Mechanism for IL-15−Driven B Cell Chronic Lymphocytic Leukemia Cycling: Roles for AKT and STAT5 in Modulating Cyclin D2 and DNA Damage Response Proteins This information is current as of September 23, 2021. Rashmi Gupta, Wentian Li, Xiao J. Yan, Jacqueline Barrientos, Jonathan E. Kolitz, Steven L. Allen, Kanti Rai, Nicholas Chiorazzi and Patricia K. A. Mongini J Immunol 2019; 202:2924-2944; Prepublished online 15 April 2019; Downloaded from doi: 10.4049/jimmunol.1801142 http://www.jimmunol.org/content/202/10/2924 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/04/14/jimmunol.180114 Material 2.DCSupplemental References This article cites 125 articles, 55 of which you can access for free at: http://www.jimmunol.org/content/202/10/2924.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Mechanism for IL-15–Driven B Cell Chronic Lymphocytic Leukemia Cycling: Roles for AKT and STAT5 in Modulating Cyclin D2 and DNA Damage Response Proteins

Rashmi Gupta,* Wentian Li,* Xiao J. Yan,* Jacqueline Barrientos,† Jonathan E. Kolitz,*,†,‡ Steven L. Allen,*,†,‡ Kanti Rai,*,‡,x Nicholas Chiorazzi,*,‡,x and Patricia K. A. Mongini*,x

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell–associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood- derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15–driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed

human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that Downloaded from both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN- primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15–driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines http://www.jimmunol.org/ provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15–induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients. The Journal of Immunology, 2019, 202: 2924–2944.

cell chronic lymphocytic leukemia (B-CLL), a disease of This led to the early conjecture that B-CLL results from a gradual the elderly with a median age at diagnosis of 69 y, de- accumulation of clonal cells defective in apoptosis (2). More re- + B velops from a nonmalignant expansion of CD5 B cells cently, heightened research on B-CLL led to the recognition that a by guest on September 23, 2021 that is referred to as monoclonal B cell lymphocytosis. Approxi- sizeable component of each clone undergoes active cycling (3, 4). mately 1–2% of people with this precursor condition require treat- Moreover, the extent of in vivo cycling is linked to patient out- ment for CLL each subsequent year (1). As the elderly population come (5, 6), with the CLL clones bearing IGHV-unmutated Ag increases, B-CLL incidence will undoubtedly rise. The personal and receptors (U-CLL) typically exhibiting faster birth rates than economic costs of living with and treating this malignancy are in- CLL clones bearing IGHV-mutated Ag receptors (M-CLL) (5). centives for continued study into its etiology and unique mecha- Importantly, cycling occurs within lymphoid tissues with a stromal nisms for growth. environment conducive to B-CLL survival and growth (5, 7). The Unlike B cell acute lymphocytic leukemia, which manifests as fact that not all tissue-localized B-CLL cells are undergoing cycling rapidly cycling, bloodborne blasts, B-CLL generally reveals itself suggests that certain stimuli must be encountered for the growth as a slow rise in relatively quiescent CD5+ B cells within blood. response.

