Evaluation of mTOR, NFκB, and BCL-2 Inhibitor Activity In Vitro on Diffuse Large B-Cell Lymphoma Cells

TL;DR

This study evaluates the effectiveness of mTOR, NFκB, and BCL-2 inhibitors on two types of diffuse large B-cell lymphoma cells in the lab.

Contribution

The study compares monotherapy and combination effects of three inhibitors on ABC and GCB DLBCL subtypes using in vitro experiments.

Findings

ABT-199 showed the strongest pro-apoptotic effect as monotherapy in Riva (ABC) cells.

AZD2014+ABT-199 and ABT-199+IMD-0354 had similar effects in Riva cells, but triple combinations did not improve further.

In Toledo (GCB) cells, AZD2014+ABT-199 was most effective in pairs, but triple combinations did not enhance the effect.

Abstract

DLBCLs constitute an aggressive type of lymphoma with varied clinical, molecular and genetic features. The cells are characterized by NFkB pathway disturbances and BCL-2 and mTOR protein deregulation, which significantly inhibit apoptosis. Hence, many treatment strategies have been established to target the functioning of these pathways. While early clinical trials have found mTOR, NFkB and Bcl-2 inhibitors to have activity in many hematological cancers, their activity as monotherapy agents may still be insufficient; therefore, combinations of these compounds with other molecules demonstrating activity in a given cancer subtype are under evaluation. In vitro studies were conducted on the Riva (ABC subtype) and Toledo (GCB subtype) cell lines. Three novel drugs were administered: AZD2014 (vistusertib)—an inhibitor of the serine–threonine kinase mTOR; IMD-0354—an NFκB inhibitor; and ABT-199 (venetoclax)—a highly selective inhibitor for BCL-2. Drugs were administered alone, in pairs and as a combination of all three agents. For the Riva cell line, ABT-199 had the strongest pro-apoptotic effect on cancer cells as monotherapy. As pairs, AZD2014+ABT-199 and ABT-199+IMD0354 demonstrated similar effects. The combination of the three drugs did not have a stronger effect than the drug pairs. For the Toledo cell line, no significant differences were noted between the drugs when used as monotherapy. In pairs, the strongest effect was observed for AZD2014+ABT-199; furthermore, this effect was not intensified by the combination of the three drugs. Our findings, including those for the BCL-2 and mTOR inhibitors, indicate that there is a need for further in vivo studies to evaluate these drugs as potentially effective treatments for DLBCL of the ABC and GCB subtypes.

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1. Introduction

Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of lymphoma whose clinical, molecular and genetic features can vary considerably [1]. A significant role in their pathogenesis is played by genetic abnormalities. However, our understanding of this heterogeneity has been significantly improved by the use of modern techniques, such as gene expression analysis using microarrays [2].

As a group, DLBCLs can be divided into activated B-cell (ABC) and germinal center B-cell (GCB) types based on gene expression analysis. It is also assumed that neoplastic cells should exhibit the characteristics of normal B lymphocytes, with varying degrees of differentiation and activation [3]. The GCB subtype demonstrates gene expression during the germline response, while the ABC subtype exhibits a similar gene expression profile to that of peripheral B lymphocytes mitogenically activated in vitro [4].

Given the latest reports regarding the various molecular subtypes of DLBCL, there is increasing discussion about subtype-specific treatment. This is particularly important given the significant intergroup differences in progression-free survival (PFS), with 5-year PFS being 74% for the GCB subtype, compared to 40% for ABC. As such, the ABC subtype is regarded as a more pressing target for new therapeutic options due to its poorer prognosis [5].

Early clinical trials of mammalian target of rapamycin (mTOR), nuclear factor kappa B (NFκB) and BCL-2 (B-cell lymphoma 2) inhibitors suggest they offer promise in different hematological cancers; however, they may still be insufficient as monotherapy [6]. Studies have found DLBCL to be characterized by NFκB pathway disturbances and BCL-2 and mTOR family deregulation. In addition, genetic profiling of DLBCL subtypes identified a greater frequency of NFκB-activating mutations in ABC- than in GCB-type disease [7]. As these changes significantly inhibit apoptosis, they have become the targets of many potential treatment strategies [7], with a key step being the discovery of small-molecule inhibitors.

AZD2014 (vistusertib) is an inhibitor of the serine–threonine kinase mTOR; however, unlike rapamycin and its derivatives, it blocks the action of both the mTORC1 and mTORC2 complexes [8]. It has been investigated in clinical trials for its potential antitumor effects, and in combination with different chemotherapies. It has demonstrated activity for treating various cancers like ovarian, breast, and lung cancers [9,10].

