LC3-Associated Phagocytosis in Bone Marrow Macrophages Suppresses AML Progression through Mitochondrial DAMP Induced Sting Activation

Abstract The bone marrow (BM) microenvironment regulates acute myeloid leukemia (AML) initiation, proliferation and chemotherapy resistance. Following cancer cell death, a growing body of evidence suggests an important role for uncleared apoptotic debris in regulating the immunologic response to, and growth of, solid tumors. LC3-associated phagocytosis (LAP) maintains tissue homeostasis by regulating immune responses, such as tumor immunity. Here we investigate the role of LAP in macrophage within the BM microenvironment of AML. We find that depletion of BM macrophages via clodronate liposomes increased AML growth in-vivo. We show that LAP is an important pathway in BM macrophage to process dead and dying cells in the AML microenvironment. We used two syngeneic leukemia models (HOXA9/Meis1 and MN1) to investigate the role of LAP on AML proliferation. AML cells were injected into LAP deficient (Atg16L1 E230-) and wild-type (Atg16L1 E230+) mice. Targeted inhibition of LAP leads to accumulation of apoptotic cells (AC) and apoptotic bodies (AB) in the tumor microenvironment resulting in accelerated leukemia growth and decreased animal survival. Mechanistically, we show, via cytokine arrays and gene analysis, that the phagocytosis of AML derived AB via LAP in BM macrophage resulted in STING pathway activation in the phagocytic cells. Furthermore, through inhibition of STING using H-151 STING inhibitor, we show that STING activation in vivo supressed leukemia growth. STING activation can lead to a type I IFN response and to recruitment of cytotoxic T-cells. We saw no increase in CD8 + T-cell numbers or activation, however, via ex vivo analysis found that STING activation is required for phagocytic functions in macrophages. Next, we found that leukemic AB can induce a STING response in BM derived macrophages and that leukemic AB have increased mitochondria content that are processed by macrophages. Moreover, we identify that mitochondrial damage associated molecular patterns (DAMPs) from leukemic AB are processed by BM macrophages via LAP. Additionally, the depletion of mitochondrial DNA (mtDNA) in AML derived AB identified that the mtDNA from leukemic AB is responsible for the induction of STING signalling in BM macrophages. In summary, we report that LAP in BM macrophage of apoptotic debris in the AML microenvironment suppresses leukemic growth, through mechanisms stimulated by AML apoptotic bodies which contain mtDNA in the BM microenvironment. This process is mediated by the activation of the STING pathway. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Absence of the Rho GTPase Activating Protein p190-B Enhances Long Term Hematopoietic Stem Cell Engraftment

Blood ◽

10.1182/blood.v112.11.614.614 ◽

2008 ◽

Vol 112 (11) ◽

pp. 614-614 ◽

Cited By ~ 1

Author(s):

Haiming Xu ◽

Hartmut Geiger ◽

Kathleen Szczur ◽

Deidra Deira ◽

Yi Zheng ◽

...

Keyword(s):

