The glycolytic enzyme alpha-enolase (ENO1) is a tumor-associated antigen and therapeutic target, known to be overexpressed in a variety of cancers compared to normal tissues. Upregulation of ENO1 at the mRNA and/or protein levels have been demonstrated in cancers of pancreas, breast, colon, gastric, prostate, head and neck, kidney, lung, ovary, and brain, with its higher expression generally associated with more advanced diseases and a shorter disease free interval. ENO1 catalyzes the reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway in the cytoplasm, while on the cell surface it functions as a plasminogen receptor. In cancer cells, ENO1 is believed to facilitate aerobic glycolysis (the Warburg effect) as well as plasminogen-dependent cell migration and invasion, thereby promoting cancer development and progression. In the clinical setting, patients with pancreatic and other cancers have been shown to harbor autoantibodies against ENO1 protein. In breast and lung cancer patients, anti-ENO1 autoantibody levels were decreased in the advanced diseases, while higher antibody levels against Ser419-phosphorylated ENO1 have been associated with longer disease free survival in pancreatic cancer patients. In another study, detection of autoantibodies to glycolytic enzymes, including ENO1 protein, was shown to precede the clinical diagnosis of breast cancer by several months. Autoantibody reactivity declined as sampling got closer to the time of diagnosis. ENO1 has also been demonstrated to elicit CD4+ and CD8+ mediated T cell responses, which correlated with the production of anti-ENO1 IgG, indicating the integrated T and B cell responses against the tumor antigen. Despite the presence of autoantibodies against ENO1, cancer patients do not exhibit signs of autoimmune diseases, suggesting a certain level of down-modulation of immune response to self-antigens in normal tissues. These findings support the notion that boosting host immunity against ENO1 tumor antigen may be safely achieved without increasing the risk of autoimmune diseases, and that it may prevent the development of cancers. To examine the feasibility and potential antitumor effects of ENO1 vaccination, Novelli et al. tested ENO1 DNA vaccine in two preclinical mouse models of pancreatic ductal adenocarcinoma (PDA) driven by constitutively activated mutant Kras oncogene, Pdx1-Cre;KrasLSL.G12D/+ mice (KC mice) and Pdx1-Cre;KrasLSL.G12D/+;p53R172H/+ mice (KPC mice). In both models, where mutant Kras is activated prenatally, animals develop a spectrum of pre-invasive ductal lesions, which mirror human pancreatic intraepithelial neoplasia (PanIN), shortly after birth. Human and mouse ENO1 are highly homologous with 95% amino acid sequence identity. Novelli and colleagues have shown that ENO1 DNA vaccine administered at 4 weeks of age followed by two booster shots given 3 weeks apart significantly delayed the development of PDA and improved the overall survival in the vaccinated animals as compared to control. ENO1 vaccination elicited anti-ENO1 antibodies that bound to the cell surface of syngeneic murine PDA cells and mediated complement dependent tumor cell killing. Higher levels of ENO1-specific Th1 and Th17 responses were demonstrated in the vaccinated mice, while the numbers of myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg) were decreased in circulation and in tumor tissues. Importantly, there were no overt signs of autoimmune diseases in any of the vaccinated animals. Safe and effective preventive measures are urgently needed to reduce cancer-associated morbidity and mortality, especially against highly aggressive cancers. Pancreatic cancer is one of the most lethal cancers in both men and women. Because pancreatic cancer is usually diagnosed at an advanced stage, the survival rate is extremely poor. Known risk factors for pancreatic cancer include cigarette smoking, obesity, chronic pancreatitis, diabetes, and family history of genetic syndromes associated with increased cancer risk, including BRCA2 gene mutation, Lynch syndrome, familial atypical multiple mole melanoma syndrome (caused by mutations in p16/CDKN2A), Peutz-Jeghers syndrome, and Von Hippel-Lindau syndrome. Other forms of aggressive cancer include triple negative breast cancer (TNBC), which accounts for 10-20% of invasive breast cancers. Risk factors for TNBC include race (African American), age (younger at diagnosis), and BRCA1 mutations. In summary, there are well-defined groups of individuals with an increased risk of pancreatic cancer or TNBC, who can be targeted for active screening and preventive intervention. The current project will examine the cancer preventive efficacy and immunogenicity of ENO1 DNA vaccine developed by Novelli et al. and determine immune correlates of protection, using genetically engineered mouse models of PDA and TNBC.