carboxyamidotriazole-orotate inhibits the growth of imatinib resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis

The Bcr/Abl kinase has been targeted for the treatment of chronic myelogenous leukaemia (CML) by imatinib mesylate. Although more common in solid tumors, increased microvessel density was also reported in chronic myelogenous leukaemia and was associated with a significant increase of angiogenic factors, suggesting that vascularity in haematologic malignancies is a controlled process and may play a role in the leukaemogenic process thus representing an alternative therapeutic target. Carboxyamidotriazole-orotate (CTO) is the orotate salt form of carboxyamidotriazole (CAI), an orally bioavailable signal transduction inhibitor that in vitro has been shown to possess antileukaemic activities (1, 2). CTO, has a reduced toxicity, increased oral bioavailability and stronger efficacy when compared to the parental compound. We have recently demonstrated that LAMA84 CML cells release exosomes and that the addition of those microvesicles to HUVEC affects several steps of in vitro angiogenesis including motility, cytokine production, cell adhesion, and cell signalling as well as in vivo angiogenesis in nude mice (3). A number of studies have recently described exosomes as new players in modulating the tumor microenvironment, promoting angiogenesis and tumor development; furthermore, neovascularization is known to exert an important role in the progression of chronic myeloid leukaemia and may represent a valid alternative target for therapy. Taking these data into account, the aims of our study were (i) to test if CTO is able to inhibit growth of human CML cells in vitro and in vivo (ii) to investigate the ability of CTO to affect tumor microenvironment by modulating exosome-stimulated angiogenesis in vitro and in vivo. Our results indicate that administration of CTO to a CML xenograft model in NOD/SCID mice may increase survival. Our in vitro studies with HUVEC demonstrated that CTO inhibits exosome stimulated motility, cytokines and cell-adhesion molecules (ICAM1 and VCAM1) expression of endothelial cells, moreover CTO inhibits exosomes activated signalling pathways and capillary-like structure formation. The matrigel plug assay that mimics physiological neo-angiogenesis, was used as in vivo model; our study showed that CTO drastically decreased exosome-stimulated angiogenesis. To investigate on the possible molecular mechanisms of the CTO-mediated antiangiogenic effect, we examined whether CTO inhibited the activation of intracellular signalling pathways involved in endothelial cell activation. Treatment of the EC with CTO blocked significantly the exosome-induced phosphorylation of signalling proteins, particularly Akt and Erk 1/2, which are the principal mediators of cell proliferation, survival, and chemotaxis in endothelial cells. Kinase-dependent and kinase-independent mechanisms are known to contribute to the abnormal adhesion and migration of CML progenitors, thus the effect of CTO on both endothelial cells and leukemic cells may concomitantly inhibit adhesion of leukaemia cells to vascular endothelium and conditions that favour leukostasis and tissue infiltration. IL8 is a member of the CXC family of chemokines, a potent proangiogenic factor and its plasma levels are found significantly higher in patients affected by chronic myelogenous leukaemia. Interestingly we showed, through the use of IL8 neutralizing antibodies and IL-8 siRNA, that IL8 was in part responsible for the effects of LAMA84R exosomes on EC activation; furthermore, treatment of EC with CTO inhibited the IL8-stimulated angiogenic phenotype. It is conceivable to hypothesize that IL8 secreted by EC stimulated with CML exosomes, may modulate both myeloid malignant cells and endothelial cells, thus generating a paracrine machinery between hematopoietic malignant cells and newly generated endothelium. In this tumor microenvironment, CTO could inhibit the angiogenic process through blocking the exosomes-mediated crosstalk, thus causing the interruption of a reciprocal stimulatory loop between leukemic and endothelial cells. As far as we are aware, this is the first study that demonstrate the inhibitory effect of an anticancer drug on angiogenesis stimulated by exosomes released from drug-resistant cancer cells; collectively, Our results indicate that CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility for CTO in leukaemia patients