Pediatric Preclinical Testing Program (PPTP) Evaluation of the mTOR Inhibitor Rapamycin

Background: Rapamycin is a specific inhibitor of the serine/threonine kinase, mTOR, that controls cap-dependent translation. Inhibition of mTOR may have direct effects on tumor cells or indirect effects through anti-angiogenic mechanisms. Methods: The PPTP includes an in vitro panel (n=27) as well as panels of xenografts (n=61) representing most of the common types of childhood solid tumors and childhood ALL. Rapamycin was tested against the in vitro panel at concentrations from 10 pM to 100 nM and against the in vivo tumor panels at a dose of 5 mg/kg IP daily X 5 (2 days off) for 42 days. Three measures of antitumor activity were used: 1) response criteria modeled after the clinical setting (e.g., partial response (PR), complete response (CR), etc.); 2) treated to control (T/C) tumor volume at day 21; and 3) a time to event (4X increase in tumor volume) measure based on the median EFS of treated and control lines (intermediate activity required EFS T/C > 2, and high activity additionally required a net reduction in median tumor volume at the end of the experiment). Results: Rapamycin was active against 10 of the 23 cell lines of the PPTP in vitro panel. The median IC 50 for the responsive lines was 1.26nM with RAMOS being the most sensitive with an IC 50 of 0.39nM. Rapamycin induced significant differences in EFS distribution in 28 of 36 (78%) of solid tumor xenografts and in 5 of 8 (63%) of the ALL xenografts. Using the PPTP time to event measure of efficacy, rapamycin had a high (4) or intermediate (11) level of activity against 15 of 31 evaluable solid tumor xenografts. Among the 8 ALL xenografts, 1 demonstrated high activity and 4 showed intermediate activity. Objective responses were observed in a rhabdoid xenograft (PR), 2 rhabdomyosarcoma xenografts (both PRs), and an osteosarcoma xenograft (maintained CR). A neuroblastoma xenograft achieved stable disease. Among the 8 xenografts in the ALL panel, rapamycin induced a PR in one T-cell ALL and a maintained CR in another T-cell ALL xenograft, with stable disease being observed in 1 B-precursor ALL xenograft. Conclusions: Rapamycin demonstrated in vitro activity against a range of cell lines, with the most sensitive being a Burkitt lymophoma line. Rapamycin demonstrated broad activity against both the solid tumor and ALL panels with tumor growth delay being observed in all the panels. Regressions were observed in the ALL panel and three of the solid tumor panels. Further work is needed to evaluate the activity of rapamycin in combination with standard chemotherapy agents and to evaluate associations between the molecular characteristics of the PPTP's preclinical models and their responsiveness to rapamycin. Methods for PPTP In Vivo Testing Stage 1 testing involves testing an agent across the entire PPTP panel of childhood cancer xenograft lines at its MTD or at a dose selected based on PK/PD studies using adult preclinical models. Solid tumor testing: For each xenograft line, 10 mice bearing SC tumors initiated treatment when the tumors were between 0.2-0.5 cm 3 . Two perpendicular tumor diameters were measured at once weekly intervals with digital vernier calipers. Assuming tumors to be spherical, volumes were calculated from the formula (π/6)×d3, where d represents the mean diameter. Acute lymphoblastic leukemia testing: For each xenograft line, 8 mice were inoculated with 3-5 x 10 6 mononuclear cells purified from the spleens of secondary recipient mice. Engraftment was monitored weekly by flow cytometry, and treatment was initiated when the proportion of human CD45+ cells in the peripheral blood reached 1%. The proportion of human CD45+ cells in the peripheral blood was monitored weekly throughout the course of treatment. Drug: Rapamycin was purchased from LC Laboratories (Woburn, MA). Rapamycin was dissolved in DMSO (5% final concentration) and diluted in 5% Tween 80 in water, and administered intraperitoneally daily for 5 for 6 consecutive weeks at a dose of 5 mg/kg. Rapamycin was provided to each testing site in coded vials for blinded testing according the PPTP's standard operating procedures.