Ixazomib

Targeted Therapy for EBV-associated B-cell Neoplasms

Abstract
Epstein-Barr virus (EBV) is directly implicated in several B-cell lymphoid malignancies. EBV associated lymphomas are characterized by prominent activation of the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-B) pathway and targeting this pathway establishes a rationale for a therapeutic approach. The ubiquitin/proteasome signaling plays an essential role in the regulation of the NF-B pathway. Ixazomib is an FDA approved, orally bioavailable proteasome inhibitor. Here we report the first preclinical evaluation of ixazomib mediated growth inhibitory effects on EBV-infected B-lymphoblastoid cell lines Raji and Daudi. Ixazomib induced apoptosis in these cell lines in a dose-dependent manner. Cell cycle analysis demonstrated ixazomib treatment induced cell cycle arrest at the G2/M phase with a concomitant decrease in G0/G1 and S phases. The results further revealed an increase in p53, p21 and p27 levels and a decrease in survivin and c-Myc protein levels. Mechanistically, ixazomib treatment resulted in the accumulation of polyubiquitinated proteins, including phosphorylated IBα with a significant reduction of p65 subunit nuclear translocation. Altogether, our pre-clinical data support the rationale for in vivo testing of ixazomib in EBV-associated B-cell neoplasms.

Introduction
Epstein-Barr virus (EBV) is directly implicated in several B-cell lymphoid malignancies (1). EBV associated B-cell neoplasms occur in both solid organ and allogeneic hematopoietic stem cell transplantations due to the necessity of immunosuppression in these patients to modulate organ rejection and graft versus host disease, respectively (2). Treatment of these B-cell neoplasms is limited to a reduction in immunosuppression, administration of immunotherapeutics, and antineoplastic chemotherapy (3). EBV associated lymphomas are characterized by prominent activation of NF-B pathway and targeting this pathway establishes a rationale for an attractive therapeutic approach (4). The ubiquitin/proteasome signaling pathway plays an important role in the regulation of NF-B pathway (5). Bortezomib (BZ) is the first FDA approved proteasome inhibitor for treating both newly diagnosed and relapsed/refractory multiple myelomas, and mantle cell lymphomas. BZ acts through inhibition of the 26S proteasome, a large protease complex that degrades ubiquitinated proteins. BZ stabilizes various cellular proteins involved in cell cycle arrest and apoptosis including p21, p27, p53, and IBα by inhibiting proteasome function (6). Stabilization of IBα results in inhibition of the NF-B signaling pathway which promotes tumor cell survival, growth, and angiogenesis(7). The major limiting factor for long-term administration of BZ through intravenous or subcutaneous routes is a risk of peripheral neuropathy (8). There was a need to develop an orally bioavailable proteasome inhibitor with a low toxicity profile to overcome this conundrum. Ixazomib is structurally a dipeptidyl leucine boronic acid and the only approved orally bioavailable proteasome inhibitor. Ixazomib inhibits proteasome activity at low concentrations by binding to the β5 subunit of the 20S catalytic core subunit of the proteasome and is currently in clinical trials for patients with relapsed/refractory multiple myeloma and solid tumors (9-11). Compared to BZ, ixazomib has demonstrated similar efficacy with better pharmacokinetic/pharmacodynamic parameters in multiple myeloma (MM).

Ixazomib in combination with lenalidomide and dexamethasone sensitize BZ resistant myeloma phenotype. From the existing literature review, there are no reports on the pre-clinical or clinical use of ixazomib in EBV-associated B-cell neoplasms. Therefore using ixazomib for targeting NF-κB activation is a novel therapeutic approach and our pre-clinical data suggest the potential ofixazomib use as monotherapy or in combination for the treatment of EBV-associated B-cell neoplasms.Cell lines and culture conditions: Human EBV-positive Burkitt’s lymphoma-derived cell lines Raji and Daudi were purchased from American Type Culture Collection (ATCC; Manassas, VA). Cells were grown in Hyclone RPMI1640 medium supplemented with 10% fetal bovine serum (Corning Life Sciences, USA) and 1% penicillin/streptomycin (Corning Life Sciences, USA) at 37°C with 5% carbon dioxide.Isolation of normal B cells: Healthy individual blood samples were procured from Biorepository Core Facility at the University of Kansas Medical Center, Kansas City, KS. Normal B cells were isolated from the blood samples using CD19 positive selection kit from StemCell Technologies, Vancouver, Canada.Reagents and antibodies: Antibodies were purchased from following: BD Biosciences San Jose, CA, USA (β-actin-612656, p65-610868, p21-556431, PARP-556494, p53-554293; Cell Signaling Technology, Beverly, MA, USA (phospho-IκBα-2859, IκBα-9247, GAPDH-5174, Survivin-28023, p27-3688; Santa Cruz Biotechnology, Dallas, TX, USA (Lamin B-6216, c-Myc-40)and Covance, Emeryville, CA, USA (polyubiquitin-SIG-39500). Propidium iodide (PI) and RNase were purchased from Sigma-Aldrich (St. Louis, MO, USA) and ixazomib from Selleck Chemicals (Houston, TX, USA).