Bone Marrow ◽

Ex Vivo ◽

Gtpase Activating Protein ◽

Self Renewal ◽

Hematopoietic Stem ◽

Proliferation And Differentiation ◽

Serial Transplantation

Abstract Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow (BM), self-renewal, proliferation and differentiation to mature blood cells. However, the molecular regulation of HSC engraftment is still poorly defined. Small Rho GTPases are critical regulator of cell migration, proliferation and differentiation in multiple cell types. While their role in HSC functions has begun to be understood, the role of their regulator in vivo has been understudied. P190-B GTPase Activating Protein (GAP), a negative regulator of Rho activity, has been implicated in regulating cell size and adipogenesis-myogenesis cell fate determination during fetal development (Sordella, Dev Cell, 2002; Cell 2003). Here, we investigated the role of p190-B in HSC/P engraftment. Since mice lacking p190-B die before birth, serial competitive repopulation assay was performed using fetal liver (FL) tissues from day E14.5 WT and p190-B−/− embryos. WT and p190-B−/− FL cells exhibited similar levels of engraftment in primary recipients. However, the level of contribution of p190-B−/− cells to peripheral blood and bone marrow was maintained between the primary and secondary recipients and still easily detectable in tertiary recipients, while the level of contribution of FL WT cells dramatically decreased with successive serial transplantion and was barely detectable in tertiary recipients. The contribution to T cell, B cell and myeloid cell reconstitution was similar between the genotypes. A pool of HSC was maintained in serially transplanted p190-B−/− animals, since LinnegScaposKitpos (LSK) cells were still present in the BM of p190-B−/− secondary engrafted mice while this population disappeared in WT controls. Importantly, this enhanced long term engraftment was due to a difference in the functional capacity of p190-B−/− HSC compared to WT HSC since highly enriched p190-B−/− HSC (LSK) demonstrated similar enhanced serial transplantation potential. Because previous studies have suggested that the loss of long term function of HSC during serial transplantation can depend, at least in part, on the upregulation of the cyclin dependent kinase inhibitor p16Ink4a (Ito et al, Nat Med 2006), the expression of p16Ink4a was examined during serial transplantation. While expression of p16Ink4a increased in WT HSC in primary and secondary recipients, p16Ink4a remained low in p190-B−/− HSC, which indicated that p190-B-deficiency represses the upregulation of p16Ink4a in HSC in primary and secondary transplant recipients. This provides a possible mechanism of p190-B-mediated HSC functions. We next examined whether p190-B-deficiency may preserve the repopulating capacity of HSC/P during ex vivo cytokine-induced culture. While freshly isolated LSK cells from WT and p190-B−/− mice exhibited comparable intrinsic clonogenic capacity, the frequency of colony-forming unit after 7 days in culture was 2 fold-higher in p190-B−/− compared with WT cultures, resulting in a net CFU expansion. Furthermore, competitive repopulation assays showed significantly higher repopulating activity in mice that received p190-B−/− cultured cells compared with WT cells equivalent to a 4.4-fold increase in the estimated frequency of repopulating units. Interestingly, p190-deficiency did not alter cell cycling rate or survival both in vivo and in vitro. Therefore, p190-B-deficiency maintains key HSC functions either in vivo or in ex vivo culture without altering cycling rate and survival of these cells. These findings define p190-B as a critical regulator of HSC functions regulating self renewal activity while maintaining a balance between proliferation and differentiation.

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CD44v6 Is Required For In Vivo Tumorigenesis Of Human AML and MM Cells: Role Of Microenvironmental Signals and Therapeutic Implications

Blood ◽

10.1182/blood.v122.21.605.605 ◽

2013 ◽

Vol 122 (21) ◽

pp. 605-605 ◽

Cited By ~ 1

Author(s):

Benedetta Nicolis di Robilant ◽

Monica Casucci ◽

Laura Falcone ◽

Barbara Camisa ◽

Bernhard Gentner ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Cells ◽

Conflicts Of Interest ◽

Human Leukemia ◽

Vegf Receptor ◽

Durable Response ◽

Wide Range ◽

Cd44v6 Expression

Abstract Background Targeting the interactions between tumor cells and their microenvironment is an exciting new frontier in cancer therapy. The biology of acute myeloid leukemia (AML) and multiple myeloma (MM) is characterized by addiction to specific signals uniquely provided within the bone marrow (BM), where tumor cells preferentially home and locally thrive. The hyaluronate receptor CD44 was shown to be required for retroviral-induced leukemogeneis in syngeneic mouse models. Conversely, CD44 mAbs interfere with human leukemia initiation in immunocompromised mice by inhibiting leukemia stem cell homing to the bone marrow (BM). The therapeutic potential of CD44 mAbs is also under clinical investigation in humans. Much less is known on the role of the differently spliced CD44 variant isoforms. The expression of exon 6 (CD44v6) conveys additional properties to standard CD44, like binding to osteopontin and cooperation with different tyrosine kinase receptors (RTKs), like VEGF receptor type II and c-Met. Interestingly, CD44v6 is the most abundantly expressed CD44 isoform in both AML and MM, where it correlates with a bad prognosis. Since CD44v6 expression is much more tumor-restricted than CD44, targeting this isoform may have a better efficacy/toxicity profile than targeting the standard molecule. Aim To preclinically validate CD44v6 as a therapeutic target in AML and MM Results By FACS analysis and RT-qPCR, we established CD44v6 over expression in a relevant fraction of leukemic blasts from AML pts (15/25, 60%) with preference for the M4-5 FAB subtypes, and in the majority of malignant plasmacells from MM pts (13/15, 87%). CD44v6 was also over expressed on THP-1, Kasumi and U937 human AML cells, and on MM.1S, XG-6 and XG-7 MM cells. To address the specific role of CD44v6 in BM homing, we pretreated MM1.S cells with either a CD44 mAb (SFF-2) or a CD44v6 mAb (VFF-18) and infused them i.v. in NSG mice. Unexpectedly, while SFF-2 almost completely inhibited early (18hrs) homing to the BM compared with an irrelevant mAb, VFF-18 had no effect. To rule out confounding variables associated with specific mAb clones, we silenced CD44v6 expression in MM1.S cells by lentiviral-mediated shRNA transduction and confirmed no difference in BM homing compared with control LV-transduced cells. Longer follow-ups (4-6 weeks) however revealed that, despite unaltered rates of in vitro proliferation, CD44v6-silenced MM1.S cells were severely hampered in their tumorigenic capacity in vivo (P<0.001). These results were confirmed by using THP-1 cells (P<0.001) and primary leukemic blasts (P<0.01). Hypothesizing that CD44v6 may be crucial for in vivo tumorigenesis by cooperating with RTKs, we set-up a co-culture system with BM-derived mesenchymal stromal cells (MSCs), which are producers of VEGF and the c-Met ligand HGF. MSCs protected a wide range of tumor cells, including primary leukemic blasts, from spontaneous apoptosis (P<0.05) and from apoptosis induced by Ara-C or daunorubicin (P<0.01), or bortezomib in the case of MM cells (P<0.001). Comparable results were obtained by using MSC supernatants, hinting to a causative soluble factor, which was neither VEGF nor HGF, as demonstrated by inhibition experiments with bevacizumab and crizotinib, respectively. Noteworthy, MSCs or their supernatants prompted a significant up-regulation of CD44v6 expression levels (P<0.01). Most importantly, preventing CD44v6 up-regulation on tumor cells by shRNA silencing restored their sensitivity to spontaneous and drug-induced apoptosis (P<0.01). Conclusions These results clearly indicate that CD44v6 is dispensable for BM homing, but responsible for AML and MM addiction to microenvironmental signals. Combining CD44v6 targeting with cytotoxic chemotherapy might interfere with this vicious circle and result in higher and/or more durable response rates. Disclosures: No relevant conflicts of interest to declare.

