Most patients with chronic lymphocytic leukemia (CLL) will require therapy at some point during their disease course, and typically receive chemoimmunotherapy as initial treatment [1
]. Standard treatments include alkylating agents (chlorambucil, CLB; bendamustine, BEN), or nucleoside analogues (fludarabine, FLU), alone or in combination with an anti-CD20 monoclonal antibody [1
]. BEN is a bifunctional alkylating agent with a mechanism unique from other alkylating agents, such as CLB [2
]. We and others have previously demonstrated that BEN is synergistic with FLU in primary CLL cells, and this synergy was related to increased DNA damage [2
However, the algorithm for therapy has recently shifted, with the advent of novel targeted therapies [1
]. These therapies include the B cell receptor (BCR) pathway inhibitors, ibrutinib (IBR) and idelalisib (IDE), which target Bruton’s tyrosine kinase (BTK) and the δ isoform of phosphatidyl-inositol 3 kinase (PI3Kδ), respectively [1
]. These agents have a unique mechanism of action, and are presently used in patients who have relapsed following chemoimmunotherapy or have a deletion (del) 17p, which is typically associated with a p53 mutation [1
]. PI3Kδ is one of four isomers of the Class I PI3Ks, and is composed of a catalytic subunit p110 and regulatory subunit p85. Following activation of the BCR, PI3K becomes phosphorylated by Syk and phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3), leading to activation of protein kinase B (AKT) [6
]. PI3Kδ activity is higher in CLL cells than in normal cells, and IDE induces apoptosis in CLL cells in vitro by inhibiting the BCR pathway [6
]. In addition, these inhibitors reduce CLL cell adherence to stromal cells in the microenvironment [7
]. When combined with rituximab, either in the first or second line settings [10
], IDE has significant activity in CLL, and autoimmune toxicity was seen more commonly in the front line setting (hepatitis, colitis and pneumonitis) [15
]. IDE has also been combined with chemotherapy, to improve antitumor activity and specificity. A placebo-controlled Phase III clinical trial of IDE with BEN/rituximab (BR) showed higher clinical efficacy than with BR alone, but with significantly more infections and marrow suppression, demonstrating a lack of tumor specificity [16
]. In vitro studies have also suggested synergy between IDE and BEN, although the mechanism remains unclear [17
In the present study we have evaluated the cytotoxicity of IDE in primary CLL cells ex vivo, demonstrating a lack of cross-resistance between this agent and chemotherapy, significant cross-resistance between IDE and IBR and a decrease in IDE activity when the microenvironment was simulated with CD40L/IL4. There was synergy between IDE and FLU, CLB or BEN, particularly in the presence of CD40L/IL4. Interestingly, synergy between BEN and IDE appeared unrelated to enhanced DNA damage but rather via transcriptional modulation. Our findings suggest a novel biological role for IDE in anti-CLL therapy.
We have demonstrated that inhibition of PI3Kδ by IDE is cytotoxic to primary CLL cells, with the sensitivity of cells differing to that of the standard chemotherapeutic agents, BEN, CLB and FLU. Indeed, some patients who were previously treated with chemoimmunotherapy showed sensitivity to IDE, consistent with clinical experience [12
]. Moreover, as previously observed ex vivo and in clinical studies, cells with a del 17p (p53 mutation), were equally sensitive to IDE [7
]. Interestingly, sensitivity to IDE correlated closely with sensitivity to IBR, suggesting a lack of cross-resistance in these patients. However, we did not specifically evaluate IDE in CLL cells from patients that had become resistant to IBR. CD40/IL4 caused significant resistance to IDE, consistent with the potent effect of CD40L on PI3Kδ expression, and had a lesser effect on sensitivity to IBR and chemotherapy.
Previous studies have demonstrated synergy between IDE and BEN [17
], IBR and BEN [18
], or MK2206 (an inhibitor of AKT) and BEN [27
]. Similarly, we have confirmed that IDE is synergistic with BEN, CLB and FLU in primary CLL cells occurring even in patients who were resistant to one agent. Interestingly, synergy was also observed in cells exposed to CD40/IL4, suggesting that the effect would be observed in the microenvironment, as well as in the peripheral blood. Synergy required the inhibition of PI3Kδ, as it did not occur in the lymphocytes of C57 BL/6 mice with dysfunctional enzyme. In addition, the synergy was not CLL specific, as it was also seen in normal peripheral blood B cells, but not in T cells.
