Background
Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma (NHL) associated with poor prognosis [
1,
2]. In recent years several studies brought evidence that implementation of high-dose cytarabine (araC) into induction therapy, e.g. by sequential chemotherapy by R(ituximab)-CHOP and R-DHAP regimens, induced higher response rate and prolonged progression-free survival compared to R-CHOP-only [
3‐
5]. Based on these results, implementation of araC into induction therapy became standard of care for all newly diagnosed younger MCL patients. Despite considerable improvement, however, most high-risk MCL patients relapse even after araC-based first-line regimen. Prognosis of relapsed/refractory (RR) MCL is dismal. Currently, there is no second-line standard-of-care for RR-MCL [
6]. Available treatment approaches for RR-MCL include cisplatin, fludarabine, cladribine, gemcitabine, temsirolimus, bortezomib, bendamustine, lenalidomide and ibrutinib-based regimen [
7‐
16].
AraC belongs among the backbone anti-leukemia agents [
17]. Both, “standard dose” araC (100-200 mg/m2 continuous i.v. infusion for 7 days), and “high dose” araC (HDAC, 2-3 g/m2, 2–4 i.v. three hour administrations every 12–24 hours) have been widely used in the therapy of acute myelogenous leukemia (AML), as well as in salvage regimen for relapsed B-NHL [
18,
19]. As mentioned above araC appears particularly effective component of multi-agent aggressive immunochemoterapy regimen used in younger MCL patients.
AraC is a prodrug, which must be 1. transported into the cell, and 2. within the cell converted into an active drug by phosphorylation by specific phosphokinases of the nucleotide salvage pathway [
20]. During “standard dose” cytarabine administration araC is transported into the cell by means of specific transporters, primarily via hENT1/SLC29A1 [
21]. During high-dose cytarabine administration araC also diffuses across plasma membrane independent of the specific transporters [
22]. The rate-limiting enzyme of the nucleotide salvage pathway is deoxycytidine-kinase (DCK), which catalyzes the first phosphorylation of araC into araCMP. AraCMP is retained in the cell and undergoes two additional consecutive phosporylations before it can be incorporated into DNA.
The molecular mechanisms of araC resistance in MCL are unknown. Resistance to araC in myeloid leukemia cells was repeatedly associated with altered expression of genes involved in nucleotide salvage pathway, including downregulation of DCK, or upregulation of key araC-inactivating enzymes, namely cytidine-deaminase (CDA) or cytoplasmic 5′nucleotidase (NT5C2) [
20‐
25].
In this study we derived araC-resistant MCL cells, studied their sensitivity to a battery of anti-cancer drugs and elucidated the molecular mechanism responsible for araC resistance in MCL.
Discussion
In this study we analyzed molecular mechanisms of araC resistance in five MCL cell lines and ten paired primary MCL samples obtained before and after araC-based therapies. In addition, we tested optimal treatment strategies for cytarabine-resistant MCL. On molecular level we identified marked and principal downregulation of DCK, the rate-limiting enzyme of nucleotide salvage pathway, in all 5 cytarabine-resistant MCL clones, and in 50% primary MCL samples obtained from patients, who progressed on or relapsed after araC-based treatments. In 50% primary MCL samples, no change of DCK expression was observed at time of lymphoma relapse or progression. Importantly, no upregulation of DCK was observed in any of the analyzed post-treatment samples. Although the analysis of the primary MCL samples indicate that the mechanisms responsible for araC resistance
in vivo are more complex than those observed
in vitro, it must be emphasized that downregulation of gene and protein DCK was indeed confirmed in a substantial part of the patients’ post-treatment samples (Table
3, Figure
7A,B). Interestingly, in one of the two MCL patients primary resistant to araC, no change of DCK expression was observed with slightly increased
ex vivo sensitivity of post-treatment MCL cells to araC (Figure
7B,C). This observation could be explained by existence of araC-resistant stem cell-like MCL cells that would reside in the niches in lymph nodes (and/or bone marrow) and produce partially araC-sensitive MCL cells mobilized in the peripheral blood. In such a case, elimination of the mobilized MCL cells, but persistence of the stem cell-like MCL compartment, would lead to stable disease, and eventual lymphoma progression (which was the actual course of the disease observed in this patient).
