mTOR inhibitors are synergistic with methotrexate: an effective combination to treat acute lymphoblastic leukemia

Abstract

We have previously demonstrated that mTOR inhibitors (MTIs) are active in preclinical models of acute lymphoblastic leukemia (ALL). MTIs may increase degradation of cyclin D1, a protein involved in dihydrofolate reductase (DHFR) synthesis. Because resistance to methotrexate may correlate with high DHFR expression, we hypothesized MTIs may increase sensitivity of ALL to methotrexate through decreasing DHFR by increasing turn-over of cyclin D1. We tested this hypothesis using multiple ALL cell lines and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with human ALL. We found MTIs and methotrexate were synergistic in combination in vitro and in vivo. Mice treated with both drugs went into a complete and durable remission whereas single agent treatment caused an initial partial response that ultimately progressed. ALL cells treated with MTIs had markedly decreased expression of DHFR and cyclin D1, providing a novel mechanistic explanation for a combined effect. We found methotrexate and MTIs are an effective and potentially synergistic combination in ALL.

Figures

Figure 1

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.

Figure 2

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.