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Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
Survivin functions as an apoptosis inhibitor and a regulator of cell division during development and tumorigenesis. Since survivin is a highly relevant target for tumor therapy, we investigated whether interference with it’s dynamic cellular localization represents a novel strategy to inhibit survivin’s cancer promoting functions. We confirmed survivin overexpression in head and neck as well as in colorectal cancers and identified an evolutionary conserved Crm1-dependent nuclear export signal (NES) in survivin. Importantly, nuclear export was required for survivin mediated protection against chemo- and radiotherapy-induced apoptosis by securing efficient interference with cytoplasmic caspases. In dividing cells, the NES was required for tethering of survivin and of the survivin/Aurora-B kinase complex to the mitotic machinery, which was inevitable for proper cell division. The clinical relevance of our findings was supported by showing that preferential nuclear localization of survivin correlated with enhanced survival in a cohort of colorectal cancer patients. Targeting survivin’s nuclear export by the application of NES-specific antibodies promoted its nuclear accumulation and inhibited its cytoprotective function. We here show that nuclear export is essential for the tumor promoting activities of survivin and encourage the identification of chemical inhibitors to specifically interfere with survivin’s nuclear export as a novel class of anticancer therapeutics.