IMD-0354 is an NFκB inhibitor that acts by directly blocking IKKβ phosphorylation, which has been found to suppress neoplastic proliferation of human cells with constitutively activated c-kit receptors [11]. It has also been observed to prevent cancer cell growth and promote apoptosis in culture. While the drug demonstrates good selectivity, its clinical application still faces challenges. It is believed to exert its anticancer effects by inhibiting cell viability and enhancing the action of chemotherapeutic agents used in treatment. It also exerts anti-inflammatory effects by blocking the NFκB pathway and cytokine production [12]. Clinically, IMD-0354 has been found to attenuate uveoretinitis, suppress corneal inflammation and angiogenesis, and restore the barrier function of various human cells.

ABT-199 (venetoclax) is a highly selective BCL-2 protein inhibitor. It has a three-fold lower affinity to BCL-2 than other proteins from the family, such as BCL-XL. The main function of BCl-2 is to inhibit the apoptosis process [13]. ABT-199 was first synthesized by Souers and colleagues, and was classified as a BH3 mimetic [14]. It binds directly to the BH3-binding groove of BCL-2, displacing any pro-apoptotic proteins from this site, thus increasing mitochondrial membrane permeability and causing the subsequent activation of caspase 9, leading to apoptosis. Venetoclax binds to BCL-XL or BCL-W only at higher doses, and does not exhibit affinity for the MCL-1 protein [14,15]. Following oral administration, maximum concentrations are achieved after approximately five to eight hours. Venetoclax binds to plasma proteins and is metabolized by cytochrome P430 3A (CYP3A) enzymes [16].

Currently, venetoclax is approved for the treatment of patients with chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). The drug is used both in monotherapy and in combination with other drugs, including with obinutuzumab (an anti-CD20 antibody) for B-cell lymphomas and with azacitidine for AML. The mechanisms of action of these three inhibitors are shown in Figure 1.

The aim of this study was to evaluate the cytotoxic effect of mTOR (AZD2014), NFκB (IMD-0354), and BCL-2 (ABT-199) inhibitors on the Riva (ABC-DLBCL subtype) and Toledo (GCB-DLBCL subtype) cell lines, both as monotherapy and in combinations of two or three inhibitors. It also evaluates the mechanisms of action of vistusertib, IMD-0354, and venetoclax pathway inhibitors on selected B-cell lymphoma cells and examines their pro-apoptotic effects. The study also assesses the activation of caspase-pathway-dependent apoptosis in response to the tested inhibitors.

2. Materials and Methods

2.1. Cell Lines

The ABC-type DLBCL cell line Riva (ACC 585) was purchased from DSMZ (Leibniz, Germany), and the GCB-type Toledo (CRL-2631) cells were purchased from ATCC (Manassas, VA, USA). The cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine and penicillin/streptomycin (50 IU/mL and 50 µg/mL) (all from PAN-Biotech, Aidenbach, Germany) at 37 °C with 5% CO_2_.

2.2. Reagents

AZD2014, IMD-0354 and ABT-199 (all from Selleckchem, Houston, TX, USA) were dissolved in diethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to prepare 10 mM stock solutions, which were stored at −20 °C. Prior to use, the stocks were diluted with RPMI to achieve the desired final concentrations. Control cells were treated with the corresponding volume of DMSO.

2.3. Cytotoxicity Assay

Cell viability was assessed by propidium iodide (PI) staining. In brief, the cultures were prepared by washing twice in cold 1% PBS (PAN-Biotech). They were then stained with 10 µg/mL PI (Sigma Aldrich, St. Louis, MO, USA) for 15 min, at room temperature, in the dark. Flow cytometry was performed with a red FL-3 fluorescent filter to measure cell fluorescence. The percentage of PI-positive cells indicates the nonviable cell population. In a pilot study, cells were incubated with the tested inhibitors at concentrations up to 300 µM (AZD2014, IMD-0354) or 100 nM (ABT-199) for 24 and 48 h. Therefore, for further studies, a 48-h time point was used, as well as the minimal dose of reagents found to induce significant cytotoxicity compared with controls.

In the final experiments, cells (0.5 × 10^6^/mL) were incubated under sterile conditions in 75 mL dishes (Nunc, Roskilde, Denmark) with the following concentrations of tested inhibitors: AZD2014 (Riva and Toledo 10 µM), IMD-0354 (Riva 5 µM and Toledo 10 µM) and ABT-199 (Riva 5 nM and Toledo 30 nM). They were also incubated with the following combinations of inhibitors: AZD2014+IMD0354, AZD2014+ABT-199, ABT-199+IMD0354, AZD2014+IMD0354+ABT-199.

