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1.
N Engl J Med ; 372(26): 2509-20, 2015 Jun 25.
Article in English | MEDLINE | ID: mdl-26028255

ABSTRACT

BACKGROUND: Somatic mutations have the potential to encode "non-self" immunogenic antigens. We hypothesized that tumors with a large number of somatic mutations due to mismatch-repair defects may be susceptible to immune checkpoint blockade. METHODS: We conducted a phase 2 study to evaluate the clinical activity of pembrolizumab, an anti-programmed death 1 immune checkpoint inhibitor, in 41 patients with progressive metastatic carcinoma with or without mismatch-repair deficiency. Pembrolizumab was administered intravenously at a dose of 10 mg per kilogram of body weight every 14 days in patients with mismatch repair-deficient colorectal cancers, patients with mismatch repair-proficient colorectal cancers, and patients with mismatch repair-deficient cancers that were not colorectal. The coprimary end points were the immune-related objective response rate and the 20-week immune-related progression-free survival rate. RESULTS: The immune-related objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair-deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair-proficient colorectal cancers. The median progression-free survival and overall survival were not reached in the cohort with mismatch repair-deficient colorectal cancer but were 2.2 and 5.0 months, respectively, in the cohort with mismatch repair-proficient colorectal cancer (hazard ratio for disease progression or death, 0.10 [P<0.001], and hazard ratio for death, 0.22 [P=0.05]). Patients with mismatch repair-deficient noncolorectal cancer had responses similar to those of patients with mismatch repair-deficient colorectal cancer (immune-related objective response rate, 71% [5 of 7 patients]; immune-related progression-free survival rate, 67% [4 of 6 patients]). Whole-exome sequencing revealed a mean of 1782 somatic mutations per tumor in mismatch repair-deficient tumors, as compared with 73 in mismatch repair-proficient tumors (P=0.007), and high somatic mutation loads were associated with prolonged progression-free survival (P=0.02). CONCLUSIONS: This study showed that mismatch-repair status predicted clinical benefit of immune checkpoint blockade with pembrolizumab. (Funded by Johns Hopkins University and others; ClinicalTrials.gov number, NCT01876511.).


Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Agents/therapeutic use , DNA Mismatch Repair , Neoplasm Metastasis/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma/secondary , Adult , Aged , Antibodies, Monoclonal, Humanized/adverse effects , Antineoplastic Agents/adverse effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Disease-Free Survival , Female , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Neoplasm Metastasis/genetics
2.
J Clin Oncol ; 33(12): 1325-33, 2015 Apr 20.
Article in English | MEDLINE | ID: mdl-25584002

ABSTRACT

PURPOSE: GVAX pancreas, granulocyte-macrophage colony-stimulating factor-secreting allogeneic pancreatic tumor cells, induces T-cell immunity to cancer antigens, including mesothelin. GVAX is administered with low-dose cyclophosphamide (Cy) to inhibit regulatory T cells. CRS-207, live-attenuated Listeria monocytogenes-expressing mesothelin, induces innate and adaptive immunity. On the basis of preclinical synergy, we tested prime/boost vaccination with GVAX and CRS-207 in pancreatic adenocarcinoma. PATIENTS AND METHODS: Previously treated patients with metastatic pancreatic adenocarcinoma were randomly assigned at a ratio of 2:1 to two doses of Cy/GVAX followed by four doses of CRS-207 (arm A) or six doses of Cy/GVAX (arm B) every 3 weeks. Stable patients were offered additional courses. The primary end point was overall survival (OS) between arms. Secondary end points were safety and clinical response. RESULTS: A total of 90 patients were treated (arm A, n = 61; arm B, n = 29); 97% had received prior chemotherapy; 51% had received ≥ two regimens for metastatic disease. Mean number of doses (± standard deviation) administered in arms A and B were 5.5 ± 4.5 and 3.7 ± 2.2, respectively. The most frequent grade 3 to 4 related toxicities were transient fevers, lymphopenia, elevated liver enzymes, and fatigue. OS was 6.1 months in arm A versus 3.9 months in arm B (hazard ratio [HR], 0.59; P = .02). In a prespecified per-protocol analysis of patients who received at least three doses (two doses of Cy/GVAX plus one of CRS-207 or three of Cy/GVAX), OS was 9.7 versus 4.6 months (arm A v B; HR, 0.53; P = .02). Enhanced mesothelin-specific CD8 T-cell responses were associated with longer OS, regardless of treatment arm. CONCLUSION: Heterologous prime/boost with Cy/GVAX and CRS-207 extended survival for patients with pancreatic cancer, with minimal toxicity.


