Interleukin-4 Stimulates Proliferation and Growth of T-Cell Acute Lymphoblastic Leukemia Cells by Activating mTOR Signaling

To the Editor

IL-4 is produced within the bone marrow (BM) microenvironment either by non-resident circulating cells, namely T lymphocytes, mast cells, and basophils, or by BM stromal cells. Importantly, IL-4 is able to induce proliferation of T-cell acute lymphoblastic leukemia (T-ALL) cells ¹. Therefore, IL-4 produced in the BM milieu might influence the progression of T-ALL by stimulating proliferation of tumor cells. However, the exact mechanisms by which IL-4 induces leukemia expansion remain unknown.

The effects of IL-4 on normal lymphocytes involve at least two signaling pathways, Jakt/STAT and PI3K/Akt(PKB) ². In addition, IL-4 activates the PI3K downstream target mTOR, which regulates cell cycle completion in activated mature T-cells ³. Constitutive activation of mTOR has been reported in T-ALL ⁴ and suggested to regulate viability, cell size and proliferation of tumor cells. However, leukemia cells depend not only on constitutive, cell-autonomous mechanisms but also on cues from the microenvironment to fully activate key signaling molecules that are essential for tumor expansion and decreased sensitivity to chemotherapy ^5,6. Therefore, we investigated whether mTOR is involved in IL-4-mediated proliferation and growth of T-ALL cells.

We previously showed that IL-4 promotes in vitro proliferation of a significant proportion of primary T-ALL samples ¹. Here, we selected twelve diagnostic patient samples that proliferated to IL-4 as assessed by ³H-thymidine incorporation, to investigate the mechanisms of IL-4-driven T-ALL cell expansion. We first evaluated the effect of IL-4 on cell cycle progression by analyzing the DNA content of primary T-ALL cells by flow cytometry. IL-4 promoted the transition from G0/G1 to S-phase and G2/M in all five samples analyzed (Figure 1A). IL-4 also induced cell size increase (cell growth) that paralleled the effect on cell cycle (Figure 1B and Supplementary Table 1).

Figure 1. IL-4 stimulates cell cycle progression of primary T-ALL cells.

(A) Primary T-ALL cells were cultured with or without 10ng/ml IL-4 for the indicated time points. The percentage of cells at each phase of the cell cycle was examined within the viable population by propidium iodide staining. Left: results from one representative patient; Right: results from all patients analyzed (n=5), 0h vs. 72h of culture with IL-4, p=0.0159 (2-tailed Mann-Whitney). Cells in medium alone did not show significant cell cycle progression (not shown). (B) Cell size of T-ALL cells cultured with or without 10ng/ml IL-4 for 48h was evaluated by flow cytometry analysis. Representative results from two of twelve patients analyzed are shown. (C-E) T-ALL cells cultured with IL-4 during the indicated periods were lysed and analyzed by immunoblot for the expression of cdk6, cdk4 and cdk2 (C), cyclin D2, cyclin E and cyclin A (D), and phosphorylation of Rb (E). (F) T-ALL cells were cultured with IL-4 for the indicated time points and in vitro kinase activity of immunoprecipitated cdk4 and cdk2 was performed using Rb-GST and Histone H1 as exogenous substrates, respectively. (G) Expression of p27kip1 was evaluated by immunoblot at the indicated time points. (H) T-ALL cells were cultured with IL-4 alone or with rapamycin, VP22 control protein or VP22/p27kip1 fusion protein. Proliferation was determined at 72h by ³H-thymidine incorporation.

Because proliferation may result not only from an effect on cell cycle progression but also from increased survival, we evaluated the effect of IL-4 on T-ALL cell viability. In accordance with previous studies ⁷, we found that IL-4 had heterogeneous effects on T-ALL cell survival. IL-4 prevented T-ALL in vitro apoptosis in 6/12 cases (50%), promoted cell death in four (33%) and had no significant effects in two cases (17%; Supplementary Table 1). Nonetheless, IL-4-mediated proliferation occurred irrespectively of the effect on cell survival, and cell cycle progression was observed both in specimens where IL-4 promoted viability and apoptosis (Supplementary Table 1). These data suggest that IL-4 promotes proliferation of primary T-ALL cells mainly via regulation of the cell cycle machinery.

