EphB Receptors Trigger Akt Activation and Suppress Fas Receptor-Induced Apoptosis in Malignant T Lymphocytes

Leukemias and lymphomas of the T cell origin are often aggressive malignancies that are associated with a wide range of molecular defects in the T cell lineage (1, 2). In pediatric T cell acute lymphoblastic leukemia (T-ALL), for example, the most common molecular abnormalities are presented by mutations or silencing of the cdkn2a/2b genes that block expression of the p15INK4 and p16INK4 cell cycle suppressors (3–5) and prevent production of the p14^ARF activator of p53 (1). Other prominent flaws in T-ALL molecular machinery also include Notch1 receptor-activating mutations (6, 7), inactivating mutations of Notch negative regulator, Fbxw7 (8–10), and of the inhibitor of PI3K signaling, the PTEN phosphatase (11). Alternatively, among lymphoid malignancies, cutaneous T cell lymphomas are characterized by frequent mutations in the nav3 gene (12), by the constitutive activation of STAT3 (13), by the overexpression of the CCR10 receptor (14), of JunB (15), and of the antiapoptotic protein Mcl-1 (16). In addition, the phosphorylation/inactivation of the proapoptotic factor Bad (16) is also common in lymphoma cells. Despite substantial progress in the understanding of the molecular biology of T cell malignancies and despite recent advances in their treatment, a large number of patients develop resistance to currently used multiagent chemotherapies and relapse (1, 17–19). These patients are typically offered an allogeneic bone marrow transplant and their best hope resides in the immunotherapeutic effect of the graft-versus-leukemia/lymphoma reaction (17, 19, 20). The graft-versus-leukemia/lymphoma response strongly relies on Fas receptor (FasR)-triggered cell death induced in malignant T cells by the Fas ligand expressed on allogeneic cytotoxic T lymphocytes and NK cells (21–23). The immunotherapeutic response may be additionally augmented by donor lymphocyte infusions (17, 20). Unfortunately, although this strategy works well in myeloid leukemias, it has proved to be of a limited efficiency in a significant portion of T-ALL patients, presumably because of the ability of malignant T cells to evade immunoelimination (2, 17, 21, 24). The evading ability may partially depend on the molecular mechanisms that reduce sensitivity to Fas stimulation and, thus, support survival of cancerous T cells.

Accumulating observations show that tyrosine kinase receptors of the Eph family are involved in the regulation of cell survival in nonhematopoietic malignancies, including the antiapoptotic action of the EphA2 receptor in breast cancer (25) and of the EphB4 receptor in colorectal cancer (26), ovarian cancer (27), head and neck carcinoma (28), mesothelioma (29), and prostate cancer (30), indirectly suggesting that proteins of the Eph group may also provide protection for malignant lymphocytes; however, little is known about the role of Eph receptors in leukemias and lymphomas.

The Eph family of receptor tyrosine kinases is represented by 16 receptors that are assigned to EphA (EphA1–10) and EphB (EphB1–6) groups based on their structural organization and ligand-binding preferences. In mammals, EphA receptors (EphA1–8, EphA10) promiscuously bind ephrin-A proteins (ephrin-A1–6), and EphB receptors (EphB1–4, EphB6) interact in a similar manner with three ephrin-Bs (ephrin-B1–3) (31). This rule, however, is not without exceptions, because EphA4 is capable of binding ephrin-Bs, and EphB2 can be activated by ephrin-A5 in addition to their conventional ephrin-A and ephrin-B ligands (32–36). In contrast, EphB6 interactions appear to be restricted to ephrin-B1 and ephrin-B2 (34, 37, 38). All ephrins are membrane-attached proteins; ephrin-As are GPI-anchored, whereas ephrin-Bs have transmembrane and cytoplasmic domains, and their membrane localization appears to be crucial for their ability to trigger Eph signaling (39). Therefore, cell–cell contact formation between Eph-and ephrin-expressing cells is typically required for Eph receptor activation (40). Ligand-activated Eph receptors initiate a complex array of signaling events and control multiple aspects of cell behavior both in developing embryos and in adult organisms (41). Eph and ephrin proteins also appear to control cellular responses in human malignancies (31, 42, 43). It has been reported that some Eph receptors and ephrin genes have their promoter regions methylated in leukemic cells, and that overexpressed EphB4 inhibits Akt activation, thus inducing apoptotic cell death in Raji B-ALL cells (44). Nevertheless, expression of some Ephs and ephrins, including EphA1, EphA2, EphA3, EphA7, EpA10, EphB6, ephrin-A4, ephrin-B1, and ephrin-B2, has been observed in ALL cell lines and chronic lymphocytic leukemia patient samples (45–51). Moreover, ephrin-A4, EphA3, and ephrin-B1 are actively involved in the regulation of cell attachment and invasive properties in chronic lymphocytic leukemia, B cell ALL, and T-ALL cells (47, 51–53). Complementing each other, these observations suggest that a disbalance in Eph receptor signaling caused by distorted expression patterns, rather than its complete blockage, is likely to contribute toward the malignant behavior of leukemic cells.

