Search

The development of the mononuclear phagocyte system requires macrophage colony-stimulating factor (CSF-1) signaling through the CSF-1 receptor (CSF1R, CD115). We examined the effect of an antibody against CSF1R on macrophage homeostasis and function using the MacGreen transgenic mouse (csf1r-enhanced green fluorescent protein) as a reporter. The administration of a novel CSF1R blocking antibody selectively reduced the CD115+Gr-1neg monocyte precursor of resident tissue macrophages. CD115+Gr-1+ inflammatory monocytes were correspondingly increased, supporting the view that monocytes are a developmental series. Within tissue, the antibody almost completely depleted resident macrophage populations in the peritoneum, gastrointestinal tract, liver, kidney, and skin, but not in the lung or female reproductive organs. CSF1R blockade reduced the numbers of tumor-associated macrophages in syngeneic tumor models, suggesting that these cells are resident type macrophages. Conversely, it had no effect on inflammatory monocyte recruitment in models, including lipopolysaccharide-induced lung inflammation, wound healing, peritonitis, and severe acute graft-versus-host disease. Depletion of resident tissue macrophages from bone marrow transplantation recipients actually resulted in accelerated pathology and exaggerated donor T-cell activation. The data indicate that CSF1R signaling is required only for the maturation and replacement of resident-type monocytes and tissue macrophages, and is not required for monocyte production or inflammatory function.

Purpose: Lysophosphatidic acid acyltransferase (LPAAT)-β catalyzes the conversion of lysophosphatidic acid to phosphatidic acid, an essential component of several signaling pathways, including the Ras/mitogen-activated protein kinase pathway. Inhibition of LPAAT-β induces growth arrest and apoptosis in cancer cell lines, implicating LPAAT-β as a potential drug target in neoplasia. Experimental Design: In this study, we investigated the effects of CT32228, a specific LPAAT-β inhibitor, on BCR-ABL-transformed cell lines and primary cells from patients with chronic myelogenous leukemia. Results: CT32228 had antiproliferative activity against BCR-ABL-positive cell lines in the nanomolar dose range, evidenced by cell cycle arrest in G2-M and induction of apoptosis. Treatment of K562 cells with CT32228 led to inhibition of extracellular signal-regulated kinase 1/2 phosphorylation, consistent with inhibition of mitogen-activated protein kinase signaling. Importantly, CT32228 was highly active in cell lines resistant to the Bcr-Abl kinase inhibitor imatinib. Combination of CT32228 with imatinib produced additive inhibition of proliferation in cell lines with residual sensitivity toward imatinib. In short-term cultures in the absence of growth factors, CT32228 preferentially inhibited the growth of granulocyte-macrophage colony-forming units from chronic myelogenous leukemia patients compared with healthy controls. Conclusion: These data establish LPAAT-β as a potential drug target for the treatment of BCR-ABL-positive leukemias.

Phosphatidic acid (PA) is an important component of mammalian target of rapamycin (mTOR) signaling and in the recruitment of Raf to the cell membrane. PA can be produced by several mechanisms, including by a series of lysophosphatidic acid acyl transferases (LPAATs). LPAAT-beta is an isoform that is overexpressed in some human cancers and its inhibition has been investigated as a potential targeted cancer therapy. We report that LPAAT-protein and enzyme activity in acute leukemia cell lines and blasts from patient samples are equivalent to levels in normal mononuclear cells. Treatment with the LPAAT-beta inhibitor CT-32228 (Cell Therapeutics, Seattle, WA) uniformly induces apoptosis in multiple leukemia cell lines. In patient samples, however, apoptosis was variably induced by CT-32228 and appeared to be related to the degree of cellular proliferation. The growth inhibitory effect of CT-32228 on normal hematopoietic progenitors was more pronounced in cells induced to proliferate by growth factors. These data suggest that CT-32228 may have potential in the treatment of acute leukemias, but that efficacy is more directly related to the degree of cell proliferation rather than to the level of LPAAT-beta expression or activity.

