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1
Van den Eynde, B., O. Peeters, O. De Backer, B. Gaugler, S. Lucas, and T. Boon. "A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma." Journal of Experimental Medicine 182, no. 3 (September 1, 1995): 689–98. http://dx.doi.org/10.1084/jem.182.3.689.
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Human melanoma MZ2-MEL expresses several distinct antigens that are recognized by autologous cytolytic T lymphocytes (CTL). Some of these antigens are encoded by genes MAGE-1, MAGE-3, and BAGE, which are expressed in a large fraction of tumors of various histological types but are silent in normal adult tissues with the exception of testis. We report here the identification of the gene coding for MZ2-F, another antigen recognized by autologous CTL on MZ2-MEL cells. This gene, which was named GAGE-1, is not related to any presently known gene. It belongs to a family of genes that are expressed in a variety of tumors but not in normal tissues, except for the testis. Antigenic peptide YRPRPRRY, which is encoded by GAGE-1, is recognized by anti-MZ2-F CTL on class I molecule HLA-Cw6. The two genes of the GAGE family that code for this peptide, namely GAGE-1 and GAGE-2, are expressed in a significant proportion of melanomas (24%), sarcomas (25%), non-small cell lung cancers (19%), head and neck tumors (19%), and bladder tumors (12%). About 50% of melanoma patients carry on their tumor at least one of the presently defined antigens encoded by the MAGE, BAGE, and GAGE genes.
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Ulloa-Montoya, Fernando, Jamila Louahed, Benjamin Dizier, Olivier Gruselle, Bart Spiessens, Frédéric F. Lehmann, Stefan Suciu, et al. "Predictive Gene Signature in MAGE-A3 Antigen-Specific Cancer Immunotherapy." Journal of Clinical Oncology 31, no. 19 (July 1, 2013): 2388–95. http://dx.doi.org/10.1200/jco.2012.44.3762.
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Purpose To detect a pretreatment gene expression signature (GS) predictive of response to MAGE-A3 immunotherapeutic in patients with metastatic melanoma and to investigate its applicability in a different cancer setting (adjuvant therapy of resected early-stage non–small-cell lung cancer [NSCLC]). Patients and Methods Patients were participants in two phase II studies of the recombinant MAGE-A3 antigen combined with an immunostimulant (AS15 or AS02B). mRNA from melanoma biopsies was analyzed by microarray analysis and quantitative polymerase chain reaction. These results were used to identify and cross-validate the GS, which was then applied to the NSCLC data. Results In the patients with melanoma, 84 genes were identified whose expression was potentially associated with clinical benefit. This effect was strongest when the immunostimulant AS15 was included in the immunotherapy (hazard ratio [HR] for overall survival, 0.37; 95% CI, 0.13 to 1.05; P = .06) and was less strong with the other immunostimulant AS02B (HR, 0.84; 95% CI, 0.36 to 1.97; P = .70). The same GS was then used to predict the outcome for patients with resected NSCLC treated with MAGE-A3 plus AS02B; actively treated GS-positive patients showed a favorable disease-free interval compared with placebo-treated GS-positive patients (HR, 0.42; 95% CI, 0.17 to 1.03; P = .06), whereas among GS-negative patients, no such difference was found (HR, 1.17; 95% CI, 0.59 to 2.31; P = .65). The genes identified were mainly immune related, involving interferon gamma pathways and specific chemokines, suggesting that their pretreatment expression influences the tumor's immune microenvironment and the patient's clinical response. Conclusion An 84-gene GS associated with clinical response for MAGE-A3 immunotherapeutic was identified in metastatic melanoma and confirmed in resected NSCLC.
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Huang, Lan-Qing, Francis Brasseur, Alfonso Serrano, Etienne De Plaen, Pierre van der Bruggen, Thierry Boon, and Aline Van Pel. "Cytolytic T Lymphocytes Recognize an Antigen Encoded by MAGE-A10 on a Human Melanoma." Journal of Immunology 162, no. 11 (June 1, 1999): 6849–54. http://dx.doi.org/10.4049/jimmunol.162.11.6849.
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Abstract From melanoma patient LB1751, cytolytic T lymphocytes (CTL) were generated that lysed specifically autologous tumor cells. To establish whether these CTL recognized one of the Ags that had previously been defined, a CTL clone was stimulated with cells expressing various MAGE genes. It produced TNF upon stimulation with target cells expressing MAGE-A10. The Ag was found to be nonapeptide GLYDGMEHL (codons 254–262), which is presented by HLA-A2.1. This is the first report on the generation of anti-MAGE CTL by autologous mixed lymphocyte-tumor cell culture (MLTC) from a melanoma patient other than patient MZ2, from whom the first MAGE gene was identified. MAGE genes are expressed in many tumors but not by normal tissues except male germline cells and placenta, which do not express HLA molecules. Therefore, the identification of an antigenic peptide derived from MAGE-A10 adds to the repertoire of tumor-specific shared Ags available for anti-tumoral vaccination trials.
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Miyashiro, Isao, Christine Kuo, Kelly Huynh, Aritoshi Iida, Donald Morton, Anton Bilchik, Armando Giuliano, and Dave S. B. Hoon. "Molecular Strategy for Detecting Metastatic Cancers with Use of Multiple Tumor-specific MAGE-A Genes." Clinical Chemistry 47, no. 3 (March 1, 2001): 505–12. http://dx.doi.org/10.1093/clinchem/47.3.505.
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Abstract Background: The human melanoma-associated antigen family A (MAGE-A) has high specificity and expression in various malignancies, but individual family members are expressed at low frequency in any one particular type of cancer. We therefore developed a method to detect mRNAs from multiple MAGE-A genes in a single reaction. Methods: Universal MAGE-A (uMAGE-A) primers and probe were designed to reverse-transcribe, amplify, and detect by electrochemiluminescence (ECL) MAGE-A mRNAs on the Origen Analyzer. The assay was performed on total RNA of melanoma (n = 9 cell lines and 24 tumors), breast cancer (n = 7 and 26), and colorectal cancer (CRC; n = 5 and 12). We also evaluated blood from melanoma (n = 50), breast cancer (n = 16), and CRC (n = 21) patients. Results: The uMAGE-A mRNA was detectable in 0.01–1 ng of cell line RNA. The identity of the uMAGE-A cDNA products was confirmed by sequencing and polyacrylamide gel electrophoresis. The uMAGE-A assay increased detection of melanoma, breast cancer, and CRC tumor by 13%, 31%, and 25%, respectively, compared with a MAGE-A1 assay, and by 17%, 19%, and 25%, respectively, compared with a MAGE-A3 assay. The uMAGE-A assay detected circulating tumor cells in the blood of melanoma (24%), breast cancer (25%), and CRC (29%) patients. Conclusions: The uMAGE-A reverse transcription-PCR/ECL assay provides a practical and sensitive approach for detection of various metastatic cancers in tissues and blood.
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Baurain, J., M. Stas, B. Neyns, G. Schuler, T. Velu, K. Thielemans, N. Van Baren, T. Dorval, M. Marchand, and P. Coulie. "Comparing immunogenicities of tumor-specific antigens administered as therapeutic vaccines in metastatic melanoma patients." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 3003. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.3003.
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3003 Background: Therapeutic vaccination of metastatic melanoma patients with detectable disease is followed by some tumor regression in only about 10% of the patients, with no clear difference observed between studies carried out with various tumor-specific antigens and vaccination modalities. For antigenic peptide MAGE-A3168–176, presented by HLA-A1 molecules and administered as peptide alone or recombinant ALVAC poxvirus, anti-vaccine T lymphocyte (CTL) responses have been observed in no progressor patient and in only half of the regressors, suggesting a poor immunogenicity of these vaccines. Methods and Results: We compiled anti-vaccine CTL responses measured in the blood of 202 metastatic melanoma patients vaccinated with various associations of 10 different tumor antigens administered as peptides, alone or with adjuvant, recombinant ALVAC poxvirus, or peptide-pulsed dendritic cells. Blood lymphocytes collected before and after vaccination were all analyzed with the same method involving in vitro restimulation in limiting dilution condition followed by labeling with tetramers for each antigen. A CTL response was deemed to have occurred if the CTL frequency increased by at least 10 times, and if the pre-vaccination frequency was lower than 2 x 10-6 of the CD8 cells. No responses were detected against peptides MAGE- A4230–239 (0/26) and MAGE-C2336–344 (0/22). Some were observed against MAGE-A3168–176 (11/81), MAGE-A1278–286 (2/22), MAGE-A3112–120 (2/55), and MAGE-A10254–262 (2/35). Responses were frequently found against NY-ESO-1157–165 (10/19), GnTVVLPDVFIRC (18/73), gp100209–217 (21/33), or Tyrosinase369–377 (11/59). For the latter four antigens, there was no correlation between the occurrence of CTL responses and that of tumor regressions. Neither did we find a correlation between the CTL responses and the expression of the antigen-encoding genes in pre- vaccination tumor samples. Conclusions: These results suggest that some of the antigenic peptides that are commonly used in melanoma vaccines are more immunogenic than others but do not induce more tumor regressions. Therefore, inducing strong CTL responses against these immunogenic peptides is probably not the most appropriate endpoint of future vaccine trials. No significant financial relationships to disclose.
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Florke Gee, Rebecca R., Helen Chen, Anna K. Lee, Christina A. Daly, Benjamin A. Wilander, Klementina Fon Tacer, and Patrick Ryan Potts. "Emerging roles of the MAGE protein family in stress response pathways." Journal of Biological Chemistry 295, no. 47 (September 13, 2020): 16121–55. http://dx.doi.org/10.1074/jbc.rev120.008029.
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The melanoma antigen (MAGE) proteins all contain a MAGE homology domain. MAGE genes are conserved in all eukaryotes and have expanded from a single gene in lower eukaryotes to ∼40 genes in humans and mice. Whereas some MAGEs are ubiquitously expressed in tissues, others are expressed in only germ cells with aberrant reactivation in multiple cancers. Much of the initial research on MAGEs focused on exploiting their antigenicity and restricted expression pattern to target them with cancer immunotherapy. Beyond their potential clinical application and role in tumorigenesis, recent studies have shown that MAGE proteins regulate diverse cellular and developmental pathways, implicating them in many diseases besides cancer, including lung, renal, and neurodevelopmental disorders. At the molecular level, many MAGEs bind to E3 RING ubiquitin ligases and, thus, regulate their substrate specificity, ligase activity, and subcellular localization. On a broader scale, the MAGE genes likely expanded in eutherian mammals to protect the germline from environmental stress and aid in stress adaptation, and this stress tolerance may explain why many cancers aberrantly express MAGEs. Here, we present an updated, comprehensive review on the MAGE family that highlights general characteristics, emphasizes recent comparative studies in mice, and describes the diverse functions exerted by individual MAGEs.
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Park, Myeng Sun, Jong Wook Park, Chang Ho Jeon, Kang Dae Lee, and Hee Kyung Chang. "Expression of Melanoma Antigen-Encoding Genes (MAGE) by Common Primers for MAGE-A1 to -A6 in Colorectal Carcinomas Among Koreans." Journal of Korean Medical Science 17, no. 4 (2002): 497. http://dx.doi.org/10.3346/jkms.2002.17.4.497.
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Lin, Yuquan, Ti Wen, Xianyi Meng, Zhenzhou Wu, Liqing Zhao, Puyue Wang, Zhangyong Hong, and Zhinan Yin. "The mouse Mageb18 gene encodes a ubiquitously expressed type I MAGE protein and regulates cell proliferation and apoptosis in melanoma B16-F0 cells." Biochemical Journal 443, no. 3 (April 16, 2012): 779–88. http://dx.doi.org/10.1042/bj20112054.
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Although many cancer vaccines have been developed against type I MAGE (melanoma antigen) genes owing to their shared tumour-specific expression properties, studies about their expression and functions are relatively limited. In the present study, we first identify a non-testis-specific type I MAGE gene, Mageb18 (melanoma antigen family B 18). Mouse Mageb18 is also expressed in digestion- and immune-related tissues as well as testis, and its expression in testis is age-dependent. Mageb18 is expressed in many mouse-derived cell lines, and DNA demethylation and histone acetylation mediate the reactivation of Mageb18 in Mageb18-negtive H22 and C6 cells. We also show that mouse Mageb18 encodes a 46 kDa protein which is predominantly localized in the cytoplasm. In testis, the endogenous MAGEB18 protein is mainly expressed in proliferative spermatogonia and primary and secondary spermatocytes, but less so in spermatids. Finally, we demonstrate that knockdown of MAGEB18 inhibits the growth of B16-F0 cells and induces apoptosis, which correlates with increased levels of TP53 (tumour protein 53), p21, Bax and caspase 3. The results of the present study thus uncover an important phenomenon that the expression of certain type I MAGE genes, at least for Mageb18, is non-testis-specific. Although they can regulate various malignant phenotypes of cancer cells, it is necessary to study further their expression pattern in normal tissues before using them to develop more effective and safer cancer vaccines.
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Ries, Jutta, Nur Mollaoglu, Takeshi Toyoshima, Eleftherios Vairaktaris, Friedrich W. Neukam, Sabine Ponader, and Emeka Nkenke. "A novel Multiple-Marker Method for the Early Diagnosis of Oral Squamous Cell Carcinoma." Disease Markers 27, no. 2 (2009): 75–84. http://dx.doi.org/10.1155/2009/510124.
