Abnormally glycosylated MUC1 establishes a positive feedback circuit of inflammatory cytokines, mediated by NF-κB p65 and EzH2, in colitis-associated cancer.

The abnormal hypoglycosylated form of the epithelial mucin MUC1 is over-expressed in chronic inflammation and on human adenocarcinomas, suggesting its potential role in inflammation-driven tumorigenesis. The presence of human MUC1 aggravates colonic inflammation and increases tumor initiation and progression in an in vivo AOM/DSS mouse model of colitis-associated cancer (CAC). High expression levels of pro-inflammatory cytokines, including TNF-α and IL-6, were found in MUC1+ inflamed colon tissues. Exogenous TNF-α promoted the transcriptional activity of MUC1 as well as over-expression of its hypoglycosylated form in intestinal epithelial cells (IECs). In turn, hypoglycosylated MUC1 in IECs associated with p65 and up-regulated the expression of NF-κB-target genes encoding pro-inflammatory cytokines. Intestinal chronic inflammation also increased the expression of histone methyltransferase Enhancer of Zeste protein-2 (EzH2) and its interaction with cytokine promoters. Consequently, EzH2 was a positive regulator of MUC1 and p65-mediated IL-6 and TNF-α gene expression, and this function was not dependent on its canonical histone H3K27 methyltransferase activity. Our findings provide a mechanistic basis for already known tumorigenic role of the hypoglycosylated MUC1 in CAC, involving a transcriptional positive feedback loop of pro-inflammatory cytokines.

mSA2 affinity-enhanced biotin-binding CAR T cells for universal tumor targeting.

Chimeric antigen receptor T cells (CAR-Ts) are promising cancer therapeutics. However, since cancer cells can lose the CAR-targeted antigen and avoid destruction, targeting multiple antigens with multiple CARs has been proposed. We illustrate here a less cumbersome alternative, anti-tag CARs (AT-CARs) that bind to tags on tumor-targeting antibodies. We have created novel AT-CARs, using the affinity-enhanced monomeric streptavidin 2 (mSA2) biotin-binding domain that when expressed on T cells can target cancer cells coated with biotinylated antibodies. Human T cells expressing mSA2 CARs with CD28-CD3ζ and 4-1BB-CD3ζ signaling domains were activated by plate-immobilized biotin and by tumor cells coated with biotinylated antibodies against the tumor-associated antigens CD19 and CD20. Furthermore, mSA2 CAR T cells were capable of mediating cancer cell lysis and IFNγ production in an antibody dose-dependent manner. The mSA2 CAR is a universal AT-CAR that can be combined with biotinylated tumor-specific antibodies to potentially target many different tumor types.

Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential.

MUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen. We purified, cloned, and characterized 13 IgGs specific for several tumor-associated MUC1 epitopes with a wide range of binding affinities. These antibodies bind hypoglycosylated MUC1 on human cancer cell lines and tumor tissues but show no reactivity against fully-glycosylated MUC1 on normal cells and tissues. We found that several antibodies activate complement-mediated cytotoxicity and that T cells carrying chimeric antigen receptors with the antibody variable regions kill MUC1(+) target cells, express activation markers, and produce interferon gamma. Fully-human and tumor-specific, these antibodies are candidates for further testing and development as immunotherapeutic drugs.

A tunable zinc finger-based framework for Boolean logic computation in mammalian cells.

The ability to perform molecular-level computation in mammalian cells has the potential to enable a new wave of sophisticated cell-based therapies and diagnostics. To this end, we developed a Boolean logic framework utilizing artificial Cys(2)-His(2) zinc finger transcription factors (ZF-TFs) as computing elements. Artificial ZFs can be designed to specifically bind different DNA sequences and thus comprise a diverse set of components ideal for the construction of scalable networks. We generate ZF-TF activators and repressors and demonstrate a novel, general method to tune ZF-TF response by fusing ZF-TFs to leucine zipper homodimerization domains. We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional repressors that show 1.3- to 16-fold repression. Using these ZF-TFs, we compute OR, NOR, AND and NAND logic, employing hybrid promoters and split intein-mediated protein splicing to integrate signals. The split intein strategy is able to fully reconstitute the ZF-TFs, maintaining them as a uniform set of computing elements. Together, these components comprise a robust platform for building mammalian synthetic gene circuits capable of precisely modulating cellular behavior.