Membranes of mammalian cells contain lysophosphatidic acid acyltransferase (LPAAT) activities that catalyze the acylation of sn-1-acyl lysophosphatidic acid (lysoPA) to form phosphatidic acid. As the biological roles and biochemical properties of the six known LPAAT isoforms have yet to be fully elucidated, we have characterized human LPAAT-beta activity using two different assays. In a membrane-based assay, LPAAT-beta used lysoPA and lysophosphatidylmethanol (lysoPM) but not other lysophosphoglycerides as an acyl acceptor, and it preferentially transferred 18:1, 18:0, and 16:0 acyl groups over 12:0, 14:0, 20:0, and 20:4 acyl groups. The fact that lysoPM could traverse cell membranes permitted additional characterization of LPAAT-beta activity in cells: PC-3 and DU145 cells converted exogenously added lysoPM and (14)C-labeled 18:1 into (14)C-labeled phosphatidylmethanol (PM). The rate of PM formation was higher in cells that overexpressed LPAAT-beta and was inhibited by the LPAAT-beta inhibitor CT-32501. In contrast, if lysoPM and (14)C-labeled 20:4 were added to PC-3 or DU145 cells, (14)C-labeled PM was also formed, but the rate was neither higher in cells that overexpressed LPAAT-beta nor inhibited by CT-32501. We propose that LPAAT-beta catalyzes the intracellular transfer of 18:1, 18:0, and 16:0 acyl groups but not 20:4 groups to lysoPA.

Lysophosphatidic acid, the substrate for lysophosphatidic acid acyltransferase β (LPAAT-β), is a well-studied autocrine/paracrine signaling molecule that is secreted by ovarian cancer cells and is found at elevated levels in the blood and ascites fluid of women with ovarian cancer. LPAAT-β converts lysophosphatidic acid to phosphatidic acid, which functions as a cofactor in Akt/mTOR and Ras/Raf/Erk pathways. We report that elevated expression of LPAAT-β was associated with reduced survival in ovarian cancer and earlier progression of disease in ovarian and endometrial cancer. Inhibition of LPAAT-β using small interfering RNA or selective inhibitors, CT32521 and CT32228, two small-molecule noncompetitive antagonists representing two different classes of chemical structures, induces apoptosis in human ovarian and endometrial cancer cell lines in vitro at pharmacologically tenable nanomolar concentrations. Inhibition of LPAAT-β also enhanced the survival of mice bearing ovarian tumor xenografts. Cytotoxicity was modulated by diacylglycerol effectors including protein kinase C and CalDAG-GEF1. LPAAT-β was localized to the endoplasmic reticulum and overexpression was associated with redistribution of protein kinase C-α. These findings identify LPAAT-β as a potential prognostic and therapeutic target in ovarian and endometrial cancer.

Phosphatidic acid (PA) is a component of cellular membranes that is also a mediator of certain cell signalling functions associated with oncogenesis. These include ras/raf/Erk and Akt/mTor [1-3]. The authors have investigated whether it would be possible to interrupt these known oncogenic pathways through the inhibition of lysophosphatidic acid acyltransferase (LPAAT), an enzyme that catalyses the biosynthesis of PA. The expression and activity of the LPAAT-beta isoform are elevated in human tumours, and the respective gene displays transforming capacity when overexpressed in vitro. Inhibition by either genetic means or by isoform-specific small molecules results in a block to cell signalling pathways and apoptosis. Furthermore, the small-molecule inhibitors of LPAAT-beta are not cytotoxic to a number of normal cell types, including primary bone marrow progenitors, indicating a differential dependence of tumour cells on LPAAT-beta function. These discoveries indicate that LPAAT-beta represents a potential novel cancer therapy target.

