AbstractChronic lymphocytic leukemia is characterized by the accumulation of B cells that are resistant to apoptosis. This resistance is induced by pro-survival stimuli from the microenvironment. TCL1 and ATM are central to the pathogenesis of the disease and associated with more aggressive disease. Their protein products have recently been shown to physically interact in leukemic cells and to impact on NF-κB signaling, which is a key regulator of apoptosis. In the present study we show that TCL1 and ATM are significantly co-expressed and up-regulated in malignant cells compared to non-malignant B cells, and that expression of TCL1 is partially deregulated by aberrant DNA-methylation. In addition, complex external stimuli induce essentially similar TCL1 and ATM time-course kinetics. In line with a coordinative regulation of NF-κB signaling by TCL1, its knockdown induced apoptosis in primary leukemia cells. These findings suggest that both genes functionally cooperate to modulate similar apoptosis-related cellular pathways.
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature B lymphocytes in the blood, lymphoid organs and bone marrow and is caused by resistance to apoptosis.1 This prolonged survival is highly dependent on pro-survival stimuli from the microenvironment including cytokines and stem-cell factor (SCF).2 The prognosis of CLL is influenced by diverse factors including the mutation status of the immunglobulin heavy chain genes (IGHV) and different cytogenetic aberrations.1 Loss of genomic material from chromosomal band 11q22-q23 harboring the ataxia telangiectasia-mutated (ATM) gene is associated with more rapid disease progression. The serine protein kinase ATM is activated by DNA double strand breaks to delay cell cycle and thereby ensure integrity of the genome.3ATM deficiency is accompanied with genomic instability and a predisposition to lymphoid malignancies.4 In a number of gene expression profiling analyses, the ATM gene showed a small but consistent and significant upregulation in CLL cells (Online Supplementary Figure S1 and references therein). Similarly, the strong overexpression of TCL1 in B cells of mice5 and men6 is associated with a more aggressive form of CLL. The TCL1 oncogene acts as a coactivator of AKT,7AP1 and NF-κB.8 It is therefore potentially involved in the resistance to apoptosis that is observed in CLL. Recently, a direct interaction of TCL1 and ATM proteins was reported in CLL in association with activation of the NF-κB pathway.9 To assess the role of both genes in the resistance to apoptosis, we characterized their expression kinetics in CLL cells stimulated with different microenvironmental support.
Design and Methods
PBMCs of CLL patients (informed consent; Ethics Committee approval 96/08 Ulm University) and healthy individuals (donors of the German Red Cross, Ulm) were isolated (Ficoll, Biochrom), CD19-selected (MACS Milteny) and controlled for purity by flow cytometry (anti-CD19-FITC, Dako).
Methyl-CpG-Immuno-precipitation (MCIp) - promoter array
Genomic DNA (2 μg) was immunoprecipitated using 30 μg recombinant MBD2–Fc fusion protein coupled to SIMAG protein-A magnetic beads (Chemicell)10 and hybridized onto custom arrays (eArray, Agilent, G4170-90012 protocol-version.1.0) with promoter tiling from -3.8 to +1.8kbp (60bp oligonucleotides, 30bp non-overlapping spacing, 10bp linker-sequence; GRCh37 hg18).
CLL cell culture
CD19-sorted CLL cells were cultured with bone marrow-derived human (“HS-5”) or mouse (“M210B4”) stromal cells (3×10 cells/6-well) or with conditioned medium (“HCM”, supernatant of HS-5 cells cultured for 3 days).
RNA (AllPrep-DNA/RNA-Mini-kit, Qiagen) was first-strand cDNA synthesized (AffinityScript-qPCR-cDNA Synthesis-Kit). QRT-PCR was performed with Absolute-QPCR-SYBR-Green-ROX-Mix (Thermo-Scientific) containing 70nM primers (Online Supplementary Table S1) with 7300-Real-time-PCR-System (Applied Biosystems) at 15 minutes 95°C, 40 cycles: 15s 95°C; 30s 60°C. Expression arrays: Illumina-Human-Sentrix-12-BeadChip.
Modeling and statistics
For analysis and visualization of previously published gene expression data, Oncomine™ (Compendia Bioscience, Ann Arbor, MI) was used. For details of network modeling please see Online Supplementary Design and Methods. In brief, genes were grouped into clusters according to their expression kinetics using Partitioning Around Medoids (PAM) clustering method to yield an appropriate number of genes. The resulting medoids represent genes corresponding to network nodes. Networks were estimated using a dynamic Bayesian network approach. The analysis employs a Markov Chain Monte Carlo algorithm for obtaining posterior edge probabilities of the network.
