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. Author manuscript; available in PMC: 2014 Aug 1.
Published in final edited form as: Am J Transplant. 2013 Jul 10;13(8):2035–2043. doi: 10.1111/ajt.12328

PI3Kδ Inhibition Augments the Efficacy of Rapamycin in Suppressing Proliferation of Epstein-Barr Virus (EBV)+ B Cell Lymphomas

S Furukawa 1,*, L Wei 1, S M Krams 1,2, C O Esquivel 1, O M Martinez 1,2
PMCID: PMC4076428  NIHMSID: NIHMS508086  PMID: 23841834

Abstract

Post-transplant lymphoproliferative disorder (PTLD) continues to be a devastating and potentially life-threatening complication in organ transplant recipients. PTLD is associated with EBV infection and can result in malignant B cell lymphomas. Here we demonstrate that the PI3K/Akt/mTOR pathway is highly activated in EBV+ B cell lymphoma lines derived from patients with PTLD. Treatment with the mTORC1 inhibitor Rapamycin (RAPA) partially inhibited the proliferation of EBV+ B cell lines. Resistance to RAPA treatment correlated with high levels of Akt phosphorylation. An mTORC1/2 inhibitor and a PI3K/mTOR dual inhibitor suppressed Akt phosphorylation and showed a greater anti-proliferative effect on EBV+ B lymphoma lines compared to RAPA. EBV+ B cell lymphoma lines expressed high levels of PI3Kδ. We demonstrate that PI3Kδ is responsible for Akt activation in EBV+ B cell lymphomas, and that selective inhibition of PI3Kδ by either siRNA, or a small molecule inhibitor, augmented the anti-proliferative effect of RAPA on EBV+ B cell lymphomas. These results suggest that PI3Kδ is a novel, potential therapeutic target for the treatment of EBV-associated PTLD and that combined blockade of PI3Kδ and mTOR provides increased efficacy in inhibiting proliferation of EBV+ cell lymphomas.

Keywords: Epstein-Barr Virus, Post-Transplant Lymphoproliferative Disorder, mTOR, PI-3Kinase/Akt Pathway

Introduction

While the use of immunosuppression is required to prevent allograft rejection, it is also associated with significantly increased susceptibility to infection and de novo malignancies in transplant recipients. In fact, post-transplant cancers are projected to surpass cardiovascular complications as the leading cause of death in transplant recipients in the next fifteen years (1). Epstein Barr virus (EBV)+ B cell lymphomas are the most serious form of post-transplant lymphoproliferative disorder (PTLD).

Management of patients with EBV-associated PTLD is complex. The initial strategy is usually to reduce or withdraw immunosuppression to allow the recipient’s immune system to recover and eliminate virally-infected lymphoma cells. However, this maneuver is often unsuccessful and places the graft in jeopardy of rejection. Other treatment approaches include chemotherapy, radiotherapy, the use of anti-B cell antibodies such as Rituxan and surgical resection when feasible (2). Determining the optimal treatment for individual patients, however, remains difficult and outcomes are mixed. Indeed, the variable efficacy of these approaches, combined with the complexity of PTLD, suggests that a more thorough understanding of the underlying molecular pathways is needed to develop more effective treatment strategies.

The mammalian target of rapamycin (mTOR) inhibitor, Rapamycin (RAPA), is an effective immunosuppressive drug that has been used in clinical transplantation as well as for treatment of human cancer (3, 4). We, and others, have suggested that RAPA and second generation Rapalogs such as everolimus and temsirolimus, may have dual benefit in transplant patients as immunosuppressives and as anti-tumor agents (5–8). However, definitive data is lacking on the efficacy of mTOR inhibitors in PTLD. Several studies report a lower overall incidence of malignancies, including PTLD, in graft recipients receiving RAPA-based maintenance therapy compared with calcineurin inhibitor-based therapies (9–13). In contrast, either no difference, or an increased incidence of PTLD in transplant recipients on RAPA-based maintenance therapy has also been reported (14–16). Thus, at present it is difficult to draw conclusions regarding the impact of RAPA on the incidence of PTLD. With respect to conversion to RAPA following diagnosis of PTLD, several small series have described a beneficial effect with complete remission often observed (17–22). Nevertheless, an unresolved issue is the variable response of mTOR-based therapies on the incidence and progression of EBV-associated PTLD.

