bluuebunny/crossref_metadata_2025_split huggingface dataset:
Palmitoyl Acyltransferase DHHC9 Is Required for Efficient Induction of Leukemia By Oncogenic NRAS
Authors: Liu Ping , Jiao Bo , Zhang Ruihong , Zhao Huanbin , Zhang Chun , Wu Min , Qiu Qingsong , Li Donghe , Zhao Xuemei , Ren Ruibao 
Published: October 2019

Abstract
Hyperactivation of RAS is common in human cancer, including hematological malignancies. Since RAS proteins are difficult to target, identification of alternative means to block RAS oncogenic signaling is critical for developing therapies against RAS-driven cancer. The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Among RAS PTMs, palmitoylation is required for the high-affinity plasma membrane binding of NRAS, HRAS and KRAS4A. NRAS mutations are common in human hematological malignancies. We have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for RAS related cancers. In previous studies, we blocked NRAS palmitoylation by mutating the palmitoylation site in NRAS. Therapeutic intervention of RAS palmitoylation requires targeting enzymes that mediate the modification reaction. Protein S-palmitoylation is catalyzed by the DHHC family of palmitoyl acyltransferases (PATs). Thus far 24 mammalian PATs have been identified. It has been shown that DHHC9 (a 364-amino acid protein encoded by ZDHHC9) is the ortholog of yeast Ras2 PAT and constitutes a mammalian PAT with specificity for H- and NRAS in vitro. Increased expression of DHHC9 has been found in various cancers. In this study we investigated the role of DHHC9 in normal hematopoiesis and NRAS leukemogenesis in vivo. We found that DHHC9 is not the only PATs for RAS palmitoylation in vivo and the Zdhhc9 knockout mice were born and grew similarly as the wild type mice. The frequency of lineage-specific populations and hematopoietic stem cell phenotypes were also similar in mice with knockout alleles of Zdhhc9 as that of wild type mice. However, loss of DHHC9 prolonged the survival of mice with myeloproliferative disease or T-cell acute lymphoblastic leukemia evoked by oncogenic NRAS expressed either from the endogenous locus or from retroviral promoter. These results demonstrate that DHHC9 is dispensable for normal hematopoiesis, but plays an important role in the pathogenesis of NRAS-induced leukemias. The findings suggest that palmitoyl acyltransferase DHHC9 may serve as a safe and effective target for developing therapies against NRAS-related cancers.

Disclosures
No relevant conflicts of interest to declare.
Carma1 Is Important for T-ALL CNS Infiltration and Survival in an Animal Model of T-ALL
Authors: Oruganti Sreenivasa Rao , Halsey Christina , Krebsbach Savannah , Asperti-Boursin Francois , Patel Sumit , Matlawska-Wasowska Ksenia , Winter Stuart S. , Cannon Judy 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) is one of the most common childhood cancer, and can arise from the T or B cell lineage (T-ALL or B-ALL respectively). T-ALL patients have almost 80% cure rate, however leukemic cell migration to the central nervous system (CNS) remains a major therapeutic challenge. Little is known about potential regulators of T-ALL cell migration to the CNS, and identifying new molecules that can drive T-ALL to the CNS may provide new targets for drug therapy to specifically block T-ALL CNS infiltration. Recently it has been shown that a chemokine receptor CCR7 is an essential regulator of T-ALL cell infiltration into the CNS. CCR7 is a homeostatic chemokine receptor expressed on T cells that controls the homing of cells to lymph nodes, including na•ve T cells, dendritic cells, and lymphoma cells. Aberrant CCR7 expression has also been associated with migration of gastric, breast and head and neck cancers. We recently identified the molecule CARMA1 as a signaling mediator that acts downstream of CCR7.
We now find that CARMA1 regulates CNS migration in T-ALL. We have established a xenograft model of T-ALL, where we can assess the engraftment of a T-ALL cell line into the NOD/SCID/IL2Rγ2 immunodeficient mice. Using luciferase expressing T-ALL cells lacking CARMA1, we imaged tumor load using the IVIS imaging system. Animals receiving T-ALL cells lacking CARMA1 show a significant delay in the timing of leukemia development as well as longer overall survival compared to animals receiving wild type T-ALL cells (n=25, P value < 0.0001). In addition, animals injected with CARMA1-knockdown T-ALL cells have lower tumor burden in the brain. In some cases, no tumor cells were found in the brains of animals receiving CARMA1-knockdown T-ALL cells. Using immunohistochemistry and flow cytometry, we also find lower tumor burden in liver and spleen of mice injected with CARMA1-knockdown T-ALL cells compared to WT cells. We confirmed the role of CARMA1 in a NOTCH1ΔE-induced T-ALL animal model. Similar to the xenograft model, we find that mice receiving CARMA1KO-Notch1ΔE survived longer than mice receiving with WT- Notch1ΔE. These results demonstrate for the first time that CARMA1 may play an important role in T-ALL induction and leukemic cell migration to the CNS.
Numerous studies have demonstrated the importance of CARMA1 in normal T cell function. We now find that CARMA1 is also involved in controlling the migration of T-ALL cells to the CNS as well as survival of animals with T-ALL. These results suggest that CARMA1 may provide a novel therapeutic target in T-ALL.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
T-Antigen Expression Causes Macrothrombocytopenia and Extensive Hemophagocytosis in Mice Lacking the Sialyltransferase ST3Gal-I Specifically in Megakaryocytes
Authors: Grozovsky Renata , Giannini Silvia , Ramsey Haley , Sola-Visner Martha , Hoffmeister Karin M 
Published: October 2019

