Anti-leukemic mechanisms of pegylated arginase I in acute lymphoblastic T-cell leukemia

Abstract

New treatments for adults with acute lymphoblastic T-cell leukemia (T-ALL) are urgently needed, as the current rate of overall remission in these patients is only about 40 percent. We recently showed the potential therapeutic benefit of the pegylated-human-arginase I (peg-Arg I) in T-ALL. However, the mechanisms by which peg-Arg I induces an anti-T-ALL effect remained unknown. Our results show the induction of T-ALL cell apoptosis by peg-Arg I, which associated with a global arrest in protein synthesis and with the phosphorylation of the eukaryotic-translation-initiation factor 2 alpha (eIF2α). Inhibition of eIF2α phosphorylation in T-ALL cells prevented the apoptosis induced by peg-Arg I, whereas the expression of a phosphomimetic eIF2α form increased the sensibility of T-ALL cells to peg-Arg I. Phosphorylation of eIF2α by peg-Arg I was mediated through kinases PERK and GCN2 and down-regulation of phosphatase GADD34. GCN2 and decreased GADD34 promoted T-ALL cell apoptosis after treatment with peg-Arg I, whereas PERK had an unexpected anti-apoptotic role. Additional results showed that phospho-eIF2α signaling further increased the anti-leukemic effects induced by peg-Arg I in T-ALL-bearing mice. These results suggest the central role of phospho-eIF2α in the anti-T-ALL effects induced by peg-Arg I and support its study as a therapeutic target.

Figure 1

Peg-Arg I induces apoptosis, an arrested translation, and phosphorylation of eIF2α in T-ALL cells. (a) Malignant T cell lines (1 × 10⁵) were cultured for 48 h in the presence or the absence of peg-Arg I (2 IU/ml) or PBS (control). Then, the levels of the apoptosis marker annexin V were determined by flow cytometry. (b) T-ALL cell lines were treated with peg-Arg I for 24 h, after which de novo translation was tested using ³⁵S-Metionine (³⁵S-Met) incorporation, as described in the methods. (c) Malignant T cell lines (1 × 10⁵/ml) were cultured for 8 h in the presence or the absence of peg-Arg I (2 IU/ml), after which phospho-eIF2α levels were detected by western blot. (d–e). A representative experiment showing the expression of phosphorylated and total eIF2α by western blot (d) and phospho-eIF2α by cytofluorescence (e) in CD5⁺ cells sorted from individual mice bearing CCRF-CEM cells for 30 days and treated with PBS (n = 6) or 0.5 mg per mouse peg-Arg I (n = 7) for 4 h. The experiment was repeated a minimum of 3 times obtaining similar results.

Figure 2

Phosphorylation of eIF2α is a central mediator in the induction of T-ALL cell apoptosis by peg-Arg I. (a) Phospho-eIF2α expression in untransfected CCRF-CEM cells (−), and CCRF-CEM cells stably transfected with eIF2α-S51S, eIF2α-S51 A or eIF2α-S51D, and treated with peg-Arg I for 8 h. (b–d) Expression of the apoptosis markers annexin V (b), cleaved poly-ADP-ribose polymerase (PARP) (c), and DiOC2(3)/propidium iodide (d) in cells from a cultured with peg-Arg I for 72 h. The experiments were repeated a minimum of three times obtaining similar results.

Figure 3

Role of eIF2α kinases in the phosphorylation of eIF2α and the cellular apoptosis induced by peg-Arg I. (a) Expression of eIF2α kinases was detected by western blot in CCRF-CEM cells treated with peg-Arg I (2 IU/ml). (b) Stably silencing of the expression of the specific eIF2α kinases was achieved after transfection of CCRF-CEM cells with plasmids coding for specific sh-RNA or sh-controls and further selection of single clones in medium supplemented with 800 μg/ml Geneticin. (c) Cells from (b) were treated with peg-Arg I (2 IU/ml) and the levels of phospho-eIF2α (3–48 h) were detected by western blot (left column) and phospho-eIF2α/eIF2α densitometric values graphed (right column). (d–e) CCRF-CEM cells previously silenced for GCN2, PERK, PKR, or HRI (d), or expressing DN-GCN or DN-PERK (e) were treated with peg-Arg I (2 IU/ml) for 48 h, after which the levels annexin V were measured by flow cytometry. Experiments were repeated a minimum of three times obtaining similar results.

Figure 4

Inhibition of GADD34 accelerates eIF2α phosphorylation and further increases cellular apoptosis induced by peg-Arg I. (a) CCRF-CEM cells were cultured in medium with or without peg-Arg I (2 IU/ml) and the expression of eIF2α phosphatase components was tested by immunoblotting. (b) Silencing of GADD34 in CCRF-CEM cells induces an earlier phosphorylation of eIF2α after treatment with peg-Arg I. (c) CCRF-CEM cells stably transfected with plasmids coding for sh-GADD34 or sh-mock were treated with peg-Arg I for 24 h, after which annexin V expression was detected using flow cytometry. (d) Expression of annexin V in CCRF-CEM cells treated for 24 h with peg-Arg I (2 IU/ml) and/or salubrinal (20 μM). Values are from 3 similar experiments.

Figure 5

Phospho-eIF2α sensitizes T-ALL to the anti-leukemic effect of peg-Arg I in vivo. (a) Survival of mice bearing CCRF-CEM cells stably transduced with eIF2α-S51S or eIF2α-S51D and that were treated with PBS (Control) (n = 10) or peg-Arg I (n = 10). Peg-Arg I extended the survival of mice injected with T-ALL cells expressing a phosphomimetic form of eIF2α. (b) Percentage of human CD5⁺ cells was established in the spleens after 30 days of the injection of CCRF-CEM cells expressing eIF2α-S51S or eIF2α-S51D. Mice were treated with 0.5 mg/mouse peg-Arg I (n = 10) or PBS (n = 10) twice a week starting on day 19 post-T-ALL injection, as described in the methods. (c) A representative hematoxylin and eosin staining histopathology from liver, spleen and bone marrow from b.