Cécile Bougeret

Coupling fission and exit of RAB6 vesicles at Golgi hotspots through kinesin-myosin interactions.

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6–Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.Actin and microtubules play important roles in Golgi structure and function but how they are connected is poorly understood. Here the authors show that KIF20A is involved in the fission process and, in association with Myosin II, serves to anchor RAB6 on Golgi/TGN membranes near microtubules nucleating sites.

New MKLP-2 inhibitors in the paprotrain series: Design, synthesis and biological evaluations

Members of the kinesin superfamily are involved in key functions during intracellular transport and cell division. Their involvement in cell division makes certain kinesins potential targets for drug development in cancer chemotherapy. The two most advanced kinesin targets are Eg5 and CENP-E with inhibitors in clinical trials. Other mitotic kinesins are also being investigated for their potential as prospective drug targets. One recently identified novel potential cancer therapeutic target is the Mitotic kinesin-like protein 2 (MKLP-2), a member of the kinesin-6 family, which plays an essential role during cytokinesis. Previous studies have shown that inhibition of MKLP-2 leads to binucleated cells due to failure of cytokinesis. We have previously identified compound 1 (paprotrain) as the first selective inhibitor of MKLP-2. Herein we describe the synthesis and biological evaluation of new analogs of 1. Our structure-activity relationship (SAR) study reveals the key chemical elements in the paprotrain family necessary for MKLP-2 inhibition. We have successfully identified one MKLP-2 inhibitor 9a that is more potent than paprotrain. In addition, in vitro analysis of a panel of kinesins revealed that this compound is selective for MKLP-2 compared to other kinesins tested and also does not have an effect on microtubule dynamics. Upon testing in different cancer cell lines, we find that the more potent paprotrain analog is also more active than paprotrain in 10 different cancer cell lines. Increased selectivity and higher potency is therefore a step forward toward establishing MKLP-2 as a potential cancer drug target.

Cell signalling associated with fibrinolytic ligand binding to human colorectal carcinoma cells

SummaryAddition of purified plasmin or plasminogen (0.1 μM) to serum-free culture media elevated cellular D-myo-inositol 1,4,5-trisphosphate (InsP3) levels in human colorectal carcinoma cells within 1 h to double those of control cells. This was accompanied by decreases in cellular phosphatidylinositol bisphosphate by 40% in cells exposed to fibrinolytic ligands for up to 1 h. The effect was not due to opening of Ca2+ channels of the type blocked by 5 μM nifedipine, and 100 μM EGTA, a Ca2+ chelator, did not suppress plasmins ability to elevate InsP3. Binding assays at 4° C with125I-labelled plasmin indicated maximum binding within 1 h suggesting that the effects of plasmin may be associated with its cell-binding function. These cells could convert exogenous plasminogen to plasmin with endogenous activation and this was accompanied by a decrease in radioactive phosphatidylinositol well below control levels (13% of control). Our results contribute to evidence for the association of plasmin-binding sites with a signalling system. A cell signalling system indirectly or directly associated with plasmin binding, would permit carcinoma cells to coordinate extracellular fibrinolysis with cell migration and motility through second messengers.

Abstract 4140: Identification of a selective MKLP2/KIF20A inhibitor with high in-vivo antitumor activity

Mitotic kinesins are essential regulators of cancer cell replication and migration. The mitotic kinesin MKLP2/KIF20A, a member of the kinesin-6 family, plays an essential role during cytokinesis and was identified as a potential new target for cancer chemotherapy1. We have previously identified Paprotrain, a new synthetic compound, as the first selective MKLP2 inhibitor2. Recently, we obtained Paprotrain analogues with higher potency on MKLP23. Herein we describe the identification and characterization of BKS0349, a new potent analogue of Paprotrain. BKS0349 compound is 10 times more potent than paprotain on MKLP2 inhibition and has shown an even more restricted specificity profile when tested on a large set of kinases. In-vitro this compound is highly cytotoxic on a wide panel of human cancer cell lines (IC50 ranged 10-70 nM, which corresponds to 1000 fold improvement of paprotrain potency) while no toxicity is observed on human normal cells such as peripheral blood mononuclear cells (PBMC) and primary hepatocytes (IC50 >50µM). BKS0349 compound is well tolerated in mice using repeated intravenous administrations (200 mg/kg, twice a week for 4 weeks). In xenografted nude mice, in-vivo treatment with BKS0349 compound demonstrates a high antitumor activity against various human cancer cell models, either sensitive or resistant to some standard-of-care treatments. In addition, human cancer cells treated in-vitro as well as in-vivo with BSK0349 compound display Golgi scattering and a mitotic arrest leading to cell death, as hallmarks of BKS0349’ mode of action. These findings show that MKLP2 is a potential new target for cancer chemotherapy and BKS0349 a good candidate to be developed for cancer treatment. 1Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance. P. Khongkow et al. Oncogene, 2015, 1-13. 2Relocation of Aurora B and Survivin from centromeres to the central spindle impaired by a kinesin-specific MKLP-2 inhibitor. S. Tcherniuk et al. Angew. Chem. Int. Ed., 2010, 49:8228-8231. 3New MKLP-2 inhibitors in the paprotrain series: design, synthesis and biological evaluations. C. Labriere et al. Bioorg and Med Chem, 2016, 24:731-734. Note: This abstract was not presented at the meeting. Citation Format: Yves Collette, Stephanie Miserey-Lenkei, Catherine Guillou, Denis Carniato, Bernard Pau, Bruno Goud, Norbert Vey, Cecile E. Bougeret. Identification of a selective MKLP2/KIF20A inhibitor with high in-vivo antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4140. doi:10.1158/1538-7445.AM2017-4140