Irene Waizenegger

Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1

Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Here we have used live cell imaging to determine the exact time when human securin is degraded in mitosis. We show that the timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, we find that both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer.

ArticleSecurin Is Required for Chromosomal Stability in Human Cells

Abnormalities of chromosome number are the most common genetic aberrations in cancer. The mechanisms regulating the fidelity of mitotic chromosome transmission in mammalian cells are therefore of great interest. Here we show that human cells without an hSecurin gene lose chromosomes at a high frequency. This loss was linked to abnormal anaphases during which cells underwent repetitive unsuccessful attempts to segregate their chromosomes. The abnormal mitoses were associated with biochemical defects in the activation of separin, the sister-separating protease, rendering it unable to cleave the cohesin subunit Scc1 efficiently. These results illuminate the function of mammalian securin and show that it is essential for the maintenance of euploidy.

Scc1/Rad21/Mcd1 Is Required for Sister Chromatid Cohesion and Kinetochore Function in Vertebrate Cells

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.

Regulation of Human Separase by Securin Binding and Autocleavage

BACKGROUND Sister chromatid separation is initiated by separase, a protease that cleaves cohesin and thereby dissolves sister chromatid cohesion. Separase is activated by the degradation of its inhibitor securin and by the removal of inhibitory phosphates. In human cells, separase activation also coincides with the cleavage of separase, but it is not known if this reaction activates separase, which protease cleaves separase, and how separase cleavage is regulated. RESULTS Inhibition of separase expression in human cells by RNA interference causes the formation of polyploid cells with large lobed nuclei. In mitosis, many of these cells contain abnormal chromosome plates with unseparated sister chromatids. Inhibitor binding experiments in vitro reveal that securin prevents the access of substrate analogs to the active site of separase. Upon securin degradation, the active site of full-length separase becomes accessible, allowing rapid autocatalytic cleavage of separase at one of three sites. The resulting N- and C-terminal fragments remain associated and can be reinhibited by securin. A noncleavable separase mutant retains its ability to cleave cohesin in vitro. CONCLUSIONS Our results suggest that separase is required for sister chromatid separation during mitosis in human cells. Our data further indicate that securin inhibits separase by blocking the access of substrates to the active site of separase. Securin proteolysis allows autocatalytic processing of separase into a cleaved form, but separase cleavage is not essential for separase activation.

The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis

Partitioning of the cytoplasm during cytokinesis or cellularisation requires syntaxin-mediated membrane fusion [1-3]. Whereas in animals, membrane fusion promotes ingression of a cleavage furrow from the plasma membrane [4,5], somatic cells of higher plants form de novo a transient membrane compartment, the cell plate, which is initiated in the centre of the division plane and matures into a new cell wall and its flanking plasma membranes [6,7]. Cell plate formation results from the fusion of Golgi-derived vesicles delivered by a dynamic cytoskeletal array, the phragmoplast. Mutations in two Arabidopsis genes, KNOLLE (KN) and KEULE (KEU), cause abnormal seedlings with multinucleate cells and incomplete cell walls [1,8]. The KN gene encodes a cytokinesis-specific syntaxin which localises to the cell plate [9]. Here, we show that KN protein localisation is unaffected in keu mutant cells, which, like kn, display phragmoplast microtubules and accumulate ADL1 protein in the plane of cell division but vesicles fail to fuse with one another. Genetic interactions between KN and KEU were analysed in double mutant embryos. Whereas the haploid gametophytes gave rise to functional gametes, the embryos behaved like single cells displaying multiple, synchronously cycling nuclei, cell cycle-dependent microtubule arrays and ADL1 accumulation between pairs of daughter nuclei. This complete inhibition of cytokinesis from fertilisation indicates that KN and KEU, have partially redundant functions and interact specifically in vesicle fusion during cytokinesis of somatic cells.

