Melanoma-associated Antigen Gene Research Articles

Nab-paclitaxel plus S-1 followed by gemcitabine-oxaliplatin as first-line alternating sequential treatment of pancreatic ductal adenocarcinoma.

Alternating sequential administration of drugs may be a promising approach to overcome chemotherapy resistance in advanced pancreatic ductal adenocarcinoma (PDAC). This study was an open-label, single-arm, and prospective trial included patients with untreated advanced PDAC. They received 2 cycles of NS regimen (nab-paclitaxel:125mg/m2, intravenously injected on days 1 and 8, plus S-1:40-60mg, orally twice per day for 1-14 days) followed by 2 cycles of GemOx regimen (gemcitabine, intravenously injected on days 1 and 8, and oxaliplatin: 130mg/m2, intravenously injected on day 1). The primary efficacy endpoint was a progression-free survival rate at 6 months (PFSR-6m). The secondary efficacy endpoints included overall survival (OS), progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and adverse events (AEs). Specific mRNA transcripts were used to explore survival associated genes. Forty-two patients received a minimum of one treatment cycle, and of these, 30 patients completed one alternating treatment consisting of 4 cycles. The PFSR-6m was 71% (95% CI = 58%-87%). The median PFS and OS were 6.53 months (95% CI = 6.03-8.43) and 11.4 months (95% CI = 9.8-14.4), respectively. Common grades 3-4 hematological AEs included neutropenia 30.9%, leukopenia 26.2%, anemia 2.4%, and thrombocytopenia in 11.9%. Patients with OS > 10 months showed high expression of HLA-DQA2 while melanoma-associated antigen genes (MAGE) were notably upregulated in patients with OS < 10 months. The alternating sequential administration of the NS and GemOx regimens may be a novel approach for first-line chemotherapy in patients with advanced PDAC requiring further study (ClinicalTrials.gov Identifier: ChiCTR1900024867).

Evolutionary and Expression Analysis of the Pig MAGE Gene Family.

The melanoma-associated antigen (MAGE) family found in eukaryotes plays a crucial role in cell proliferation and differentiation, spermatogenesis, neural development, etc. This study explored the validation and evolution of MAGE genes in eukaryotic genomes and their distribution and expression patterns in pigs. In total, 249 MAGE genes were found on 13 eukaryotic species. In total, 33, 25, and 18 genes were located on human, mouse, and pig genomes, respectively. We found eight, four, and three tandemly duplicated gene clusters on the human, mouse, and pig genomes, respectively. The majority of MAGE genes in mammals are located on the X chromosome. According to the phylogenetic analysis, the MAGE family genes were classified into 11 subfamilies. The NDN gene in zebrafish (DreNDN) was the root of this evolutionary tree. In total, 10 and 11 MAGE genes on human and mouse genomes, respectively, exhibited a collinearity relationship with the MAGE genes on pig genomes. Taking the MAGE family genes in pigs, the MAGE subfamilies had similar gene structures, protein motifs, and biochemical attributes. Using the RNA-seq data of Duroc pigs and Rongchang pigs, we detected that the expression of type I MAGE genes was higher in reproductive tissues, but type II MAGE genes were predominantly expressed in the brain tissue. These findings are a valuable resource for gaining insight into the evolution and expression of the MAGE family genes.