Activation of both Eph receptors and FasRs occurs in the area of cell–cell contact formation (40, 54), and cytotoxic CD8⁺ T cells express not only Fas ligand, but also ephrin-B proteins on their membranes (23, 55–57), suggesting that Eph receptors and FasR are likely to be costimulated in the course of the graft-versus-leukemia response and raising a possibility that a combination of ephrin-B–interacting Eph receptors expressed in malignant T cells may interfere with FasR signaling and FasR-mediated apoptotic cell death.

We report in this article that the EphB3 receptor is expressed by all tested malignant T lymphocytes, where it is usually accompanied by another EphB receptor, EphB6, and show that stimulation with their common ephrin-B ligands strongly suppresses FasR-initiated apoptosis in these cells. This prosurvival response is associated with ephrin-B–induced activation of apoptosis suppressor, Akt. In agreement with previously reported Akt properties (58), ephrin-B stimulation also inhibits FasR-mediated activation of both caspase-8 and -3 proteases that are responsible for the propagation of the apoptotic signal (59). Moreover, inhibition of Akt, but not of the Ras-MAPK pathway or Src kinases, blocks the prosurvival action of EphBs, suggesting that these receptors are likely to rely on Akt signaling in their antiapoptotic responses in cancerous T lymphocytes.

Overall, our work demonstrates a new role and a new mechanism of action for EphB receptors in the suppression of FasR-mediated cell death. Our observations also indicate that EphB receptors may significantly contribute toward the immunoevasive properties of T cell leukemias and, therefore, may serve as promising targets for novel therapeutic approaches, aiming to enhance the efficiency of the graft-versus-leukemia response.

T-ALL cell lines H9 and E6.1 were purchased from ATCC (Manassas, VA). Molt-16 cells were kindly provided by Dr. John DeCoteau (Department of Pathology, Royal University Hospital, University of Saskatchewan, Saskatoon, SK, Canada). DND-41, ALL-SIL, TALL-1, and KOPT-K1 cells were from Dr. Andrew Weng’s collection and were previously characterized (6). All cell lines were cultured in RPMI-1640 (HyClone, Logan, UT) supplemented with FBS and Pen-Strep (Invitrogen, Carlsbad, CA) at 37°C, 5% CO₂. Primary T-ALL samples were obtained from two independent pediatric patients with T-ALL at the time of diagnostic marrow aspiration after informed consent and with approval from local Institutional Review Board (Alberta Children’s Hospital, Calgary, AB, Canada). Mononuclear cells from bone marrow aspirates were prepared on Percoll (Pharmacia, New York, NY) gradient as directed by the manufacturer. The diagnoses of T-ALL in these cases were confirmed by flow cytometry as a part of clinical diagnostic workup. T-ALL cells were cultured in the LGM-3 medium (Lonza, Walkersville, MD).

