Antitumor effects of duvelisib on Epstein–Barr virus‐associated lymphoma cells

Abstract Epstein–Barr virus (EBV) is a ubiquitous oncogenic virus that is associated with B cell lymphomas, including Burkitt lymphoma and Hodgkin lymphoma. Previous studies have shown that the phosphatidylinositol 3‐kinase (PI3K)/Akt pathway is activated in EBV‐associated lymphomas and can be a novel therapeutic target. An oral dual inhibitor of PI3Kγ and PI3Kδ, duvelisib, is in clinical trials for the treatment of lymphoid malignancies. In this study, we evaluated how duvelisib affects the activity of the PI3K/Akt signaling pathway and if it has antitumor effects in EBV‐associated lymphoma cell lines. We found that the PI3K/Akt signaling pathway was activated in most of the B and T cell lymphoma cell lines tested. Additionally, duvelisib treatment inhibited cellular growth in the tested cell lines. Overall, B cell lines were more susceptible to duvelisib than T and NK cell lines in vitro regardless of EBV infection. However, the additional influence of duvelisib on the tumor microenvironment was not assessed. Duvelisib treatment induced both apoptosis and cell cycle arrest in EBV‐positive and ‐negative B cell lines, but not in T cell lines. Furthermore, duvelisib treatment reduced the expression of EBV lytic genes (BZLF1 and gp350/220) in EBV‐positive B cell lines, suggesting that duvelisib suppresses the lytic cycle of EBV induced by B cell receptor signaling. However, duvelisib did not induce a remarkable change in the expression of EBV latent genes. These results may indicate that there is therapeutic potential for duvelisib administration in the treatment of EBV‐associated B cell lymphomas and other B cell malignancies.


Introduction
-3B, and -3C, respectively) and latent membrane proteins (LMP) 1 and 2 (LMP1, LMP2, respectively). Conversely, EBV-positive Burkitt lymphoma is usually characterized by a type I latency pattern and primarily expresses EBNA-1 [1]. Patients with EBV-associated lymphoid malignancies treated with cytotoxic chemotherapy are often refractory to it. Therefore, understanding the underlying molecular pathways of EBV-associated lymphoid malignancies is necessary to develop more effective treatment strategies. LMP1 is considered a major EBV oncoprotein that mediates activation of multiple cellular signaling pathways, such as c-Jun-N terminal kinases (JNK), tumor necrosis factor (TNF) receptor associated factor TRAF, and nuclear factor kappa beta (NF-κB) signaling [5,6]. In 2003, Dawson et al. demonstrated that LMP1 can also activate phosphatidylinositol 3-kinase (PI3K) and induce subsequent activation of serine/threonine protein kinase (Akt) to promote cell survival [7]. Subsequently, several other studies have shown that constitutive activation of the PI3K/ Akt pathway via LMP1 is a key element of LMP1-mediated transformation. This suggests that the PI3K/Akt pathway can be a therapeutic target for the treatment of EBVassociated lymphoid malignancies [8][9][10][11]. Furthermore, a recent study using genome-wide CRISPR/Cas9 screens in EBV-transformed B cells has revealed that the EBV oncoprotein LMP2A also activates the PI3K/Akt pathway [12].
The PI3K/Akt signaling pathway has been associated with many virus-associated cancers. Moreover, it is known to regulate numerous biological activities, including cellular growth, survival, and proliferation [13,14]. The PI3Ks are divided into three classes: I, II, and III. Of the three classes of PI3K isoforms, class I PI3K comprises four different catalytic isoforms including PI3Kα, β, γ, and δ [15]. While PI3Kα and β are ubiquitously expressed in all mammalian tissues, PI3Kγ and PI3Kδ have more selective roles. PI3Kγ has a particular role in T cell activation and PI3Kδ expression is largely restricted to hematopoietic cells, and has a crucial role in mediating B-cell receptor (BCR) signaling, proliferation/survival, and migration [16]. Furthermore, PI3Kδ is aberrantly activated in a variety of B cell malignancies such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia [17,18]. Therefore, PI3Kδ has emerged as a promising therapeutic target in hematological malignancies. In fact, in a phase III clinical trial testing the oral selective PI3Kδ inhibitor idelalisib in combination with rituximab, the survival of CLL patients improved considerably [19].
Duvelisib is a molecule previously found to inhibit the PI3K/Akt signaling pathway, thereby inhibiting BCR signaling, diminishing chemotaxis, and inhibiting cytokineinduced CLL cell proliferation with minimal apoptosis [20,21]. The duvelisib molecule closely resembles the chemical structure of idelalisib, but biochemically targets PI3Kγ in addition to PI3Kδ [22]. It is expected that dual inhibition of PI3Kγ and PI3Kδ by duvelisib may be another therapeutic target for the treatment of CLL and may overcome resistance formed against idelalisib [23]. Furthermore, clinical studies of duvelisib in indolent non-Hodgkin lymphoma and CLL have shown clinical activity [20,24]. However, the effects of PI3Kγ or PI3Kδ inhibitors on EBV-associated lymphoma cells have not been investigated. In this study, we evaluated the activity of the PI3K/Akt signaling pathway and antitumor effects of duvelisib on EBV-associated lymphoma cell lines.

