UPF1 regulates myeloid cell functions and S100A9 expression by the hnRNP E2/miRNA-328 balance

BackgroundTherapeutically targeting tumor myeloid cells has emerged as a novel and complementary strategy to existing cancer immunotherapy approaches. The interaction of tumor expressed CD47 with SIRP alpha (signal regulatory protein-alphaa, SIRPA) on macrophages, dendritic cells and neutrophils inhibits key immune effector mechanisms. Targeting SIRPa-CD47 represents a novel approach to enhance anti-tumor immunity by augmenting or reactivating critical tumor clearance mechanisms.H5F9, an antibody against CD47, has shown promising therapeutic activities in patients with MSD, AML and NHL. However, agents targeting CD47 present hematological toxicities and present a huge antigen sink leading to not achieving an optimum therapeutic window. Our approach is to target SIRP alpha, the receptor of CD47 and focus therapeutic targeting to relevant mechanisms related to phagocytosis and myeloid cell activation and at the same time avoid undesired effects of blocking CD47. SIRP gamma, a very close relative of SIRP alpha is expressed on T cells and also binds to CD47. It has been shown that blockade of SIRP gamma-CD47 interaction inhibits T cell proliferation and blocks trans-endothelial T cell migration. Hence, our aim is to generate SIRP alpha selective antibodies that do not cross-react with SIRP gamma and have minimal impact on T cell functions.MethodsUsing Apexigen’s APXiMAB™ proprietary antibody discovery platform, we have generated two novel anti-SIRP alpha antibodies (APX701 & APX702) with differentiated properties as compared to other approaches targeting the CD47/SIRP alpha axis. We have used ELISA, FACS based cell binding and blocking assays, and functional assays including in vitro phagocytosis and antibody-dependent cell phagocytosis (ADCP) in combination with tumor-opsonizing antibody to select APX701 & APX702.ResultsOur novel preclinical-stage APX701 & APX702 antibodies have demonstrated the following attributes: high binding affinity to human SIRP alpha (APX701 Kd = 0.95nM, APX702 Kd = 0.88nM), no binding to SIRP gamma, efficient blockade of SIRP alpha binding to CD47(APX701 IC50 = 1.04nM, APX702 IC50 = 0.80nM), potent macrophage mediated phagocytosis, enhancement of ADCP mediated by tumor-opsonizing antibody and favorable developability CMC profiles. In comparison with the benchmark antibody OSE-172, APX701 & APX702 showed potent phagocytosis activity and ADCP enhancement in all donors tested while OSE-172 induced phagocytosis in only 50% of the donors. This may result from the fact that APX701 and APX702 bind to all major SIRP alpha variants (V1, V2 & V8; covering ~92% population) while OSE 172 only binds to SIRPalpha V1 (~50% population).ConclusionsAPX701 and APX702 demonstrate differentiated anti-SIRPalpha activities by enhancing myeloid cell-mediated anti-tumor immunity and reactivating critical tumor clearance mechanisms within the tumor microenvironment.

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Innate immune deficiencies in patients with COVID-19

10.1101/2021.03.29.21254560 ◽

2021 ◽

Author(s):

Marine Peyneau ◽

Vanessa Granger ◽

Paul-Henri Wicky ◽

Dounia Khelifi-Touhami ◽

Jean-François Timsit ◽

...

Keyword(s):