Latent Membrane Protein 1 and 2a (LMP1 and LMP2a) are two major EBV latent cycle proteins that function as constitutively active mimics of CD40 and the B cell receptor, respectively (23, 24). We previously showed that LMP1 and LMP2a drive activation of the PI3K/Akt pathway and thereby promote cell survival and growth in EBV-infected B cells (25–27). PI3K is a lipid kinase that is critical for propagating growth factor signals for cell growth, proliferation, and survival and the serine/threonine kinase Akt is an important target of PI3K in these processes (28). Of the eight isoforms of PI3K in mammals, the class IA PI3Ks is known to be responsible for Akt activation (29). Class IA PI3K are heterodimers consisting of a p110 α, β, or δ catalytic subunit and a p85 regulatory subunit. Whereas p110α and β are expressed in all cells, p110δ is expressed mainly in leukocytes but is also expressed in many cancers (30, 31). mTOR is a serine threonine kinase that acts as a central node to integrate signals received from the PI3K and Ras pathways to coordinate protein and lipid biosynthesis and growth factor induced cell cycle progression (32). In mammals two mTOR complexes exist, mTORC1 and mTORC2, each composed of mTOR, a common regulatory subunit mLST8, and at least a third subunit that determines downstream substrates. mTORC1 is RAPA-sensitive, is activated downstream of Akt, and can promote growth through suppression of 4E-BP1, an inhibitor of cap-dependent translation, as well as through activation of p70S6 Kinase 1 (S6K1), a protein kinase required for G1 cell cycle progression. In contrast, mTORC2 is generally considered RAPA-insensitive, and phosphorylates Akt at serine 473 (Ser473), thereby increasing Akt activity. Recent studies suggest that mTORC1 activation causes a negative feedback through S6K1 that reduces the activity of PI3K. Thus, inhibition of mTORC1 by RAPA can result in a rebound effect leading to increases in PI3K activation (33). This raises the possibility that the PI3K/AKT pathway may influence susceptibility of EBV+ B cell lymphomas to RAPA and Rapalogs, and further suggests that blocking both PI3K and mTOR could provide augmented efficacy in tumors with dysregulated PI3K/Akt activation.

In the present study, we aimed to identify molecular mediators within the PI3K/Akt/mTOR signaling axis that govern proliferation of EBV+ PTLD lymphomas. We also evaluated the effects of RAPA and various inhibitors that target the PI3K/Akt/mTOR pathway on the proliferation of PTLD-derived EBV+ B lymphoma cell lines. Our results indicate that PI3Kδ, in particular, plays a central role in proliferation of EBV+ B cell lymphomas and that targeting PI3Kδ augments the efficacy of mTOR-based treatment of EBV+ B cell lymphomas in PTLD.

Materials and Methods

Reagents

Primary antibodies to the following molecules were obtained: β-actin (Sigma St. Louis, MO, USA), Akt, phospho-Akt (Ser473), phospho-Akt (Thr308), S6K1, phospho-S6K1, p110α, p110β (Cell Signaling Technology, Danvers, MA, USA), and p110δ (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies, including HRP-conjugated polyclonal goat anti-rabbit IgG and HRP-conjugated polyclonal donkey anti-mouse IgG, were obtained from Jackson Immunoresearch Labs, Inc. (West Grove, PA, USA). Recombinant proteins, PI3Kα, β, and δ, were obtained from SignalChem Pharmaceuticals Inc. (Richmond, BC, Canada). RAPA, PI3K inhibitor GDC-0941, and PI3K/mTOR dual inhibitor NVP-BEZ235 were obtained from LC Laboratories (Woburn, MA, USA). PI3Kα inhibitor A66, PI3Kδ inhibitor CAL-101 (now GS-1101), and mTOR inhibitor AZD8055 were obtained from Selleck (Houston, TX, USA) and the PI3Kβ inhibitor TGX-221 was obtained from Chemdea (Rodgewood, NJ, USA).