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Objective: Melanoma associated antigens-A (MAGE-A) expression is highly specific to cancer cells. Thus, they can be the most suitable targets for the diagnosis of malignancy. The aim of this study was to evaluate the sensitivity of multiple MAGE-A expression analysis for the diagnosis of oral squamous cell carcinoma (OSCC).Methods: Total of 70 OSSC and 20 normal oral mucosal (NOM) samples of otherwise healthy volunteers were examined for the expression of 10 different single antigens out of 12 different MAGE-A subtypes by highly sensitive reverse transcriptase polymerase chain reaction (RT-PCR) methods. The results were correlated to clinicopathological parameters of tumor samples.Results: Expression of MAGE-A was restricted to OSCC. The expression frequency of single antigen was between 10% and 55%. However, expression rate was increased up to 93% by the elevated number of genes examined. A significant correlation was found between the expression of MAGE-A and malignancy (p = 0.0001). In addition, multiple MAGE-A detection has also correlated to the incidence of lymph node metastasis, grading and advanced clinical stages.Conclusions: Analysis of multiple MAGE-A expression is more sensitive than the analysis of a single MAGE-A for the diagnostic evaluation of OSCC. Multiple MAGE-A expression analysis may be a very sensitive method to be used for the diagnosis even in the early stage of OSCC.
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Fon Tacer, Klementina, Marhiah C. Montoya, Melissa J. Oatley, Tessa Lord, Jon M. Oatley, Jonathon Klein, Ramya Ravichandran, et al. "MAGE cancer-testis antigens protect the mammalian germline under environmental stress." Science Advances 5, no. 5 (May 2019): eaav4832. http://dx.doi.org/10.1126/sciadv.aav4832.
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Ensuring robust gamete production even in the face of environmental stress is of utmost importance for species survival, especially in mammals that have low reproductive rates. Here, we describe a family of genes called melanoma antigens (MAGEs) that evolved in eutherian mammals and are normally restricted to expression in the testis (http://MAGE.stjude.org) but are often aberrantly activated in cancer. Depletion of Mage-a genes disrupts spermatogonial stem cell maintenance and impairs repopulation efficiency in vivo. Exposure of Mage-a knockout mice to genotoxic stress or long-term starvation that mimics famine in nature causes defects in spermatogenesis, decreased testis weights, diminished sperm production, and reduced fertility. Last, human MAGE-As are activated in many cancers where they promote fuel switching and growth of cells. These results suggest that mammalian-specific MAGE genes have evolved to protect the male germline against environmental stress, ensure reproductive success under non-optimal conditions, and are hijacked by cancer cells.
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Kasuga, Chinatsu, Yukiko Nakahara, Shigeo Ueda, Cynthia Hawkins, Michael D. Taylor, Christian A. Smith, and James T. Rutka. "Expression of MAGE and GAGE genes in medulloblastoma and modulation of resistance to chemotherapy." Journal of Neurosurgery: Pediatrics 1, no. 4 (April 2008): 305–13. http://dx.doi.org/10.3171/ped/2008/1/4/305.
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Object Cancer testis antigens (CTAs) were initially identified by their ability to elicit autologous T-cell–mediated immune responses in patients with melanoma. The CTA genes are widely expressed in a variety of human cancers, such as melanoma, breast cancer, lung cancer, esophageal cancer, and hepatocellular carcinoma; however, their expression in pediatric brain tumors, such as medulloblastoma (MB), has not been the subject of in-depth analysis. The MAGE proteins are members of the CTA family and have been shown to correlate with tumor development, aggressive clinical course, or resistance to chemotherapeutic agents. The authors undertook this study to examine the expression and role of MAGE proteins in human MB cell lines and specimens. Methods From a transcriptional profiling study in which 47,000 genes in MB cell lines were examined, the authors identified members of the MAGE and GAGE families as being highly expressed. A series of MB tumors was examined using both immunohistochemistry and Western blot analysis with antibodies to the MAGE-A family, MAGE-A1, and GAGE proteins. Results Western blot analysis showed expression of these 3 proteins (MAGE-A family, MAGE-A1, and GAGE) in 62, 46, and 84%, respectively, of MB specimens examined. In addition, a correlation was observed between the expression of MAGE and GAGE genes and resistance of MB cells to chemotherapeutic agents. The functional significance of this correlation was examined in MAGE knockdown studies, and increased drug-induced cytotoxicity was observed in UW426 MB cells following treatment with chemotherapeutic drugs. Cleaved caspase-3 was found in UW426/MAGE small interfering (si)RNA–inhibited cells treated with cisplatin, but not in UW426 cells treated with cisplatin alone at the same concentration. Conclusions These data show that MAGE and GAGE family members are expressed in MB cell lines and specimens, and that inhibition of MAGE and GAGE genes by siRNA increases apoptosis of MB cells and sensitizes them to certain chemotherapeutic agents such as cisplatin and etoposide.
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Wu, Yunyan, Meixiang Sang, Fei Liu, Jiandong Zhang, Weijing Li, Zhenhua Li, Lina Gu, Yang Zheng, Juan Li, and Baoen Shan. "Epigenetic modulation combined with PD-1/PD-L1 blockade enhances immunotherapy based on MAGE-A11 antigen-specific CD8+T cells against esophageal carcinoma." Carcinogenesis 41, no. 7 (June 12, 2020): 894–903. http://dx.doi.org/10.1093/carcin/bgaa057.
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Abstract Cancer testis antigens (CTAs) are promising targets for T cell-based immunotherapy and studies have shown that certain CT genes are epigenetically depressed in cancer cells through DNA demethylation. Melanoma-associated antigen A11 (MAGE-A11) is a CTA that is frequently expressed in esophageal cancer and is correlated with a poor esophageal cancer prognosis. Consequently, MAGE-A11 is a potential immunotherapy target. In this study, we evaluated MAGE-A11 expression in esophageal cancer cells and found that it was downregulated in several tumor cell lines, which restricted the effect of immunotherapy. Additionally, the specific recognition and lytic potential of cytotoxic T lymphocytes (CTLs) derived from the MAGE-A11 was determined. Specific CTLs could kill esophageal cancer cells expressing MAGE-A11 but rarely lysed MAGE-A11-negative tumor cells. Therefore, induction of MAGE-A11 expression is critical for CTLs recognition and lysis of esophageal cancer cells. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine increased MAGE-A11 expression in esophageal cancer cells and subsequently enhanced the cytotoxicity of MAGE-A11-specific CD8+T cells against cancer cell lines. Furthermore, we found that PD-L1 expression in esophageal cancer cells affected the antitumor function of CTLs. programmed death-1 (PD-1)/PD-L1 blockade could increase the specific CTL-induced lysis of HLA-A2+/MAGE-A11+ tumor cell lines treated with 5-aza-2′-deoxycytidine. These findings indicate that the treatment of tumor cells with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine augments MAGE-A11 expression in esophageal cancer cells. The combination of epigenetic modulation by 5-aza-2′-deoxycytidine and PD-1/PD-L1 blockade may be useful for T cell-based immunotherapy against esophageal cancer.
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Valiño-Rivas, Lara, Leticia Cuarental, Mateo Agustin, Holger Husi, Pablo Cannata-Ortiz, Ana B. Sanz, Harald Mischak, Alberto Ortiz, and Maria Dolores Sanchez-Niño. "MAGE genes in the kidney: identification of MAGED2 as upregulated during kidney injury and in stressed tubular cells." Nephrology Dialysis Transplantation 34, no. 9 (December 11, 2018): 1498–507. http://dx.doi.org/10.1093/ndt/gfy367.
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Abstract Background Mutations in Melanoma Antigen-encoding Gene D2 (MAGED2) promote tubular dysfunction, suggesting that MAGE proteins may play a role in kidney pathophysiology. We have characterized the expression and regulation of MAGE genes in normal kidneys and during kidney disease. Methods The expression of MAGE genes and their encoded proteins was explored by systems biology multi-omics (kidney transcriptomics and proteomics) in healthy adult murine kidneys and following induction of experimental acute kidney injury (AKI) by a folic acid overdose. Changes in kidney expression during nephrotoxic AKI were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry. Factors regulating gene expression were studied in cultured tubular cells. Results Five MAGE genes (MAGED1, MAGED2, MAGED3, MAGEH1, MAGEE1) were expressed at the mRNA level in healthy adult mouse kidneys, as assessed by RNA-Seq. Additionally, MAGED2 was significantly upregulated during experimental AKI as assessed by array transcriptomics. Kidney proteomics also identified MAGED2 as upregulated during AKI. The increased kidney expression of MAGED2 mRNA and protein was confirmed by qRT-PCR and western blot, respectively, in murine folic acid- and cisplatin-induced AKI. Immunohistochemistry located MAGED2 to tubular cells in experimental and human kidney injury. Tubular cell stressors [serum deprivation and the inflammatory cytokine tumour necrosis factor-like weak inducer of apoptosis (TWEAK)] upregulated MAGED2 in cultured tubular cells. Conclusions MAGED2 is upregulated in tubular cells in experimental and human kidney injury and is increased by stressors in cultured tubular cells. This points to a role of MAGED2 in tubular cell injury during kidney disease that should be dissected by carefully designed functional approaches.
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Oh, Chaeun, Hwa-Ryeon Kim, Sumin Oh, Je Yeong Ko, Yesol Kim, Keunsoo Kang, Young Yang, et al. "Epigenetic Upregulation of MAGE-A Isoforms Promotes Breast Cancer Cell Aggressiveness." Cancers 13, no. 13 (June 25, 2021): 3176. http://dx.doi.org/10.3390/cancers13133176.
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After decades-long efforts to diagnose and treat breast cancer, the management strategy that has proved most successful to date is molecular-subtype-specific inhibition of the hormone receptors and HER2 that are expressed by individual cancers. Melanoma-associated antigen (MAGE) proteins comprise >40 highly conserved members that contain the MAGE homology domain. They are often overexpressed in multiple cancers and contribute to cancer progression and metastasis. However, it remains unclear whether the biological activity arising from MAGE gene expression is associated with breast cancer subtypes. In this study, we analyzed the RNA-sequencing (RNA-seq) data of 70 breast cancer cell lines and found that MAGEA12 and MAGEA3 were highly expressed in a subset of these lines. Significantly, MAGEA12 and MAGEA3 expression levels were independent of hormone receptor expression levels but were closely associated with markers of active histone modifications. This indicates that overexpression of these genes is attributable to epigenetic deregulation. RNA-seq of MAGEA12-depleted cells was then used to identify 382 candidate targets of MAGEA12 that were downregulated by MAGEA12 depletion. Furthermore, our gain-of-function experiments showed that MAGEA12 overexpression promoted aggressive behaviors of malignant breast cancer cells, including enhancing their cell migration and invasion. These changes were associated with increased epigenetic deregulation of the MAGEA12 signature genes. Thus, MAGEA12 may play an important role in breast cancer malignancy. Taken together, our findings suggest that MAGEA12 could be a promising therapeutic target in breast cancer, and its overexpression and epigenetic changes could serve as subtype classification biomarkers.
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Faiena, Izak, Samuel Aaron Funt, Stephanie H. Astrow, Tanya B. Dorff, Sabina Adhikary, Tristan Gorgan, Jonathan E. Rosenberg, David Elashoff, Allan J. Pantuck, and Alexandra Drakaki. "Differential expression of melanoma-associated antigen A3/6 and associated immune molecules prior to and post treatment with immune checkpoint inhibitors (ICI) in patients with mUC." Journal of Clinical Oncology 38, no. 6_suppl (February 20, 2020): 433. http://dx.doi.org/10.1200/jco.2020.38.6_suppl.433.
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433 Background: MAGE is an attractive target for IO given its expression restricted to carcinomas and the testes. Engineered T-cell targeting MAGE A3/6 has shown promise. However, successful treatment is dependent on circumventing the potentially hostile immune microenvironment in metastatic tumors. In this study, we aim to assess the effects of ICI on the immune microenvironment in patients who progressed on ICI. Methods: We obtained FFPE tissue from 16 patients with mUC across 3 institutions. Samples from the primary or metastatic tumor prior to treatment with PD1/PD-L1 inhibitors (ICI) were paired with samples from metastatic sites post-treatment. Differential mRNA expression was assessed using NanoString PanCancerIO360 panel for the main genes of interest ( MAGEA3/6, CD274, HLADP, HLAA, B2M and PDCD1) in addition to immune profiling of the tumor samples. Also, IHC was done on the paired samples to assess DE for the genes of interest using an H-score analyzed via the paired T-test. p-values <0.05 were considered significant and FDR corrected. Results: MAGEA3/A6 mRNA expression was unchanged in the post-treatment samples. In addition, time to progression was not influenced by the mRNA DE. Similarly, IHC expression was not decreased post-treatment. The other genes of interest were also similarly expressed pre and post treatment. Immune cell localization genes CPA3,TPSAB1/B2, HDC, MS4A2 were significantly downregulated post treatment. Genes implicated in cancer cell killing ( MX1,KIT) were similarly downegulated whereas CD276, TLR1 were upregulated. Expression in the post treatment tissue of PTPN1, CES3, ACVR1C, and BAD were associated with increased progression. LY9, PTPRC, CTLA4, HLA-DMA, IL10RA, HLA-DMB, ITGAL, CD48 were associated with decreased progression. Conclusions: Prior ICI treatment does not decrease expression of MAGE A3/6, suggesting that salvage treatments targeting this antigen remain a viable strategy. Suggestion of immune microenvironment alterations were also noted. More work is necessary to understanding the tumor microenvironment in order to design rational treatment combinations and sequences.
