Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, it is expected the development of a potent, selective small molecule inhibitor of Mps1 would greatly facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 co-crystal structures of novel, selective small molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit, and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells possessing extra centrosomes, an abnormality found in some cancers, Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.
The MYC protooncogene is frequently deregulated in human cancers. Here, by screening a kinase-directed library of small inhibitory RNAs, we identify glycogen synthase kinase 3 (GSK3) as a gene whose inactivation potentiates TNF-related apoptosisinducing ligand death receptor-mediated apoptosis specifically in MYC-overexpressing cells. Small inhibitory RNA-induced silencing of GSK3 prevents phosphorylation of MYC on T58, thereby inhibiting recognition of MYC by the E3 ubiquitin ligase component FBW7. Attenuating the GSK3-FBW7 axis results in stabilization of MYC, up-regulation of surface levels of the TNF-related apoptosis-inducing ligand death receptor 5, and potentiation of death receptor 5-induced apoptosis in vitro and in vivo. These results identify GSK3 and FBW7 as potential cancer therapeutic targets and MYC as a critical substrate in the GSK3 survivalsignaling pathway. The results also demonstrate paradoxically that MYC-expressing tumors might be treatable by drug combinations that increase rather than decrease MYC oncoprotein function. The MYC protooncogene encodes a basic helix-loop-helix leucine zipper transcription factor that plays a central role in promoting the development of many human cancers (1, 2). In addition to driving cancer cell growth and proliferation, MYC is also capable of sensitizing cells to apoptosis (3,4), an activity that we recently proposed might be exploitable pharmacologically to selectively kill MYC-expressing tumors (5). We showed that MYC activation results in up-regulation of cell surface levels of death receptor 5 (DR5; also known as TRAIL-R2, TNFRSF10B), an apoptosis-inducing receptor for the cytokine TNF-related apoptosis-inducing ligand (TRAIL) (reviewed in refs. 6 and 7). MYCoverexpressing cells can therefore be killed preferentially over isogenic normal cells by agonists of DR5 apoptotic signaling. This MYC-induced apoptotic sensitivity may be a primary mechanism underlying TRAIL's unusual ability, unique among the TNF family of death ligands, to induce apoptosis in tumor cells preferentially over normal cells (5,(8)(9)(10)(11).Recombinant human TRAIL and agonistic antibodies against its two death-inducing receptors, DR4 and DR5, are currently undergoing development as cancer therapeutics. However, because many tumors, including MYC-expressing tumors, are resistant or only weakly sensitive to their effects (10), it would be desirable to identify agents that potentiate TRAIL-induced apoptosis. Here, to this end, we screened a library of small inhibitory RNAs (siRNAs) directed primarily against the protein kinase superfamily to identify genes whose inactivation potentiates DR5-mediated apoptosis specifically in MYC-expressing cells. This screen can be thought of as a sensitized synthetic lethal genetic screen (12, 13) in which the phenotypic output, lethality, is sensitized not only by a genetic alteration, MYC activation, but also by an environmental condition, i.e., by the presence of a suboptimal dose of DR5 agonistic antibody. Among the genes identified in th...
Mad1 is a member of theMad-dependent repression is mediated by a complex of proteins that is tethered to Mad proteins through mSin3 (24, 25). Several proteins have been identified in this complex, including N-CoR, SAP30, and histone deacetylases (26 -32). The presence of the latter suggests that the Mad repressor complex functions at least in part by affecting chromatin structure. This complex can dominantly interfere with activator-induced transcription (22,23). Together, these findings support the concept that the Myc/Max/Mad network affects cellular behavior through the regulation of specific target genes. Whether Myc and Mad proteins target the same genes or whether Myc-responsive genes exist which are not affected by Mad and vice versa is not resolved at present.Myc protein expression is tightly linked to cellular proliferation and the forced expression of Myc stimulates cell cycle progression and inhibits differentiation (for review, see Refs. 1 and 33). In addition, deregulated expression of myc genes as a result of chromosomal aberrations is frequently found in human tumors and Myc cooperates with an activated form of Ras in the transformation of rat embryo fibroblasts (for review, see Ref. 34). The important function of Myc proteins in promoting proliferation is further supported by the findings that the homozygous deletion of either c-myc or N-myc is embryonically lethal in mice (35-37). Furthermore, Myc can also promote apoptosis under specific circumstances (for review, see Ref. 38). Thus, Myc proteins are critical components of a regulatory network underlying cell growth control and have been suggested to be part of a molecular switch that affects growth, differentiation, and apoptosis.In contrast to Myc proteins, Mad proteins are expressed in differentiating, terminally differentiated, and resting cells (5, 6, 39 -42). Constitutive expression of Mad proteins interferes with cellular growth and with re-entry of resting cells into the cell cycle (7, 8, 26, 43-45). Furthermore, these proteins inhibit * This work was supported by a predoctoral fellowship from the Cusanus Werk (to S. G.), by Grant Lu 466/6-2 from the Deutsche Forschungsgemeinschaft, and by a grant from the Fonds der Chemischen Industrie (to B. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Present address: DLR Projektträ ger des BMBF, 53175 Bonn, Germany.§ These authors contributed equally to this work. ¶ To whom correspondence should be addressed: Inst. fü r Molekularbiologie, OE 5250, Medizinische Hochschule Hannover, Carl-Neuberg Str. 1, 30625 Hannover, Germany. Tel.: 49-511-532-5954; Fax: 49-511-532-4283; E-mail: luscher.bernard@mh-hannover.de 1 The abbreviations used are: bHLHZip, basic region/helix-loop-helix/ leucine zipper; CAD, carbamoyl-phosphate synthase/aspartate transferase/dihydroorotase; CDK, cyclin-dependent kinase; CMV, cytomegaloviru...
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