Innate Immunity ◽

Myeloid Cell ◽

Innate Immune ◽

Activation Markers ◽

Icu Patients ◽

Cell Functions ◽

Hla Dr ◽

Immune Deficiencies ◽

Severe Infections

AbstractCOVID-19 can cause acute respiratory distress syndrome (ARDS), leading to death in a significant number of individuals. Evidence of a strong role of the innate immune system is accumulating, but the precise cells and mechanism involved remain unclear. In this study, we investigated the links between circulating innate phagocyte phenotype and functions and severity in COVID-19 patients. Eighty-four consecutive patients were included, 44 of which were in intensive care units (ICU). We performed an in-depth phenotyping of neutrophil and monocyte subpopulations and measured soluble activation markers in plasma. Additionally, myeloid cell functions (phagocytosis, oxidative burst, and NETosis) were evaluated on fresh cells from patients. Resulting parameters were linked to disease severity and prognosis. Both ICU and non-ICU patients had circulating neutrophils and monocytes with an activated phenotype, as well as elevated concentrations of soluble activation markers (calprotectin, myeloperoxidase, neutrophil extracellular traps, MMP9, sCD14) in their plasma. ICU patients were characterized by increased CD10low CD13low immature neutrophils, LOX-1+ and CCR5+ immunosuppressive neutrophils, and HLA-DRlow CD14low downregulated monocytes. Markers of immature and immunosuppressive neutrophils were strongly associated with severity and poor outcome. Moreover, neutrophils and monocytes of ICU patients had impaired antimicrobial functions, which correlated with organ dysfunction, severe infections, and mortality. Our study reveals a marked dysregulation of innate immunity in COVID-19 patients, which was correlated with severity and prognosis. Together, our results strongly argue in favor of a pivotal role of innate immunity in COVID-19 severe infections and pleads for targeted therapeutic options.One Sentence SummaryOur study reveals a marked dysregulation of innate immunity in COVID-19 patients, which correlates with severity and prognosis.

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Loss of miR-1469 expression mediates melanoma cell migration and invasion

PLoS ONE ◽

10.1371/journal.pone.0256629 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0256629

Author(s):

Mallory J. DiVincenzo ◽

Zoe Barricklow ◽

Emily Schwarz ◽

Maribelle Moufawad ◽

J. Harrison Howard ◽

...

Keyword(s):

Disease Progression ◽

Cell Lines ◽

Melanoma Cell ◽

Myeloid Cell ◽

Migration And Invasion ◽

Specific Gene ◽

Primary Cutaneous Melanoma ◽

Cell Functions ◽

Invasive Capacity ◽

A375 Melanoma Cell Line

Tumor ulceration is considered one of the most prognostically significant findings in primary cutaneous melanoma, associated with decreased disease-free and overall survival. However, the unique features associated with ulcerated melanoma that contribute to a poor prognosis in affected patients remain poorly defined. microRNAs are small, non-coding RNAs that function to inhibit expression of specific gene targets, therefore altering the functions of cells in which they are expressed. miR-1469 is a novel miR with significantly decreased expression in ulcerated melanoma tissue relative to non-ulcerated tumors. We hypothesized that loss of miR-1469 expression in melanoma contributes to altered tumor cell functions mediating disease progression. Transfection of a miR-1469 mimic resulted in a significant reduction in the migratory and invasive capacity of the CHL1 and MEL39 melanoma cell lines (>58.1% reduction, p < 0.0332), as well as the invasive capacity of the A375 melanoma cell line (>50% reduction, p < 0.0021). Expression of myeloid cell leukemia-1 (MCL1), a miR-1469 target gene, was reduced in the A375 and MEL39 cell lines by immunoblot. No significant differences in viability, resistance to apoptotic stimuli, or proliferation were observed following transfection. These findings together demonstrate how migration and invasion are specific functions through which miR-1469 expression in melanoma cells can contribute to the differences in disease progression associated with tumor ulceration.

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Identification of Novel MYB Target Genes

Blood ◽

10.1182/blood.v112.11.3580.3580 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3580-3580

Author(s):

Liang Zhao ◽

Faisal Al- Owaidi ◽

Diwakar R Pattabiraman ◽

Emily Verrier ◽

Anna Tsykin ◽

...