*The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY 11030; specimens used for this study and edited the paper. P.K.A.M. conceived the exper- †Department of Medicine, Northwell Health, Manhasset, NY 11030; ‡Department of iments, performed certain experiments, analyzed data, and wrote the paper. Medicine, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; and x Address correspondence and reprint requests to Dr. Patricia K. A. Mongini at the Department of Molecular Medicine, Zucker School of Medicine at Hofstra/Northwell, current address: Pathogen and Microbiome Institute, Room 156, P.O. Box 4073, Hempstead, NY 11549 Northern Arizona University, Flagstaff, AZ 86011. E-mail address: patricia. ORCIDs: 0000-0002-3717-5803 (X.J.Y.); 0000-0003-2700-4784 (J.E.K.); 0000- [email protected] 0002-3482-3182 (S.L.A.); 0000-0002-5804-2823 (K.R.); 0000-0003-1023- The online version of this article contains supplemental material. 6650 (N.C.); 0000-0002-4966-7741 (P.K.A.M.). Abbreviations used in this article: ATM, ataxia telangiectasia mutated; B-CLL, Received for publication August 17, 2018. Accepted for publication March 13, 2019. B cell chronic lymphocytic leukemia; 53BP1, p53-binding protein 1; ChIP-seq, This work was supported by research funds from Dr. Betty Diamond (The Center for chromatin immunoprecipitation sequencing; chr, chromosome; CLL, chronic lympho- Autoimmune, Musculoskeletal and Hematopoietic Diseases, The Feinstein Institute, cytic leukemia; Ct, threshold cycle; CTCF, CCCTC-binding factor; DDR, DNA Manhasset, NY), the National Cancer Institute, National Institutes of Health (NIH) damage response; F, forward; GAS, g-activated sequence; M-CLL, CLL clone (Grant CA08155C4 to N.C.), and the National Institute of Arthritis and Musculo- bearing IGHV-mutated Ag receptors; MDC1, mediator of DNA damage checkpoint skeletal and Skin Diseases, NIH (Grant AR061653 to P.K.A.M.). protein 1; MFI, median fluorescence intensity; ODN, oligodeoxynucleotide; PB, peripheral blood; Q-PCR, quantitative PCR; qRT-PCR, quantitative RT-PCR; R, R.G. performed most in vitro experiments, analyzed data, and edited the paper. W.L. reverse; TBP, TATA box–binding TF; TF, transcription factor; TP53BP1,tumor performed the bioinformatics analysis of ATM, MDC1,andTP53BP1 promoters. protein 53BP1; TSS, transcription start site; U-CLL, CLL clone bearing IGHV- X.J.Y. led the IGHV gene characterization of all B cell chronic lymphocytic leukemia unmutated Ag receptors. (B-CLL) clones. J.B. and J.E.K. provided B-CLL blood specimens and clinical information. S.L.A. and K.R. provided B-CLL blood specimens and clinical infor- Ó mation and edited the paper. N.C. led the acquisition and characterization of B-CLL Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801142 The Journal of Immunology 2925

CpG oligodeoxynucleotides (ODN) and IL-15 are two candidate cells did show that IL-15 enhanced the phosphorylation of Shc, stimuli that manifest notable synergy in driving the in vitro cycling ERK, JAK1, JAK3, and STAT5 but not STAT3 or STAT1 (29). of many, albeit not all, blood-derived B-CLL clones (8). Indeed, More recently, it was reported that IL-15 independently activated clonal potential for in vitro ODN + IL-15–driven growth was p38 and STAT5 in naive mouse peritoneal B-1a cells but not statistically linked to clinical outcome in patients with U-CLL (8). conventional B cells (31). Nevertheless, even M-CLL clones, which typically succumb to In this study, ODN-primed B-CLL cells with heightened IL-15R apoptosis following culture with ODN alone (9), show sustained expression are used to examine both early and late events following viability and often extended cycling (six to eight divisions) upon IL-15 signaling. We assess the involvement of PI3K/AKT and culture with both ODN and IL-15 (8). The recent documentation STAT5 pathways in IL-15–driven growth. Furthermore, we in- of IL-15–producing cells within B-CLL–infiltrated