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Bozkurt, Süreyya, Filiz Yarimcan, Hüseyin Ayhan, Hacer Kotan, Hüma Tuğçe Sezgin, Elif Çınar, Ceren Aynacı, Remzi Okan Akar, and Veysel Sabri Hançer. "Investigation of PTGS2, MAGE-A3, CALR, KRT19 and TMPRSS4 expressions in HCT116 colon cancer and PC3 prostate cancer cell lines." Genetics & Applications 4, no. 2 (December 24, 2020): 37. http://dx.doi.org/10.31383/ga.vol4iss2pp37-42.
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Cancer is a disease arising from DNA alterations that dysregulate gene structure and function. These deregulated genes can also play a role in tumor invasion and metastasis or resistance to treatment. In this study, we determined the gene expression during transcription of PTGS2 (Prostaglandin-endoperoxide synthase 2), MAGE-A3 (Melanoma-associated antigen 3), CALR (Calreticulin), KRT19 (Cytokeratin 19), and TMPRSS4 (Transmembrane protease, serine 4) in HCT116 colon cancer cell line and PC3 prostate cancer cell line. After RNA isolation and cDNA conversion, DNA amplification was performed with Real-Time PCR. We determined the altered transcriptional expression level of those genes. In HCT116 colon cancer cell line, expression of the TMPRSS4 gene, MAGEA3 gene and KRT19 gene was found as increased and expression of the CALR gene and the PTGS2 gene was found as decreased. Especially a 93.70-fold increase in expression of the KRT19 gene was found in HCT116 colon cancer cell line. In PC3 prostate cancer cell lines, TMPRS4 gene expression and MAGEA3 gene expression were found as increased. But there was 50 fold decrease in PTGS2 gene expression.
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Miller, AR, WH McBride, SM Dubinett, GJ Dougherty, JD Thacker, H. Shau, DB Kohn, RC Moen, MJ Walker, and R. Chiu. "Transduction of human melanoma cell lines with the human interleukin-7 gene using retroviral-mediated gene transfer: comparison of immunologic properties with interleukin-2." Blood 82, no. 12 (December 15, 1993): 3686–94. http://dx.doi.org/10.1182/blood.v82.12.3686.3686.
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Abstract Two human melanoma cell lines were transduced with the human interleukin (IL)-7 and IL-2 genes using retroviral-mediated gene transfer. Stable, high-level cytokine expression was achieved. The in vitro growth of transduced tumors was unaltered. Neither of the IL-2- transduced melanoma cell lines grew in athymic mice, whereas one IL-7- transduced melanoma line showed retarded in vivo growth. This is consistent with animal studies suggesting a predominantly T-cell response to IL-7-transduced tumors and a more nonspecific response to IL-2-transduced tumors. Both IL-7- and IL-2-transduced melanoma cell lines could induce cytotoxic lymphocytes in mixed lymphocyte-tumor cultures. The expression of putative melanoma antigens (MAGE)-1 and MAGE-3 was unaltered by cytokine transduction. In one cell line, IL-7 transduction resulted in a marked inhibition of the immunosuppressive peptide transforming growth factor (TGF)beta 1. The results allow a comparison of immunobiologic properties of IL-7- and IL-2-transduced human melanoma cell lines in consideration of their use in genetically engineered tumor vaccines. IL-7 transduction results in stable cytokine expression and phenotypic alterations that appear to be favorable for enhanced immunogenicity and it deserves clinical testing.
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Miller, AR, WH McBride, SM Dubinett, GJ Dougherty, JD Thacker, H. Shau, DB Kohn, RC Moen, MJ Walker, and R. Chiu. "Transduction of human melanoma cell lines with the human interleukin-7 gene using retroviral-mediated gene transfer: comparison of immunologic properties with interleukin-2." Blood 82, no. 12 (December 15, 1993): 3686–94. http://dx.doi.org/10.1182/blood.v82.12.3686.bloodjournal82123686.
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Two human melanoma cell lines were transduced with the human interleukin (IL)-7 and IL-2 genes using retroviral-mediated gene transfer. Stable, high-level cytokine expression was achieved. The in vitro growth of transduced tumors was unaltered. Neither of the IL-2- transduced melanoma cell lines grew in athymic mice, whereas one IL-7- transduced melanoma line showed retarded in vivo growth. This is consistent with animal studies suggesting a predominantly T-cell response to IL-7-transduced tumors and a more nonspecific response to IL-2-transduced tumors. Both IL-7- and IL-2-transduced melanoma cell lines could induce cytotoxic lymphocytes in mixed lymphocyte-tumor cultures. The expression of putative melanoma antigens (MAGE)-1 and MAGE-3 was unaltered by cytokine transduction. In one cell line, IL-7 transduction resulted in a marked inhibition of the immunosuppressive peptide transforming growth factor (TGF)beta 1. The results allow a comparison of immunobiologic properties of IL-7- and IL-2-transduced human melanoma cell lines in consideration of their use in genetically engineered tumor vaccines. IL-7 transduction results in stable cytokine expression and phenotypic alterations that appear to be favorable for enhanced immunogenicity and it deserves clinical testing.
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Bhat, Smitha S., Shreya Das Mahapatra, Sindhu R, Sarana Rose Sommano, and Shashanka K. Prasad. "Virtual Screening and Quantitative Structure–Activity Relationship of Moringa oleifera with Melanoma Antigen A (MAGE-A) Genes against the Therapeutics of Non-Small Cell Lung Cancers (NSCLCs)." Cancers 14, no. 20 (October 15, 2022): 5052. http://dx.doi.org/10.3390/cancers14205052.
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In the last decade, there have been significant advancements in the treatment of non-small cell lung cancer, including remarkable gains in detection, diagnosis, and therapy. The emergence of molecular targeted therapies, immunotherapeutic inhibitors, and antiangiogenesis medicines has largely fueled improvements in combination therapy and systemic treatments, all of which have dramatically ameliorated patient outcomes. The Moringa oleifera bioactive compounds have been effective in the suppression of cancers, making them the therapeutic agents of choice for the current investigation to treat MAGE-A presented in NSCLC. The ligand entrants were screened for their pharmacological properties, and 2,2-diphenyl-1,3-benzodioxole was stipulated as the lead candidate. 2,2-Diphenyl-1,3-benzodioxole exhibited better pharmacological properties and superior binding with branched-chain amino acids, making it an ideal candidate to address MAGE-A. The study concluded that addressing MAGE-A to impede their activity and antigenicity can be exploited as immunotarget(s).
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Lu, Yong-Chen, Linda L. Parker, Tangying Lu, Zhili Zheng, Mary Ann Toomey, Donald E. White, Xin Yao, et al. "Treatment of Patients With Metastatic Cancer Using a Major Histocompatibility Complex Class II–Restricted T-Cell Receptor Targeting the Cancer Germline Antigen MAGE-A3." Journal of Clinical Oncology 35, no. 29 (October 10, 2017): 3322–29. http://dx.doi.org/10.1200/jco.2017.74.5463.
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Purpose Adoptive transfer of genetically modified T cells is being explored as a treatment for patients with metastatic cancer. Most current strategies use genes that encode major histocompatibility complex (MHC) class I–restricted T-cell receptors (TCRs) or chimeric antigen receptors to genetically modify CD8+ T cells or bulk T cells for treatment. Here, we evaluated the safety and efficacy of an adoptive CD4+ T-cell therapy using an MHC class II–restricted, HLA-DPB1*0401–restricted TCR that recognized the cancer germline antigen, MAGE-A3 (melanoma-associated antigen-A3). Patients and Methods Patients received a lymphodepleting preparative regimen, followed by adoptive transfer of purified CD4+ T cells, retrovirally transduced with MAGE-A3 TCR plus systemic high-dose IL-2. A cell dose escalation was conducted, starting at 107 total cells and escalating at half-log increments to approximately 1011 cells. Nine patients were treated at the highest dose level (0.78 to 1.23 × 1011 cells). Results Seventeen patients were treated. During the cell dose-escalation phase, an objective complete response was observed in a patient with metastatic cervical cancer who received 2.7 × 109 cells (ongoing at ≥ 29 months). Among nine patients who were treated at the highest dose level, objective partial responses were observed in a patient with esophageal cancer (duration, 4 months), a patient with urothelial cancer (ongoing at ≥ 19 months), and a patient with osteosarcoma (duration, 4 months). Most patients experienced transient fevers and the expected hematologic toxicities from lymphodepletion pretreatment. Two patients experienced transient grade 3 and 4 transaminase elevations. There were no treatment-related deaths. Conclusion These results demonstrate the safety and efficacy of administering autologous CD4+ T cells that are genetically engineered to express an MHC class II–restricted antitumor TCR that targets MAGE-A3. This clinical trial extends the reach of TCR gene therapy for patients with metastatic cancer.
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Mollaoglu, Nur, Eleftherios Vairaktaris, Emeka Nkenke, Friedrich W. Neukam, and Jutta Ries. "Expression of MAGE-A12 in Oral Squamous Cell Carcinoma." Disease Markers 24, no. 1 (2008): 27–32. http://dx.doi.org/10.1155/2008/359840.
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Melanoma associated-A antigens (MAGE-A) are silent in normal tissues except testis. However, they are activated in a variety of different tumors. Thus, their expression is highly specific to cancer cells. Reverse transcription-nested polymerase chain reaction (RT-nPCR) is a highly sensitive technique that has been used successfully for the detection ofMAGE genes in tissue samples. The aim of the study is to analyze the expression rate of MAGE-A12 in oral squamous cell carcinoma (OSCC) using a high sensitive RT-nPCR. Total of 57 tissue samples obtained from patients with OSCC and 20 normal oral mucosal (NOM) probes of otherwise healthy volunteers were included to this study. No expression of MAGE-A12 was observed in the non-neoplastic NOM tissues. MAGE-A12 was expressed in 49.1% of the investigated tumor samples. The correlation between malignant lesion and MAGE-A12 detection was significant (p < 0.001). It is concluded that results of this study may indicate MAGE-A12 as a useful additional diagnostic marker especially for the early detection of OSCC distinguishing neoplastic transformation and detection of occult and/or rare disseminated cancer cells. In addition, MAGE-A12 expression in OSCC may also determine a new immunotherapeutic target and might be warranted to develop vaccine for OSCC.
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Takeuchi, Hiroya, Donald L. Morton, Christine Kuo, Roderick R. Turner, David Elashoff, Robert Elashoff, Bret Taback, Akihide Fujimoto, and Dave S. B. Hoon. "Prognostic Significance of Molecular Upstaging of Paraffin-Embedded Sentinel Lymph Nodes in Melanoma Patients." Journal of Clinical Oncology 22, no. 13 (July 1, 2004): 2671–80. http://dx.doi.org/10.1200/jco.2004.12.009.
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PurposeDetection of micrometastases in sentinel lymph nodes (SLNs) is important for accurate staging and prognosis in melanoma patients. However, a significant number of patients with histopathology-negative SLNs subsequently develop recurrent disease. We hypothesized that a quantitative realtime reverse transcriptase polymerase chain reaction (qRT) assay using multiple specific mRNA markers could detect occult metastasis in paraffin-embedded (PE) SLNs to upstage and predict disease outcome.Patients and MethodsqRT was performed on retrospectively collected PE SLNs from 215 clinically node-negative patients who underwent lymphatic mapping and sentinel lymphadenectomy for melanoma and were followed up for at least 8 years. PE SLNs (n = 308) from these patients were sectioned and assessed by qRT for mRNA of four melanoma-associated genes: MART-1 (antigen recognized by T cells-1), MAGE-A3 (melanoma antigen gene-A3 family), GalNAc-T (β1→4-N-acetylgalactosaminyl-transferase), and Pax3 (paired-box homeotic gene transcription factor 3).ResultsFifty-three (25%) patients had histopathology-positive SLNs by hemotoxylin and eosin and/or immunohistochemistry. Of the 162 patients with histopathology-negative SLNs, 48 (30%) had nodes that expressed at least one of the four qRT markers, and these 48 patients also had a significantly increased risk of disease recurrence by a Cox proportional hazards model analysis (P < .0001; risk ratio, 7.48; 95% CI, 3.70 to 15.15). The presence of ≥ one marker in histopathology-negative SLNs was also a significant independent prognostic factor by multivariate analysis for overall survival (P = .0002; risk ratio, 11.42; 95% CI, 3.17 to 41.1).ConclusionMolecular upstaging of PE histopathology-negative SLNs by multiple-marker qRT assay is a significant independent prognostic factor for long-term disease recurrence and overall survival of patients with early-stage melanoma.
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Mikhaylova, I. N., H. M. Treshalina, I. A. Utyashev, M. V. Kiselevsky, A. A. Lushnikova, and I. Zh Shubina. "ANALYSIS OF CANCER-TESTIS ANTIGENS AS POTENTIAL MARKERS FOR DISSEMINATION OF PRIMARY HUMAN SKIN MELANOMA." Siberian journal of oncology 20, no. 3 (June 29, 2021): 98–106. http://dx.doi.org/10.21294/1814-4861-2021-20-2-98-106.