DCK catalyzes the first phosphorylation (=activation necessary for their cytotoxic activity) not only of araC into araCMP, but also of most nucleoside analogs (both pyrimidine and purine-derived) commonly used in anti-cancer therapy. Using DAVID bioinformatic analyzer
purine/pyrimidine metabolism, and
B-cell receptor signaling were among the functional cathegories associated with the most downregulated and upregulated genes, respectively. In accordance with these results we subsequently showed that all R clones were cross-resistant to both pyrimidine analog gemcitabine, and to purine analogs fludarabine and cladribine (all of which are activated by DCK). Sensitivity of R clones to other types of anti-cancer molecules including genotoxic cytostatics (cisplatin, doxorubicin, bendamustine), targeted drugs (temsirolimus, bortezomib) or biological agents (monoclonal anti-CD20 antibody rituximab) remained unaffected, or was even augmented in the case of BTK inhibitor ibrutinib. The reason, why ibrutinib more effectively eliminated araC-resistant MCL cells remained elusive, but might be at least partially explained by the observed upregulation of B-cell receptor signaling in R clones compared to CTRL cells (Additional file
1: Figure S1).
The results of our
in vitro and
in vivo tests combined with the observed decreased expression of DCK in all araC-resistant MCL clones and in 50% post-treatment primary MCL samples suggest that the resistance of MCL cells to high-dose araC is caused by suppressed araC activation by DCK due to markedly decreased DCK expression. DCK has low substrate preference and phosphorylates both, purines and pyrimidines, including synthetic analogs cytarabine, fludarabine, gemcitabine and cladribine [
27‐
29]. The fact that above-mentioned nucleoside analogs are substrates of DCK explains the observed cross-resistance of R clones to all tested nucleoside analogs, both purine- and pyrimidine-derived. Retained sensitivity to other classes of anti-MCL agents (i.e. other than nucleoside analogs) with diverse molecular mechanisms of their respective antitumor activities suggests that no major additional molecular alteration was involved in the development of araC resistance.
Prognosis of patients with relapsed/refractory MCL (RR-MCL) is dismal. Currently there is no standard-of-care for RR-MCL patients. Second-line treatment approaches include fludarabine, gemcitabine, cladribine, cisplatin, bortezomib, temsirolimus, bendamustine, lenalidomide and ibrutinib-based regimen. We have proved
in vitro and
in vivo on a mouse xenograft model of MCL that treatment of patients, who progress on or relapse after high-dose araC-based regimen should not rely on nucleoside analogs, namely on the currently used agents fludarabine, gemcitabine and cladribine, since all of them must be phosphorylated by DCK to exert their anti-lymphoma activity. Instead, other classes of anti-lymphoma drugs should be applied in case of araC failure, i.e. in the setting of anticipated araC-resistance. Some of these agents have only recently been approved for the therapy of relapsed/refractory (RR-) MCL, temsirolimus in Europe, bortezomib and ibrutinib in USA. It might be speculated that high-dose therapy (given before autologous stem cell transplant) based on other agents than nucleoside analogs might prove more beneficial especially for those patients with suboptimal responses after induction araC-based immunochemotherapy (e.g. patients, who achieve partial remission, or patients with detectable minimal residual disease). In addition to the currently approved agents, bendamustine represents another extremely promising drug in MCL. Recently it was demonstrated that bendamustine potentiates the effect of araC by augmenting the level of intracellular ara-CTP, and the R-BAC (rituximab, bendamustine, araC) regimen was shown to be effective even in patients resistant to araC thus providing a treatment option even for the elderly and/or frail patients [
16,
30,
31]. It might be speculated that the increased level of ara-CTP might partially offset the anticipated downregulation of DCK thereby explaining, why the combination of bendamustine and araC was shown to be effective even in patients, who relapsed after araC-based therapies [
30].
Conclusions
Our data from the cell lines and primary MCL samples clearly demonstrate that acquired resistance of MCL cells to araC is associated with downregulation of mRNA and protein expression of DCK, enzyme of the nucleotide salvage pathway responsible for phosphorylation of most nucleoside analogs used in anti-cancer therapy. In translation, the results suggest that 1. nucleoside analogs should not be used for the second-line therapy of MCL patients, who fail after araC-based regimen; 2. non-nucleoside analogs should be employed in this setting, including cisplatin, ibrutinib, temsirolimus, bortezomib or bendamustine; 3. ibrutinib appears particularly effective in eliminating araC-resistant MCL cells.