Keyword(s):

Target Genes ◽

Cellular Proliferation ◽

Myeloid Cell ◽

Cell System ◽

Suppressor Activity ◽

Gm Csf ◽

Cell Functions ◽

Tumour Suppressor Activity ◽

Myeloid Cell Line ◽

Positive Regulators

Abstract The MYB oncogene encodes a transcription factor, Myb, which is essential for normal haemopoiesis and also for the proliferation of most acute leukaemias (reviewed in ref. 1). While a number of Myb target genes have been reported previously, these do not completely account for key elements of MYB’s activity, including its pro-leukaemic and differentiation-suppressing functions. We hypothesised that this reflects the fact that previous screens may have not been sufficiently comprehensive and/or employed the most appropriate cell systems. Thus we have embarked upon a program to identify and validate Myb targets critical for these functions. Here we report results from extensive expression profiling studies using a conditionally myb-transformed myeloid cell line, ERMYB (2). We have used ~44,000-element Illumina Beadchips in conjunction with a kinetic profiling strategy that selects candidates based on rapid, statistically-significant and consistent responses to both activation and inactivation of Myb. This has resulted in the identification of a substantial number of candidate Myb-activated and -repressed genes (381 and 502, respectively). In addition, we have used this cell system to identify candidate Myb-regulated microRNAs. Inspection of the list of candidate Myb-activated genes revealed several previously-described Myb targets including myc, bcl2, gstm1 and mpo, providing additional confidence in our approach. Our focus to date has been on novel candidates that may mediate myb’s ability to enhance proliferation, suppress differentiation and possibly suppress apoptosis. Q-RT-PCR was used as an initial validation step for a number of such targets; to date 13/14 genes identified by array screening have been confirmed by this method in ERMYB cells. A second approach to validation is to confirm correlation with Myb over-expression in a second cell system (FDB-1) (3). As in primary cells, enforced Myb expression can suppress differentiation and promote proliferation of these cells in the presence of GM-CSF (4). Amongst the Myb-activated genes are gfi1 and nucleostemin/gnl3, which are involved in stem-cell functions, cellular proliferation and in the case of gfi1, lineage-specific functions. Strikingly, candidate Myb-repressed genes include several important positive regulators of haemopoietic differentiation and/or negative regulators of proliferation, namely gata3, sfpi1/pu.1, cebpb, junb, klf’s-3,-6 -13 and btg1. Most of these genes have evolutionarily conserved internal or proximal candidate Myb binding sites. Our progress in validating these by chromatin immunoprecipitation will be presented. Finally, we have identified a number of microRNAs that are potentially regulated by Myb. These include members of the miR-17–92 cluster and mir-146b, which appear to be activated and repressed by Myb, respectively. These have been validated by Q-PCR for both the mature miR and the precursor pri-miR transcript. Interestingly, the mir-17–92 cluster has been strongly implicated in oncogenesis and cell cycle regulation (5), while miR-146a/b may have tumour suppressor activity.

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CD13 is dispensable for normal hematopoiesis and myeloid cell functions in the mouse

Journal of Leukocyte Biology ◽

10.1189/jlb.0210065 ◽

2010 ◽

Vol 88 (2) ◽

pp. 347-359 ◽

Cited By ~ 24

Author(s):

B. Winnicka ◽

C. O'Conor ◽

W. Schacke ◽

K. Vernier ◽

C. L. Grant ◽

...

Keyword(s):

Myeloid Cell ◽

Cell Functions

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230 Preclinical efficacy of CLEC-1 antagonist as novel myeloid immune checkpoint therapy for oncology

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.230 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A245-A245

Author(s):

