bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Use Of Two Photon Microscopy To Image Vaso-Occulsion In Pulmonary Microvessels Of Living Mice With Sickle Cell Disease
Authors: Ralainirina Natacha , Sonderegger Ferron Lynn , Pei Hong , Chodaczek Grzegorz , Linden Joel 
Published: October 2019

Abstract
Although sickle cell anemia is initiated by red cell pathology, it is accompanied by an inflammatory immune response involving platelets and white blood cells that contribute to vaso-occlusvie episodes including painful vaso-occlusive crises (pVOC) and acute chest syndrome (ACS). In order to better understand the cellular and molecular bases of vaso-occlusion we are in the process of developing procedures to image microvessels in the lung, liver and spleen of living mice by 2-photon microscopy, a procedure that is based on excitation of a fluorophore by two photons simultaneously. The two-photon technique utilizes infrared light that efficiently penetrates tissues up to 200 microns with low phototoxicity allowing time-lapse imaging. Two-photon intravital microscopy can be used to study the behavior of intravascular cells during vaso-occlusive events.
Mice are prepared for lung intravital microscopy by the intraperitoneal injection of a mixture of ketamine and xylazine. Additional anesthesia is added during experimentation. The trachea is opened and the mouse intubated. The chest is opened to allow access to the left lobe of the lung through a window that is a few millimeters in diameter. PBS is applied to keep the lung moist. A custom built suction device is placed on the lung and covered with a cover glass at the same time pressure is exerted to seal the organ and the glass cover together. Throughout the procedure, the mouse is held at a temperature of 37°C. Once surgery is completed, a mixture of antibodies coupled to fluorophores is given by retro-orbital injection. In order to minimize photobleaching we used antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555 or Alexa Fluor 647.
We are able to visualize and quantify interactions between red blood cells, white blood cells, and endothelial cells as well as the expression of adhesion molecules on endothelial cells in real time. During pVOC triggered by hypoxia, cell adhesion of neutrophils, lymphocytes and monocytes to the endothelium is observed that is associated with an increase in endothelial expression of ICAM-1 and V-CAM. We label endothelial cells with anti-CD31, lymphocytes with anti-CD45, monocytes with anti-Ly6C and neutrophils with anti-Ly6G. Platelets are labeled with anti-CD41 or anti-CD62P, NK cells with anti-NKp46, and macrophages with anti-F4/80 and anti-CD1d. We are able to quantify cell shape, rolling, adhesion and movement.
Our preliminary results demonstrate that it is possible in real time to image the sequence of events occurring during pulmonary vasoocclusion in sickle cell disease. In conclusion, intravital 2-photon microscopy holds great potential for enabling us to better understand inflammatory responses within the blood vessels of living SCD mice.

Disclosures:
No relevant conflicts of interest to declare.
Systems Biology and Epigenetic Mechanisms in Erythropoiesis
Authors: Socolovsky Merav 
Published: October 2019

Abstract
Irreversible rapid cellular decisions are often controlled by network motifs known as bistable switches. We identified a cell-cycle regulated bistable switch that controls activation of the erythroid transcriptional program during early S phase of the last generation of erythroid colony-forming-unit progenitors (CFUe). This switch drives a rapid, multi-layered commitment event that activates GATA-1 transcription, renders the cells dependent on erythropoietin, and brings about chromatin reconfiguration at erythroid gene loci. In addition, it triggers an unusual process of genome-wide DNA demethylation, the first known example of such a process in somatic cell development. Approximately 25 to 30 percent of all methylation marks are lost from essentially all genomic elements during erythroid terminal differentiation. The bistable switch activating erythroid transcription consists of two linked double-negative feedback interactions of the erythroid transcriptional repressor PU.1, which antagonizes both S phase progression, and the erythroid master transcriptional regulator GATA-1. During operation of the switch, a rapid S phase-dependent decline in PU.1 activates GATA-1 transcription. The dependence of this switch on S phase progression coincides with a dramatic change in the nature of S phase itself, which becomes shorter and 50 percent faster. The accelerated intra-S phase DNA synthesis rate is essential for the loss of genome-wide DNA methylation, which in turn is required for the rapid induction of erythroid genes.

