The dual PI3K and mTOR inhibitor NVP-BEZ235 exhibits anti-proliferative activity and overcomes bortezomib resistance in mantle cell lymphoma cells
Mantle cell lymphoma (MCL) is one of the most difficult B-cell lymphomas to be treated. The phos- phatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is constitutively activated in MCL and plays a critical role in tumor growth and survival. However, single targeted agent mTOR has limited efficacy in treating MCL. Here, we investigate for the first time potential efficacy of NVP-BEZ235 (BEZ235) in treating MCL by simultaneously targeting Akt and mTOR.
In this study, phosphorylated Akt and mTOR level were elevated in tissue samples from MCL patients and in MCL cell lines. We also generated bortezomib-resistant MCL cell lines and found increased phos- phorylation of Akt and mTOR. Individual inhibition of PI3K or mTOR had limited anti-proliferative effects, whereas dual inhibition with BEZ235 effectively inhibited cell growth. The effect of BEZ235 was syner- gistic and sensitized the cells to the cytotoxic effects of conventional agents. Furthermore, BEZ235 could overcome acquired resistance to bortezomib in MCL cells and suppress the activated Akt/mTOR pathway. Therefore, these data suggest that the Akt/mTOR pathway plays a key role in the growth and survival of MCL cells and that these proteins may need to be simultaneously targeted for effective treatment of the disease. Our findings suggest that BEZ235 may be an effective agent for the treatment of MCL.
1. Introduction
Mantle cell lymphoma (MCL) is a subtype of B-cell non-Hodgkin lymphoma with an aggressive clinical behavior [1]. The major- ity of MCL patients respond to current first-line chemotherapies, including CHOP or hyper-CVAD. However, most patients eventu- ally relapse, whereupon they often exhibit chemoresistance and have poor prognosis with an overall survival of about 3–5 years [2,3]. MCL is commonly characterized by the chromosomal translo- cation t(11;14)(q13;q32), which leads to aberrant expression of cyclin D1 and the proliferation and progression of MCL [4]. Vari- ous signaling pathways are also reportedly associated with MCL, including NF-nB, Wnt, B-cell receptor (BCR), and phosphatidyli- nositol 3-kinase (PI3K)/Akt pathways [5–8]. Therefore, novel and effective treatment strategies for MCL are needed to target these aberrantly activated signaling pathways. Recently, the proteasome inhibitor, bortezomib, was approved for the treatment of refractory or relapsed MCL [9]. Although phase II clinical trials demonstrated that bortezomib achieves durable responses in 30–50% of MCL patients [10,11], other studies have found that MCL commonly fails to respond to this treatment, resulting in a low rate of complete remission [12].
The PI3K/Akt pathway plays an important role in various cellu- lar processes, including growth regulation, apoptosis, and survival in many cancers [13]. Activation of this pathway has frequently been observed in both aggressive blastoid and classic MCL, where it strongly contributes to the pathogenesis and survival of MCL patients [5,14]. Moreover, BCR and Wnt activation lead to down- stream signaling cascades that involve the PI3K/Akt signaling pathway [15,16]. Given these findings, Akt may be an attractive molecular target for MCL therapies.
One important target of Akt is mammalian target of rapamycin (mTOR), which is involved in two distinct functional complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [17]. mTORC1 regulates ribosomal protein S6 kinase (S6K) and eukary- otic translation initiation factor 4E-binding-protein-1 (4E-BP1), which in turn regulate cyclin D1, Bad, and Bcl-2 [17,18]. Thus, mTOR inhibitors (e.g., rapamycin and its analogs) have been proposed as potential therapeutic agents for several cancer types [19]. However, mTORC2 has rapamycin insensitive-components and phosphory- lates Akt at serine 473, thereby attenuating the suppression of mTORC1 by rapamycin [20,21]. Accordingly, mTOR inhibition alone may initially show an anti-proliferative effect but the cells can acquire resistance via mTORC2. To avoid activation of this feedback loop, a novel agent capable of simultaneously inhibiting mTORC1/2 and Akt is required.