Vanessa Gauttier ◽

Marion Drouin ◽

Sabrina Pengam ◽

Javier Saenz ◽

Bérangère Evrard ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Cells ◽

Immune Cell ◽

Human Tumor ◽

Myeloid Cell ◽

Lectin Receptors ◽

Knock Out ◽

Cell Functions ◽

Preclinical Efficacy

BackgroundC-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) tempers T cell’s responses in vivo by limiting antigen cross-presentation by cDC1. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment, in particular tumor-associated macrophages.MethodsMacrophages were generated from monocytes of healthy volunteers for phagocytosis assays. MC38 and Hepa 1.6 murine tumor cells were implanted in Clec1a KO or KI mice for immunotherapeutic treatment evaluation.ResultsUsing newly developed anti-human CLEC-1 monoclonal antibodies (mAbs), we found that antagonist anti-CLEC-1 mAbs with the capacity to block CLEC-1/CLEC-1Ligand interaction, as opposed to non-antagonist CLEC-1 mAbs, increase the phagocytosis of CLEC-1Ligand-positive human tumor cells by human macrophages, in particular when opsonized by tumor-associated antigen mAbs (Rituximab, Cetuximab, Trastuzumab) or with anti-CD47 mAb (Magrolimab). In-vivo, CLEC-1 knock-out (KO) mice (n=19) display significant prolonged survival in monotherapy as compared to wild-type littermates (n=12) in an orthotopic hepatocellular carcinoma (HCC) model and anti-tumor memory responses was demonstrated by tumor rechallenge in cured mice. CLEC1 KO mice also illustrate significant eradication of MC38 colorectal tumors in combination with chemotherapy promoting CLEC-1Ligand expression by tumor cells (n=16 with Gemcitabine or n=11 with Cyclophosphamide). HCC tumor microenvironment analysis after 2 weeks of tumor implantation shows significantly higher number of CD8+ and memory CD8+ T cells with reduced PD1 expression in CLEC1 KO animals (n=16 versus n=12 for KO vs WT mice respectively). Finally, we recently generated human CLEC-1 knock-in mice expressing the extracellular human CLEC1 domain fused to the intracellular mouse CLEC1 tail and confirmed preclinical efficacy in vivo with anti-human CLEC1 antagonist mAb in monotherapy in the orthotopic HCC model.ConclusionsThese data illustrate that CLEC-1 inhibition represents a novel therapeutic target for immuno-oncology modifying T cell immune responses and tumor cell phagocytosis by macrophages.

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Mir-590-5p, Mir-219-5p, Mir-15b and Mir-628-5p Are Commonly Regulated by IL-3, GM-CSF and G-CSF in Acute Myeloid Leukemia,

Blood ◽

10.1182/blood.v118.21.3520.3520 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3520-3520

Author(s):

Amanda Favreau ◽

Erin Cross ◽

Pradeep Sathyanarayana

Keyword(s):

