Temsirolimus and methotrexate are synergistic and produce durable remission. NOD/SCID mice were xenografted with human ALL from patient samples. After establishment of ALL, mice were randomized to treatment with vehicle, temsirolimus (CCI), methotrexate, or a combination of temsirolimus and methotrexate. Temsirolimus was dosed with 2 schedules, 5 mg/kg 5 days a week and 20 mg/kg weekly. Disease was evaluated at weekly intervals by FACS of peripheral blood for anti–human CD19 and anti–human CD45. (A) Comparison of arms from xenografts generated from sample 359 showing weekly changes in blast count (WBC in mm³ × % blasts). As similar results were found for both dosing schedules only the 20 mg/kg weekly dosing is depicted in panel A. Each series of vertical bars represents average blast count in animals for a particular treated arm at a given timepoint. Control animals died after 3 weeks. Mice treated with temsirolimus or methotrexate alone had improvement in disease but eventual progression. Mice treated with both drugs had complete resolution of peripheral blasts by day 21. After 49 days (depicted by black arrow in panel B) all drugs were stopped. One-half of the mice were killed and no mouse receiving combination therapy had measurable disease. The remaining mice were followed for 2 months and killed. (B) Time to progression on different arms by Kaplan-Meier analysis from sample 359 (top) and 240 (bottom). (C) Immunoblots of splenocytes from sample 240 mice treated with temsirolimus (“Treated”) or vehicle control (“Control”) for various lengths of time, showing decreased cyclin D1 (top row), DHFR (second row), and phospho-S6 (pS6; third row), comparing treated to control animals. In addition, mice that were treated with temsirolimus until relapse (“Treated after Relapse”) had increased expression of cyclin D1 after relapse. 240 samples depicted in panel C from left to right: control 7 days, control 14 days, control 21 days, control 30 days, treated 7 days, treated 14 days, treated 21 days, and treated 30 days.