Biochemical correlates of mTOR inhibition by the rapamycin ester CCI-779 and tumor growth inhibition

The rapamycin ester, CCI-779, potently inhibits cell growth in vitro, inhibits tumor growth in vivo, and is currently in Phase I clinical trials. To further understand the relationship between plasma systemic exposure and inhibition of the target Ser/Thr kinase, mTOR/FRAP, two assays have been developed. The first assay involves determination of the 4E suppressor protein (4E-BP1) bound to eukaryotic initiation factor 4E (eIF4E), and the second is direct Western analysis of phosphorylation of residue Thr⁷⁰ of 4E-BP1. Under normal growth conditions in vitro, rapamycin caused rapid association of 4E-BP1 with eIF4E within 1 h in Rh30 and GC³ human tumor cells. Association was persistent up to 16 h. In mice, administration of rapamycin (5 or 20 mg/kg) caused rapid association of 4E-BP1 with eIF4E within 4 h in both human colon adenocarcinoma GC³, and rhabdomyosarcoma Rh30 xenografts. Using phospho-specific antibody against Thr⁷⁰ of 4E-BP1, rapid and persistent dephosphorylation within 30 min of exposure to rapamycin was detected in Rh18 rhabdomyosarcoma cells. Evaluation of CCI-779 against Rh18 xenografts showed this tumor to be growth inhibited at daily dose levels of ≥8.7 mg/kg. Because immunoblotting may be more suitable for assaying tumor biopsy tissue, a "blinded" comparison between the effect of CCI-779 on Thr⁷⁰ phosphorylation and growth inhibition of human tumor xenografts was undertaken. Mice were treated daily for 5 days with CCI-779 (20 mg/kg/day) or with drug vehicle, and tumor diameters were measured. Tumors were excised 1 h after the final administration and frozen, and phospho Thr⁷⁰ was determined by Western blot analysis. The correlation coefficient for decreases in Thr⁷⁰ phosphorylation and growth inhibition was high (r², 0.99). The results indicate that an assay of decreases in phosphorylation of Thr⁷⁰ of 4E-BP1 may be a useful surrogate for determining the inhibition of mTOR activity in tumor specimens.