bluuebunny/crossref_metadata_2025_split huggingface dataset:
Harnessing Invariant Natural Killer T Cells for Cancer Therapy
Authors: Dhodapkar Madhav V. 
Published: October 2019

Abstract
Abstract SCI-20
Natural killer T (NKT) cells are distinct innate lymphocytes that recognize lipid ligands in the context of nonclassical MHC molecules called CD1d. NKT cells have been broadly classified into two major types based on their T-cell receptors (TCRs). One group, termed type I invariant, or iNKT cells, bears a noncanonical conserved TCR (Va24/Vb11 in humans, and Va14 in mice), while others, termed type II NKT cells, express diverse TCRs. Recent studies have identified several naturally occurring lipid ligands recognized by these cells. iNKT cells are efficiently activated by α-galactosylceramide (α-GalCer), a lipid ligand first isolated from a marine sponge. Activation of iNKT cells in vivo leads to activation of NK cells, as well as downstream activation of adaptive immunity, leading to strong antitumor effects in mice. Several hematologic malignancies express CD1d and are attractive targets of NKT-targeted therapeutic approaches. Progression from premalignant gammopathy to clinical myeloma is associated with loss of the interferon-γ-secreting effector function of NKT cells. Adoptive transfer of α-GalCer-loaded dendritic cells (DCs) led to activation and expansion of circulating NKT cells in vivo in patients with advanced cancer. However, the functional properties of NKT cells were found to be impaired. Lenalidomide leads to enhanced activation and cytokine secretion by NKT cells in a ligand-dependent manner in culture. This led us to treat patients with early multiple myeloma with three cycles of low-dose lenalidomide and α-GalCer-loaded DCs. Preliminary studies from this trial show that this combination leads to activation of diverse innate cells, including NKT and NK cells, and upregulation of NKG2D and CD56. Activation of these innate lymphocytes also led to an increase in serum-soluble interleukin-2 receptor and induction of eosinophilia. Together, these data demonstrate the capacity of this combination to recruit several innate lymphocytes and mediate antitumor effects. In contrast to conventional MHC I/II restricted T cells, understanding the diversity and functional properties of lipid-reactive T cells is still in its infancy. The biology of NKT cells may have an impact, due to the cells' immune-regulatory properties, in diverse inflammatory states, including cancer, atherosclerosis, and allergy. This research was supported in part by funds from the Translational Research Program of the Leukemia and Lymphoma Society.

Disclosures:
Dhodapkar: KHK: Research Funding; Celgene: Research Funding. Off Label Use: Lenalidomide in asymptomatic myeloma.
The Role of Iron in Toxicity of Stored Red Blood Cell Units
Authors: Hod Eldad A. , Brittenham Gary M. , Spitalnik Steven L. 
Published: October 2019

Abstract
Abstract SCI-46
Accumulating evidence suggests that transfusion of red blood cells (RBCs) stored for longer durations is associated with increased rates of infection, morbidity, and mortality in hospitalized patients. During refrigerated storage in vitro, RBCs undergo cumulative biochemical and biomechanical changes that reduce their survival in vivo. After transfusion, storage-damaged RBCs are rapidly cleared from the circulation by reticuloendothelial macrophages, usually within the first hour. The associated hemoglobin iron is then rapidly catabolized and returned to the plasma at a pace that can exceed the rate of uptake by the physiologic iron transporter, transferrin, thereby producing plasma non-transferrin-bound iron. Our overarching hypothesis is that the rapid clearance of storage-damaged RBCs is responsible for some of the adverse effects of transfusion, specifically by delivering a large bolus of toxic hemoglobin-derived iron to macrophages. In particular, plasma non-transferrin-bound iron can produce oxidative damage to lipids, proteins, and DNA, thereby activating endothelial cells and platelets to increase thrombotic risk. Furthermore, increased macrophage iron and plasma non-transferrin-bound iron can enhance infectious risk by providing sources of iron to microbial pathogens both within macrophages and within the systemic circulation. Our studies in healthy human volunteers demonstrate that transfusions of “older” RBCs (stored for 40–42 days), but not “fresh” RBCs (stored for less than seven days), significantly increase circulating non-transferrin-bound iron levels in vivo (1) and enhance bacterial growth in vitro in posttransfusion serum samples. Animal studies (2) confirm these findings and provide additional evidence that transfusion exacerbates infection in vivo with Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus. In addition, murine studies suggest that rapid transfusion of the equivalent of two units of older, stored RBCs produces a proinflammatory cytokine response, which was not seen after slow transfusion of one unit of older, stored RBCs to healthy human volunteers. Studies in dogs are currently under way to test whether the RBC transfusion rate and dose affects the proinflammatory response. In conclusion, transfusion of older, stored RBCs results in delivery of a potentially toxic dose of iron to macrophages that both increases macrophage intracellular iron and produces plasma non-transferrin-bound iron. This iron toxicity may increase infectious and thrombotic risks and potentially exacerbate an existing proinflammatory response.