A number of mechanisms may account for this synergy. First, there might be an interaction between the BCR and DNA repair pathways, although we did not observe an effect of BEN on p-AKT levels or of IDE on p-ATM production. Second, synergy may be related to increased BEN-induced DNA damage by IDE. However, this also appeared unlikely, as the combination of BEN and IDE did not produce a synergistic increase in γH2AX formation, to parallel the changes seen in cytotoxicity. Third, the synergy may be related to alterations in transcription by IDE. We observed transcriptional suppression with IDE and IBR treatments in CLL cells in vitro, which was most marked when RNA synthesis was initially increased by CD40/IL4. Similarly, a recent study in mantle cell lymphoma also showed that IDE can inhibit protein synthesis which correlated with a reduction in cell size and growth [28
]. While a detailed analysis of the genes effected by IDE in CLL has not yet been carried out, a recent clinical study has demonstrated that the CLL cells of patients receiving IBR show primarily a decrease in genes involved in receptor/cytokines signaling and those expressed in proliferating cells, although increased expression of a subset of genes was also observed [29
]. Interestingly, in contrast to the synergy seen with IDE, the global transcription inhibitor DRB produced antagonism with BEN indicating that inhibition or induction of specific proteins was required for the synergy between BEN/IDE. In this regard, it has been demonstrated that IDE can reduce Mcl-1 levels in CLL cells stimulated with anti-IgM antibody in vitro, although the degree of protection is inconsistent [17
]. CD40/IL4 may preferentially induce BclxL
and Bfl-1 expression over Mcl-1 in CLL, and these are also likely reduced by inhibition of the BCR pathway [30
]. Finally, IDE may reduce cell membrane levels of the activation marker CD69 in CLL, and CD69 has been associated with drug resistance [18
Our comprehensive analysis confirmed that IDE alone produces a measurable increase in γH2AX in CLL cells, suggesting the induction of DNA double strand breaks [23
]. ATM is autophosphorylated upon DNA double strand break induction, and phosphorylates serine 139 on H2AX to form γH2AX; Mediator Of DNA Damage Checkpoint 1 (MDC1) then binds to γH2AX, to prolong its half-life whereby γH2AX plays a key role in subsequent DNA repair [25
]. However, recent studies have demonstrated that γH2AX has multiple functions apart from DNA repair, including roles in cell division, development and senescence [31
]. Surprisingly, despite the increase in γH2AX with IDE, we did not detect DNA breaks by comet assay, indicating either that there were insufficient breaks for detection or that IDE may directly induce γH2AX formation. IDE might induce small numbers of DNA breaks in CLL cells by inhibiting the repair spontaneously formed DNA breaks, as has been observed with 2’-deoxycoformycin (pentostatin) [33
]. However, this appears unlikely, as IDE did not inhibit the repair of irradiation-induced DNA breaks, as measured by γH2AX removal or comet assay. DNA breaks could also be induced by IDE through effects of the drug on activation-induced cytidine deaminase (AID). IDE increases the transcription of activation-induced cytidine deaminase (AID) in mouse B cells grown in CD40L/IL4, leading to class switch recombination and somatic hypermutations at the immunoglobulin gene site, and off-target translocations across the genome [34
]. However, γH2AX formation with IDE was also observed in a variety of non-lymphoid cell lines (fibroblasts, HeLa cells and U251MG cells) indicating that the effect does not require a functional BCR PI3Kδ signaling axis. A similar effect in CLL cells was seen with IBR, and patients receiving IBR have demonstrated histone modifications, with loss of H3K27ac and H3K27me3, and changes in transcription, reflecting both increased and decreased RNA synthesis for different genes [29
]. We cannot exclude the possibility that chromatin modifications may utilize short-lived DNA strand break intermediates that require γH2AX to maintain DNA integrity. It is noteworthy that Singh et al (2015) recently demonstrated interplay of high mobility group AT-hook 2 (HMGA2), ATM, and H2AX in transcription initiation [36
]. However, McManus et al. (2005) characterized the occurrence of ATM-dependent but damage-independent γH2AX phosphorylation in all phases of the mammalian cell cycle [37
]. Whether γH2AX expression increases in CLL cells after clinical treatment and whether histone modification occur following in vitro treatment requires further study [31
]. As our IDE/DRB data indicates the involvement of specific transcriptional targets, future work would involve determining whether IDE alters H3K27ac and H3K27me3 levels at specific gene loci. ChipSeq studies comparing transcriptional events accompanying IDE-mediated transcriptional activation and/or repression would pinpoint new IDE-responsive targets. Furthermore, as we identified IDE-dependent but PI3Kδ-independent changes in γH2AX, which we inferred as IDE-induced chromatin modulation events, ChipSeq analysis using splenic cells from PI3Kδ-deficient/proficient mice would glean IDE drug on-target versus off-target effects; data with profound significance to CLL patients experiencing BCR drug toxicity and/or resistance.
