MGMT autoantibodies as a potential prediction of recurrence and treatment response biomarker for glioma patients.

BACKGROUND: Cancer-specific autoantibodies found in serum of cancer patients have been characterized as potential predictors of the high risk of recurrence and treatment response. The objective of this study is to investigate the clinical utility of serum O-6-methylguanine-DNA methyltransferase (MGMT) autoantibodies as novel biomarkers for prediction of recurrence and treatment response for glioma through MGMT peptides microarray. METHODS: A total of 201 serum samples of glioma patients with various WHO grade and 311 serum samples of healthy donors were examined for the detection of MGMT autoantibodies by peptides microarray. The clinical value of MGMT autoantibodies was studied through univariable and multivariable analyses. RESULTS: Autoantibodies to MGMT peptides were detected in sera from glioma patients and five highly responsive autoantibodies to peptides were identified in the glioma group. The positive rate of MGMT autoantibody to 20 peptides in glioma groups is compared with healthy individuals, the positive rate of MGMT-02 (45%), MGMT-04 (27%), MGMT-07 (21%), MGMT-10 (13%), and MGMT-18 (24%) were significantly elevated in patients with glioma. MGMT autoantibody and its protein expression exhibited a significant correlation. The levels of MGMT autoantibodies decreased on the 30th day after operation, reaching preoperative levels, similar to those when tumor recurrence developed. Univariable and multivariable analyses revealed that the only preoperative autoantibodies to MGMT-02 peptide were independently correlated with recurrence-free survival. Preoperative seropositive patients were more likely than seronegative patients to have shorter recurrence times and to be resistant to chemoradiotherapy or chemotherapy with temozolomide. CONCLUSION: Monitoring the levels of preoperative serum autoantibodies to MGMT-02 peptide was useful for predicting patients at high risk of recurrence and treatment response.

Biological characterisation and application of human MTH1 and monoclonal antibody preparation.

Human MutT homolog 1 (MTH1) hydrolyses oxidised nucleotide triphosphates, thereby preventing them from being incorporated into DNA; MTH1 has been found to be elevated in many types of cancers, including lung, stomach cancer, melanoma and breast cancer. Thus, tumour­targeted hMTH1 may be valuable for developing novel anticancer therapies. In the present study, we prepared human MTH1 protein and its monoclonal antibody (mAb). The hMTH1 gene was cloned into the prokaryotic expression vector pET28a and optimally expressed in the E. coli Transetta (DE3) strain. Using an Ni­NTA column and a G­50 gel filtration column, 20.1 mg of active hMTH1 was obtained from 1,000 ml of bacterial culture, and the purity was over 98%, as detected by high­performance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC50) of TH287 (hMTH1 inhibitor) was determined to be 3.53±0.47 nM using the recombinant hMTH1 protein (rhMTH1). The enzyme activity assay showed the Michaelis constant (Km) and the catalytic constant (kcat) of the protein were 106.13±48.83 µM and 3.64±0.58 sec­1, respectively. The anti­hMTH1 mAb was obtained via the hybridoma technique and validated by western blot analysis. In addition, an immunofluorescence assay (IFA) and ELISA determined that the mAb could efficiently bind to natural hMTH1 expressed on the human breast cancer cell line MCF­7. Taken together, the results showed the rhMTH1 is an active protein and has practical applications for inhibitor selection, and our prepared hMTH1 mAb will provide a valuable tool for the further characterisation of hMTH1 and antitumour medicinal development in future.

Immunotherapy holds the key to cancer treatment and prevention in constitutional mismatch repair deficiency (CMMRD) syndrome.

Monoallelic germline mutations in one of the DNA mismatch repair (MMR) genes cause Lynch syndrome, with a high lifetime risks of colorectal and endometrial cancer at adult age. Less well known, is the constitutional mismatch repair deficiency (CMMRD) syndrome caused by biallelic germline mutations in MMR genes. This syndrome is characterized by the development of childhood cancer. Patients with CMMRD are at extremely high risk of developing multiple cancers including hematological, brain and intestinal tumors. Mutations in MMR genes impair DNA repair and therefore most tumors of patients with CMMRD are hypermutated. These mutations lead to changes in the translational reading frame, which consequently result in neoantigen formation. Neoantigens are recognized as foreign by the immune system and can induce specific immune responses. The growing evidence on the clinical efficacy of immunotherapies, such as immune checkpoint inhibitors, offers the prospect for treatment of patients with CMMRD. Combining neoantigen-based vaccination strategies and immune checkpoint inhibitors could be an effective way to conquer CMMRD-related tumors. Neoantigen-based vaccines might also be a preventive treatment option in healthy biallelic MMR mutation carriers. Future studies need to reveal the safety and efficacy of immunotherapies for patients with CMMRD.