Flow Cytometry: FACS analysis was carried out as described previously (12). Briefly, normal B cells, Raji and Daudi cells were treated with various concentrations of ixazomib for 48 hours, cells were harvested, washed with phosphate buffered saline (PBS) and incubated with the -FITC-Annexin V antibody (556419 BD Biosciences San Jose, CA, USA) and TO-PRO-3 stain (Molecular Probes, Eugene, OR, USA). Samples were analyzed using BD Accuri C6 Plus flow cytometer (BD Biosciences San Jose, CA, USA).Cell cycle analyses: After indicated treatments, cells were harvested, washed with PBS, and fixed in ethanol overnight at -20C. The cell cycle analysis was performed as previously described (12).Cell fractionation and immunoblot analyses: After indicated treatments cells were harvested, and nuclear and cytosolic fractionations were made using NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology, Rockford, IL USA). For Immunoblot analyses, total cell lysates were made with a non-denaturing lysis buffer with protease inhibitors (Complete Mini, Roche Diagnostics, and Indianapolis, USA), protein concentrations were determined using BCA protein assay kit (Pierce Biotechnology, Rockford, IL USA) and samples separated by SDS-PAGE as previously described (12). The blots were scanned using the Odyssey IR scanner (Li-cor Biosciences, Lincoln, NE, USA).Immunofluorescence: After designated treatments, cells were cytospun onto glass slides (Wescor Cytopro Cytocentrifuge, Logan, UT, USA), fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton-X-100 in PBS, and blocked with 5% BSA/PBS. The slides were then incubated with p65 antibody for 2 hours, washed with PBS and incubated with secondary antibody conjugated with Dylight-594 for an hour (D1-2594, Vector Laboratories, Burlingame, CA, USA) and finally mounted with Vectashiled antifade mounting medium with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA). Images were acquired using with an Eclipse E1000 microscope (Nikon).Significant differences in values between treated and untreated lymphoma cells in different experimental conditions were determined by one-way ANOVA analysis with Dunnett’s test method (SigmaPlot, version 13.0; Systat Software). Significance was defined as P values < 0.05.

Results
We determined the apoptotic effect of ixazomib in EBV-associated lymphoma cell lines Raji and Daudi. The normal B cells and the lymphoma cell lines were treated with increasing concentrations (0 to 100 nM) of ixazomib for 48 hours. After treatment, cells were washed with PBS and incubated with FITC-Annexin V and To-Pro-3 stain. The percentage of apoptotic cells was then determined by flow cytometry. As demonstrated in Figure 1A, Raji and Daudi both cell lines showed induced apoptosis in a dose-dependent manner (p < 0.005) (Figure 1A) butno significant effect on normal B cells. The tested two cell lines are sensitive to ixazomib. Altogether, these results suggest that ixazomib induces apoptosis in EBV-associated lymphoma cell lines.Ixazomib induces G2/M cell Cycle arrest in EBV-associated lymphoma cellsWe also determined the effect of ixazomib on the cell cycle status in Raji and Daudi cells. Due to the high percentage of apoptotic cells at higher concentrations and extended treatment periods, we restricted the treatment duration to 24 hours. After 24 hours of treatment, cells were washed, fixed, and then cell cycle analysis was performed using flow cytometry. The addition of ixazomib inhibited the growth of cells in a dose-dependent manner in both cell lines (Figure 1B). Indeed at 100 nM of ixazomib concentration, the proportion of cells actively replicating DNA (S phase) decreased by 10% and the fraction of 2N DNA (G0/G1) reduced significantly by 25% compared to the control. Then we had cells at 4N DNA (G2/M) which increased markedly by more than 30% (Figure 2). These results suggest that ixazomib treatment inhibits the cellular DNA synthesis leading to cell cycle arrest at the G2/M phase of the EBV-associated lymphoma cell lines.After observing induced apoptosis and cell cycle arrest with ixazomib treatment, we explored the changes in the key regulatory molecules involved in the process of cell cycle and apoptosis. Both cell lines Raji and Daudi were treated with ixazomib (0 to 100 nM) and incubated for 24 hours. As demonstrated in Figure 2A and B, both cell lines showed increased polyubiquitinated proteins with induction of key cell cycle regulators p53, p21 and p27 in a dose-dependent manner (Figure 2 A, B). Compared to control, the anti-apoptotic protein amounts of survivin and c-Myc were decreased in ixazomib treated lymphoma cells. Also, we observed cleaved PARP in a dose-dependent manner in both cell lines (Figure 2 A, B). These observations clearly confirm the effect of ixazomib in cell cycle regulation and apoptosis by differentially regulating p53, p21, p27, survivin, c-Myc, and PARP activity.

