Development of p21 activated kinase-targeted multikinase inhibitors that inhibit thyroid cancer cell migration.

CONTEXT: The p21 activated kinases (PAKs) are a family of serine/threonine kinases that are downstream effectors of small GTPase Cdc42 and Rac. PAKs regulate cell motility, proliferation, and cytoskeletal rearrangement. PAK isoform expression and activity have been shown to be enhanced in cancer and to function as an oncogene in vivo. PAKs also have been implicated in cancer progression. OBJECTIVE: In thyroid cancer, we have previously determined that PAK overactivation is common in the invasive fronts of aggressive tumors and that it is functionally involved in thyroid cancer cell motility using molecular inhibitors. We report the development of two new PAK-inhibiting compounds that were modified from the structure OSU-03012, a previously identified multikinase inhibitor that competitively blocks ATP binding of both phosphoinositide-dependent kinase 1 (PDK1) and PAK1. RESULTS: Seventeen compounds were created by combinatorial chemistry predicted to inhibit PAK activity with reduced anti-PDK1 effect. Two lead compounds were identified based on the ability to inhibit PAK1 activity in an ATP-competitive manner without discernible in vivo PDK1 inhibitory activity in thyroid cancer cell lines. Both compounds reduced thyroid cancer cell viability. Although they are not PAK-specific on a multikinase screening assay, the antimigration activity effect of the compounds in thyroid cancer cells was rescued by overexpression of a constitutively active PAK1, suggesting this activity is involved in this biological effect. CONCLUSIONS: We have developed 2 new multikinase inhibitors with anti-PAK activity that may serve as scaffolds for further compound development targeting this progression-related thyroid cancer target.

Sorafenib and Mek inhibition is synergistic in medullary thyroid carcinoma in vitro.

Clinical trials using kinase inhibitors have demonstrated transient partial responses and disease control in patients with progressive medullary thyroid cancer (MTC). The goal of this study was to identify potential combinatorial strategies to improve on these results using sorafenib, a multikinase inhibitor with activity in MTC, as a base compound to explore signaling that might predict synergystic interactions. Two human MTC cell lines, TT and MZ-CRC-1, which harbor endogenous C634W or M918T RET mutations, respectively, were exposed to sorafenib, everolimus, and AZD6244 alone and in combination. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and poly (ADP-ribose) polymerase (PARP) cleavage assays were performed to measure cell survival and apoptosis. Western blots were performed to confirm activity of the compounds and to determine possible mechanisms of resistance and predictors of synergy. As a solitary agent, sorafenib was the most active compound on MTT assay. Western blots confirmed that sorafenib, everolimus, and AZD6244 inhibited their anticipated targets. At concentrations below its IC(50), sorafenib-treated TT and MZ-CRC-1 cells demonstrated transient inhibition and then re-activation of Erk over 6 h. In concordance, synergistic effects were only identified using sorafenib in combination with the Mek inhibitor AZD6244 (P<0.001 for each cell line). Cells treated with everolimus demonstrated activation of Akt and Ret via TORC2 complex-dependent and TORC2 complex-independent mechanisms respectively. Everolimus was neither additive nor syngergistic in combination with sorafenib or AZD6244. In conclusion, sorafenib combined with a Mek inhibitor demonstrated synergy in MTC cells in vitro. Mechanisms of resistance to everolimus in MTC cells likely involved TORC2-dependent and TORC2-independent pathways.