Acute Myeloid Leukemia ◽

Mirna Expression ◽

Myeloid Leukemia ◽

Specific Inhibitor ◽

Myeloid Cell ◽

Gm Csf ◽

Cell Functions ◽

Novel Mirna ◽

Acute Myeloid ◽

Myeloid Progenitors

Abstract Abstract 3520 IL-3, GM-CSF and G-CSF are predominant regulators for growth and differentiation of myeloid progenitors. Interestingly, they all signal via a common JAK2-STAT5 pathway in myeloid progenitor compartments. However, the specific mechanism through which JAK2-STAT5 responds differentially to early-acting and lineage restricted cytokines, particularly in leukemic and stem/progenitor cells, is largely unresolved. Aberrations in IL-3, GM-CSF and G-CSF induced signaling are frequently reported in acute myeloid leukemia (AML). microRNA (miRNA) play several crucial roles during hematopoiesis that include lineage decisions, stem cell progenitor transitions, niche control and other cell functions. Recent investigations have linked aberrant miRNA expression with AML. We hypothesized that a unique response of leukemic myeloid progenitors to IL-3, GM-CSF, and G-CSF are possibly mediated in part by distinct regulation at the miRNA level. Therefore, herein, by utilizing a unique leukemic myeloid cell line, AML-193, that responds to both early and late acting cytokines, we profiled IL-3, GM-CSF and G-CSF regulated miRNA signatures in leukemic myeloid progenitors. For miRNA profiling, AML-193 cells were initially exposed to IL-3 for 3 days followed by GM-CSF for 3 more days and subsequently to G-CSF for 3 days. We then profiled miRNA expression induced by IL-3, GM-CSF and G-CSF in AML-193 cells by treating the respective cohorts post growth factor deprivation with corresponding cytokines. Using SA Bioscience's complete Human V2.0 miRNA Genome Array platform for real-time qPCR-based miRNA expression profiling, we investigated miRNA signatures regulated by IL-3, GM-CSF and G-CSF for n=704 miRNAs in AML-193 cells. Frequencies of false positives were avoided using stringent filters. IL-3 specifically regulated 54 miRNAs and those miRNAs that were highly regulated included miR-362-3p, miR-590-3p, miR-340, miR-24-2, miR-1183 and miR-99a. GM-CSF specifically regulated miRNAs included let-7f, let-7a*, miR-195, miR-122, miR-376-c and miR-33a. G-CSF specifically regulated set included miR-21*, miR-192*, miR-32*, miR-7-1*, miR-545* and miR-37-4a*. Interestingly, 301 miRNAs were commonly regulated by IL-3, GM-CSF and G-CSF. Among the commonly regulated miRNAs, the ones that were subjected to high levels of regulation included miR-590-5p, miR-219-5p, miR-92-a1*, miR-378*, miR-548-3p, miR-29a*, miR-590-3p, miR-203, miR-363, miR-454, miR-340, miR-196a, miR-152, miR-10b, miR-24-2*, miR-10a, miR-182, miR-27a*, and miR-199a-3p. Interestingly, the commonly regulated miRNAs demonstrated a directional regulation in the order of IL-3>GM-CF>G-CSF. Analysis of the potential targets of significantly regulated miRNAs revealed important functional roles in myeloid cell development and differentiation. Conserved targets of miR-590-5p included BMPR2, PCBP2 and KLF3. Targets for miR-219-5p included Smad4 and GADD45b. Targets for miR-362-3p included Sox17, an inhibitor of the Wnt signaling pathway. Furthermore, to identify the JAK2-STAT5 responsiveness of miR-219-5p, miR-362-3p, and miR-590-5p, a STAT5 specific inhibitor (N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide) was used prior to cytokine treatment. Interestingly, IL-3 and GM-CSF mediated induction of miR-590-5p, miR-219-5p and miR-362-3p were not significantly affected by STAT5 inhibitor. However, the G-CSF induction of miR-219-5p and miR-590-5p were significantly inhibited by the STAT5 specific inhibitor. In summary, we have discovered for the first time novel miRNA profiles regulated by IL-3, GM-CSF and G-CSF in an acute myeloid leukemia progenitor cell model. Importantly, we have established a cytokine regulated miRNome for leukemic myeloid progenitors and set the stage for future investigations in leukemic stem cells to delineate the pathological roles of dysregulated miRNAs in AML. In addition, our study displayed that G-CSF induced a subset of miRNAs that are principally regulated via the STAT5 circuit in AML. Further, these novel miRNA signatures may have therapeutic implications for targeting dysregulated miRNAs by antagomir strategy or miRNA replacement therapy, paving the way for the development of novel miRNA-based therapeutic interventions in AML. Disclosures: No relevant conflicts of interest to declare.

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Pleiotropic consequences of Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses

Blood ◽

10.1182/blood-2004-01-0207 ◽

2004 ◽

Vol 104 (4) ◽

pp. 1191-1197 ◽

Cited By ~ 71

Author(s):

Anita Mangla ◽

Anupriya Khare ◽

Varanasi Vineeth ◽

Nagesh Narayan Panday ◽

Asok Mukhopadhyay ◽

...

Keyword(s):