Disclosures:
No relevant conflicts of interest to declare.
Microenvironmental Factors and The Role Of Tumor Necrosis Factor Receptor Type 1 (TNFR-1) In Chronic Lymphocytic Leukemia
Authors: Duerr Claudia , Schulz Angela , Ohl Sibylle , Zenz Thorsten , Stilgenbauer Stephan , Lichter Peter , Seiffert Martina 
Published: October 2019

Abstract
B-cell Chronic Lymphocytic Leukemia (CLL) represents the most common leukemia among adults in the western world and remains still incurable. CLL is characterized by the accumulation of malignant B-cells in the peripheral blood and the lymphoid organs due to apoptosis resistance. Removing the cells from their physiological microenvironment spontaneously drives them into apoptosis in vitro unless they are supported by bystander cells (e.g. stromal cells or accessory leukocytes) or soluble factors such as cytokines produced by these cells.
In order to identify differences in the biology of healthy B-cells and the leukemic clones we performed comparative microarray analyses of both cell types before and after cultivation in high cell density that allows cell-cell interactions of CLL cells with accessory leukocytes. These studies revealed the establishment of an inflammatory microenvironment in CLL characterized by the transcriptional upregulation of several cytokines. Antibody array analyses of culture supernatants and blood serum samples confirmed the upregulation of these proteins and their relevance for CLL. The 11 most deregulated cytokines were quantified in the serum of 250 CLL patients of the German CLL8 study cohort and 50 age- and sex-matched controls in order to identify novel predictive or prognostic markers. Bioinformatical analyses of the data are currently ongoing.
Among other proteins, we found Tumor Necrosis Factor Receptor Type 1 (TNFR-1) to be significantly upregulated in the malignant B-cells under survival-maintaining cultivation as well as in CLL serum, but not in healthy controls (Figure 1). To exploit the difference in TNFR-1 biology, we treated CLL cells with TNF-α in combination with wogonin which is known to block the survival supporting pathway propagated by TNFR-1 upon TNF-α binding. This combinational treatment strategy effectively triggered

Disclosures:
No relevant conflicts of interest to declare.
Self-Complementary AAV Vectors Delivered Intramuscularly Do Not Break Tolerance In a Partial Knockout Model Of Hemophilia B
Authors: Rogers Geoffrey L. , Herzog Roland W 
Published: October 2019