In summary, we have demonstrated marked synergy between IDE and BEN in CLL cells, both when quiescent and under conditions to simulate the microenvironment. These beneficial outcomes have been seen clinically, but were associated with increased immunosuppression and associated infections at the commonly-used drug doses [16
]. Based on our results, we would suggest that future clinical studies with idelalisib and chemotherapy should use an initial step-up in drug dosages to determine the optimum regimen for therapeutic efficacy. While a number of mechanisms have been proposed for the synergy, further studies are required to identify the transcriptional changes with IDE in CLL cells in vitro that are required for this activity. Finally, we have demonstrated BCR- and PI3Kδ- independent induction of γH2AX formation via BCR chemo-inhibition in both CLL and solid tumor cells and determine the mechanism for this phenomenon and its biological significance.
4. Materials and Methods
4.1. Patient Samples, Cell Lines, Culture Conditions
Patients were selected from the Manitoba CLL Clinic to ensure variation in prior treatments and prognostic markers. B cell isolation from CLL patients was performed as previously described [38
]. Informed consent was obtained from all participants, and the study was authorized by the human research ethics board at the University of Manitoba (Ethics# HS19803 (H2019:217)). PBMCs were isolated from age-matched volunteers without CLL using the same protocol as for CLL cell isolation except without B cell selection [39
]. Freshly isolated cells were used for all experiments and drug exposures were carried out in serum-free hybridoma media (SFM) (Life Technologies, Carlsbad, CA, USA). For the microenvironment simulation experiments, 50 ng/mL each of CD40L and IL4 (R & D Systems, Minneapolis, MN, USA reconstituted in sterile phosphate-buffered saline (PBS), Life Technologies) were added to cells in SFM and allowed to incubate for 1 h prior to drug addition. HeLa, U251MG, and 293T were from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media (Life Technologies, Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum, 1× Pen/Strep, and 1× Glutamax (Life Technologies). The Burkitt’s lymphoma cell line, BJAB [40
], were grown in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 1× Pen/Strep, and 1× Glutamax and 10% fetal bovine serum. Human primary fibroblasts (HPF) were from the Coriell Institute and were cultured in DMEM/F12 media (Life Technologies) and supplemented with 15% fetal bovine serum, 1× Pen/Strep, and 1× Glutamax. Drug exposures for all cell types were carried out at 37 °C and 5% CO2
in a humidified atmosphere.
BEN, CLB, FLU, and DRB were purchased from Millipore Sigma and IDE and IBR were purchased from Selleckchem (Houston, TX, USA). All agents were reconstituted to 100 mM in dimethyl sulfoxise (DMSO) (MilliporeSigma, Burlington, VT, USA), aliquoted and stored at −80 °C, except DRB which was reconstituted to 50 mM and stored at −20 °C. Aliquots were then diluted in SFM prior to use. DMSO alone treatment was used in all experiments as a negative control and, unless otherwise stated, for normalization.
4.3. Flow Cytometry and Drug Synergy
For synergy and dose-response determinations, CLL cells were treated in 96-well plates with 6 increasing concentrations of drug for ~18 or ~72 h. Concentration ranges were selected and optimized to ensure that the exponential phase of the dose-response curve was obtained for the majority of the samples. Ranges were 0–160 µM for BEN, 0–80 µM for IDE and CLB, 0–20 µM for IBR and 0–10 µM for FLU. Synergy experiments with DRB were performed using 3 or 4 increasing doses of drug for ~18 h with the concentration ranges 0–40 µM for BEN and IDE, and 0–20 µM for DRB. The concentration of DMSO was constant between samples. After 18 or 72 h incubation, cell death was determined by flow cytometry by annexin-V-FITC and 7AAD (BD Pharmingen, San Jose, CA, USA) [41
]. Cells were stained with for 15 mins with AV/7AAD and analyzed by flow cytometry using a NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA) with a 96-well plate adapter. Cells were considered alive when they were double-negative for AV and 7AAD. When CD19 and CD3 were analyzed in the non-CLL donor PBMCs, anti-CD19-APC or anti-CD3-APC (BD Pharmingen) were added in a triple stain with AV/7AAD. Isotype control antibodies were also run for CD19 (anti-mouse-IgG1κ, BD Pharmingen) and CD3 (anti-mouse-IgG2aκ, BD Pharmingen).
