bluuebunny/crossref_metadata_2025_split huggingface dataset:
Depletion and Restoration of the Intravascular Nitrite (NO2-) Reserve Modulate Susceptibility to Liver Vaso-Occlusive Infarction in Sickle Cell Transgenic Mice.
Authors: Hsu Lewis L. , Shiva Sruti , Noguchi Audrey , Curtis Erin , MacArthur Peter , Raat Nicolaas J.H. , Diwan Bhalchandra A. , Schechter Alan N. , Noguchi Constance Tom , Gladwin Mark T. 
Published: October 2019

Abstract
Multiple studies link low bioavailability of nitric oxide (NO) and major complications of sickle cell disease, especially pulmonary hypertension and acute vaso-occlusive pain episodes. A gender dimorphism has been suggested in human studies, with lower levels of NO availability observed in males. These studies support efforts to develop NO-based therapeutics and the newly characterized small molecule nitrite represents an ideal candidate. Nitrite is a circulating anion that is reduced to NO during physiological and pathological hypoxia and mediates potent cellular cytoprotection at physiologic concentrations in the setting of ischemia and reperfusion injury. Supporting a role for nitrite in sickle cell pathogenesis, we found that nitrite levels are depleted in transgenic mice expressing exclusively sickle hemoglobin (sickle mice), with whole blood nitrite levels 50% lower compared to control mice, and are further reduced by 19% in male sickle mice compared with females. Thus, the murine model demonstrates a sexual dimorphism in nitrite levels, consistent with data on human NO bioavailability. We then examined hepatic vaso-occlusion by challenging sickle mice with 2 hours of moderate normobaric hypoxia (10% oxygen), restoring them to normoxia, and then 18 hours later drawing blood to assay plasma alanine aminotransferase (ALT) levels as a quantitative measure of tissue injury. Sickle mice showed a 4- fold rise in ALT with this hypoxia-reoxygenation (HR) challenge; wild-type controls or non-sickling colony control mice with the same HR challenge had no change in ALT. Sexual dimorphism was again evident such that female mice were less susceptible to HR challenge than males. Therapeutic nitrite supplementation, administered either as 2.4 nanomole/g intraperitoneal injection during HR or oral supplementation (estimated 4 micromole/g/day) for 7 days before HR challenge, abrogated the ALT rise in sickle mice. Pharmacokinetic data indicated that nitrite supplementation at these injected and oral doses raise the nitrite levels in blood and tissue, without affecting methemoglobin levels. These studies provide additional mechanistic evidence for a gender contribution to sickle cell endothelial dysfunction and vasculopathy, and suggest that reduced NO bioavailabity in SCD results in dysregulated nitrite homeostasis. Depleted nitrite reserves are associated with enhanced injury with hypoxia-induced vaso-occlusion and therapeutic restoration of nitrite levels, both by intraperitoneal delivery and in the diet, reduces this injury.
MKL1 Promotes Megakaryocytic Differentiation Via Stimulation of Serum Response Factor Target Genes.
Authors: Cheng Ee-chun , Renda Matthew J. , Wang Lin , Krause Diane S. 
Published: October 2019