Subject(s)
Cancer Vaccines/administration & dosage , Carcinoma, Pancreatic Ductal/therapy , Cyclophosphamide/administration & dosage , GPI-Linked Proteins/biosynthesis , Listeria monocytogenes/metabolism , Pancreatic Neoplasms/therapy , Aged , Aged, 80 and over , Antineoplastic Agents, Alkylating/administration & dosage , Antineoplastic Agents, Alkylating/adverse effects , Cancer Vaccines/adverse effects , Carcinoma, Pancreatic Ductal/immunology , Combined Modality Therapy , Cyclophosphamide/adverse effects , Female , GPI-Linked Proteins/genetics , Humans , Listeria monocytogenes/genetics , Male , Mesothelin , Middle Aged , Pancreatic Neoplasms/immunology , Survival Rate , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology
3.
Int J Cancer ; 136(1): 127-37, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-24832153

ABSTRACT

Cancer immunotherapy induces a variety of autoinflammatory responses, including those against the thyroid gland, which can be exploited to predict clinical outcomes. Considering the paucity of information about thyroid autoimmunity in patients receiving cancer vaccines, we designed our study to assess the development of thyroglobulin antibodies (TgAbs) in patients treated with GVAX (vaccine made of a tumor cell type transfected with GM-CSF) and/or ipilimumab and correlated seroconversion with survival. Using both in house and commercial ELISA assays, we measured TgAbs in patients with pancreatic (No. = 53), prostate (No. = 35) or colon (No. = 8) cancer, before and after treatment with GVAX only (No. = 34), GVAX plus ipilimumab (No. = 42) or ipilimumab (No. = 20), and correlated their levels with patient's survival, disease status and T-cell surface markers. Antibodies to thyroperoxidase, myeloperoxidase, proteinase 3, insulin and actin were also measured. TgAbs specifically developed after GVAX, independent of the underlying cancer (81% in prostate, 75% colon cancer and 76% pancreatic cancer) and co-administration of ipilimumab (75% in GVAX only and 78% in GVAX plus ipilimumab). This TgAbs seroconversion could be detected mainly by the in house assay, suggesting that the thyroglobulin epitopes recognized by the antibodies induced by GVAX are different from the epitopes seen in the classic form of Hashimoto thyroiditis. Notably, TgAbs seroconversion was associated with significantly prolonged survival (p = 0.01 for pancreas and p = 0.005 for prostate cancer). In conclusion, GVAX immunotherapy induces the appearance of TgAbs that recognize a unique antigenic repertoire and associate with prolonged survival.


Subject(s)
Cancer Vaccines/administration & dosage , Colonic Neoplasms/therapy , Pancreatic Neoplasms/therapy , Prostatic Neoplasms/therapy , Thyroglobulin/immunology , Antibodies, Monoclonal/administration & dosage , Antibodies, Neoplasm/blood , Antineoplastic Agents/administration & dosage , Autoantibodies/blood , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cancer Vaccines/immunology , Cell Line, Tumor , Cohort Studies , Colonic Neoplasms/blood , Colonic Neoplasms/immunology , Colonic Neoplasms/mortality , Combined Modality Therapy , Humans , Ipilimumab , Male , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/mortality , Prostatic Neoplasms/blood , Prostatic Neoplasms/immunology , Prostatic Neoplasms/mortality , RNA, Messenger/genetics , RNA, Messenger/metabolism , Survival Analysis , Thyroglobulin/genetics , Thyroglobulin/metabolism , Thyrotropin/blood , Vaccination
4.
Cancer Immunol Res ; 2(10): 949-61, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25116755