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Src-like Adaptor Protein Promotes the CD103+ dendritic Cell Homeostasis in the Lung

Blood ◽

10.1182/blood.v128.22.1339.1339 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1339-1339

Author(s):

Namit Sharma ◽

Pan Zhongda ◽

Tracy Lauren Smith ◽

Savar Kaul ◽

Emilie Ernoult ◽

...

Keyword(s):

Bone Marrow ◽

Immune Response ◽

Immune Cell ◽

Conflicts Of Interest ◽

Adaptor Protein ◽

Cell Receptor ◽

Apoptotic Cells ◽

Direct Role

Abstract Dendritic cells (DCs) along with mast cells function as sentinels for the innate immune system and perform as antigen presenting cells (APCs) to mount an adaptive immune response against invading pathogen. FLT3 receptor tyrosine kinase signaling has been shown to regulate the homeostatic mechanisms of subsets of DCs particularly, CD103+DCs compared to CD11b+DCs. CD103+DCs are regarded as APCs with superior capabilities to mount an effective immune response, thus understanding their homeostasis mechanism(s)/function is of paramount importance to devise effective therapeutics including DC vaccines. The Src-like adapter protein (SLAP) has been shown to dampen the signaling downstream of receptor tyrosine kinases including FLT3, cKit, and immune cell receptors including T cell receptor, B cell receptor, and Granulocyte-monocyte colony stimulating factor receptor via by recruiting c-Cbl, an ubiquitin ligase. Here, we report that SLAP deficient mice (KO) have reduced numbers of CD103+DC in lung while equal numbers in liver and kidney compared to control mice. To further confirm reduced CD103+DC in the lung, efferocytosis assays that are dependent upon CD+103 DC in lung epithelium to cleanse the apoptotic cells were performed. Flow cytometric quantification of CD103+DCs that uptake fluorescently labeled apoptotic cells administered via intranasal route and migrate to mediastinal lymph nodes confirmed reduced number of CD103+DCs in SLAP KO mice. Further analysis of DC progenitor populations showed reduced pre-DC progenitor in the lung in SLAP KO mice while bone marrow compartment showed equal progenitor populations including pre-DC and common dendritic progenitors suggesting the role of SLAP in localized FLT3 signaling in the lung. Consistently, DCs in lymphoid compartment including spleen, thymus, inguinal and popliteal lymph node did not show any defects. Upon further dissecting the cellular mechanism, SLAP KO DCs showed increased apoptosis while having similar proliferation potential in vivo at steady state.Bone marrow progenitors from SLAP KO mice failed to generate mature DCs in the presence of FLT3 ligand in vitrodue to enhanced apoptosis at early time points. Also, submaximal inhibition of FLT3 with an inhibitor, quizartinib partially rescues the apoptotic phenotype of SLAP KO bone marrow progenitors suggesting a cell-intrinsic role of SLAP in the survival of DCs. Biochemical analysis revealed that SLAP is directly recruited to the juxta-membrane residues of the FLT3 receptor in an inducible manner suggesting a direct role of SLAP in the regulation of FLT3 signaling. Phosphoflow analysis of DCs generated in the combined presence of GMCSF and FLT3 ligands showed that SLAP promotes the signaling to SHP2 while perturbs signaling to the mTOR pathway. Together these results suggest that SLAP is a critical regulator of CD103+DCs homeostasis in selective peripheral organs including the lung. Disclosures No relevant conflicts of interest to declare.