Abstract
Our studies demonstrate a critical role for MKL1 (megakaryoblastic leukemia 1) in the molecular regulation of megakaryocytopoiesis. MKL1 is part of the fusion protein formed by the t (1; 22) translocation, which is found uniquely in Acute Megakaryoblastic Leukemia (AMKL). The translocation fuses the RBM15 (also known as OTT) gene on chromosome 1 with the MKL1 (also known as MAL) gene on chromosome 22. Previous studies in muscle cells show that MKL1 is a positive cofactor for the transcription factor serum response factor (SRF), and works via the Rho-A pathway to turn on immediate early genes and muscle specific genes. Using qRT-PCR we show that MKL1 mRNA is markedly up-regulated during megakaryocyte (MK) differentiation of primary murine bone marrow and fetal liver cells. When we overexpress MKL1 in the human erythroleukemia (HEL) cell line and differentiate the cells to become MK by phorbol ester (TPA), there is far greater MK differentiation than in control HEL cells. Via analysis of Wright-Geimsa stained cytospins, MKL1 overexpression increases the average percentage of mature MK from 26% to 48%. Using flow cytometry, platelet glycoprotein V positive cells increase from 14% to 43% on average. The percentage of cells with greater than 4N ploidy increases from 13% to 34%. In order to assess the mechanisms by which MKL1 promotes MK differentiation, we tested whether SRF expression was required for the effects of MKL1. The stimulatory effects of MKL1 are strongly abrogated when cells are transfected with siRNA against SRF, proving that MKL1 acts via SRF to stimulate MK differentiation. Interestingly, SRF siRNA also causes a statistically significant decrease in ploidy in control cells stimulated with TPA. By microarray analyses using both Affymetrix and Illumina platforms, enforced MKL1 expression upregulates many cytoskeletal genes and adhesion molecules, enhances the expression of platelet specific genes such as glycoprotein V (consistent with the FACS data), and accelerates the loss of expression of genes associated with erythropoiesis, such as erythrocyte membrane protein band 4.2. These data indicate that MKL1 enhances MK differentiation by promoting endoduplication as well as increasing expression of platelet-specific genes and of multiple cytoskeletal proteins required for proplatelet formation.
Homeostatic Role of the Krüppel-Like Factor 4 (KLF4) in Proliferation and Differentiation of Primitive Hematopoietic Progenitor Cells.
Authors: Park Chun Shik , Yamada Takeshi , Lacorazza H. Daniel 
Published: October 2019

Abstract
Abstract 1497
Poster Board I-520
KLF4 is a tumor suppressor in the gastrointestinal tract known to induce cell cycle arrest in a cell context dependent manner. We recently reported that KLF4 maintains quiescence of T lymphocytes downstream of T-cell receptor signaling (Yamada et al., Nature Immunology, 2009). The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with Oct3/4, c-Myc and Sox2 suggests that KLF4 restricts proliferation of undifferentiated cells. In spite of a redundant role of KLF4 in fetal liver hematopoietic stem cells (HSC), its role in the maintenance of adult bone marrow HSCs has not been studied yet. To study the role of KLF4 in the hematopoietic system we used gain- and loss-of-function mouse models. Retroviral transfer of KLF4 into wild type bone marrow (BM) cells led to significant reduction of colony forming units (CFU) in methylcellulose cultures due to increased apoptosis and lower proliferation. Then, Mx1-Cre was used to induce deletion of Klf4-floxed mice by polyI:C administration. Analysis of peripheral blood cells up to 6-9 months post polyI:C administration showed significant reduction of monocytes, as previously reported, and expansion of CD8+CD44+ T cells due to their increased proliferative potential. BM cells from Klf4-deficient mice exhibited increased number of myeloid progenitor cells measured by flow cytometry (Lin-Sca-1-c-kit+FcRII/III+CD34+ cells), CFU and CFU-S8. Cytoablation with 5-fluorouracil (5-FU) showed lower nadir of peripheral white blood cells in Klf4-deficient mice compared to control mice. In spite of normal multilineage reconstitution in BM transplants experiments, competitive reconstitution with Klf4-deficient and normal BM cells resulted in reduced contribution of Klf4-deficient cells to peripheral blood, likely due to homing and proliferative differences. Collectively, our data shows that KLF4 has an important role in function of hematopoietic stem and progenitor cells.

