Inhibition of anaplastic lymphoma kinase (ALK) activity provides a therapeutic approach for CLTC-ALK-positive human diffuse large B cell lymphomas

ALK positive diffuse large B-cell lymphomas (DLBCL) are a distinct lymphoma subtype associated with a poor outcome. Most of them feature a t(2;17) encoding a clathrin (CLTC)-ALK fusion protein. The contribution of deregulated ALK-activity in the pathogenesis and maintenance of these DLBCLs is not yet known. We established and characterized the first CLTC-ALK positive DLBCL cell line (LM1). LM1 formed tumors in NOD-SCID mice. The selective ALK inhibitor NVP-TAE684 inhibited growth of LM1 cells in vitro at nanomolar concentrations. NVP-TAE684 repressed ALK-activated signalling pathways and induced apoptosis of LM1 DLBCL cells. Inhibition of ALK-activity resulted in sustained tumor regression in the xenotransplant tumor model. These data indicate a role of CLTC-ALK in the maintenance of the malignant phenotype thereby providing a rationale therapeutic target for these otherwise refractory tumors.     

BAL1 and Its Partner E3 Ligase,BBAP, Link Poly(ADP-Ribose) Activation,Ubiquitylation, and Double-Strand DNA Repair Independent of ATM,MDC1, and RNF8

The BAL1 macrodomain-containing protein and its partner E3 ligase, BBAP, are overexpressed in chemotherapy-resistant lymphomas. BBAP selectively ubiquitylates histone H4 and indirectly promotes early 53BP1 recruitment to DNA damage sites. However, neither BBAP nor BAL1 has been directly associated with a DNA damage response (DDR), and the function of BAL1 remains undefined. Herein, we describe a direct link between rapid and short-lived poly(ADP-ribose) (PAR) polymerase 1 (PARP1) activation and PARylation at DNA damage sites, PAR-dependent recruitment of the BAL1 macrodomain-containing protein and its partner E3 ligase, local BBAP-mediated ubiquitylation, and subsequent recruitment of the checkpoint mediators 53BP1 and BRCA1. The PARP1-dependent localization of BAL1-BBAP functionally limits both early and delayed DNA damage and enhances cellular viability independent of ATM, MDC1, and RNF8. These data establish that BAL1 and BBAP are bona fide members of a DNA damage response pathway and are directly associated with PARP1 activation, BRCA1 recruitment, and double-strand break repair.     

IL-12 and IL-18 are increased and stimulate IFN-gamma production in sarcoid lungs

Sarcoidosis is a systemic chronic granulomatous disease of unknown cause. Recent investigations revealed that the cytokine profile in inflamed lesions of sarcoidosis is Th1 dominant. To obtain better immunopathologic understanding of sarcoidosis, we examined the expression of IL-12 and IL-18 and their roles in IFN-gamma production in pulmonary sarcoidosis. Sarcoid cases had significantly elevated levels of IL-12 (p40 and p70) and IL-18 in bronchoalveolar lavage (BAL) fluids compared with healthy subjects. IL-12 p70 and IL-18 were immunohistochemically expressed in the epithelioid cells and giant cells of sarcoid granulomas. Significant induction of IFN-gamma, IL-12 p70, and IL-18 was observed from sarcoid BAL fluid cells with LPS stimulation, whereas LPS tended to induce only IL-12 p70 in BAL fluid cells from healthy subjects. Sarcoid cases had significantly greater IFN-gamma induction with LPS stimulation than healthy subjects did. IL-18 mRNA expression was observed in freshly isolated sarcoid BAL fluid cells as well as in LPS-stimulated sarcoid BAL fluid cells, but IFN-gamma and IL-12 mRNA expression was observed only in LPS-stimulated BAL fluid cells. Treatment with anti-IL-12- and anti-IL-18-neutralizing Abs significantly inhibited IFN-gamma production from LPS-stimulated BAL fluid cells of sarcoid cases. Coadministration of rIL-12 or rIL-18 induced greater IFN-gamma production in sarcoid BAL fluid cells than in normal BAL fluid cells. We concluded that bioactive IL-12 and IL-18 were produced in sarcoid BAL fluid cells and synergistically induced IFN-gamma production, indicating important cytokines in the Th1 response of sarcoidosis.     

Gastrointestinal lymphomas in a North American population: clinicopathologic features from one major Central-Midwestern United States tertiary care medical center

