Fine-mapping of the cell-division cycle, notably the identification of mitotic kinase signaling pathways, provides novel opportunities for cancer-drug discovery. As a key regulator of multiple steps during mitotic progression across eukaryotic species, the serine/threonine-specific Polo-like kinase 1 (Plk1) is highly expressed in malignant cells and serves as a negative prognostic marker in specific human cancer types . Here, we report the discovery of a potent small-molecule inhibitor of mammalian Plk1, BI 2536, which inhibits Plk1 enzyme activity at low nanomolar concentrations. The compound potently causes a mitotic arrest and induces apoptosis in human cancer cell lines of diverse tissue origin and oncogenome signature. BI 2536 inhibits growth of human tumor xenografts in nude mice and induces regression of large tumors with well-tolerated intravenous dose regimens. In treated tumors, cells arrest in prometaphase, accumulate phosphohistone H3, and contain aberrant mitotic spindles. This mitotic arrest is followed by a surge in apoptosis, detectable by immunohistochemistry and noninvasive optical and magnetic resonance imaging. For addressing the therapeutic potential of Plk1 inhibition, BI 2536 has progressed into clinical studies in patients with locally advanced or metastatic cancers.

Although the MAPK pathway is frequently deregulated in cancer, inhibitors targeting RAF or MEK have so far shown clinical activity only in BRAF- and NRAS-mutant melanoma. Improvements in efficacy may be possible by combining inhibition of mitogenic signal transduction with inhibition of cell-cycle progression. We have studied the preclinical pharmacology of BI 847325, an ATP-competitive dual inhibitor of MEK and Aurora kinases. Potent inhibition of MEK1/2 and Aurora A/B kinases by BI 847325 was demonstrated in enzymatic and cellular assays. Equipotent effects were observed in BRAF-mutant cells, whereas in KRAS-mutant cells, MEK inhibition required higher concentrations than Aurora kinase inhibition. Daily oral administration of BI 847325 at 10 mg/kg showed efficacy in both BRAF- and KRAS-mutant xenograft models. Biomarker analysis suggested that this effect was primarily due to inhibition of MEK in BRAF-mutant models but of Aurora kinase in KRAS-mutant models. Inhibition of both MEK and Aurora kinase in KRAS-mutant tumors was observed when BI 847325 was administered once weekly at 70 mg/kg. Our studies indicate that BI 847325 is effective in in vitro and in vivo models of cancers with BRAF and KRAS mutation. These preclinical data are discussed in the light of the results of a recently completed clinical phase I trial assessing safety, tolerability, pharmacokinetics, and efficacy of BI 847325 in patients with cancer. Mol Cancer Ther; 15(10); 2388-98. ©2016 AACR.

Previously, we reported that the major stress-inducible heat shock protein 70 (Hsp70) acts as a recognition structure for natural killer (NK) cells, if localized on the cell surface of tumor cells. Incubation of purified NK cells with lowdose interleukin (IL)-2 (100 IU/mL) plus recombinant Hsp70-protein or the immunogenic 14-mer Hsp70-peptide TKDNNL-LGRFELSG 450-463 , termed TKD (2 g/mL), enhances the cytolytic activity against Hsp70 membrane-positive (CXϩ) but not against Hsp70-negative (CXϪ) tumor cells. Here, we show that the cytolytic activity against Hsp70-positive tumor cells is inducible by incubation of unseparated peripheral blood mononuclear cells (PBMNC) with low-dose IL-2 plus TKD. Cell sorting experiments revealed that within the PBMNC population CD94ϩ/CD3Ϫ NK cells, and not CD94Ϫ/CD3ϩ T cells, mediate the cytotoxic activity against Hsp70-positive tumor cells. The antitumoral effect of PBMNC stimulated either with IL-2 plus TKD or with IL-2 alone was assessed in tumor-bearing severe combined immunodeficiency/beige mice. A single intravenous (iv) injection of 40 ϫ 10 6 IL-2 plus TKD-stimulated PBMNC (containing 5.2 ϫ 10 6 NK cells) on day 4 results in a 60% reduction in tumor size, from 3.89 g to 1.56 g. In contrast, the adoptive transfer of the identical amount PBMNC stimulated with low-dose IL-2 only (containing 4.4 ϫ 10 6 NK cells) reduces the tumor size only less than 10% (3.64 g). A phenotypic characterization of the excised tumors revealed that predominantly Hsp70-positive tumor cells were eliminated by TKD-activated PBMNC. Kinetic studies demonstrate that the in vivo cytolytic capacity of TKD-stimulated PBMNC is dependent on the effector to target cell ratio. An iv injection of effector cells on day 1 or 2 after tumor cell inoculation results in significantly smaller tumors (0.77 g or 0.89 g) on day 21 as compared with mice that were immunoreconstituted on day 4 or 8 (1.39 g or 2.23 g). The tumor size of nonimmunoreconstituted control animals was 3.55 g.

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Background. The serine/threonine kinase Aurora B is involved in the regulation of several mitotic processes, including chromosome condensation, congression and segregation as well as cytokinesis. These essential functions of Aurora B and its overexpression in many cancer types render this protein kinase an attractive target for anticancer drug development. Methods. BI 811283 was profiled in enzymatic kinase assays as well as in proliferation assays on various human cancer cell lines. Cell cycle status was assessed by DNA content analysis (Cellomics ArrayScan, FACScalibur). Histone H3 phosphorylation was determined by immunofluorescence (Cellomics ArrayScan). Apoptosis was detected by Western blotting for cleaved PARP and microscopic enumeration of DAPI-stained cells showing nuclear fragmentation. Senescent cells were identified by staining for SA-ß-Gal activity. Results. BI 811283 inhibited human Aurora B kinase activity with an IC50 value of 9 nM, Aurora A and C kinases with 70 nM and 17 nM, respectively. In a panel of 46 additional kinases representative of the human kinome, BI 811283 at 1000 nM inhibited 7/46 kinases by more than 50%. EC50 values for inhibition of proliferation of >20 human cancer cell lines were in the range of 2 to 14 nM. In the non-small cell lung cancer cell line NCI-H460, treatment with BI 811283 resulted in a rapid (<1 h) inhibition of histone H3 phosphorylation. Within 48 h of treatment, the fraction of polyploid cells increased from <5% to >80%, paralleled by a marked increase in cell volume. An increase of cleaved poly (ADP-ribose) polymerase and a concomitant increase in the fraction of cells with nuclear fragmentation from <1% to 7% was observed after 72 h and 96 h of treatment. Within 96 h, the fraction of senescent cells markedly increased from <3% to 25% of the population. Conclusions. BI 811283 is a potent and selective Aurora kinase inhibitor that inhibits proliferation of cancer cells independent of tissue origin or oncogenome status. Treated cells exhibit a polyploid phenotype characteristic for Aurora B inhibition and show hallmarks of senescence as well as a slow onset of apoptosis in a small fraction of cells. In vivo activity of BI 811283 has been demonstrated in multiple cancer xenograft models in nude mice (see accompanying poster). Phase I clinical trials are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1080.