NBS1 is required for macrophage homeostasis and functional activity in mice.

Nijmegen breakage syndrome 1 (NBS1) is a component of the MRE11 complex, which is a sensor of DNA double-strand breaks and plays a crucial role in the DNA damage response. Because activated macrophages produce large amounts of reactive oxygen species (ROS) that can cause DNA lesions, we examined the role of NBS1 in macrophage functional activity. Proliferative and proinflammatory (interferon gamma [IFN-γ] and lipopolysaccharide [LPS]) stimuli led to increased NBS1 levels in macrophages. In mice expressing a hypomorphic allele of Nbs1, Nbs1(∆B/∆B), macrophage activation-induced ROS caused increased levels of DNA damage that were associated with defects in proliferation, delayed differentiation, and increased senescence. Furthermore, upon stimulation, Nbs1(∆B/∆B) macrophages exhibited increased expression of proinflammatory cytokines. In the in vivo 2,4-dinitrofluorobenzene model of inflammation, Nbs1(∆B/∆B) animals showed increased weight and ear thickness. By using the sterile inflammation by zymosan injection, we found that macrophage proliferation was drastically decreased in the peritoneal cavity of Nbs1(∆B/∆B) mice. Our findings show that NBS1 is crucial for macrophage function during normal aging. These results have implications for understanding the immune defects observed in patients with NBS and related disorders.

Screening diagnostic candidates for schistosomiasis from tegument proteins of adult Schistosoma japonicum using an immunoproteomic approach.

BACKGROUND: Schistosomiasis is one of the world's most prevalent zoonotic diseases and a serious worldwide public health problem. Since the tegument (TG) of Schistosoma japonicum is in direct contact with the host and induces a host immune response against infection, the identification of immune response target molecules in the schistosome TG is crucial for screening diagnostic antigens for this disease. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an immunoproteomics approach used TG proteins as screening antigens to identify potential diagnostic molecules of S. japonicum. Ten spots corresponding to six proteins were identified that immunoreacted with sera from S. japonicum-infected rabbits but not sera from uninfected rabbits and their specific IgG antibody levels declined quickly after praziquantel treatment. Recombinant phosphoglycerate mutase (PGM) and UV excision repair protein RAD23 homolog B (RAD23) proteins were expressed and their diagnostic potential for schistosomiasis was evaluated and compared with schistosome soluble egg antigen (SEA) using ELISA. The results showed high sensitivity and specificity and low crossreactivity when rSjPGM-ELISA and rSjRAD23-ELISA were used to detect water buffalo schistosomiasis. Moreover, antibodies to rSjPGM and rSjRAD23 might be short-lived since they declined quickly after chemotherapy. CONCLUSION/SIGNIFICANCE: Therefore, the two schistosome TG proteins SjPGM and SjRAD23 were identified as potential diagnostic markers for the disease. The two recombinant proteins might have the potential to evaluate the effectiveness of drug treatments and for distinguishing between current and past infection.

DNA mismatch repair enzymes: genetic defects and autoimmunity.

DNA mismatch repair (MMR) is one of the several DNA repair pathways conserved from bacteria to humans. The primary function of MMR is to eliminate the mismatch of base-base insertions and deletions that appear as a consequence of DNA polymerase errors at DNA synthesis. The genes encoding the DNA MMR enzymes (MMREs) are highly conserved throughout evolution. In humans, there are two sets of MMREs, corresponding to homologues of the bacterial MutLS systems. The human MutS enzymes consist of MSH2, MSH3 and MSH6, and the human MutL enzymes include MLH1, MLH3, PMS1 and PMS2. Since the beginning of this century, a few reports on autoantibodies to some MMREs have been reported in autoimmune inflammatory myopathy, cancer and hematological disorders. This review charts the functional structures of MMREs, their genetic defects and associated disorders, and autoimmunity to MMREs, including our recent data that was the first to analyze autoantibodies against all seven kinds of MMREs in systemic autoimmune diseases, including idiopathic inflammatory myopathies.

