While individual targeting of each kinase may show modest suppressive activity, the combination of crizotinib/BKM120 demonstrated superior efficacy in inhibiting multiple aspects of tumor cell growth (viability, migration, colony formation) and (PDX). significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent. Malignant mesothelioma (MM) is a slow growing, solid tumor that primarily originates in pleural (80%), peritoneal (20%) and pericardial cavities (1%)1,2. Various etiological factors contribute to the onset of MM such as exposure to asbestos or erionite, Simian virus 40 (SV40), genetic predisposition and radiation therapy3,4,5. The current standard therapy for MM consists of Fluoxymesterone surgical resection, combination chemotherapy with cisplatin and pemetrexed, and potentially Fluoxymesterone radiation6,7. Despite advances in chemotherapy, MM has very poor prognosis and median survival of less than one year which is unacceptably low8. Therefore, there is a pressing need for more efficacious therapies for MM. RTKs are known to play a crucial role in tumor growth and metastasis. Some RTKs were originally discovered as oncogenes and are known to provide key signals that lead to transformation, tumor growth and metastasis9. Several studies have demonstrated that RTKs including epidermal growth factor receptor (EGFR), MET, insulin growth factor receptor (IGFR) Rabbit Polyclonal to RPL19 and vascular endothelial growth factor receptor (VEGFR) are overexpressed in MPM10,11,12,13. Previously we demonstrated that the MET/HGF axis is activated in MPM through overexpression, amplification and mutations of MET. SU11274, a small molecule inhibitor of MET is known to decrease cell proliferation of mesothelioma cells14. Crizotinib (PF02341066, Pfizer) is an orally available, potent, ATP competitive, small molecular inhibitor of MET, anaplastic lymphoma kinase (ALK) and c-Ros Oncogene 1 (ROS1). Its affinity for MET is greater than that for ALK or ROS1. FDA has approved its use for the treatment of NSCLC15. Phosphatidylinositol 3-kinase (PI3K) is a key downstream signaling molecule of MET and other RTKs. It is a cellular proto-oncogene and an essential lipid kinase, that plays an important role in the regulation of cell proliferation, survival and motility16. Several preclinical studies have shown that this pathway is hyper activated in mesothelioma17,18. BKM120 is a potent inhibitor of class I PI3Ks, currently in Phase I and II clinical trials for patients with a variety of solid tumors. Another PI3K inhibitor we investigated in this study is GDC-0980, a potent small molecule inhibitor of class I PI3K isoforms and mTOR. In the present study, we have investigated the effects of crizotinib and BKM120, singly or in combination, on MPM tumor growth using both and models. Apart from BKM120 similar results were also observed with GDC-0980. While single use of BKM120 inhibited growth of MPM tumor in a PDX mouse model, the combined treatment with crizotinib and BKM120 was highly synergistic. Results Synergistic suppression of MPM cell proliferation using MET and PI3K inhibitors Cell viability was determined following treatment with increasing concentrations of BKM120 for 72?h and results are presented in Fig. 1. Most of the MPM cell lines used were sensitive to treatment with BKM120 with IC50 values ranging from 0.79C1.51?M. However, Met-5A, a control mesothelial cell line and H28 were less sensitive to BKM120 (Fig. 1A). Open in a separate window Figure 1 Effect of MET and PI3K inhibitors on proliferation of human mesothelioma cells and Synergistic anti-tumor activity of these inhibitors.(A) Mesothelioma cell lines H2373, H2596, H513, Fluoxymesterone H2052, H2461, H28, and Met-5A were treated with BKM120 for 72?h. Viability was measured by Alamar Blue assay. The data shown represents the average??SEM. (B) Combination Index plot and Isobologram for combination of Crizotinib and BKM120 in H2596 cells. The left side panel shows the CI plot for the combinations of drugs where synergy (identified by a Combination Index 1) over a range of drug concentrations. The green triangle in the isobologram represents concentrations of both drugs that inhibit cellular proliferation by 90% (Fraction affected?=?0.9). A combination index (CI) value of 0.08 was calculated using CompuSyn software. The line represents an additive affect, where CI?=?1. (C) Combination Index plot and Isobologram for combination of Crizotinib and GDC-0980 in H2596 cells. The left side panel shows the CI plot for Fluoxymesterone the combinations of drugs where synergy (identified by a Combination Index 1) over a range of drug concentrations. The green triangle in the isobologram represents concentrations of both drugs that inhibit cellular proliferation by 90% (Fraction.