Abstract
Changes in glycans expression have been associated with defects in blood platelet counts. However, the role of posttranslational modifications on platelet production is poorly understood. Six genes encoding sialyltransferases (ST)3Gal-I to -VI that form a2-3 sialic acid linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. We and others have shown previously that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor. Loss of ST3Gal-I activity has been associated with core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T antigen). We here investigated the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency by generating St3gal1loxP/PF4+ mice that lack ST3Gal-I specifically in the megakaryocyte (MK) lineage. Blood platelet counts were reduced by ~50% in St3gal1loxP/PF4+ mice, compared to control mice. Other blood cell counts were normal in St3gal1loxP/PF4+ mice. The clearance rate of St3gal1-null platelets was increased by ~15%, as determined by in vivo platelet biotinylation. Bone marrow MK numbers were normal in St3gal1loxP/PF4+ mice, compared to control mice, indicating that mechanisms other than clearance regulate circulating platelet counts in St3gal1loxP/PF4+ mice. Both St3gal1loxP/PF4+ platelets and bone marrow MKs had increased T antigen expression, as evidenced by flow cytometry using peanut agglutinin (PNA) binding. St3gal1loxP/PF4+ mice had increased bone marrow macrophage numbers, as evidenced by immunohistochemistry and flow cytometry using the macrophage marker F4/80. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), as determined by immunohistochemistry and flow cytometry, indicative of an activated macrophage state. Consistently, St3gal1loxP/PF4+ bone marrow smears stained with May-Grunwald/Giemsa revealed increased hemophagocytosis. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes normalized blood platelet counts and size, and significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice. Together, our data indicates that platelet production in the bone marrow is reliant on correct glycosylation on MK surface proteins and that the intimate interaction between MKs and macrophages play an important role in regulating platelet production and bone marrow homeostasis.

Disclosures
No relevant conflicts of interest to declare.
The Efficacy of Low-Dose Cytosine Arabinoside Therapy for Patients of Down Syndrome with Transient Abnormal Myelopoiesis
Authors: Kanzawa Takahiro , Suzuki Kyogo , Sakaguchi Hirotoshi , Yoshida Nao , Kato Koji 
Published: October 2019

