Synergistic Action of a RAF Inhibitor and a Dual PI3K/mTOR Inhibitor in Thyroid Cancer

Abstract

In thyroid cancer clinical trials, agents targeting VEGF receptors (VEGFR) and RET, among other kinases, have led to partial responses but few complete or durable responses. The RAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways are frequently activated in differentiated and medullary thyroid cancer (DTC and MTC) and may provide therapeutic targets for these diseases. We tested a novel drug combination targeting RAF, phosphoinositide 3-kinase (PI3K), and mTOR, plus VEGFR2 and RET, in thyroid cancer preclinical models with defined genetic backgrounds. RAF265, an ATP-competitive pan-RAF inhibitor active against VEGFR2, and BEZ-235, a PI3K inhibitor also active against Torc1 and Torc2, were tested alone and in combination in a panel of thyroid cancer lines. We tested RAF265 and BEZ-235 for kinase inhibition, growth inhibition and cell-cycle alterations, and inhibition of signaling targets and tumor growth in xenograft models. Both drugs potently inhibited their kinase targets in the extracellular signal-regulated kinase (ERK) and PI3K pathways. In addition, RAF265 had significant RET inhibitory activity (IC₅₀ = 25-50 nmol/L for RET(C634W)). The combination strongly inhibited proliferation of DTC and MTC cell lines with mutations in RAS, BRAF, PTEN, and RET. Synergy was shown for B-CPAP (BRAF(V600E)) and TT cells (RET(C634W)). The combination of both drugs significantly inhibited growth of CAL62 (KRAS(G12R/G12R)) and TT xenografts, thoroughly inhibiting ERK and PI3K pathway signaling. Combined blockade of ERK and PI3K signaling potently inhibits growth in preclinical models representing the key genotypes seen in refractory thyroid cancer. These targets and therapies are promising for further development in both differentiated and medullary thyroid cancers.