2.4. Apoptosis Assay

Apoptosis, i.e., programmed cell death expressed by externalization of phosphatidylserine, was assessed with the Annexin V Apoptosis Detection Kit (Becton Dickinson, San Jose, CA, USA). After incubation, the cells were rinsed twice with cold PBS and re-suspended in 85 μL of binding buffer. To each sample, 5 μL of Annexin V–FITC and 10 μL of PI (10 μg/mL) were added, and the samples were kept in the dark for 15 min. Fluorescence was detected using flow cytometry with an FL-1 filter, and level of apoptosis was determined based on the percentage of Annexin-V-positive cells.

2.5. Assessment of Mitochondrial Membrane Potential (ΔΨ)

Changes in mitochondrial membrane potential were detected using MitoTracker™ Red CMXRos (MolecularProbes, Eugene, OR, USA). The dye, initially prepared as a 1 mM stock solution, was diluted in culture medium to a final concentration of 50 nM, and 2.5 mL was applied to the cell cultures. After incubating for 15 min at 37 °C in a humidified 5% CO_2_ atmosphere, the reduction in the ΔΨ was detected using flow cytometry with an FL3 filter (BD Pharmingen, San Diego, CA, USA).

2.6. Expression of Caspase-3, -8, and -9

After incubation, the cells underwent fixation and permeabilization on ice for 20 min using a Cytofix/Cytoperm^TM^. Subsequently, the cells were washed twice and incubated at room temperature for 30 min with anti-caspase-3 antibodies in Perm/Wash^TM^ buffer (all from BD Pharmingen, San Diego, CA, USA). Immediately after the final wash, flow cytometry analysis was performed using the FL1 filter. Caspase-8 and -9 activity were evaluated using the corresponding FAM-LETD-FMK FLICA^®^ Caspase 8 and FAM-LEHD-FMK FLICA^®^ Caspase 9 kits (Immunochemistry Technologies, Davis, CA, USA) according to the manufacturer’s protocols.

2.7. Flow Cytometry Analysis

Fluorescence was measured using a FACSCanto II (Becton Dickinson, San Jose, CA, USA) flow cytometer. FITC, PE and Cy-5 signals were detected using standard FL1 (green, λ = 530 ± 20 nm), FL2 (orange, λ = 560–600 nm), and FL3 (red, λ > 600 nm) emission filters, respectively. For each sample, 10,000 cells were collected.

2.8. Statistical Analysis

All statistical analyses were performed using GraphPad Prism 9.5 (San Diego, CA, USA). Data distribution was confirmed using the Shapiro–Wilk normality test. Following this, the differences between the treatment groups were evaluated by one-way ANOVA and Tukey’s post hoc analysis. A p < 0.05 was considered statistically significant, and the data are shown as the mean ± standard error of the mean (SEM) of five independent experiments.

3. Results

In Riva cells, the strongest apoptosis induction was observed for ABT-199, with a mean of 42.3% ANX-V-positive cells. Similar levels of induction were noted for the two-drug combinations, i.e., AZD2014+ABT-199 (57.6%) and ABT-199+IMD-0354 (45.7%). Each of these combinations differed significantly from AZD2014 (23.3%) and IMD-0354 (9.2%) used alone, but none of them differed from ABT-199. Additionally, the two-drug combinations AZD2014+ABT-199 and ABT-199+IMD-0354 demonstrated significantly higher apoptosis than AZD2014+IMD-0354 (20.8%). Although the triple combination of AZD2014+IMD-0354+ABT-199 resulted in the highest percentage of ANX-V/PI-positive cells (64.2%), this was not significantly higher than ABT-199 alone or either of the ABT-199 two-drug combinations. Notably, the triple combination of AZD2014+IMD-0354+ABT-199 induced apoptosis more strongly than either AZD2014 or IMD-0354 used alone, as well as combined AZD2014+IMD-0354 (Figure 2A).