Abstract 999: Colocalization of MAGE-A3 and MAGE-A4 in bladder cancer

Abstract The treatment of bladder cancers has improved with new immunotherapy such as PD-L1. However, not all bladder cancers respond to PD-L1 immunotherapy which is why new targets are being assessed. Melanoma-associated antigen gene A (MAGE-A) family proteins are expressed in a variety of tumors, with each MAGE-A protein having unique roles in cancer pathogenesis. One advantage of targeting MAGE-A family members is the lack of expression in normal tissues which makes them well-suited for targeted cancer immunotherapy. The challenge with screening the MAGE-A family is to find a specific antibody since the 12-member family has over 60% sequence in homology. In this study, we evaluate multiple MAGE-A3 and MAGE-A4 antibodies using CytoSections, a reproducible alternative to control tissue with an expression of target biomarkers. A series of antibodies specific to each MAGE-A3 and MAGE-A4 protein were discovered and used to screen twenty-five bladder cancer tissues. The results showed that MAGE-A3 was present in 9 of the 25 bladder cancers, while MAGE-A4 was present in 10 of the 25 bladder cancers evaluated; in the meantime, 7 bladder cancers were positive for both targets after double immunofluorescence staining with MAGE-A3 or A4 specific antibodies. The data presented here could be an important development in treatment protocols for pre-screening patients who are at risk. Our findings show that MAGE-A3 and MAGE-A4 can be both present in bladder cancer, suggesting that targeting both genes may be necessary for the success of immunotherapy treatment. Citation Format: Bailey Gilmore, Rachel Gonzalez, Aubrey Su, Dehe Kong, Eden Zewdu, Jina Yom, Tianli Qu, Zhaoying Guo, Xiaomin Hu, Qi Ren, Ranran Zhang, Eric Christenson, Yan Ma, Dezhong Yin, Patrick Yin, Wei Fu, Xuan Liu, Krishnan Allampallam. Colocalization of MAGE-A3 and MAGE-A4 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 999.

IFNα and 5-Aza-2'-deoxycytidine combined with a dendritic-cell targeting DNA vaccine alter tumor immune cell infiltration in the B16F10 melanoma model.

DNA vaccines containing a fusion of the gene encoding chemokine MIP-3α (CCL20), the ligand for CCR6 on immature dendritic cells (DCs), to melanoma-associated antigen genes have enhanced anti-tumor immunity and efficacy compared to those lacking the chemokine gene. Previous work has shown that type-I interferon (IFNα or IFN) and 5-Aza-2'-deoxycytidine (5Aza) significantly enhance the therapeutic benefit of DNA vaccines as measured by reduced tumor burden and improved mouse survival. Here, we explored mouse intratumoral immune correlates underlying the therapeutic benefit of this combination regimen (vaccine, IFN, and 5Aza) as compared to vaccine alone and IFN and 5Aza without vaccine, focusing on chemokine mRNA expression by qRT-PCR and inflammatory cellular infiltration into the tumor microenvironment (TME) by flow cytometry and immunohistochemistry (IHC). The combination group significantly upregulated intratumoral mRNA expression of key immune infiltration chemokines XCL1 and CXCL10. Flow cytometric analyses of tumor suspensions exhibited greater tumor infiltration of CD8+ DCs, CCR7+ DCs, and NK cells in the combination group, as well as reduced levels of myeloid-derived suppressor cells (MDSCs) in vaccinated groups. The mice receiving combination therapy also had greater proportions of effector/memory T-cells (Tem), in addition to showing an enhanced infiltration of Tem and central memory CD8+ T-cells, (Tcm). Tem and Tcm populations both correlated with smaller tumor size. Immunohistochemical analysis of tumors confirmed that CD8+ cells were more abundant overall and especially in the tumor parenchyma with combination therapy. Efficient targeting of antigen to immature DCs with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This treatment creates an environment of increased inflammatory chemokines that facilitates the trafficking of CD8+ DCs, NK cells, and CD8+ T-cells, especially memory cells, while reducing the number of MDSCs. Importantly, in the combination group, CD8+ cells were more able to penetrate the tumor mass in addition to being more numerous. Further analysis of the pathways engaged by our combination therapy is expected to provide additional insights into melanoma pathogenesis and facilitate the development of novel treatment strategies.