ABSTRACT: BACKGROUND: Gastrointestinal (GI) lymphomas are very common types of extranodal lymphomas, and we hypothesize there are regional differences in subtype, distribution in the GI tract, and epidemiological features among the different populations. METHODS: We retrospectively evaluated the clinical, molecular and histologic features of North American primary and secondary GI lymphomas diagnosed from 2000?2009 seen at our institution. We utilized immunohistochemistry and fluorescence in situ hybridization to further evaluate a subset of the gastric lymphomas. RESULTS: Extranodal marginal zone lymphomas of mucosal associated lymphoid tissue (MALTs) and diffuse large B cell lymphomas (DLBCLs) were the most common subtypes of GI lymphomas. Select gastric DLBCLs (N?=?6) and MALTs (N?=?13) were further examined for API2-MALT1 and IGH translocations, and P16 and P53 protein expression. Gastric MALTs showed frequent API2-MALT1 (38%) but not IGH translocations (0%), and the DLBCLs showed neither translocation. Expression of P16 and P53 proteins and the proliferative index were compared between high grade gastric lymphomas (gastric DLBCLs) and low grade gastric lymphomas (gastric MALTs). P53 overexpression (P?=?0.008) and a high proliferation index [Ki-67] (P?=?0.00042) were significantly associated with gastric DLBCL, but no statistically significant difference was observed in P16 expression (p?=?0.108) between gastric DLBCL and gastric MALT. CONCLUSION: Our study revealed that GI lymphomas from a Central-Midwestern North American population showed differences and similarities to non-North American cohorts. In addition, API2-MALT1, P16 and P53 abnormalities occurred frequently in gastric lymphomas from this North American population. Virtual slides: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1415505838687793.     

Interleukin-18, a proinflammatory cytokine

Although IL-18 is an inducer of IFN-gamma and Th1 responses. IL-18 is a proinflammatory cytokine by several criteria. In several animal models. antibodies that neutralize endogenous IL-18 reduce the severity of disease. Endotoxin lethality is prevented by anti-IL-18. Even in models that are interferon-y independent, neutralization of IL-18 prolongs survival. Anti-IL-18 also protects the liver against cellular injury induced by toxins or activated T cells. In models of hepatic melanoma metastasis. IL-18 blockade reduces the adherence of malignant cells by preventing IL-18 upregulation of vascular endothelial adhesion-1 molecule expression. IL-18 and IL-12 act synergistically to stimulate I cells and natural killer cells to produce IFN-gamma but neutralization of IL-18 prevents IL-12 induction of IFN-gamma. IL-18. like several cytokines. can be used to enhance host defense against tumors in mice a mechanism that is most often IFN-gamma-dependent. Nevertheless. it is the proinflammatory portfolio of IL-18 which likely contributes to enhanced host defenses. In models or arthritis, lung injury or inflammatory bowel disease, neutralization of IL-18 reveals the important role of this cytokine in mediating inflammation.     

Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma

By implanting nondisrupted pieces of human lung tumor biopsy tissues into SCID mice, it has been possible to establish viable grafts of the tumor, as well as the tumor-associated microenvironment, including inflammatory cells, fibroblasts, tumor vasculature, and the extracellular matrix. Using this xenograft model, we have evaluated and characterized the effects of a local and sustained release of human rIL-12 (rhIL-12) from biodegradable microspheres. In response to rhIL-12, the human CD45+ inflammatory cells present within the xenograft mediate the suppression or the complete arrest of tumor growth in SCID mice. Analysis of the cellular events reveals that human CD4+ and CD8+ T cells are induced by rhIL-12 to produce and secrete IFN-gamma. Serum levels of human IFN-gamma in mice bearing rhIL-12-treated tumor xenografts correlate directly with the degree of tumor suppression, while neutralizing Abs to human IFN-gamma abrogate the IL-12-mediated tumor suppression. Gene expression profiling of tumors responding to intratumoral rhIL-12 demonstrates an up-regulation of IFN-gamma and IFN-gamma-dependent genes not observed in control-treated tumors. Genes encoding a number of proinflammatory cytokines, chemokines (and their receptors), adhesion molecules, activation markers, and the inducible NO synthase are up-regulated following the introduction of rhIL-12, while genes associated with tumor growth, angiogenesis, and metastasis are decreased in expression. NO contributes to the tumor killing because an inhibitor of inducible NO synthase prevents IL-12-induced tumor suppression. Cell depletion studies reveal that the IL-12-induced tumor suppression, IFN-gamma production, and the associated changes in gene expression are all dependent upon CD4+ T cells.     

Comparative analysis of IFN-gamma B7.1 and antisense TGF-beta gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma

Cancer progression is attributed in part to immune evasion strategies that include lack of co-stimulation, down-regulation of cell surface MHC molecules, and secretion of immunosuppressive factors, such as transforming growth factor-beta (TGF-beta). Gene therapy has been employed to counter these mechanisms of immune evasion by transference of B7.1, IFN-gamma or antisense TGF-beta genes into tumor cells, resulting in cell surface expression of B7.1, upregulation of MHC class I and class II molecules, or elimination of tumor-derived TGF-beta, respectively. Although each of these transgenes has been shown to alter tumorigenicity in murine models, a direct comparison of their efficacy has not been performed. In this study, we have employed a very aggressive, poorly immunogenic and highly metastatic mammary model, 4T1, to compare the efficacy of B7.1, IFN-gamma and antisense TGF-beta gene transfer in stimulating an anti-tumor response. We demonstrate that both IFN-gamma and antisense TGF-beta gene expression significantly reduced the tumorigenicity of these cells compared to mock transduced cells, with IFN-gamma having a greater effect. In contrast, B7.1 gene transfer did not affect the tumorigenicity of 4T1 cells. The anti-tumor response directed against antisense TGF-beta-expressing 4T1 tumors was mediated by CD4+ and CD8+ T cells. However, CD8+ T cells, and not CD4+ T cells, appeared to mediate the anti-tumor response against IFN-gamma-expressing tumors. Treatment of tumor-bearing animals with IFN-gamma or antisense TGF-beta gene-modified tumor cell vaccines reduced the number of clonogenic metastases to the lungs and liver compared to treatment with mock-transduced cells. Finally, in a residual disease model in which the primary tumor was excised and mice were vaccinated with irradiated tumor cells, treatment of mice with vaccinations consisting of 4T1 cells expressing both antisense TGF-beta and IFN-gamma genes was the most effective in prolonging survival.     

Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo.

The host response to Gram-negative LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of chemokines. The expression and function of chemokines in vivo appears to be highly selective, though the molecular mechanisms responsible are not well understood. All CXC (IFN-gamma-inducible protein (IP-10), macrophage inflammatory protein (MIP)-2, and KC) and CC (JE/monocyte chemoattractant protein (MCP)-1, MCP-5, MIP-1alpha, MIP-1beta, and RANTES) chemokine genes evaluated were sensitive to stimulation by LPS in vitro and in vivo. While IL-10 suppressed the expression of all LPS-induced chemokine genes evaluated in vitro, treatment with IFN-gamma selectively induced IP-10 and MCP-5 mRNAs, but inhibited LPS-induced MIP-2, KC, JE/MCP-1, MIP-1alpha, and MIP-1beta mRNA and/or protein. Like the response to IFN-gamma, LPS-mediated induction of IP-10 and MCP-5 was Stat1 dependent. Interestingly, only the IFN-gamma-mediated suppression of LPS-induced KC gene expression was IFN regulatory factor-2 dependent. Treatment of mice with LPS in vivo also induced high levels of chemokine mRNA in the liver and lung, with a concomitant increase in circulating protein. Hepatic expression of MIP-1alpha, MIP-1beta, RANTES, and MCP-5 mRNAs were dramatically reduced in Kupffer cell-depleted mice, while IP-10, KC, MIP-2, and MCP-1 were unaffected or enhanced. These findings indicate that selective regulation of chemokine expression in vivo may result from differential response of macrophages to pro- and antiinflammatory stimuli and to cell type-specific patterns of stimulus sensitivity. Moreover, the data suggest that individual chemokine genes are differentially regulated in response to LPS, suggesting unique roles during the sepsis cascade.     

ALVAC-mediated gene transfer is efficient in lymphoid malignancies of T-and early B-cell origin, but not in tumors arising from mature B-cells

Natural attenuation of ALVAC virus in mammals makes it an attractive vector for cancer vaccine therapy of immunocompromised hosts, such as patients with lymphoid malignancies. However, the transduction efficiency of ALVAC constructs in lymphoid tumors has not yet been characterized. We studied a wide spectrum of human T- and B-cell leukemia and lymphomas and found significant heterogeneity of the ALVAC-mediated gene product expression in these tumors. While ALVAC-B7.1, ALVAC-B7.2, or ALVAC-luciferase vectors effectively expressed recombinant genes in malignancies arising from T- or early B-cell precursors, negative or low expression of ALVAC recombinant genes occurred in tumors arising from mature B-cells. We showed that ALVAC-encoded B7.1 or B7.2 was continuously expressed on the infected, and subsequently irradiated, leukemia cells, and only cells with ALVAC-mediated expression of costimulatory molecules (but not unmodified leukemia cells or those infected with the ALVAC-parental vector) induced significant proliferation and IFN-gamma production by alloreactive T-cells. These data provide the rationale for clinical studies using the ALVAC vector system for gene transfer into lymphoid tumors of T- and early B-cell origin to render them more immunogenic, while alternative strategies should be considered for immunotherapy of mature B-cell malignancies.     

B cells amplify IFN-gamma production by T cells via a TNF-alpha-mediated mechanism

Aside from being the precursors of the Ab-secreting cells, B cells are engaged in other immune functions such as Ag presentation to T cells or cytokine production. These functions may contribute to the pathogenic role of B cells in a wide range of autoimmune diseases. We demonstrate that B cells acquire the capacity to amplify IFN-gamma production by CD4 and CD8 T cells during the course of the Th1 inflammatory response to Toxoplasma gondii infection. Using the two following different strategies, we observed that B cells from T. gondii-infected mice, but not from naive mice, induce higher IFN-gamma expression by splenic host T cells: 1) reconstitution of B cell-deficient mice with B cells expressing an alloantigen different from the recipients, and 2) adoptive transfer of B and T cells into RAG-/- mice. In vitro assays allowing the physical separation of T and B cells demonstrate that Ag-primed B cells enhance IFN-gamma production by T cells in a contact-dependent fashion. Using an OVA-transgenic strain of T. gondii and OVA-specific CD4 T cells, we observed that the proinflammatory effect of B cells is neither Ag specific nor requires MHCII expression. However, TNF-alpha expressed on the surface of B cells appears to mediate in part the up-regulation of IFN-gamma by the effector T cells.