In contrast, for Toledo cells, no significant differences in apoptosis induction were observed between the inhibitors when tested individually (approximately 7% ANX-V-positive cells for each drug). Among the two-drug combinations, AZD2014+ABT-199 exerted the strongest pro-apoptotic effect (24.4%), which was significantly higher than either AZD2014 or ABT-199 used alone and the combination of AZD2014+IMD-0354 (3.0%). Moreover, the triple combination of AZD2014+IMD-0354+ABT-199 showed a stronger pro-apoptotic effect (54.2%) compared to AZD2014, IMD-0354 or ABT-199 used alone, and AZD2014+IMD-0354; however, it did not demonstrate any significant advantage over AZD2014+ABT-199 or ABT-199+IMD-0354 (Figure 2B).

In order to understand the mechanism of action related to the pro-apoptotic effect of the studied inhibitors and their combinations, the next stage analyzed the fall in mitochondrial membrane potential and the expression of caspase-3, -8 and -9. The two cell lines demonstrated similar percentages of cells with lower mitochondrial membrane potential (Figure 3A,B).

In the Riva cells, ABT-199 elicited the greatest caspase-3 activation. AZD2014 activated caspase-8 and -9 more strongly than ABT-199, but not significantly so. Among the two-drug combinations, ABT-199+AZD2014 achieved the highest caspase-3, -8, and -9 activation, exceeding those produced by single-agent treatments. Although the triple combination of AZD2014+IMD-0354+ABT-199 generated the strongest overall caspase activation, its effect was comparable to that of ABT-199+AZD2014 (Figure 4A–C).

In turn, in Toledo cells, no significant changes in caspase-3, -8 and -9 expressions were observed following treatment with individual agents. Among the two-drug combinations, ABT-199+AZD2014 induced the strongest caspase-3 activation, significantly exceeding the effects of both ABT-199 and AZD2014 used alone. Interestingly, the triple drug combination did not exert a stronger effect on caspase-3 activation than ABT-199+AZD2014. In turn, ABT-199+IMD-0354 and the triple combination of AZD2014+IMD-0354+ABT-199 induced the strongest caspase-8 and -9 activation, with no significant difference between them (Figure 4D–F).

4. Discussion

DLBCL is an aggressive disease associated with complex mechanisms of lymphocyte maturation and differentiation, regulated at the molecular level. Despite significant progress, therapeutic outcomes remain unsatisfactory. In recent years, increasing emphasis has been placed on modern therapy against molecular targets, including combinations of drugs affecting various signaling pathways, i.e., chemotherapy-free treatment. This work investigated the effects of three modern drugs that inhibit pathways involved in the development of DLBCL as monotherapy, dual-drug and triple-drug regimens. The study used two cell lines: Riva, corresponding to the ABC subtype of DLBCL, which has a worse prognosis, and Toledo, corresponding to the GCB subtype of DLBCL. No such in vitro study on the anticancer effects of such combinations of new drugs has been published to date.

The mechanism of action of ABT-199 (venetoclax) is primarily based on inhibition of the anti-apoptotic activity of BCL-2 family proteins. In the Riva cell line, venetoclax demonstrated the strongest antitumor activity, both alone and in combination with each of the tested drugs; this difference was significant with IMD-0354, but insignificant with AZD2014. The three-drug combination did not yield a significantly stronger pro-apoptotic effect then the dual regimens AZD2014+ABT-199 and ABT-199+IMD-0354. This confirms that venetoclax is by far the most effective of the tested drugs in vitro for the ABC subtype of DLBCL. Induction of apoptosis by venetoclax was confirmed in 2013 by Soures et al. [14], who noted significant features indicative of apoptosis initiation. A single dose of ABT-199 administered to three patients with refractory DLBCL resulted in tumor cell lysis within 24 h. These findings suggest that selective pharmacological inhibition of BCL-2 may be an effective treatment option for patients diagnosed with BCL-2-dependent lymphoproliferative disorders.

The Phase 2 CAVALLI study evaluated the efficacy and safety of venetoclax added to standard R-CHOP therapy in 206 previously untreated patients with BCL-2 + DLBCL [17]. The CR rate at the end of treatment was 69% in the overall population. After a median follow-up of 32.2 months, insignificantly longer PFS was observed for venetoclax plus R-CHOP compared to R-CHOP in the overall study population. Despite a higher incidence of grade 3/4 hematologic adverse events (86%), this did not significantly increase the number of deaths (2%). In first-line treatment of DLBCL, the addition of venetoclax to R-CHOP yielded increased myelosuppression and potentially improved efficacy, particularly in high-risk subgroups of patients with BCL-2 expression.