The anti-FasR–activating Ab, 7C11, was purchased from Beckman-Coulter (Mississauga, ON, Canada). Resazurin stain, anti–phospho-Akt (Ser⁴⁷³), ephrin-B1–Fc, and ephrin-B2–Fc were from R&D Systems (Minneapolis, MN). Anti-human Fc was purchased from Pierce Biotechnology (Rockford, IL). Abs for caspase-3, EphB3, and Erk2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The Ab for caspase-8 was from Cell Signaling Technology (Danvers, MA). The Akt inhibitors Akti-1/2 and perifosine were purchased from Calbiochem (La Jolla, CA) and BioVision (Mountain View, CA), respectively. The MEK1/2 inhibitors U0126 and PD98059 were very kindly provided by Dr. Michael Sherman (Department of Biochemistry, Boston University School of Medicine, Boston, MA). SU6656, an Src family inhibitor, and anti-EphB6 were from Sigma-Aldrich (Oakville, ON, Canada). Random primers, Superscript II reverse transcriptase, and Elongase Enzyme Mix were obtained from Invitrogen.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada) according to the manufacturer’s instructions. The first-strand cDNA was obtained and PCR was performed as previously described (51). The PCR primers for EphB2, EphB3, EphB4, EphB6, and β-actin were described previously (51, 60, 61) and synthesized by Sigma-Genosys (Oakville, ON, Canada). EphB1 (5′-CAAGACTGTGGCAACCATCACCG-3′, and 5′-GTGACTGACTTATCTGGACCCTC-3′) and EphA4 (5′-CTGCTGGCAGAAGGAGAGGAGC-3′, and 5′-CTGGTGCGTGATGGCTGTGATAC-3′) primers were also synthesized by Sigma-Genosys. PCR products were resolved by agarose gel electrophoresis, stained with ethidium bromide, visualized in ultraviolet light, and quantitated in triplicates using the Carestream Molecular Imaging Software densitometry tool. Expression of the Eph receptor transcripts in each cell line is presented as a percentage relative to matching β-actin controls.

The activating anti-FasR Ab, 7C11, in combination with ephrin-B1–Fc, ephrin-B2–Fc, or human IgG (hIgG; Sigma) was immobilized in 96-well plates overnight, at 4°C. hIgG was used to maintain a constant protein concentration and to provide a specificity control for the Fc fragment of the ephrin fusion proteins. The wells were rinsed with serum-free medium and seeded with 6 × 10⁴ cells/well to achieve a single-cell layer. ALL-SIL cells were seeded at 3 × 10⁴ cells/well. Cells were incubated at 37°C and 5% CO₂ in serum-free RPMI-1640. To monitor survival, we stained cells with resazurin, following the manufacturer’s instructions, and fluorescence was measured using a microplate reader (Excitation 530 nm, Emission 590 nm). Error bars represent SD based on the analysis of triplicates. In some experiments, H9 and Molt-16 cells were incubated in the presence of inhibitors of Akt (Akti-1/2 or perifosine), MEK1/2 (U0126 or PD98059), or Src kinases (SU6656), as previously described (62–65). Matching volumes of corresponding solvents were used as controls.

Ephrin-B2–Fc was precomplexed with goat anti-human Fc for 30 min on ice (3 μg/ml ephrin-B2–Fc with 4.5 μg/ml anti-human Fc), and cells were stimulated at 37°C. Control cells were incubated at 37°C with precomplexed hIgG. In some experiments, ephrin-B2–Fc or hIgG was coimmobilized with activating anti-FasR Ab onto 24-well plates overnight at 4°C. Wells were washed with serum-free RPMI-1640; cells were loaded and incubated at 37°C for the indicated times. After stimulation, cells were lysed with 0.2% NP-40 lysis buffer and lysates were cleared by centrifugation. Alternatively, cells were lysed in boiling 1× SDS sample buffer to produce total lysates. Cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose (Amersham, Arlington Heights, IL) for Western blotting.

To monitor FasR-mediated apoptosis, the TUNEL-based APO-BRDU Apoptosis Detection Kit (BD Biosciences, Mississauga, ON, Canada) was used according to the supplier’s directions. The activating anti-FasR Ab in combination with ephrin-B2–Fc or hIgG, or IgG alone, were immobilized onto 24-well plates overnight at 4°C. Plates were rinsed with serum-free medium and 0.5 × 10⁶ of H9 cells, 0.75 × 10⁶ of Molt-16 cells, 0.6 × 10⁶ of DND-41 cells, or 0.35 × 10⁶ of primary cells from T-ALL patient samples were loaded per well. Cells were incubated at 37°C for 6 (H9), 7 (Molt-16), 21 (DND-41), or 24 h (patient’s cells), fixed in 1% formaldehyde, and permeabilized with 70% ethanol. Permeabilized cells were labeled with BrdUTP using terminal deoxytransferase for 1 h at 37°C, stained with FITC-conjugated anti-BrdU Ab, and analyzed by flow cytometry. Gating was performed to exclude cell debris during flow cytometry analysis.

To monitor the cell surface expression of the FasR, we treated cells with precomplexed ephrin-B2–Fc or hIgG for 5 or 18 h. Stimulated cells were stained with FITC-conjugated anti-FasR (BD Biosciences) or matching isotype control (BD Biosciences). Isotype control staining was confined to the first log. The presence of the FasR was determined by flow cytometry as described above.