We next evaluated the mechanisms by which IL-4 mediated cell cycle progression in T-ALL cells. IL-4 did not affect the expression of cyclin-dependent kinases cdk6, cdk4 and cdk2 (Figure 1C). In contrast, cyclins were upregulated by IL-4 in a sequential manner (Figure 1D). The early G1 molecule cyclin D2 peaked around 12-24h of culture with IL-4, whereas expression of cyclins E and A, which are associated with late G1 and S-phase, reached a plateau at later time points (48 and 72h). These effects were paralleled by hyperphosphorylation of Rb, a critical substrate of cyclin/cdk activity in the cell (Figure 1E), indicating that IL-4 induced cyclin/cdk activity. To confirm these results we performed in vitro kinase assays with cdk4 and cdk2 immunoprecipitated from IL-4-stimulated primary T-ALL cells. IL-4 clearly augmented cdk activity (Figure 1F). In addition, IL-4 induced the downregulation of the cdk inhibitor p27^kip1 (Figure 1G). This event was mandatory for IL-4-mediated cell cycle progression, because forced expression of p27^kip1 completely abrogated IL-4 mediated proliferation (Figure 1H).

Because mTOR-dependent signaling has been associated with regulation of cell cycle and size, we next evaluated whether IL-4 activated mTOR in the T-ALL cell line TAIL7, whose biological features are similar to those from primary leukemia cells ⁸. IL-4 induced phosphorylation of mTOR downstream targets p70^S6K, S6 and 4E-BP1 in TAIL7 cells (Figure 2A). As expected, IL-4-mediated phosphorylation of these molecules was inhibited by treatment with the mTOR-specific antagonist rapamycin (Figure 2B). These data strongly indicate that IL-4 activates mTOR signaling in T-ALL cells. To evaluate the functional consequences of IL-4-mediated mTOR activation, we cultured T-ALL cells with IL-4 in the presence of rapamycin. At the molecular level, inhibition of mTOR prevented IL-4-dependent p27^kip1 downregulation (Figure 2C). Accordingly, rapamycin completely blocked IL-4-mediated proliferation (Figure 1H), cell cycle progression (Figure 2D,E) and growth (Figure 2F,G) of both TAIL7 and primary T-ALL cells.

Figure 2. IL-4-mediated activation of mTOR pathway is critical for cell cycle progression of T-ALL cells.

(A) TAIL7 T-ALL cells were stimulated with 10ng/ml IL-4 for the indicated time points, lysed and analyzed by immunoblot for phosphorylation of the indicated mTOR downstream targets. (B,C) TAIL7 cells were cultured with 10ng/ml IL-4 in the presence or absence of rapamycin (rapa) and phosphorylation of the indicated mTOR downstream target proteins (B) or expression of p27^kip1 (C) was evaluated at 72h. TAIL7 (D,F) or primary (E,G) T-ALL cells were cultured in the presence of IL-4 with or without rapamycin and analyzed for cell cycle distribution (D,E) and cell growth (F,G) at the indicated time points. (D,E) Percentage of cells in G0/G1 (lower left region), S-phase (upper region) and G2/M (lower right region) were identified using propidium iodide and BrdU + anti-BrdU-FITC. (F,G) Cell growth was determined by FSC versus SSC flow cytometry analysis. Percentage of large-sized (FSC high) cells was estimated relative to the control, small-size bulk population. Results in this figure are representative of at least two independent experiments.

In summary, we demonstrated that IL-4 mediates proliferation of T-ALL cells via mTOR-dependent regulation of cell cycle progression. In showing that mTOR is activated by a BM microenvironmental factor that positively stimulates leukemia T-cells, our observations strengthen the emphasis on mTOR as a key molecular target in T-ALL, and suggest that inhibition of IL-4 signaling may also have therapeutic potential in T-cell leukemia.