NVP-BEZ235 (BEZ235) is a dual inhibitor of PI3K and mTORC1/2 that has shown anti-proliferative effects in solid cancers and some hematopoietic malignancies, including multiple myeloma (MM), T- cell acute lymphoblastic leukemia (T-ALL), and follicular lymphoma [22–25]. Based on these in vitro studies, BEZ235 is currently being tested in phase I/II clinical trials in patients with advanced breast cancer [26].
Here, we show that BEZ235 can down-regulate activation of Akt and mTOR in MCL cells, and it can overcome acquired resistance to bortezomib. This is the first study evaluating the anti-proliferative effects of BEZ235 in MCL cells, and our findings seem to suggest that clinical trials may be warranted to examine the use of this drug to enhance therapeutic outcomes in MCL patients.
2. Materials and methods
2.1. Patient samples, cell lines, and reagents
Paraffin-embedded tissues from MCL patients or patients with reactive tonsils were obtained from the Korea Cancer Center Hospital. Four human MCL cell lines (Grnata519, Jeko1, Mino and Rec1) and two bortezomib-resistant MCL cell lines (Jeko1/BTZ and Mino/BTZ) were used in this study. The Jeko1, Mino and Rec1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 media with 10% fetal bovine serum (FBS). To generate bortezomib-resistant MCL cell lines, we continuously exposed Jeko1 and Mino cells to increasing concentrations of bortezomib over a period of 6 months. The resulting stable cell lines were designated Jeko1/BTZ and Mino/BTZ. Granta519 cells were purchased from DSMZ (Braunschweig, Germany) and maintained in DMEM media with 10% FBS. NK-92, Ramos and Raji cells were used for comparison with MCL cells. We are grateful to Novartis (Basel, Switzerland) for providing us with NVP- BKM120 (PI3K inhibitor), RAD001 (mTOR inhibitor) and NVP-BEZ235 (dual PI3K/Akt and mTOR inhibitor). Bortezomib was purchased from LC Laboratories (Woburn, MA, USA). Doxorubicin and vincristine were obtained from Sigma–Aldrich (St. Louis, MO, USA).
2.2. Cell viability assays
Cells (6 × 105 per well) were seeded in 6-well plates and treated with BKM120, RAD001 or BEZ235. To evaluate the effects of BKM120, RAD001 or BEZ235 alone, cells were harvested and counted using an automatic cell counter (Digital Bio Technology, Korea).
To analyze the combined effects of BEZ235 and other agents, Mino and Rec1 cells (4 × 104 per well) were seeded in 96-well plates and treated with various con- centrations of BEZ235 plus bortezomib, doxorubicin or vincristine. After 48 h, the combination index (CI) was evaluated using CalcuSyn software (Biosoft, Cambridge, UK). A CI value of <1 indicated synergy. To evaluate the effect of BEZ235, Jeko/BTZ, Mino/BTZ and their parental cell lines (4 × 105 cells per well) were seeded in 96-well plates and treated with various doses of BEZ235. After 48 h, MTT reagent was added to each well, and the plate was incubated for 3 h at 37 ◦C. The resulting crystals were solubilized with 100 µl SDS, and the absorbance was read at 595 nm. 2.3. Cell cycle analysis and apoptosis assays For cell cycle analysis, BEZ235-treated cells were harvested and fixed overnight in 70% ethanol. Fixed cells were washed and incubated with RNase followed by propidium iodide (PI; Sigma) staining. For apoptosis assays, cells were treated with BEZ235, bortezomib, doxorubicin or vincristine for 48 h and then co-stained with Annexin V (BD Biosciences, San Jose, CA, USA) and PI. Cell cycle progression and apo- ptosis were analyzed using flow cytometry (FACSCalibur; BD Biosciences). Annexin V-positive and PI-negative cells were considered apoptotic. 2.4. Western blotting Cells were harvested and lysed, and lysates were incubated for 30 min on ice before centrifugation at 13,000 rpm for 10 min at 4 ◦C. Protein concentrations were measured using a protein assay (Bio-Rad, Richmond, CA, USA). Equal amounts of pro- tein (30 µg) were separated on an SDS-PAGE gel and transferred to PVDF membranes (Perkin-Elmer, Boston, MA, USA). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): phospho-Akt (Ser473), phospho-mTOR (Ser2448), phospho-p70S6K (Thr389) and phospho-S6 (Ser235/236). Antibodies for Akt, mTOR and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The results were visualized using enhanced chemiluminescence reagent (ECL; GE Healthcare, Piscataway, NJ, USA). 