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Targeting Nucleolin for Reversal of Chemotherapy Resistance in Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-127073 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5058-5058

Author(s):

Jianda Hu ◽

Yanxin Chen ◽

Zhengjun Wu ◽

Lingyan Wang ◽

Jingjing Wen ◽

...

Keyword(s):

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

Drug Sensitivity ◽

De Novo ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Mrna Level ◽

Chemotherapy Resistance

Chemotherapy resistance is considered to be the principal cause of ineffective treatment in acute lymphoblastic leukemia (ALL). Nucleolin (NCL) is high expression andplays oncogenic roles in most cancers. However, less research on the role of NCL in hematologic malignancies was noted. Our previous studies have showed that overexpression of NCL was associated with worse prognosis in the patients with acute leukemia and NCL expressionwashigher in resistant HL-60/ADR than in sensitive HL-60 cells. The potential mechanisms of NCL in chemotherapy resistance have yet to be revealed. Here we presented that expression of NCL was associated positively with chemotherapy resistance and poor prognosis in ALL. Overexpressed NCL at both mRNA and protein level was relevant to a poorer overall survival (OS) and relapse free survival (RFS), indicating NCL as an independent prognostic marker in ALL. mRNA level of NCL in de novo ALL was quantitatively higher than in complete remission(CR) status, and refractory/relapse ALL had the highest level. Upon above clinical data, we further investigated the mechanism(s) by which NCL regulated drug resistance in ALL cells. Remarkably, NCL expression was higher in resistant ALL cells relative to sensitive parental cells. When treated with ADM, NCL level was decreased in sensitive parental cells while unchanged in resistant cells. Overexpressing NCL suppressed drug sensitivity, altered drug effluxion and decreased intracellular drug accumulation, while inhibition of NCL led to a completely reversed appearance, more intracellular Adriamycin(ADM) mean fluorescence intensity (MFI) and percentage of ADM accumulated cells population. Overexpression of NCL increased significantly the IC50 of ADM. The IC50 of ADM on Jurkat-NCL-overexpression(OE), Jurkat-NCL-knockdown(KD), Molt-4-NCL-OE, Molt-4-NCL-KD, Nalm-6-NCL-OE, Nalm-6-NCL-KD were 1.362±0.271μg/ml, 0.077±0.010μg/ml, 4.863±0.733μg/ml, 0.081±0.018μg/ml, 0.237±0.042μg/ml and 0.046±0.002μg/ml, respectively (P <0.05). Involvement of ATP-binding cassette (ABC) transporters was proved in NCL mediated drug resistance. Silencing NCL resulted in a decrease of P-gp, MRP1, LRP and BCRP in ALL cells, and NCL overexpression increased the MRP1, LRP and BCRP. The Akt/mTOR and ERK signaling pathways were involved in this procedure. Notably, co-IP assays confirmed the NCL-Ras, NCL-ERK and NCL-BCRP interaction. For intervention study, aptamer AS1411, a NCL inhibitor, could reduce drug resistance in ALL cell lines and primary ALL cells.Moreover, AS1411 treatment decreased BCRP protein expression. Furthermore, the ALL leukemia models that nude mice engrafted with Nalm-6 cells and NCG mice engrafted with Luc+ Nalm-6 cells were established, then treated with ADM plus AS1411 or control CRO for comparison drug sensitivity and survival. Growth of subcutaneous xenograft tumors was inhibited in those treated with AS1411 or ADM, compared to their respective controls treated with CRO or PBS. The stronger inhibition effect was observed in those treated with AS1411 combined with ADM. For Luc+Nalm-6 derived ALL model, leukemia progression was suppressed in mice treated with AS1411 and AS1411 combined with ADM. AS1411and ADM, especially combination of AS1411 and ADM, could improve survival of the leukemic mice compared to those treated with PBS. The results showed that NCL targeted by AS1411 sensitized ADM treatment and prolonged survival in vivo. In summary, our findings revealed NCL as a survival predictor and the novel role of NCL in ALL chemo-resistance. NCL may be a potential target for improving outcome in ALL. Disclosures No relevant conflicts of interest to declare.