ABSTRACT

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccines are bioactive, but limited by disease burden and immune tolerance. Cyclophosphamide augments vaccine activity in tolerant neu mice and in patients with metastatic breast cancer. HER2-specific monoclonal antibodies (mAb) enhance vaccine activity in neu mice. We hypothesized that cyclophosphamide-modulated vaccination with HER2-specific mAb safely induces relevant HER2-specific immunity in neu mice and patients with HER2+ metastatic breast cancer. Adding both cyclophosphamide and the HER2-specific mAb 7.16.4 to vaccination maximized HER2-specific CD8+ T-cell immunity and tumor-free survival in neu transgenic mice. We, therefore, conducted a single-arm feasibility study of cyclophosphamide, an allogeneic HER2+ GM-CSF-secreting breast tumor vaccine, and weekly trastuzumab in 20 patients with HER2+ metastatic breast cancer. Primary clinical trial objectives were safety and clinical benefit, in which clinical benefit represents complete response + partial response + stable disease. Secondary study objectives were to assess HER2-specific T-cell responses by delayed type hypersensitivity (DTH) and intracellular cytokine staining. Patients received three monthly vaccinations, with a boost 6 to 8 months from trial entry. This combination immunotherapy was safe, with clinical benefit rates at 6 months and 1 year of 55% [95% confidence interval (CI), 32%-77%; P = 0.013] and 40% (95% CI, 19%-64%), respectively. Median progression-free survival and overall survival durations were 7 months (95% CI, 4-16) and 42 months (95% CI, 22-70), respectively. Increased HER2-specific DTH developed in 7 of 20 patients [of whom 4 had clinical benefit (95% CI, 18-90)], with a trend toward longer progression-free survival and overall survival in DTH responders. Polyfunctional HER2-specific CD8+ T cells progressively expanded across vaccination cycles. Further investigation of cyclophosphamide-modulated vaccination with trastuzumab is warranted.


Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/therapy , Cancer Vaccines/therapeutic use , Receptor, ErbB-2/metabolism , Adult , Aged , Animals , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/adverse effects , Antineoplastic Agents/adverse effects , Breast Neoplasms/immunology , Breast Neoplasms/metabolism , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/adverse effects , Cancer Vaccines/immunology , Cell Line, Tumor , Combined Modality Therapy , Cyclophosphamide/administration & dosage , Cyclophosphamide/adverse effects , Feasibility Studies , Female , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Hypersensitivity, Delayed/immunology , Mice, Transgenic , Middle Aged , Neoplasm Metastasis , Receptor, ErbB-2/immunology , Survival Analysis , Trastuzumab
5.
Ann Surg Oncol ; 21(12): 3931-7, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24943235

ABSTRACT

BACKGROUND: Despite recent advances in earlier detection and improvements in chemotherapy, the 5-year survival rate of patients with metastatic colorectal carcinoma remains poor. Immunotherapy is a potentially effective therapeutic approach to the treatment of colorectal carcinoma. Preclinical studies have supported the antitumor activity of immunization with a granulocyte-macrophage colony-stimulating factor (GM-CSF) producing murine colon tumor cell vaccine. METHODS: A novel colorectal cancer vaccine composed of irradiated, allogeneic human colon cancer cells and GM-CSF-producing bystander cells was developed and tested in combination with a single intravenous low dose of cyclophosphamide in a phase 1 study of patients with metastatic colorectal cancer. RESULTS: A total of nine patients were enrolled onto and treated in this study. Six patients had a history of colorectal adenocarcinoma hepatic metastases and underwent curative metastasectomy, while three other patients had unresectable stage IV disease. This study demonstrates the safety and feasibility of this vaccine administered in patients with metastatic colorectal cancer. At last follow-up, the six patients who underwent curative metastasectomy survived longer than 36 months, and four of these six patients were without disease recurrence. Immunologic correlate results suggest that the GM-CSF-producing colon cancer vaccine enhances the production of anti-MUC1 antibodies. CONCLUSIONS: This vaccine is feasible and safe. Future investigation of the efficacy and antitumor immunity of this vaccine is warranted.