Methods
Cell culture
JEKO-1, GRANTA-519 and REC-1 were purchased from German Collection of Microorganisms and Cell Cultures (DSMZ), MINO was from American Tissue Culture Collection (ATCC), HBL-2 was a kind gift of prof. Dreyling (University of Munich, Germany). Cell lines were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
Reagents
Cytarabine, fludarabine, gemcitabine, cladribine, cyclophosphamide, doxorubicin and cisplatin were from Clinical Dept. of Hematology, University Hospital in Prague, Czech Republic. Temsirolimus, bortezomib, bendamustine and ibrutinib were purchased from Selleck Chemicals. Rituximab was kindly provided by Roche, Czech Republic.
Establishment of araC-resistant clones
MCL cell lines were incubated in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 15% fetal bovine serum with increasing concentrations of cytarabine up to 50 μM.
Proliferation assays
Proliferation was estimated using WST-8 Quick Cell Proliferation Assay Kit (BioVision) according to the manufacturer instructions. Briefly, 5.000 cells were seeded into 96-well plate on day 1. Drugs were added on day 1. Proliferation was measured on day 1 and then since day 4 daily. Antiproliferative activity of each drug was analyzed at several concentrations.
Absorbance of the triplicate samples was measured on ELISA reader after 3 hour incubation with WST-8 reagent at 37 grades Celsius in the thermostat. Maximal absorbance (MAXu) obtained from the untreated cells during the particular experiment was arbitrary set as 100%. Absorbance of medium without cells was used as background (B). For each cell population (both, unexposed and drug-exposed) and for each measurement (M1, M2, M3…MX) the proliferation curve was calculated as follows: (MX - B)/(MAXu - B). As a consequence, the proliferation curve of untreated cells always peaks 100%, while proliferation curves of drug-exposed cells can terminate below or above 100%.
51Cr release assay for the assessment of the impact that CD20 mAbs have on rituximab-mediated complement mediated cytotoxicity (CMC) and antibody dependent cellular cytotoxicity (ADCC)
CTRL MCL cells and R clones were labeled with 51Cr at 37°C, 5% CO2 for 2 hrs. 51Cr-labeled cells were then placed in 96-well plates at a cell concentration of 1 × 105 cells/well (complement-mediated cytotoxicity (CMC) assay) or 1 × 104 cells/well (antibody-dependent cell cytotoxicity (ADCC) assay). Cells were then exposed to rituximab (10 mg/ml) or isotype antibody (10 mg/ml) and human serum (for CMC assay, 1:4 dilution) or peripheral blood mononuclear cells (PBMCs) (for ADCC assay, 40:1 effector: target ratio) for six hrs at 37°C and 5% CO2. 51Cr release was measured from the supernatant by standard gamma counting and the percentage of lysis was calculated. PBMCs were obtained from healthy donors (Roswell Park Cancer Institute IRB-approved protocol CIC-016) and isolated by Histopaque-1077 ultracentrifugation of peripheral whole blood and used at an effector: target ratio of 40:1 for ADCC assays. Pooled human serum was used as the source of complement for CMC assays.
Gene expression profiling and data analysis
A biological duplicate of each araC-resistant MCL clone (R) was compared to a biological duplicate of the original araC-sensitive (CTRL) cell line. In total, five R clones were compared to five corresponding CTRL cell lines using two microarray chips. Total RNA was extracted by RNeasy Mini Kit (Qiagen), and its quality verified using the Agilent 2100 Bioanalyzer (Agilent Technology). Extracted RNA was amplified using the Illumina RNA Amplification Kit (Ambion). Amplified RNA was hybridized to the Illumina HumanRef-8 and HumanRef-12 BeadChips (Illumina). Subsequent data analysis was performed in R-software, mainly in limma package from Bioconductor (
http://www.bioconductor.org). Multiple testing correction was performed using Benjamini & Hochberg method. The filtered group of genes with fold change at least ±1.5-fold and adjusted p value < 0.05 were annotated and arranged into biologically relevant categories using The Database for Annotation, Visualization and Integrated Discovery (DAVID,
http://david.abcc.ncifcrf.gov).