H9 cells (3 × 10⁷) were stimulated with 3 μg/ml precomplexed ephrin-B2 or hIgG for 4 min or 2 h. Molt-16 cells (5.6 × 10⁷) were stimulated in the same manner as H9 for 10 min. After stimulation, cells were pelleted and snap-frozen in liquid nitrogen. Cell pellets were lysed with 80 μl lysis buffer (20 mM Tris-HCL pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM Na₃VO₄, 1 mM NaF, 1 μg/ml leupeptin, 1 g/ml aprotinin, 1 mM PMSF), incubated on ice for 10 min, and then spun in a microcentrifuge at maximum speed for 10 min at 4°C. An 80-μl aliquot of this supernatant was mixed with 10 μl of the activation mix (50% glycerol, 500 μM ATP, 60 mM MgCl₂, 0.05% v/v Brij-35, and 0.25 mg/ml BSA) and incubated on the chip for 2 h at 37°C in a humidity chamber. After incubation, slides were washed once in PBS-Triton, then submerged in stain (PRO-Q Diamond Phosphoprotein Stain; Invitrogen) with agitation for 1 h. Arrays were then washed in tubes containing destain (20% acetonitrile [EMD Biosciences, VWR distributor, Mississauga, ON, Canada] and 50 mM sodium acetate [Sigma] at pH 4.0) for 10 min three times with the addition of new destain each time. A final wash was done with distilled water. Arrays were dried and read using a GENEPIX professional 4200A microarray scanner (MDS Analytical Technologies, Toronto, ON, Canada) at 532–560 nm with a 580-nm filter to detect dye fluorescence. Images were collected using the GENEPIX 6.0 software (MDS Analytical Technologies). Signal from images was then captured and statistical analysis performed using a variation of previously published microarray analysis techniques (66, 67).

E6.1 cells were treated for 15 min with precomplexed ephrin-B2–Fc (3 μg/ml) or hIgG as a control and analyzed using the Human Phospho-Kinase Array kit (R&D Systems) according to the manufacturer’s instructions. In brief, treated cells were lysed and the lysates were cleared by centrifugation. Cleared lysates were loaded onto the provided membranes precoated with capture Abs, and the presence of bound phospho-proteins was determined by Western blotting with a mixture of detection Abs. The signal intensities for Akt and GSK3 phosphorylation were determined in triplicates by densitometry and normalized on the provided positive control. The status of protein phosphorylation in ephrin-B2–treated cells is reported relative to the IgG-treated control.

Rapidly accumulating observations suggest that both ephrin membrane proteins and EphA receptors play an important role in lymphoid malignancies, predominantly through the regulation of attachment and invasive properties of leukemic cells (47, 51–53). Meanwhile, little is known about the potential involvement of the ephrin-B–interacting Ephs, EphB1–4, EphB6, and EphA4 (34), collectively referred to as EphrinB receptors.

To monitor the expression of specific EphrinB receptors, we used the RT-PCR approach. Aliquots of the RT-PCR products were resolved by electrophoresis; the amount of cDNA in each sample was determined by densitometry and normalized on a matching β-actin control. The results of this analysis have demonstrated that all human T-ALL cell lines assessed and also the human malignant lymphoblastic T cells, H9, express the EphB3 and EphB6 transcripts, whereas expression of other EphBs varies, and EphA4 is missing in six cell lines from seven tested (Fig. 1A). Pediatric T-ALL represents only approximately 10% of childhood ALLs (68), and a limited number of patient samples were available for our analysis. Nevertheless, EphB3 expression has been observed in both examined independent patient samples (patients 1, 2) and one of them lacked EphA4. The EphB6 transcript was present in one of two samples (Fig. 1A).

Because EphB3 and EphB6 were present in the majority of analyzed malignant T cells, their expression was also assessed on a protein level by Western blotting. Although, unfortunately, amounts of material in T-ALL patient samples were not sufficient for Western blot analysis, both proteins were easily detected with specific Abs in all T cell lines, thus supporting our RT-PCR data (Fig. 1B, 1C). Interestingly, we could not detect the EphA4 protein in ALL-SIL cells using a similar approach (not shown), despite the presence of the EphA4 mRNA (Fig. 1A), suggesting that the protein is likely to be expressed at a very low level, if expressed at all.