Introduction: Soft tissue sarcomas (STS) are heterogeneous mesenchymal tumors which are both morbid and lethal. G100 is a stable oil-in-water emulsion of glucopyranosyl lipid adjuvant, a highly potent toll-like receptor 4 (TLR4) agonist, which has been utilized for intratumoral (IT) injections and as vaccine adjuvants without significant toxicity. We hypothesized that IT G100 would induce a robust local and potentially systemic anti-tumor immune response in the STS microenvironment, leading to improved outcomes. Methods: 15 metastatic STS patients who had a superficial injectable lesion were treated with weekly IT G100 for 8-12 weeks; 12 patients received concurrent radiation for 2 weeks at the start, while 3 got IT G100 alone for 6 weeks prior to radiation. Biopsies and blood were collected pre and post treatment, and flow cytometry was performed on fresh tumor samples. T-cell receptor (TCR) deep sequencing of tumor-infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC) was performed on 7 patients. RECIST v1.1 and the Common Terminology Criteria for Adverse Events were used to monitor clinical outcomes. Results: Patients had a median of 3 (0~5) prior lines of therapy and mean tumor size of 5.6cm (1~20cm). No grade 3 or higher treatment-related toxicity was observed, and local tumor control was achieved in 93% (14/15). 6 (40%) had stable disease after treatment, and 1 (P06) had complete regression of injected tumor. This tumor had a high percentage of infiltrating pre-treatment immune cells (12% CD45+ on flow cytometry versus 2.7% for all other tumors). TCR sequencing showed that the increase in clonality of PBMC after treatment was greater in P06 (389%) compared to 6 other patients (mean 34%). There was also higher overlap in TCR sequence between TIL versus PBMC after treatment (13% versus 22%), suggesting systemic expansion of tumor-specific T-cells. In 7 patients evaluable for tumor-associated macrophages (tumors with >1000 CD45+CD11b+ cells), 71% had a shift from an M2 to M1 phenotype. In all patients who received G100 alone, there was an increase in T-cell infiltration into tumor after treatment. In one patient (P14), the proportion of CD3+ live cells in tumor went from Conclusion: IT G100 provides a potentially viable agent for local control of metastatic STS. With or without radiation, G100 appears to shift the tumor microenvironment into a more inflammatory state with significant infiltration of T cells. The increase in clonality in PBMC and TIL, as well as increased overlap of tumor-associated versus peripheral TCR sequences, suggest induction of a tumor-specific response. Combination of G100 with other immunomodulators may further enhance the adaptive anti-tumor response. Citation Format: Yongwoo D. Seo, Edward Y. Kim, Ernest U. Conrad, Ryan B. O'Malley, Sara Cooper, Bailey Donahue, Lee D. Cranmer, Hailing Lu, Frank Hsu, Elizabeth T. Loggers, Taylor Hain, Darin J. Davidson, Lynn Bonham, Venu G. Pillarisetty, Gabrielle M. Kane, Stanley R. Riddell, Robin L. Jones, Seth M. Pollack. Intratumoral injection of the toll-like receptor 4 agonist G100 induces a T-cell response in the soft tissue sarcoma microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2947. doi:10.1158/1538-7445.AM2017-2947

Tumor-infiltrating inflammatory cells comprise a major part of the stromal microenvironment and support cancer progression by multiple mechanisms. High numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and are coupled with the angiogenic switch and malignant progression. However, the specific roles and regulation of heterogeneous tumor myeloid populations are incompletely understood. CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also linked to poor outcomes. To characterize myeloid cell function in tumors, we combined confocal intravital microscopy with depletion of CSF-1R-dependent cells using a neutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-driven polyoma middle T antigen breast cancer model. The depleted cells shared markers of tumor-associated macrophages and dendritic cells (M-DCs), matching the phenotype of tumor dendritic cells that take up antigens and interact with T cells. We defined functional subgroups within the M-DC population by imaging endocytic and matrix metalloproteinase activity. Anti-CSF-1R treatment altered stromal dynamics and impaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr-1(+) neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate in metastatic lungs differed significantly from that in mammary tumors, the reduction in metastasis may result from the impact on primary tumors. The combination of functional analysis by intravital imaging with cellular characterization has refined our understanding of the effects of experimental targeted therapies on the tumor microenvironment.

11017Background: Tumor associated macrophages (TAM) are critical for immune evasion in many tumors. We hypothesized that combining G100, a TLR4 agonist that stimulates innate immunity and increases...