The N-terminal domain of the Schaaf–Yang syndrome protein MAGEL2 likely has a role in RNA metabolism

MAGEL2 encodes the L2 member of the melanoma-associated antigen gene (MAGE) protein family, truncating mutations of which can cause Schaaf-Yang syndrome, an autism spectrum disorder. MAGEL2 is also inactivated in Prader–Willi syndrome, which overlaps clinically and mechanistically with Schaaf–Yang syndrome. Studies to date have only investigated the C-terminal portion of the MAGEL2 protein, containing the MAGE homology domain that interacts with RING-E3 ubiquitin ligases and deubiquitinases to form protein complexes that modify protein ubiquitination. In contrast, the N-terminal portion of the MAGEL2 protein has never been studied. Here, we find that MAGEL2 has a low-complexity intrinsically disordered N-terminus rich in Pro-Xn-Gly motifs that is predicted to mediate liquid–liquid phase separation to form biomolecular condensates. We used proximity-dependent biotin identification (BioID) and liquid chromatography–tandem mass spectrometry to identify MAGEL2-proximal proteins, then clustered these proteins into functional networks. We determined that coding mutations analogous to disruptive mutations in other MAGE proteins alter these networks in biologically relevant ways. Proteins identified as proximal to the N-terminal portion of MAGEL2 are primarily involved in mRNA metabolic processes and include three mRNA N 6-methyladenosine (m6A)-binding YTHDF proteins and two RNA interference-mediating TNRC6 proteins. We found that YTHDF2 coimmunoprecipitates with MAGEL2, and coexpression of MAGEL2 reduces the nuclear accumulation of YTHDF2 after heat shock. We suggest that the N-terminal region of MAGEL2 may have a role in RNA metabolism and in particular the regulation of mRNAs modified by m6A methylation. These results provide mechanistic insight into pathogenic MAGEL2 mutations associated with Schaaf–Yang syndrome and related disorders.

Epigenetic Upregulation of MAGE-A Isoforms Promotes Breast Cancer Cell Aggressiveness.

Simple SummaryBreast cancer is a heterogeneous disease that has complex causes and mechanisms of development. Currently, patient treatment options depend on the breast cancer molecular subtype, which is classified based on the presence or absence of hormone receptors and HER2. However, this classification system has limitations in terms of predicting responsiveness to anticancer drugs and patient outcomes. In this study, we present a new approach to classifying molecular breast cancer subtypes: it is based on changes in histone modifications in the promoter region of the MAGEA12 locus, which we found related closely to MAGEA12 expression and MAGEA12-associated malignancy of breast cancer cells.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.

Downregulation of Brain Enriched Type 2 MAGEs Is Associated With Immune Infiltration and Poor Prognosis in Glioma.

Melanoma associated antigen (MAGE) is an extensively studied family of tumor-associated genes that share a common MAGE homology domain (MHD). Based upon their expression pattern, MAGE genes have been broadly classified into type 1 MAGEs (T1Ms) and type 2 MAGEs (T2Ms) categories. Interestingly, several T2Ms are highly expressed in the brain and involved in the regulation of neuronal development, differentiation, and survival. Available literature suggests possible tumor suppressor functions of a few T2Ms, while information available about their expression, regulation, and clinical significance in glioma is scanty. This prompted us to perform a comprehensive analysis of T2M expression in glioma. Gene expression data from glioma datasets: Oncomine, TCGA, and REMBRANDT study, were used to assess the mRNA expression of T2M genes (MAGED1, MAGED2, MAGED3, MAGED4, MAGED4B, MAGEE1, MAGEE2, MAGEF1, MAGEH1, MAGEL2, NSMCE3, and NDN), and their association with clinical characteristics and composition of the tumor microenvironment. Further, mutation, copy number alteration, and DNA methylation data from TCGA were assessed for determining potential mechanisms of T2Ms expression in glioma. Expression analysis revealed overexpression of MAGED subfamily genes in glioma, while other genes of this family exhibited reduced expression in advanced grades of this malignancy. Further, the expression of T2Ms exhibited varying extent of positive correlations with each other. Amongst downregulated T2Ms, MAGEH1 expression exhibited negative correlations with DNA methylation. Additionally, genes associated with MAGEH1 were enriched in Myc and Hedgehog signaling. Furthermore, T2Ms downregulation was associated with immune infiltration in glioma tissues and poor overall survival of glioma patients. In multivariate Cox regression analysis, MAGEH1 emerged as an independent prognosticator in lower grade glioma. Conclusively, these results suggest that expression of T2Ms is associated with important clinical and molecular features in glioma. Mechanistic studies may further provide novel insights into their role in glioma progression.