Subject(s)
Adenocarcinoma/therapy , Cancer Vaccines/therapeutic use , Colorectal Neoplasms/therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/therapy , Macrophage Colony-Stimulating Factor/administration & dosage , Neoplasm Recurrence, Local/therapy , Adenocarcinoma/immunology , Adenocarcinoma/mortality , Adenocarcinoma/secondary , Adult , Aged , Antineoplastic Agents, Alkylating/administration & dosage , Clinical Trials, Phase I as Topic , Colorectal Neoplasms/immunology , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Combined Modality Therapy , Cyclophosphamide/administration & dosage , Feasibility Studies , Female , Follow-Up Studies , Humans , Immunotherapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/immunology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/mortality , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Recurrence, Local/immunology , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Prognosis , Safety , Survival Rate , Tumor Cells, Cultured
7.
Ann Surg Oncol ; 20 Suppl 3: S725-30, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24046118

ABSTRACT

BACKGROUND: Low total lymphocyte count (TLC) and lymphocyte-to-neutrophil ratio have been found to be poor prognostic indicators in several different tumor types at various stages. Although immune-based therapies are under rapid development, it is not known whether baseline complete blood counts, particularly lymphocytes, are associated with the clinical outcomes of patients receiving immunotherapies. METHODS: We performed a retrospective analysis of complete blood count for 59 patients enrolled onto a phase II trial evaluating the integration of an adjuvant immunotherapy-irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting allogeneic pancreatic tumor vaccine (GVAX)-with standard chemoradiation. RESULTS: After adjusting for nodal status, individuals with a TLC of <1,500 cells/mm(3) (10 patients) had significantly higher risk, both in terms of overall survival (OS) [adjusted hazard ratio 2.63, 95 % confidence interval (CI) 1.22-5.67, p = 0.013] and progression-free survival (adjusted hazard ratio 3.07, 95 % CI 1.03-6.93, p = 0.003), compared to those with a TLC of ≤ 1,500 cells/mm(3) (49 patients). Adjuvant chemoradiation significantly reduced lymphocyte counts from baseline values. Patients with suppression of their lymphocytes to <500 cells/mm(3) after chemoradiation also had shorter disease-free and OS. CONCLUSIONS: Immunosuppressive conditions associated with surgical procedures and chemoradiation may affect the efficacy of immunotherapy.


Subject(s)
Cancer Vaccines/therapeutic use , Carcinoma, Pancreatic Ductal/mortality , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Immunotherapy , Pancreatic Neoplasms/mortality , Adjuvants, Immunologic , Adult , Aged , Aged, 80 and over , Antimetabolites, Antineoplastic/therapeutic use , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/therapy , Clinical Trials, Phase II as Topic , Combined Modality Therapy , Female , Fluorouracil/therapeutic use , Follow-Up Studies , Humans , Lymphocyte Count , Male , Middle Aged , Neoplasm Staging , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/therapy , Prognosis , Radiotherapy Dosage , Retrospective Studies , Survival Rate
8.
J Immunother ; 36(7): 382-9, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23924790