Cell lines

The EBV+ B cell lines derived from the blood (MF4, VB5, JB7) or lymph nodes (AB5) of patients diagnosed with PTLD were generated in our lab as previously described (5, 34). The Burkitt’s lymphoma line BL41 was kindly provided by Dr. Elliot Kieff (Harvard Medical School). Cell lines were maintained in a 5% CO2 humidified 37°C incubator in RPMI 1640 (Mediatech Inc., Waterford, CT, USA) supplemented with 10% heat-inactivated FCS (Serum Source International, Charlotte, NC, USA), and 50 units/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA).

Cellular proliferation assay

Cells (2 × 105 cells/ml) were cultured for 72 h in quadruplicate in 96-well flat bottom plates in serial dilutions of small molecule inhibitors or equivalent amounts of vehicle (DMSO; 1:1000). Cell proliferation was assessed by MTS assay using the Cell Titer 96 AQueous One Solution Cell Proliferation Assay reagent (Promega, Madison, WI, USA) according to the manufacturer’s directions. Data were converted to a percentage of the absorbance at 490 nm of control cells that were treated with DMSO or transfected with control siRNA.

Western blotting

Cells were harvested, lysates prepared and Western blot analysis performed as previously described (25).

siRNA knockdown of p110 expression

Silencer Select Validated siRNA for p110α, β, δ, and a negative control siRNA were obtained from Ambion (Grand Island, NY, USA). VB5 cells (3 × 106) were transfected with 100 pmol siRNA using the Amaxa Nucleofector in 100 μl of cell line nucleofector kit V solution (Lonza, Basel, Switzerland) according to the manufacturer’s directions. Cells were pulsed with program Y-001. Transfected cells were incubated in a 6-well plate or 96-well plate at 5 × 105 cells/ml. After 48 h, cells were collected and subjected to western blot analysis for confirmation of target-specific knockdown and Akt phosphorylation. For the analysis of cell proliferation, RAPA or vehicle (DMSO) was added to the cell culture 6 h after transfection, and proliferation was measured at 72h after transfection using the MTS assay as described above.

Purification and stimulation of human T cells from peripheral blood

Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors by Ficoll density gradient centrifugation. T cells were isolated from PBMC by negative selection using the MACS pan T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). Purity of isolation was determined by immunofluorescent staining for CD3 expression and flow cytometric analysis of the T cell-enriched fraction. T cells (2.5 × 105 cells/well) were plated in 96-well plate pre-coated with 5 μg/ml anti-CD3 and anti-CD28 antibodies, and cultured for 72 h in serial dilutions of small molecule inhibitors or equivalent amounts of vehicle (DMSO). T cell proliferation was assessed by MTS assay.

Statistical Analysis

Statistical analysis of proliferation results were performed using Student’s t test, and P values of <0.05 were considered statistically significant.

Results

PI3K/Akt and mTORC2 are constitutively activated in PTLD-derived EBV+ B cell lymphomas

We evaluated the effect of RAPA on the proliferation of four EBV+ B lymphoma cell lines (VB5, AB5, JB7, and MF4) derived from patients with PTLD. RAPA inhibited the proliferation of all EBV+ B lymphoma cell lines in a dose-dependent manner (Figure 1A). However, the effect of RAPA on cell proliferation was partial, and complete inhibition of proliferation was not observed even at high concentrations of RAPA (Figure 1A). Interestingly, the maximum efficacy of RAPA was variable amongst EBV+ B lymphoma cell lines, with the VB5 cell line particularly resistant to RAPA-mediated inhibition compared to the other cell lines. The maximum reduction of cell proliferation by RAPA was 29%, 51%, 64%, and 66% in VB5, AB5, JB7, and MF4 cell lines, respectively.

Figure 1. Constitutive activation of PI3K/Akt pathway in PTLD-derived EBV+ B cell lymphomas.