The necdin interactome: evaluating the effects of amino acid substitutions and cell stress using proximity-dependent biotinylation (BioID) and mass spectrometry.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by the loss of function of a set of imprinted genes on chromosome 15q11-15q13. One of these genes, NDN, encodes necdin, a protein that is important for neuronal differentiation and survival. Loss of Ndn in mice causes defects in the formation and function of the nervous system. Necdin is a member of the melanoma-associated antigen gene (MAGE) protein family. The functions of MAGE proteins depend highly on their interactions with other proteins, and in particular MAGE proteins interact with E3 ubiquitin ligases and deubiquitinases to form MAGE-RING E3 ligase-deubiquitinase complexes. Here, we used proximity-dependent biotin identification (BioID) and mass spectrometry (MS) to determine the network of protein-protein interactions (interactome) of the necdin protein. This process yielded novel as well as known necdin-proximate proteins that cluster into a protein network. Next, we used BioID-MS to define the interactomes of necdin proteins carrying coding variants. Variant necdin proteins had interactomes that were distinct from wildtype necdin. BioID-MS is not only a useful tool to identify protein-protein interactions, but also to analyze the effects of variants of unknown significance on the interactomes of proteins involved in genetic disease.

A new tool for early diagnosis of rheumatoid arthritis using combined biomarkers; synovial MAGE-1 mRNA and serum anti-CCP and RF.

Introductionrheumatoid arthritis (RA) is a common autoimmune disease with unknown etiology and pathogenesis. Biomarkers have the potential to aid in the clinical diagnosis of the disease, or to provide means of detecting early signs of the disease. Evaluating Melanoma associated antigen genes (MAGE-1) mRNA expression rate in synovial fluid cells and serum levels of anti-cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) for RA early diagnosis.Methodsa total of 213 subjects were enrolled in the study, 135 RA patients and 78 normal subjects with traumatic knee joints (control group). Serum RF and anti-CCP were estimated quantitatively using ELISA. MAGE-1 mRNA expression rate was analyzed by RT-PCR.Resultsa significant increase in serum levels of RF IgM and anti-CCP in RA patients compared to the controls. A positively significant correlation was found between serum anti-CCP and RF IgM. The expression rate of MAGE-1 mRNA was 100% in RA patients versus the controls (0%). The specificity and the sensitivity of the three biomarkers was 100%.Conclusionthe high expression rate of MAGE-1 in synovial fluid cells of RA patients is encouraging its utilization as a diagnostic biomarker for RA. The combined use of MAGE-1 transcript in synovial fluid cells, serum RF and anti-CCP is recommended for improving early diagnostic ability of RA.

Transcriptome Alterations of Isolated Primary Bone Marrow Endothelial Cells Provide Insigt to MGUS and Multiple Myeloma Pathobiology

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.

Possible predictive role of cancer/testis antigens in breast ductal carcinoma in situ.