ABSTRACT

Preclinical reports support the concept of synergy between cancer vaccines and immune checkpoint blockade in nonimmunogenic tumors. In particular, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) antibodies have been successfully combined with GM-CSF cell-based vaccines (GVAX). Ipilimumab (anti-CTLA-4) has been tested as a single agent in patients with pancreatic ductal adenocarcinoma (PDA) resulting in a delayed response at a dose of 3 mg/kg. Our study evaluated ipilimumab 10 mg/kg (arm 1) and ipilimumab 10 mg/kg + GVAX (arm 2). A total of 30 patients with previously treated advanced PDA were randomized (1:1). Induction doses were administered every 3 weeks for a total of 4 doses followed by maintenance dosing every 12 weeks. Two patients in arm 1 showed evidence of stable disease (7 and 22 wk) but none demonstrated CA19-9 biochemical responses. In contrast, 3 patients in arm 2 had evidence of prolonged disease stabilization (31, 71, and 81 wk) and 7 patients experienced CA19-9 declines. In 2 of these patients, disease stabilization occurred after an initial period of progression. The median overall survival (OS) (3.6 vs. 5.7 mo, hazards ratio: 0.51, P = 0.072) and 1 year OS (7 vs. 27%) favored arm 2. Similar to prior ipilimumab studies, 20% of patients in each arm had grade 3/4 immune-related adverse events. Among patients with OS > 4.3 months, there was an increase in the peak mesothelin-specific T cells (P = 0.014) and enhancement of the T-cell repertoire (P = 0.031). In conclusion, checkpoint blockade in combination with GVAX has the potential for clinical benefit and should be evaluated in a larger study.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Antineoplastic Agents/administration & dosage , Cancer Vaccines/administration & dosage , Carcinoma, Pancreatic Ductal/therapy , Pancreatic Neoplasms/therapy , Adult , Aged , CTLA-4 Antigen/immunology , Carcinoma, Pancreatic Ductal/diagnostic imaging , Carcinoma, Pancreatic Ductal/immunology , Combined Modality Therapy , Female , GPI-Linked Proteins/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Humans , Ipilimumab , Male , Mesothelin , Middle Aged , Pancreatic Neoplasms/diagnostic imaging , Pancreatic Neoplasms/immunology , Radiography , T-Lymphocytes/immunology , Transfection
9.
J Immunol ; 186(7): 3847-57, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21346233

ABSTRACT

We previously reported that CD8(+) T cells are directed predominantly toward the immunodominant Her-2/neu (neu) epitope RNEU(420-429) in nontolerized FVB/N but not tolerized HER-2/neu (neu-N) mice. In this study, we screened overlapping peptides of the entire neu protein and identified six new epitopes recognized by vaccine-induced neu-N-derived T cells. Evaluation of individual nondominant responses by tetramer staining and IFN-γ secretion demonstrate that this repertoire is peripherally tolerized. To address the role that the complete CD8(+) T cell repertoire plays in vaccine-induced antitumor immunity, we created a whole-cell vaccine-expressing neu cDNA that has been mutated at the RNEU(420-429) anchor residue, thereby abrogating activation of immunodominant epitope responses. Studies comparing the mutated and nonmutated vaccines indicate that nondominant CD8(+) T cells can induce antitumor immunity when combined with regulatory T cell-depleting agents in both neu-N and FVB/N mice. Collectively, these studies demonstrate that the neu-directed T cell repertoire is not intrinsically incapable of eradicating tumors. Rather, they are suppressed by mechanisms of peripheral tolerance. Thus, these studies provide new insights into the function of the complete T cell repertoire directed toward a clinically relevant tumor Ag in tumor-bearing hosts.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/administration & dosage , Epitopes, T-Lymphocyte/metabolism , Immunodominant Epitopes/administration & dosage , Mammary Neoplasms, Experimental/immunology , Mammary Neoplasms, Experimental/therapy , Receptor, ErbB-2/deficiency , Animals , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Cancer Vaccines/genetics , Cancer Vaccines/metabolism , Cell Line, Tumor , Dose-Response Relationship, Immunologic , Epitopes, T-Lymphocyte/administration & dosage , Epitopes, T-Lymphocyte/genetics , Gene Expression Regulation, Neoplastic/immunology , Immune Tolerance/genetics , Immunodominant Epitopes/genetics , Immunodominant Epitopes/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mammary Neoplasms, Experimental/genetics , Mice , Mice, Transgenic , NIH 3T3 Cells , Rats , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism
10.
Appl Environ Microbiol ; 70(2): 967-72, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14766578