Primary MCL sample acquisition, real-time RT-PCR analysis, and apoptosis measurement
All primary MCL samples were obtained from patients with MCL at diagnosis (D1-D10), and at the relapse or during progression after failure of high-dose araC-based front-line therapies (R1-R10). Samples were obtained from patients, who signed informed consent according to the Declaration of Helsinki. Mononuclear cells were isolated from all PBMC and PE samples by the standard Ficoll-Hypaque gradient centrifugation. Mononuclear cells were then CD19 sorted on magnetic columns using CD19 microbeads (Miltenyi Biotec). The purity of MCL population after sorting was > 95% in all cases as verified by flow-cytometry. Total RNA was isolated from CD19-sorted PBMC or PE cells stored in RNAlater solution using RNeasy Mini Kit (Qiagen, Hilden, Germany) and from fresh-frozen paraffin-embedded (FFPE) lymph node samples using High Pure RNA Paraffin Kit (Roche Diagnostics GmbH, Germany) according to the manufacturer’s instructions. cDNA synthesis was carried out from 1 μg of total RNA with the High-Capacity cDNA Reverse Transcription Kit (random primers) (Applied Biosystems). Real-time RT-PCR was performed using TaqMan Gene Expression Assays on the ABI 7900HT detection system (Applied Biosystems). The reference gene was GAPDH. Ex vivo apoptosis of primary MCL cells was determined using Annexin-V-PE (Apronex, Czech Republic) and flow cytometry (BD FACS Canto II) according to the manufacturer’s instructions after 24 hours exposure to 25 μM araC, 100 μM fludarabine and 25 μM gemcitabine.
Experimental therapy of MCL xenografts
In vivo studies were approved by the institutional Animal Care and Use Committee. Immunodeficient NOD.Cg-
Prkdc
scid
Il2rg
tm1Wjl
/SzJ mice (Jackson Laboratory) were maintained in individually ventilated cages. JEKO-1 cell line-based mouse model of MCL was used for experiments [
32]. JEKO-1 cells were harvested, suspended in PBS, and injected (1 × 10
6/mouse) i.v. into tail vein of 8- to 12-week-old female mice on DAY 1. Therapy was initiated on DAY 8. Each cohort of mice contained 6–8 animals. The mice received treatment as follows: temsirolimus 1 mg s.c. 1 x weekly (3 cycles), cyclophosphamide 3 mg i.p. 1 × weekly (3 cycles), bendamustine 0.5 mg i.p. two subsequent days (day 1 + day 2) every two weeks (2 cycles), bortezomib 25 μg i.p. 2 × weekly (3 cycles), cisplatin 180 μg i.p. every two weeks (2 cycles), gemcitabine 10 mg i.p. 1 × weekly (3 cycles), fludarabine 1 mg three subsequent days (day 1–3) weekly (3 cycles), rituximab 250 μg s.c. 1 × weekly (3 cycles). The data were analysed in GraphPad Software.
Two-dimensional electrophoresis
IPG strips (pH 4.0-7.0, 24 cm; ReadyStrip, Bio-Rad) were rehydrated overnight in 450 μL of sample, representing 1.5 mg of protein. Isoelectric focusing was performed for 70 kVh using Protean IEF cell (Bio-Rad). Six replicates were run for each cell type. Focused strips were equilibrated and reduced in equilibration (6 M urea, 50 mM Tris pH 8.8, 30% glycerol, 2% SDS) supplemented with DTT (450 mg per 50 mL) for 15 min and then alkyled in equilibration buffer with added iodacetamide (1.125 mg iodacetamide per 50 mL). SDS-PAGE electrophoresis was performed in a Tris-glycine-SDS system using a 12-gel Protean Dodeca Cell apparatus (Bio-Rad) with buffer circulation and external cooling (20°C). Gels were run at a constant voltage of 80 V per gel for 30 min and then at a constant voltage of 200 V for 6 h. Gels were washed in deionized water to remove redundant SDS and with colloidal Coomassie Brilliant Blue (SimplyBlue™ Safestain, Invitrogen, Carlsbad, CA, USA) overnight.
Gel image analysis and extraction of peptides
Stained gels were scanned with GS 800 calibrated densitometer (Bio-Rad) and image analysis was performed with Progenesis™ software (Nonlinear Dynamics, Ltd., Newcastle upon Tyne, UK) in semi-manual mode with 6 gel replicates for each cell type. Normalization of gel images was based on total spot density, and integrated spot density values (spot volumes) were then calculated after background subtraction. Average spot volume values (averages from the all 6 gels in the group) for each spot were compared between the groups. Protein spots were considered differentially expressed if their average normalized spot volume difference was > 2-fold. As determined by the Student’s t-test, a p-value < 0.05 was considered to indicate a statistically significant difference.