Antibodies to DNA repair proteins in headache with neurological deficits and cerebrospinal fluid lymphocytosis (HaNDL) patients.

Autoimmune mechanisms have been implicated in the pathogenesis of headache and neurological deficits with cerebrospinal fluid lymphocytosis (HaNDL). Pooled sera of five HaNDL patients and 30 controls (10 multiple sclerosis patients, 10 migraine patients, 10 healthy controls) were screened by protein macroarray. All sera were also individually subjected to immunoprecipitation with neuroblastoma cells and the bound antigens were identified by mass spectrometry. Antibodies to three DNA repair proteins (mitogen-activated protein kinase-4, DNA-dependent protein kinase catalytic subunit, DNA excision repair protein ERCC-6) were identified by both macroarray and immunoprecipitation methods in 3/5 HaNDL sera, but in none of the controls. The presence of DNA repair protein antibodies indicates DNA damage and provides further support for the inflammatory etiology of HaNDL.

Autoantibodies to DNA mismatch repair enzymes in polymyositis/dermatomyositis and other autoimmune diseases: a possible marker of favorable prognosis.

Objective. Myositis-specific autoantibodies(MSAs) are useful tools for identifying clinical subsets of patients with idiopathic inflammatory myopathies(IIMs). There have been few reports on antibodies to some DNA mismatch repair enzymes (MMREs) inpatients with IIMs. This study was undertaken to determine the frequencies and clinical associations of antibodies to 7 types of MMREs (MLH1, MLH3, MSH2,MSH3, MSH6, PMS1, and PMS2) in patients with IIMs and other systemic autoimmune diseases.Methods. Clinical data and serum samples were collected from 239 Japanese patients with IIMs (147 with adult dermatomyositis, 13 with juvenile dermatomyositis,57 with polymyositis, and 22 with myositis overlap syndrome). One hundred patients with other diseases, including 40 with systemic lupus erythematosus(SLE), were assessed as disease controls. The presence of anti-MMRE antibodies in serum was examined by immunoprecipitation, enzyme-linked immunosorbenassay, and immunoprecipitation/Western blotting.Results. Anti-MMRE antibodies were found in 1 patients with IIMs and 3 patients with SLE. They were restricted to MLH1, PMS1, MSH2, and PMS2, with simultaneous positivity for more than one of these antibodies occurring in some patients. Nine IIM patients with anti-MMREs also had other MSAs and their associated clinical features. All patients with anti-MMREs were still living at the time of the present analysis.Conclusion. Anti-MMRE antibodies, which often co exist with other MSAs, may be serologic markers forgood prognosis in IIMs.

A polymorphism in the TH 2 locus control region is associated with changes in DNA methylation and gene expression.

BACKGROUND: Genomewide association and epigenetic studies found a region within the RAD50 gene on chromosome 5q31 to be associated with total serum IgE levels and asthma. In mice, this region harbors a locus control region for nearby TH 2 cytokines, which is characterized by four Rad50 DNase I hypersensitive sites (RHS4-7). Among these, RHS7 seems to have the strongest impact on TH 2 differentiation. We investigated whether within the human homolog of RHS7, functional polymorphisms exist, which could affect DNA methylation or gene expression in the 5q31 locus and might have an influence on asthma status or IgE regulation. METHODS: The human RHS7 region was fine mapped using 1000 genomes database information. In silico analysis and electrophoretic mobility shift assays were used to assess SNP function. Allele-specific effects on DNA methylation were evaluated in cord blood (n = 73) and at age of 4.5 years (n = 61) by pyrosequencing. Allele-specific effects on RAD50, IL4, and IL13 expression were analyzed in 100 subjects. Associations with asthma and IgE levels were investigated in the MAGICS/ISAAC II population (n = 1145). RESULTS: Polymorphism rs2240032 in the RHS7 region is suggestive of allele-specific transcription factor binding, affects methylation of the IL13 promoter region and influences RAD50 and IL4 expression (lowest P = 0.0027). It is also associated with total serum IgE levels (P = 0.0227). CONCLUSION: A functional relevant polymorphism in the TH 2 locus control region, equivalent to RHS7 in mice, affects DNA methylation and gene expression within 5q31 and influences total serum IgE on the population level.

Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1ß production.

Double-stranded DNA (dsDNA) in the cytoplasm triggers the production of interleukin 1ß (IL-1ß) as an antiviral host response, and deregulation of the pathways involved can promote inflammatory disease. Here we report a direct cytosolic interaction between the DNA-damage sensor Rad50 and the innate immune system adaptor CARD9. Transfection of dendritic cells with dsDNA or infection of dendritic cells with a DNA virus induced the formation of dsDNA-Rad50-CARD9 signaling complexes for activation of the transcription factor NF-κB and the generation of pro-IL-1ß. Primary cells conditionally deficient in Rad50 or lacking CARD9 consequently exhibited defective DNA-induced production of IL-1ß, and Card9(-/-) mice had impaired inflammatory responses after infection with a DNA virus in vivo. Our results define a cytosolic DNA-recognition pathway for inflammation and a physical and functional connection between a conserved DNA-damage sensor and the innate immune response to pathogens.

Immunochemistry screening for Lynch syndrome in colorectal adenocarcinoma using an initial two antibody panel can replace a four antibody panel.

AIM: The current practice in immunochemistry staining for Lynch syndrome (LS) is to use a four-antibody panel, (MLH1, MSH2, MSH6, PMS2) to screen for the four Mismatch Repair (MMR) gene expressions involved. We hypothesised that testing two antibodies (MSH6 and PMS2), followed by the other two only when there is loss of expression of the first two antibodies, would be equally effective as a four antibody panel in detecting LS. This hypothesis is based on the biochemical binding properties of the MMR proteins. METHODS: We tested this hypothesis on a patient cohort consisting of all cases of colorectal cancer that were stained for MMR gene expression at Auckland City Hospital (Auckland, New Zealand) from the years 2000 to 2010 (inclusive), providing a series of 410 cases for this study. Exclusions were made based on heterogeneous staining pattern and unsatisfactory staining results on MSH6 and PMS2, which left n=400 included in the study. RESULTS: The MMR gene protein stains were regarded as demonstrating loss of expression (LOE) when there was no uptake in the nucleus of the tumour cells, with a positive internal control. The results from our analysis supported our hypothesis. Seventy-four cases showed LOE of MSH6 or PMS2. One of them showed LOE of all four MMR proteins. For the remaining 326 cases, there was no LOE of all four MMR proteins. CONCLUSION: Our study gives further evidence that an initial two-antibody panel consisting of PMS2 and MSH6 would be as effective as a four-antibody panel in detecting DNA MMR gene protein LOE. This study has implications for significant cost cutting and improved efficiency in detection of DNA MMR gene protein LOE in LS.

Mek1 stabilizes Hop1-Thr318 phosphorylation to promote interhomolog recombination and checkpoint responses during yeast meiosis.

Red1, Hop1 and Mek1 are three yeast meiosis-specific chromosomal proteins that uphold the interhomolog (IH) bias of meiotic recombination. Mek1 is also an effector protein kinase in a checkpoint that responds to aberrant DNA and/or axis structure. The activation of Mek1 requires Red1-dependent Hop1-Thr(T)318 phosphorylation, which is mediated by Mec1 and Tel1, the yeast homologs of the mammalian DNA damage sensor kinases ATR and ATM. As the ectopic expression of Mek1-glutathione S-transferase (GST) was shown to promote IH recombination in the absence of Mec1/Tel1-dependent checkpoint function, it was proposed that Mek1 might play dual roles during meiosis by directly phosphorylating targets that are involved in the recombination checkpoint. Here, we report that Mek1 has a positive feedback activity in the stabilization of Mec1/Tel1-mediated Hop1-T318 phosphorylation against the dephosphorylation mediated by protein phosphatase 4. Our results also reveal that GST-Mek1 or Mek1-GST further increases Hop1-T318 phosphorylation. This positive feedback function of Mek1 is independent of Mek1's kinase activity, but dependent on Mek1's forkhead-associated (FHA) domain and its arginine 51 residue. Arginine 51 directly mediates the interaction of Mek1-FHA and phosphorylated Hop1-T318. We suggest that the Hop1-Mek1 interaction is similar to the Rad53-Dun1 signaling pathway, which is mediated through the interaction of phosphorylated Rad53 and Dun1-FHA.