Disclosures:
No relevant conflicts of interest to declare.
Acute Myeloid Leukemia Cells Depend on VEGF, PDGFR and SCF Receptor Signaling: Leukemia Regression with Pazopanib.
Authors: Gars Eric , McGee Christie S , Bosse Raphael C , Wise Elizabeth , Meacham Amy , Cogle Christopher R. 
Published: October 2019

Abstract
Abstract 1057
Acute myeloid leukemia (AML) cells depend on endothelial cells for survival and proliferation. By targeting endothelial cells with a novel vascular disrupting agent, we recently demonstrated regression of AML (Madlambayan, et al., Blood 2010). In an effort to discover a more selective, anti-vascular therapy for leukemia, we hypothesized that targeting endothelial cell-derived paracrine and leukemia cell-derived autocrine growth factors would result in regression of disease. In particular, our strategy focused on vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and stem cell factor (SCF), which are known to promote leukemia cell proliferation. Receptors for each of these growth factors are potently inhibited by pazopanib, which is an orally available tyrosine kinase inhibitor. The aim of our study was to determine the dependence of leukemia cell survival and proliferation on the combined receptor signaling of VEGF, PDGF and SCF. Leukemia cell lines (KG-1, HL60 and K562) were incubated at various durations with varying concentrations of pazopanib. Leukemia cell proliferation was quantified using XTT assay. Apoptosis induction was analysed by flow cytometry (Annexin V and PI staining). Pazopanib effectively impaired proliferation in leukemia cells in vitro in a dose and time dependent fashion. During a 16-hour incubation of leukemia cells, pazopanib showed a 50% lethal concentration (LC50) of 22.57 μM (r = .986), 41.6 μM (r = .991), and 81.97 μM (r = .996) for HL60, K562, and KG-1 cells, respectively. Staining with Annexin V and PI identified apoptosis as the main cause of cell death after exposure to pazopanib. The IC50 of apoptosis for HL60 cells was 132.5 ± 2.7 μM (p = 0.0002). These results indicate that leukemia cells depend on the combined signaling of VEGF, PDGF, and SCF, and suggest that selective inhibition by pazopanib may be a promising therapeutic for AML. Furthermore, pazopanib is orally available and associated with minimal side effects, thus representing an attractive candidate for further testing in AML.

Disclosures:
No relevant conflicts of interest to declare.
Liposomal Targeted Delivery Overcomes Immunostimulatory Effects of Oligonucleotide Based Therapy In Chronic Lymphocytic Leukemia.
Authors: Yu Bo , Mao Yicheng , Bai Li-Yuan , Herman Sarah E.M. , Ramanunni Asha , Jin Yan , Mo Xiaokui , Cheney Carolyn , Chan Kenneth K. , Jarjoura David , Marcucci Guido , Lee Robert , Lee L.James , Byrd John C. , Muthusamy Natarajan 
Published: October 2019

Abstract
Abstract 1475

Therapeutic application of several CpG-containing anti-sense oligodeoxyribonucleotides (ODN) and siRNAs are limited due to their potent immune stimulatory properties. To resurrect these promising agents for clinical application, we have developed an antibody based liposomal delivery strategy that overcomes the immunostimulatory properties without compromising the target down modulation. We show here G3139, an archetypical anti-sense ODN for Bcl-2, induced activation of NF-kB, which results in the up-regulation of its downstream anti-apoptotic targets such as Bcl-2 (22.5%, n=10, p=0.04) and Mcl-1 (35.0%, n=4, p<0.005) and co-stimulatory markers such as CD86 (47.5%, n=10, p<0.05) and HLA-DR (175%, n=10, p<0.005) as well as cytokine flare in B chronic lymphocytic leukemia (CLL) cells. These adverse immunostimulatory responses are abrogated by Rituximab mediated immunoliposomal delivery of G3139, resulting in significant Bcl-2 down-regulation and ∼60% enhanced sensitivity to fludarabine induced cytotoxicity (n=4, p<0.001). Consistent with the in-vitro observations, significant in-vivo therapeutic efficacy was demonstrated in a SCID mouse xenograft leukemia/lymphoma model. A total of 74 female 6- to 8-week-old SCID mice were engrafted with 2×106 Raji cells through tail vein injection on Day 0. The 10-day treatment was initiated via i.p. injection on Day 4. Fludarabine and G3139 were injected into the mice at the dose of 100mg/kg/day and 5mg/kg on alternative days respectively. Hind limb paralysis was considered as the key endpoint while other early removal criteria were also considered. The median survival time for placebo controls was 15 to 18 days without liposomal G3139 treatment. Although the in-vivo experiment is still in progress, CD20-targeting ILP-G3139 has already shown significant therapeutic efficacy with >80% survival rate (n=14) at day 58 of the study as a single agent or in combination with fludarabine treatment, compared to the mice treated with the control Her-ILP-G3139 that showed hind-limb paralysis before Day 45 in all mice. These results indicate potential use of targeted delivery vehicles to resurrect and improve the clinical efficacy of immunostimulatory oligonucleotide therapeutics for B-cell malignancies.

