The knowledge about Taspase and its particular activation me

The knowledge about Taspase1 and its particular activation mechanism was subsequently used to design a dominant-negative variant of Taspase1 (dnTASP1). This mutant combines the S291A and C163E mutations. Therefore, such a mutant monomer — per se unable to become activated itself — binds with the modified docking head to other Taspase1 monomers and thereby blocks the autoproteolytic step. Co-immunoprecipitation and in vitro experiments revealed that neither dnTASP1 homodimers nor dnTASP1::Taspase1 heterodimers display any autoproteolytic or substrate hydrolysis activity (Fig. 4AB). DnTASP1 should represent per se a therapeutic protein in the context of t(4;11) leukemia, because the co-expression of dnTASP1 together with the leukemogenic AF4–MLL oncoprotein resulted in the disappearance of the unprocessed AF4–MLL fusion protein (Fig. 7A). This is due to the fact that unprocessed AF4–MLL fusion protein (p328) is a substrate of the two E3-ligases SIAH1 and SIAH2. Both E3 ligases mediate polyubiquitinylation, which in turn leads to a rapid proteasomal degradation of the AF4–MLL oncoprotein (Bursen et al., 2004). By contrast, Taspase1-hydrolyzed AF4–MLL (p178N and p180C) forms a heterodimer which serves as a platform for the assembly of a high molecular weight complex that is highly stable and leukemogenic (Benedikt et al., 2011). We attempted to validate this hypothesis by analyzing the effects of dnTASP1 on cells that bear a t(4;11) translocation. For this purpose, we used our recently established Sleeping Beauty vectors to transfect SEM cells (Kowarz et al., 2015). After a 4week period of ‘pulsed selection’ to obtain transgenic SEM cells, we analyzed the two transgenic cell populations for their growth behavior. The induction of the dnTASP1 transgene led to a significant and reproducible reduction of cell growth in a doxycyclin-dependent manner (Fig. 8), while control experiments (Luciferase) did not reveal such effects. These data are supporting our notion about the role of dnTASP1 for AF4–MLL. Taspase1 is presumably not only important in the context of normal MLL functions or leukemia, but also for solid tumors. Several cell lines deriving from different solid tumors overexpress Taspase1 (Takeda et al., 2006). This suggests that Taspase1 has a potential function in tumor cells although only MLL germline gamma-Secretase inhibitor IX are expressed. Solid tumors may require more cleaved MLL complexes e.g., to enhance transcriptional processes. This important concept has been recently successfully validated for breast cancer cells (Dong et al., 2014). Several groups — including our own — have already tried to identify potential Taspase1 inhibitors (Lee et al., 2009; Chen et al., 2012). However, all these screening strategies failed so far. The reason for this is not quite clear but may be due to the inhibitory chloride anion that disables Taspase1 in the absence of substrate. Conversely, our data imply to use an allosteric inhibition approach in order to target Taspase1 by small molecules. Taspase1 could potentially be inhibited by targeting the ‘docking zone’, e.g., by blocking the movement of the E295 residue. However, such experiments would require to crystallize Taspase1 monomers (e.g., the W173A mutant). Since there is no guarantee for good crystals, we suggest to use our established in vivo FRET reporter assay for initial screening experiments. Since this cellular system may respond to any kind of Taspase1 inhibition, it will presumably speed up any kind of screening effort to identify first lead structures.
Author Contributions
Funding This study was supported by grant DKS 2011.09 from the German Children Cancer Aid to RM, and by research grants Ma1876/10-1 and Ma1876/11-1 from the DFG to RM. RM is PI within the CEF on Macromolecular Complexes funded by DFG grant EXC 115.
Role of Funding Resources
Conflict of Interest
Acknowledgements
Introduction ERBB2, also known as HER2 or v-erb-B2 erythroblastic leukemia viral oncogene homolog 2 among other names, is an oncogenic cell surface receptor tyrosine kinase and an important cancer therapeutic target. ERBB2 amplification or overexpression occurs in 20–30% of breast cancer and is a strong predictor of poor disease prognosis (Ross et al., 2009; Slamon et al., 1987). ERBB2 overexpression also occurs in several other types of cancer (Junttila et al., 2003; Lassus et al., 2004; Saffari et al., 1995; Tanner et al., 2005). A number of ERBB2-targeting agents have been developed, including monoclonal antibodies, small molecule tyrosine kinase inhibitors, and antibody-cytotoxic agent conjugates (Incorvati et al., 2013), but they are limited by drug resistance and high drug cost. For example, trastuzumab, a humanized monoclonal antibody, has been the leading agent for treatment of ERBB2-positive breast cancer, but about half of patients do not respond to trastuzumab-based therapy (Pohlmann et al., 2009; Romond et al., 2005; Vogel et al., 2002). Moreover, trastuzumab, which is produced in mammalian cells, costs over $50,000 for a full course of treatment. On the other hand, it is now widely recognized that combination of agents with different targeting mechanisms improves treatment outcome.