2.5. Immunohistochemistry (IHC) Formalin-fixed, paraffin-embedded tissue samples were cut into 4 µm thick sec- tions, placed on slides, dried and deparaffinized. Slides were incubated for 30 min in methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase activity. Antigen retrieval was performed with citrate buffer in a steamer for 30 min. Immunoreactions were performed using a Labvision autostainer (Thermo Scientific, Bremen, Germany) and primary antibodies against phospho-Akt, phospho-mTOR and phospho-p70S6K, followed by detection using a Labvision horseradish perox- idase polymer detection system (Thermo Scientific). Immunostained slides were observed using a light microscope (Olympus, Japan). 3. Results 3.1. Akt and mTOR are activated in MCL tissues and cells To determine the status of Akt and mTOR signaling in MCL, we assessed the phosphorylation levels of Akt, mTOR and p70S6K in MCL tumor specimens and cell lines. IHC staining revealed that 8 of 10 MCL samples (80%) showed strong staining for phospho-Akt in tumor cells, whereas such positivity was seen for only a few scattered lymphocytes among the reactive tonsil samples. Sim- ilarly, strong staining for phospho-mTOR and phospho-p70S6K was observed in 60% (6/10) and 70% (7/10) of the MCL tissue samples, while such staining was not seen in the reactive tonsils samples (Fig. 1A). In addition, Western blotting analysis revealed that these proteins were strongly phosphorylated in MCL cell lines (Granta519, Jeko, Mino and Rec1) compared with other lymphoma cell lines (NK-92, Ramos and Raji) (Fig. 1B). These results indicate that Akt and its down-stream proteins are constitutively activated in MCL tumor tissues and cell lines, suggesting that inhibition of Akt and mTOR may efficiently suppress the growth of MCL. 3.2. Inhibition of Akt or mTOR limits anti-proliferative activity in MCL cells We evaluated the effect of inhibiting PI3K or mTOR individu- ally in MCL cells. First, we incubated MCL cell lines with the PI3K inhibitor NVP-BKM120 (BKM120) and confirmed that it decreased phospho-Akt levels without changing that of total Akt in all four MCL cell lines (Fig. 2A). We also found that phospho-mTOR and its down-stream proteins were either unchanged or markedly increased compared with untreated cells (Fig. 2A). We then evalu- ated the effect of BKM120 on the proliferation and survival of Mino and Rec1 cells, and found that BKM120 inhibited cell proliferation by only 20% (Fig. 2B). These data suggest that BKM120-induced mTOR activation may confer resistance to BKM120 in these cells. Next, we examined the effects of the mTOR inhibitor RAD001 on the MCL cell lines and found that levels of phospho-mTOR, phospho-70S6K and phospho-S6K were decreased compared with untreated cells (Fig. 3A). In contrast, the phospho-Akt level was increased in three of the four cell lines, with Granta519 cells being the exception (Fig. 3A). In addition, RAD001 treatment failed to inhibit MCL cell growth (Fig. 3B). These data suggest that mTOR inhibition may turn on phospho-Akt activation, thereby limiting the anti-proliferative effects of mTORC1. Collectively, our results suggest that dual inhibition of Akt and mTOR is necessary to inter- fere with the feedback loop between Akt and mTOR for effective MCL treatment. 3.3. The dual inhibitor NVP-BEZ235 blocks Akt/mTOR signaling and greatly inhibits MCL cell growth Because individual inhibition of PI3K and mTOR had limited effects on MCL cell proliferation, we examined the effect of dual inhibition of PI3K and mTOR using the novel compound NVP- BEZ235. As shown in Fig. 4A, BEZ235 dramatically decreased phospho-Akt, phospho-mTOR and the phosphorylation of their down-stream effectors in all four tested MCL cell lines. MCL is characterized by the translocation t(11;14)(q13;q32) resulting in over-expression of cyclin D1 and G1 phase cyclins, which are regulated by the Akt/mTOR pathway [27]. We found that the BEZ235-induced decrease of phospho-Akt and phospho-mTOR levels in Mino and Rec1 cells increased the accumulation of cells in G1 phase compared with untreated cells (Fig. 4B). Treating cells for 48 h with BEZ235 significantly decreased cell proliferation in all MCL cell lines (Fig. 4C). Taken together, these results show that the dual PI3K-mTOR inhibitor BEZ23 effectively attenuates phos- phorylation of the Akt/mTOR pathway and inhibits cell growth via G1 phase arrest efficiently than treatment with either of the single inhibitors tested herein. 