From the previous results, it is clearly evident that ixazomib treatment leads to accumulation of polyubiquitinated proteins. Based on previous literature, it is clear that EBV induced NF-κB activation plays a crucial role in the transformation of lymphoma cells (1). To confirm, we have examined the expression status of pIκBα (S32/36) after 100 nM of ixazomib treatment in a time-dependent manner for up to 8 hours. We observed the inhibition of pIκBα proteasomal degradation which is a key regulatory subunit of NF-κB (Figure 3A). However, no changes were detected in protein amounts of IκBα and p65 in a time-dependent manner in both tested cell lines (Figure 3 A). These results suggest that ixazomib blocks proteasomal degradation of pIKBα and inhibits the NF-κB pathway in EBV-associated lymphoma cell line.Removal of pIBα subunit from the p65 complex leads to the activation of NF-κB signaling. Inhibition of pIBα degradation by the proteasome inhibitor ixazomib, decreases p65 translocation from the cytosol into the nucleus. Based on the time course results, we chose to use the 6th hour as the time point and 100 nM as the treatment concentration for immunofluorescence and western blot analysis to show the cellular localization of p65 (red color). In both cell lines, immunofluorescence showed an increased cytosolic accumulation of p65 after 6 hours (Figure 3B). Western blot analysis from nuclear and cytosolic fractionations showed that ixazomib inhibited the p65 translocation from cytoplasm to the nucleus in a time-dependent manner (Figure 3C), which is concomitant with increased pIBα in both tested cell lines (Figure 3A). The purity of fractions was assessed using GAPDH as cytosolic and lamin B as the nuclear fraction controls. In conclusion, these results reveal that ixazomib treatment inhibits the NF-κB pathway by actively reducing p65 translocation to the nucleus in EBV-associated lymphoma cell lines.

Discussion
Inhibition of proteasome activity disrupts the regulation and stability of intracellular proteins like cell cycle regulators, pro-apoptotic proteins and many others (5). BZ is a known and well-characterized proteasome inhibitor, which is clinically used to treat several hematological malignancies. The major limiting factor of BZ is long-term administration through intravenous or subcutaneous routes with the associated risk of peripheral neuropathy (8). Carfilzomib is another FDA approved proteasome inhibitor but administered intravenously similar to BZ (11). Ixazomib is the only alternative proteasome inhibitor to overcome this BZ mediated clinical toxicity (11). Ixazomib is the first clinically available oral proteasome inhibitor and has been approved for use as a single agent or in combination with other drugs in myeloma therapy (13). A phase 1 study was conducted to determine the pharmacokinetic parameters in multiple myeloma patients (13). Based on these results, several phase 2 and 3 clinical trials have been conducted in relapsed/refractory multiple myeloma patients. Results indicate progression-free survival is longer in ixazomib treated patients compared to a placebo group. A recent double-blind, phase3 clinical trial demonstrated ixazomib plus lenalidomide and dexamethasone resulted in a significantly longer progression-free survival with limited toxic effects in multiple myeloma patients compared to the control arm (13). In this study, we tested the preclinical efficacy of the next generation orally administrated proteasome inhibitor ixazomib in EBV-associated lymphoma cell lines. We found that ixazomib significantly enhances apoptosis and cell cycle arrest by stabilizing p53 and inhibiting the NF-B pathway by interfering p65 translocation to the nucleus.