Cancer/testis antigens (CTAs) are a large family of tumor-associated antigens expressed in human tumors of different histological origin, but not in normal tissues, with the exception of the testes and placenta. Numerous immunohistochemical studies have reported associations between CTA expression and a negative estrogen receptor (ER) status in breast tumors, and demonstrated that CTAs are frequently expressed in tumors with higher nuclear grade. The expression of CTAs has not been studied as extensively in ductal carcinoma in situ (DCIS) as it has been in invasive breast cancer. The present retrospective study included archived paraffin-embedded specimens from 83 patients diagnosed with DCIS in the period between January 2007 and December 2014. The follow-up time for local recurrence ranged between 1 and 8 years (mean, 5.02 years). Antigens from the melanoma-associated antigen gene (MAGE) family, namely multi-MAGE-A, MAGE-A1, MAGE-A10 and New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, were evaluated by immunostaining and their subcellular location was investigated. Presence of tumor-infiltrating lymphocytes (TILs) was evaluated on all sections, together with the histopathological variables of DCIS. Specific tested antigens exhibited associations with histopathological parameters for DCIS and all demonstrated statistically significant associations with nuclear staining, simultaneous cytoplasmic and nuclear staining, and local recurrence. Antigen MAGE-A10 demonstrated a significant association with higher expression of ER (P=0.005) and higher tumor nuclear grade (P=0.001), cytoplasmic staining (P=0.029) and antigen NY-ESO-1 with higher tumor size (P=0.001), expression of TILs (P=0.001) and R1 resection (P=0.001). A χ2 test revealed significant associations between simultaneous cytoplasmic and nuclear staining and local recurrence (P=0.005), central necrosis (P=0.016), and the expression of ER (P=0.003) and progesterone receptor (PR) (P=0.010). Additional analysis revealed an association between antigen MAGE-A10 and TILs (P=0.05). Additional analysis of TILs indicated that they were significantly associated with tumor grade (P=0.023), central necrosis (P<0.001), ER (P=0.003) and PR (P=0.029). Overall, CTAs from the MAGE family (MAGE-A1, multi-MAGE-A and MAGE-A10) and NY-ESO-1 associate with histopathological predictive variables of DCIS. The expression of antigens NY-ESO-1 and MAGE-A10 could serve an important role in the treatment of patients with negative histopathological predictive variables, but further analysis is required. Simultaneous cytoplasmic and nuclear protein expression of MAGE-A family and NY-ESO-1 CTAs may represent an independent marker for local recurrence. Taken together, the present data suggest that CTAs are not perfect indicators of invasiveness for DCIS, but could inform treatment strategies for patients when taken in combination with other histopathological predictive variables. However, this was a small study and further larger studies will be necessary to confirm the current findings.

Abstract 3569: Humanized mouse model of MAGE-A1-targeted anti-melanoma T cell therapy

Abstract Adoptive cellular therapy (ACT) has resulted in durable anti-tumor responses for both solid and hematological malignancies, mediated by the persistence and activation of transferred T cells. Unfortunately most patients, especially those with solid tumors, continue to progress despite persistent T cells. Thus revealing challenges specific to the hostile microenvironment of solid tumors and a critical need to identify factors that limit ACT efficacy. Furthermore, serial patient biopsies are challenging to obtain and accessible peripheral blood does not reflect relevant cell interactions at the tumor site. Xenograft models that lack infiltrating human macrophages, NK cells and lymphocytes cannot faithfully recapitulate critical interactions within the tumor microenvironment. To overcome this limitation, we initiated ACT in the MISTRG mouse that supports hematopoiesis after transplantation of human CD34+ stem cells resulting in functional human innate and adaptive immune cells. Co-transplantation of the melanoma cell line (Me275) results in tumor infiltration by human macrophages (TAMs) supporting tumor progression. The cancer/testis antigen Melanoma associated Antigen Gene (MAGE)-A1 is an attractive ACT target due to broad expression in tumors and limited expression in normal tissues rendering off-tumor tissue toxicities unlikely. We have identified a high-affinity HLA-A*02:01-restricted TCR that recognizes MAGE-A1278-286 peptide (TCRMA1) and confers effector functions in transduced CD8 and CD4 T cells when co-transduced with CD8αβ. Ex vivo analysis of MISTRG T cell cytolysis and proliferation suggest that CD4+ T cell help is required in this model. After two weeks of in vitro expansion, CD4+ and CD8+ T cells from humanized MISTRG spleens were transduced and transferred into MISTRG littermates harboring MAGE-A1+ Me275. Although TCRMA1 treated mice subtly controlled the growth of Me275, the changes were not significant. However, compared to an irrelevant TCR, only TCRMA1 CD8+ T cells localized to tumor and persisted in blood and spleen 8 and 28 days post transfer. TCRMA1 CD8 T cells displayed an effector phenotype, with high levels of PD-1 expression characteristic of recently activated and/or exhausted T cells. These results suggest that TCRMA1 cells recognized the tumor and were consequently activated, but could not overcome suppression in the microenvironment, mirroring human studies. Additional studies are being initiated to overcome the specific factors associated with the TAMs that limit in vivo T cell function. This model provides an ideal environment to test next generation T cell therapies, such as addition of intrinsic co-stimulation, to enhance responses against solid tumors. Humanized MISTRG mice are a powerful tool to study suppression of ACT in vivo, offering streamlined approaches to overcome hostile tumor microenvironments before clinical translation. Citation Format: Megan S. McAfee, Trisha Sippel, Daniel Hunter, Jean Campbell, Thomas Schmitt, Robert Pierce, Anthony Rongvaux, Aude Chapuis. Humanized mouse model of MAGE-A1-targeted anti-melanoma T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3569.