We transfected 5x10 CLL-PBMCs using 1 μg siRNA targeting TCL1A (Silencer-Select-Pre-designed-and-Validated siRNA, Ambion) and 100μl B-cell solution-B using program U-015 (Amaxa NucleofectorII). ON-TARGETplus SMARTpool siRNA, MCL-1 (Dharmacon) and Silencer-Negative-Control #1 (Ambion) were used as controls. After transfection, cells were cocultured with HS-5 cells (2.6x10 cells/24-well). CLL cell survival was analyzed by staining 7-AAD, Annexin V and propidium iodide (PI) and CD19-APC using FACSCalibur flow cytometer (BD Biosciences).
Results and Discussion
TCL1A and ATM (Figure 1A) are central players in the pathogenesis of CLL. We therefore studied transcription of TCL1A and ATM and discovered a strikingly strong correlation of their expression levels in primary cells (Figure1B). In order to rule out confounding effects caused by deletion of the critical region in 11q22-q23 harboring the ATM and the miR34b-5p genes that target TCL1A,11,12 only CLL cells without cytogenetic aberrations or only harboring a single deletion of 13q14.3 were analyzed (Table 1). To ask whether both genes are synchronously deregulated via epigenetic aberrations we determined DNA-methylation levels at ATM and TCL1A promoters using Methyl-CpG Immonuprecipitation (MCIp) followed by hybridization onto custom promoter arrays (position of oligonucleotides: Figure 1A). Within the ATM promoter no aberrant DNA-methylation was detected in CLL samples (n=13) compared to non-malignant B cells from age-matched healthy controls (n=6; Figure 1C). However, the TCL1A promoter displayed hypomethylation in CLL cells (Figure 1D) significantly correlating with TCL1A transcriptional upregulation (Figure 1F). These results are in agreement with previous reports13,14 and suggest that the small but significant ATM overexpression in CLL (Online Supplementary Figure S1 and references therein) is independent of promoter DNA-methylation in CLL cells that do not carry deletion of 11q22-q23 (Figure 1E). In contrast, TCL1A might partially be up-regulated in CLL cells by DNA hypomethylation (Figure 1F).
Given the significant coexpression of TCL1A and ATM we investigated whether both genes display similar expression kinetics when CLL cells are exposed to different stimuli in vitro, both by murine and human cells.15 Therefore we subjected primary CLL cells to different culturing conditions, and both TCL1A and ATM were synchronously downregulated in CLL cells upon culture (Figure 2 A and B). This is unlikely to be caused by miR34a/b localized in 11q and targeting TCL1A,12 as these miRs are not dysregulated upon coculture.17 While stimulation with nurse-like cells has been shown not to impact on expression of TCL1A,18 here expression levels of ATM and TCL1A consistently increased after 8 h of culture independent of the stimulus applied. Coexpression dynamics of both genes were validated in 6 CLL patient samples exposed to the different culture conditions at serial time-points using qRT-PCR (Figure 2C-F). Strikingly, we observed a highly similar expression pattern for ATM and TCL1A in all patient samples with exception of CLL#5 in HS5 coculture (Figure 2F). CLL#5 had no different clinical history or cytogenetic aberrations compared to the remaining patient cohort (Table 1).
TCL1A and ATM were among the 8 most significantly deregulated genes between stimulated and non-stimulated conditions (Idler, Bhattacharya et al., manuscript in preparation). This allowed us to compare their coregulation to the 6 remaining candidate genes using dynamic Bayesian net-works, ranking all expression profiled genes according to i) deregulation in the stimulated versus non-stimulated culture conditions and ii) coregulation upon different culture conditions (see supplementary materials and methods for detailed description of analysis). From the top 0.5% ranked candidates (n=243) all genes functionally associated with GO-terms “apoptosis”, “cell death” and “survival” in IPA (Ingenuity Systems, www.ingenuity.com; n=66 genes) and DAVID19 (n=53 genes) were selected. Thirty-five genes present in both databases were grouped via gene expression kinetics into 8 clusters using Partitioning Around Medoids (PAM) clustering to yield an appropriate number of genes for network modeling. Medoids that best represent kinetics of these clusters (HSPA1B, ZFP36, VHL,
ATM, NFKB1, INPP5D, FOS, ATM and TCL1A) were employed as nodes in a dynamic Bayesian network analysis (Figure 2G). Intriguingly, of all gene-interrelations deducted from expression kinetics, the connection of ATM and TCL1A was the strongest (Figure 2G, H). This suggested a coregulation of both genes in primary CLL cells when applying different stimuli. The Markov Chain Monte Carlo algorithms underlying the network estimation were initiated with either a random or an empty network, which yielded similar results (Figure 2H) and showed that the estimated networks are stable.