Abstract
Self-complementary adeno-associated viral (scAAV) vectors have become a popular tool for AAV gene therapies due to their increased transgene expression relative to single-stranded (ss)AAV vectors, at least for models in which the transgene can fit within these restrictive constructs. The most recent clinical trial for hemophilia B used a scAAV8 vector. However, recent reports by our lab and others have suggested that these vectors are also more immunogenic than their single-stranded brethren. We previously showed that, during hepatic gene transfer, the innate immune response is greater with a scAAV vector. Moreover, Wu et al. demonstrated an enhanced T and B cell response to a secreted form of HIV’s gag protein when it was delivered via scAAV. While this was beneficial for their vaccine model, it could potentially be problematic for therapeutic gene transfer applications. To investigate whether this phenomenon could deleteriously impact gene therapy for hemophilia B, we used a partial knockout model of hemophilia B. This mouse model has the murine factor IX gene knocked out, and then a truncated form of human factor IX (hF.IX) is inserted. Specifically, the mice we used have a late stop codon (LS) at amino acid 338; while hF.IX can be detected in the liver, this mutated protein is not released into circulation (crm-). During muscle gene transfer with AAV2, LS mice have an interesting response: while they form antibodies to hF.IX, there is no CD8+ T cell infiltrate detected in the transduced tissue. We then set out to determine whether this partial tolerance could be disrupted using a scAAV vector. Using AAV1, which is a more relevant serotype for muscle gene therapy, we injected single-stranded and self-complementary vectors (1011 vg/mouse) and measured the resulting immune responses. Surprisingly, unlike with AAV2, the LS mice completely tolerated AAV1-mediated intramuscular delivery of hF.IX. They did not make detectable anti-hF.IX IgG1, and their plasma had no Bethesda activity through 4 weeks post-injection. In contrast, hF.IX null mice (HBKO) made 10708±3869 ng anti-hF.IX IgG1 and developed inhibitory activity of 22.5±26 Bethesda units 4 weeks after delivery of a scAAV vector. Consequently, LS mice had circulating hF.IX levels of 148.9±18.64 (ssAAV1) and 60.12±8.71 ng/mL (scAAV1), while hF.IX was undetectable in the plasma of HBKO mice at this time point. The CD8 response was similarly lacking in LS mice, with splenic responses to hF.IX detected by ELISPOT reduced by ∼2-10 fold relative to HBKO mice. Infiltrating CD8 cells were detected in the muscle of HBKO but not LS mice. In conclusion, our data suggest that the underlying mutation within the patient is a more important risk factor for immune responses to the transgene than the molecular form of the AAV genome.

Disclosures:
Herzog: Genzyme: AAV technology, AAV technology Patents & Royalties.
In Vivo Targeting Of Acute Myeloid Leukemia Using CpG-Stat3 siRNA Results In T Cell-Dependent Tumor Eradication
Authors: Hossain Dewan Md Sakib , Santos Cedric Dos , Zhang Qifang , Kozlowska Anna , Liu Hongjun , Gao Chan , Moreira Dayson , Swiderski Piotr , Jozwiak Agnieszka , Kline Justin , Forman Stephen J. , Bhatia Ravi , Kuo Ya-Huei , Kortylewski Marcin 
Published: October 2019

Abstract
Signal Transducer and Activator of Transcription-3 (STAT3) is an oncogene and immune checkpoint commonly activated in cancer cells and in tumor-associated immune cells. As a focal point of downstream signaling from numerous cytokines and growth factors present in the tumor microenvironment, STAT3 is an attractive but also elusive pharmacological target. We previously developed an immunostimulatory strategy based on targeted Stat3 gene silencing in Toll-like Receptor (TLR9)-positive hematopoietic cells using CpG-siRNA conjugates. Here, we assessed therapeutic effect of systemic STAT3 blocking/TLR9 triggering in disseminated acute myeloid leukemia (AML). We used a model of mouse Cbfb/MYH11/Mpl-induced leukemia, which mimics human inv(16) AML. Our results demonstrate that intravenously delivered CpG-Stat3 siRNA, but not control oligonucleotides, can eradicate established AML and impair leukemia-initiating potential. These antitumor effects require host’s effector T cells but not TLR9-positive antigen-presenting cells. Instead, CpG-Stat3 siRNA has direct immunogenic effect on AML cells in vivo leading to up regulation of MHC class II, co-stimulatory molecules and proinflammatory mediators, such as IL-12 and IFNγ, while down regulating expression of co-inhibitory PD-L1 molecule. Systemic injections of CpG-Stat3 siRNA generate potent tumor antigen specific immune responses, increase the ratio of tumor-infiltrating CD8+ T cells to T regulatory cells (Tregs) in various organs and result in CD8+ T cell-dependent regression of leukemia. Targeted STAT3 inhibition/TLR9 triggering also enhances immunogenicity of primary patients’ AML cells which gain ability to stimulate T cell proliferation. Our findings underscore the potential of using targeted STAT3 inhibition/TLR9 triggering to break tumor tolerance and induce potent immunity against AML and potentially other TLR9-positive blood cancers.