Genome-Wide RNAi Screen Identify Melanoma-Associated Antigen Mageb3 Involved in X Chromosome Inactivation

Xist (inactivated X chromosome specific transcript) is a prototype long noncoding RNA in charge of epigenetic silencing of one X chromosome in each female cell in mammals. In a genetic screen, we identify Mageb3 and its homologs Mageb1 and Mageb2 as genes functionally required for Xist-mediated gene silencing. Mageb1–3 are previously uncharacterized genes belonging to the MAGE (melanoma-associated antigen) gene family. Mageb1–3 are expressed in undifferentiated ES cells and early stages of in vitro differentiation, a critical time window of X chromosome inactivation. Mageb3 showed both cytoplasmic and nuclear localization without enrichment on the inactive X (Xi). Mageb3 interacted with Polycomb group ring finger 3 (Pcgf3), a RING finger protein involved in recruiting Polycomb activities onto Xi. Mageb3 overexpression stabilized Pcgf3 protein. Mageb1–3 gene knockout affected H3K27me3 enrichment and the spreading of gene silencing along Xi. These data suggested that Mageb3 might regulate the recruitment of the Polycomb complex onto Xi and subsequent H3K27me3 modification through Pcgf3. Moreover, the nucleolar enrichment of Mageb3 was diminished when nuclear matrix factor hnRNP U is overexpressed, implying the interaction between Mageb3 and nuclear matrix, which is another possible mechanism for Mageb3 to regulate X chromosome inactivation.

Depletion of Mageb16 induces differentiation of pluripotent stem cells predominantly into mesodermal derivatives

The Melanoma-associated Antigen gene family (MAGE) generally encodes for tumour antigens. We had identified that one of the MAGE gene members, Mageb16 was highly expressed in undifferentiated murine embryonic stem cells (ESCs). While the role of Mageb16 in stemness and differentiation of pluripotent stem cells is completely unknown, here, in our current study, we have demonstrated that Mageb16 (41 kDa) is distributed in cytosol and/or in surface membrane in undifferentiated ESCs. A transcriptome study performed at differentiated short hairpin RNA (shRNA)-mediated Mageb16 knockdown (KD) ESCs and scrambled control (SCR) ESCs until a period of 22 days, revealed that Mageb16 KD ESCs mainly differentiated towards cells expressing mesodermal and cardiovascular lineage - gene markers. Gene markers of other mesoderm-oriented biological processes such as adipogenesis, osteogenesis, limb morphogenesis and spermatogenesis were also significantly enriched in the differentiated Mageb16 KD ESCs. The expression levels of contractile genes were higher in differentiated Mageb16 KD ESCs when compared to differentiated SCR and wild ESCs, suggesting a higher cardiomyogenic potential of Mageb16 depleted ESCs. Further analysis indicates that regulative epigenetic networks and nucleocytoplasmic modifications induced by the depletion of Mageb16, may play a probable role in differentiation.

Complex roles of NRAGE on tumor.