spleens (8) vestigate whether these receptor-proximal pathways influence and lymph nodes (10) and in proximity to pseudofollicles (8) mRNA levels for cyclin D2 (important for the G1 to S phase strengthens the possibility that IL-15 fosters B-CLL growth in transition) as well as mRNA/protein for several DNA damage patients. Similar to leukemic incidence, the frequency of IL-15+ response (DDR) molecules important in cell cycle checkpoint stromal cells rises with age (11, 12). Furthermore, CpG DNA is control and DNA repair: ataxia telangiectasia mutated (ATM), available in lymphoid tissues, as microbes drain into these sites, p53-binding protein 1 (53BP1), and mediator of DNA damage and stressed or apoptotic cells are locally produced (8). Indeed, checkpoint protein 1 (MDC1) (32, 33). Recently, IL-15 was the characteristic specificity of B-CLL Ag receptors for microbes reported to repress these DDR mediators in cycling normal CD8+ and stressed/apoptotic cells (13–15) should enhance B-CLL cell T cell clones (34). A pressing issue is whether this property ex- internalization of CpG DNA (16). These observations provide tends to B-CLL cell clones cycling in response to ODN + IL-15. Downloaded from ample reason to suspect that ODN + IL-15 synergy contributes Importantly, B-CLL evolution is linked to genetic anomalies that to B-CLL growth in patients, prompting us to investigate the compromise the DDR (35–37). If so, mechanisms that curtail mechanisms involved. DNA repair should significantly increase the likelihood that rep- Recently, we demonstrated that this synergy in part reflects a licating B-CLL cells survive DNA damage and ensuing mutations. 20-h ODN priming period, during which both IL-15 receptors,

IL-15Ra and CD122 (IL-2/15Rb), are significantly upregulated Materials and Methods http://www.jimmunol.org/ through pathways involving NF-kB (17). Subsequent CD122/gc Ethics statement signaling is critical for both IL-15–facilitated B-CLL cell cycle entry and continued cycling (17). In the current study, we focus on These studies were approved by the Institutional Review Board of Northwell Health (08-202A). Before blood collection, written informed illuminating the proximal and downstream effects of IL-15 en- consent from CLL-bearing patients was obtained in accordance with the gagement with these upregulated receptors on ODN-primed Declaration of Helsinki. B-CLL cells. Most prior insights into IL-15 signaling have come from CLL patient samples and characterization NK and CD8+ T cell studies (reviewed in Ref. 18). In the B-CLL specimens were obtained from patient peripheral blood (PB) before above lymphocytes, IL-15 engagement with the IL-2/15Rb treatment, excepting CLL-430 and CLL675 at 12 and 39 mo after treatment. by guest on September 23, 2021 (CD122)/ɣc signaling complex triggers the activation of cytokine A characterization of the B-CLL cohort employed in this study is shown in Table I. Additional clinical information and laboratory results on a fraction receptor-associated tyrosine kinases JAK1 and JAK3 and down- of this study’s cohort were detailed elsewhere (8). When used, the term stream activation of both STAT5 and PI3K/AKT pathways (18, 19). B-CLL clone connotes a CD19+/CD5+ B-CLL population expressing a Upon JAK phosphorylation, STAT5 transcription factors (TF) uniform IGHV sequence; it does not exclude the presence of intraclonal form dimers and undergo nuclear translocation. The evidence of variants with differing characteristics. + severe impairments in NK and CD8 T cell development in mice B-CLL cell isolation from patient blood and culture conditions with genetic deficiency of IL-15 or either STAT5 isoform STAT5A → Purified B-CLL cells were isolated from blood by negative selection using or STAT5B (18, 20) indicates the importance of this IL-15 RosetteSep Human B Cell Enrichment Cocktail (STEMCELL Technolo- STAT5 pathway. Within the cell nucleus, each STAT5 isoform