The Meiosis I-to-Meiosis II Transition in Mouse Oocytes Requires Separase Activity

Faithful segregation of homologous chromosomes during the first meiotic division is essential for further embryo development. The question at issue is whether the same mechanisms ensuring correct separation of sister chromatids in mitosis are at work during the first meiotic division. In mitosis, sister chromatids are linked by a cohesin complex holding them together until their disjunction at anaphase. Their disjunction is mediated by Separase, which cleaves the cohesin. The activation of Separase requires prior degradation of its associated inhibitor, called securin. Securin is a target of the APC/C (Anaphase Promoting Complex/Cyclosome), a cell cycle-regulated ubiquitin ligase that ubiquitinates securin at the metaphase-to-anaphase transition and thereby targets it for degradation by the 26S proteasome. After securin degradation, Separase cleaves the cohesins and triggers chromatid separation, a prerequisite for anaphase. In yeast and worms, the segregation of homologous chromosomes in meiosis I depends on the APC/C and Separase activity. Yet, it is unclear if Separase is required for the first meiotic division in vertebrates because APC/C activity is thought to be dispensable in frog oocytes. We therefore investigated if Separase activity is required for correct chromosome segregation in meiosis I in mouse oocytes.

A Novel RAF Kinase Inhibitor with DFG-Out–Binding Mode: High Efficacy in BRAF-Mutant Tumor Xenograft Models in the Absence of Normal Tissue Hyperproliferation

BI 882370 is a highly potent and selective RAF inhibitor that binds to the DFG-out (inactive) conformation of the BRAF kinase. The compound inhibited proliferation of human BRAF–mutant melanoma cells with 100× higher potency (1–10 nmol/L) than vemurafenib, whereas wild-type cells were not affected at 1,000 nmol/L. BI 882370 administered orally was efficacious in multiple mouse models of BRAF-mutant melanomas and colorectal carcinomas, and at 25 mg/kg twice daily showed superior efficacy compared with vemurafenib, dabrafenib, or trametinib (dosed to provide exposures reached in patients). To model drug resistance, A375 melanoma–bearing mice were initially treated with vemurafenib; all tumors responded with regression, but the majority subsequently resumed growth. Trametinib did not show any efficacy in this progressing population. BI 882370 induced tumor regression; however, resistance developed within 3 weeks. BI 882370 in combination with trametinib resulted in more pronounced regressions, and resistance was not observed during 5 weeks of second-line therapy. Importantly, mice treated with BI 882370 did not show any body weight loss or clinical signs of intolerability, and no pathologic changes were observed in several major organs investigated, including skin. Furthermore, a pilot study in rats (up to 60 mg/kg daily for 2 weeks) indicated lack of toxicity in terms of clinical chemistry, hematology, pathology, and toxicogenomics. Our results indicate the feasibility of developing novel compounds that provide an improved therapeutic window compared with first-generation BRAF inhibitors, resulting in more pronounced and long-lasting pathway suppression and thus improved efficacy. Mol Cancer Ther; 15(3); 354–65. ©2016 AACR.

Efficacy of the highly selective focal adhesion kinase inhibitor BI 853520 in adenocarcinoma xenograft models is linked to a mesenchymal tumor phenotype

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, has attracted interest as a target for pharmacological intervention in malignant diseases. Here, we describe BI 853520, a novel ATP-competitive inhibitor distinguished by high potency and selectivity. In vitro, the compound inhibits FAK autophosphorylation in PC-3 prostate carcinoma cells with an IC50 of 1 nmol/L and blocks anchorage-independent proliferation of PC-3 cells with an EC50 of 3 nmol/L, whereas cells grown in conventional surface culture are 1000-fold less sensitive. In mice, the compound shows long half-life, high volume of distribution and high oral bioavailability; oral dosing of immunodeficient mice bearing subcutaneous PC-3 prostate adenocarcinoma xenografts resulted in rapid, long-lasting repression of FAK autophosphorylation in tumor tissue. Daily oral administration of BI 853520 to nude mice at doses of 50 mg/kg was well tolerated for prolonged periods of time. In a diverse panel of 16 subcutaneous adenocarcinoma xenograft models in nude mice, drug treatment resulted in a broad spectrum of outcomes, ranging from group median tumor growth inhibition values >100% and tumor regression in subsets of animals to complete lack of sensitivity. Biomarker analysis indicated that high sensitivity is linked to a mesenchymal tumor phenotype, initially defined by loss of E-cadherin expression and subsequently substantiated by gene set enrichment analysis. Further, we obtained microRNA expression profiles for 13 models and observed that hsa-miR-200c-3p expression is strongly correlated with efficacy (R2 = 0.889). BI 853520 is undergoing evaluation in early clinical trials.