NRAGE, also known as Dlxin-1or MAGE-D1, is a member of type II melanoma-associated antigen (MAGE) and plays an essential role in life activities, including differentiation, apoptosis, and cell cycle. Studies increasingly found that NRAGE is closely related to the tumor events, such as tumor occurrence, invasion, and metastasis. However, complex and contradictory functions of NRAGE in different circumstances are observed, suggesting that NRAGE is unique from other MAGE gene family members. This review summarizes recent findings concerning the structure and biological functions of NRAGE, which may provide a basis for a more comprehensive understanding of and further research on NRAGE.

Structures of Two Melanoma-Associated Antigens Suggest Allosteric Regulation of Effector Binding.

The MAGE (melanoma associated antigen) protein family are tumour-associated proteins normally present only in reproductive tissues such as germ cells of the testis. The human genome encodes over 60 MAGE genes of which one class (containing MAGE-A3 and MAGE-A4) are exclusively expressed in tumours, making them an attractive target for the development of targeted and immunotherapeutic cancer treatments. Some MAGE proteins are thought to play an active role in driving cancer, modulating the activity of E3 ubiquitin ligases on targets related to apoptosis. Here we determined the crystal structures of MAGE-A3 and MAGE-A4. Both proteins crystallized with a terminal peptide bound in a deep cleft between two tandem-arranged winged helix domains. MAGE-A3 (but not MAGE-A4), is predominantly dimeric in solution. Comparison of MAGE-A3 and MAGE-A3 with a structure of an effector-bound MAGE-G1 suggests that a major conformational rearrangement is required for binding, and that this conformational plasticity may be targeted by allosteric binders.

Chemo/Photoacoustic Dual Therapy with mRNA-Triggered DOX Release and Photoinduced Shockwave Based on a DNA-Gold Nanoplatform.

A multifunctional nanoparticle based on gold nanorod (GNR), utilizing mRNA triggered chemo-drug release and near-infrared photoacoustic effect, is developed for a combined chemo-photoacoustic therapy. The constructed nanoparticle (GNR-DNA/FA:DOX) comprises three functional components: (i) GNR as the drug delivery platform and photoacoustic effect enhancer; (ii) toehold-possessed DNA dressed on the GNR to load doxorubicin (DOX) to implement a tumor cell specific chemotherapy; and (iii) folate acid (FA) modified on GNR to guide the nanoparticle to target tumor cells. The results show that, upon an effective and specific delivery of the nanoparticles to the tumor cells with overexpressed folate receptors, the cytotoxic DOX loaded on the GNR-DNA nanoplatform can be released through DNA displacement reaction in melanoma-associated antigen gene mRNA expressed cells. With 808 nm pulse laser irradiation, the photoacoustic effect of the GNR leads to a direct physical damage to the cells. The combined treatment of the two modalities can effectively destroy tumor cells and eradicate the tumors with two distinctively different and supplementing mechanisms. With the nanoparticle, photoacoustic imaging is successfully performed in situ to monitor the drug distribution and tumor morphology for therapeutical guidance. With further in-depth investigation, the proposed nanoparticle may provide an effective and safe alternative cancer treatment modality.

Cancer/testis antigen expression as a predictor for epidermal growth factor receptor mutation and prognosis in lung adenocarcinoma