The prognostic impact of O6-methylguanine DNA methyltransferase and epidermal growth factor receptor expressions on primary gliosarcoma: a clinicopathologic and immunohistochemical study of seven cases at a single institution.

CONTEXT: Gliosarcoma is an uncommon variant of glioblastoma characterized by a biphasic tissue pattern of glial and mesenchymal differentiation. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from O6-guanine in DNA. Lack of MGMT protein expression immunohistochemically is related to drug responses in patients of malignant glioma treated with alkylating agents. Epidermal growth factor receptor (EGFR) is the most frequently amplified gene in glioblastoma and associated with tumor invasiveness, angiogenesis, poor survival, and resistance to radiation therapy. AIMS: To elucidate the relationship between the statuses of the MGMT as well as EGFR proteins and the prognosis. The study was undertaken on samples received at the Department of Pathology from 2003 to 2009. MATERIALS AND METHODS: Clinicopathologic and immunohistochemical study of seven cases was performed. RESULTS: This series included three men and four women with a mean age of 49.3 years at first surgery. The median progression-free survival (PFS) was 22.2 months and 8.6 months for primary tumors with 0 to 1+ and 2+ to 3+ MGMT staining, respectively; the median overall survival (OS) was 27.5 months and 14.2 months for primary tumors with 0 to 1+ and 2+ to 3+ MGMT staining, respectively. The median PFS was 17.2 months and 11.2 months for primary tumors with 0 to 1+ and 2+ to 3+ EGFR staining, respectively; the median OS was 20.4 months and 17.7 months for primary tumors with 0 to 1+ and 2+ to 3+ EGFR staining, respectively. CONCLUSIONS: The series showed that MGMT and EGFR protein expressions were both unfavorable prognostic factors for patients with gliosarcoma.

Immunohistochemistry for PMS2 and MSH6 alone can replace a four antibody panel for mismatch repair deficiency screening in colorectal adenocarcinoma.

AIMS: Currently, testing for mismatch repair deficiency in colorectal cancers is initiated by performing immunohistochemistry with four antibodies (MLH1, PMS2, MSH2 and MSH6). If any one of these stains is negative the tumour is considered microsatellite unstable and, if clinical circumstances warrant it, the patient is offered genetic testing for Lynch's syndrome. Due to the binding properties of the mismatch repair heterodimer complexes, gene mutation and loss of MLH1 and MSH2 invariably result in the degradation of PMS2 and MSH6, respectively, but the converse is not true. We propose that staining for PMS2 and MSH6 alone will be sufficient to detect all cases of mismatch repair deficiency and should replace routine screening with all four antibodies. METHODS: The electronic database of the department of Anatomical Pathology, Royal North Shore Hospital, Sydney, Australia, was searched for all colorectal carcinomas on which a four panel immunohistochemical microsatellite instability screen was performed. An audit of the slides for concordant loss of MLH1-PMS2 and MSH2-MSH6 was then undertaken. Unusual or discordant cases were reviewed and, in some cases, re-stained to confirm the staining pattern. RESULTS: Of 344 cases of colorectal cancer which underwent four antibody immunohistochemistry, 104 displayed loss of at least one mismatch repair protein. Of these, 100 showed concordant mismatch repair loss (i.e., loss of MLH1 and PMS2 or loss of MSH2 and MSH6). The four discordant cases comprised two single negative cases (1 MSH6 negative/MSH2 positive case, 1 PMS2 negative/MLH1 positive) and two triple negative (both MLH1/PMS2/MSH6 negative). The microsatellite instability (MSI) group showed a relatively high median age (69.3 years) due to the departmental policy of testing all cases with possible MSI morphology regardless of age. CONCLUSIONS: The sensitivity and specificity of a two panel test comprised of PMS2 and MSH6, compared to a four panel test, is 100%. No false negatives or positives were identified. We conclude that the two panel test should replace a four panel protocol for immunohistochemical screening for mismatch repair deficiency.

Differential expression of hMLH1 in sporadic human colorectal cancer tumors and distant metastases.