ABSTRACT

Genetic characterization of a signal transduction pathway requires the isolation of mutations in the pathway. Characterization of these mutated genes and their loci enumerates the components of the pathway and leads to an understanding of the role of each gene locus in the pathway under study. We have designed and developed a strategy based on resistance to the chemical flucytosine for the identification of mutations in a given pathway. In this study, the Escherichia coli codA gene, which encodes the enzyme cytosine deaminase, was fused to the light-intensity-regulated gene promoter psbDII. Cytosine deaminase converts 5'-fluorocytosine to the toxic product 5-fluorouracil. Wild-type cells containing an intact signal transduction pathway that regulates the psbDII promoter will die in the presence of this chemical. Cells that carry mutations in the pathway that inactivate the psbDII promoter will not express the codA gene and, consequently, will live on 5'-fluorocytosine, allowing the isolation and subsequent characterization of mutations in this signaling pathway. Utilizing this selection method, we have successfully isolated and characterized mutations in the psbDII pathway. This selection scheme can be used with a tissue-specific or phase-specific promoter fused to the codA gene to direct the timing of expression of codA to obtain mutants defective in temporal or cell-specific expression of a particular pathway. This scheme also allows the isolation of mutants even when a clearly identifiable phenotype is not available. The selection scheme presented here extends the molecular tools available for the genetic dissection of signal transduction pathways.


Subject(s)
Cyanobacteria/genetics , Mutation , Selection, Genetic , Signal Transduction , Cyanobacteria/drug effects , Cyanobacteria/growth & development , Cyanobacteria/metabolism , Cytosine Deaminase/genetics , Cytosine Deaminase/metabolism , Drug Resistance, Bacterial , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Flucytosine/pharmacology , Gene Expression Regulation, Bacterial , Photosystem II Protein Complex/genetics , Photosystem II Protein Complex/metabolism , Promoter Regions, Genetic , Transcription, Genetic
11.
Curr Biol ; 12(20): 1728-37, 2002 Oct 15.
Article in English | MEDLINE | ID: mdl-12401167

ABSTRACT

BACKGROUND: The inducible production of antimicrobial peptides is a major immune response in Drosophila. The genes encoding these peptides are activated by NF-kappaB transcription factors that are controlled by two independent signaling cascades: the Toll pathway that regulates the NF-kappaB homologs, Dorsal and DIF; and the IMD pathway that regulates the compound NF-kappaB-like protein, Relish. Although numerous components of each pathway that are required to induce antimicrobial gene expression have been identified, less is known about the mechanisms that either repress antimicrobial genes in the absence of infection or that downregulate these genes after infection. RESULTS: In a screen for factors that negatively regulate the IMD pathway, we isolated two partial loss-of-function mutations in the SkpA gene that constitutively induce the antibacterial peptide gene, Diptericin, a target of the IMD pathway. These mutations do not affect the systemic expression of the antifungal peptide gene, Drosomycin, a target of the Toll pathway. SkpA encodes a homolog of the yeast and human Skp1 proteins. Skp1 proteins function as subunits of SCF-E3 ubiquitin ligases that target substrates to the 26S proteasome, and mutations affecting either the Drosophila SCF components, Slimb and dCullin1, or the proteasome also induce Diptericin expression. In cultured cells, inhibition of SkpA and Slimb via RNAi increases levels of both the full-length Relish protein and the processed Rel-homology domain. CONCLUSIONS: In contrast to other NF-kappaB activation pathways, the Drosophila IMD pathway is repressed by the ubiquitin-proteasome system. A possible target of this proteolytic activity is the Relish transcription factor, suggesting a mechanism for NF-kappaB downregulation in Drosophila.


Subject(s)
Cysteine Endopeptidases/metabolism , Drosophila melanogaster/immunology , Insect Proteins/genetics , Multienzyme Complexes/metabolism , SKP Cullin F-Box Protein Ligases , Signal Transduction , Ubiquitin/metabolism , Amino Acid Sequence , Animals , Anti-Infective Agents/chemistry , Anti-Infective Agents/metabolism , Cell Cycle Proteins/genetics , Crosses, Genetic , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Ethyl Methanesulfonate , Female , Humans , Immunologic Deficiency Syndromes/genetics , Insect Proteins/chemistry , Male , Molecular Sequence Data , Mutagenesis , Mutagens , Proteasome Endopeptidase Complex , Recombinant Proteins/biosynthesis , S-Phase Kinase-Associated Proteins , Sequence Homology, Amino Acid
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