3.4. BEZ235 synergistically enhances the cytotoxic effects of conventional agents used against MCL Because clinical experiences in the therapeutic management of MCL patients indicate that drug combinations can enhance response rates over those obtained with single treatments, we investigated whether BEZ235 synergistically interacted with three other chemotherapeutic agents (bortezomib, doxorubicin and vincristine) used in treating MCL patients. As shown in Fig. 5A, MTT assays revealed that BEZ235 showed synergistic effects with the tested drugs in Mino and Rec1 cells as determined by CI values < 1.0 (Fig. 5B). Consistent with these findings, combined treatment with BEZ235 and the conventional agents induced effective apoptosis but treatment of MCL cells with conventional agents alone did not cause increased cell death (Fig. 5C). These results indicate that BEZ235 can enhance the cytotoxic effect of conventional agents, thus implying that BEZ235 could be a sensitizer for therapeutic regimens against MCL. 3.5. BEZ235 suppresses PI3K/Akt/mTOR activation and cell growth in bortezomib-resistant MCL cells As MCL patients frequently fail to respond to treatment with bortezomib [12]. We established bortezomib-resistant MCL cell lines (Jeko/BTZ and Mino/BTZ) for further testing. As shown in Fig. 6A, Western blotting revealed that the levels of phospho- Akt and its down-stream target proteins were up-regulated in both Jeko/BTZ and Mino/BTZ cells, compared with levels in the parental Jeko and Mino cells. Moreover, when the bortezomib- resistant MCL cells were treated with bortezomib, they showed elevation of phospho-Akt and its down-stream proteins (Fig. 6B). These results indicate that elevated Akt and mTOR phosphoryla- tion may be associated with bortezomib resistance in MCL cells. However, BEZ235 treatment completely abrogated Akt and mTOR phosphorylation and inhibited cell proliferation in the bortezomib- resistant cell lines (Fig. 6B and C). These data indicate that BEZ235 can efficiently attenuate activation of Akt and mTOR signaling in bortezomib-resistant cells and potentially sensitizes them to the anti-tumor effects of conventional treatments. 4. Discussion In the current study, we demonstrate that simultaneous inhi- bition of Akt and mTOR markedly suppressed proliferation of MCL cell. BEZ235, a dual inhibitor of PI3K/Akt and mTOR, had a much stronger anti-proliferative effect in MCL cells compared with com- parable single inhibitors. In addition, it effectively down-regulated Akt/mTOR signaling in bortezomib-resistant MCL cells. Therefore, our findings suggest that dual inhibition of Akt and mTOR by BEZ235 could potentially lead to better therapies for cases of aggressive MCL. At present, CHOP and CHOP-like regimens are the standard ini- tial treatments for MCL [28]. However, most MCL patients relapse and become refractory to these conventional therapies, indicating the need for new targeted therapies for MCL [29]. Recent strategy for MCL therapies has included the use of an anti-CD20 antibody (rituximab), a proteasome inhibitor (bortezomib), mTOR targeting inhibitors and immunomodulatory agents [30–33]. These agents are generally combined with conventional therapy to improve the clinical outcome of MCL but the results to date have been unsatisfactory. Howard et al. has been reported that combined treatment with rituximab and CHOP yields no significant differ- ence in progression-free survival compared with CHOP alone [34]. Bortezomib was approved for treating relapsed MCL in 2006 [9] but many patients exhibited initial resistance or subsequently became refractory to bortezomib [12,31]. A few factors have been sug- gested to contribute to proteasome inhibitor resistance, including accumulation of anti-apoptotic Bcl-2 family members, mutation of the proteasome β5 subunit, increased heat shock protein lev- els and Akt pathway activation [35–38]. Previously, relatively little was known about the causes of bortezomib resistance in MCL. Here, we generated