John Joseph Hunter

Localization of human TACC3 to mitotic spindles is mediated by phosphorylation on Ser558 by Aurora A: a novel pharmacodynamic method for measuring Aurora A activity.

Aurora A is a serine/threonine protein kinase essential for normal mitotic progression. Aberrant increased expression of Aurora A, which occurs frequently in human cancers, results in abnormal mitoses leading to chromosome instability and possibly tumorigenesis. Consequently, Aurora A has received considerable attention as a potential target for anticancer therapeutic intervention. Aurora A coordinates several essential mitotic activities through phosphorylation of a variety of proteins, including TACC3, which modulates microtubule stabilization of the mitotic spindle. Recent studies identified a conserved serine in Xenopus (Ser(626)) and Drosophila (Ser(863)) TACC3 orthologues that is phosphorylated by Aurora A. We show that this conserved serine on human TACC3 (Ser(558)) is also phosphorylated by Aurora A. Moreover, phosphorylation of TACC3 by Aurora A in human cells is essential for its proper localization to centrosomes and proximal mitotic spindles. Inhibition of Aurora A with the selective small molecule inhibitor MLN8054 in cultured human tumor cells resulted in mislocalization of TACC3 away from mitotic spindles in a concentration-dependent manner. Furthermore, oral administration of MLN8054 to nude mice bearing HCT-116 human tumor xenografts caused a dose-dependent mislocalization of TACC3 away from spindle poles that correlated with tumor growth inhibition. As TACC3 localization to mitotic spindles depends on Aurora A-mediated phosphorylation, quantifying TACC3 mislocalization represents a novel pharmacodynamic approach for measuring Aurora A activity in cancer patients treated with inhibitors of Aurora A kinase.

Noninvasive Bioluminescence Imaging of Normal and Spontaneously Transformed Prostate Tissue in Mice

Several transgenic mouse models of prostate cancer have been developed recently that are able to recapitulate many key biological features of the human condition. It would, therefore, be desirable to employ these models to test the efficacy of new therapeutics before clinical trial; however, the variable onset and non-visible nature of prostate tumor development limit their use for such applications. We now report the generation of a transgenic reporter mouse that should obviate these limitations by enabling noninvasive in vivo bioluminescence imaging of normal and spontaneously transformed prostate tissue in the mouse. We used an 11-kb fragment of the human prostate-specific antigen (PSA) promoter to achieve specific and robust expression of firefly luciferase in the prostate glands of transgenic mice. Ex vivo bioluminescence imaging and in situ hybridization analysis confirmed that luciferase expression was restricted to the epithelium in all four lobes of the prostate. We also show that PSA-Luc mice exhibit decreased but readily detectable levels of in vivo bioluminescence over extended time periods following androgen ablation. These results suggest that this reporter should enable in vivo imaging of both androgen-dependent and androgen-independent prostate tumor models. As proof-of-principle, we show that we could noninvasively image SV40 T antigen-induced prostate tumorigenesis in mice with PSA-Luc. Furthermore, we show that our noninvasive imaging strategy can be successfully used to image tumor response to androgen ablation in transgenic mice and, as a result, that we can rapidly identify individual animals capable of sustaining tumor growth in the absence of androgen.

Detection of Activity Centers in Cellular Pathways Using Transcript Profiling

Abstract We present a new computational method for identifying regulated pathway components in transcript profiling (TP) experiments by evaluating transcriptional activity in the context of known biological pathways. We construct a graph representing thousands of protein functional relationships by integrating knowledge from public databases and review articles. We use the notion of distance in a graph to define pathway neighborhoods. The pathways perturbed in an experiment are then identified as the subgraph induced by the genes, referred to as activity centers, having significant density of transcriptional activity in their functional neighborhoods. We illustrate the predictive power of this approach by performing and analyzing an experiment of TP53 overexpression in NCI-H125 cells. The detected activity centers are in agreement with the known TP53 activation effects and our independent experimental results. We also apply the method to a serum starvation experiment using HEY cells and investigate the predicted activity of the transcription factor MYC. Finally, we discuss interesting properties of the activity center approach and its possible applications beyond the comparison of two experiments.

Computational discovery and experimental validation of novel drug targets in immuno-oncology

The past few years have witnessed a renaissance in the field of immuno-oncology largely due to the clinical success in targeting the immune checkpoints CTLA-4 and PD-1. Towards identification of novel immune checkpoint drug targets we developed a dedicated predictive discovery platform. The B7/CD28 discovery platform is a predictive model based on genomic and protein features along with expression patterns of known B7/CD28 proteins. The platform has been tested and validated extensively and has demonstrated its validity by identifying non-novel immune checkpoints such as TIGIT and VISTA, which were not used in the design stage. The B7/CD28 predictive platform was employed to identify several novel immune checkpoint candidates which are currently in different validation stages. In this poster, we will describe our discovery approach as well as our validation path. In addition, we will present experimental data demonstrating the immuno-modulatory function and expression patterns of several of our novel immune checkpoints. These experimental results serve as an additional confirmation to the accuracy of our B7/CD28 predictive discovery platform and shed light on the therapeutic potential of the novel immune checkpoints identified using this unique discovery approach.

Abstract 5027: Identification of novel immune checkpoints as potential targets for cancer immunotherapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Members of the B7/CD28 family of immune checkpoints, such as CTLA4, PD1 and PDL-1, play critical roles in immune cell regulation and have emerged as promising drug targets for cancer immunotherapy. We hypothesize that additional immune checkpoints play a role as negative immune regulators and thus may serve as targets for therapeutic mAbs. Utilizing Compugens predictive discovery platform, we identified novel members of this family that may serve as immune checkpoints. The therapeutic relevance of three of these proteins, CGEN-15001T, CGEN-15022, and CGEN-15049, was confirmed following the validation of their immunomodulatory properties and their expression in various cancers. Two of these proteins, CGEN-15001T and CGEN-15022, are the basis of a license and collaboration agreement recently signed with Bayer as targets for cancer immunotherapy. Here we present results obtained for an additional novel immune checkpoint, CGEN-15049. Following its ectopic expression on cancer cell lines, CGEN-15049 inhibits the activity of NK cells and cytotoxic T cells (CTLs). The fusion protein, consisting of the extracellular domain of CGEN-15049 fused to an IgG Fc domain, displays robust inhibition of T cell activation and enhances iTregs differentiation. IHC studies indicate that CGEN-15049 is expressed in tumor cells of numerous types of cancers, as well as in tumor infiltrating immune cells. Based on its immunomodulatory activities on immune cell types with key roles in cancer immune evasion, together with its expression pattern in cancer tissues, CGEN-15049 may serve as mAb target for cancer immunotherapy. Citation Format: Galit Rotman, Ofer Levy, Amir Toporik, Gady Cojocaru, Liat Dassa, Ilan Vaknin, Shirley Sameah-Greenwald, Inbal Barbiro, Jinhong Fan, Susan Watson, John Hunter, Eyal Neria, Zurit Levine. Identification of novel immune checkpoints as potential targets for cancer immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5027. doi:10.1158/1538-7445.AM2014-5027