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Purpose of the study: to analyze characteristics of cancer-testis antigens (Ctas) as potential biomarkers for dissemination of primary human skin melanoma (sm).Material and Methods. Recent publications from Pubmed, scopus and elibrary databases were analyzed for the available appropriate literature review. In total, 176 papers reported the description of Ctas and encoding genes and their potential for prognosis of primary sm dissemination. The authors included 52 of them in the given review.Results. Two sections of the paper comprise clinically significant characteristics of Ctas and their genes, including overexpression, which is selective for the heterogeneous tumor cell populations and mediated by humoral and/or cellular immune reactions; the association of tumor process and activation of Cta genes by demethylation of promotor sites, which is correlated with tumor progression; and the conditions required for effective immunotherapy involving Ctas and/or their genes.Conclusion. At present, there are no standards or clinical recommendations for the Cta-based prognosis of the early dissemination of primary skin melanoma. Therefore, it is important to study and analyze the Cta and encoding gene characteristics that reveal the connection between primary sm progression and tumor genesis including the role of circulating tumor cells (ctc), similar to stem cells, which have epithelial-mesenchymal transition (emt) phenotype, for clinical diagnostics of early sm dissemination. As a result of the study, the following Ctas could be considered as significant biomarkers of the early sm dissemination: mage-a1, mage-a4 and ny-eso-1, which expression correlates with the clinical pathological description of the disease progression, as well as with the relapse-free period and overall survival of the patients; magea3, which expression correlates with spag5 activation and Cd8+ t-cell abundance; ssx, a marker for stem cell migration including identification of the cells with emt and/or ctcs; and prame, signaling marker for dissemination of the uveal melanoma.
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Tüting, Thomas, Cara C. Wilson, Dina M. Martin, Yvette L. Kasamon, Jennifer Rowles, Debora I. Ma, Craig L. Slingluff, et al. "Autologous Human Monocyte-Derived Dendritic Cells Genetically Modified to Express Melanoma Antigens Elicit Primary Cytotoxic T Cell Responses In Vitro: Enhancement by Cotransfection of Genes Encoding the Th1-Biasing Cytokines IL-12 and IFN-α." Journal of Immunology 160, no. 3 (February 1, 1998): 1139–47. http://dx.doi.org/10.4049/jimmunol.160.3.1139.
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Abstract DNA-based immunization strategies designed to elicit cellular antitumor immunity offer an attractive alternative to protein- or peptide-based approaches. In the present study we have evaluated the feasibility of DNA vaccination for the induction of CTL reactivity to five different melanoma Ags in vitro. Cultured, monocyte-derived dendritic cells (DC) were transiently transfected with plasmid DNA encoding human MART-1/Melan-A, pMel-17/gp100, tyrosinase, MAGE-1, or MAGE-3 by particle bombardment and used to stimulate autologous PBMC responder T cells. CTL reactivity to these previously identified melanoma Ags was reproducibly generated after two or three stimulations with genetically modified DC. Co-ordinate transfection of two melanoma Ag cDNAs into DC promoted CTL responders capable of recognizing epitopes from both gene products. Coinsertion of genes encoding the Th1-biasing cytokines IL-12 or IFN-α consistently enhanced the magnitude of the resulting Ag-specific CTL reactivity. Importantly, DC transfected with a single melanoma Ag cDNA were capable of stimulating Ag-specific CTL reactivity restricted by multiple host MHC alleles, some of which had not been previously identified. These results support the inherent strengths of gene-based vaccine approaches that do not require prior knowledge of responder MHC haplotypes or of relevant MHC-restricted peptide epitopes. Given previous observations of in situ tumor HLA allele-loss variants, DC gene vaccine strategies may elicit a greater diversity of host therapeutic immunity, thereby enhancing the clinical utility and success of such approaches.
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Reu, Frederic J., Soo In Bae, Leonid Cherkassky, Douglas W. Leaman, Daniel Lindner, Normand Beaulieu, A. Robert MacLeod, and Ernest C. Borden. "Overcoming Resistance to Interferon-Induced Apoptosis of Renal Carcinoma and Melanoma Cells by DNA Demethylation." Journal of Clinical Oncology 24, no. 23 (August 10, 2006): 3771–79. http://dx.doi.org/10.1200/jco.2005.03.4074.
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Epigenetic editing of gene expression by aberrant methylation of DNA may help tumor cells escape attack from the innate and acquired immune systems. Resistance to antiproliferative effects and apoptosis induction by interferons (IFNs) was postulated to result from silencing of IFN response genes by promoter hypermethylation. Treatment of human ACHN renal cell carcinoma (RCC) and A375 melanoma cells with the DNA demethylating nucleoside analog 5-AZA-2′-deoxycytidine (5-AZA-dC) synergistically augmented antiproliferative effects of IFN- alpha (α) 2 and IFN-beta (β). Either 5-AZA-dC or an antisense to DNA methyltransferase 1 (DNMT1) overcame resistance to apoptosis induction by IFNs with up to 85% apoptotic cells resulting from the combinations. No similar potentiation occurred in normal kidney epithelial cells. IFN response genes were augmented more than 10 times in expression by 5-AZA-dC. Demethylation by 5-AZA-dC of the promoter of the prototypic, apoptosis-associated IFN response gene XAF1 was confirmed by methylation-specific polymerase chain reaction. siRNA to XAF1 inhibited IFN-induced apoptosis; conversely, overexpression of XAF1 overcame resistance to apoptosis induction by IFN-β. As occurred with apoptosis-resistant melanoma cells in vitro, tumor growth inhibition in the nude mouse of human A375 melanoma xenografts resulted from treatment with 5-AZA-dC in combination with IFN-β, an effect not resulting from either single agent. The importance of epigenetic remodeling of expression of immune-modifying genes in tumor cells was further suggested by identifying reactivation of the cancer-testis antigens MAGE and RAGE in ACHN cells after DNMT1 depletion. Thus, inhibitors of DNMT1 may have clinical relevance for immune modulation by augmentation of cytokine effects and/or expression of tumor-associated antigens.
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Cai, Pan, Yan Lu, Zhifeng Yin, Xiuhui Wang, Xiaoxiao Zhou, and Zhuokai Li. "Trophinin Is an Important Biomarker and Prognostic Factor in Osteosarcoma: Data Mining from Oncomine and the Cancer Genome Atlas Databases." BioMed Research International 2021 (July 6, 2021): 1–9. http://dx.doi.org/10.1155/2021/6885897.
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Osteosarcoma (OS) is a type of bone malignancy with a high rate of treatment failure. To date, few evident biomarkers for the prognostic significance of OS have been established. Oncomine was used to integrate RNA and DNA-seq data from the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and the published literature. The correlation of the gene Trophinin (TRO) and different types of cancers was generated using the Cancer Cell Line Encyclopedia (CCLE) online tool. Prognostic values of featured Melanoma Antigen Gene (MAGE) members were further assessed by establishing the overall survival using the Kaplan-Meier plotter. Moreover, the online tool, Database for Annotation, Visualization and Integrated Discovery version (DAVID), was used to understand the biological meaning list of the genes. MAGEB10, MAGED2, TRO, MAGEH1, MAGEB18, MAGEB6, MAGEB4, MAGEB1, MAGED4B, MAGED1, MAGEB2, and MAGEB3 were significantly overexpressed in sarcoma. TRO was further demonstrated to be distinctively upregulated in osteosarcoma cell lines and associated with shorter overall survival. TRO may play an important role in the development of OS and may be a promising potential biomarker and prognostic factor.
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Nardiello, Tricia, Anna Mei, Michael Mangone, and Hearn J. Cho. "CT7 (MAGE-C1) Promotes Survival and Interacts with STAT1 and PIASy in Multiple Myeloma Cells." Blood 118, no. 21 (November 18, 2011): 1807. http://dx.doi.org/10.1182/blood.v118.21.1807.1807.
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Abstract Abstract 1807 The type I Melanoma Antigen GEnes MAGE-A3 and CT7 (MAGE-C1) are detected in more than 75% of primary multiple myeloma specimens, and their expression is correlated with proliferation and progression of disease. We previously showed that MAGE-A3 inhibits apoptosis in human myeloma cell lines (HMCL) and primary cells in part through ubiquitinylation of the prototypical tumor suppressor p53, which targets it for proteasomal degradation and inhibits its pro-apoptotic transcriptional program (Nardiello et al, Clin Cancer Res, 2011; 17:4309). However, silencing of MAGE-A3 in HMCL and primary cells that lacked functional p53, either through deletions or mutations, also resulted in apoptosis. Silencing of CT7 alone did not affect survival of HMCL that lacked p53, but it did increase their sensitivity to chemotherapy-induced apoptosis. Many type I MAGE proteins, including MAGE-A3, bind to the RING domain protein TRIM28/Kap1 through their highly conserved MAGE Homology Domain (MHD) to form E3 ubiquitin ligase complexes, but it was unknown if CT7 also associated with TRIM28. These results lead to the hypothesis that CT7 is a survival factor for myeloma cells that can act through p53-independent mechanisms. To investigate the biochemical activity of CT7 and identify non-p53 pathways regulated by type I MAGE in myeloma cells, we analyzed protein-protein interactions with CT7 by two methods. We immunoprecipitated (IP'ed) CT7 from lysates of HMCL followed by sequencing of co-IP'ed proteins by mass spectroscopy and we performed yeast two-hybrid screening with a bait construct containing the MHD of CT7. CT7 was reciprocally co-IP'ed from HMCL lysates with TRIM28, demonstrating that it, too, associated with this RING domain protein. These methods also revealed two novel interactions with CT7. CT7 reciprocally co-IP'ed with STAT1, a transcription factor that plays a critical role in receptor-mediated signaling for cytokines such as interferon α/β. CT7 also interacted with Protein Inhibitor of Activated STAT y (PIASy), a modified RING domain protein that negatively regulates STAT proteins through its Small Ubiquitin-like MOdifier (SUMO) ligase activity, which sequesters target proteins out of the nucleus and into the cytoplasm. Surprisingly, phosphorylated STAT1 (pSTAT1) was detected in lysates from unstimulated HMCL, indicating a tonic level of activation, but most or all of the pSTAT1 was in the cytoplasm, suggesting that its transcriptional activity was being blocked through exclusion from the nucleus. Phospho-STAT1 also appeared to preferentially associate with the CT7/TRIM28 complex. Phospho-STAT1 did not appear to be ubiquitinylated in these HMCL, but SUMO2/3 modification of pSTAT1 was detected. These results suggest that CT7 negatively regulates pSTAT1 activity in HMCL by sequestering the transcription factor out of the nucleus through SUMO modification. This may be a direct result of CT7-mediated SUMOylation in partnership with PIASy, or indirectly due to CT7/TRIM28-mediated ubiquitinylation events that result in activation of a SUMO ligase such as PIASy. These findings suggest the possibility that complexes containing type I MAGE proteins may also have SUMO ligase activity and identify STAT1 as a novel non-p53 biochemical pathway regulated by these genes. Disclosures: No relevant conflicts of interest to declare.
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Thompson, Jeffrey C., Christiana Davis, Charuhas Deshpande, Wei-Ting Hwang, Seth Jeffries, Alexander Huang, Tara C. Mitchell, Corey J. Langer, and Steven M. Albelda. "Gene signature of antigen processing and presentation machinery predicts response to checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma." Journal for ImmunoTherapy of Cancer 8, no. 2 (October 2020): e000974. http://dx.doi.org/10.1136/jitc-2020-000974.
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BackgroundLimited data exist on the role of alterations in HLA Class I antigen processing and presentation machinery in mediating response to immune checkpoint blockade (ICB).MethodsThis retrospective cohort study analyzed transcriptional profiles from pre-treatment tumor samples of 51 chemotherapy-refractory advanced non-small cell lung cancer (NSCLC) patients and two independent melanoma cohorts treated with ICB. An antigen processing machinery (APM) score was generated utilizing eight genes associated with APM (B2M, CALR, NLRC5, PSMB9, PSME1, PSME3, RFX5, and HSP90AB1). Associations were made for therapeutic response, progression-free survival (PFS) and overall survival (OS).ResultsIn NSCLC, the APM score was significantly higher in responders compared with non-responders (p=0.0001). An APM score above the median value for the cohort was associated with improved PFS (HR 0.34 (0.18 to 0.64), p=0.001) and OS (HR 0.44 (0.23 to 0.83), p=0.006). The APM score was correlated with an inflammation score based on the established T-cell-inflamed resistance gene expression profile (Pearson’s r=0.58, p<0.0001). However, the APM score better predicted response to ICB relative to the inflammation score with area under a receiving operating characteristics curve of 0.84 and 0.70 for PFS and OS, respectively. In a cohort of 14 high-risk resectable stage III/IV melanoma patients treated with neoadjuvant anti-PD1 ICB, a higher APM score was associated with improved disease-free survival (HR: 0.08 (0.01 to 0.50), p=0.0065). In an additional independent melanoma cohort of 27 metastatic patients treated with ICB, a higher APM score was associated with improved OS (HR 0.29 (0.09 to 0.89), p=0.044).ConclusionOur data demonstrate that defects in antigen presentation may be an important feature in predicting outcomes to ICB in both lung cancer and melanoma.
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Cho, Hearn Jay, Anna Huo-Chang Mei, Kaity Tung, Jessie Han, Deepak Perumal, Jonathan J. Keats, Daniel Auclair, et al. "MAGE-A3 Promotes Chemotherapy Resistance and Proliferation in Multiple Myeloma through Regulation of BIM and p21Cip1." Blood 132, Supplement 1 (November 29, 2018): 4464. http://dx.doi.org/10.1182/blood-2018-99-118692.