two bortezomib-resistant MCL cell lines and investigated the mechanism of resistance in these cells. We found that the resistant cells activated Akt and its down-stream pro- teins compared with the parent cells. Furthermore, when these cells were treated with bortezomib, they showed additional eleva- tion in Akt and mTOR phosphorylation, which was associated with increased cell proliferation (Supplementary data in the online ver- sion at doi:10.1016/j.leukres.2012.02.010). These findings indicate that activation of Akt and its down-stream mTOR/p70S6K pathway may contribute to bortezomib resistance, suggesting that simulta- neous inhibition of Akt and mTOR may be essential to overcoming bortezomib resistance. The PI3K/Akt pathway contributes to regulating a variety of extracellular signals and plays an important role in tumor survival and cell proliferation [13]. Gene expression profiling and proteomic analyses have indicated that MCL pathogenesis is associated with constitutive activation of the Akt/mTOR pathway [39,40]. Recently, Psyrri et al. reported that amplification of the PIK3CA gene and inac- tivation of the PTEN were frequently found in MCL patients and cell lines [41]. Here, we show Akt, mTOR and p70S6K are highly phos- phorylated in most MCL patients and cell lines. Together, these data support the notion that targeting the PI3K/Akt/mTOR pathway may be a successful strategy for treating MCL. The PI3K inhibitor LY294002 up-regulates p27 and suppresses cyclin D1, leading to G1 phase arrest in MCL cell lines [14]. More- over, several PI3K isoform targeting inhibitors have been shown to reduce Akt phosphorylation and apoptosis in MCL cell lines [42,43]. However, these agents have not yet been approved for clinical use. Thus, further investigation is needed to define the possibility of clinically available agent in MCL. Rapamycin and its analogs are effective inhibitors of mTORC1- dependent S6K and 4E-BP1 phosphorylation [44]. Given these facts, phase II clinical trial demonstrated that the synthetic mTOR inhibitor, temsirolimus, prolongs overall patient survival [45]. However, rapamycin analogs inhibit mTORC1, the existence of the negative feedback loop between S6K and Akt may promote cell survival and chemoresistance, thus decreasing their therapeutic efficacy [46]. Further investigations will be needed to define addi- tional clinically relevant agents for the treatment of MCL. Here, we observed insufficient growth inhibition in MCL cells when BKM120 or RAD001 were used to suppress the phosphorylation of PI3K or mTORC1, respectively. Therefore, we hypothesized that use of a dual Akt and mTOR inhibitor would avoid activation of PI3K/Akt feedback loops. The new orally available agent, BEZ235, has been shown to inhibit both PI3K/Akt and mTOR signaling and inhibits tumor growth in various cancers [24,25]. BEZ235 potently reduces the kinase activity of both p110 PI3K and mTORC1/2 and is currently undergoing in phase I/II clinical trials for use in advanced solid tumors [26]. Additionally, BEZ235 triggers apoptosis through cas- pase cleavage in T-ALL and MM [22,23]. Our results in MCL cells demonstrated that BEZ235 reduced Akt phosphorylation and activation of down-stream proteins, thus inhibiting cell proliferation. However, we did not observe signif- icant caspase activation or apoptosis in the BEZ235-treated MCL cells (data not shown). We therefore evaluated the feasibility of co-treating cells with BEZ235 plus various cytotoxic agents con- ventionally used to treat MCL. Our results revealed that combined treatment of BEZ235 plus conventional MCL chemotherapeutic agents synergized in MCL cells, suggesting that BEZ235 may be a promising agent to improve the current MCL treatment strategies. In conclusion, this study supports the hypothesis that constitutive Akt and mTOR activity is critical for MCL progression and survival. Our study on BEZ235 suggests dual inhibition of Akt and mTOR has strong therapeutic potential for MCL, either alone or in combination with conventional chemotherapeutics. Moreover, BEZ235 has anti-proliferative effects in both bortezomib-sensitive and -insensitive MCL cell lines, suggesting that it may be useful Dactolisib for MCL patients with bortezomib-refractory or relapsed MCL.