gies, Vancouver, BC), as described earlier (8), stored frozen in the vapor binds a similar DNA core motif, TTC(T/C)N(G/A)GAA (20), phase of liquid N2, and upon defrosting, subjected to Ficoll-Hypaque often called a g-activated sequence (GAS) because of shared centrifugation for viable cell recovery. Data on the purity of such B-CLL similarity to the binding sites of IFN-g–activating STAT1 and B cell preparations were presented elsewhere (8, 17) as were details pertaining to culture conditions and stimuli: CpG DNA (ODN) and rhu– other STAT molecules (20). IL-15–induced activation of the PI3K/ IL-15 (8, 17). CFSE-labeled cells were employed to reveal the cycling AKT pathway is dependent upon recruitment of Shc to a JAK- history of gated viable and dead B-CLL as described (8). In most of the phosphorylated site on the CD122 cytoplasmic domain and sub- latter experiments, cultures were pulsed with a known number of fluo- sequent enlistment of adaptor proteins Grb2 and Gab2 (18, 21, 22). rescent standardizing beads immediately prior to harvesting to compute the absolute B-CLL yield per culture (8). Downstream PI3K/AKT-mediated activation of mTOR is important for T/NK cell enlargement, metabolism, and proliferation (23, 24), Modulating reagents and linkages between activated PI3K/AKT and sustained STAT5 Neutralizing anti-human CD122 (IL2/15Rb) (mouse IgG1 clone TU27; signaling appear to drive IL-15–dependent viability and growth of BioLegend) was used at a final concentration of 10 mg/ml. LY294002 CD8+ T cells (25). Studies primarily performed with IL-2, which (Selleckchem), an inhibitor of PI3K p110a, b, and ɣ isoforms (38) also uses the IL-2/15Rb(CD122)/gc complex for signaling, revealed (Selleckchem), was used at a final dose of 20 mM. STAT5 inhibitor, that downstream events in T cells include diminished expression of pimozide (also known as STAT Inhibitor III) (Calbiochem), and STAT5 Inhibitor II (CAS 285986-31-4) (Calbiochem or Cayman Chemical) were proapoptotic molecules and augmented expression of anti-apoptotic tested at doses near their reported IC50 values: pimozide (5 mM) (39) and proteins, cyclins, and molecules of the mTOR pathway (19, 26). STAT5 Inhibitor II (47 mM) (40). Pimozide blocks STAT5 phosphorylation Although IL-15 strongly potentiates the in vitro growth of both through an undefined mechanism (39); STAT5 Inhibitor II suppresses normal B cells (27, 28) and B-CLL cells (8, 29, 30) receiving STAT5 signaling via specific binding to the STAT5 SH2 domain, precluding dimerization (40). In inhibition studies, aliquots of the above molecules in activation signals from CD40, BCR, or TLR9 ligands, few studies DMSO vehicle (or vehicle alone) were pulsed into ODN-primed cultures have investigated the mechanisms involved, as discussed recently 15 min prior to the IL-15 pulse. JAK1/2 inhibitor ruxolitinib (INCB018424; (17). Nonetheless, a past study involving CD40-activated B-CLL IC50 = 3 nM) (41) and JAK3 . JAK1 . JAK2 inhibitor tofacitinib citrate 2926 MECHANISM FOR CpG DNA AND IL-15 SYNERGY IN B-CLL GROWTH

(CP-690550) (Selleckchem) with IC50 of 1, 20, and 112 nM, respectively no. NM_002467.4) was used with the following primers: F = 59-GCTGCTTA- (41), were used at 50 nM final concentration. All pharmacologic inhibitors GACGCTGGATTT-39 and R = 59-TAACGTTGAGGGGCATCG-39. were reconstituted in DMSO vehicle and stored at 280˚C in stock aliquots prior to use. Western blotting assessments of ATM, 53BP1, and MDC1 protein Immunofluorescent assays for phosphorylated AKT, STAT5, Lysates from B-CLL cells prior to and following 4 d of culture with ODN 6 STAT3, and STAT1 IL-15 were generated with RIPA lysis buffer containing EDTA + protease/ 2 Levels of activated AKT and the above STAT family members were phosphatase inhibitors (Roche Applied Bioscience) and stored at 80˚C as measured by intracellular staining with specific fluorochrome-conjugated reported (42). Using 3–8% Tris acetate gradient