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Abstract The type I Melanoma Antigen Gene (MAGE) A3 is a promising functional target in multiple myeloma (MM). MAGE-A3 expression in primary MM specimens is associated with proliferation and progression of disease. Previous laboratory studies showed that MAGE-A3 inhibits p53-dependent and independent apoptosis and cell cycle regulation in human myeloma cell lines (HMCL) and primary cells. We investigated the mechanisms of these oncogenic activities in laboratory models and through analysis of gene expression and clinical outcome data. Gene expression profiling (GEP) by RNA sequencing (RNAseq) of p53+/+ HMCL MM.1r and H929 after MAGE-A knockdown identified a set of 201 differentially expressed genes (DEG, p<0.05). Of the top DEGs, eight were BH3-only Bcl-2 family members (BAX, BCL2L11/BIM, BBC3/PUMA, BMF, BCL2L14/BCLG) or apoptotic pathway genes (TNFRSF10B/TRAILR2, CASP3, TRIAP1). Four of the top DEGs were cell cycle regulation genes (TRAF4, CDKN1A/p21CIP1, TRIM5, MAP2K6/MEK6) and three were DNA binding/damage repair genes (APOBEC3B, DDB2, ARID3A). We interrogated protein expression after MAGE knockdown and demonstrated significantly increased levels of pro-apoptotic BIM, but other Bcl-2 proteins were either not altered or not detected. BIM protein stability is regulated by serine phosphorylation, which leads to subsequent ubiquitinylation and degradation. MAGE knockdown resulted in higher levels of ser69- and ser77-phosphorylated BIM, indicating that MAGE does not affect phosphorylation but likely mediates further post-translational modification and degradation. We also detected increased levels of the endogenous cyclin-dependent kinase (CDK) inhibitor p21Cip1, which correlated with the GEP results. Depletion of MAGE-A in HMCL increased the sensitivity to induction of apoptosis by the alkylating agent melphalan but not to proteasome inhibition, suggesting that inhibition of BIM expression may contribute to chemotherapy resistance. To assess the clinical significance of these mechanisms, we analyzed RNAseq data from the iA9 release of the Multiple Myeloma Research Foundation CoMMpass database of more than 650 newly diagnosed MM patients based on high or low MAGEA3expression, which revealed a set of significantly DEGs (p<0.05) that included several MAGE family members and related X-linked genes. Gene set enrichment analysis demonstrated associations with cell cycle and DNA replication pathways, similar to that observed in HMCL. We ranked subjects based on MAGEA3 mRNA expression levels and correlated the highest (219 subjects) and lowest quartiles (212 subjects) with clinical outcome, which showed that the MAGEA3high group had worse overall survival (Hazard Ratio = 2.5, p<0.01, data not shown). This survival difference was even more striking in the subgroup of subjects that had undergone high dose melphalan chemotherapy and autologous stem cell transplantation (ASCT, fig.). MAGEA3 high subjects (39 subs) had worse progression-free (p<0.1) and overall survival (p<0.01) compared to the lowest (37 subs). These results demonstrate that MAGE-A3 negatively regulates BIM at both the transcriptional and post-translational levels, which favor survival and resistance to chemotherapy. MAGE-A3 also inhibits p21Cip1transcription and protein expression, likely through ubiquitinylation and degradation of p53 protein, which promotes passage through the early G1 checkpoint and proliferation. These mechanisms are a biochemical basis for MAGE-A3-mediated resistance to chemotherapy-induced apoptosis and the associations with progression of disease and tumor proliferation. These results provide a rationale to investigate methods to inhibit MAGE-A3 activity in MM. Disclosures Cho: Janssen: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agenus Inc.: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; J & J: Consultancy; Genentech Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chari:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; The Binding Site: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Array Biopharma: Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jagannath:Medicom: Speakers Bureau; Merck: Consultancy; Multiple Myeloma Research Foundation: Speakers Bureau; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy.
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Cho, Hearn Jay, Anna Mei, Jami Fukui, Kaity Tung, Violetta V. Leshchenko, Alessandro Lagana, Manishkumar Patel, et al. "MAGE-a Mediate Resistance to Chemotherapy in Multiple Myeloma through Regulation of Bcl-2 Proteins." Blood 128, no. 22 (December 2, 2016): 3277. http://dx.doi.org/10.1182/blood.v128.22.3277.3277.
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Abstract Type I Melanoma Antigen Genes of the MAGE-A family are commonly expressed in multiple myeloma (MM). Their frequency of expression is higher in patients who have relapsed after chemotherapy compared to newly diagnosed patients, with a significant association with progression of disease. MAGE were also associated with high-risk disease in gene expression profiling studies. They belong to the Cancer-Testis group of oncofetal genes that are normally restricted to germ cells and trophoblastic tissue, and are aberrantly expressed in a broad range of human cancers. Type I MAGE proteins partner with the RING domain protein Kap1 to form ubiquitin ligase complexes, and in laboratory models of solid tumors they ubiquitinylate cancer-associated targets involved in metabolism, autophagy, and metastasis. We demonstrated that expression of MAGE-A genes, especially MAGE-A3, was correlated with progression of disease in MM and that MAGE-A inhibit p53-dependent and independent apoptosis in MM cells. These results lead us to investigate the hypothesis that MAGE-A promote resistance to chemotherapy-induced apoptosis in MM cells. We performed gene expression profiling by RNAseq of primary tumor specimens from an open label phase 2 clinical trial of panobinostat, a pan-HDAC inhibitor approved for treatment of relapsed MM, in combination with lenalidomide and dexamethasone (NCT01651039). These GEP data were analyzed for differential expression based on progression free survival (PFS) < 90 days (short PFS) versus PFS > 90 days (long PFS). MAGE-A1 was the most overexpressed gene in the short PFS group compared to long (p<0.005), and this correlation was confirmed at the protein level (p<0.05). We then silenced MAGE-A expression in human myeloma cell lines (HMCL) by transduction of a MAGE-A-specific shRNA lentivirus, which resulted in nearly complete loss of MAGE-A protein at 24-48 hrs. HMCL were transduced with MAGE-A shRNA lenti or a negative control construct containing a non-target shRNA sequence, and untreated cells served as an additional negative control. Silencing of MAGE-A increased sensitivity to cell death induced by panobinostat. Similar results were seen with melphalan, an alkylating agent, and bortezomib, a proteasome inhibitor, both of which are approved for treatment of MM. These results indicate that MAGE-A play a critical role in resistance to chemotherapy-induced apoptosis in relapsed myeloma patients. To investigate the mechanisms of inhibition of apoptosis, we analyzed the impact of MAGE-A on Bcl-2 family members, including Bim, Bid, and PUMA, BH3-only family members that initiate apoptosis, Mcl-1, which plays a role in chemotherapy-induced apoptosis in myeloma cells, and the prototypical survival proteins Bcl-2 an Bcl-xl. HMCL were transduced with MAGE-A shRNA lenti or controls and cells were harvested after disappearance of MAGE-A protein. Heavy membrane preparations were made from these cells and probed by Western blot for Bcl-2 proteins. Loss of MAGE-A resulted in higher levels of Bim compared to controls. Of note, the short isoform of Bim (Bim S), which is the most potent activator of apoptosis, demonstrated relatively higher expression compared to the long and extra long isoforms (Bim L and EL). Phosphorylation of Bim at Ser69 and 77 by Erk1/2 and other kinases leads to ubiquitinylation and proteasomal degradation. In the absence of MAGE-A, phosphorylated Bim is stabilized, suggesting MAGE-A inhibits apoptosis by promoting ubiquitinylation and degradation of phospho-Ser69/77-Bim. These results demonstrate that MAGE-A/Kap1 is a key regulator of Bim that promotes survival of MM cells. Loss of MAGE-A also resulted in increased protein levels of Mcl-1 and decreased protein expression of Bcl-2. No significant changes in Bid or Bcl-xl proteins were observed and PUMA was not detected under these conditions. These results support a model in which MAGE-A mediates resistance to panobinostat and other chemotherapeutic agents through differential regulation of protein expression of Bim, Mcl-1, and Bcl-2. Therefore, MAGE-A is a promising novel therapeutic target, and inhibition of its anti-apoptotic activity may directly induce apoptosis or increase sensitivity to chemotherapy-induced apoptosis. Disclosures Cho: Ludwig Institute for Cancer Research: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Agenus, Inc.: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Chari:Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen Inc.: Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Array Biopharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Research Funding. Jagannath:Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Merck: Consultancy.
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Adamik, Juraj, Deena Maurer, Paul Munson, Alexis Combes, Philippe Pierre, Matthew Krummel, Rafael Argüello, and Lisa Butterfield. "299 Immuno-metabolic signatures of dendritic cells associate with T-cell responses in melanoma patients." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A326. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0299.
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BackgroundThe therapeutic efficacy of Dendritic cells (DC) vaccines remains low and there is an unmet need for more effective vaccine design to achieve durable clinical outcomes. Our study analyzed the transcriptomic and energetic metabolism profile of an adenoviral-based DC vaccine targeted against three commonly shared melanoma antigens: Tyrosinase, MART-1 and MAGE-A6 from 35 subjects enrolled in a Phase I study of autologous DC vaccines in late-stage melanoma.1 To further investigate the immuno-metabolic features of monocyte-derived DC vaccines, we are employing a novel flow cytometry-based method, called SCENITH2 to integrate functional metabolic states with multiparametric DC immune phenotypes.Methods iDC were generated from HD and patient monocytes using GM-CSF+IL-4 for 5d. DC were matured (mDC) using IFN?+LPS for additional 24 hrs. Tolerogenic DC (Tol DC) were generated using vitamin-D3 and dexamethasone. Seahorse® was used to measure DC metabolic profile. Cytek/Aurora spectral flow cytometry was used for multiparametric-phenotypic and metabolic analysis by SCENITH™.ResultsMelanoma patient mDC used for autologous vaccine generation showed significantly altered metabolic gene signatures associated with enhanced oxidative phosphorylation (OXPHOS) and lipid metabolism pathways as compared to HD mDC. Furthermore, increased enrichment for mitochondrial respiration genes involved in the TCA cycle, electron transport chain and fatty acid oxidation (FAO) correlated with inferior tumor antigen-specific T cell responses and clinical outcome in patients. Seahorse analyses confirmed that HD and good outcome patient DC demonstrated the highest maturation-induced reduction in maximal oxygen consumption rate/OXPHOS and exogenous FAO. Interestingly, while the glycolytic rate of non-responding patient DC was the lowest, overall, we observe only a moderate increase in glycolytic capacity during DC maturation. SCENITH analysis showed that unlike monocytes, which are primarily glycolytic, differentiated mono-derived iDC and mDC utilize both glycolysis and mitochondrial respiration. Interestingly, under tolerogenic (Tol) differentiation conditions Tol iDC shift from glucose dependence into FAO and/or glutaminolysis while Tol mDC strongly depend on OXPHOS. Consistent with dependence on mitochondrial respiration, Tol mDC exhibit reduced HIF1a levels together with enhanced p-AMPK:p-mTOR ratio. Additionally, we show that the altered metabolism of Tol mDC is linked to retention of CD14-monocyte antigen with reduced DC markers HLA-DR, CD86, CD206, CD11c, CD33, with increased PD-L1 and ILT3 expression. Furthermore, we show that unlike HD mDC, tolerogenic and melanoma patient-derived mDC populations exhibit similar metabolic and immune characteristics.ConclusionsWe demonstrate that metabolic profile of DCs is tightly associated to the immuno-stimulatory potential of DC vaccines from cancer patients. Using SCENITH, we linked phenotypic and functional metabolic changes associated to immune signatures that correspond to heathy and immuno-suppressed DC differentiation.Ethics ApprovalThe clinical trial reported was fully approved by the Univ. Pittsburgh PRC and IRB (PRO12010416, #09–021) and had FDA IND #15044 and NCT01622933.ReferencesButterfield LH, Vujanovic L, Santos PM, Maurer DM, Gambotto A, Lohr J, Li C, Waldman J, Chandran U, Lin Y, et al. Multiple antigen-engineered DC vaccines with or without IFNalpha to promote antitumor immunity in melanoma. Journal for immunotherapy of cancer 2019;7:113.Argüello RJ, Combes AJ, Char R, Bousiquot E, Gigan JP, Camosseto V, Samad B, Tsui J, Yan P, et al. SCENITH: A flow cytometry based method for functional profiling energy metabolism with single cell resolution. BioRxiv 2020.03.10.985796; doi: https://doi.org/10.1101/2020.03.10.985796. Cell Metabolism. (Sneak and Peek: under review 10.2139/ssrn.3565001)
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Chang, Yao-Jen, Zhifu Kang, Jiayuan Bei, Shu-Jen Chou, Mei-Yeh Jade Lu, Yu-Lun Su, Sheng-Wei Lin, Hsin-Hui Wang, Steven Lin, and Ching-Jin Chang. "Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits." International Journal of Molecular Sciences 23, no. 12 (June 20, 2022): 6839. http://dx.doi.org/10.3390/ijms23126839.
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TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited TRIM28 using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The TRIM28 KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in TRIM28 KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.
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Bräunig, Sandro, Dimitra Zacharaki, Hongzhe Li, Hooi Ching Lim, Stefan Lang, Thoas Fioretos, and Stefan Scheding. "Transcriptome Alterations of Isolated Primary Bone Marrow Endothelial Cells Provide Insigt to MGUS and Multiple Myeloma Pathobiology." Blood 134, Supplement_1 (November 13, 2019): 4373. http://dx.doi.org/10.1182/blood-2019-128024.