Figure 1

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(A) EBV+ B cell lymphoma cell lines from PTLD patients (VB5, AB5, JB7 and MF4) were treated with Rapamycin (RAPA; 0.01 – 100 nM) for 72 h. Cell proliferation was assessed by MTS assay. Data are shown as the % control ± SEM of quadruplicate cultures. (B) Cell lysates of EBV-negative Burkitt’s lymphoma line (BL41) and EBV+ PTLD lines (AB5, MF4, VB5 and JB7) were separated by SDS-PAGE, transferred to nitrocellulose, and probed for phospho-Akt (Thr308 and Ser473) and total Akt by Western blotting. Akt phosphorylation was quantified with densitometric analysis using ImageJ software, normalized to total Akt level, and represented relative to the level of VB5. Data are expressed as means ± SEM of three independent experiments. Representative blot is shown. (C) EBV+ PTLD lines were treated with 100 nM RAPA for the indicated amounts of time. Cells were then harvested and lysed. Cell lysates were separated by SDS-PAGE, transferred to a nitrocellulose, and subjected to immunoblot analysis for p-S6K1, S6K1, p-Akt (T308 and S473) and Akt. Densitometry was calculated with ImageJ and is indicated numerically below each set of blots.

We previously demonstrated that the PI3K/Akt pathway is activated in EBV+ B lymphoma cells (26). To address whether dysregulated PI3K/Akt activation influences the efficacy of RAPA, we assessed Akt phosphorylation in the PTLD-derived EBV+ B cell lines. Akt is activated by phosphorylation at two distinct residues, Thr308 and Ser473, and phosphorylation of Thr308 and Ser473 is regulated by PI3K/PDK1 and mTORC2, respectively. Western blot analyses revealed constitutive Akt phosphorylation at both Thr308 and Ser473 in all EBV+ B cell lines (Figure 1B) compared to the EBV-negative Burkitt’s lymphoma line BL41. While the levels of Akt phosphorylation were variable amongst cell lines, the VB5 cell line shown above to be more resistant to RAPA, had the highest level of Akt phosphorylation (Figure 1B). Taken together, these data indicate that in addition to PI3K, mTORC2 is also activated in EBV+ B cell lymphomas.

We next examined the effect of RAPA on activation of the Akt/mTOR pathway. We observed constitutive phosphorylation of the mTORC1 substrate, S6K1, in all EBV+ B lymphoma cell lines (Figure 1C). This up-regulated S6K1 phosphorylation was completely inhibited when cells were treated with RAPA (Figure 1C, top panels). In contrast, RAPA had only a small effect on Akt phosphorylation at either residue Thr308, or Ser473, in any of the cell lines (Figure 1C, lower panels). Akt is known to activate multiple downstream pathways other than mTORC1, such as FOXO, BAD and glycogen synthase kinase 3 (GSK-3) (35), which also play a role in regulating apoptosis and promoting cell proliferation. Thus, the partial efficacy of RAPA on EBV+ B cell lymphomas may be attributed to the fact that RAPA blocks only the mTORC1 component of Akt downstream signaling.

Combined inhibition of PI3K and mTOR is more effective than mTORC1 inhibition alone in suppressing proliferation of PTLD-derived EBV+ B cell lymphomas

Because the mTORC1 inhibitor RAPA only partially inhibited proliferation of EBV+ B lymphoma cell lines, we asked whether dual mTORC1 and mTORC2 inhibition could provide augmented inhibition. We examined the anti-proliferative effect of the mTOR inhibitor AZD8055 that targets both mTORC1 and mTORC2, and the PI3K/mTOR dual inhibitor NVP-BEZ235 that blocks PI3K, as well as mTORC1 and mTORC2. Both inhibitors showed more potent anti-proliferative efficacy (Figure 2A and 2B) than RAPA (Figure 1A) against each of the cell lines, including the RAPA-resistant cell line VB5. Proliferation was reduced by 73%, 84%, 69%, and 77% with 1 μM AZD8055, and by 64%, 83%, 77%, and 68% with 1 μM NVP-BEZ235, in the AB5, MF4, VB5, and JB7 cell lines, respectively.

Figure 2. Effect of PI3K/mTOR inhibition on proliferation of PTLD-derived EBV+ B cell lymphomas.