gels, equivalent lysate Ser473 m mAbs: 1) AF 647 rabbit anti-pAKT mAb (catalog no. 2337; Cell protein amounts (20 or 30 g) and high m.w. standards were separated and subsequently transferred to PDVF membranes. For detection of ATM, Signaling Technology) with rabbit IgG control (each at 0.05 mg/ml) or Ser473 53BP1, MDC1, and loading control b-actin protein, blots were sequen- alternatively AF 647– or AF 488–conjugated mouse anti-pAKT (BD Biosciences) with mouse IgG1 control (Santa Cruz Biotechnology, Dallas, tially exposed (with intervening stripping) to Abs specific for ATM (mouse TX) (each at 1.5 mg/ml). 2) AF 647 mouse anti-STAT5Y694/699 (mAb clone anti-ATM mAb [clone 2C1; GeneTex, Irvine, CA]); 53BP1 (rabbit anti- 47; BD Biosciences) with mouse IgG1 control (Santa Cruz Biotechnology) 53BP1 polyclonal IgG [NB100-304; Novus Biologicals; Littleton, CO]); (3 mg/ml). Of note, nonreceptor tyrosine JAKs activate STAT5A and MDC1 (mouse anti-MDC1 mAb, which recognizes the N terminus of b STAT5B by phosphorylation at critical Y694 and Y699 residues, respec- MDC1 (clone P2B11; LSBio, Seattle, WA; MilliporeSigma), and -actin (anti-actin mAb [Novus Biologicals]). HRP goat anti-mouse IgG (human tively; both are recognized by mAb clone 47 and cannot be distinguished Ig adsorbed) (SouthernBiotech, Birmingham, AL) or HRP-conjugated anti- in this study. For brevity, we will refer to positive binding as representing pSTAT5Y694. 3) PE mouse anti-pSTAT3Y705 mAb or PE isotype control rabbit IgG (Cell Signaling Technology, Danvers, MA) and SuperSignal (BD Biosciences) and 4) AF 488 mouse anti-pSTAT1Y701 or AF 488 Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA) were used for detection. For quantifying ATM, 53BP1, and MDC1 isotype control (BD Biosciences). Downloaded from In early experiments, cytoplasmic pAKTSer473 was assessed in the fol- levels, densitometry assessments were made of exposed films, and values were normalized on the basis of b-actin loading control. lowing manner: harvested cells (precultured with medium or ODN) were suspended in RPMI 1640 medium without additives and allowed to sta- Flow cytometric measurements of pSTAT5, ATM, MDC1, and bilize at 37˚C for 15 min. Subsequently, cells were exposed to IL-15 53BP1 protein in cycling B-CLL (15 ng/ml) for 30 min prior to being washed, fixed for 10 min (37˚C) with 2% electron microscopy–grade formaldehyde and phosphatase/ CFSE-labeled B-CLL, stimulated for 5–6 d with ODN 6 IL-15, were protease inhibitors, permeabilized with Phosflow Perm/Wash Buffer (BD harvested, treated with fixable V450–Pacific Blue viability stain, fixed, and http://www.jimmunol.org/ Biosciences), exposed to AF 647–labeled mAb, refixed with 2% parafor- permeabilized to permit both cytoplasmic and nuclear staining as de- maldehyde, and analyzed for fluorescence by flow cytometry of viability- scribed above. Assessments of pSTAT5 involved fluorochrome-conjugated gated cells. In later measurements of cytoplasmic and nuclear levels of primary mAb as delineated above. Two-stage staining assays were used to both pAKT, pSTAT5, pSTAT3, and pSTAT1, cells were pretreated with monitor levels of total ATM, MDC1, and 53BP1. For ATM and MDC1, fixable V450–Pacific Blue viability stain, fixed with formaldehyde as primary mAbs were validated anti-ATM mAb (clone 2C1; Genetex) and above, and permeabilized with ice-cold methanol (30 min on ice) prior to anti-MDC1 (clone P2B11; LSBio, Seattle, WA) or IgG1 control (MOPC- washing in Phosflow Perm/Wash Buffer and staining as above. For time- 21; BD Pharmingen): second stage detection Ab: RPE-conjugated goat course experiments, fixed cells were maintained in the perm/wash buffer F(ab9)2 anti-mouse IgG (H+L), human Ig adsorbed (Southern Biotech). prior to simultaneous staining of all B-CLL specimens. Flow cytometric For 53BP1, primary affinity-purified rabbit anti-53BP1 polyclonal IgG analysis employed either Fortessa or LSR II flow cytometers (BD Biosciences) (NB100-304; Novus Biologicals) or rabbit IgG control (Santa Cruz Bio- and FlowJo data analysis. technology) was followed by secondary PE-labeled goat F(ab9)2 anti-rabbit by guest on September 23, 2021 IgG (H+L), human/mouse Ig adsorbed (Southern Biotech). Because cul- Quantitative RT-PCR of ATM, TP53BP1, and MDC1 mRNA tures stimulated with ODN versus ODN + IL-15 have significantly dif- from B-CLL ferent yields after 5–6 d of culture, cell counts were made after harvesting, and equivalent B-CLL numbers were used for staining. Total mRNA was isolated from washed B-CLL cells with RNeasy (Qiagen) and cDNA prepared with oligo(dT) primers as described (42). Specific Bioinformatics analysis of the promoter regions for ATM, cDNA was amplified in triplicate assays with Eurogentec MasterMix 53BP1, and MDC1 (AnaSpec, Fremont, CA), 2.5 mM specific probe (from human Universal ProbeLibrary of Roche Applied Science [Indianapolis, IN]), and intron- Data providing insights into occupied STAT binding sites in several human spanning, optimized forward (F) and reverse (R) primers (each at 10 mM) cell lines (http://genome.ucsc.edu/ENCODE/cellTypes.html) were derived (ProbeFinder version 2.50 for human [Roche Diagnostics]) using TaqMan from Genome Browser images of “TF ChIP-seq (161 TF) from ENCODE Q-PCR (Applied Biosystems, Foster City, CA) with parameters as recently 2012 with Factorbook Motifs” track. The available ENCODE chromatin reported (42). Probe accession numbers and primer sequences were ob- immunoprecipitation sequencing (ChIP-seq) data are a track in the Uni- versity of California Santa Cruz’s Genome Browser (http://genome.ucsc. tained from the RefSeq database (https://www.ncbi.nlm.nih.gov/refseq/). . . Amplification was extended to 45 cycles to reveal the plateau of maximal edu/) under the title “Regulation ENCODE regulation Txn Factor substrate use; threshold cycle (Ct) values for data analysis represented Chip” for the reference genome version GRCh37/hg19. Four STAT TFs are thresholds within the linear region (routinely between 20 and 36). Fold covered in the ENCODE ChIP-seq data: STAT1 (for cell lines GM12878, change was calculated by comparing ΔCt of treated versus ΔCt of un- HeLa-S3, K562), STAT2 (for K562), STAT3 (for GM12878, HeLa-S3, treated groups; analysis by RQ Manager 1.2 (Applied Biosystems) MCF 10A), and STAT5A (for GM12878, K562). We also investigated with endogenous control for calculation of ΔCt as GAPDH (Universal the binding data for TATA box–binding TF (TBP) (for cell lines GM12878, ProbeLibrary probe 60; accession no. NM_002046.3) with the following H1-hESC, HeLa-S3, HepG2, K562) and CCCTC-binding factor (CTCF) primers: F = 59-AGCCACATCGCTCAGACAC-39 and R = 59- insulator (for most of the cell lines). GCCCAATACGACCAAATCC-39. For quantitative PCR (Q-PCR) of ATM Monitored for ATM was the region on chromosome (chr)11: cDNA, probe 55 (accession no. NM_000051.3) was employed together 108093559-108239826, including a transcription start site (TSS) from the with the following primers: F = 59-TTTCTTACAGTAATTGGAGC- GM12878 B-lymphoblastoid cell line and the K562 cell line. ATTTTG-39 and R = 59-GGCAATTTACTAGGGCCATTC-39 (Synthe- Monitored for TP53BP1 were two regions: 1) chr15: 43699412-43785354, sized by Eurofins mwg\operon). For Q-PCR of mediator of MDC1 cDNA, representing two transcripts (variants/isoforms 1 and 2) found in the HeLa-S3 probe 76 (accession no. NM_014641.2) was employed, together with the cervical tumor cell line, and 2) chr15: 43699412-43802707 (3.7 kb from TSS), following primers: F = 59-GCAGCTTCCAGACAACAGGT-39 and R = 59- associated with an alternative transcript (variant/isoform 3) found both in K562 GTGTCAAAAGGCTGGGTCTC-39, which correspond to sequences be- [line from chronic myelogenous leukemia with some T cell properties (43)] ginning at 2204 and 2290 nts from the MDC1 transcriptional start site, and in MCF10A-Er-Src mammary gland