Dose Of Agonistic anti-Fas Antibody Research Articles

Abstract 4965: Oncoprotein PMLRARα directly suppresses Fas-mediated apoptosis through forming an apoptotic inhibitory complex with c-FLIP in vivo

Abstract Objective: Many genotoxic therapies, including radiation, depend on intact Fas signaling to eradicate cancer cells. Defective Fas signaling is an important cause of cancer resistance to therapy. Restoring Fas apoptosis or sensitizing cancer cells to Fas-mediated apoptosis would improve the efficacy of many cancer therapies. To elucidate a role for specific regulators of Fas signaling in cancer cells, we sought to identify potential modulators of Fas expressed in cancers and target them to selectively sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods: Liquid chromatography tandem mass spectrometry was used to identify Fas-associated proteins; co-immunoprecipitation and Western blot were used to detect interactions of PMLRARα, PML, c-FLIP and Fas, and to examine the components of death-inducing signaling complex (DISC) and caspase-8 cleavage. Deletional mutagenesis was used to map the interaction domains. PML shRNA lentivirus and As2O3 were used to knock down PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis. Mice were transfected with PMLRARα, monitored for survival, and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results: We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase-8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P<.001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Livers from PMLRARα-transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with control vector-transfected mice. Conclusions: PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. These data suggest that PMLRARα is a cancer specific Fas-binding inhibitor of Fas-mediated apoptosis and thus, can contribute to cancer development and resistance to therapy. The newly discovered PMLRARα-Fas and PML-Fas complexes can be sites for modulation of apoptosis. By neutralizing the effect of death receptor inhibitors, such as PMLRARα, we can improve responses to many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4965. doi:1538-7445.AM2012-4965

PML and PMLRARα Interact with Fas to Regulate Fas-Mediated Apoptosis In Vivo

Abstract 2451 Background:Altered Fas signaling leads to resistance to various anticancer therapies. The presence of at least low level expression of Fas is required for the transformed phenotype of many cells. In order for Fas to offer this complex regulation and to prevent inadvertent Fas activation, we propose that Fas signaling is subjected to tight regulation and this regulation could produce resistance to apoptosis of cancer cells. Methods:Cell lines NB4 (PMLRARα positive), HL60, U937/PR9 (PMLRARα expression driven by metalothionine promoter), or transfected HEK293 cells were collected and cell extracts were prepared for immunoprecipitation and Western blot analysis with the indicated antibodies. Cellular fractionation was performed using the nuclear/cytosol fractionation kit (BioVision, Mountain View, CA), according to the manufacturer’s instructions. We expressed PMLRARα in C57B6 female mice by transfection of PMLRARα plasmid or empty-vector plasmid (12 mice per group) by tail vein inoculation using a hydrodynamic transfection method. Twenty-four hours after transfection, mice were challenged intraperitoneally with 2 μg/g mouse weight of agonistic Fas antibody Jo2, and monitored for survival. Liver tissues were stained with TUNEL assay and antibodies targeting apoptosis proteins. Differences between two groups were assessed using the 2-tailed student’s t test and a P value < 0.05 was considered to be statistically significant. Results:In this study, we identified, by mass spectrometry analysis of Fas-associated proteins, the proapoptotic promyelocytic leukemia protein (PML) as a Fas-interacting protein. PML is a ubiquitously expressed tumor suppressor, which is downregulated or mutated in over 50% of all cancers. PML promotes apoptosis especially under conditions of cell stress. The function of PML is blocked by its dominant-negative form promyelocytic leukemia - retinoic acid receptor α (PMLRARα). Our focus on PMLRARα showed that it interacts with Fas in acute promyelocytic leukemia-derived cell lines and APL primary cells. Because PMLRARα is considered to be a nuclear transcription factor, we fractionated cell compartment proteins and identified PMLRAR α-Fas complexes present only in the cytoplasmic fraction. Binding of PML to Fas was mapped to the B-box domain of PML and death domain of Fas. PMLRARα blockage of apoptosis was shown in U937/PR9 and NB4 cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase 8 from the Fas death-inducing signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P<.001). The protected mice tissues contained Fas-PMLRARα-cFLIP complexes. Knockdown of PML in 293 cells compared to non-targeted knockdown control cells showed lower rates of CH-11-induced apoptosis, suggesting a facilitating role of PML in Fas mediated apoptosis. To examine the prevalence of PML-Fas complexes in non cancer tissues, we screened over 6 mouse tissues and all contained PML-Fas complexes. Conclusion:Together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL through the accumulation of c-FLIP- Fas complexes. Furthermore, the common expression of PML-Fas complexes suggests Fas signaling is routinely regulated in the cytoplasm by PML, but susceptible to the blocking effects of PMLRARα in cancer. Disclosures:Karp:Schering Merck: Research Funding.

PMLRARα binds to Fas and suppresses Fas-mediated apoptosis through recruiting c-FLIP in vivo

AbstractDefective Fas signaling leads to resistance to various anticancer therapies. Presence of potential inhibitors of Fas which could block Fas signaling can explain cancer cells resistance to apoptosis. We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PML–retinoic acid receptor α (PMLRARα). We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)–derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase 8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P < .001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Taken together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. The presence of PML-Fas complexes across different tissues implicates that PML functions in apoptosis regulation and tumor suppression are mediated by direct interaction with Fas.

Peptides Compete in Human Herpesvirus 8 K1-Fas Complexes Associated with Hyperplasia and Restore Apoptosis.