Towards understanding the coregulation of TCL1A and ATM in CLL, sets of transcription factors (TFs) bound at the gene bodies of the most deregulated genes HSAP1B, ZFP36, NFKB1, VHL, INPP5D, FOS, TCL1A and ATM were identified in publicly available data of chromatin immunoprecipitations from 95 different cell lines and culture conditions20 (visualization and analysis via the University of California Santa Cruz (UCSC) genome browser: genome.ucsc.edu). Most transcription factors were bound at the ATM gene (75 TFs), which was therefore used as reference. All genes showed a significant overlap in the bound TFs compared to ATM (on average 70%) but also carried specific TFs that were not detectable at the ATM gene (Figure 2 I, red portion). However, all of the TFs binding to TCL1A were also detectable at the ATM gene (Figure 2 I, right two columns). While this full overlap of the set of transcription factors is suggestive that TCL1A is the target of similar signaling pathways as ATM, the analyzed data are not specific to B or CLL cells and should be viewed with caution.
In line with a functional connection of TCL1A and ATM, a direct interaction of their gene products has recently been shown in CLL and linked to activated NF-κB signal-ing,9 a pathway centrally involved in CLL pathogenesis21 and apoptosis.22 We therefore tested whether knockdown of TCL1A causes apoptosis of primary CLL cells (n=5). In comparison to a non-target control siRNA and a siRNA targeting MCL123 used as positive control, knockdown of oncogenic TCL1A caused a significant reduction in CLL cell survival. This suggests a role of TCL1A in the resistance to apoptosis described for CLL cells23 (Figure 2J; for validation see Online Supplementary Figure S2).
In summary, ATM and TCL1A are significantly coexpressed and display similar kinetics when exposed to different external stimuli. This suggests that both genes are involved in similar cellular pathways. In line with previous reports that TCL1A knockdown reduces NF-κB activation,9 knockdown of TCL1A in primary CLL cells induced apoptosis. The joint deregulation of TCL1A and ATM might therefore contribute to the massive accumulation of malignant cells in CLL patients.
We would like to thank Martina Seiffert for helpful discussions, Clemens Philippen and Manuela Zucknick for statistical support and CLL patients for generous donation of primary material.
Funding: This work was funded by in part by the Deutsche Krebshilfe (109539), Virtual Helmholtz Institute VH-VI-404, Helmholtz Systems Biology Initiative “SBCancer” and the Deutsche Carreras Leukämie Stiftung (DJCLS-R11/01).
- ↵The online version of this article has a Supplementary Appendix.
- Authorship and Disclosures: Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.
- Received May 24, 2012.
- Accepted July 18, 2012.
- Zenz T, Mertens D, Kuppers R, Dohner H, Stilgenbauer S. From pathogenesis to treatment of chronic lymphocytic leukaemia. Nat Rev Cancer. 2010; 10(1):37-50. PubMedGoogle Scholar
- Ghia P, Caligaris-Cappio F. The indispensable role of microenvironment in the natural history of low-grade B-cell neoplasms. Adv Cancer Res.. 2000; 79:157-73. PubMedhttps://doi.org/10.1016/S0065-230X(00)79005-1Google Scholar
- Lee JH, Paull TT. Activation and regulation of ATM kinase activity in response to DNA double-strand breaks. Oncogene.. 2007; 26(56):7741-8. PubMedhttps://doi.org/10.1038/sj.onc.1210872Google Scholar
- Lavin MF. Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer. Nat Rev Mol Cell Biol.. 2008; 9(10):759-69. PubMedhttps://doi.org/10.1038/nrm2514Google Scholar
- Bichi R, Shinton SA, Martin ES, Koval A, Calin GA, Cesari R. Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proc Natl Acad Sci USA.. 2002; 99(10):6955-60. PubMedhttps://doi.org/10.1073/pnas.102181599Google Scholar
- Pekarsky Y, Santanam U, Cimmino A, Palamarchuk A, Efanov A, Maximov V. Tcl1 expression in chronic lymphocytic leukemia is regulated by miR-29 and miR-181. Cancer Res.. 2006; 66(24):11590-3. PubMedhttps://doi.org/10.1158/0008-5472.CAN-06-3613Google Scholar