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An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽

10.1182/blood.v118.21.2333.2333 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2333-2333

Author(s):

Brian D. Adams ◽

Shangqin Guo ◽

Haitao Bai ◽

Changchun Xiao ◽

E. Premkumar Reddy ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Hematopoietic Recovery ◽

Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

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Heterogeneity in the Making of Blood

Blood ◽

10.1182/blood.v126.23.sci-27.sci-27 ◽

2015 ◽

Vol 126 (23) ◽

pp. SCI-27-SCI-27

Author(s):

David T. Scadden

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Niche ◽

Cell Selection ◽

Hematopoietic Progenitor ◽

Marrow Space ◽

And Function ◽

Osteolineage Cells

It is increasingly clear that the bone marrow is comprised of a heterogeneous complex of niches for hematopoietic cells, some for stem cells in the perivascular space and some for progenitors. We have used two approaches to define the role of specific cells in the marrow. First, single cell selection and characterization based on in vivo proximity to HSPC. This method has defined a subset of endosteal lining cells that can be immunophenotypically defined and isolated and reveals IL-18 as a regulator of hematopoietic progenitor quiescence. Second, candidate cell depletion that revealed mature osteolineage cells expressing osteocalcin as regulating the production of thymic emigrants through the expression of Dll4. Deletion of these cells reduces the number and function of T-biased lymphoid progenitors in the marrow space as well as thymic populations and mature T cells in the blood. These data suggest that specific niche subsets can be defined and through them, novel molecular regulators of HSPC function. The bone marrow niche is a heterogeneous composite of distinctive niches. Disclosures No relevant conflicts of interest to declare.

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Microenvironment Induced Myelophthisis Caused By CYP26 Deficiency

Blood ◽

10.1182/blood-2018-99-115331 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1297-1297

Author(s):

Laura Palau ◽

Jessica M. Synder ◽

Traci Beth Topping ◽

Cathryn Hogarth ◽

Nina Isoherranen ◽

...

Keyword(s):

Histological Analysis ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Myeloproliferative Neoplasm ◽

Primary Myelofibrosis ◽

Differentiation Potential ◽

Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) reside in a complex microenvironment that enforces the balance between self-renewal and differentiation. The exact physiologic mechanisms by which the niche controls HSC fate remain elusive. Retinoic acid (RA) is a powerful morphogen that controls stem cell behavior across a variety of systems. In bone marrow (BM), mesenchymal stroma cells (MSCs) express cytochrome P450 (CYP)26 enzymes and can inactivate endogenous and pharmacological retinoids (Ghiaur G et al PNAS 2013, Su M et al. PlosOne 2015, Alonso S et al. 2016). Stromal CYP26 activity is required to maintain human HSCs ex vivo (Ghiaur G et al PNAS 2013). Here we set to study the role of CYP26 in HSC homeostasis in vivo. For this, we induced CYP26 knockout via injection of Tamoxifen in ROSA26CreERT CYP26A1loxP/loxPCYP26B1loxP/loxPmice (CYP26KO) and ROSA26CreERT wildtype mice (CTR). After 5 daily tamoxifen injections, the knockout was confirmed at DNA and RNA level in multiple tissues of CYP26KO mice. Within 4 weeks, CYP26KO mice showed profound leukocytosis (5.97 ±0.37 vs. 21.12 ±3.81 k/mm3, n=4, p<0.01 CTR vs. CYP26KO) with neutrophilia and monocytosis compared to CTR mice. By 6 weeks, they experience profound weight loss, became moribund and had to be sacrificed. At that time, they had massive splenomegaly and lymphadenopathy as well as brittle/pale bones compared to CTR (Figure A). Histological analysis revealed presence of extramedullary hematopoiesis with a predominantly myeloid infiltrate in the spleen (confirmed by flow cytometry analysis) and lymph nodes but also multiple clusters megakaryocytes (Figure B). The mice also displayed decreased BM cellularity (11.25 ±1.01 vs. 5.62 ±0.36 x106/femur, n=5, p<0.01, CTR vs. CYP26KO), increased frequency of CFU-C (44.33 ±3.72 vs. 69.83 ±8.10 CFU/25000 BM mononuclear cells (MNCs), n=10, p=0.02, CTR vs. CYP26KO) with a myeloid bias (26.76±3.89 vs. 44.57 ±3.21 %CFU-GM/G/M, n=10, p<0.01, CTR vs. CYP26KO). When cellularity was taken into account, total CFU-C per femur was comparable between the two groups. The mice also had increased frequency of LSK cells in the BM (0.26 ±0.12 vs 0.73 ±0.18 % LSK of MNCs, n=2, CTR vs. CYP26KO) and an increased frequency of circulating CFU-C in the peripheral blood (37 ±4.04 vs. 179.5 ±43.06 per 200 ml of blood, n=4, p=0.01, CTR vs. CYP26KO). On histological analysis, the BM is dominated by a myeloid infiltrate and shows a striking decrease in radial bone diameter (Figure C,D). MSCs derived from CYP26KO mice have lower levels of CXCL12 and SCF and have impaired osteoblast differentiation potential compared to MSCs derived from control mice (Figure E). These findings were confirmed with ex vivo generated CYP26KO MSCs via retroviral mediated Cre recombination of CYP26A1loxP/loxPCYP26B1loxP/loxPstroma cells. More so, preliminary studies suggest that the hematopoietic phenotype observed depends on cell extrinsic presence of CYP26 activity as transplantation of CYP26A1loxP/loxPCYP26B1loxP/loxP BM cells into wildtype BoyJ recipients did not reproduce the phenotype upon injection with Tamoxifen of the recipient mice in spite of 100% donor derived hematopoiesis. Alternatively, transplant of wildtype cells into CYP26A1loxP/loxPCYP26B1loxP/loxPrecipients, did reproduce the phenotype after injection of Tamoxifen. In conclusion, we show here that dysregulated RA homeostasis in the BM impairs MSCs function and result in egress of hematopoiesis to extramedullary sites. These results come to complement data from RARγKO (Walkley CR et al Cell 2007) and SMRTmRIDmice models (Hong S-H et al PNAS 2013) and suggest a pivotal role of RA signaling in pathogenesis of myeloproliferative neoplasm and myelofibrosis. To what extent these findings correlate with human primary myelofibrosis is currently under investigation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Novel Pleiotropic Effects of Thrombin in Regulation of Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells – Identification of Negative PAR1 and PAR3 Receptor Crosstalk.