Figure 2

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(A and B) EBV+ PTLD lines were treated for 72 h with the indicated final concentration of the mTOR inhibitor AZD8055 or the PI3K/mTOR dual inhibitor NVP-BEZ235. Cell proliferation was assessed by MTS assay. Data are shown as the % control ± SEM of quadruplicate cultures. (C) EBV+ PTLD lines were treated with 100 nM RAPA, 1 μM AZD8055, 1 μM NVP-BEZ235, or DMSO equivalent for 2 h. Cells were then harvested and lysed. Cell lysates were separated by SDS-PAGE, transferred to a nitrocellulose, and subjected to immunoblot analysis for p-S6K1, S6K1, p-Akt (T308 and S473) and Akt. Densitometry was calculated with ImageJ and is indicated numerically below each set of blots.

We next examined the effect of these inhibitors on Akt phosphorylation. RAPA had only a modest effect on Akt phosphorylation (Figure 2C). In contrast, NVP-BEZ235 markedly suppressed phosphorylation of S6K1 as well as Akt at both Thr308 and Ser473 (Figure 2C), indicating that NVP-BEZ235 more broadly blocks downstream Akt signaling and thereby effectively inhibits the proliferation of EBV+ B lymphoma cells. AZD8055, an mTORC1/mTORC2 dual inhibitor, also reduced phosphorylation of S6K1, Akt-Ser473 and Akt-Thr308, although AZD8055 does not block PI3K activity directly. Since phosphorylation of Akt-Ser473 is proposed to stabilize that of Akt-Thr308 (36), our results suggest that AZD8055 inhibits Akt-Ser473 phosphorylation and thereby indirectly reduces Akt-Thr308 phosphorylation. Importantly, these dual inhibitors showed potent anti-proliferative effects against all EBV+ B lymphoma cell lines tested, regardless of their responsiveness to RAPA. Taken together, these results suggest that blocking mTORC1 and mTORC2, or PI3K and mTOR, provides superior anti-proliferative benefit compared to blocking mTORC1 alone for PTLD-derived EBV+ B lymphoma cells.

PI3Kδ is highly expressed and is required for activation of the PI3K/Akt pathway in PTLD-derived EBV+ B cell lymphomas

To clarify which PI3K isoform is responsible for Akt activation in EBV+ B cell lymphoma cells, we first evaluated the expression of PI3K isoforms by immunoblotting using antibodies specific for each p110 isoform. All three isoforms of class IA PI3Ks, α, β, and δ, were detected in EBV+ B cell lymphoma cell lines (Figure 3A). By comparing the band intensity between cell lysates and recombinant proteins standards, we determined that PI3Kδ was highly expressed in EBV+ B cell lymphomas, whereas the relative expression levels of PI3Kα and PI3Kβ were much lower.

Figure 3. Expression and function of PI3K isoforms in PTLD-derived EBV+ B cell lymphomas.

Figure 3

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(A) Expression of PI3K isoforms in EBV+ PTLD lines was assessed by western blotting. Cell lysates (20 μg protein) were separated by SDS-PAGE, transferred to a nitrocellulose, and subjected to immunoblot analysis using specific antibodies for p110α, β, and δ isoform. Actin was used as a loading control. Purified recombinant PI3K proteins (10 ng) were used as controls. (B) EBV+ PTLD line VB5 cells were transfected with p110α, β, δ siRNA or control siRNA by Amaxa nucleofection. Cells were collected and lysed 48 h after nucleofection. Lysates were separated by SDS-PAGE, transferred to a nitrocellulose, and subjected to immunoblot analysis for p110α, β, δ, p-Akt (T308 and S473), and total Akt. Actin was used as a loading control. Densitometry was calculated with ImageJ and normalized to actin (p110 isoforms) or total Akt (p-Akt). Data are indicated numerically below each set of blots. (C) VB5 cells were transfected with p110α, β, δ siRNA or control siRNA, and incubated at 5 × 105/ml in 96-well plates. After 6 h, cells were treated with the indicated final concentrations of RAPA for additional 66 h. Cell proliferation was assessed by MTS assay. Data are shown as the % control ± SEM of quadruplicate cultures. Statistical significance compared to control by unpaired t test are indicated as follows: *P<0.01, **P<0.001, ***P<0.0001.