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Multiple myeloma (MM) is characterized by an abnormal clonal expansion of plasma cells in the bone marrow, production of monoclonal immunoglobulins and finally organ damage (CRAB). The premalignant precursor of MM is Monoclonal gammopathy of undetermined significance (MGUS) and one percent of all MGUS patients progress to MM yearly. The bone marrow microenvironment is thought to play an important role in plasma cell growth, migration, and survival mainly via cytokine secretion and cell-cell interactions. Endothelial cells (ECs) are a major component in the bone marrow microenvironment, they regulate trafficking and homing of hematopoietic progenitor and stem cells. In MM increased bone marrow angiogenesis and recruitment of endothelial progenitors to the bone marrow niche has been reported. However, the specific EC contribution to myelomagenesis is not yet known. This study therefore aimed to investigate transcriptome alterations in prospectively isolated bone marrow ECs from MGUS and MM patients to identify possible disease-stage related changes. We isolated primary ECs from MGUS and MM patients undergoing diagnostic bone marrow aspirations and age-matched healthy donors by FACS. RNA from Lin- CD45- CD71- CD235a- CD271- CD31+ cells of MGUS (n=4) and MM (n=7) patients and healthy donors (n=6) was extracted. Sequencing was done using the Illumina® NextSeq 500/550 High Output Kit v2.5 (300 cycles). Gene expression analysis was performed in R. Differential gene expression analysis (DEseq2) identified 1,507 genes with p adjusted values below 1e-2 that were significantly differentially expressed between the three groups. Hierarchical clustering was done following Ward's method (ward.D2). Unsupervised clustering on the data showed that one MGUS-EC sample clustered with the healthy controls, and that one healthy control sample clustered with the MGUS samples. We therefore decided to restrict the analysis to those samples that clearly clustered separately, to be able to better depict the MGUS-, MM- and healthy EC specific profiles. Further clustering of differential expressed genes into 8 clusters revealed two especially interesting expression patterns. One cluster (#4) contained 102 genes that where higher expressed in the healthy controls with lower expression in MGUS and lowest expression in MM Samples. These genes thus reflect the downregulation during progression from a healthy bone marrow microenvironment to a reduced expression MGUS and further downregulation in MM. Another cluster (#6) showed the opposite pattern, with 105 genes being low or not expressed in healthy controls while the expression was higher in MGUS and highest in MM. Gene sets where further analyzed in the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. Cluster 4 showed a high number of downregulated transmembrane genes. Six genes of the major histocompatibility complex conserved site where identified might indicate a possible immunomodulating effect in disease progression. Furthermore, within cluster 4 we identified a cluster of genes involved in cell adhesion and receptor binding. Cluster 6 most strikingly showed a group of 6 genes of the melanoma-associated antigen (MAGE) gene family that were upregulated with disease progression. MAGE genes which belong to the cancer-testis group of germline genes have previously been reported in MM, as being involved in tumorigenesis, and plasma cell MAGE expression has been associated with chemotherapy resistance. Furthermore, cluster 6 contained a high number of extracellular matrix genes, and genes for proteins having an extracellular region, respectively, hinting towards a differential microenvironment composition upon MM development. Taken together RNA sequencing analysis of prospectively isolated bone marrow endothelial cells identified genes that were specifically upregulated/suppressed in MM-ECs compared to MGUS-ECs and healthy donor-ECs. These genes thus represent potential gene candidates involved in the disruption of normal microenvironment function, thus leading to disease development and progression. Accordingly, studies are underway to investigate selected transcriptional deregulation EC-MM microenvironmental functions in the context of the disease. Disclosures No relevant conflicts of interest to declare.
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Meneveau, Max, Pankaj Kumar, Kevin Lynch, Karlyn Pollack, and Craig Slingluff. "611 RNA-sequencing reveals a unique immune transcriptional landscape in the vaccine sites of patients with circulating T-cell responses to cancer immunization." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A646—A647. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0611.
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BackgroundVaccines are a promising therapeutic for patients with advanced cancer, but achieving robust T-cell responses remains a challenge. Melanoma-associated antigen-A3 (MAGE-A3) in combination with adjuvant AS15 (a formulation of Toll-Like-Receptor (TLR)-4 and 9 agonists and a saponin), induced systemic CD4+ T-cell responses in 50% of patients when given subcutaneously/intradermally. Little is known about the transcriptional landscape of the vaccine-site microenvironment (VSME) of patients with systemic T-cell responses versus those without. We hypothesized that patients with systemic T-cell responses to vaccination would exhibit increased immune activation in the VSME, higher dendritic cell (DC) activation/maturation, TLR-pathway activation, and enhanced Th1 signatures.MethodsBiopsies of the VSME were obtained from participants on the Mel55 clinical trial (NCT01425749) who were immunized with MAGE-A3/AS15. Biopsies were taken 8 days after immunization. T-cell response to MAGE-A3 was assessed in PBMC after in-vitro stimulation with recMAGE-A3, by IFNγ ELISPOT assay. Gene expression was assessed by RNAseq using DESeq2. Comparisons were made between immune-responders (IR), non-responders (NR), and normal skin controls. FDR p<0.01 was considered significant.ResultsFour IR, four NR, and three controls were evaluated. The 500 most variable genes were used for principal component analysis (PCA). Two IR samples were identified as outliers on PCA and excluded from further analysis. There were 882 differentially expressed genes (DEGs) in the IR group vs the NR group (figure 1A). Unsupervised clustering of the top 500 DEGs revealed clustering according to the experimental groups (figure 1B). Of the 10 most highly upregulated DEGs, 9 were immune-related (figure 1C). Gene-set enrichment analysis revealed that immune-related pathways were highly enriched in IRs vs NRs (figure 1D). CD4 and CD8 expression did not differ between IR and NR (figure 2A), though both were higher in IR compared to control. Markers of DC activation/maturation were higher in IR vs NR (figure 2B), as were several Th1 associated genes (figure 2C). Interestingly, markers of exhaustion were higher in IR v NR (figure 2D). Expression of numerous TLR-pathway genes was higher in IR vs NR, including MYD88, but not TICAM1 (figure 2E).Abstract 611 Figure 1Gene expression profiling of vaccine site samples from patients immunized with MAGE-A3/AS15. (A) Volcano plots showing the distribution of differentially expressed genes (DEGs) between immune responders (IR) and non-responders (NR), IR and control, and NR and control. (B) Heatmap of the top 500 most differentially expressed genes demonstrating hierarchical clustering of sequenced samples according to IR, NR, and control. (C) Table showing the 10 most highly up and down-regulated genes in IR compared to NR. 9 of the top 10 most highly up-regulated genes are related to the immune response. (D) Enrichment plots from a gene set enrichment analysis highlighting the upregulation of immune related pathways in IR compared to NR. Gene set enrichment data was generated from the Reactome gene set database and included all expressed genes. Significance was set at FDR p <0.01Abstract 611 Figure 2Expression of T-cell markers in IR vs NR vs Control samples in the vaccine site microenvironment (VSME). (A) T-cell markers showing similar expression in IR vs NR but higher expression in IR vs control. (B) Markers of dendritic cell activation and maturation in the VSME which are higher in IR vs control but not IR vs NR. (B) Transcription factors and genes associated with Th1/Th2 responses within the VSME. (D) Genes associated with T-cell exhaustion at the VSME. (E) Expression of TLR pathway genes in the VSME. Expression data is provided in terms of normalized counts. Bars demonstrate median and interquartile range. N=9. IR = immune responder, NR = non-responder, TLR = Toll-like Receptor. * = <0.01, ** < 0.001, *** <0.0001, **** < 0.00001ConclusionsThese findings suggest a unique immune-transcriptional landscape in the VSME is associated with circulating T-cell responses to immunization, with differences in DC activation/maturation, Th1 response, and TLR signaling. Thus, immunologic changes in the VSME are useful predictors of systemic immune response, and host factors that modulate immune-related signaling at the vaccine site may have concordant systemic effects on promoting or limiting immune responses to vaccines.Trial RegistrationSamples for this work were collected from patients enrolled on the Mel55 clinical trial NCT01425749.Ethics ApprovalThis work was completed after approval from the UVA institutional review board IRB-HSR# 15398.
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Shin, Gyu-Tae, Ji Eun Park, and Min-Jeong Lee. "MAGEH1 interacts with GADD45G and induces renal tubular cell apoptosis." PLOS ONE 16, no. 11 (November 17, 2021): e0260135. http://dx.doi.org/10.1371/journal.pone.0260135.
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Background Melanoma-associated antigen H1 (MAGEH1) is a protein that belongs to melanoma-associated antigen (MAGE) superfamily. Growth arrest and DNA damage 45G (GADD45G) is a member of the DNA damage-inducible gene family which responds to environmental stresses. We have previously shown that GADD45G is a protein that promotes apoptosis of renal tubular cells in response to a nephrotoxic injury. In this study, we show evidence that MAGEH1 interacts with GADD45G and is involved in the induction of nephrotoxin-induced apoptosis of renal tubular cells. Methods Primary human renal tubular epithelial (HRE) cells and human kidney 2 (HK-2) cells were used in this study. To produce stable cell lines in which MAGEH1 expression was silenced, HRE cells were transduced with a lentiviral vector encoding a single guide RNA construct targeting the MAGEH1 gene. To knockdown GADD45G expression in HRE cells, a vector containing short hairpin RNA (shRNA) was used. We used short interfering RNAs (siRNA) to achieve transient silencing of genes in HK-2 cells. Recombinant adenoviruses were synthesized to overexpress MAGEH1 and GADD45G proteins. Human protein microarray was used to identify proteins that binds to GADD45G. Co-immunoprecipitation assays were then performed to confirm microarray results. Cell death was induced by cyclosporine A (CsA). Real-time quantitative PCR assay was used to evaluate gene expression levels. The degree of apoptosis and necrosis of cultured cells was evaluated by flow cytometry. Expression levels of caspases were examined using western blot analysis. Results We found that GADD45G bound to one protein spotted in the protein microarray, which was subsequently identified as MAGEH1. We confirmed the interaction between GADD45G and MAGEH1 protein using the co-immunoprecipitation assay. MAGEH1 gene expression was not altered by CsA-induced cytotoxic injury, whereas GADD45G gene expression was increased significantly upon CsA treatment. MAGEH1 expression was significantly downregulated in GADD45G knockdown HRE stable cells suggesting that MAGEH1 expression may be dependent on GADD45G expression. CsA-induced apoptosis was significantly reduced in MAGEH1 knockdown HRE stable cells which led to an increased survival of these cells. Similar results were observed in GADD45G knockdown HRE stable cells. Accordingly, CsA-induced apoptosis was significantly decreased in MAGEH1 siRNA and GADD45G siRNA transfected HK-2 cells. CsA-induced activation of caspase-7 and caspase-9 was inhibited in MAGEH1 knockdown HRE stable cells, and similarly in GADD45G knockdown HRE stable cells. Conclusions To the best of our knowledge, this is the first study to show that MAGEH1 interacts with GADD45G and that MAGEH1 is involved in caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.
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Hoon, D. S., D. Yuzuki, M. Hayashida, and D. L. Morton. "Melanoma patients immunized with melanoma cell vaccine induce antibody responses to recombinant MAGE-1 antigen." Journal of Immunology 154, no. 2 (January 15, 1995): 730–37. http://dx.doi.org/10.4049/jimmunol.154.2.730.
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Abstract The MAGE-1 gene was recently characterized to encode an immunogenic tumor Ag on several types of human tumors, including melanoma. This Ag is expressed in a wide variety of human tumors and not in normal cells, except testicular tissue, as assessed through specific mRNA analysis. In this study we cloned the MAGE-1 gene exon 3 region from a colon carcinoma cell line and expressed it in Escherichia coli. The recombinant MAGE-1 protein was affinity purified. By using Western blot analysis, IgG and IgM anti-MAGE-1 Abs were detected in the sera of melanoma patients. Fifty-three patients immunized with a melanoma cell vaccine (MCV) were assessed for anti-MAGE-1 IgG responses by using a MAGE-1 Ag-specific ELISA. The MCV consisted of three melanoma cell lines that expressed MAGE-1. Comparisons of anti-MAGE-1 IgG response pre-MCV treatment with 12- to 16-wk post-MCV treatment were made. Fifty-seven percent of the patients immunized with the MCV showed significant enhancement of IgG response to recombinant MAGE-1 protein. Patients who responded had no particular HLA-A or -B allele expression pattern. Melanoma patients immunized with whole cell MCV containing MAGE-1 can enhance anti-MAGE-1 IgG Abs. Recombinant MAGE-1 protein can be used to assess patient response to MAGE-1 and will be investigated as a potential cancer vaccine against a wide variety of human tumors that express MAGE-1.
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Howard, Scott C., Ansu Kumar, Michael Castro, Himanshu Grover, Subrat Mohapatra, Shweta Kapoor, Anuj Tyagi, et al. "Biosimulation Using the Cellworks Computational Omics Biology Model (CBM) Identifies Immune Modulation As a Key Pathway for Predicting Azacitidine (AZA) Response in Myelodysplastic Syndromes (MDS)." Blood 138, Supplement 1 (November 5, 2021): 3690. http://dx.doi.org/10.1182/blood-2021-153539.