Tyrosine Kinase ◽

Inflammatory Responses ◽

Apoptotic Cell ◽

Myeloid Cell ◽

Polymorphonuclear Neutrophil ◽

Neutrophil Granulocytes ◽

Oxygen Intermediates ◽

Cell Functions ◽

Bruton Tyrosine Kinase

AbstractBruton tyrosine kinase (Btk), a non-receptor-associated tyrosine kinase of the Tec family, appears to participate in many myeloid cell functions. We show that macrophages from X-linked immunodeficient (XID) mice lacking functional Btk cannot generate efficient bursts of reactive oxygen intermediates (ROIs). The induction of apoptotic cell death by inflammatory stimuli is also enhanced in XID macrophages. Phagocytosis of bacterial particles is only marginally affected in them. In vivo, XID mice show reduced severity of inflammatory diseases in models of experimental autoimmune encephalomyelitis (EAE), dextran sulfate sodium (DSS)-induced colitis, and carrageenan-induced acute edema. Also, polymorphonuclear neutrophil granulocytes (PMNs) in XID mice show poor ROI and nitric oxide (NO) induction, along with a reduction in PMN recruitment to peritoneal inflammation. XID mice show reduction in PMN numbers in peripheral blood, and their bone marrow shows a reduction in the numbers of both monocytic and granulocytic lineages, extending to the earliest progenitor populations. Thus, Btk is likely to play a significant role at multiple points during the development and functioning of the myeloid lineages, affecting the outcome of many infectious as well as noninfectious inflammatory events in vivo. (Blood. 2004;104:1191-1197)

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Fibg390-396A Limits Metastatic Potential Via a Mechanism Independent of Fibrin-Myeloid Cell Interactions, but Prospectively through the Disruption of FXIII-Mediated Fibrin Crosslinking

Blood ◽

10.1182/blood-2019-131644 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2385-2385

Author(s):

Brenton J Francisco ◽

Balkrishan Sharma ◽

Leah A. Rosenfeldt ◽

Duaa Mureb ◽

Joseph S. Palumbo

Keyword(s):

Factor Xiii ◽

Germ Line ◽

Myeloid Cells ◽

Myeloid Cell ◽

Metastatic Potential ◽

Binding Motif ◽

Experimental Metastasis ◽

Cell Functions ◽

Polymer Formation ◽

Immunocompetent Mice

Previous studies have shown that fibrin(ogen) supports metastasis. In order to better define the mechanisms coupling fibrin(ogen) to metastasis, we compared metastatic potential in immunocompetent mice carrying specific structure/function alterations in fibrinogen and controls. FibrinogenAEK mice, which have a germ-line mutation in the thrombin cleavage site that essentially "locks" fibrinogen in its soluble state, exhibited diminished metastatic potential relative to FibWT mice. Notably, previous studies have established that factor XIII is also a significant determinant of metastasis. Taken together, these studies suggest that stable fibrin polymer formation is important in the metastatic process. However, FibAEK retained significant metastatic potential relative to mice with complete fibrinogen deficiency, indicating that fibrinogen possesses prometastatic properties in the absence of polymer formation. Fibrin(ogen) has also been shown to mediate inflammatory cell functions through direct engagement of leukocyte integrins independently of its role in platelet aggregation. Given previous studies demonstrating that myeloid cells are promote metastatic potential, we hypothesized that fibrin(ogen)-mediated leukocyte engagement represents one mechanism by which fibrinogen drives metastasis. Consistent with this view, mice expressing a mutant fibrinogen lacking the y chain binding motif for the leukocyte integrin αMβ2 (Fibγ390-396A) had diminished metastatic potential in both spontaneous and experimental metastasis assays. Furthermore, fate analyses revealed that Fibγ390-396A results in diminished survival of newly-formed metastatic foci. The early reduction in metastatic potential observed in Fibγ390-396A mice suggests that fibrin(ogen) promotes metastasis by recruiting myeloid cells to the early metastatic niche. In order to explore this hypothesis, we performed experimental metastasis assays in immunocompetent mice in which macrophages or neutrophils were specifically depleted using either clodronate or an anti-Ly6G antibody. In contrast to mice carrying the Fibγ390-396A mutation, depletion of macrophages or neutrophils had no significant impact on the early survival of metastatic foci. These studies suggest that expression Fibγ390-396A limits metastatic potential via a mechanism independent of interactions with myeloid cells. In addition to disrupting fibrin-αMβ2 interactions, elimination of the γ chain 390-396a binding motif has also been shown to limit factor XIII binding to fibrinogen. Given the established importance of FXIII in metastasis, it is conceivable that even subtle alterations in the kinetics of fibrin cross-linking resulting from expression of Fibγ390-396A limits metastatic potential. Disclosures Palumbo: Ionis Pharmaceuticals: Research Funding.

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