Cell proliferation
Cells (2 × 10 5 /mL) were cultured for 48 and 72 h in the presence of duvelisib or idelalisib at concentrations from 0.1 to 5 μmol/L. Viable cells were determined using a Countess ™ Automated Cell counter (Invitrogen, Carlsbad, CA). Subsequent experiments were performed in triplicate at minimum.

Detection of PI3K/Akt signaling by immunoblotting
Collected cell extracts were diluted in sample buffer, and prepared for SDS-PAGE. Equal amounts of cellular protein extracts were loaded on 4-15% polyacrylamide gel. βactin was used as a loading control. The separated protein bands were transferred to PVDF membranes, prior to antibody incubation. Membranes were incubated with primary antibodies at 4°C with gentle shaking, overnight. The primary antibodies were as follows: Akt, phospho-Akt (Thr308), PI3Kγ, PI3Kδ, and caspase-3 (Cell Signaling Technology, Beverly, MA), poly ADP ribose polymerase (PARP) and βactin (Sigma-Aldrich, St. Louis, MO).

Apoptosis detection with Annexin V by flow cytometry
Apoptosis was assessed using an Annexin V-PE/7-AAD Apoptosis Detection Kit (BD Pharmingen Biosciences, San Diego, CA), following manufacturer's instructions. Briefly, cells (2 × 10 5 /mL) were seeded in 24-well plates and treated with 5 μmol/L of duvelisib or DMSO for 48 h. Cells were then washed in PBS and incubated with Annexin V-phycoerythrin (PE) in a buffer containing 7-aminoactinomycin D (7-AAD). Apoptotic cells (7-AAD negative, PE Annexin V positive) were analyzed using flow cytometry.

Cell cycle analysis
Cells (2 × 10 5 /mL) were treated with 5 μmol/L of duvelisib or DMSO for 48 h. Cells were then fixed with ice-cold 70% ethanol, and then washed with PBS. Fixed cells were re-suspended in 50 μg/mL propidium iodide (PI) solution (Sigma-Aldrich) with DNase-free RNase, and then analyzed using flow cytometry. Experiments were performed in triplicate.

Real-time RT-PCR
RNA was extracted from 1 × 10 6 cells from the culture medium with the QIAamp RNeasy Mini Kit (Qiagen, Hilden, Germany). Contaminating DNA was removed using the RNase-Free DNase set (Qiagen). The expression of two lytic (BZLF1 and glycoprotein 350/220) and six latent (EBNA1, EBNA2, LMP1, LMP2, EBER1, and BARTs) EBV genes was quantified by one-step multiplex real-time RT-PCR using QuantiFast Multiplex RT-PCR kits (Qiagen) and an Mx3000P instrument (Stratagene, La Jolla, CA) as described previously [36][37][38]. The relative expression of EBV genes were determined by normalization to β2microglobulin mRNA.  Fig. 1A and B). Cell growth inhibition of EBV-negative T cell lines Jurkat, MOLT4, and EBV-positive NK cell line KAI3, was also observed at 1 or 5 μmol/L of duvelisib. However, no growth inhibition was observed in EBV-positive SNT16 and EBV-negative KHYG1. Overall, B cell lines were more susceptible to duvelisib than T and NK cell lines, and further experiments with duvelisib were focused on EBVpositive and -negative B cell lines.