C57BL/6 mice (8–10 weeks old) were purchased from and maintained in a pathogen-free facility at the GMC, University of Manitoba, according to the Canadian Council on Animal Care guidelines. Mice were either WT or expressed a mutated inactive form of p110δ (p110δD910A) [42
]. Spleens from 2 WT or 4–6 p110δ-deficient mice were crushed, filtered, washed with RPMI (Hyclone, Logan, UT, USA) with 2% Penicillin and Streptomycin (Pen/Strep, Life Technologies, Carlsbad, CA, USA), centrifuged at 4 °C, and resuspended in 2 mL of ammonium-chloride-potassium (ACK) lysis buffer (150 mM NH4
CL, 10 mM KHCO3
, and 0.1 mM Na EDTA pH 7.2–7.4) for 2 mins at room temperature. 15 mL Media was then added, samples were centrifuged and resuspended in 1× PBS (Thermo Fisher, Waltham, MA, USA) with 2% FBS (Life Technologies, Carlsbad, CA, USA). B cells were isolated using the EasySep™ Mouse Pan-B Cell Isolation Kit (StemCell Technologies, Vancouver, Canada) as per the manufacturer’s instructions. Isolated splenic B cells were then stimulated for 24 h with CD40L (1µg/mL) and IL4 (5ng/mL) in RPMI with 1% Penicillin/Streptomycin. 2 × 106
cells/mL were then treated with drug, incubated in 100 μL for ~72h at 37 °C and 5% CO2
in a humidified atmosphere, and cell death was then measured. Experiments were performed twice with similar results. All mice experiments were approved by University of Manitoba animal care committee (protocol approval number: B2017-0130) under title “Phosphoinositide-dependent signalling pathways controlling B lymphocyte activation” on 1 April 2019 for period from 30 May 2019 to 29 May 2020.
4.5. DNA Damage Analysis
DSBs were measured for immunoreactivity to anti-γH2AX antibody after cells were treated for ~18 h with drug. Cellular irradiation (IR; 20 Gy at ~17 h post cell seeding) using a RS-2000 Rad Source (Rad Source Technologies, Inc., Buford, GA, USA) served as a damage-positive control. Thirty min post-irradiation, samples were washed in PBS (MilliporeSigma), resuspended in 70% ethanol (MilliporeSigma), and stored at −20 °C for at least 1 h (up to 4 days). Cells were then washed 3 times with cell staining buffer (Biolegend, San Diego, CA, USA) and 1.75 µL of Alexa 647conjugated anti-H2AXγ antibody or Alexa647-Mouse-IgG1 Isotype control (Biolegend) was added to 50 µL of cell staining buffer at room temperature in the dark for 30 min. Samples were washed and analyzed via a NovoCyte flow cytometer.
Following 24 h treatment with IDE, adherent non-B lineage cells (Hela, U251MG and HPF) were treated followed by immunostaining with 1/1000 diluted Alexa 647-conjugated anti-H2AXγ antibody in 3% bovine serum albumin (BSA)-PBS [43
]. Images were captured via epifluorescence microscopy. A minimum of 30 cells per treatment or cell line were scored and the average number of γH2AX-immunostained foci per cell was tabulated and graphed.
4.6. Comet Assay
The alkaline comet assay was performed as previously described [43
], with the following modifications. Cells were treated for ~18 h. At ~17 h, untreated cells underwent 20Gy irradiation as a positive control for DNA damage. Cells were embedded in agarose on 96-well slides and underwent lysis, alkali unwinding and gel electrophoresis as per manufacturer’s protocols (Trevigen, Gaithersburg, MD, USA). Comets were visualized via SYBR green staining on the Cytation V using Gen5 software (BioTek Instruments, Inc., Winooski, VT, USA).
4.7. IR Recovery, Dose Response, and Synergy
For synergy and dose-response determination, CLL cells were treated in 96-well plates with 6 increasing concentrations of IDE from 0–80 μM. The concentration of DMSO was constant between samples. Plates were then irradiated with 0, 1, 2, 5, 8, 10 or 20 Gy and cell death and γH2AX were measured after 72 h. In IR recovery experiments, CLL patient cells were stimulated with 50 ng/mL of CD40L and IL4, or with PBS as a control. DMSO or 10 μM of IDE was then added to the cells, the plates were incubated for ~18 h, and the cells were analyzed for γH2AX and comet assay. Simultaneously with the IDE incubation starting at the beginning of drug treatment, at 3 h, at 0.5 h, or immediately prior to γH2AX or comet assay analysis, cells were irradiated with 10 Gy and allowed to recover.