Disclosures:
No relevant conflicts of interest to declare.
Novel Targeting Strategies for Leukemia-Initiating Cells in Myeloid Neoplasms
Authors: Sauvageau Guy , Pabst Caroline , Lemieux Sebastien , Hébert Josée , Krosl Jana 
Published: October 2019

Abstract
In this session we will present results from our chemo-genomic screens designed to categorize more accurately human acute myeloid leukemia (AML) subsets based on both their genetic make-up (RNA and exon Next Generation Sequencing) and their response to clinically-approved chemicals. The success of this project relies on our recent development of newly defined culture conditions that support the majority of leukemia stem cells in short-term cultures (Pabst et. al., submitted) and availability of a large collection of clinically annotated specimens (J.H., BCLQ). Integration of chemical and genetic data is possible through our novel bioinformatics tools developed for this purpose (Lemieux et. al., submitted). In addition to the generation of more accurate prognostic tools, these studies set the stage for drug repositioning and personalized medicine for human AML.

Disclosures:
No relevant conflicts of interest to declare.
ceRNAs and ceRNA Networks in Normal and Malignant Hematopoiesis and Their Therapeutic Implications
Authors: Pandolfi Pier Paolo 
Published: October 2019

Abstract
We will discuss exciting new data regarding the role of pseudogenes, long intergenic non-coding RNAs (lincRNAs) and miRNAs in the pathogenesis of leukemia and lymphoma, as also studied in vivo in the mouse, and their therapeutic implications. We will also focus on competing endogenous RNAs (ceRNAs), circular(circ)-ceRNAs and pseudo-ceRNAs and give important attention to how understanding their cross-talk and the ceRNA language will facilitate efforts to deconvolute ceRNA networks and their role in the pathogenesis of hematopoietic malignancies.

Disclosures:
No relevant conflicts of interest to declare.
Platelets, Tumor Cell Invasiveness, and Metastasis
Authors: Hynes Richard O. , Begum Shahinoor , Labelle Myriam 
Published: October 2019

Abstract
Platelets have long been known to promote metastasis, and multiple mechanisms have been proposed to explain this phenomenon, including adhesion, coagulation, and protection against natural killer (NK) cells or turbulence. One mechanism that has been little explored is the possibility that platelets might secrete growth factors or provide other stimuli that could enhance the malignant properties of tumor cells. We have shown that pretreatment of carcinoma cells with platelets induces an EMT-like transformation in their properties in vitro and renders them much more metastatic after introduction into mice. TGF-β, produced by platelets and released on their activation is essential for both the in vitro and the in vivo effects. However, TGF-β alone is insufficient; platelet-tumor cell contact is also required and this contact activates NFkB signaling, which synergizes with the TGF-β signaling. Both signals are required for the enhancement of metastasis. In addition to enhancing migration and invasion in vitro, platelets enhance extravasation in vivo. Earlier work has shown that both P-selectin (expressed on platelets) and L-selectin (expressed on leukocytes) are essential for efficient metastasis, and aggregates of tumor cells, platelets, and leukocytes can be observed at sites of tumor cell arrest and extravasation. It has also been demonstrated by others that leukocytes can enhance extravasation and metastatic seeding. Therefore, we have been interested in the question of the relative roles of platelets and leukocytes in these processes. Which cell types are recruited at the sites of metastatic seeding? Does one cell type depend on another? Which cell types enhance metastasis? What roles do the platelets play in recruiting the other cell types? The involvement of platelets in enhancing metastasis also raises questions about the effects of platelets on circulating tumor cells (CTCs). Could platelets enhance the metastatic capacity of CTCs? Could it be the case that only those CTCs that are associated with platelets and/or leukocytes are functionally involved in seeding metastases? Such aggregates are not scored in most current assays for CTCs and will require new investigative approaches. Platelet participation in metastasis also raises the possibility of therapeutic interventions targeting platelet-specific targets and the paracrine interactions between them and other cells.