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Abstract Background: DNA methyltransferase inhibition (DNMTi) with the hypomethylating agents (HMA) azacitidine (AZA) or decitabine, remains the mainstay of therapy for the majority of high-risk Myelodysplastic Syndromes (MDS) patients. Nevertheless, only 40-50% of MDS patients achieve clinical improvement with DNMTi. There is a need for a predictive clinical approach that can stratify MDS patients according to their chance of benefit from current therapies and that can identify and predict responses to new treatment options. Ideally, patients predicted to be non-responders (NR) could be offered alternative strategies while being spared protracted treatment with HMA alone that has a low likelihood of efficacy. Recently, an intriguing discovery of immune modulation by HMA has emerged. In addition to the benefits of unsilencing differentiation genes and tumor suppressor genes, HMA's reactivate human endogenous retroviral (HERV) genes leading to viral mimicry and upregulation of the immune response as a major mechanism of HMA efficacy. Although the PD-L1/PD1 blockade plus HMA has been recognized as a beneficial combination, there are no established markers to guide decision-making. We report here the utility of immunomic profiling of chromosome 9 copy number status as a significant mechanism of immune evasion and HMA resistance. Methods: 119 patients with known clinical responses to AZA were selected for this study. Publicly available data largely from TCGA and PubMed was utilized for this study. The aberration and copy number variations from individual cases served as input into the Cellworks Computation Omics Biology Model (CBM), a computational biology multi-omic software model, created using artificial intelligence heuristics and literature sourced from PubMed, to generate a patient-specific protein network map. Disease-biomarkers unique to each patient were identified within protein network maps. The Cellworks Biosimulation Platform has the capacity to biosimulate disease phenotypic behavior and was used to create a disease model and then conduct biosimulations to measure the effect of AZA on a cell growth score comprised of a composite of cell proliferation, viability, apoptosis, metastasis, and other cancer hallmarks. Biosimulation of drug response was conducted to identify and predict therapeutic efficacy. Results: Although AZA treatment increased tumor associated antigens and interferon signaling, it also increased PD-L1 expression to inactivate cytotoxic CD8(+) T cells. Copy number alterations of the chromosome 9p region were found to significantly drive PD-L1 expression with multiple genes such as CD274, IFNA1, IFNA2, JAK2, PDCD1LG and KDM4C playing a role in PD-L1 regulation further increasing immune suppression (Figure 1). Among 6 cases of chromosome 9p aberration in this dataset, 9p amp (n=2) were clinical non-responders (NR) while 9p del (n=4) were responders (R) to AZA. In principle, checkpoint immunotherapy could improve outcomes for patients with 9p abnormalities. Additionally, copy number variation loss of key genes located on chromosome 16 involved in antigen processing and presentation such as CIITA, CTCF, IRF8, PSMB10, NLRC5, and SOCS1 were found to negatively impact AZA sensitivity (NR=4; R=0); these patients would also be unlikely to respond to checkpoint immunotherapy. Also, aberrations in melanoma antigen gene (MAGE) family proteins (NR=2; R=O), and STT3A (NR=1; R=5) were found to impact AZA efficacy by decreasing antigen processing on tumor cells. Conclusion: Based on the results from the Cellworks Biosimulation Platform applied to the CBM, copy number variants of chromosome 9p and 16 can be converted into CBM-derived biomarkers for response to checkpoint immunotherapy in combination with HMA. Our results support a future prospective evaluation in larger cohorts of MDS patients. Figure 1 Figure 1. Disclosures Howard: Servier: Consultancy; Cellworks Group Inc.: Consultancy; Sanofi: Consultancy, Other: Speaker fees. Kumar: Cellworks Group Inc.: Current Employment. Castro: Bugworks: Consultancy; Exact sciences Inc.: Consultancy; Guardant Health Inc.: Speakers Bureau; Cellworks Group Inc.: Current Employment; Caris Life Sciences Inc.: Consultancy; Omicure Inc: Consultancy. Grover: Cellworks Group Inc.: Current Employment. Mohapatra: Cellworks Group Inc.: Current Employment. Kapoor: Cellworks Group Inc.: Current Employment. Tyagi: Cellworks Group Inc.: Current Employment. Nair: Cellworks Group Inc.: Current Employment. Suseela: Cellworks Group Inc.: Current Employment. Pampana: Cellworks Group Inc.: Current Employment. Lala: Cellworks Group Inc.: Current Employment. Singh: Cellworks Group Inc.: Current Employment. Shyamasundar: Cellworks Group Inc.: Current Employment. Kulkarni: Cellworks Group Inc.: Current Employment. Narvekar: Cellworks Group Inc.: Current Employment. Sahni: Cellworks Group Inc.: Current Employment. Raman: Cellworks Group Inc.: Current Employment. Balakrishnan: Cellworks Group Inc.: Current Employment. Patil: Cellworks Group Inc.: Current Employment. Palaniyeppa: Cellworks Group Inc.: Current Employment. Balla: Cellworks Group Inc.: Current Employment. Patel: Cellworks Group Inc.: Current Employment. Mundkur: Cellworks Group Inc: Current Employment. Christie: Cellworks Group Inc.: Current Employment. Macpherson: Cellworks Group Inc.: Current Employment. Marcucci: Abbvie: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings.
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Asai, Takashi, Yan Liu, Silvana Di Giandomenico, Anthony Deblasio, Silvia Menendez, Jack Antipin, Boris Reva, Rachel Wevrick, and Stephen D. Nimer. "Necdin Regulates Hematopoietic Stem Cell Quiescence and Sensitivity to Genotoxic Stress." Blood 114, no. 22 (November 20, 2009): 379. http://dx.doi.org/10.1182/blood.v114.22.379.379.
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Abstract Abstract 379 Necdin, a member of MAGE (melanoma antigen) family proteins, is a growth suppressing protein that was first identified in post mitotic neurons. The gene encoding necdin is one of several deleted in individuals with Prader-Willi syndrome, a neurobehavioural disorder associated with an increased risk of myeloid leukemia. It is reported that necdin interacts with p53 and represses p53-mediated apoptosis in neurons, but its role in hematopoiesis is largely unknown. Recently, we defined a critical role of p53 in regulating hematopoietic stem cell quiescence, and identified necdin as a target gene of p53, that is highly expressed in LT-HSCs (Liu Y et al., Cell Stem Cell, 2009). To define the role of necdin in hematopoiesis, we have analyzed the hematopoietic compartment of necdin-null mice. As necdin-null mice die perinatally, we first investigated fetal hematopoiesis and found no alteration in the frequency of fetal liver HSCs, defined as Lin-Sca1+Mac1+CD48-CD150+ within the fetal liver cells. Although necdin-null fetal liver HSCs increase serial replating capability in methylcellulose and maintain stemness in long-term stromal based cultures better than wild type HSCs, necdin-null fetal liver HSCs repopulate lethally irradiated recipient mice similar to wild type HSCs, in primary, secondary, and tertiary serial bone marrow transplantation assays. In addition, necdin-null HSCs show almost comparable repopulating ability as wild type HSCs, after secondary competitive bone marrow transplantation assays. These imply that necdin is dispensable for HSC self renewal. On the other hand, BM-derived necdin-null HSCs show decreased quiescence 4 months after transplantation, and increased proliferation as indicated by in vivo BrdU incorporation assays. Furthermore, recipient mice repopulated with necdin-null HSCs show enhanced sensitivity both to weekly 5-FU administration and to total body irradiation, as manifested by increased mortality. This suggests that the decreased quiescence of necdin-null HSCs leads to their depletion under conditions of genotoxic stress. Gene expression profiling studies have identified several deregulated signaling pathways in the necdin-null HSCs. Expression of several p53 target genes is altered in irradiated necdin-null HSCs, which may account for their enhanced radiosensitivity. We are now investigating these necdin target genes to clarify how necdin functions to critically regulate HSC quiescence. We are also determining whether targeting necdin could be a therapeutic approach to eliminate quiescent leukemia stem cells, using a murine CML model. Disclosures: No relevant conflicts of interest to declare.
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Zewdu, Eden B., Rachel M. Gonzalez, Yan Ma, Derek Ling, Xiaomin Hu, Qi Ren, Ranran Zhang, et al. "Abstract 2803: A renewable IHC control tool CytoSections࣪ for defining MAGEA3, MAGEA4, and MAGEA9 antibody specificity." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2803. http://dx.doi.org/10.1158/1538-7445.am2022-2803.
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Abstract Melanoma-associated antigen gene A (MAGEA) family proteins are expressed in a variety of tumors with each MAGEA protein having unique roles in cancer pathogenesis. The MAGEA members lack of expression in normal tissue and their role in cancer make them well suited for targeted cancer immunotherapy. Thus, knowing which MAGEA protein expression exists in a tumor is important. However, the MAGEA family shares sequence similarity that makes it difficult to find antibodies that are specific to just one family member. It is also challenging to source tissue generally with HIPAA regulation and even more so to get tissue representation of all 12 MAGEA family members. Here we present CytoSections as an alternative to patient control tissues. CytoSections are formalin fixed, paraffin embedded (FFPE) section of over-expression cell pellets. These cells were transfected with expression plasmids coding for genes of individual MAGES family. In this study, CytoSections are used to screen antibodies to all 12-member MAGEA family of proteins. The MAGEA family members were initially shown positive by targeted protein expression in western and immunohistochemistry using DDK tag antibody. Then multiple MAGEA3, MAGEA4, MAGEA9 antibodies were assessed by IHC to determine their specificity to their intended target. Using MAGEA family as a pilot project, we show that CytoSections is a verified, reproducible, and renewable alternative to human control tissues and serves as an ideal tool for antibody specificity assessment. Citation Format: Eden B. Zewdu, Rachel M. Gonzalez, Yan Ma, Derek Ling, Xiaomin Hu, Qi Ren, Ranran Zhang, Li-Hui Lei, Krishnan Allampallam, Dezhong Yin, Xuan Liu, Wei Fu. A renewable IHC control tool CytoSections࣪ for defining MAGEA3, MAGEA4, and MAGEA9 antibody specificity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2803.
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Gaugler, B., B. Van den Eynde, P. van der Bruggen, P. Romero, J. J. Gaforio, E. De Plaen, B. Lethé, F. Brasseur, and T. Boon. "Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes." Journal of Experimental Medicine 179, no. 3 (March 1, 1994): 921–30. http://dx.doi.org/10.1084/jem.179.3.921.
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Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, that codes for one of these antigens named MZ2-E. We show here that antigen MZ2-D, which is present on the same tumor, is encoded by another member of the MAGE gene family named MAGE-3. Like MAGE-1, MAGE-3 is composed of three exons and the large open reading frame is entirely located in the third exon. Its sequence shows 73% identity with MAGE-1. Like MZ2-E, antigen MZ2-D is presented by HLA-A1. The antigenic peptide of MZ2-D is a nonapeptide that is encoded by the sequence of MAGE-3 that is homologous to the MAGE-1 sequence coding for the MZ2-E peptide. Competition experiments using single Ala-substituted peptides indicated that amino acid residues Asp in position 3 and Tyr in position 9 were essential for binding of the MAGE-1 peptide to HLA-A1. Gene MAGE-3 is expressed in many tumors of several types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma and breast carcinoma, but not in normal tissues except for testes. It is expressed in a larger proportion of melanoma samples than MAGE-1. MAGE-3 encoded antigens may therefore have a wide applicability for specific immunotherapy of melanoma patients.
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DePietro, Paul, Mary Nesline, Yong Hee Lee, RJ Seager, Erik Van Roey, Shuang Gao, Vincent Giamo, et al. "77 Prevalence of secondary immunotherapeutic targets in the absence of established immune biomarkers in solid tumors." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A86. http://dx.doi.org/10.1136/jitc-2021-sitc2021.077.
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BackgroundImmune checkpoint inhibitor-based therapies have achieved impressive success in the treatment of several cancer types. Predictive immune biomarkers, including PD-L1, MSI and TMB are well established as surrogate markers for immune evasion and tumor-specific neoantigens across many tumors. Positive detection across cancer types varies, but overall ~50% of patients test negative for these primary immune markers.1 In this study, we investigated the prevalence of secondary immune biomarkers outside of PD-L1, TMB and MSI.MethodsComprehensive genomic and immune profiling, including PD-L1 IHC, TMB, MSI and gene expression of 395 immune related genes was performed on 6078 FFPE tumors representing 34 cancer types, predominantly composed of lung cancer (36.7%), colorectal cancer (11.9%) and breast cancer (8.5%). Expression levels by RNA-seq of 36 genes targeted by immunotherapies in solid tumor clinical trials, identified as secondary immune biomarkers, were ranked against a reference population. Genes with a rank value ≥75th percentile were considered high and values were associated with PD-L1 (positive ≥1%), MSI (MSI-H or MSS) and TMB (high ≥10 Mut/Mb) status. Additionally, secondary immune biomarker status was segmented by tumor type and cancer immune cycle roles.ResultsIn total, 41.0% of cases were PD-L1+, 6.4% TMB+, and 0.1% MSI-H. 12.6% of cases were positive for >2 of these markers while 39.9% were triple negative (PD-L1-/TMB-/MSS). Of the PD-L1-/TMB-/MSS cases, 89.1% were high for at least one secondary immune biomarker, with 69.3% having ≥3 markers. PD-L1-/TMB-/MSS tumor types with ≥50% prevalence of high secondary immune biomarkers included brain, prostate, kidney, sarcoma, gallbladder, breast, colorectal, and liver cancer. High expression of cancer testis antigen secondary immune biomarkers (e.g., NY-ESO-1, LAGE-1A, MAGE-A4) was most commonly observed in bladder, ovarian, sarcoma, liver, and prostate cancer (≥15%). Tumors demonstrating T-cell priming (e.g., CD40, OX40, CD137), trafficking (e.g., TGFB1, TLR9, TNF) and/or recognition (e.g., CTLA4, LAG3, TIGIT) secondary immune biomarkers were most represented by kidney, gallbladder, and sarcoma (≥40%), with melanoma, esophageal, head & neck, cervical, stomach, and lung cancer least represented (≥15%).ConclusionsOur studies show comprehensive tumor profiling that includes gene expression can detect secondary immune biomarkers targeted by investigational therapies in ~90% of PD-L1-/TMB-/MSS cases. While genomic profiling could also provide therapeutic choices for a percentage of these patients, detection of secondary immune biomarkers by RNA-seq provides additional options for patients without a clear therapeutic path as determined by PD-L1 testing and genomic profiling alone.ReferenceHuang R S P, Haberberger J, Severson E, et al. A pan-cancer analysis of PD-L1 immunohistochemistry and gene amplification, tumor mutation burden and microsatellite instability in 48,782 cases. Mod Pathol 2021;34: 252–263.