Blood ◽

10.1182/blood.v114.22.336.336 ◽

2009 ◽

Vol 114 (22) ◽

pp. 336-336

Author(s):

Marcin Wysoczynski ◽

Rui Liu ◽

Mariusz Z Ratajczak

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Metastatic Potential ◽

Cell Interaction ◽

Lymphocytic Leukemia ◽

Coagulation Cascade ◽

Receptor Crosstalk

Abstract Abstract 336 Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescence and childhood that frequently infiltrates bone marrow (BM) to this degrees that it may mimic acute lymphocytic leukemia. We identified chemokines and growth factors (e.g., SDF-1, HGF) that play an important role in RMS metastasis (Blood 2002;100:2597, Cancer Res. 2003;63:7926, Cancer Res. 2007;67:2131). Novel evidence however accumulates that metastatic process for many tumors may be modulated by the components of coagulation cascade (CC) (e.g., thrombin, activated platelets). Thus, we become interested on a role of CC in modulating metastasis of RMS cells. First, we learned that RMS cells express tissue factor (TF) and thus may activate coagulation by generation of thrombin. Thrombin activated in tumor microenvironment activates platelets that release microvesicles. We observed that platelet derived microvesicles (PMV) transfer to RMS cells several platelet integrin receptors (e.g., α2β3) important for RMS cell interaction with endothelium, and thus increase their adhesive potential to endothelial cells. To support this, we noticed that RMS cells covered with PMV showed higher metastatic potential after intravenous injection into immunodeficient SCID mice. We also found that PMV also directly chemoattracted RMS cells and activated MAPKp42/44 and AKT. Next we learned that all 10 human RMS cell lines investigated in our studies express functional PAR1 and PAR3 receptors. To support this, we observed in thrombin stimulated RMS cells phosphorylation of MAPKp42/44 and MAPKp38. To our surprise however, in in vitro experiments thrombin decreased RMS chemotactic response to conditioned media from bone marrow fibroblast and PMVs. Furthermore, we didn't observe any effect of thrombin on proliferation, survival and expression of pro-angiogenic factors in RMS cells. Thrombin also decreased adhesion of RMS cells to fibronectin and bone marrow stroma cells. In contrast PAR1 specific agonist TRAP-6 stimulated proliferation of RMS cells. Different responsiveness to thrombin and TRAP-6 stimulation could be explained by negative modulatory role of PAR3 receptor in response to stimulation by thrombin. Thus, to learn more on a role of PAR1 and PAR3 in RMS proliferation/metastasis we knock-down both receptors by employing shRNA strategy. We observed that PAR1-/- receptor RMS cells that express intact PAR3 cells formed in vivo smaller tumors as compared to unmodified control cells. On the other hand, PAR3-/- RMS cells that express functional PAR1 began to proliferate robust in response to thrombin. In conclusion, we demonstrate that RMS-expressed TF activates prothrombin and that thrombin is a novel, underappreciated, pro-metastatic factor for these cells. Activated in tumor proximity by thrombin, platelets release PMVs that chemoattract and transfer several platelet-derived receptors/adhesion molecules to RMS cells that are crucial for adhesion/interaction with the endothelium. Conversely, by decreasing the responsiveness of RMS cells to local chemoattractants and decreasing adhesiveness of RMS cells, thrombin promotes their release from the primary tumor into circulation. Consequently, RMS cells that are covered by PMVs release into circulation and respond to chemoattractants in distant organs for metastasis. Finally, our data also supports a negative regulatory role of thrombin-PAR3 axis in proliferation of RMS. Disclosures: No relevant conflicts of interest to declare.

