Hoyoung Maeng

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.

Mechanism of Fas Signaling Regulation by Human Herpesvirus 8 K1 Oncoprotein

BACKGROUND Herpesvirus 8 (HHV-8) oncoprotein K1 is linked to lymphoproliferation and suppression of apoptosis mediated by the Fas death receptor. Expression of K1 in transgenic mice induces accumulation of lymphoid tissue cells and lymphoma. METHODS To examine how K1 and Fas interact to suppress apoptosis, K1-Fas binding was studied in human embryonic kidney (HEK) and lymphoma (BJAB) cells that expressed wild-type K1 or a K1 Ig domain deletion mutant and were treated with Fas ligand (FasL) or an agonistic Fas antibody, using immunoprecipitation and Western blot analysis. Cleavage of caspase-3 and apoptosis was compared in liver samples from mice that were transfected with empty vector vs with plasmids expressing wild-type K1 or a K1 Ig deletion mutant and treated with agonistic Fas antibody for 7 hours. These studies used immunohistochemical staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. All statistical tests were two-sided. RESULTS Immunoprecipitation and Western blot analysis of transfected HEK and BJAB cells revealed that wild-type K1 but not Ig-deleted K1 binds to Fas and prevents Fas activation by FasL or by an agonistic Fas antibody. More mice that were transfected with wild-type K1 (7 of 10) than mice transfected with empty vector (3 of 13) or the K1 Ig deletion mutant (0 of 6) survived treatment with the agonistic Fas antibody. Compared with vector-transfected mice, livers of wild-type K1-transfected mice contained fewer cells in which caspase-3 was cleaved (87.6% vs 58.0%, difference = 29.6%, 95% confidence interval [CI] = 19.2% to 40.0%; P = .003) and fewer apoptotic cells (83.7% vs 34.2%, difference = 49.5%, 95% CI = 39.8% to 59.2%; P = .003). CONCLUSIONS K1 blocks Fas signaling by directly binding to Fas through the Ig-like domain of K1 and preventing binding of FasL. The relative resistance of cancer cells to Fas-mediated apoptosis may be due to the inhibition of Fas by Ig domain-containing proteins.

Long-term results of a phase II trial of lenalidomide plus prednisone therapy for patients with myelofibrosis

Lenalidomide, with or without prednisone, is an active therapy for patients with myelofibrosis (MF). We provide an update of a phase II study of lenalidomide plus prednisone in patients with MF, after median follow up of 9 years. Forty patients were enrolled in the study and all patients were evaluable for response. Response to the treatment was reevaluated using IWG response criteria published in 2013: quality of response improved over time and overall response rate was 35%. Response in splenomegaly was seen in 39% of patients and anemia response in 32%. The median time to treatment failure (TTF) in all patients was 8.2 months and the median duration of response was 34.6 months. Response was highly durable in some patients: six patients (15%) had TTF for more than 60 months (5 years) and three patients are still on the treatment beyond 109 months (9 years). Complete and partial responses were seen in one and five patients, respectively, but achieving deeper response was not necessary for the response to be durable. New clinical studies are needed to explore safe and well tolerated lenalidomide-based combination strategies for patients with MF.

Kaposi's sarcoma human herpesvirus K1 interferes with FAS-mediated apoptosis and stimulates clonal growth and lymphoid hyperplasia.

Background Infection with human herpesvirus 8 (HHV-8), also known as Kaposi sarcoma associated herpesvirus, is associated with the development of primary effusion lymphoma and Kaposi sarcoma. A transmembrane protein of HHV-8, K1, is readily expressed in these tumors, and the expression of K1 alone causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1-expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was previously shown to be involved in activation of nuclear factor-kappa B (NF-κB). Moreover, we have recently shown that K1 suppresses Fasmediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survive a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations in Fas signaling.

Human herpesvirus 8 K1-derived peptides disrupt the inhibitory FAS-K1 complex and restore FAS receptor-mediated apoptosis

2667 Background: Human herpesvirus 8 (HHV-8) infection is associated with the development of primary effusion lymphoma, Kaposi sarcoma, and multicentric Castleman disease. The K1 gene of HHV-8 is expressed in tumor cells as a transmembrane protein with an immunoglobulin-like domain in its ectodomain and an immunoreceptor tyrosine-based activation motif (ITAM). We demonstrated that K1 protein activates nuclear factor-kappa B (NF-κB), and K1 expression in transgenic mice stimulated accumulation of lymphatic cells and development of lymphoma. How K1 blocks apoptosis and induces hyperplasia and lymphomas is not known. We hypothesized that K1 contributes to lymphoma development partly by suppressing apoptosis, and that this suppression combined with its NF-κB activation produces lymphoma.
 Results: We found that K1 binds to Fas and in turn, inhibits Fas-mediated apoptosis. We mapped the region that K1 uses to bind to Fas as an immunoglobulin (Ig) chain-like domain by expressing deletion mutants of K1. Overexpression of an Ig domain-containing protein CD79b competed with K1-Fas binding in a dose-dependent manner. Two 20-amino acid peptides (N251, N253) representing the Ig domain of K1 competed with K1-Fas binding in immunoprecipitation/immunoblotting analysis. The N251 and N253 peptides (100 μM) enhanced anti-Fas antibody (CH-11, 50 ng/mL)-induced apoptosis of BJAB lymphoma cells that expressed K1 but that of vector-transfected BJAB cells. Ig-deleted K1 (K1dIg)-transfected mice were not protected (0/6), and K1-transfected mice were protected (7/10, P