Abstract 2965Poster Board II-941The long-term expression of human herpesvirus 8 (HHV-8) K1 produces hyperplasia of lymph nodes, splenomegaly, and lymphomas in mice. The mechanism of how K1 causes hyperplasia and lymphomas. The immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to activate of Akt and nuclear factor kappa B (NF-kB). However, we have recently shown that K1 suppresses Fas-mediated 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 altered Fas signaling.Examination of mice expressing K1 via a ubiquitous promoter showed that K1 transgenic mice (n=10) had 90% developed lymphoid hyperplasia (at least 3 lymph nodes >3 mm) and 60% developed lymphomas after 18 months, while all (26) control nontransgenic mice remained free of lymph node hyperplasia, splenomegaly, and lymphoma. Some K1 mice developed liver or mesenteric tumors (4 of 10 mice). The spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-K1 transgenic control mice. Hematoxylin and eosin staining of spleen sections showed lymphocyte expansion in the periarteriolar lymphocyte sheath with disruption of normal spleen architecture. Anti-kappa and anti-lambda light chain antibodies revealed the presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but no foci were present in 4 control non-transgenic mice. Moreover, K1 protein was expressed in approximately 10% of splenic cells after staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein confers resistant to Fas-mediated apoptosis, splenic cells of 6-month-old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4±1% of splenocytes from K1 mice versus 17±2% of control splenocytes showed morphology of apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11±0.6% vs. 50±6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2-induced apoptosis compared with splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 out of 12 K1 transgenic (30%) and 13 out of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.We mapped the region that K1 uses to bind to Fas to 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 mM) enhanced anti-Fas antibody (CH-11, 50 ng/mL)-induced apoptosis of BJAB lymphoma cells that expressed K1 but not that of vector-transfected BJAB cells. K1 splenocytes incubated with N251 and N253 showed enhanced rates of Fas-mediated apoptosis over control peptide-treated K1, and peptides did not enhance the apoptosis rates of control non-K1 expressing splenocytes.This analysis indicates a key role of K1 in lymphoid hyperplasia and lymphoma and that K1 direct binding to Fas occurs in vivo, which is effectively targeted by competing peptides to restore apoptosis. Disclosures:No relevant conflicts of interest to declare.

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 Protein K1 Interference with Fas-Mediated Apoptosis Induces Clonal Growth Which Leads to Lymphoid Hyperplasia.

Background: Human herpesvirus 8 (HHV-8) infection is causally associated with the development of primary effusion lymphoma and Kaposi s sarcoma. A transmembrane protein of HHV-8, K1, is readily expressed in those 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 shown previously to be involved in activation of Nuclear Factor kappa B (NF -B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived 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 of Fas signaling.Results: Gross examination of thoracic and abdominal cavities of transgenic mice with K1 expression driven by a ubiquitous promoter were sacrificed at 18 months of age revealed enlarged cervical, mediastinal, renal, mesenteric lymph nodes and spleens. Peyer s patches were also enlarged and readily visible on the outer surface of the ileum. Of 10 K1 mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 200 mg vs. 94 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with antikappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 1% of splenocytes from K1 mice versus 17 2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11 0.6% vs. 50 6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.Conclusion: These results over all confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation of this phenomenon. Interference of K1 with Fas-mediated apoptosis disrupts the normal life cycle of lymphocytes.

Human Herpesvirus 8 Protein K1 Interferes with Fas-Mediated Apoptosis Induces Clonal Growth and Lymphoid Hyperplasia

Background: Expression of Human herpesvirus 8 (HHV-8) K1 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 shown previously to be involved in activation of Nuclear Factor kappa B (NF-k-B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived 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 of Fas signaling.Results: At 18 months, 10 K1 transgenic mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 ± 200 mg vs. 94 ± 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with anti-kappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 ± 1% of splenocytes from K1 mice versus 17 ± 2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11 ± 0.6% vs. 50 ± 6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis. We mapped the region that K1 uses to bind to Fas as an immunoglobulin (Ig) chainlike domain by expressing deletion mutants of K1. Overexpression of an Ig domaincontaining 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 mM) enhanced anti-Fas antibody (CH-11, 50 ng/mL)-induced apoptosis of BJAB lymphoma cells that expressed K1 but not 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 < 0.01) against the lethal effects of agonistic anti-Fas (Jo2) antibody. K1dIg expressed in mice did not form complexes with Fas, suggesting that the Ig domain is essential for K1-Fas binding and suppression of apoptosis.Conclusion: These results confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation. K1 blocks Fas-mediated apoptosis and competing peptides can reinstate apoptosis.

Human Herpesvirus 8 Protein K1 Interference with Fas-Mediated Apoptosis Induces Clonal Growth Which Leads to Lymphoid Hyperplasia.

Background: Human herpesvirus 8 (HHV-8) infection is causally associated with the development of primary effusion lymphoma and Kaposi's sarcoma. A transmembrane protein of HHV-8, K1, is readily expressed in those 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 shown previously to be involved in activation of Nuclear Factor kappa B (NFκ-B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived 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 of Fas signaling.Results: Gross examination of thoracic and abdominal cavities of transgenic mice with K1 expression driven by a ubiquitous promoter were sacrificed at 18 months of age revealed enlarged cervical, mediastinal, renal, mesenteric lymph nodes and spleens. Peyer's patches were also enlarged and readily visible on the outer surface of the ileum. Of 10 K1 mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 ± 200 mg vs. 94 ± 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with anti-kappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4±1% of splenocytes from K1 mice versus 17±2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11±0.6% vs. 50±6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 - induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.Conclusion: These results over all confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation of this phenomenon. Interference of K1 with Fas-mediated apoptosis disrupts the normal life cycle of lymphocytes.