Somatic defects in the mismatch repair system constitute an important pathway in colorectal carcinogenesis. We have examined the expression of mismatch repair proteins in sporadic stage IV colorectal tumors and their derived metastases. Sporadic tumors were further examined for differences in expression between the tumor transition zone and the invasive front. Expression of hMSH2, hMLH1, and hPMS2 was screened immunohistochemically in 92 stage IV tumors and derived liver metastases. In cases with loss of mismatch repair protein expression, lymph node metastases were also examined. Clinicopathological parameters and Ki-67 staining indexes were evaluated and compared. Four tumors displayed a complete loss of hMLH1/hPMS2 expression at the transition zone; however, three of these expressed both proteins at the invasive front and in liver and lymph node metastases. A further four were predominantly hMLH1/hPMS2 negative at the transition zone, but with distinct subclones of hMLH1/hPMS2-expressing cells at the transition zone. All of these tumors expressed hMLH1/hPMS2 at the invasive front and in liver metastases, with three also expressing hMLH/hPMS2 in lymph node metastases. No significant difference in the proliferative index was observed for the hMLH1/hPMS2-compromised group. In stage IV tumors re-expression of hMLH1/hPMS2 occurred, leading to different patterns of expression within the primary tumor and between tumor and metastases. This may have functional importance for the chemosensitivity of metastases compared to the primary tumor.

DNA double strand break repair defects, primary immunodeficiency disorders, and 'radiosensitivity'.

PURPOSE OF REVIEW: It is important to assess 'radiosensitivity' in patients suspected of immunodeficiency because underlying DNA double strand break (DSB) repair defects have considerable impact on V(D)J recombination, class switching and lymphocyte maturation, leading to increased infections and cancer risk. In addition, the phenotype of 'radiosensitivity' may identify patients with increased toxicity to radiation and chemotherapeutic agents and could impact upon their preparation for stem cell transplantation. To date, the gold standard for evaluating 'radiosensitivity' has been the colony-survival assay (CSA), which reflects the efficiency of DNA repair of DSBs as it impacts upon replication and cell survival. Other methods measure other aspects of DNA repair; however, their limited specificity often leads to false negatives for predicting 'radiosensitivity', especially clinical radiosensitivity. Lastly, clinical awareness of an overarching syndrome of DSB repair disorders, XCIND, could help to raise diagnostic levels of suspicion and, thereby, identify additional patients with new forms of immunodeficiency, cancer susceptibility and radiosensitivity. RECENT FINDINGS: Within the past year, three new radiosensitivity disorders of DSB repair have been described, involving deficiencies of RNF168, RAD50, and DNA-PKcs. These are truly translational advances because they validate laboratory models and allow new patients to be identified. SUMMARY: Recognizing compromised genome stability is important but difficult. We review the evidence for correlations between DSB repair, abnormal colony formation, clinical radiosensitivity and other laboratory methods.

Two Prp19-like U-box proteins in the MOS4-associated complex play redundant roles in plant innate immunity.

Plant Resistance (R) proteins play an integral role in defense against pathogen infection. A unique gain-of-function mutation in the R gene SNC1, snc1, results in constitutive activation of plant immune pathways and enhanced resistance against pathogen infection. We previously found that mutations in MOS4 suppress the autoimmune phenotypes of snc1, and that MOS4 is part of a nuclear complex called the MOS4-Associated Complex (MAC) along with the transcription factor AtCDC5 and the WD-40 protein PRL1. Here we report the immuno-affinity purification of the MAC using HA-tagged MOS4 followed by protein sequence analysis by mass spectrometry. A total of 24 MAC proteins were identified, 19 of which have predicted roles in RNA processing based on their homology to proteins in the Prp19-Complex, an evolutionarily conserved spliceosome-associated complex containing homologs of MOS4, AtCDC5, and PRL1. Among these were two highly similar U-box proteins with homology to the yeast and human E3 ubiquitin ligase Prp19, which we named MAC3A and MAC3B. MAC3B was recently shown to exhibit E3 ligase activity in vitro. Through reverse genetics analysis we show that MAC3A and MAC3B are functionally redundant and are required for basal and R protein-mediated resistance in Arabidopsis. Like mos4-1 and Atcdc5-1, mac3a mac3b suppresses snc1-mediated autoimmunity. MAC3 localizes to the nucleus and interacts with AtCDC5 in planta. Our results suggest that MAC3A and MAC3B are members of the MAC that function redundantly in the regulation of plant innate immunity.