Rapamycin is active against B-precursor leukemia in vitro and in vivo, an effect that is modulated by IL-7-mediated signaling

Abstract

A balance between survival and apoptotic signals regulates B cell development. These signals are tightly regulated by a host of molecules, including IL-7. Abnormal signaling events may lead to neoplastic transformation of progenitor B cells. Signal transduction inhibitors potentially may modulate these abnormal signals. Inhibitors of the mammalian target of rapamycin (mTOR) such as rapamycin have been used as immunosuppressive agents. We hypothesized that rapamycin might demonstrate activity against B-precursor acute lymphoblastic leukemia. We have found that rapamycin inhibited growth of B-precursor acute lymphoblastic leukemia lines in vitro, with evidence of apoptotic cell death. This growth inhibition was reversible by IL-7. One candidate as a signaling intermediate cross-regulated by rapamycin and IL-7 was p70 S6 kinase. Rapamycin also demonstrated in vivo activity in E mu-ret transgenic mice, which develop pre-B leukemia/lymphoma: E mu-ret transgenic mice with advanced disease treated daily with rapamycin as a single agent showed a >2-fold increase in length of survival as compared with symptomatic littermates who received vehicle alone. These results suggest that mammalian target of rapamycin inhibitors may be effective agents against leukemia and that one of the growth signals inhibited by this class of drugs in precursor B leukemic cells may be IL-7-mediated.

Figures

Fig. 1.

Rapamycin inhibits proliferation of ALL cells. Eμ-ret transgenic mouse-derived cell lines 83, T309, 289, and 420.2 were cultured with 1 unit/ml IL-7 and increasing concentrations of rapamycin (0-100 ng/ml). After 3 days of incubation, cell proliferation was assessed by using MTT. All measurements were performed in triplicate. Bars represent mean of the treated and control mice (relative absorbance of triplicate cultures, as described in Materials and Methods); error bars represent SEM.

Fig. 2.

Induction of apoptosis by rapamycin in ALL cell line T309. Eμ-ret transgenic mouse-derived pro-B cell line T309 was treated with increasing concentrations of rapamycin (rap) for 3 days. Cells were assessed for apoptotic response by labeling with FITC-conjugated annexin V. Percentages of apoptotic cells are indicated. Flow cytometric histograms show untreated (a), 1 ng/ml rapamycin (b), and 100 ng/ml rapamycin (c). Peak on the right in a-c represents FITC-conjugated annexin V-positive cells.

Fig. 3.

IL-7-stimulated proliferation of ALL cell lines in the absence of stroma. Cell lines 83, 781, T309, 289, and 420.2 were cultured with increasing concentrations of IL-7 (0-30 units/ml). After 4-5 days of incubation, cell proliferation was assessed by using MTT. All measurements were performed in triplicate. Bars represent mean of the treated and control mice (absolute absorbance of triplicate cultures, as described in Materials and Methods); error bars represent SEM.

Fig. 4.

IL-7 rescue of rapamycin-induced growth inhibition of ALL cells. Cells were cultured with increasing concentrations of rapamycin (0, 1, or 100 ng/ml) and IL-7 (0, 1, or 10 units/ml) for 3 days. Cell proliferation was assessed by using MTT. All measurements were performed in triplicate. Bars represent mean of the treated and control mice (relative absorbance of triplicate cultures, as described in Materials and Methods); error bars represent SEM.

Fig. 5.

Effect of rapamycin and IL-7 on phospho-p70 S6 kinase (Thr-389). Line 289 ALL cells (5 × 106) were cultured with 10 units/ml IL-7 and 100 ng/ml rapamycin (Rap) for 4 h. (Upper) Immunoblot of phospho-p70 S6 kinase (Thr-389). (Lower) Total p70 S6 kinase protein from the immunoblot in Upper. Each lane was loaded with 100 μg of protein.

Fig. 6.

Prolonged survival in mice with advanced ALL receiving rapamycin as a single agent. Eμ-ret transgenic mice with overt disease were treated daily i.p. with rapamycin (5 mg/kg per dose), as described in Materials and Methods. This Kaplan-Meier analysis of event-free survival (EFS) shows the fraction of animals surviving after the onset of disease. Rapamycin doubled the mean survival time of leukemic mice. The average time from diagnosis to death was 32 days in treated mice (n = 34) and 15 days in untreated mice (n = 23) (P < 0.00001).

Fig. 7.

The mTOR inhibitor rapamycin reduces leukemic adenopathy significantly. (Left) Leukemic Eμ-ret transgenic mouse with overt disease. This mouse has significant cervical and axillary adenopathy, as indicated by arrows. (Right) The same mouse 14 days post treatment with rapamycin. The mouse shows significant reduction in visible tumor burden, as indicated by arrows.