FasR-mediated apoptotic cell death plays an important role in the graft-versus-leukemia/lymphoma response and is critical for the survival of patients who undergo bone marrow transplantation (19, 21–23). T-ALL cells, however, often develop immunoevasive properties and protect themselves from the cytotoxic action of allogeneic lymphocytes (17, 21). Because Eph receptors have been shown to initiate antiapoptotic responses in nonlymphoid malignancies (25–30), we examined the ability of EphB receptors to interfere with FasR-initiated cell death in malignant T cells. To assess the effect of EphBs, we stimulated H9 and Molt-16 cells with the activating anti-FasR Ab, 7C11, alone or in the presence of increasing concentrations of the common ligand for EphB3 and EphB6, ephrin-B2 (34, 38). Because an ephrin-B2–Fc fusion protein, with the extracellular portion of the ligand fused to the Fc fragment of hIgG, was used, hIgG was introduced as a specificity control. Costimulation of EphB receptors strongly suppressed FasR-mediated response and supported cell survival, monitored in these experiments by resazurin staining (Fig. 2A, 2B). The ability of ligand-activated EphB receptors to inhibit FasR-induced apoptosis was further verified by the TUNEL assay, and antiapoptotic responses were observed in both H9 and Molt-16 cell lines, and in the sample from T-ALL patient 1 (Fig. 2C). Although a lower level of apoptosis was detected in the patient 2 sample, it was also inhibited by the costimulation with ephrin-B2 (data not shown).

T-ALL cells E6.1 lack the activity of the negative regulator of the PI3K-Akt prosurvival pathway, the PTEN phosphatase. In agreement, FasR stimulation produced a relatively limited death response in these cells; nevertheless, this response was efficiently inhibited by ephrin-B2 costimulation (Fig. 2D). Moreover, costimulation of two other T-ALL cell lines, ALL-SIL and KOPT-K1, also revealed the protective property of EphBs (Fig. 2E), and although the prosurvival effect could not be observed in T-ALL cells DND-41 by resazurin staining, it could be easily detected in the TUNEL assay, most probably because of the higher sensitivity of this approach (Fig. 2F).

Additional experiments with another EphB3 and EphB6 ligand, ephrin-B1 (34, 37), have, once again, shown the ability of EphB proteins to attenuate FasR cytotoxicity (Fig. 3A–C), indicating that ephrin-B1– and ephrin-B2–activated EphB receptors trigger the antiapoptotic action and may be potentially responsible for the immunoevasive behavior of malignant T lymphocytes.

To determine whether EphB receptors may suppress FasR responses by downregulating its cell surface expression, we stained ephrin-B2–stimulated H9 and Molt-16 with anti-FasR Ab and monitored FasR presence by flow cytometry. Although untreated cells appeared to express FasR at a very high level, no effect of ephrin-B2 on its expression could be observed (Fig. 4A), suggesting that EphBs were likely to affect FasR action at the level of cytoplasmic signaling.

Eph receptors are known to control cell behavior through the initiation of an intricate combination of signaling responses in both normal and malignant cells (31, 41, 69). To assess the overall effect of EphB receptors on the phosphorylation-dependent signaling, we stimulated H9 cells with ephrin-B2 and monitored the resulting responses using a human peptide array for kinome analysis recently developed in analogy to the previously described bovine-specific array (66, 67). The obtained data confirmed the ability of EphB receptors to initiate antiapoptotic signaling in malignant T cells; relevant array-based observations are summarized in Table I. Interestingly, although ephrin-B2 stimulation produces no effect on a central prosurvival event, PI3K-PDK1–dependent Akt activation after a short, 4-min stimulation in H9 cells, it induces a reduction in PTEN phosphorylation at the Tyr²⁴⁰ and Tyr³¹⁵ residues, which is supposed to inactivate this inhibitor of the PI3K-PDK1-Akt signaling (Table I). Complementing PTEN inactivation, Akt phosphorylation on activating Thr³⁰⁸, which is mediated by the PI3K pathway, could be observed in these cells after a prolonged, 2-h treatment (Table I). Moreover, the results of the kinome arrays consistently indicate that EphB receptors support Akt activation through phosphorylation on the Ser⁴⁷³ residue, which is known to be performed by the mTOR kinase acting in the context of the mTORC2 complex (70). EphB receptors appeared to trigger predominantly the prosurvival responses, because no significant effect of ephrin-B2 stimulation on other signaling events central to the T cell lineage, including JNK2-, p38-, Lck-, or Fyn-related phosphorylation (71–74), could be observed (Table I). In agreement, an independent assessment of ephrin-B2–stimulated Molt-16 cells in this array showed that EphBs trigger a statistically significant, 2.0-fold increase in Ser⁴⁷³ phosphorylation of Akt (p < 0.002). Analysis of the E6.1 cell line, using the R&D Systems Human Phospho-Kinase array, also revealed a 2.7-fold increase in Akt phosphorylation on Ser⁴⁷³ induced by ephrin-B2, and this was accompanied by the phosphorylation of one of the Akt targets, the GSK3 kinase (75), being increased 4.9-fold (p values < 0.05).