- Pekarsky Y, Koval A, Hallas C, Bichi R, Tresini M, Malstrom S. Tcl1 enhances Akt kinase activity and mediates its nuclear translocation. Proc Natl Acad Sci USA.. 2000; 97(7):3028-33. PubMedhttps://doi.org/10.1073/pnas.97.7.3028Google Scholar
- Pekarsky Y, Palamarchuk A, Maximov V, Efanov A, Nazaryan N, Santanam U. Tcl1 functions as a transcriptional regulator and is directly involved in the pathogenesis of CLL. Proc Natl Acad Sci USA.. 2008; 105(50):19643-8. PubMedhttps://doi.org/10.1073/pnas.0810965105Google Scholar
- Gaudio E, Spizzo R, Paduano F, Luo Z, Efanov A, Palamarchuk A. Tcl1 inter-acts with Atm and enhances NF-κB activa-tion in hematologic malignancies. Blood. 2012; 119(1):180-7. PubMedhttps://doi.org/10.1182/blood-2011-08-374561Google Scholar
- Gebhard C, Schwarzfischer L, Pham TH, Schilling E, Klug M, Andreesen R. Genome-wide profiling of CpG methylation identifies novel targets of aberrant hypermethylation in myeloid leukemia. Cancer Res.. 2006; 66(12):6118-28. PubMedhttps://doi.org/10.1158/0008-5472.CAN-06-0376Google Scholar
- Herling M, Patel KA, Weit N, Lilienthal N, Hallek M, Keating MJ. High TCL1 levels are a marker of B-cell receptor pathway responsiveness and adverse outcome in chronic lymphocytic leukemia. Blood. 2009; 114(21):4675-86. PubMedhttps://doi.org/10.1182/blood-2009-03-208256Google Scholar
- Cardinaud B, Moreilhon C, Marcet B, Robbe-Sermesant K, LeBrigand K, Mari B. miR-34b/miR-34c: a regulator of TCL1 expression in 11q- chronic lymphocytic leukaemia?. Leukemia. 2009; 23(11):2174-7. PubMedhttps://doi.org/10.1038/leu.2009.125Google Scholar
- Mikeska T, Carney DA, Seymour J F,, Dobrovic A. No evidence for DNA methy-lation of the ATM promoter CpG island in chronic lymphocytic leukemia. Leuk Lymphoma.. 2012; 53(7):1420-2. PubMedhttps://doi.org/10.3109/10428194.2011.653640Google Scholar
- Yuille MR, Condie A, Stone EM, Wilsher J, Bradshaw PS, Brooks L. TCL1 is activated by chromosomal rearrangement or by hypomethylation. Gene Chromosome Canc.. 2001; 30(4):336-41. https://doi.org/10.1002/gcc.1099Google Scholar
- Kurtova AV, Balakrishnan K, Chen R, Ding W, Schnabl S, Quiroga MP. Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apop-tosis: development of a reliable and repro-ducible system to assess stromal cell adhe-sion-mediated drug resistance. Blood. 2009; 114(20):4441-50. PubMedhttps://doi.org/10.1182/blood-2009-07-233718Google Scholar
- Longo PG, Laurenti L, Gobessi S, Sica S, Leone G, Efremov DG. The Akt/Mcl-1 path-way plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells. Blood. 2008; 111(2):846-55. PubMedhttps://doi.org/10.1182/blood-2007-05-089037Google Scholar
- Sivina M, Hartmann E, Vasyutina E, Boucas JM, Breuer A, Keating MJ. Stromal cells modulate TCL1 expression, interacting AP-1 components and TCL1-targeting micro-RNAs in chronic lymphocytic leukemia. Leukemia. 2012; 26(8):1812-20. PubMedhttps://doi.org/10.1038/leu.2012.63Google Scholar
- Burger JA, Quiroga M P,, Hartmann E, Burkle A, Wierda WG, Keating MJ. High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation. Blood. 2009; 113(13):3050-8. PubMedhttps://doi.org/10.1182/blood-2008-07-170415Google Scholar
- Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Proto.. 2009; 4(1):44-57. Google Scholar
- Consortium E. A user's guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol.. 2011; 9(4):e1001046. PubMedhttps://doi.org/10.1371/journal.pbio.1001046Google Scholar
- Hewamana S, Alghazal S, Lin TT, Clement M, Jenkins C, Guzman ML. The NF-κB subunit Rel A is associated with in vitro survival and clinical disease progression in chronic lymphocytic leukemia and repre-sents a promising therapeutic target. Blood. 2008; 111(9):4681-9. PubMedhttps://doi.org/10.1182/blood-2007-11-125278Google Scholar
- Coscia M, Pantaleoni F, Riganti C, Vitale C, Rigoni M, Peola S. IGHV unmutated CLL B cells are more prone to spontaneous apoptosis and subject to environmental prosurvival signals than mutated CLL B cells. Leukemia. 2011; 25(5):828-37. PubMedhttps://doi.org/10.1038/leu.2011.12Google Scholar
- Caligaris-Cappio F, Hamblin TJ. B-cell chronic lymphocytic leukemia: a bird of a different feather. J Clin Oncol.. 1999; 17(1):399-408. PubMedGoogle Scholar