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Ex Vivo Manufacture of Rapamycin-Resistant Human Th1/Tc1 Cells.

Blood ◽

10.1182/blood.v114.22.501.501 ◽

2009 ◽

Vol 114 (22) ◽

pp. 501-501

Author(s):

Emily Bunce ◽

Justin Taylor ◽

Jason Foley ◽

Bruce Levine ◽

Carl H. June ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Human Lymphocytes ◽

Type I ◽

Culture Vessel ◽

Human T Cells ◽

Ifn Γ

Abstract Abstract 501 Introduction: Previously, we found that ex vivo usage of rapamycin and a type I polarizing cytokine could be utilized to generate murine Th1/Tc1 cells that persisted in vivo and mediated increased GVHD. The type I cytokine profile and increased in vivo persistence of this population may prove useful in autologous transplant settings. In recent experiments using culture flasks and polarization with IFN-α, we found that human T cells similarly can adopt a Th1/Tc1 phenotype in rapamycin; furthermore, such human T cells have increased in vivo persistence when transferred into immune-deficient murine hosts. In this project, our goal was to evaluate whether human Th1/Tc1 cell generation in rapamycin varied depending on use of culture flasks or more clinically relevant, closed system polyolefin culture bags. Methods: Human lymphocytes were collected by steady-state apheresis and subsequent counter flow centrifugal elutriation. CD3+ T cells were purified and co-stimulated using anti-CD3/anti-CD28 tosyl-activated P450 magnetic beads. T cells were cultured under four conditions: recombinant human (rh) IL-2 alone; rhIL-2 plus rapamycin; rhIL-2 plus rhIFN-α; or rhIL-2 plus rhIFN-α plus rapamycin. After 6 days of culture in flasks or bags, cells were harvested and co-stimulated; cytokine production was measured by Luminex assay, and cell surface markers were assessed using flow cytometry. Cells were further characterized using an in vivo xenogeneic cytokine storm model of GVHD (x-GVHD). Specifically, immune deficient Rag2/γc knockout mice were irradiated, injected with ex vivo expanded T cells, and challenged with lipopolysaccharide (LPS) injection on day 6 after adoptive transfer to induce lethal levels of TNF-α. Results: Relative to T cells expanded in only IL-2, further addition of IFN-α yielded T cells with increased secretion of IFN-γ (33.5 vs. 5.7 ng/ml, p=0.01) and reduced secretion of IL-4 (0.6 vs.18.7 pg/ml, p=0.02). Inclusion of both IFN-α and rapamycin also yielded cells with preferential secretion of IFN-γ relative to IL-4; IFN-γ and IL-4 secretion values did not vary significantly between flask and bag conditions. However, relative to flask expanded T cells, Th1/Tc1 cells expanded in bags had increased co-expression of T central memory molecules CD62L and CCR7 (median percentage of co-expression increased from 20% to 40%, p<0.05). Because of the increased type I polarity of T cells expanded in bags in the presence of IFN-α, we hypothesized that such cells would mediate increased x-GVHD relative to T cells expanded in bags in the absence of IFN-α. However, contrary to our hypothesis, lethality after LPS challenge was actually reduced in recipients of IFN-α exposed human T cells (p<0.05). Given these results, we reasoned that IFN-α might also induce production of the counter-regulatory cytokine, IL-10. Indeed, relative to T cells expanded in IL-2 alone, further addition of IFN-α increased IL-10 secretion from 679 to 5982 pg/ml (without rapamycin; p<0.05) and from 85 to 2302 pg/ml (with rapamycin; p<0.05). Intra-cellular flow cytometry demonstrated that Th1/Tc1 cell co-expression of IFN-γ and IL-10 occurred at the single cell level. Conclusions: Ex vivo manufacture of human Th1/Tc1 cells in clinical grade polyolefin bags resulted in T cells with a differential cytokine phenotype relative to T cells cultured in flasks. Using bags, addition of IFN-α to culture yielded increased T cell capacity to secrete both IFN-γ and IL-10; this phenotype occurred independent of whether the culture also included rapamycin. The observed co-expression of IFN-γ and IL-10 supports a model whereby human Th1/Tc1 cells can simultaneously express pro- and anti-inflammatory cytokines. Th1/Tc1 cell co-expression of IFN-γ and IL-10 appeared to moderate inflammation in vivo because recipients of such bag-cultured T cells had reduced lethal x-GVHD relative to recipients of flask-cultured T cells. As such, we conclude that type of culture vessel (bag vs. flask), presence of a polarizing cytokine (IFN-α), and level of IL-10 secretion are important variables to consider for the ex vivo manufacture of human Th1/Tc1 cells. Disclosures: No relevant conflicts of interest to declare.