Disclosures:
No relevant conflicts of interest to declare.
The Classical and Alternative NF-κB Activity Supports Cell Survival and Confers Chemo-Sensitivity Of Chronic Lymphocytic Leukemia Cells
Authors: Feng Guo , Xu Jingjing , Sun Aining , Chen Zi-Xing 
Published: October 2019

Abstract
Constitutive classical NF-κB activation protects chronic lymphocytic leukemia (CLL) B-cells from spontaneous cell death and plays a critical role in the chemo-resistance. RelB, representing the alternative NF-κB activity, functions specifically in lymphoid organogenesis and B-cell maturation. RelB indeed plays a tumor-supportive role and confer radiation-resistance in solid tumors. However, the involvement of RelB in CLL is unrevealed. Here, we analyzed the NF-κB activation in CLL bone marrow (BM). Both the RelA and RelB activity could be detected in CLL B-cells from BM, in spite of inevitable variability. High RelB activity, concomitant with high RelA activity, was linked to an unfavorable prognosis of CLL. CLL-derived BM stromal cells (BMSCs) were functionally similar to disease-free BMSCs in their capability to support CLL B-cells survival. The migration and adhesion ability of CLL B-cells was not affected by the RelB activity. High RelB activity, together with the RelA activity, maintained the basal survival of cells. The induction of RelA and RelB expression in the nuclear was responsible for the better survival of CLL B-cells supported by BMSCs. In addition, the presence of high RelB activity in CLL B-cells was correlated with the sensitivity to proteasome inhibitor but not fludarabine. Taken together, we provided evidences that not only RelA but also RelB, subunits of NF-κB family, plays an important role in the cellular behavior of CLL cells from BM. The strength of RelB activity influenced the prognosis of CLL patients.

Disclosures:
No relevant conflicts of interest to declare.
A6 Peptide Is Selectively Cytotoxic For Chronic Lymphocytic Leukemia Cells
Authors: Zhang Suping , Lai Hsien , Liu Grace , Rassenti Laura , Choi Michael Y. , Howell Stephen B , Finlayson Malcolm , Kipps Thomas J. 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL) cells express high levels of CD44, a cell-surface glycoprotein receptor for hyaluronic acid (HA). We found that a mAb specific for CD44 was directly cytotoxic for leukemia B cells, but had little effect on normal B cells. Moreover, this anti-CD44 mAb could induce CLL cells that expressed the zeta-associated protein of 70 kDa (ZAP-70) to undergo caspase-dependent apoptosis, independent of complement or cytotoxic effector cells (Proc Natl Acad Sci, USA 2013, PMID: 23530247). The cytotoxic effect of this mAb was not mitigated when the CLL cells were co-cultured with mesenchymal stromal cells (MSCs) or hyaluronic acid or when they were stimulated via ligation of the B-cell receptor with anti-µ.
A6 (Angstrom Pharmaceuticals) is an 8-amino acid peptide that has marked homology with a linear sequence of CD44. A6 can bind CD44 within a region of the ligand-binding domain, leading to inhibition of the migration and metastatic potential of CD44-expressing cancer cells in vitro and in vivo (Mol Cancer Ther, 2011 PMID: 21885863).
We evaluated the cytotoxic activity of A6 against primary leukemia cells of patients with CLL (n = 22). We found that A6 peptide also was directly cytotoxic for CLL cells isolated from different patients in a dose-dependent manner at concentrations that may be achieved in vivo. The A6 peptide appeared less cytotoxic for CLL cells than the intact anti-CD44 mAb, but still had greater direct cytotoxicity for CLL cells that expressed ZAP-70 than for CLL cells that were ZAP-70 negative. Furthermore, the A6 peptide had negligible effect on the viability of lymphocytes isolated from the blood of healthy donors (n = 3). Because clinical studies have found the A6 peptide to be well-tolerated and without dose-limiting toxicity in patients with solid tumors who have been treated to date (N = 40), a clinical study is planned to evaluate the safety and activity of the A6 peptide in the treatment of patients with CLL.

