MTIs can be effectively combined with chemotherapy. Aliquots of 2 × 10⁴ cells from 3 ALL lines were treated with sirolimus (rap) and 7 chemotherapeutics for 72 hours. (A) MTT data for each drug combination in 1 representative cell line. represent untreated, ▧ represent MTI alone, ■ represent cytotoxic alone, and ▩ represent combined effect. All data are normalized to untreated baseline (= 1) with a value greater than 1 representing relative cell proliferation and less than 1 inhibition. Each group of 4 bars represents a combination with a different cytotoxic agent. MTIs had at least an additive effect when combined with methotrexate (MTX), dexamethasone (DEX), L-asparaginase (L-ASP), etoposide (VP-16), and doxorubicin (DOX). The combination of MTIs with vincristine (VCR) and Ara-C (cytarabine) did not add a benefit over either single agent alone. Doses depicted in panel A: sirolimus (0.3 ng/mL), MTX (5 nM), DEX (5 μM), L-ASP (1 μg/μL), VP-16 (1 nM), DOX (1 nM), VCR (1 nM), ARA-C (0.1 μg/mL). Next, aliquots of cells from 9 ALL lines were treated with methotrexate and 2 MTIs (temsirolimus (CCI) and sirolimus). (B) MTT data for temsirolimus and methotrexate in 1 cell line (289), demonstrating a synergistic effect at multiple drug doses. The other cell lines tested showed similar results. (C) Chou and Talalay median effects analysis results for one representative cell line (289), showing a combination index (CI) less than 1 at ED50 (median effective dose to inhibit 50% of cells), ED75, and ED90. (D) A representative example of annexin-V and 7-AAD staining in one cell line (289), demonstrating a synergistic increase in cell death and apoptosis with combined treatment. Doses depicted in panel D: CCI (7.5 ng/mL) and MTX (12.5 nM). Error bars represent SD.