line. Monitored for MDC1 was the respectively. For Q-PCR of tumor protein 53BP1 (TP53BP1) probe 24 region on chr6: 30667583-30685458, including a TSS, in the HeLa-S3, (accession no. NM_001141980.1) was used with the following primers: MCF10A Er, K562, and GM12878 cell lines. F=59-GGACAGAACCCGCAGATTT-39 and R = 59-CCTGTCTGACT- Statistical analyses GACCCCTTT-39. For cyclin D2, probe 49 (accession no. NM_001759.3) was used with the following primers: F = 59-GGACATCCAACCCTACATGC-39 and Tests used for determining statistical significance are indicated in figure R=59-CGCACTTCTGTTCCTCACAG-39. For MYC, probe 66 (accession legends. Typically, a two-sided t test was employed for data with a normal The Journal of Immunology 2927 distribution. If comparisons involved absolute values from pairs of one or more time points during the 20-h period after IL-15, untreated/treated cultures with the same B-CLL, a paired test was used. If whereas only one of nine of the parallel unprimed cultures (left) groups of B-CLL were different, the test was unpaired. Unpaired t test was did so. Box plots of pooled data in Fig. 1C represent 60-min also employed when data were normalized by providing a value of 1 to cultures not receiving the treatment in question. In cases when the data IL-15–boosted pAKT levels in primed versus unprimed B-CLL distribution did not pass the Shapiro–Wilk normality test, the nonpara- clones as fold increase above respective levels without IL-15. metric Mann–Whitney rank sum test or the Wilcoxon signed rank test was ODN priming had a statistically significant effect at facilitating , used. Statistical significance was determined when p 0.05; determina- IL-15–induced pAKT (p = 0.04). Thus, IL-15 signals provide a tions were made with either Sigma-Plot 13 or Excel. way for B-CLL cells to sustain AKT function following transient Results activation of this pathway by CpG DNA (44). IL-15 promotes AKT phosphorylation in B-CLL cells primed by IL-15 promotes STAT5 activation in ODN-primed B-CLL CpG DNA (ODN) Fig. 2A presents fluorescence histograms of pSTAT5Y694/699 levels We sought evidence that IL-15 could activate the AKT pathway by within unprimed and ODN-primed M-CLL1031 and U-CLL1158 using flow cytometry to examine intracellular pAKTSer473 levels cells, following a 60-min pulse with IL-15 or medium. Although within B-CLL cells (Table I) precultured with medium (unprimed) baseline STAT5 activation was evidenced in unprimed B-CLL or ODN for 20 h (primed). Fig. 1A shows representative fluo- (pSTAT5 fluorescence above IgG control), pSTAT5 levels were rescence histograms of two such clones, U-CLL996 and greatest in ODN-primed B-CLL exposed to IL-15. Summarized U-CLL1148, 6 a 30(60)-min IL-15 pulse. In both primed B-CLL results from time-course analyses in Fig. 2B show that pSTAT5

cells, IL-15 prompted a rise in activated ATK, whereas unprimed is consistently elevated in all ODN-primed B-CLL following Downloaded from B-CLL cells were less affected by IL-15. Consistent with the 20–40 h of IL-15 exposure, albeit not all clones manifest elevated transient nature of AKT activation following ODN exposure to pSTAT5 at 60 min postcytokine. Unlike for AKT phosphorylation B-CLL cells (44), we found that pAKT levels in ODN-primed (Fig. 1B), there was no indication that STAT5 is activated more B-CLL with no cytokine exposure were no greater than levels in rapidly in U-CLL versus M-CLL clones (Fig. 2B). Box plots of unstimulated B-CLL (Fig. 1A). Plots of the time course of IL-15– pooled data from these time-course experiments (Fig. 2C) show induced AKT activation in a larger cohort of B-CLL clones that IL-15 elicits significantly greater STAT5 activation within ODN- http://www.jimmunol.org/ (Fig. 1B) indicate that eight of nine ODN-primed B-CLL cultures primed versus unprimed B-CLL cells (p =0.008andp = 0.006, at (right) manifest an IL-15–induced boost in pAKT expression at 20 and 40 h post–IL-15, respectively).