BACKGROUND Lysophosphatidic acid acyltransferase-β (LPAAT-β) tumor expression is an emerging prognostic, diagnostic, and therapeutic target in early epithelial ovarian cancer (EOC). The significance of tumor overexpression of LPAAT-β was investigated in a large number of advanced- and early-stage EOC patients. METHODS LPAAT-β expression was analyzed by immunohistochemistry (IHC) in 158 ovarian tumors, including 68 advanced and 90 low-stage tumors, representing all grades and histologies (including 33 borderline tumors). In advanced-stage patients, tissue from multiple sites was evaluated to assess differential expression of LPAAT-β in local tumor and distant metastases. RESULTS LPAAT-β was overexpressed in 90 (57%) of all 158 ovarian tumors. Forty-nine (72%) of 68 advanced tumors overexpressed LPAAT-β. LPAAT-β was associated with the presence of carcinoma versus borderline histology (67% vs. 18%, P < .0001), high histologic grade [according to the Silverberg Grading Scheme] (Grade 1, 25%; Grade 2, 21%; and Grade 3, 54%; P < .0001), and with papillary-serous histology. In an analysis of the 125 carcinoma patients, LPAAT-β increased with but was not significantly associated with advanced clinical stage (P = .1431). LPAAT-β expression was associated with shortened progression-free survival (PFS) (5-year PFS, 32% for LPAAT-β-positive vs. 60% for LPAAT-β-negative; P = .0318) and decreased overall survival (OS) (5-year OS, 54% for LPAAT-β-positive vs. 74% for LPAAT-β-negative; P = .0173). CONCLUSIONS LPAAT-β is highly expressed in advanced ovarian tumors and is associated with aggressive histology and decreased PFS and OS. LPAAT-β is an intriguing prognostic tool for the identification of high-risk EOC and a potential target for directed therapy that warrants further study. Cancer 2006. © 2006 American Cancer Society.

Purpose: Lysophosphatidic acid acyltransferase-β (LPAAT-β) is a transmembrane enzyme critical for the biosynthesis of phosphoglycerides whose product, phosphatidic acid, plays a key role in raf and AKT/mTor-mediated signal transduction. Experimental Design: LPAAT-β may be a novel target for anticancer therapy, and, thus, we examined the effects of a series of inhibitors of LPAAT-β on multiple human non–Hodgkin's lymphoma cell lines in vitro and in vivo. Results: We showed that five LPAAT-β inhibitors at doses of 500 nmol/L routinely inhibited growth in a panel of human lymphoma cell lines in vitro by >90%, as measured by [3H]thymidine incorporation. Apoptotic effects of the LPAAT-β inhibitors were evaluated either alone or in combination with the anti-CD20 antibody, Rituximab. The LPAAT-β inhibitors induced caspase-mediated apoptosis at 50 to 100 nmol/L in up to 90% of non–Hodgkin's lymphoma cells. The combination of Rituximab and an LPAAT-β inhibitor resulted in a 2-fold increase in apoptosis compared with either agent alone. To assess the combination of Rituximab and a LPAAT-β inhibitor in vivo, groups of athymic mice bearing s.c. human Ramos lymphoma xenografts were treated with the LPAAT-β inhibitor CT-32228 i.p. (75 mg/kg) daily for 5 d/wk × 4 weeks (total 20 doses), Rituximab i.p. (10 mg/kg) weekly × 4 weeks (4 doses total), or CT-32228 plus Rituximab combined. Treatment with either CT-32228 or Rituximab alone showed an approximate 50% xenograft growth delay; however, complete responses were only observed when the two agents were delivered together. Conclusions: These data suggest that Rituximab, combined with a LPAAT-β inhibitor, may provide enhanced therapeutic effects through apoptotic mechanisms.

PS-341 (bortezomib, Velcade™) is a promising novel agent for treatment of advanced multiple myeloma (MM); however, 65% of patients with relapsed refractory disease in a phase II study did not respond. Lysophosphatidic acid (LPA) is a phospholipid which mediates tumor cell migration and invasion. Recent studies have shown that inhibition of LPAAT-β inhibits both Ras/Raf/Erk and PI3K/Akt signaling cascades. We have previously shown that lysophosphatidic acid acyltransferase (LPAAT)-β inhibitor CT-32615 triggers caspase-dependent apoptosis, and can overcome resistance to conventional therapeutics (ie, dexamethasone, doxorubicin, melphalan) in MM cells. In this study, we determined whether CT-32615 could also overcome resistance to PS-341. We first characterized molecular mechanisms of resistance to PS-341 in DHL-4 lymphoma cells. DHL-4 cells express low levels of caspase-3 and caspase-8; furthermore, no cleavage in caspase-8, caspase-9, caspase-3, poly ADP-ribose polymerase (PARP), or DNA fragmentation factor (DFF) 45 is triggered by PS-341 treatment. We have previously shown that PS-341 treatment triggers phosphorylation of c-Jun NH2-terminal kinase (JNK), which subsequently induces caspase-dependent apoptosis; conversely, JNK inhibition blocks PS-341-induced apoptosis. Here we show that PS-341 does not induce phosphorylation of SEK-1, JNK, and c-Jun in DHL-4 cells, suggesting that it does not trigger a stress response. Importantly, CT-32615 inhibits growth of DHL-4 cells in a time- and dose-dependent fashion: a transient G2/M cell cycle arrest induced by CT-32615 is mediated via downregulation of cdc25c and cdc2. CT-32615 triggers swelling and cell membrane destruction in DHL-4 cells, without caspase/PARP cleavage or TUNEL-positivity, suggesting a necrotic response. Our studies therefore demonstrate that LPAAT-β inhibitor CT-32615 triggers necrosis even in PS-341-resistant DHL-4 cells, providing the framework for its evaluation to overcome clinical PS-341 resistance and improve patient outcome.

