Michael H. Kagey

Mediator and cohesin connect gene expression and chromatin architecture.

Transcription factors control cell-specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. Here we report that mediator and cohesin physically and functionally connect the enhancers and core promoters of active genes in murine embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with cohesin, which can form rings that connect two DNA segments. The cohesin-loading factor Nipbl is associated with mediator–cohesin complexes, providing a means to load cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by mediator and cohesin. Mediator and cohesin co-occupy different promoters in different cells, thus generating cell-type-specific DNA loops linked to the gene expression program of each cell.

Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells.

Embryonic stem (ES) cells have a unique regulatory circuitry, largely controlled by the transcription factors Oct4, Sox2, and Nanog, which generates a gene expression program necessary for pluripotency and self-renewal. How external signals connect to this regulatory circuitry to influence ES cell fate is not known. We report here that a terminal component of the canonical Wnt pathway in ES cells, the transcription factor T-cell factor-3 (Tcf3), co-occupies promoters throughout the genome in association with the pluripotency regulators Oct4 and Nanog. Thus, Tcf3 is an integral component of the core regulatory circuitry of ES cells, which includes an autoregulatory loop involving the pluripotency regulators. Both Tcf3 depletion and Wnt pathway activation cause increased expression of Oct4, Nanog, and other pluripotency factors and produce ES cells that are refractory to differentiation. Our results suggest that the Wnt pathway, through Tcf3, brings developmental signals directly to the core regulatory circuitry of ES cells to influence the balance between pluripotency and differentiation.

Abstract 369: Therapeutic targeting of the Wnt antagonist DKK1 with a humanized monoclonal antibody in oncology indications

Wnt signaling is a fundamental pathway that is dysregulated in oncology. The Wnt antagonist DKK1 is expressed in a variety of tumor types which frequently correlates with a poor prognosis, including overall survival. DKK1 has known oncogenic activity by stimulating proliferation, metastasis, and angiogenesis, and recently been implicated in contributing to an immunosuppressive tumor microenvironment. The neutralization of DKK1 is hypothesized to have efficacy from both a direct antitumor effect and through an immune stimulated response. Here we describe the characterization of DKN-01, a humanized monoclonal therapeutic antibody to DKK1. DKN-01 binds DKK1 with high affinity and selectivity, disrupts the interaction of DKK1 with the LRP6 co-receptor, and neutralizes DKK1 activity in a cell based assay. In vivo, DKN-01 has efficacy both as a monotherapy and in combination with chemotherapies in a non-small cell lung (NSCLC) cancer A549 xenograft model. Results suggest that DKN-01 has an antiangiogenic effect and may stimulate a NK cell mediated antitumor response. Clinically, DKN-01 is being evaluated in relapsed/refractory esophageal cancer patients in combination with paclitaxel, and preliminary results demonstrate promising activity. Archival patient tumor samples are currently being analyzed genetically and by IHC for DKK1 and β-catenin staining for biomarker identification. Taken together, our results suggest that DKN-01, a novel therapeutic, has clinical efficacy by disrupting Wnt signaling, which results in a direct anti-tumor effect and stimulates a pro-inflammatory tumor response. Citation Format: Michael H. Kagey, Yinyuan Wu, Xinjun Zhang, Cynthia A. Sirad, Shane E. Mulligan, Xi He, Christopher K. Mirabelli. Therapeutic targeting of the Wnt antagonist DKK1 with a humanized monoclonal antibody in oncology indications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 369. doi:10.1158/1538-7445.AM2017-369