Disclosures:
No relevant conflicts of interest to declare.
Platelets and the Tumor Cell Microenvironment
Authors: Byzova Tatiana V. , Kerr Bethany A. , Feng Weiyi , McCabe Patrick 
Published: October 2019

Abstract
The link between tumors and the hemostatic system, first of all platelets, has been acknowledged by numerous studies. Platelets activation at the tumor microenvironment and subsequent intravascular clotting are often associated with cancer complications. Recent clinical studies demonstrated that platelet inhibitors substantially decrease risk of tumor metastasis and mortality in cancer patients, thereby emphasizing clinical importance of platelets. However, the precise function of platelets in cancer progression remains enigmatic. Growing tumors secrete a plethora of growth factors and cytokines (tumor secretome) aiming to change its micro- and macro-environment. These factors promote angiogenesis, induce inflammation, and even stimulate changes in bones prior to metastasis. Tumor secretome signals are likely transmitted through the circulation; however, these signals need to be protected from degradation or utilization prior to reaching their target. Due to their ability to intake and secrete back a wide spectrum of proteins, platelets could function as a mobile storage compartment for the tumor secretome. Using platelet infusion and depletion during tumor growth, we assessed the role of platelets in tumor growth and tumor-induced bone remodeling. We demonstrated that platelet infusion stimulates communications between bone marrow and the tumor, while platelet depletion had the opposite effects. Importantly, tumor-induced changes in bone structure were completely abolished in the absence of platelets. Using platelets from knockout mice with various platelet defects, we demonstrated that platelet secretion (but not aggregation) was crucial for their function in tumor progression. In particular, the function of platelet α-granules is essential for the pro-tumorigenic role of platelets. Analysis of the tumor secretome revealed that 77 percent of tumor-secreted factors are highly enriched within platelets over plasma. Several of these factors are essential mediators of bone remodeling. Thus, our data show that platelet α-granule sequestration and secretion represent a cellular mechanism mediating communication between the bone microenvironment and the primary tumor.

Disclosures:
No relevant conflicts of interest to declare.
Selecting T-Cell Subsets for Adoptive T-Cell Therapy to Optimize Potency and Persistence
Authors: Riddell Stanley , Turtle Cameron , Hudecek Michael , Sommermeyer Daniel , Jensen Michael C. 
Published: October 2019

Abstract
Adoptive T-cell therapy with tumor-reactive T cells is emerging as a highly effective strategy for eliminating even the most advanced chemotherapy refractory malignancies. Endogenous T cells specific for tumor-associated antigens can sometimes be isolated and expanded from the patient’s blood or tumor infiltrate, or more expeditiously can be engineered by gene transfer to express a T-cell receptor specific for a tumor associated MHC/peptide complex or a synthetic chimeric antigen receptor (CAR) specific for a tumor associated cell surface molecule. The remarkable regression of advanced acute lymphocytic leukemia and lymphoma in patients treated with T cells engineered to express CD19-specific CARs illustrates the potential for this approach to transform clinical care. Therapeutic activity is variable in individual patients, however, and this appears to correlate with the ability of transferred, tumor-reactive T cells to persist and proliferate in vivo, and to retain effector function. These attributes may reflect both the qualities of the T cells that are isolated or engineered for therapy, and the local tumor microenvironment that may contain regulatory T cells; cells that express ligands that engage inhibitor receptors on effector T cells or cytokines that inhibit effector T-cell proliferation. The CD4+ and CD8+ T cell pools in normal individuals contain a variety of naïve, memory, and regulatory T-cell subsets that differ in epigenetic, transcriptional, and functional properties. Because most clinical protocols have used polyclonal peripheral blood mononuclear cells as recipients for CAR gene transfer, the composition of T-cell products that are being administered is highly variable, particularly when the T cells are obtained from cancer patients that have received prior cytotoxic chemotherapy that can skew the phenotypic composition of the peripheral T-cell pool. As a consequence, transferring tumor-targeting receptors into polyclonal unselected cell populations provides poor control over the cellular composition of the final T-cell product, which may in part explain the marked differences in efficacy and toxicity that have been observed in the clinic, and may complicate regulatory approval of these novel therapies. Methods to derive T cells from distinct naïve and memory T-cell subsets have been developed, enabling the rapid production of therapeutic T cells of uniform composition. The results of preclinical studies that illustrate the improved potency of defined T-cell products that are engineered with tumor-specific CARs, and the clinical implementation of this approach in B-cell malignancies will be presented.