Disclosures:
Howell: Angstrom Phamaceuticals: Membership on an entity’s Board of Directors or advisory committees. Finlayson:Angstrom Phamaceuticals: Employment.
Tocilizumab Overcomes Chemo-Resistance Of CLL Cells
Authors: Liu Fengting , Jia Li , Wang Ping , Farren Tim , Agrawal Samir G 
Published: October 2019

Abstract
IL-6 in the cancer microenvironment plays a crucial role in the constitutive activation of STAT3, which contributes to cancer cell progression. IL-6 binds to a heterodimer of membrane-bound receptors, IL-6R (CD126) and gp130, that triggers phosphorylation of JAK2 and downstream STAT3. The phosphorylated STAT3 acts as a transcription factor, binding target DNA and promoting tumorigenesis, cell proliferation and survival. IL-6 signaling pathways have been extensively studied as therapeutic targets in both cancer and autoimmune conditions. The anti-IL6R antibody Tocilizumab is a humanized anti CD126 antibody, approved by the FDA (Food and Drug Administration) for treatment of rheumatoid arthritis, that can block IL-6 classic and trans-signalling pathways. We investigated IL-6R expression in CLL cells and analysed the correlation of IL-6R expression with chemo-sensitization and the potential of targeting IL-6R to overcome drug resistance in vitro.
41 CLL patients were studied. Membrane-bound and soluble IL-6 receptors, CD126 and gp130, were measured by flow cytometry or ELISA. Constitutive activation of STAT3 and the expression of Bcl-2 family proteins were detected by western blotting. Cell cycle distribution was assessed with Ki67 and cell viability indicated by propidium iodide (PI) negative staining.
CLL cells were found to express variable levels of membrane-bound receptors of IL-6, CD126 (average 22%, range 3-60%) and gp130 (average 12%, range 3-25%), which was significantly higher than normal B-cells (CD126 average 1.7%, range 0-6.3%; gp130 average 1.3%, rage 0-7.9%). Although in vitro Chlorambucil-induced cell death was variable among the cases, there was a strong correlation with CD126 expression (Figure 1): higher CD126 expression was associated with increased resistance to Chlorambucil.
Treatment of CLL cells with Tocilizumab led to inhibition of constitutive STAT3 activation decreased Mcl-1, Bcl-xl expression and cells exited from G0 phase of the cell cycle. Tocilizumab did not induce cell death in CLL cells. Combined treatment with both Chlorambucil and Tocilizumab significantly increased cell death (p < 0.01). Interestingly, higher CD126 expression was highly significantly correlated with the increased cell death seen with combined Chlorambucil and Tocilizumab (p < 0.0001, Figure 2)
These results suggest that inhibiting IL6 signal pathways via IL-6R blockade with Tocilizumab is a new approach in CLL, leading to increased chemo-sensitisation to standard chemotherapeutic agents. Although CLL cells with higher CD126 expression exhibited more drug resistance in vitro, they also showed better response to Tocilizumab. Targeting the combination of Tocilizumab with anti-cancer therapy to CLL patients with high IL-6R expression might achieve clinical therapeutic efficacy.

Disclosures:
No relevant conflicts of interest to declare.
Arsenic Trioxide Induces Apoptosis In Chronic Lymphocytic Leukemic Cells Through Down-Regulation Of Survivin Via The p53-Dependent Signaling Pathway
Authors: Zhang Xiaohui , Lv Meng , Jiang Qian , Zhu Honghu , Qin Yazhen , Huang Xiaojun 
Published: October 2019

Abstract
Arsenic trioxide induces cell apoptosis in a variety of tumor cells. However, the molecular mechanism underlying the killing effects of arsenic trioxide on tumor cells is not fully understood. Here, we demonstrate a signal pathway of arsenic trioxide treatment that leads to apoptosis in a B-cell chronic lymphocytic leukemia (WSU-CLL) cell line. Arsenic trioxide treatment significantly inhibited proliferation and induced apoptosis of WSU-CLL cells in a time- and dose-dependent manner. This anti-tumor effect of arsenic trioxide on WSU-CLL cells was mediated through the down-regulation of survivin and up-regulation of p53. Knock-down of intracellular survivin with siRNA potentiated the inhibitory effect of arsenic trioxide on proliferation in WSU-CLL cells. Also, knock-down of p53 by a specific siRNA prevented the down-regulation of survivin by arsenic trioxide and reduced the cytotoxic effect of arsenic trioxide to WSU-CLL cells. These results indicate that arsenic trioxide may be used to as novel therapeutic agent for chronic lymphocytic leukemia, which down-regulates survivin expression via a p53-dependent signaling pathway.

Disclosures:
No relevant conflicts of interest to declare.