4.8. Western Blots
Primary CLL cells were treated with 10 µM IDE and/or 40 µM BEN for ~18 h with or without CD40L/IL4 treatment and pelleted. Protein extracts were prepared and underwent western analysis as per [43
]. Blots were immunostained with antibodies listed in Table S1
, followed by appropriate horseradish peroxidase–conjugated secondary antibodies (Table S1
) and detected using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA). Antibodies to actin and vinculin (Table S1
), and Ponceau staining (MilliporeSigma) of the transferred membrane were used as protein-loading controls. Blots were visualized using a LAS 500 Imager (GE Healthcare, Chicago, IL, USA). Densitometric analysis was performed via ImageJ. Protein levels were first normalized to a loading control and then phospho-protein levels were normalized to their non-phosphorylated counterparts.
4.9. Transcriptional Analysis
Primary CLL cells were treated with either CD40L/IL4 or PBS as a control and incubated for 18 h. 10 μM IDE, 5 μM IBR, 30 μM DRB, or 25 μM BEN were also added either immediately following CD40L/IL4 treatment or after 12 h (for the 6 h drug treatment time point) to cells for a final volume of 100 μL in a 96- well plate. Following drug treatment, the Click-iT® RNA Alexa Fluor® 488 HCS Assay (Life Technologies) was performed as per the manufacturers’ instructions with the following modifications (in consultation with manufacturer). One h prior to the end of drug treatment, 100 μL of 2 mM 5-ethynyl uridine (EU) was added directly to the 100 μL of cells and incubated under normal culture conditions. The cells were then stained for 30 mins at 4 °C with the eBioscience (San Diego, CA, USA) Fixable Viability Dye eFluor™ 780 prior to fixation with 3.7% formaldehyde (MilliporeSigma) and permeabilization with 0.5% Triton® X-100 (MilliporeSigma). After incubation with Click-iT® reaction cocktail and washing with Click-iT® reaction rinse buffer, cells were resuspended in 100 μl of PBS (MilliporeSigma) and analyzed by flow cytometry using a NovoCyte flow cytometer. Cells stained without the viability dye, the Alexa Fluor® azide, or both were run as controls. Click-iT® median fluorescence intensity (MFI) was measured in the viable cells. AV/7AAD was run in parallel as a control for viability. All wash steps were performed in PBS followed by microplate centrifugation at 250 g and removal of the supernatant.
4.10. IGHV Mutational Analysis
The immunoglobulin heavy chain variable region (IGHV)
mutational status of the patients was determined from RNA, as previously described [44
4.11. FISH Analysis
Fluorescence in situ hybridization (FISH) analysis was carried out on fresh or stored cells, as previously described [41
4.12. Statistical and Synergy Analysis
Graphical representation and statistical analysis were performed using MS Excel and GraphPad Prism. Drug synergy was assessed using Combenefit software [19
] and GraphPad Prism [39
]. Combenefit plots represent the average difference in cell viability or γH2AX production compared to that predicted to the single dose-response curves for each agent (blue—synergy, green—additivity, red—antagonism) [19
]. Statistical tables from Combenefit show 95% confidence intervals (* p
< 5 × 10−2;
, *** p
). Synergy was also assessed according to the method of Chou and Talalay, where the CI is defined as: CI = (d1x
) + (d2x
); where d1x
are doses of drug 1 and drug 2, respectively, required to produce a given reduction in cell viability (x) when given in combination, and D1x
are doses of drug 1 and drug 2, respectively, required to produce the same effect in single-agent treatments [46
]. CI < 1, =1 and >1 are interpreted as synergy, additivity and antagonism, respectively. The doses chosen for CI analysis were averaged from the closest values available in the drug combination matrix to the clinically relevant dose for each drug (5 μM for IDE, 10 and 20 μM for BEN, 5 μM for CLB, and 2.5 and 5 μM for FLU) [20
]. A p
-value < 0.05 was considered significant.
To determine the correlation between the IC50 of the drugs and the average CI values, the Pearson correlation coefficient (r) was calculated and the p-value was determined using a two-tailed T test with a 95% CI. To test the significance of the Click-iT® (Life Technologies) data, a paired two-tailed t-test was performed with a 95% confidence interval (* p < 5 × 10−2; ** p < 1 × 10−2, *** p < 1 × 10−3).