Plocabulin, a novel tubulin-binding agent, inhibits angiogenesis by modulation of microtubule dynamics in endothelial cells.

BACKGROUND: Vascular supply of tumors is one of the main targets for cancer therapy. Here, we investigated if plocabulin (PM060184), a novel marine-derived microtubule-binding agent, presents antiangiogenic and vascular-disrupting activities. METHODS: The effects of plocabulin on microtubule network and dynamics were studied on HUVEC endothelial cells. We have also studied its effects on capillary tube structures formation or destabilization in three-dimensional collagen matrices. In vivo experiments were performed on different tumor cell lines. RESULTS: In vitro studies show that, at picomolar concentrations, plocabulin inhibits microtubule dynamics in endothelial cells. This subsequently disturbs the microtubule network inducing changes in endothelial cell morphology and causing the collapse of angiogenic vessels, or the suppression of the angiogenic process by inhibiting the migration and invasion abilities of endothelial cells. This rapid collapse of the endothelial tubular network in vitro occurs in a concentration-dependent manner and is observed at concentrations lower than that affecting cell survival. The in vitro findings were confirmed in tumor xenografts where plocabulin treatment induced a large reduction in vascular volume and induction of extensive necrosis in tumors, consistent with antivascular effects. CONCLUSIONS: Altogether, these data suggest that an antivascular mechanism is contributing to the antitumor activities of plocabulin.

Lurbinectedin Inactivates the Ewing Sarcoma Oncoprotein EWS-FLI1 by Redistributing It within the Nucleus.

There is a great need to develop novel approaches to target oncogenic transcription factors with small molecules. Ewing sarcoma is emblematic of this need, as it depends on the continued activity of the EWS-FLI1 transcription factor to maintain the malignant phenotype. We have previously shown that the small molecule trabectedin interferes with EWS-FLI1. Here, we report important mechanistic advances and a second-generation inhibitor to provide insight into the therapeutic targeting of EWS-FLI1. We discovered that trabectedin functionally inactivated EWS-FLI1 by redistributing the protein within the nucleus to the nucleolus. This effect was rooted in the wild-type functions of the EWSR1, compromising the N-terminal half of the chimeric oncoprotein, which is known to be similarly redistributed within the nucleus in the presence of UV light damage. A second-generation trabectedin analogue lurbinectedin (PM01183) caused the same nuclear redistribution of EWS-FLI1, leading to a loss of activity at the promoter, mRNA, and protein levels of expression. Tumor xenograft studies confirmed this effect, and it was increased in combination with irinotecan, leading to tumor regression and replacement of Ewing sarcoma cells with benign fat cells. The net result of combined lurbinectedin and irinotecan treatment was a complete reversal of EWS-FLI1 activity and elimination of established tumors in 30% to 70% of mice after only 11 days of therapy. Our results illustrate the preclinical safety and efficacy of a disease-specific therapy targeting the central oncogenic driver in Ewing sarcoma. Cancer Res; 76(22); 6657-68. ©2016 AACR.

PM060184, a new tubulin binding agent with potent antitumor activity including P-glycoprotein over-expressing tumors.

PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganization and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.

c-Jun N-terminal kinase phosphorylation is a biomarker of plitidepsin activity.

Plitidepsin is an antitumor drug of marine origin currently in Phase III clinical trials in multiple myeloma. In cultured cells, plitidepsin induces cell cycle arrest or an acute apoptotic process in which sustained activation of c-Jun N-terminal kinase (JNK) plays a crucial role. With a view to optimizing clinical use of plitidepsin, we have therefore evaluated the possibility of using JNK activation as an in vivo biomarker of response. In this study, we show that administration of a single plitidepsin dose to mice xenografted with human cancer cells does indeed lead to increased phosphorylation of JNK in tumors at 4 to 12 h. By contrast, no changes were found in other in vitro plitidepsin targets such as the levels of phosphorylated-ERK, -p38MAPK or the protein p27KIP1. Interestingly, plitidepsin also increased JNK phosphorylation in spleens from xenografted mice showing similar kinetics to those seen in tumors, thereby suggesting that normal tissues might be useful for predicting drug activity. Furthermore, plitidepsin administration to rats at plasma concentrations comparable to those achievable in patients also increased JNK phosphorylation in peripheral mononuclear blood cells. These findings suggest that changes in JNK activity provide a reliable biomarker for plitidepsin activity and this could be useful for designing clinical trials and maximizing the efficacy of plitidepsin.

Molecular pharmacology and antitumor activity of Zalypsis in several human cancer cell lines.

Zalypsis is a new synthetic alkaloid tetrahydroisoquinoline antibiotic that has a reactive carbinolamine group. This functionality can lead to the formation of a covalent bond with the amino group of selected guanines in the DNA double helix, both in the absence and in the presence of methylated cytosines. The resulting complex is additionally stabilized by the establishment of one or more hydrogen bonds with adjacent nucleotides in the opposite strand as well as by van der Waals interactions within the minor groove. Fluorescence-based thermal denaturation experiments demonstrated that the most favorable DNA triplets for covalent adduct formation are AGG, GGC, AGC, CGG and TGG, and these preferences could be rationalized on the basis of molecular modeling results. Zalypsis-DNA adducts eventually give rise to double-strand breaks, triggering S-phase accumulation and apoptotic cell death. The potent cytotoxic activity of Zalypsis was ascertained in a 24 cell line panel. The mean IC(50) value was 7nM and leukemia and stomach tumor cell lines were amongst the most sensitive. Zalypsis administration in four murine xenograft models of human cancer demonstrates significant tumor growth inhibition that is highest in the Hs746t gastric cancer cell line with no weight loss of treated animals. Taken together, these results indicate that the potent antitumor activity of Zalypsis supports its current development in the clinic as an anticancer agent.