To further confirm the ability of EphB receptors to support activating Akt phosphorylation, we stimulated H9 cells with ephrin-B2 for both short and long time periods, and followed Akt phosphorylation by Western blotting with anti–phospho-Akt Abs, recognizing specifically the activating phosphorylation at Thr³⁰⁸ or Ser⁴⁷³. Surprisingly, despite the kinome array data showing an increase in Thr³⁰⁸ phosphorylation in response to the prolonged ephrin-B2 stimulation, we could not detect any similar effect by Western blotting (not shown), probably because of insufficient Ab sensitivity. However, our Western blotting with the anti–phospho-Akt(Ser⁴⁷³) Ab has demonstrated that EphB receptors are, indeed, responsible for the stimulation-dependent Akt(Ser⁴⁷³) phosphorylation in malignant T cells both in the absence and presence of active FasR signaling (Fig. 4B–D). This phosphorylation could be easily observed after only 3 min of stimulation and was also detectable after 2 h of ephrin-B2 treatment (Fig. 4B, 4C). In agreement with the results of the kinome array, no effect of EphB receptors on Src family members could be detected using anti–phospho-Src, which specifically recognizes Src family kinases phosphorylated on the activating tyrosine (Fig. 4E). This, once again, suggested that the prosurvival action of EphB receptors may be mediated specifically by Akt.

FasR-triggered death signaling relies on the activation of the caspase proteolytic cascade, typically initiated by the cleavage of caspase-8 and leading to the processing of effector caspases, including caspase-3 (59). Because Akt has been shown to prevent FasR-mediated activation of caspase-8 and -3 in T cells (58), we monitored the effect of ephrin-B2 costimulation on the status of these proteins. Our analysis has shown that, in line with the ability of ephrin-B2–stimulated EphBs to activate Akt and inhibit FasR-initiated apoptotic response, costimulation with ephrin-B2 at least partially suppresses the proteolytic activation of both caspases (Fig. 5). This matches very well the partial protective effect of EphB receptors observed in the TUNEL assay and appears to be efficient in supporting cell survival (Fig. 2), suggesting overall, that by operating through Akt, EphB receptors may actively interfere with FasR proapoptotic signaling.

Akt activity is known to be crucial for cell survival and, in particular, for suppressing FasR-induced apoptosis (58). Because our experiments have shown that EphBs block the apoptotic cell death and prevent FasR signaling, while also promoting Akt activating phosphorylation on Ser⁴⁷³, it appeared that EphB receptors may rely on Akt in their prosurvival signaling.

To examine the requirement for Akt for the prosurvival action of EphB receptors, we treated H9 and Molt-16 cells with the Akt inhibitors, Akti-1/2 and perifosine, or with inhibitors of signaling molecules known to play a crucial role in T cell biology, including MEK and Src family kinases. Although the MEK1/2 inhibitors, U0126 and PD98059, and the Src family inhibitor, SU6656, efficiently suppressed their related phosphorylation events (Fig. 6A, 6C, 6E, 6G), they failed to produce any consistent significant effect on the EphB receptor-induced antiapoptotic response (Figs. 6B, 6D, 6F, 7A). In contrast, treatments with both Akt inhibitors strongly suppressed the ability of EphBs to rescue H9 and Molt-16 cells (Fig. 7B, 7C), thus supporting the central role for Akt signaling in the antiapoptotic functions of EphB receptors in T cell malignancies.