Disclosures:
No relevant conflicts of interest to declare.
F-627, a G-CSF Dimer, Stimulated a More Rapid Neutrophil Recovery In Cyclophosphamide-Treated Monkeys Compared to Monomer Rhg-CSFs.
Authors: Hu Zheng Tao , Huang Zhi Hua , Cen Xiao Bo , Wang Tao , Zhuang Kun , Yang Hong Zhou , Zhang Dan Ping , Sun Nai Chau , Sun Rou Yun , Wang Li , Huang Yu Liang , Yan Xiao Qiang 
Published: October 2019

Abstract
Abstract 1485
Recombinant human granulocyte colony-stimulating factors (rhG-CSF) including filgrastim, lenograstim and pegfilgrastim are widely used to treat chemotherapy-induced neutropenia. However, it remains a challenge to manage severe neutropenia in cancer patients after high dose chemotherapy. The activation of G-CSFR by the G-CSF requires dimerization of two receptor chains bound to two G-CSF ligands. We hypothesized that a G-CSF dimer might generate a faster in vivo response, thus to benefit patients with severe neutropenia. F-627 is a recombinant human G-CSF dimer expressed in mammalian cells. In vitro, F-627 was able to activate STAT3 and to stimulate the proliferation of 32D-GCSFR and M-NSF60 cell lines. To evaluate the efficacy of F-627 in chemotherapy-induced neutropenia, 36 adult cynomolgus monkeys (3/sex) were injected with cyclophoshamide (CY, i.v., 60 mg/kg on day 0 and 65 mg/kg on day 1). Animals were randomized to receive (s.c.) either carrier, or F-627 on day 5 and day 10, at 25, 60 and 150 μg/kg, or rhG-CSF at 10 μg/kg /day (daily injection), or pegfilgrastim at 60 μg/kg on day 5 and day 10. Pharmacokinetics and pharmacodynamics (PK/PD) were evaluated. Significant absolute neutrophil count (ANC) increase was observed in animals treated with F-627 at 25 μg/kg compared to carrier-treated monkeys. At nadir, F-627 at 60 μg/kg generated optimal ANC response with 6.9- and 3.0-fold higher ANC compared to pegfilgrastim- (0.17 × 109/L) and rhG-CSF- (0.39 × 109/L) treated monkeys, respectively. The PK parameters of F627 at 60 ug/kg including MRT, Cmax, Tmax, AUC and CL were comparable to that of pegfilgrastim-treated monkeys, despite the relative G-CSF molar dose in F-627-treated animals was 2.5x lower than the pegfilgrastim-treated animals. The results demonstrate that the G-CSF dimer generated a faster response in CY-treated monkeys compared to the G-CSF monomer-treated animals, suggesting that F-627 could benefit cancer patients with severe neutropenia after high-dose chemotherapy.