To address whether p110δ is functionally involved in Akt activation, we next examined the effect of p110 isoform-specific knockdown. Transfection with p110 isoform-specific siRNA significantly down-regulated (~90%) the corresponding p110 isoform, but did not affect the expression of other isoforms (Figure 3B, left panel). Moreover, knockdown of the p110δ isoform markedly decreased the level of Akt phosphorylation at both Thr308 and Ser473 (Figure 3B, right panel). In contrast, knockdown of either p110α, or p110β, had no effect on Akt phosphorylation. These results suggest that PI3Kδ plays a predominant role in dysregulated PI3K/Akt activation in EBV+ B lymphoma cells.

Next we examined the effect of p110δ knockdown on cell proliferation. Transfection with p110δ siRNA alone (Fig 3C, 0 nM RAPA) had minimal effect on EBV+ B cell lines compared to cells transfected with control siRNA. However, knockdown of p110δ in combination with RAPA significantly inhibited proliferation of VB5 cells compared to RAPA alone (Figure 3C, P<0.01 at 0.1–100 nM RAPA). These results indicate that PI3Kδ inhibition alone is not sufficient to effect proliferation of EBV+ B cell lymphomas, but that combined inhibition of PI3Kδ and mTORC1 mediates synergistic anti-proliferative effects on EBV+ B cell lymphomas.

The PI3Kδ inhibitor CAL-101 augments the effect of RAPA on proliferation of PTLD-derived EBV+ B cell lymphomas

To further investigate the contribution of specific p110 isoforms to proliferation of EBV+ B cell lymphomas, we utilized p110 isoform-selective small molecule inhibitors. First, we examined the effect of PI3K inhibitors on Akt phosphorylation in VB5 cells. The PI3Kδ selective inhibitor CAL-101 suppressed Akt phosphorylation, as did the PI3K-pan inhibitor GDC-0941 (Figure 4A). In contrast, the PI3Kα and β selective inhibitors, A66 and TGX-221 respectively, were less effective. These results are consistent with findings in the siRNA experiments discussed above and indicate an important role for PI3Kδ in activation of Akt in EBV+ B cells.

Figure 4. Effect of PI3Kδ inhibitor on proliferation of PTLD-derived EBV+ B cell lymphomas.

Figure 4

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(A) EBV+ PTLD line VB5 cells were treated with 1 μM PI3K-pan inhibitor GDC-0941, 1 μM PI3Kδ selective inhibitor CAL-101, 1 μM PI3Kα inhibitor A66, or 1 μM PI3Kβ inhibitor TGX-221 for 2 h. Cells were then harvested and lysed. Cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblot analysis for p-Akt (T308 and S473) and Akt. Densitometry was calculated with ImageJ and is indicated numerically below each set of blots. (B) EBV+ PTLD lines were incubated with the indicated final concentration of CAL-101 for 72 h. Cell proliferation was assessed by MTS assay. Data are shown as the % control ± SEM of quadruplicate cultures. (C) VB5 cells were incubated with RAPA (0–100 nM) in the presence or absence of CAL-101 (0, 0.1, 1 or 10 μM) for 72 h. Cell proliferation was assessed by MTS assay. Data are shown as the % control ± SEM of quadruplicate cultures. Statistical significance compared to the group without CAL-101 treatment by an unpaired t test are indicated as follows: *P<0.01, **P<0.001, ***P<0.0001 (D) VB5 cells were treated with 100 nM RAPA and/or 1 μM CAL-101 for 2 h. Cell lysates were separated by SDS-PAGE, transferred to a nitrocellulose, and subjected to immunoblot analysis for p-Akt (T308 and S473), Akt, p-S6K1 and S6K1. Densitometry was performed as in (A). (E) EBV+ PTLD lines were incubated with 10 nM RAPA and/or 1 μM CAL-101 for 72 h. Cell proliferation was assessed by MTS assay. Data are shown as the % control ± SEM of quadruplicate cultures. Statistical significance by an unpaired t test between RAPA or CAL-101 treated group and group treated with a combination of RAPA and CAL are indicated as follows: *P<0.01, **P<0.001, ***P<0.0001.