FasR-initiated cell death plays an important role in the elimination of residual malignant T cells by cytotoxic T lymphocytes and NK cells in the course of the graft-versus-leukemia/lymphoma response after bone marrow transplantation (19, 21–23). This response is very effective in some malignancies, including myeloid leukemia; however, it has proved to be inefficient in T-ALL patients (2, 17, 21). Our work shows that malignant T cells may be at least partially protected from FasR-triggered apoptosis by the action of ephrin-B–activated Eph receptors. These receptors are typically represented by EphB receptors (EphB1–4, EphB6) and EphA4 (34). Our analysis suggests that malignant T cells consistently express EphB3 and most often EphB6, whereas frequently missing EphA4, which, according to previously published data and our observations (Supplemental Table I), is expressed in normal human T lymphocytes, in addition to EphB3 and EphB6 (46, 76, 77). Interestingly, EphB6 appears not to be strictly required for the antiapoptotic effect, because stimulation with ephrin-B2 rescues patient 1 T-ALL cells from FasR-induced death despite its absence, suggesting that EphB3 alone may be sufficient, especially when overexpressed (Figs. 1A, 2C). Nevertheless, our observations showing that EphB6 frequently accompanies the EphB3 receptor in T-ALL cells, and that stimulation with EphB6 and EphB3 common ligands, ephrin-B1 and ephrin-B2 (34), suppresses the apoptotic response, indirectly suggest that it may act in cooperation with EphB3 to enhance the antiapoptotic effect of ephrin-B stimulation. This model matches well the unusual properties of the EphB6 receptor, which is an atypical member of the Eph family, carrying alterations in the conserved motifs of its kinase domain and lacking tyrosine kinase activity (78, 79). Because of its kinase-deficient nature, EphB6 relies on the assistance provided by the catalytically active members of the EphB group and has been shown to interact with EphB1 and EphB4 (37, 80). Therefore, it is possible that EphB6 may form heteroreceptor complexes with EphB3 and support its antiapoptotic signaling in malignant T cells. It is also possible that the EphB3 and EphB6 receptors may have prosurvival functions in normal T cells, whereas EphA4, which also interacts with ephrin-B2 (34), may interfere with their antiapoptotic action, and EphA4 absence may provide a survival advantage to malignant lymphocytes. Interestingly, while this article was in preparation, another group reported a ligand-dependent inhibitory effect of a different member of the EphA group, the EphA2 receptor, on prosurvival signaling in cancer cells (81). This presents a possibility that EphA4 may exhibit a similar behavior in T lymphocytes, thus moderating the efficiency of EphB receptor prosurvival response. These intriguing models of Eph receptor cross talks are currently under active investigation.

The antiapoptotic action of EphB receptors is accompanied by Akt activation in cancerous T cells through its phosphorylation on Ser⁴⁷³. Akt has been previously reported to interfere with the initiation of FasR signaling in T lymphocytes (58), and in agreement with the ability of ligand-activated EphB receptors to trigger Akt activation, ephrin-B2 stimulation also effectively suppresses FasR-dependent induction of the caspase cascade that plays a central role in FasR-mediated apoptotic response (59).

The initiation of FasR signaling can be also suppressed by the FLIP protein, which structurally resembles caspase-8, but lacks proteolytic activity (82). Therefore, we examined whether EphB receptors may block apoptotic cell death through the induction of FLIP expression; however, we failed to observe any effect of ephrin-B stimulation on the level of FLIP (not shown), which further supported a model where EphB receptors predominantly rely on Akt for the prosurvival action. In line with this, the general importance of Akt for the ability of EphBs to inhibit FasR-induced apoptosis was further confirmed by our observations that Akt, but not MEK or Src family inhibitors, block their prosurvival effect in malignant T cells.

Overall, our observations indicate that EphB receptors may act in malignant T lymphocytes, in particular, in pediatric T-ALL cells, to support their survival through Akt activation and, eventually, inhibition of FasR-initiated signaling and apoptotic cell death. Because FasR-mediated apoptosis plays a crucial role in the immunoelimination of T-ALL cells in the course of the graft-versus-leukemia response, our work also suggests that inhibitors of EphB receptors or EphB receptor-blocking Abs may represent promising new therapeutic approaches, especially when combined with allogeneic bone marrow transplantation. As it appears to be the case with other therapies targeting specific cell surface receptors (83–85), the efficiency of these potential EphB receptor-interfering approaches would depend on the balance of therapeutic benefits and negative side effects, and require a detailed evaluation and careful fine-tuning in relevant animal models.

The authors have no financial conflicts of interest.