Many sarcomas and leukemias carry non-random chromosomal translocations encoding mutant fusion transcription factors that are essential to their molecular pathogenesis. These novel, tumor-specific proteins provides a unique opportunity for the development of highly selective anticancer drugs that has yet to be exploited. A particularly clear example is provided by Ewing's Sarcoma Family Tumors (ESFT) which contain a characteristic t(11;22) translocation leading to expression of the oncogenic fusion protein EWS-FLI1. EWS-FLI1 is a disordered protein that precluded standard structure-based small molecule inhibitor design. Using surface plasmon resonance screening, we discovered a lead compound, NSC635437. A derivative compound, YK-4-279, blocks RHA binding to EWS-FLI1, induces apoptosis in ESFT cells, and reduces the growth of ESFT orthotopic xenografts. These findings provide proof of principle that inhibiting the interaction of mutant cancer-specific transcription factors with the normal cellular binding partners required for their oncogenic activity provides a promising strategy for the development of uniquely effective, tumor-specific anticancer agents.

We analyzed 28 thymic epithelial tumors (TETs) using next-generation sequencing and identified a missense mutation (chromosome 7 c.74146970T>A) in GTF2I at high frequency in type A thymomas, a relatively indolent subtype. In a series of 274 TETs, we detected the GTF2I mutation in 82% of type A and 74% of type AB thymomas but rarely in the aggressive subtypes, where recurrent mutations of known cancer genes have been identified. Therefore, GTF2I mutation correlated with better survival. GTF2I β and δ isoforms were expressed in TETs, and both mutant isoforms were able to stimulate cell proliferation in vitro. Thymic carcinomas carried a higher number of mutations than thymomas (average of 43.5 and 18.4, respectively). Notably, we identified recurrent mutations of known cancer genes, including TP53, CYLD, CDKN2A, BAP1 and PBRM1, in thymic carcinomas. These findings will complement the diagnostic assessment of these tumors and also facilitate development of a molecular classification and assessment of prognosis and treatment strategies.

Ezrin is a multifunctional protein that connects the actin cytoskeleton to the extracellular matrix through transmembrane proteins. High ezrin expression is associated with lung metastasis and poor survival in cancer. We screened small molecule libraries for compounds that directly interact with ezrin protein using surface plasmon resonance to identify lead compounds. The secondary functional assays used for lead compound selection included ezrin phosphorylation as measured by immunoprecipitation and in vitro kinase assays, actin binding, chemotaxis, invasion into an endothelial cell monolayer, zebrafish and Xenopus embryonic development, mouse lung organ culture and an in vivo lung metastasis model. Two molecules, NSC305787 and NSC668394, that directly bind to ezrin with low micromolar affinity were selected based on inhibition of ezrin function in multiple assays. They inhibited ezrin phosphorylation, ezrin–actin interaction and ezrin-mediated motility of osteosarcoma (OS) cells in culture. NSC305787 mimicked the ezrin morpholino phenotype, and NSC668394 caused a unique developmental defect consistent with reduced cell motility in zebrafish. Following tail vein injection of OS cells into mice, both molecules inhibited lung metastasis of ezrinsensitive cells, but not ezrin-resistant cells. The small molecule inhibitors NSC305787 and NSC668394 demonstrate a novel targeted therapy that directly inhibits ezrin protein as an approach to prevent tumor metastasis.

Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITCtubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.

The signaling/oncogenic activity of beta-catenin can be repressed by activation of the vitamin D receptor (VDR). Conversely, high levels of beta-catenin can potentiate the transcriptional activity of 1,25-dihydroxyvitamin D3 (1,25D). We show here that the effects of beta-catenin on VDR activity are due to interaction between the activator function-2 (AF-2) domain of the VDR and C terminus of beta-catenin. Acetylation of the beta-catenin C terminus differentially regulates its ability to activate TCF or VDR-regulated promoters. Mutation of a specific residue in the AF-2 domain, which renders the VDR trancriptionally inactive in the context of classical coactivators, still allows interaction with beta-catenin and ligand-dependent activation of VDRE-containing promoters. VDR antagonists, which block the VDRE-directed activity of the VDR and recruitment of classical coactivators, do allow VDR to interact with beta-catenin, which suggests that these and perhaps other ligands would permit those functions of the VDR that involve beta-catenin interaction.