Next, we examined the effect of the small molecule PI3Kδ inhibitor, CAL-101, on proliferation of EBV+ B cell lymphomas. CAL-101 reduced the proliferation of all EBV+ B lymphoma cell lines in a dose-dependent manner, but the effect was moderate with ~50% inhibition achieved at 10 μM CAL-101. However, CAL-101 significantly enhanced the efficacy and potency of RAPA to inhibit proliferation of VB5 cells (Figure 4C). To determine why treatment with CAL-101 alone only partially inhibited proliferation of VB5 cells, we analyzed the effect of CAL-101 on S6K1 phosphorylation. CAL-101 showed moderate inhibition of S6K1 phosphorylation in VB5 cells (Figure 4D, lower panel). In contrast, the combined use of CAL-101 and RAPA suppressed both S6K1 and Akt phosphorylation (Figure 4D, upper and lower panels). Furthermore, the addition of CAL-101 significantly augmented the ability of RAPA to inhibit proliferation in all EBV+ B lymphoma cell lines (Figure 4E) including the RAPA-resistant VB5 cell line. These results suggest that regardless of RAPA susceptibility, EBV+ B lymphoma cell proliferation can be effectively inhibited by adjunctive use of CAL-101 with RAPA. Thus, targeting PI3Kδ augments the efficacy of RAPA in inhibition of EBV+ B cell lymphoma proliferation.

Inhibition of PI3Kδ spares T cell function following RAPA treatment

We next investigated whether inhibition of the PI3K/Akt/mTOR pathway influences the T cell response. RAPA induced partial, but marked, inhibition of human T cell proliferation induced by stimulation with anti-CD3 and anti-CD28 antibodies (Figure 5A), consistent with its role as a potent immunosuppressive agent. The mTOR inhibitor AZD8055 and the PI3K/mTOR dual inhibitor NVP-BEZ235 showed more pronounced anti-proliferative activity than RAPA on human T cells, suggesting that the addition of pan PI3K or mTORC2 targeting not only enhances the efficacy against EBV+ B lymphoma proliferation, but also increases the immunosuppressive effect (Figure 5A). In contrast, the PI3Kδ selective inhibitor CAL-101 had a more modest on T cell proliferation. Furthermore, adjunctive use of CAL-101 did not markedly augment the inhibitory effect of RAPA on T cell proliferation (Figure 5B). Taken together, these results suggest that PI3Kδ inhibition can increase the anti-lymphoma efficacy of RAPA while largely sparing the T cell response.

Figure 5. Effect of PI3K/mTOR inhibitors on T cell proliferation.

Figure 5

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(A) T cells isolated from healthy donors were stimulated with anti-CD3 and anti-CD28 antibodies for 72 h, in the presence of the indicated final concentrations of RAPA, AZD8055, NVP-BEZ235, and CAL-101. Cell proliferation was assessed by MTS assay. Data are shown as % inhibition of cell proliferation compared with control DMSO-treated cells ± SEM of triplicate cultures. (B) T cells isolated from healthy donor were stimulated with anti-CD3 and anti-CD28 antibodies, and incubated with RAPA (1–100 nM) in the presence or absence of CAL-101 (0, 0.1, or 1 μM) for 72 h. Cell proliferation was assessed by MTS assay. Data are shown as % inhibition of cell proliferation compared with control cells in the absence of CAL-101 ± SEM of triplicate cultures and are representative of three experiments.

Discussion

Post-transplant malignancies are emerging as a leading cause of death in organ transplant recipients, particularly as long-term graft survival improves and younger patients benefit from transplantation. Major gaps in our knowledge concerning the pathogenesis of EBV+ B cell lymphomas and selection of the most efficacious treatments from the current available options have hindered optimal management of PTLD. In this study we investigated the underlying molecular pathways that contribute to proliferation of EBV+ B cell lymphomas, and propose potential new therapeutic strategies for the treatment of EBV-associated PTLD. We demonstrate that complete inhibition of proliferation of EBV+ B cell lymphomas is difficult to achieve at doses of RAPA utilized in transplant recipients. Furthermore, some variability in sensitivity to RAPA was evident amongst EBV+ B lymphoma cell lines, with the VB5 cell line particularly refractory to RAPA treatment. Our results with RAPA are in agreement with other studies reporting that RAPA and Rapalogs significantly inhibit proliferation of EBV+ B lymphoblastoid cell lines (5, 37). However, our findings that the susceptibility to RAPA differs considerably amongst EBV+ B cells may provide insight into the mixed reports on the impact of RAPA on the incidence of PTLD in transplant recipients. The reason for this variability is unknown but it is clear that a good predictive marker for lymphoma susceptibility to RAPA would help to determine the optimal therapeutic approach. Given that Akt phosphorylation was significantly up-regulated in the RAPA-resistant VB5 cells, the extent of Akt phosphorylation may be linked to the responsiveness of the B lymphoma cell line to RAPA. However, further analyses of a larger panel of PTLD-derived cell lines would be required to develop accurate markers that predict which patients will benefit from the use of RAPA.