Abstract
<Introduction>
Transient abnormal myelopoiesis (TAM) occurs in about 10% of neonates with Down syndrome. While TAM is generally considered as self-limiting disease, substantial number of patients suffer from serious complications including liver fibrosis or pericardial effusion and eventually leads to fatal clinical outcomes. Low-dose cytosine arabinoside (CA) therapy has been reported to be effective for clinical symptoms of TAM patients, but it is still controversial as to which patient should be treated. We have retrospectively reviewed the clinical outcomes of TAM patients in our institution to confirm the efficacy as well as the safety of low-dose CA therapy by comparing the clinical outcomes before and after introduction of CA.
<Patients>
Before introduction of CA, we have experienced 20 TAM patients of 13 male and 7 female between September 1992 and November 2008. The median gestational age of those was 37w4d (range, 31w5d - 39w2d) , median peak value of white blood cell (WBC) count was 48×109/L (range, 19.8 - 399×109/L), and median peak direct bilirubin(DB) level was 0.9 mg/dL (range, 0.1 - 18.4 mg/dL), respectively. Sixteen of twenty patients (80%) had various complications including dyspnea, hepatosplenomegaly, pericardial effusion, and ascites.
Since 2009, we have introduced low dose CA (0.4 - 1.0mg/kg/day) for those with high WBC count (>100×109/L) until WBC count decrease to 10×109/L . Three patients received chemotherapy, but one patient with high WBC count did not, because consent was not obtained. The median gestational age of those was 36w4d (range, 33w6d - 39w2d) , median peak value of WBC count was 106×109/L (range, 101 - 267×109/L), and median peak DB level was 1.95mg/dL (range, 1.0 - 11.0 mg/dL), respectively. All of them had various complications including dyspnea, hepatosplenomegaly, pericardial effusion, liver fibrosis and ascites.
<Results>
Before introduction of low dose CA therapy, five of six patients (83%) with high WBC count (>100×109/L) died of liver failure(4 of 5 patients) or AML, and one of 14 patients (7%) with low WBC count (<100×109/L) died of pneumonia at the age of eight without any relation to TAM. After introduction of CA, all three patients who received chemotherapy are alive without serious complications related to TAM or chemotherapy. Their blast cells in the peripheral blood disappeared promptly and liver dysfunction as well as biomarker of liver fibrosis is getting normalized. One patient who did not receive chemotherapy died of liver failure in spite of supportive care.
<Conclusion>
It is suggested that low dose CA therapy is safely conducted and is effective to improve the clinical outcomes of TAM patients with high WBC count. Prospective analysis is mandatory to confirm our findings by multi-institutional basis.

Disclosures
No relevant conflicts of interest to declare.
Role of Microbes in B-Cell Lymphopoiesis and Early Ig Repertoire Development
Authors: Wesemann Duane R. , Shrestha Akritee , Magee Jennifer , Chen Yuezhou , Silver Jared , Granato Alessandra 
Published: October 2019

Abstract
Progenitor (pro-) and precursor (pre-) B cells express the recombination-activating gene (RAG1/RAG2) endonuclease, which initiates the V(D)J recombination reaction that assembles Immunoglobulin heavy (IgH) and light (IgL) chain variable region exons from germline gene segments. Expression of productively assembled IgH μ chains and IgL (Igκ or Igλ) chains generates IgM molecules that form the B-cell antigen binding receptor (BCR) with a diverse primary repertoire of binding specificities. As the newly assembled and expressed IgM interacts with local antigens, RAG expression can continue, allowing continued Igκ V(D)J recombination that can replace the previously assembled VκJκ exon with one that generates a new specificity. This "receptor editing" process provides an antigen-mediated regulatory checkpoint that helps shape the pre-immune BCR repertoire. As the principal location of early B-cell development, the bone marrow antigenic environment is the main force affecting receptor editing. However, the extent to which self-antigens outside of the bone marrow — as well as environmental antigens — may shape the primary Ig repertoire is not known. Here we show that early B-cell development takes place in peripheral sites, including the small intestinal mucosa, in young mice. Cells in these sites co-express RAG with cytoplasmic Igκ and Igμ, indicating active receptor editing; and, correspondingly, they have significant differences in Vκ usage compared to RAG2-expressing bone marrow B-lineage cells and other RAG2-expressing B-lineage cells from other peripheral sites. In addition, the luminal microbial environment appears to also affect Igλ/Igκ ratios. We conclude that early B-cell development can occur in the periphery, including the intestinal mucosa, where commensal microbes may influence BCR diversification and gut pre-immune Ig repertoires.

