High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions.

Polo-like kinase 1 (Plk1) is widely established as one of the most promising targets in oncology. Although the protein kinase domain of Plk1 is highly conserved, the polo-box domain (PBD) of Plk1 provides a much more compelling site to specifically inhibit the localization and target binding of Plk1. We recently identified, via fluorescence polarization assay, the natural product derivative, Poloxin, as the first smallmolecule inhibitor specifically targeting the function of the Plk1 PBD. In this study, we characterized its mitotic phenotype and its function in vitro and in vivo. Poloxin induces centrosome fragmentation and abnormal spindle and chromosome misalignment, which activate the spindle assembly checkpoint and prolong mitosis. Notably, centrosomal fragmentation induced by Poloxin is partially attributable to dysfunctional Kizuna, a key substrate of Plk1 at centrosomes. Moreover, Poloxin strongly inhibits proliferation of a panel of cancer cells by inducing mitotic arrest, followed by a surge of apoptosis. More important, we report, for the first time to our knowledge, that the PBD inhibitor, Poloxin, significantly suppresses tumor growth of cancer cell lines in xenograft mouse models by lowering the proliferation rate and triggering apoptosis in treated tumor tissues. The data highlight that targeting the PBD by Poloxin is a powerful approach for selectively inhibiting Plk1 function in vitro and in vivo.

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Salt-inducible kinases (SIKs) are key metabolic regulators. The imbalance in SIK function is associated with the development of diverse cancers, including breast, gastric, and ovarian cancers. Chemical tools to clarify the roles of SIK in different diseases are, however, sparse and are generally characterized by poor kinome-wide selectivity. Here, we have adapted the pyrido[2,3-d]pyrimidin-7-one-based p21-activated kinase (PAK) inhibitor G-5555 for the targeting of SIK, by exploiting differences in the back-pocket region of these kinases. Optimization was supported by high-resolution crystal structures of G-5555 bound to the known off-targets, MST3 and MST4, leading to a chemical probe, MRIA9, with dual SIK/ PAK activity and excellent selectivity over other kinases. Furthermore, we show that MRIA9 sensitizes ovarian cancer cells to treatment with the mitotic agent paclitaxel, confirming earlier data from genetic knockdown studies and suggesting a combination therapy with SIK inhibitors and paclitaxel for the treatment of paclitaxel-resistant ovarian cancer.

Caspase activation is a hallmark of apoptosis. However, the molecular mechanisms underlying the regulation of caspase-8 activation within the extrinsic death pathway are not well understood. In this study, we demonstrate that procaspase-8 is phosphorylated in mitotic cells by Cdk1/cyclin B1 on Ser-387, which is located at the N terminus of the catalytic subunit p10. This phosphorylation of procaspase-8 on Ser-387 occurs in cancer cell lines, as well as in primary breast tissues and lymphocytes. Furthermore, RNA interferencemediated silencing of cyclin B1 or treatment with the Cdk1 inhibitor RO-3306 enhances the Fas-mediated activation and processing of procaspase-8 in mitotic cells. A nonphosphorylatable procaspase-8 (S387A) facilitates Fas-induced apoptosis during mitosis. Our findings suggest that Cdk1/cyclin B1 activity shields human cells against extrinsic death stimuli and unravel the molecular details of the cross talk between cell cycle and extrinsic apoptotic pathways. Finally, this new mechanism may also contribute to tumorigenesis.Intact apoptotic machinery is essential to maintain the integrity and homeostasis of multicellular organisms (8). The evasion of apoptosis is a hallmark of cancer, as demonstrated by the inability of cancer cells to respond appropriately to signals that normally control unrestricted growth (11). In mammalian cells, the apoptotic response is mediated by either the intrinsic or the extrinsic pathway, depending on the origin of the death stimulus. After stimulation of the death receptor Fas (APO-1/CD95) the death-inducing signaling complex (DISC) assembles, which contains the oligomerized receptor, the adaptor molecule FADD, two isoforms of procaspase-8 (procaspase-8a and -8b), procaspase-10, and c-Flip L/S/R (28, 37). In accordance with the induced proximity model, immediately after DISC formation, procaspase-8, which consists of two death effector domains (DED) and a protease domain containing the p18 and p10 subunits, is proteolytically processed (28). This autoprocessing follows a sequential order of events: while the first cleavage step occurs at Asp-374 and results in the formation of the subunits p43/p41 and p12, the second cleavage at Asp-216 and Asp-384 produces the enzymatically active subunits p18, p10, and the prodomain p26/p24 (5, 14, 27, 40). The mature caspase-8 heterotetramer p18 2 -p10 2 then translocates from the DISC to the cytosol, where it cleaves several substrates, such as Bid, and effector caspases to initiate the apoptotic cascade (22).Increasing evidence highlights the functional importance of procaspase-8 for carcinogenesis; several findings suggest that the impairment of procaspase-8 function by genetic and epigenetic mechanisms correlates with the malignant potential of different types of cancer (6,12,43,44). In the present study, we investigated the functional correlation of the cell cycle with the extrinsic death pathway. The cyclin-dependent kinase 1 (Cdk1) in complex with cyclin B1 (Cdk1/cyclin B1) is one of the key mitotic kinases. The kinase ac...