Clinical use of human granulocyte-colony stimulating factor (hG-CSF) to treat various diseases involving neutropenia has been previously shown to (1) successfully increase circulating neutrophils, (2) reduce condition-related infections, and (3) cause few side effects in patients. To alleviate the symptoms of neutropenia, the patient must receive frequent injections of recombinant hG-CSF. Permanent ways to deliver stable levels of the molecule to the patient are being investigated. Among them, the transplantation of hG-CSF-secreting cells has been proposed and performed successfully in rodents, using fibroblast cell lines and primary muscle cells. We thus investigated whether similar results could be obtained by intramuscular myoblast transplantation in a large animal model. When 1-3 x 10(8) myoblasts were injected into three Macaca mulatta, hG-CSF was detected at high levels (300-900 pg/ml), which in turn led to a four- to fivefold increase in circulating neutrophils. However, both the concentrations of hG-CSF and neutrophil levels were found to decrease over time. Nonetheless, neutrophils were found at higher levels from the fourth week until the end the experiment (up to 29 weeks) in G-CSF monkeys compared with control animals. These results show that transplantation of hG-CSF-secreting myoblasts may indeed be a therapeutic option for the treatment of neutropenic patients.

Gene transfer to skeletal muscle was examined as a means of gene therapy for neutropenia. A recombinant retrovirus containing a human granulocyte colony-stimulating factor (G-CSF) gene was introduced into primary human or rat myoblasts, which were then shown to produce biologically active G-CSF. Transplantation of G-CSF-producing rat myoblasts into the muscle of syngeneic rats resulted in a 15-fold increase in absolute neutrophil counts. This increase correlated with detection of circulating human G-CSF protein throughout the 6-month duration of the experiment. These results clearly demonstrate long-term production of therapeutically relevant amounts of a human protein by normal cells in vivo.

Mus dunni endogenous virus (MDEV) is transcriptionally inactive in cultured M. dunni cells but can be activated by treatment of the cells with either hydrocortisone or 5-iodo-2′-deoxyuridine (24). Activation can be easily measured because MDEV can package Moloney murine leukemia virus (Mo-MLV)-based retrovirus vectors. Once activated, MDEV will continue to replicate in M. dunni cells and can infect many other cell types (4). MDEV is endogenous to M. dunni wild mice (also known as Mus terricolor) at a proviral copy number of one to two per genome, and there is at least one other element in dunni cells that hybridizes to MDEV probes, but it has a different restriction map (4). MDEV has not been found in the genomes of laboratory mice or in mammalian genera other than Mus (4). It is unknown whether MDEV causes pathology or is ever activated in M. dunni mice. MDEV does not interfere with known MLVs, indicating that it uses a different receptor for cell entry (26). MDEV also does not interfere with some nonmurine retroviruses, such as gibbon ape leukemia virus (GALV) (4). The endogenous cat retrovirus RD114 was found to interfere at a low level with MDEV, but this interference was observed in only one cell line (G355 cat glial cells) (4). Furthermore, this interference was weak and varied from one experiment to another, making it unclear whether MDEV and RD114 share a receptor in G355 cells. Other members of the RD114 interference group, such as spleen necrosis virus and Mason-Pfizer monkey virus, have not been found to interfere with MDEV (4). The MDEV receptor is widely expressed among different species, as indicated by the ability of a retroviral vector pseudotyped by MDEV to transduce cells from many species (4). Molecular clones of MDEV were obtained to study its genome and receptor usage (4). However, the clones were unable to produce infectious virus after transfection into permissive cells. Here we describe the correction of a clone that renders it infectious and show that the resulting virus is in the same interference group as the biological isolates. In addition, we have determined the entire sequence of MDEV and describe some unique features of the MDEV genome.