Disclosures
No relevant conflicts of interest to declare.
Molecular Modification of the Microenvironment by the Tumor Clone
Authors: Ghobrial Irene M. 
Published: October 2019

Multiple Myeloma (MM) is a disease of plasma cells with specific localization in the bone marrow. Recent studies have shown that MM is consistently preceded by Monoclonal Gammopathy of Undetermined Significance (MGUS). This has given rise to the concept of a myeloma precursor disease and raised questions about the biologic events leading to progression of these precursor states to symptomatic MM. The genomic complexity in MM was recently corroborated by next generation sequencing which revealed heterogeneity in tumors with tumor progression in a branching rather than in a linear manner, leading to substantial clonal diversity and coexistence of wide genetic heterogeneity. Although many factors regulating tumor progression are tumor cell autonomous, they are insufficient to induce progression and metastasis, and a permissive microenvironment is required for frank malignancy to emerge. Indeed, studies have shown that the tumor microenvironment is a key regulator in many steps of the invasion-metastasis cascade including tumor oncogenesis, egress, protection in the circulation, preparation of the metastatic niche, organ-specific homing, and the permissive role of the microenvironment in tumor colonization. These studies provide proof of bidirectional interactions that occur between tumor cells and the nearby microenvironment that are permissive for tumor initiation and progression, establishing a positive-feedback loop that may be self-amplifying. Although the bone marrow (BM) microenvironment is commonly referred to as the “non-tumor” entity, it has to be kept in mind that it is a complex network including a broad range of cells and factors that act as the "malignant niche" that is permissive for tumor growth in MM. Indeed, the BM microenvironment consists of three components: the cellular component (hematopoietic and non-hematopoietic cells, including mesenchymal stromal cells, osteoclasts, osteoblasts, endothelial cells, and immune cells such as T-regs, myeloid derived suppressor cells and plasmacytoid dendritic cells); the extracellular matrix component; and the soluble component (such as exosomes, cytokines, circulating free miRNA and growth factors). Multiple biological processes are affected by interactions between abnormal plasma cells and the BM microenvironment including homing to the BM, spread to secondary BM sites by the bloodstream, generation of paracrine factors, osteoclastogenesis, inhibition of osteogenesis, immune cell dysregulation, and angiogenesis lead to the progression of MM from early MGUS stages to overt metastatic myeloma.

Disclosures
Ghobrial: BMS: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Off Label Use: CXCR4 inhibitors, SDF-1 inhibitors.
MYd88 Blockade Caused Reduction of PDL1 on Tumor Infiltrating MDSCs in a Murine Model of Melanoma
Authors: Forghani Parvin , Waller Edmund K 
Published: October 2019

Abstract
Introduction: Myeloid differentiation primary response gene 88 (Myd 88) is an important adaptor molecule for the activation of NADPH oxidase and regulation of arginase-1, which are responsible for the suppressive function of myeloid-derived suppressor cells (MDSCs). Blockade of Myd88 signaling induces antitumor effects in mice by skewing the immunosuppressive function of myeloid-derived suppressor cells. As the PD-L1/PD-1 axis has been characterized as a potent inhibitor of immune activation, particularly through inhibition of effector T cell function, we characterized the effect of Myd88 on checkpoint expression on tumor-infiltrating MDSC/T cells in a murine model melanoma.
Methods: Pathogen-free 8-10-week-old WT(B6-background) and Myd 88−/− mice that been backcrossed to a C57BL/6 genetic background were challenged with 1 × 106 B16 (F1) tumor cells s.c. On day 14, mice were sacrificed and spleen and tumors were removed and digested into single-cell suspensions, blocked with anti-FcR mAbs and analyzed for surface and intracellular staining by flow cytometry. We analyzed CD11b+/Gr-1+hi/int myeloid cells subsets and T cells in the blood, spleen and tumors of mice by flow cytomery.
Results: The growth of B16 melanoma tumor was significantly slower in Myd 88−/− mice compared with WT mice. No significant difference between two groups was found in the frequency of absolute number of MDSC subsets and expression of PDL1 check-point marker on spleen-derived MDSC subsets. In contrast CD4(+) and C8(+) T cells residing in spleens of Myd88(-/-) mice showed increased expression of TNF-α/IFN-α and GrZB compared with T cells from wild-type mice following short-term activation with PMA/iono. Of note, the frequencies and absolute numbers of infiltrating CD11b+/Gr1+ MDSC in tumor-bearing Myd 88−/− mice were lower than those in WT mice. Also we found that viable CD11b+/Gr1+ MDSC subsets from WT mice expressed higher level of PD-L1 compared with MDSCs from Myd 88−/− mice in concordance with the reduced expression of PD-1 on tumor infiltrating CD4+ T cells in Myd 88−/− mice. Collectively, the profile of PD-L1 and PD-1 expression in tumor microenvironments is favorably altered to enhance adaptive immune response in myd 88 KO vs WT mice harboring B16 melanoma.
Conclusion: The results of this study provide further evidence that blocking Myd 88 signaling increases anti tumor immunity against melanoma, and that the enhanced immunity can be explained, in part, by reduction of expression PDL1/PD1 immune checkpoint molecules. Considering the importance of tumor-infiltrating MDSCs in regulating anti tumor immunity in the tumor microenvironment, our findings could provide insight into the design of new therapeutics targeting Myd 88. Further experiments are needed to show how alteration in profile of PDL1 checkpoint expression on MDSCs influences anti-tumor T cell responses.