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Distinct Role of Antigen-Specific Tc1 and Tc17 Cells in Tumor Eradication In Vivo

Blood ◽

10.1182/blood.v118.21.1910.1910 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1910-1910

Author(s):

Yu Yu ◽

Hyun Il Cho ◽

Dapeng Wang ◽

Kane Kaosaard ◽

Claudio Anasetti ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Growth ◽

Tumor Immunity ◽

Conflicts Of Interest ◽

B16 Melanoma ◽

Host Cells ◽

Tumor Bearing ◽

Tc17 Cells

Abstract Abstract 1910 Background: Adoptive cell transfer (ACT) of tumor-reactive T cells is one of the most promising approaches for the treatment of established melanoma. Recently, limited studies provide some evidence that Th/Tc17 cells may also have potent anti-tumor activities, but the conclusion is far from reach. Methods: Human gp10025-specific Tc1 or Tc17 cells were generated from pmel-1 transgeneic mice and used as cell source for ACT. Luciferase-transduced B16 melanoma was intravenously injected into C57BL/6 mice to establish lung-metastasis. After 7 days, tumor-bearing mice were lethally irradiated and transferred gp-10025 specific Tc1 or Tc17 cells in the combination of syngeneic bone marrow. Survival of those tumor-bearing mice was monitored daily, and tumor growth was monitored weekly using in vivo bioluminescent imaging (BLI). Donor T-cell expansion and cytokine secretion from the spleen and lung of tumor bearing mice were analyzed using flow cytometry and ELISPOT assays. To evaluate the role of IFNγ in anti-tumor immunity, we used a B16 melanoma cell line that was transduced with a plasmid encoding a dominant-negative IFNγ receptor (B16-IFNγRDN), and IFNγR knockout mice as tumor-bearers. Results: As expected, irradiation and transfer of syngeneic bone marrow had little or no effect on established melanoma. Adoptive transfer of tumor-specific Tc17 cells significantly suppressed the tumor growth, whereas Tc1 cells induced long-term regression of established melanoma. After ACT, Tc1 cells maintained their phenotype to produce IFNγ. However, Tc17 cells largely preserved their ability to produce IL-17, but a subset of them secreted IFNγ, indicating the plasticity of Tc17 cells in vivo. Mechanistically, Tc1 cells executed their anti-tumor immunity primarily through the direct effect of IFNγ on melanoma cells because Tc1 cells had essentially no effect on B16-IFNγRDN tumor. However, Tc1 cells had a similar therapeutic effect on IFNγR knockout as wild type mice, indicating that IFNγ signaling in host cells was not critical. In contrast, despite the fact that Tc17 cells also secreted IFNγ, Tc17-mediated anti-tumor immunity was independent of the effect through IFNγ. Ironically, IFNγ was inhibitory to Tc17-mediated anti-tumor activity. Conclusions: Taken together, these studies demonstrate that both Tc1 and Tc17 cells can mediate effective anti-tumor immunity, but Tc1 is superior to Tc17. These findings also demonstrated for the distinct effect mechanisms of antigen-specific Tc1 and Tc17 cells in anti-tumor response, and direct IFNγ signaling on tumor cells is a key effect to eradicate established tumors mediated by Tc1 cells. Disclosures: No relevant conflicts of interest to declare.

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