Disclosures:
No relevant conflicts of interest to declare.
The Immune-Inhibitory Receptor Osteoactivin Is up-Regulated In Monocyte-Derived Dendritic Cells Upon Exposure to Tyrosine Kinase Inhibitors
Authors: Gutknecht Michael , Schwarzbich Mark-Alexander , Salih Julia , Kanz Lothar , Salih Helmut R. , Schmidt Susanne M. , Grünebach Frank 
Published: October 2019

Abstract
Abstract 1733
Targeted therapies with tyrosine kinase inhibitors (TKI) have significantly improved the treatment of cancer patients. Ex vivo generated dendritic cells (DC) are commonly used in immunotherapeutic strategies due to their unique ability to initiate adaptive immune responses, and multiple approaches presently aim to combine targeted therapies with immunotherapy. However, as many kinases targeted by TKI are, besides governing tumor cell growth, also involved in the activation of DC, TKI therapy may cause immunoinhibitory side effects. Osteoactivin (GPNMB, DC-HIL) is a type I transmembrane glycoprotein that is detected abundantly in DC but not in monocytes. Its expression on antigen-presenting cells can inhibit T cell activation by binding syndecan-4 (SD-4) on T cells. Here we investigated the effect of the BCR/ABL TKI imatinib, dasatinib and nilotinib, which are approved for the treatment of CML, on the expression of osteoactivin and DC functions. DC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Imatinib, nilotinib or dasatinib were added to the culture medium every second day starting from the first day of culture. In some experiments, toll-like receptor (TLR) ligands (L) (LPS (TLR4L), pam3Cys (TLR2L), poly I:C (TLR3L) or R848 (TLR7/8L) were added on day 6 of culture for maturation of DC. We found that DC generated in the presence of therapeutic concentrations of all three TKI displayed an altered phenotype. Imatinib caused significantly reduced expression of the typical DC markers CD1a, CD83 and the co-stimulatory molecule CD86. Nilotinib reduced the expression of CD1a, CD83, CD86 and the DC-specific C-type lectin receptor DC-SIGN (CD209). Dasatinib impaired expression of CD1a, CD83, CD86, CD80 and DC-SIGN. Most notably, we observed excessive up-regulation of osteoactivin on DC upon treatment with all three TKI. Interestingly, incubation with the immunosuppressive and anti-inflammatory cytokine IL-10 also resulted in osteoactivin over-expression. In line with osteoactivin up-regulation, exposure to TKI resulted in reduced stimulatory capacity of DC in MLR with allogenic T cells that could be restored by addition of blocking anti-osteoactivin antibody. In summary, our data demonstrate that up-regulation of osteoactivin is critically involved in the inhibition of DC function upon TKI exposure. These findings are of great importance for future combinatory approaches using TKI and DC-based immunotherapy and indicate that inhibition of osteoactivin expression or function may serve as a novel strategy to enhance the efficacy of immunotherapeutic interventions in cancer patients.

Disclosures:
No relevant conflicts of interest to declare.
A Screen for Drugs Showing Specific Co-Resistance or Hyper-Sensitivity Upon the Acquisition of Ara-C Resistance In AML
Authors: Rathe Susan K , Largaespada David 
Published: October 2019