Disclosures
No relevant conflicts of interest to declare.
Tumor Cell Thrombin/PAR-1 Signaling Drives Pancreatic Ductal Adenocarcinoma Growth and Dissemination
Authors: Flick Matthew J. , Rewerts Cheryl , Cruz Carolina , Palumbo Joseph S. , Luyendyk James P. , Yang Yi , Konieczny Stephen F. 
Published: October 2019

Abstract
Pancreatic ductal adenocarcinoma (PDAC) accounts for ~85% of diagnosed pancreatic cancers and is among the most lethal malignancies. The 5-year survival rate for pancreatic cancer patients has improved only marginally in the last 40 years (3% → 7%), with effectively no change in survival profile for patients with metastatic disease (2%). High mortality is linked to the aggressive and invasive nature of the malignancy and poor efficacy of limited treatment options, which collectively highlight the need for novel treatment strategies. Notably, analyses of pancreatic cancer in patients and animal models have demonstrated that PDAC is associated with robust coagulation system activity. Previous work has shown that patient PDAC tumor cells often express high levels of tissue factor (TF) and protease-activated receptor (PAR)-1. To determine the potential contribution of tumor cell derived-TF and PAR-1 to PDAC growth and metastasis, a novel tumor cell line (termed KPC2) was derived from mice in which PDAC tumorigenesis was induced by activation of two established pancreatic cancer alleles, KrasG12D and Trp53R172H. In transplant studies, tumor growth and experimental metastasis were evaluated using KPC2 cells in which TF or PAR-1 expression was suppressed by shRNA knockdown. In addition, the interplay of tumor-derived TF and PAR-1 with host factors in promoting tumor growth and experimental metastasis were evaluated in mice with genetically imposed deficits in coagulation system components. TF knockdown (to ~10% of the parental line) in KPC2 cells resulted in a significant diminution of both primary tumor growth and experimental metastasis. This reduction appeared to be linked to thrombin activity as primary tumor growth and experimental metastasis of parental KPC2 cells were significantly reduced in fIIlow mice (which constitutively express 10% of normal prothrombin) relative to wild-type mice. PAR-1 knockout mice displayed similar KPC2 growth and experimental metastasis to wild-type animals indicating that stromal cell-derived PAR-1 was not significant determinant. In stark contrast, shRNA-mediated knockdown of PAR-1 in KPC2 (to ~10% of the parental line) cells resulted in significantly diminished tumor growth and experimental metastasis. Diminished tumor growth was linked to reduced expression of the macrophage chemokine MCP-1 and the metalloproteinase MMP9 by the tumor cells as well as reduced thrombin-stimulated ERK phosphorylation. Our results suggest that a major mechanism of PDAC growth and dissemination is through TF/thrombin-driven PAR-1 signaling on tumor cells.

Disclosures
No relevant conflicts of interest to declare.