Effect of idelalisib on EBV-positive and negative cell lines
To confirm the effects of the selective PI3Kδ inhibitor idelalisib on B, T, and NK cell lines, 0.1-5 μmol/L of idelalisib was used to treat cells. Viable cells were counted after 72 h of treatment (Fig. 2). Growth of EBV-positive and -negative B cell lines was inhibited by idelalisib in a dose-dependent manner. On the other hand, no or modest growth inhibition was observed in T and NK cell lines.

Duvelisib induces apoptosis in EBV-positive and -negative B cell lines
To investigate whether duvelisib induces apoptosis, Annexin V/7-AAD staining of various duvelisib-treated cells was evaluated. Duvelisib treatment increased the number of apoptotic cells (Annexin V-positive and 7-AAD-negative), compared to DMSO control treated cells in six of seven tested B cell lines (Fig. 4A). However, duvelisib did not induce apoptosis in B cell line P3HR1 and T cell lines (Jurkat and MOLT4). The cleavage of caspase-3 and PARP was also investigated in B and T cell lines to identify apoptosis after duvelisib treatment for 48 h. Duvelisib treatment induced cleavage of PARP in BJAB, Akata (+), and Mutu I (Fig. 4B). Decreased levels of full-length caspase-3 were observed in BJAB and Akata (−), whereas cleaved caspase-3 was not observed in any cell lines. The combined results of flow cytometry and immunoblotting suggest that duvelisib induces apoptosis in EBV-negative and -positive B cell lines, whereas it induces no apoptosis in T cell lines.

Duvelisib induces G1 cell cycle arrest in B and T cell lines
We next examined the effect of duvelisib on cell cycle distribution in EBV-positive and -negative B cells using flow cytometry analysis with PI staining. Duvelisib treatment induced an increase in cells in the G1 phase and a decrease in cells in the S and G2/M phases in all cell lines tested except P3HR1 (Fig. 5). These results indicate that duvelisib induces G1 cell cycle arrest in B and T cell lines resulting in inhibition of proliferation.

Duvelisib suppresses the expression of lytic EBV genes in B cell lines
We next investigated the effect of duvelisib on the expression of eight viral genes in B cell lines. The expression of BZLF1, which is an immediate-early lytic EBV gene, significantly decreased after duvelisib treatment in all EBVpositive B cell lines tested (Fig. 6). Furthermore, the expression of the late lytic EBV gene gp350/220 decreased significantly in Akata (+), Mutu I, and P3HR1. In contrast, the expression of LMP1, a latent oncogene of EBV, was decreased in Mutu I. However, this effect was not observed in other cell lines (Fig. 6). Duvelisib treatment did not induce remarkable change in the expression of the other five latent genes (EBNA1, EBNA2, LMP2, EBER1, and BARTs) (data not shown). Taken together, these results suggest that duvelisib suppresses the lytic cycle of EBV, but has little effect on the latent cycle.