However, venetoclax treatment still results in varying degrees of drug resistance in DLBCL, which also limits its potential use [18]. Clinical trials in patients diagnosed with acute myeloid leukemia have found that venetoclax resistance can be overcome by combining it with other modern drugs, such as hypomethylating agents [19]. Similar results were obtained in the present study, in which the antitumor activity of venetoclax was found to be strongest when combined with both an mTOR inhibitor (AZD2014) and an NFκB inhibitor (IMD-0354). For the Toledo cell line (GCB subtype of DLBCL), all drugs demonstrated similar pro-apoptotic effects on tumor cells when administered alone: in contrast to the ABC subtype, venetoclax did not demonstrate stronger activity than the other drugs. When the drugs were administered in pairs, however, AZD2014 with ABT-199 achieved stronger effects than AZD2014+IMD-0354 and the agents used alone. In addition, the three-drug combination was not more potent than either the AZD2014+ABT-199 or the ABT-199+IMD-0354 drug pair.

AZD2014 has demonstrated broad antiproliferative activity in multiple cell lines. In the present study, the drug achieved a cytotoxic effect when combined with venetoclax, with this effect being significantly stronger than venetoclax used alone. Previous studies report that the addition of AZD2014 overcame resistance to the PI3Kβ/δ inhibitor AZD818; it also completely prevented DLBCL cell growth in patients and in in vivo mouse xenograft models derived from the cell line [20]. Similarly, the combination of Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib and AZD2014 was found to have positive synergistic cytotoxic effects against ABC-subtype DLBCL cell lines in vitro. The simultaneous inhibition of BTK and mTOR also induced apoptosis in vivo in a xenograft model, resulting in tumor regression [21].

Following the successful results achieved by combining a BTK inhibitor with AZD2014, a Phase 1b study was initiated in patients with R/R DLBCL based on the combination of a newer, covalent BTK inhibitor, acalabrutinib, with AZD2014 [22]. In this study, the overall response rate (ORR) was 12% (i.e., 3/25). No subset of DLBCL patients demonstrating clinical benefit was revealed by cell subtyping or next-generation sequencing; however, the circulating tumor DNA dynamics were found to correlate with the radiological response. These data suggest that AZD2014 does not synergize sufficiently to enhance the clinical activity of acalabrutinib monotherapy. AZD2014 was also evaluated in a Phase 2 study in 37 patients with R/R DLBCL [22]; of these, 30 patients received AZD2014 and six received AZD2014 plus rituximab for up to six cycles (28-day cycles). With monotherapy, two partial remissions (PRs) were achieved, with their durations of response being 57 and 62 days. Of the total group, 19% had stable disease within six cycles. In the monotherapy group, median progression-free survival was 1.69 months and median OS was 6.58 months. The median duration of response or stable disease throughout the study period was 153 days. AZD2014 was well tolerated; in 36 patients, 86% of adverse events were grade 1–2 [23].

In the present study, the NFκB inhibitor IMD-0354 proved to be the least active agent, both alone and in combination with other tested drugs. Its activity in combination with ABT-199 was comparable to that of ABT-199 plus AZD2014 in the Riva cell line, which corresponds to the ABC subtype of DLBCL. IMD-0354 is a small-molecule IKKβ inhibitor that can effectively inhibit the NFκB pathway. Furthermore, while IMD-0354 can inhibit various cancer cells in culture, its poor solubility limits its clinical application. In humans, the NFκB pathway is involved in several biological processes, such as the development of inflammation, cell proliferation, and resistance to infection. Previous studies have found IMD-0354 to effectively inhibit cancer cell growth in vitro, in a similar way to preclinical models [24]. In addition to its anti-inflammatory effects, IMD-0354 has also been reported to have cytostatic effects on cancer cells, including breast cancer and melanoma [25].

Many studies indicate NFκB activation to be a hallmark of most human cancers, including leukemias and lymphomas [26]. Therefore, studies on IMD-0354 have also included patients with lymphatic malignancies. Kanduri et al. report that IMD-0354 induced apoptosis (mean 26%, range 8–48%) in CLL cells, regardless of IGHV mutation status, with a dose-dependent cytotoxic effect; the treatment also significantly reduced NFκB DNA-binding activity in CLL cells, and the authors also identified differences in the expression levels of pro- and anti-apoptotic genes following treatment [27]. IMD-0354 has not yet been evaluated in patients with DLBCL, either as monotherapy or in combination with other novel agents.

5. Conclusions

In vitro studies offer great potential for predicting clinical interactions between administered drugs, and are perfect for identifying combinations for testing in vivo. Our present findings, including those for the BCL-2 and mTOR inhibitors, support their further development as potential treatments for ABC- and GCB-subtype DLBCL.