare.
Discovery and Validation of a Novel Class of Small Molecule Inhibitors of the CDC7 Kinase: Modulation of Tumor Cell Growth in Vitro and In Vivo.
Authors: Frattini Mark G. , Shum David , O'Dwyer Kristen M , Brentjens Renier J. , Maslak Peter , Heaney Mark , Jurcic Joseph G , Djaballah Hakim , Kelly Thomas J
Published: October 2019

Abstract 3771

Poster Board III-707

High throughput screening of compounds comprising the Memorial Sloan Kettering chemical library resulted in several confirmed hits against the recombinant Cdc7:Dbf4 heterodimeric kinase, a key regulator in the initiation of DNA replication and the G1 to S phase transition. Chemoinformatic analysis of the hits revealed an enrichment in one chemical cluster made up of several naturally occurring compounds, of which the most potent compound, CKI-7, was selected for further investigation. First, CKI-7 was found to be a non competitive inhibitor for ATP and prompted us to prolife it against a panel of 200 known kinases in order to assess its selectivity profile. The results were as predicted and very few kinases were specifically affected. Second, CKI-7 cytotoxic activity was assessed against a panel of well established cancer cell lines representing both hematopoietic and solid tumor malignancies as well as against a panel of primary hematopoietic cells derived from leukemia patients (both chemotherapy naïve and relapsed/refractory samples) and was found to be a very effective agent with potencies in the low nanomolar range. Subsequent studies using an isogenic pair of cell lines with one over expressing the Bcl_xL anti-apoptotic protein further confirmed the induction of the intrinsic apoptotic pathway via caspase-3 activation in the absence and attenuation of the activity in the presence of Bcl_xL. This was further demonstrated through standard cell cycle synchronization studies revealing that exposure to the Cdc7 inhibitor results in an S phase arrest, cell cycle dependent caspase-3 activation, and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a Cdc7 substrate biomarker assay. Third, the physicochemical properties of this class of naturally occurring compounds also prompted us to investigate their effect on several multidrug resistence (MDR) over-expressing cell lines. We found that CKI-7 was not a substrate for the efflux pumps demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. Based of the above observations, in vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilizing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Taken together, our data confirm that Cdc7 is a new promising target for cancer therapy, and that the newly discovered inhibitor CKI-7, a naturally occurring selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent.

Disclosures:

No relevant conflicts of interest to declare.