The cell transforms to a cancerous cell by gaining the sustainable control of different cellular functions such as apoptosis, differentiation, growth, replication, angiogenesis, tissues invasion and metastasis, which are mediated by different molecular network mechanisms (14). The ubiquitin-proteasome degradation pathway (UPP) is one such cellular function, UPP has been demonstrated to be involved in cell cycle progression, apoptosis, transcription, inflammation as well as immune surveillance (15). These essential functional roles of UPP have made it an important druggable pathway to treat different types of cancer (16). Proteasome signaling playsacritical role in NF-B activation and regulation. Further, B-cell lymphoid malignancies are directly influenced by EBV, which are known to activate the NF-B pathway (17). In the present study, we examined the proteasome inhibitor ixazomib effects on apoptosis, cell cycle checkpoints in EBV associated lymphoma cell lines Raji and Daudi. Ixazomib treatment significantly induced apoptosis in these two cell lines in a dose-dependent manner. Our results are in line with the data of Chauhan et al., where they demonstrated ixazomib treatment induced apoptosis in human multiple myeloma cell lines (18). Furthermore, ixazomib induced apoptosis results are comparable to BZ in EBV-associated B-cell lymphoma cells (1).Several other studies have reported that proteasome inhibitors lead to stabilization of polyubiquitinated proteins. Here in we observed the same with ixazomib in a dose-dependent manner. p53, a tumor suppressor and a cell cycle regulator, is one such specific molecule studied for polyubiquitinated protein stabilization (19). Upon ixazomib treatment, both the studied cell lines showed polyubiquitinated proteins and the stabilization of p53 protein, which lead to differential regulation of cell cycle regulators such as p21, p27 and anti-apoptotic molecules like c-Myc and survivin (20). G1-S and G2-M cell cycle transitions are controlled by multiple proteins. Transcription of a plethora of cell cycle promoting genes is maintained in an inactive state by interaction or transcription inhibition by p21 and p27 (21). In different cancer entities, it is established that p53 activation transcriptionally represses c-Myc required for cell cycle regulation and apoptosis (22). Survivin is a downstream regulatory molecule of the NF-κB signaling axis which is also a transcriptionally repressed anti-apoptotic gene by p53. (23). Cleavage of Poly (ADP-ribose) polymerase (PARP) is a known molecular event for activated apoptosis signaling (24). Our data suggest that ixazomib induces expression of cell cycle regulators p21 and p27, where they inhibit the cell cycle at G1-S and G2-M. Further, reduced anti-apoptotic proteins c-Myc and survivin with the association of PARP cleavage is a clear indication of its positive role in apoptosis.

As discussed, NF-κB plays a critical role in proteasome-mediated cellular functions and consists of NF-κB1/p50, NF-κB2/p52, RelA/p65, RelB and c-Rel as family members (Visual Overview). NF-κB forms various homo- and hetero-dimeric complexes. Activation of NF-κB signaling mediates through translocation of the p65 complex into the nucleus. NF-κB activation is regulated by IκBα which masks the nuclear localization signal motif of p65. When IκB kinases (IKKα, IKKβ, and IKKγ) phosphorylate IκBα, it also simultaneously ubiquitinated and degraded by proteasome machinery (Visual Overview). Thus, the free NF-κB dimers can translocate to the nucleus, where they dysregulate different tumorigenic genes (25). Ixazomib treatment stabilized the phosphorylated IκBα protein amounts without altering the total IκBα protein amount in both treated cell lines Raji and Daudi. Further, it is clearly evident that in both these cell lines RelA/p65 accumulated in the cytoplasm rather than in the nucleus. Secondly, RelA/p65 nuclear presence decreased markedly in the treated cells. As expected, the screened NF-κB regulated genes c-Myc and survivin were also downregulated (22-23). Collectively these preclinical findings suggest that inhibition of NF-B signaling using ixazomib may be an attractive therapeutic approach in patients with EBV-associated B-cell neoplasms.