Discussion
In this study, we evaluated the antitumor effects of duvelisib on EBV-associated lymphoma cell lines and found that B cell lines appeared to be more susceptible to duvelisib than T and NK cell lines regardless of the presence of EBV. It was expected that the presence of EBV might be associated with susceptibility to duvelisib because activation of the PI3K/Akt pathway via LMP1 is considered to be one of the key elements of EBV associated malignancies. Compared to EBV-negative Akata cell lines, inhibition of Akt phosphorylation by duvelisib was more clearly observed in EBV-positive cell lines. However, no remarkable differences were observed in susceptibility to duvelisib between EBV-positive and negative B cell lines. It might be possible that LMP1 or other EBV proteins have only a small effect on PI3K/Akt pathway activation in these cell lines. LCL, which is classified as type III EBV latency, was the most susceptible to duvelisib among B cell lines tested. However, the association between latency pattern and susceptibility was not clear in this study. Interestingly, P3HR1 was insensitive to duvelisib despite that fact that it harbors an EBNA2-deficient EBV strain. One study has shown that EBNA2 upregulates Akt activation by inducing miR-21 expression [39]. Although the phosphorylated form of Akt was detected in P3HR1 by immunoblotting, cellular proliferation of R3HR1 might be less dependent on the PI3K/Akt signaling pathway than other EBV-positive B cell lines.
Most of the B cell lines used in this study were cells originating from Burkitt lymphoma. To our knowledge, this is the first study to show the antitumor effects of duvelisib on Burkitt lymphoma cell lines because antitumor effects of duvelisib have been exclusively investigated in CLL [20,21,23]. Regarding idelalisib, a selective PI3Kδ inhibitor, a recent study has shown that antiproliferative effects on EBV-positive and -negative Burkitt lymphoma cell lines (Namalwa and Ramos, respectively) were equivalent to its effects on CLL cell lines [40]. While c-MYC deregulation is a hallmark of Burkitt lymphoma, synergy between constitutive PI3K/Akt signaling pathway activation and c-MYC has been shown. This suggests that the PI3K/ Akt signaling pathway can be a therapeutic target in Burkitt lymphoma [41].
It was expected that duvelisib would have antitumor effects on T or NK cell lines as well as B cell lines because duvelisib is a dual inhibitor of PI3Kγ and PI3Kδ. Compared to idelalisib, a selective PI3Kδ inhibitor, duvelisib showed slightly more cell growth inhibition of T cell lines such as Jurkat or MOLT4. However, cell growth inhibition by duvelisib was modest in T or NK cell lines. Overall, the antitumor effects of idelalisib and duvelisib were similar in the cell lines that were tested. Furthermore, duvelisib did not induce apoptosis in T cell lines. On the other hand, G1 cell cycle arrest was observed in all B and T cell lines tested except P3HR1. Duvelisib treatment could inhibit T cell proliferation by inducing cell cycle arrest. However, its antitumor effects on T cells were limited because apoptosis was not induced.
We found that duvelisib treatment reduced the expression of BZLF1 and gp350/220 mRNA in EBV-positive B cell lines, suggesting that duvelisib suppresses the lytic cycle of EBV. In EBV-positive B cell lines, BCR signaling induces BZLF1 activation, and previous studies have shown that PI3K inhibitors such as wortmannin and idelalisib inhibit the EBV lytic cycle [42,43]. Our results are in line with these previous studies, and duvelisib may have specific effects on EBV-positive B cell lines. In general, the EBV latent cycle is associated with tumorigenesis, and among EBV latent proteins, LMP1 is considered to be a major EBV oncoprotein [5]. Induction of the EBV lytic cycle by agents like proteasome inhibitors or histone deacetylase inhibitors could be therapeutically beneficial to EBV-associated malignancies [44,45]. Furthermore, previous reports have shown that induction of the EBV lytic cycle by chemotherapeutic agents enhanced antiviral nucleoside drug susceptibility to EBV positive tumor cells, suggesting that combination of chemotherapy and antiviral drug may provide a new therapeutic approach for EBV-associated malignancy [46,47]. Conversely, some reports have shown that the EBV lytic cycle may play a role in tumorigenesis in EBV-associated malignancies [48,49]. However, it is unclear whether inhibition of the EBV lytic cycle could be a therapeutic target of EBV-associated malignancies. In this study, suppression of the EBV lytic cycle was observed in P3HR1, which was insusceptible to duvelisib, and EBV-negative B cell lines showed similar susceptibility to duvelisib. It might be possible that inhibition of the EBV lytic cycle does not play an important role in antitumor effects of duvelisib on EBV-positive cell lines.
In conclusion, we demonstrated that the PI3K/Akt signaling pathway was activated in EBV-positive and -negative B cell lines. Further, duvelisib treatment induced apoptosis and cell cycle arrest. Although some progress has been achieved with treatments for EBV-associated lymphomas such as anti-CD20 antibody and adoptive EBV-specific cytotoxic T lymphocyte transfer, the effects of these new therapies are still restricted. Our results may indicate that duvelisib has therapeutic potential for the treatment of EBV-associated B cell lymphomas.