Abstract
Abstract 1844
Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. We hypothesized that the changes required by the cells to become Ara-C resistant might also make them more susceptible to elimination by other drugs. We previously developed two highly resistant murine AML cell lines, B117H and B140H, by introducing increasing concentrations of Ara-C to their parental cell lines, B117P and B140P, respectively. The Ara-C resistant derivatives, B117H and B140H, can tolerate Ara-C concentrations ∼1000X that of their drug sensitive parental cell lines. We previously demonstrated that the down-regulation of deoxycytidine kinase was one mechanism of resistance in the Ara-C resistant B117H and B140H cells, a mechanism that has also been documented in human cases of refractory disease. A high throughput drug screen was conducted to determine if an increase in Ara-C resistance would result in increased sensitivity to other drugs. The screen evaluated 446 FDA approved drugs, which included a variety of drug types, including chemotherapy drugs, analgesics, steroids, antidepressants, immunosuppressives, and antipsychotics. Despite the diverse nature of the parental cell lines and their Ara-C resistant derivatives, two drugs (prednisolone and AM404), showed an increase in sensitivity in both Ara-C resistant cell lines. Follow-up drug assays were performed on both prednisolone and AM404 to establish the 50% inhibitory concentration (IC50) in all four cell lines. Both Ara-C resistant cell lines displayed a greater than 3-fold reduction in the IC50 of prednisolone as compared to their parental lines, and the IC50 of AM404 demonstrated a greater than 2-fold reduction. Prednisolone has been shown to induce differentiation in murine AML cells in vitro, and high-dose methylprednisolone has been found to induce differentiation in various subtypes of childhood AML. The mechanism of differentiation by prednisolone is theorized to be independent of glucocorticoid receptor binding. We believe this model will be useful in elucidating the role of prednisolone in leukemic differentiation, and in suggesting new mechanisms to target refractory AML cells.

Disclosures:
No relevant conflicts of interest to declare.
Local Treg Immunomodulation Cures Metastatic Lymphoma Including CNS Sites
Authors: Marabelle Aurelien , Kohrt Holbrook E , Brody Joshua , Torchia James Anthony , Zhou Gang , Luong Richard , Rajapaksa Ranjani , Tse Victor , Levy Ronald 
Published: October 2019

Abstract
Abstract LBA-2

Background:
CD4+CD25+FOXP3+ regulatory T-cells (Tregs) protect cancers from the immune system. They express important functional molecules such as CTLA-4 and Ox40 on their surface. Anti-CTLA4 monoclonal antibody (mAb) is the first drug to improve survival of refractory metastatic melanoma. Its recent approval by the FDA/EMEA inaugurates a paradigm shift in cancer therapy where the immune system is targeted rather than the tumor itself.

Scientific question:
Does local immunomodulation of tumor specific Tregs trigger a systemic anti-tumor immune response and cure disseminated lymphoma?

Results:
The Tregs that infiltrate the tumor site preferentially express CTLA4 and OX40 compared to their counterparts in the blood and other lymphoid organs, both in mice and in humans. Upon injection of low doses of anti-CTLA4 and anti-OX40 together with CpG, a TLR-9 agonist, directly into the tumor, tumor-specific Tregs are depleted from the tumor-infiltrate. This immunomodulatory combination therapy triggers an anti-tumor immune response able to cure mice with established disseminated disease. The triple combination is uniquely required as neither CpG alone nor mAbs without CpG are effective. The local Treg immunomodulation with anti-CTLA4+anti-OX40+CpG in a single sub-cutaneous tumor eradicates disease in mice with established CNS lymphoma, even with leptomeningeal and spinal cord metastases, whereas chemotherapies or mAb therapy directed against the tumor cells have little effect in the CNS site. Moreover, these cured mice have a long term intra-cranial anti-tumor immunity since they are protected against late intra-cranial re-challenge.

Significance:
Immunomodulatory antibodies are currently under clinical development for cancer treatment, and their major side effect is the triggering of auto-immune diseases. We show here that injecting very little doses of these antibodies in combination with CpG at one tumor site is sufficient to trigger a systemic anti-tumor response able to eradicate distant sites, including in the CNS which is usually considered a sanctuary site for conventional systemic therapies.

Impact:
We recently have published positive results of intra-tumoral CpG in patients with follicular Lymphoma (Brody,Levy, et al. JCO, 2010). Anti-CTLA4 has just been FDA approved in patients with metastatic melanoma, and anti-Ox40 antibodies are currently being tested in phase I/II clinical trials. Therefore, the combination described here can be tested in patients with injectable sites of lymphoma, even if they have CNS disease.

Disclosures:
No relevant conflicts of interest to declare.