Inhibition of the spindle assembly checkpoint kinase TTK enhances the efficacy of docetaxel in a triple-negative breast cancer model.

BACKGROUND: Triple-negative breast cancers (TNBC) are considered the most aggressive type of breast cancer, for which no targeted therapy exists at the moment. These tumors are characterized by having a high degree of chromosome instability and often overexpress the spindle assembly checkpoint kinase TTK. To explore the potential of TTK inhibition as a targeted therapy in TNBC, we developed a highly potent and selective small molecule inhibitor of TTK, NTRC 0066-0. RESULTS AND CONCLUSIONS: The compound is characterized by long residence time on the target and inhibits the proliferation of a wide variety of human cancer cell lines with potency in the same range as marketed cytotoxic agents. In cell lines and in mice, NTRC 0066-0 inhibits the phosphorylation of a TTK substrate and induces chromosome missegregation. NTRC 0066-0 inhibits tumor growth in MDA-MB-231 xenografts as a single agent after oral application. To address the effect of the inhibitor in breast cancer, we used a well-defined mouse model that spontaneously develops breast tumors that share key morphologic and molecular features with human TNBC. Our studies show that combination of NTRC 0066-0 with a therapeutic dose of docetaxel resulted in doubling of mouse survival and extended tumor remission, without toxicity. Furthermore, we observed that treatment efficacy is only achieved upon co-administration of the two compounds, which suggests a synergistic in vivo effect. Therefore, we propose TTK inhibition as a novel therapeutic target for neoadjuvant therapy in TNBC.

In vitro evaluation of DNA-DNA hybridization as a two-step approach in radioimmunotherapy of cancer.

The specific delivery of radioisotopes to a tumor at minimal radiation of normal tissue is the ultimate aim of radioimmunotherapy. In this respect a two-step pretargeting regimen generally leads to an improved tumor to normal tissue uptake ratio compared to direct administration of radioimmunoconjugates. In this paper, in vitro studies are described in which the specific hybridization of complementary DNA fragments is the recognition mechanism in a pretargeting regimen comprising tumor cell saturation with a monoclonal antibody (MoAb)-oligonucleotide conjugate, followed by administration of the radiolabeled complementary oligonucleotide. Complementary oligodeoxynucleotides (15-mers; melting temperature, 68 degrees C) were prepared on a DNA synthesizer. The 5'-end was derivatized with a functional group for labeling with iodine, and the 3'-end was substituted with an amino function suitable for conjugation to an antibody (or attachment of a biotin residue). Both terminal modifications ensure stability of the oligonucleotides against exonucleases because the unconjugated form is stable for 24 h and the conjugated form is stable for several days when incubated in human plasma at 37 degrees C. Antibody-DNA conjugates were prepared by introduction of sulfhydryl groups into the oligonucleotide, followed by conjugation to maleimide-substituted MoAbs. Typically, 3 oligonucleotides were conjugated to an IgG, and 4-6 were conjugated to an IgM with preservation of immunoreactivity. Histochemistry on fresh frozen sections of breast cancer tissue demonstrated qualitatively the specificity of our two-step procedure. In vitro experiments with human tumor cell lines and tumor-specific MoAbs showed that, after saturation with tumor-specific MoAb-DNA conjugates, quantitative hybridization of the tumor cell-bound oligonucleotides occurred at a 30-fold excess of the labeled complementary oligonucleotide: hybridization was complete after 30 min of incubation. No reaction was observed with an irrelevant MoAb-DNA conjugate. The oligonucleotide was neither taken up by tumor cells or endothelial cells nor hybridized to a significant extent with human genomic DNA. These data indicate the feasibility of this two-step approach in radioimmunotherapy.

sLex is not responsible for the interaction of sLex-positive memory T lymphocytes with E-selectin.

E-selectin in an adhesion molecule that is transiently and exclusively expressed on endothelial cells in response to inflammatory cytokines. In addition, E-selectin participates in the initial interaction of leucocytes with activated endothelial cells. This role of E-selectin in cell adhesion has made it a potential target for modulation of inflammatory processes that, for example, are occurring in autoimmune diseases such as rheumatoid arthritis. Although on granulocytes the ligand for E-selectin has been identified as the tetrasaccharide sialyl Lewis x (sLex), the molecular nature of this ligand on T lymphocytes has not yet been identified. In the present study, it was shown by fluorescence-activated cell sorter (FACS) analysis with the anti-sLex antibody CSLEX1 that T lymphocytes stimulated with phytohaemagglutanin (PHA), interleukin-2 (IL-2) and transforming growth factor-beta 1 (TGF-beta 1) expressed sLex. Furthermore, in a cell adhesion assay these activated T cells of the memory phenotype bound specifically to E-selectin-transfected Chinese hamster ovary (E-CHO) cells. This adhesion could be blocked with an anti-E-selectin antibody but not with CSLEX1. In the same assay, the interaction of sLex-positive U937 cells with the E-CHO cells could be inhibited both with anti-E-selectin and CSLEX1 antibodies. From these results it can be inferred that sLex on activated T lymphocytes is not responsible for the interaction with E-selectin. Rather, these results suggest that stimulated T lymphocytes express an additional E-selectin ligand(s) with much higher avidity for E-selectin than sLex.