Immune therapy targeting cancer/testis (CT) antigens improve the survival in several types of solid tumours. The expression of CT antigens is related to poor survival in non-small-cell lung cancer (NSCLC). The epidermal growth factor receptor (EGFR) mutation is the best predictive factor for the sensitivity to tyrosine kinase inhibitors in lung adenocarcinoma. The aim of this study was to elucidate the correlation between the expression of CT antigens and clinicopathological factors, including the EGFR mutation, and to analyse the prognosis in lung adenocarcinoma. Data were collected from a total of 281 lung adenocarcinoma patients who underwent surgery. Among them, 125 cases, whose specimens were too small to extract sufficient DNA and/or RNA, and 2 cases with the coexistence of another histological lung cancer were excluded. A total of 154 patients were reviewed. The expression of CT antigens (melanoma-associated antigen gene [MAGE]-A4 and KK-LC-1) and the EGFR-activating mutation (L858R point mutation in exon 21 and inframe deletion in exon 19) was evaluated by using polymerase chain reaction amplification. The expression of MAGE-A4 and KK-LC-1 was detected in 14 (9%) and 54 patients (35%) with adenocarcinoma. The EGFR-activating mutation was found in 64 patients (42%). Univariate and multivariate analyses demonstrated that tumours expressing at least one CT antigen were associated with no EGFR mutation (odds ratio = 0.3; 95% confidence interval, 0.14-0.71; P < 0.01). A survival analysis was performed in 135 patients who underwent complete resection and the 5-year overall survival rate was 71.1% in those with any expression of CT antigens and 83.2% in those without expression of the genes (P < 0.04). Two different therapeutic targets, EGFR-activating mutation and CT antigen, have a negative relationship with each other.

Characterization of Melanoma-Associated Antigen-A Genes Family Differential Expression in Non-Small-Cell Lung Cancers

BackgroundThe melanoma-associated antigen (MAGE) genes families are found in different cancers, including non-small-cell lung cancers. These genes are silent in normal tissues, except for the testis. The goal of this study was to investigate the differentially expressed profile of the different MAGE genes subclass in non-small-cell lung cancer (NSCLC) tumoral tissue. MethodsFormalin-fixed paraffin embedded NSCLC resected tissues were collected from 31 patients hospitalized in our referral hospital, and 29 patients were diagnosed with either squamous cell carcinoma (SCC) or adenocarcinoma (ADC). We used a nested reverse transcriptase–polymerase chain reaction, which comprised independent amplification of MAGE-A1, MAGE-A2, MAGE-A3/6, MAGE-A4, and MAGE-A12, to detect expression frequency of the MAGE-A family in lung tissue biopsies at both tumoral and nontumoral parts of patients' tissues. ResultsFrom 29 patients with diagnosis of either SCC (n = 16) or ADCs (n = 13), 58 samples were prepared. From 58 blocks sampled for this experiment, 37 tumoral tissue samples and 22 nontumoral tissue samples expressed at least one of the MAGE-A genes. MAGE-A4 gene had the highest incidence among all MAGE-A genes in both tumoral and nontumoral gens. In SCC and ADCs, the data showed expression of at least one of the MAGE-A genes in 59.4% and 69.2% of tumoral and nontumoral tissues, respectively. ConclusionThe detection of the MAGE-A genes expression could be a molecular tumor marker for early diagnosis and potential targets for active immunotherapy in NSCLC, particularly in ADCs and SCC. Besides, the frequency of different subtypes of MAGE genes may vary in different regions of world.

Necdin Protects Embryonic Motoneurons from Programmed Cell Death

NECDIN belongs to the type II Melanoma Associated Antigen Gene Expression gene family and is located in the Prader-Willi Syndrome (PWS) critical region. Necdin-deficient mice develop symptoms of PWS, including a sensory and motor deficit. However, the mechanisms underlying the motor deficit remain elusive. Here, we show that the genetic ablation of Necdin, whose expression is restricted to post-mitotic neurons in the spinal cord during development, leads to a loss of 31% of specified motoneurons. The increased neuronal loss occurs during the period of naturally-occurring cell death and is not confined to specific pools of motoneurons. To better understand the role of Necdin during the period of programmed cell death of motoneurons we used embryonic spinal cord explants and primary motoneuron cultures from Necdin-deficient mice. Interestingly, while Necdin-deficient motoneurons present the same survival response to neurotrophic factors, we demonstrate that deletion of Necdin leads to an increased susceptibility of motoneurons to neurotrophic factor deprivation. We show that by neutralizing TNFα this increased susceptibility of Necdin-deficient motoneurons to trophic factor deprivation can be reduced to the normal level. We propose that Necdin is implicated through the TNF-receptor 1 pathway in the developmental death of motoneurons.