Disclosures:
Riddell: Cell Medica: Consultancy, Membership on an entity’s Board of Directors or advisory committees; ZetaRx: Consultancy.
Therapeutic Targeting of the Bcl2 Family
Authors: Letai Anthony , Davids Matthew S. , Chonghaile Triona Ni , Deng Jing , Patel Luv 
Published: October 2019

Abstract
Many, perhaps most, cancer chemotherapy agents kill cancer cells via the mitochondrial pathway of apoptosis that is controlled by the Bcl-2 family of proteins. Bcl-2 family proteins regulate commitment to cell death by controlling mitochondrial outer membrane permeabilization (MOMP). To better understand how cancer cells commit to apoptosis, and what drugs might make them commit to apoptosis, we have studied perturbing mitochondria with BH3 peptides that are derived from pro-death Bcl-2 family proteins. Using this provocative test, which we call BH3 profiling, we are able to measure how close a cell is to the threshold of apoptosis, a property we call “priming”. Priming corresponds to sensitivity to chemotherapy. Moreover, BH3 profiling can be used to detect dependence on Bcl-2 and Bcl-xL for survival, which predicts cytotoxic response to small molecule antagonists such as ABT-199 and ABT-263. In acute lymphoblastic leukemia, we find that dependence on either Bcl-2 or Bcl-xL varies from case to case, with very important consequences for sensitivity to ABT-199 and ABT-263. In chronic lymphocytic leukemia, ABT-199 has already demonstrated significant clinical activity that corresponds to its on-target activity in mitochondria in vitro. We have been testing how this in vitro mitochondrial activity in BH3 profiling assays might be translated into a useful clinical predictive biomarker. Finally, we can measure how short term incubation with many kinds of drugs, including targeted pathway inhibitors, can increase cancer cell priming, including for primary lymphoid malignancy cells. This short term increase in priming predicts subsequent cancer cell death, including in clinical treatment. We call this method “Dynamic BH3 Profiling” and are exploring how it might best be utilized in the clinic.

Disclosures:
Letai: Dana-Farber Cancer Institute: Patents & Royalties; AbbVie: Consultancy.
Leukocyte Adhesion and Activation in Atherothrombosis
Authors: Gawaz Meinrad 
Published: October 2019

Abstract
Platelets interact with a variety of cells, including leukocytes and progenitor cells. Upon activation, platelets release chemokines such as CXCL12/SDF-1 that modulate important functional aspects of vascular inflammation and atherogenesis. Platelet-derived SDF-1 and CyPA regulate chemotaxis, migration, and cell activation in the microenvironment of accumulating platelets. Previously we have shown that platelets are the major source of CXCL12/SDF-1. Platelet-derived SDF-1 provides a strong signal for recruitment of circulating progenitor cells and leukocytes towards vascular and myocardial injury. Patients with severe coronary artery disease have enhanced levels of plasma and platelet-associated SDF-1 that correlates with an enhanced number of circulating progenitor cells and the development of cardiovascular diseases. In patients with both acute myocardial infarction platelet-SDF-1 and an increase in circulating platelet/progenitor cell aggregates, the combination is associated with myocardial recovery and improved prognosis, indicating that platelet-SDF-1 is critical for repair and regeneration of vascular or myocardial injury. Administration of a stable recombinant SDF-1 fusion protein reduces infarct size and preserves myocardial infarction in mice. We showed that platelet-derived SDF-1 is an important survival factor both for platelets (in an autocrine manner) and for surrounding cells. SDF-1 released from activated platelets binds to its receptor CXCR4 and induces internalization of the SDF-1/CXCR4 complex. SDF-1-ligation of CXCR4 augments surface expression of CXCR7; a process coupled via ERK1/2 and cyclophilin A. SDF-1α caused downstream phosphorylation of Erk1/2 and cyclophilin A (CyPA). NIM-811 a CyPA-PPIase-activity inhibitor significantly abolished SDF-1α-driven CXCR7 surface exposure. Moreover, Cypa-/-murine platelets failed to show SDF-1α/CXCR4-mediated CXCR7 translocation. SDF-1α-induced ubiquitination of CXCR7 was essential for its surface translocation and was dependent on Erk1/2 and CyPA-PPIase activity, inhibited by U0126 and NIM-811, respectively. In contrast to wild-type, Cypa-/- murine platelets failed to exhibit a dynamic change in CXCR7 ubiquitination status upon SDF-1α exposure and thus failed in subsequent CXCR7 externalization. SDF-1/CXR4-driven CXCR7 translocation inhibits activation-induced apoptosis of platelets and prolongs platelet survival in vitro and in vivo. Similarly, platelet SDF-1 interacts with monocytes, reduces apoptosis and promotes differentiation of monocytes into macrophages. Inhibition of platelet release and adhesion to endothelial cells reduces monocyte migration and differentiation into macrophages in vitro and attenuates vascular inflammation and atheroprogression in apoE-deficient mice. In conclusion, platelets and platelet interaction with leukocytes are critical in regulation of vascular inflammation and atheroprogression and may offer promising targets for modulating inflammation in disease states.