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Cheng, Sonia, Wei Liu, Moises Mercado, Shereen Ezzat, and Sylvia L. Asa. "Expression of the melanoma-associated antigen is associated with progression of human thyroid cancer." Endocrine-Related Cancer 16, no. 2 (June 2009): 455–66. http://dx.doi.org/10.1677/erc-09-0002.
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Thyroid cancer exhibits a spectrum from relatively indolent tumors to tumors that are invasive, metastatic, or progress to poorly differentiated carcinoma. Microarray expression analysis of thyroid cancer cell lines has implicated a member of the melanoma-associated (MAGE) family of cancer–testis antigens in thyroid cancer development and progression. We performed this study to validate the role of MAGE in human thyroid cancers. A tissue microarray (TMA) of samples from 375 patients with thyroid cancer was analyzed with immunohistochemistry (IHC) to localize MAGE. Western blotting of fractionated proteins from MAGE-transfected cells was used to confirm intracellular localization of proteins. Automated analysis of TMA samples was evaluated and subjected to statistical analysis. MAGE immunoreactivity was identified in nuclear and cytoplasmic compartments of normal and malignant tissues. Specificity of staining was proved by fractionation studies that confirmed MAGE expression in nucleus and cytoplasm. Normal thyroid tissue exhibited weak cytoplasmic and strong nuclear MAGE reactivity. Tumors exhibited an increase in cytoplasmic MAGE scores that correlated with clinical behavior: larger tumors had higher MAGE scores, and there was a positive and significant correlation between MAGE cytoplasmic score and the number of histologically proven lymph node metastases. There was a statistically significant negative correlation between cytoplasmic MAGE and the percentage of p53-positive nuclei. Our data confirm gene-profiling evidence that members of the MAGE family play a role in thyroid cancer progression. The use of TMA analyses identifies IHC techniques that are translatable to the clinical setting for prognostic assessment of patients with thyroid cancer.
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Schuler-Thurner, Beatrice, Erwin S. Schultz, Thomas G. Berger, Georg Weinlich, Susanne Ebner, Petra Woerl, Armin Bender, et al. "Rapid Induction of Tumor-specific Type 1 T Helper Cells in Metastatic Melanoma Patients by Vaccination with Mature, Cryopreserved, Peptide-loaded Monocyte-derived Dendritic Cells." Journal of Experimental Medicine 195, no. 10 (May 13, 2002): 1279–88. http://dx.doi.org/10.1084/jem.20012100.
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There is consensus that an optimized cancer vaccine will have to induce not only CD8+ cytotoxic but also CD4+ T helper (Th) cells, particularly interferon (IFN)-γ–producing, type 1 Th cells. The induction of strong, ex vivo detectable type 1 Th cell responses has not been reported to date. We demonstrate now that the subcutaneous injection of cryopreserved, mature, antigen-loaded, monocyte-derived dendritic cells (DCs) rapidly induces unequivocal Th1 responses (ex vivo detectable IFN-γ–producing effectors as well as proliferating precursors) both to the control antigen KLH and to major histocompatibility complex (MHC) class II–restricted tumor peptides (melanoma-antigen [Mage]-3.DP4 and Mage-3.DR13) in the majority of 16 evaluable patients with metastatic melanoma. These Th1 cells recognized not only peptides, but also DCs loaded with Mage-3 protein, and in case of Mage-3DP4–specific Th1 cells IFN-γ was released even after direct recognition of viable, Mage-3–expressing HLA-DP4+ melanoma cells. The capacity of DCs to rapidly induce Th1 cells should be valuable to evaluate whether Th1 cells are instrumental in targeting human cancer and chronic infections.
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Manici, Simona, Tiziana Sturniolo, Maria Adele Imro, Juergen Hammer, Francesco Sinigaglia, Christoph Noppen, Giulio Spagnoli, et al. "Melanoma Cells Present a MAGE-3 Epitope to CD4+ Cytotoxic T Cells in Association with Histocompatibility Leukocyte Antigen DR11." Journal of Experimental Medicine 189, no. 5 (March 1, 1999): 871–76. http://dx.doi.org/10.1084/jem.189.5.871.
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In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4+ T cells from the blood of a healthy donor. CD4+ T cells strongly recognized MAGE-3281–295 and, to a lesser extent, MAGE-3141–155 and MAGE-3146–160. Moreover, CD4+ T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4+ T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3–positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4+ T cells recognized MAGE-3281–295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4+ T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4+ T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4+ T cell epitopes.
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Valmori, Danila, Uzi Gileadi, Catherine Servis, P. Rod Dunbar, Jean-Charles Cerottini, Pedro Romero, Vincenzo Cerundolo, and Frédéric Lévy. "Modulation of Proteasomal Activity Required for the Generation of a Cytotoxic T Lymphocyte–defined Peptide Derived from the Tumor Antigen MAGE-3." Journal of Experimental Medicine 189, no. 6 (March 15, 1999): 895–906. http://dx.doi.org/10.1084/jem.189.6.895.
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We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201–associated tumor peptide antigen MAGE-3271–279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271–279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271–279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271–279–specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymatic activities.
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Traversari, C., P. van der Bruggen, I. F. Luescher, C. Lurquin, P. Chomez, A. Van Pel, E. De Plaen, A. Amar-Costesec, and T. Boon. "A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E." Journal of Experimental Medicine 176, no. 5 (November 1, 1992): 1453–57. http://dx.doi.org/10.1084/jem.176.5.1453.
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We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.
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Bolli, Martin, Thomas Kocher, Michel Adamina, Ulrich Guller, Peter Dalquen, Philippe Haas, Martina Mirlacher, et al. "Tissue Microarray Evaluation of Melanoma Antigen E (MAGE) Tumor-Associated Antigen Expression." Annals of Surgery 236, no. 6 (December 2002): 785–93. http://dx.doi.org/10.1097/00000658-200212000-00011.
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Lendvai, Nikoletta, Sacha Gnjatic, Erika Ritter, Yao-Tseng Chen, Christina Coughlin, Robert H. Vonderheide, Ruben Niesvizky, et al. "Host Immune Responses Against CT Antigens in Multiple Myeloma Patients." Blood 108, no. 11 (November 16, 2006): 3492. http://dx.doi.org/10.1182/blood.v108.11.3492.3492.
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Abstract The type I Melanoma Antigen GEne (MAGE) proteins belong to the Cancer-Testis (CT) family of tumor-associated antigens and are widely expressed in solid and hematologic malignancies. They are immunogenic and frequently elicit spontaneous immune responses in patients with CT antigen-expressing tumors, particularly in malignant melanoma. In melanoma patients, there is high concordance between humoral and cellular immunity. Based on these findings, CT antigens are widely investigated as potential antigenic targets for tumor-specific therapeutic vaccines. We previously showed that the type I MAGE proteins CT7 (MAGE-C1), CT10 (MAGE-C2) and MAGE-A3 were commonly detected in primary myeloma specimens, and expression of CT7 and MAGE-A3 was correlated with abnormally elevated plasma cell proliferation. These findings suggest that type I MAGE may be rational targets for vaccine therapy in multiple myeloma. Therefore, it is important to determine if type I MAGE elicit cellular or humoral immune responses in myeloma patients. To investigate this hypothesis, we assessed cellular immunity against CT7 and humoral immunity against a broad panel of CT antigens. To quantify CT7-specific cellular immunity, expanded, polyclonal pools of T cells from the bone marrow, the tumor microenvironment, were co-cultured in interferon gamma (IFNγ) ELISpot assays with autologous antigen-presenting cells (APC) transduced with in vitro transcribed mRNA coding for CT7 or control antigens. CT antigen-specific humoral immunity was examined by ELISA assay using patient serum or plasma and recombinant CT antigens. This analysis demonstrated that 2/9 patients exhibited specific T cell immunity against CT7 in their bone marrow lymphocytes as measured by IFNγ secretion. These same two patients had positive titers for other CT antigens; one for MAGE-A1 (another type I MAGE), the other for SSX-1 (a structurally distinct CT antigen). Interestingly, neither patient had positive serology for CT7. Serum from 16 other myeloma patients did not have detectible antibody titers for a broad panel of CT antigens. These results show that CT antigens are immunogenic in myeloma patients, with cellular responses against CT7 and humoral responses against MAGE-A1 and SSX-1. However, unlike other types of cancer, there appears to be discordance between humoral and cellular immunity against CT7 in multiple myeloma. This may be due in part to the significant derangements of humoral immunity in this disease. These results support further investigation of immunologic therapies targeting type I MAGE in myeloma, especially therapeutic vaccine strategies.
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Srisawat, Wanwisa, Boondarika Nambooppha, Kidsadagon Pringproa, Atigan Thongtharb, Worapat Prachasilchai, and Nattawooti Sthitmatee. "A Preliminary Study of the Cross-Reactivity of Canine MAGE-A with Hominid Monoclonal Antibody 6C1 in Canine Mammary Gland Tumors: An Attractive Target for Cancer Diagnostic, Prognostic and Immunotherapeutic Development in Dogs." Veterinary Sciences 7, no. 3 (August 10, 2020): 109. http://dx.doi.org/10.3390/vetsci7030109.
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Melanoma-associated antigen-A (MAGE-A), a family of cancer/testis antigens, has been recognized as a potential target molecule for cancer immunotherapy. However, there has been very little information available with regard to this antigen in dogs. This study aimed to investigate the expression of MAGE-A in canine mammary gland tumors (CMTs) using immunohistochemistry and immunoblotting with human monoclonal MAGE-A antibody 6C1. The present study has provided evidence of cross-reactivity of the canine MAGE-A expression with the human MAGE-A antibody in CMTs. The MAGE-A antigens were expressed in moderate- and high-grade malignant CMTs (22.22%, 2/9), but no expression was observed in benign CMTs. The immunohistochemical staining of canine MAGE antigen in CMT cells showed nuclear and nuclear–cytoplasmic expression patterns that may be involved with the mitotic cell division of tumor cells. Molecular weights of the canine MAGE-A antigen presented in this study were approximately 42–62 kDa, which were close to those of other previous studies involving humans and dogs. The findings on this protein in CMTs could supply valuable oncological knowledge for the development of novel diagnostic, prognostic and immunotherapeutic tumor markers in veterinary medicine.
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Kanda, Mitsuro, Hisaharu Oya, Soki Hibino, Hideki Takami, Dai Shimizu, Masahiko Koike, Shuji Nomoto, et al. "Overexpression of melanoma-associated antigen D4 as an independent prognostic factor in squamous cell carcinoma of the esophagus." Journal of Clinical Oncology 32, no. 3_suppl (January 20, 2014): 71. http://dx.doi.org/10.1200/jco.2014.32.3_suppl.71.
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71 Background: To pursue an urgently needed treatment target for esophageal cancer (EC), we investigated the function of the recently discovered melanoma-associated antigen (MAGE)-D4 in squamous cell EC. Methods: MAGE-D4 mRNA expression was analyzed in nine EC cell lines using quantitative reverse transcription-polymerase chain reaction (RT-PCR). In 65 surgical specimens of squamous cell EC with no prior neoadjuvant therapy, MAGE-D4 mRNA expression in EC tissues and corresponding normal tissues was analyzed and compared, and evaluated in terms of clinicopathological factors. In representative cases, MAGE-D4 protein distribution was analyzed immunohistochemically. Results: The heterogeneity of MAGE-D4 mRNA expression was confirmed in EC cell lines by quantitative RT-PCR. In surgical specimens, MAGE-D4 mRNA expression was significantly higher in EC tissues than in corresponding normal tissues (P < 0.001). Patients with the highest MAGE-D4 mRNA expression in EC tissues (top quartile, n=17) had significantly shorter overall survival than patients with low expression (2-year survival: 44% and 73%, respectively, P = 0.006). Univariate analysis identified age (≥ 65 years), lymphatic involvement and high MAGE-D4 mRNA expression as significant prognostic factors; high MAGE-D4 mRNA expression was also an independent prognostic factor in multivariable analysis (hazard ratio: 2.194; P = 0.039), and was significantly associated with Brinkman index (P = 0.008) and preoperative carcinoembryonic antigen level (P = 0.002). Immunohistochemical MAGE-D4b expression was consistent with MAGE-D4mRNA profiling. Conclusions: Our results suggest that MAGE-D4 overexpression influences tumor progression and MADE-D4 can be a prognostic marker and a potential molecular target in squamous cell EC.