The FAK inhibitor BI 853520 exerts anti-tumor effects in breast cancer

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that regulates a plethora of downstream signaling pathways essential for cell migration, proliferation and death, processes that are exploited by cancer cells during malignant progression. These well-established tumorigenic activities, together with its high expression and activity in different cancer types, highlight FAK as an attractive target for cancer therapy. We have assessed and characterized the therapeutic potential and the biological effects of BI 853520, a novel small chemical inhibitor of FAK, in several preclinical mouse models of breast cancer. Treatment with BI 853520 elicits a significant reduction in primary tumor growth caused by an anti-proliferative activity by BI 853520. In contrast, BI 853520 exerts effects with varying degrees of robustness on the different stages of the metastatic cascade. Together, the data demonstrate that the repression of FAK activity by the specific FAK inhibitor BI 853520 offers a promising anti-proliferative approach for cancer therapy.

Abstract 4558: In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091

The programmed cell death-1 (PD-1) receptor provides inhibitory checkpoint signals to activated T cells upon binding to its ligands, PD-L1 and PD-L2, which are expressed on antigen-presenting cells and cancer cells leading to suppression of T-cell effector function and tumor immune evasion. Blockade of the PD-1 axis using either anti-PD-1 or anti-PD-L1 approved monoclonal antibodies (mAbs) results in improved T-cell effector function and anti-tumor immune responses. Durable tumor responses occur in 15-30% of cancer patients. BI 754091, a humanized IgG4 mAb with high affinity against hPD-1 blocks the interaction between PD-1 and PD-L1 or PD-L2. BI 754091 was characterized in a panel of binding, blocking and functional cell-based assays. In addition, efficacy and safety was assessed in mice and in cynomolgus monkeys, respectively. The ability of BI 754091 to stimulate cytokine production in exhausted human T cells in vitro was tested in an autologous assay system with antigen-specific memory CD4+ T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of BI 754091 or isotype control. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on their surface. Furthermore, PD-L1 and PD-L2 were expressed on the dendritic cells. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure for T-cell activation. BI 754091 showed a potent dose dependent T-cell activation. The average fold increase of IFNγ was 7.9 as compared to isotype control, with an average EC50 of 0.9 nM. The in vivo activity of BI 754091 was determined in MC-38 tumor-bearing mice, using a mouse strain where parts of the extracellular domain of murine PD-1 was replaced by the corresponding human PD-1 domain (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice). A dose of 10 mg/kg BI 754091, given either as single treatment or in a twice weekly schedule, induced significant tumor growth inhibition (median TGI of 83% and 90%, respectively) and complete responses (CRs) in some tumors (3 CRs out of 10 and 2 CRs out of 10, respectively). BI 754091 binds to PD-1 from cynomolgus monkeys with comparable affinities as to human PD-1, thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754091 were well tolerated without adverse immune-related effects. BI 754091 is currently undergoing clinical investigations (NCT02952248). Citation Format: Markus Zettl, Melanie Wurm, Otmar Schaaf, Inigo Tirapu, Sven Mostbock, Markus Reschke, Stephan-Michael Schmidbauer, Lee Frego, Ivo C. Lorenz, Michael Thibodeau, Diann Blanset, Elisa Oquendo Cifuentes, Jurgen Moll, Norbert Kraut, Eric Borges, Anne Vogt, Jonathon Sedgwick, Irene C. Waizenegger. In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4558.