We also demonstrated that PI3K/mTOR inhibition is superior to RAPA alone in attenuating proliferation of EBV+ B lymphomas. PI3K/mTOR dual inhibitors can achieve complete suppression of the PI3K/Akt pathway, thus these results might not be surprising. On the other hand, the use of PI3K/mTOR inhibitors may raise concerns about toxicity since PI3Kα and PI3Kβ are ubiquitously expressed, and are involved in numerous cellular functions. Knockout mice of PI3Kα or PI3Kβ are embryonic lethal (38, 39), and mice with heterozygous mutations in PI3Kα displayed reduced somatic growth and impaired insulin and glucose tolerance (40). Furthermore, we show that PI3K/mTOR dual inhibition has a profound effect on the T cell response. Clearly, further diminution of T cell function is not ideal in the setting of PTLD, thus it is difficult to propose that PI3K/mTOR inhibitors are beneficial for treatment of EBV-associated PTLD. In contrast, the expression of PI3Kδ is generally restricted to hematopoietic cells, and knockout of the δ isoform results in viable mice displaying alteration mainly in B cell function (41, 42). PI3δ acts downstream of several important molecules expressed on B cells including the B cell antigen receptor, CD40, BAFF-R, and the IL-4 receptor (43). In EBV+ B cell lymphomas, as studied here, it is possible that PI3Kδ is also activated by viral proteins such as LMP1 since it has been shown that LMP1 activates PI3Kδ in epithelial cells (44) and we demonstrated that LMP1 activates PI3K in EBV+ B cell lines (27). In contrast to its activity on B cells, CAL-101 seems to spare normal T cells and natural killer cells from cytotoxicity, and does not diminish antibody-dependent cellular cytotoxicity (45). Here we demonstrate that CAL-101 augments the anti-lymphoma efficacy of RAPA with minimal additional effect on T cells, a scenario that could be beneficial for generating an anti-tumor immune response. CAL-101 is currently in clinical trials for chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (46, 47), and has shown promising clinical activity in refractory CLL (48). Toxicities and side effects have been minimal, so far, in the limited clinical studies with CAL-101 though some patients were reported to develop transient lymphocytosis. Collectively, these results suggest that CAL-101 can be a potent adjunctive drug for prevention and treatment of EBV+ B cell lymphomas.

In summary, we demonstrate that the PI3K/Akt/mTOR pathway is constitutively active in EBV+ B lymphomas, and that PI3Kδ plays a critical role in this activation. We also demonstrate that the small molecule PI3Kδ inhibitor, CAL-101, and RAPA synergistically suppress the proliferation of EBV+ B lymphomas. These results provide potential new therapeutic strategies for the treatment of EBV-associated PTLD.

Acknowledgments

This work was supported by NIH RO1AI41769 (OMM), the Lucile Salter Packard Foundation, and the Arnold and Barbara Silverman Fund. We thank Dr. Olivia Hatton for her help with the siRNA assay.

Abbreviations

AMPK

AMP-activated kinase

CLL

chronic lymphocytic leukemia

EBV

Epstein-Barr Virus

GSK-3

glycogen synthase kinase 3

LMP1

latent membrane protein 1

LMP2a

latent membrane protein 2A

mTOR

mammalian target of rapamycin

mTORC1 or 2

mTOR complex 1 or 2

PBMC

peripheral blood mononuclear cells

PI3K

phosphoinositide-3-kinase

PTLD

Post-Transplant Lymphoproliferative Disorder

RAPA

rapamycin

Ser

Serine

S6K1

p70S6 kinase

Thr

Threonine

Footnotes

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

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