Disclosures:
No relevant conflicts of interest to declare.
Ras Effector Pathways Differentially Support Proliferation and Survival of Acute Myeloid Leukemia
Authors: Eckfeldt Craig E , Pomeroy Emily J , Lee Robin DW , Hazen Katherine S , Moriarity Branden S , Largaespada David A 
Published: October 2019

Abstract
Mutations that lead to activation of Ras oncogenes and their effector pathways are common in acute myeloid leukemia (AML) making Ras signaling an attractive therapeutic target. To date, efforts to therapeutically target Ras directly have proven difficult and clinical outcomes have been disappointing. The rational development of strategies for targeting Ras effector pathways in AML have been hindered by a limited understanding of the how specific Ras pathways maintain AML proliferation and survival. To determine the roles of specific Ras effector pathways in AML we disrupted signaling through canonical Ras effector pathways (RAF-MEK-ERK, PI3K-AKT-mTOR, and RAL) in murine and human AML cells. Consistent with previously published data (Kim et al. Blood. 2009;113:1086), we found that suppression of NRAS(V12) expression in a tetracycline-repressible NRAS(V12) and Mll-AF9-driven murine AML model (tNM AML) led to AML remission in leukemic mice in vivo and almost completely suppressed leukemic-colony formation in vitro (20-fold reduction, p <0.01). In the tNM model suppression of the NRAS oncogene led to apoptosis of AML cells, but had little effect on cell cycle progression. Surprisingly, pharmacologic inhibition of the major oncogenic Ras effector pathways, RAF-MEK-ERK and PI3K-AKT-mTOR, alone or in combination suppressed AML cell growth by inducing G0/G1 cell cycle arrest in both tNM AML cells and human AML cell lines, but did not promote apoptosis in either. Conversely, pharmacologic inhibition of RALB signaling in both mouse and human AML cells induced apoptosis, but had little effect on cell cycle progression. Notably, these effects were seen in both NRAS mutant (tNM and THP1) and NRAS wild-type (MOLM13) AML cells. In preclinical studies, the RALB inhibitor Dinaciclib and the dual PI3K/mTOR inhibitor BEZ235 synergized to suppress AML growth in vitro, and had a potent anti-leukemic activity against tNM AML in vivo. We are currently evaluating the effects of a dominant negative mutant of RALB, RALB(N28), on AML growth and survival, and investigating whether RALB signaling supports AML survival through TBK1, NF-kB, and BCL2 family members as has been shown for KRAS-driven epithelial cancers (Barbie et al. Nature. 2009;462:108). By elucidating the distinct roles for RAS effector pathways in maintaining AML proliferation and survival, we have uncovered a critical role for RALB signaling in AML survival. This work suggests that combination therapy targeting both Ras effectors that promote proliferation (PI3K/mTOR) and survival (RALB) may provide a rational treatment approach for AML.

Disclosures
Largaespada: NeoClone Biotechnology: Consultancy, Equity Ownership; Discovery Genomics, Inc.: Consultancy, Equity Ownership.