Protein digestion and peptide extraction
Spots containing differentially expressed proteins were excised from the gels, cut into small pieces and washed 3 times with 25 mM ammonium bicarbonate in 50% acetonitrile (ACN). The gels were then dried in a SpeedVac Concentrator (Eppendorf, Hamburg, Germany). Sequencing grade modified trypsin (Promega, Madison, WI, USA) (6 ng/μl in 25 mM ammonium bicarbonate in 5% ACN) was added. Following overnight incubation at 37°C, the resulting peptides were extracted with 50% ACN.
MS analysis and protein identification
Peptide samples were spotted on a steel target plate (Bruker Daltonics, Bremen, Germany) and allowed to dry at room temperature. Matrix solution (3 mg α-cyano-4-hydroxycinnamic acid in 1 ml of 50% ACN containing 0.1% trifluoroacetic acid) was then added. MS was performed on an Autoflex II MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) using a solid nitrogen laser (337 nm) and FlexControl software in reflectron mode with positive ion mass spectra detection. The mass spectrometer was externally calibrated with Peptide Calibration Standard II (Bruker Daltonics). Spectra were acquired in the mass range 800–3,000 Da. The peak lists were generated using FlexAnalysis and searched against Swiss-Prot (2012_07 version, 536 789 sequences) using Mascot software. The peptide mass tolerance was set to 100 ppm, taxonomy Homo sapiens, missed cleavage was set to 1, fixed modification for cysteine carbamidomethylation, and variable modifications for methionine oxidation and protein N-terminal acetylation. Proteins with Mascot score over the threshold 56 for p < 0.05 calculated for the used settings were considered as identified. If the score was lower, the identity of protein candidate was confirmed by MS/MS.
Western blot analysis
Cells were lysed in NHT buffer (140 mM NaCl, 10 mM HEPES, 1.5% Triton X-100, pH 7.4). Protein concentration in the collected supernatants was determined by the Bradford assay (Bio-Rad). Lysate samples (50 μg) were combined with SDS loading buffer containing 2-mercaptoethanol and boiled for 5 min. Quadruplicate samples were separated on 12% SDS-PAGE minigels in Tris-glycine buffer (Bio-Rad). Electrophoresis was performed at a constant voltage for 30 min at 45 V per gel, and then at 90 V per gel until the dye front reached the gel bottom. Proteins were transferred onto 0.45 μm PVDF membranes (Milipore, Billerica, MA, USA) in a semi-dry blotter (Hoefer, San Francisco, CA, USA) at 0.8 mA/cm2. Membranes were incubated in PBS (Invitrogen) containing 0.1% Tween-20 and 5% non-fat dried milk for 1 h. GAPDH or Actin were used as the loading controls. As primary antibodies anti-deoxycytidine kinase mouse monoclonal antibody (sc 81245 Santa Cruz Biotechnology, Sanat Cruz, CA, USA) diluted 1:200 or polyclonal anti-GAPDH produced in rabbit (Sigma-Aldrich, G9545) diluted 1:10,000 were used. After thorough washing in blocking buffer, a secondary horseradish peroxidase-conjugated anti-mouse (sc2005) or anti-rabbit antibody (sc2313) (both from Santa Cruz Biotechnology) was added (1:10,000). The signal was detected using LumiGLO Reserve, (KPL, Gaithersburg, MD, USA) or Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and membranes were exposed to X-ray films (Kodak, Rochester, NY, USA).
Acknowledgements
Financial Support: IGA-MZ NT13201-4/2012, GACR14-19590S, GA-UK 446211, GA-UK 253284 700712, GA-UK 595912, GA-UK 1270214, UNCE 204021, PRVOUK-27/LF1/1, PRVOUK P24/LF1/3, SVV-2013-266509 and BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University in Vestec“ (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
PK and JP conceived of the study and participated in drafting of the manuscript. MK carried out gene expression analysis and in vivo experiments, OV, LL and JP carried out proteomic analysis and western blotting. BM, DV, JM, PV and LL participated in in vitro experiments. CM carried out chrome-releasing assays. VK performed the statistical analysis. FH and MV participated in the design of the study and helped to review the manuscript. RJ and MT carried out analysis of primary MCL samples. All authors read and approved the final manuscript.