Table I. Characterization of B-CLL clones used for culture

% Positived CLL Age RAI WBC Count 3 103/ml Clonea (y)b Sex Stagec IGHV M IGHV Gene (% Lymphocytes) FISH CD25 CD38 by guest on September 23, 2021 275 78 Female 0 M 3-30*03 31 (78) 21% del13q ND ND 321 60 Male 1 U 4-34 104 (77) 53% Tri-12 14 9 348 58 Female 1 M 3-7 184 (93) ND 47 6 430 68 Male 2 U 1-69*01 288 (85) 97% del13q; 10% del11q; ATM M 99 5 515 48 Male 2 U 4-39 224 (89) NEG ND 59 618 52 Female 3 M 3-72 and 3-74 14 (70) 10% del13q; 43% Tri-12 ND ND 675 65 Female 0 U 3-23*01 11 (61) 80% del13q; 19% del11q 91 45 693 65 Female 0 M 3-7 224 (87) NEG 95 2 770 66 Female 1 U 3-15*01 113 (79) 89% del13q; 3 ATM M 100 9 791 65 Female 0 U 3-21*01 242 (89) 76% del13q ND 14 992 62 Male 0 M 3-53*02 6 (17) 30% del13q ND 44 996 84 Male 0 U 1-69 178 (87) NEG 49 46 1031 73 Female 1 M 4-39 225 (91) NEG 2 3 1058 63 Female 1 U 3-11 and 4-34 131 (89) 97% del13q ND 13 1158 69 Female 1 U 3-15*01 247 (88) 97% del13q; 48% del11q 18 15 1239 67 Female 0 U 3-30*03 159 (90) NEG 95 7 1328 53 Male 0 M 4-61*01 23 (77) 85% del13q ND 3 1444 72 Female 0 U 4-31*03 84 (76) 80% del13q ND 4 1306 46 Female 1 U 4-39*01 47 (75) NEG ND 64 1692 56 Male 1 U 2-70*01 153 (88) 53% Tri-12 ND 27 1953 54 Male 1 U 3-30*03 82 (89) ND ND ND 1961 61 Male 2 U 3-15*07 450 (92) ND ND ND 1993 52 Male ND U 3-11*01 72 (83) ND ND ND 2018 68 Male 0 M 3-84*01 85 (73) 61% del13q; 61% Tri-12 ND ND 2166 73 Female 0 M 3-7 53 (75) 52% Tri-12 78 0 2245 59 Female 3 U 1-69*04 374 (77) NEG ND 0 2255 60 Male 1 M 3-33 191 (92) 92% del13q ND 0 2258 31 Male 2 U 3-33 97 (77) 60% del11q ND ND 2277 64 Female 0 U 1-69 48 (83) ND ND ND 2278 92 Female ND M 3-23 85 (87) 58% Tri-12 ND 0 aB-CLL clone is used to connote a CD19+/CD5+ B-CLL population expressing a uniform IGHV sequence; it does not exclude the presence of subclones. Most of the clonal populations were taken from patients prior to therapy; for the two exceptions, CLL430 and CLL675, PB samples were obtained from patients 12 and 39 mo after last therapy. bNEG. cRAI clinical stage at the time of test sample acquisition. dFraction of CD19+ cells expressing CD25 (IL-2Ra) or CD38. del, deletion; FISH, fluorescence in situ hybridization; M, mutated; NEG, negative; Tri-12, trisomy-12; U, unmutated. 2928 MECHANISM FOR CpG DNA AND IL-15 SYNERGY IN B-CLL GROWTH Downloaded from http://www.jimmunol.org/

FIGURE 1. IL-15 augments AKT activation in ODN-primed B-CLL. B-CLL cells were precultured for 20 h in medium 6 ODN prior to adding IL-15. After varying intervals, pAKTSer473 levels were monitored by immunofluorescent staining and flow cytometry. (A) Fluorescence histograms of cytoplasmic pAKT in viable-gated U-CLL996 cells at 30 min post–IL-15 (left column) or cytoplasmic + nuclear levels of pAKT in U-CLL1158 cells at 60 min post–IL- 15 (right column) (see Materials and Methods for procedural differences). Inserted values represent ratio of MFI (RMFI) in anti-pAKT–stained versus isotype control–stained cells). (B) Fold increase in pAKT fluorescence (ratio: +IL-15/no IL-15) within medium-precultured cells (left) and ODN-primed cells (right) at varying intervals (15–1200 min) post–IL-15. Center legend shows individual B-CLL tested (U-CLL = open symbols; M-CLL = closed symbols). (C) Bar graphs summarizing fold increase in pAKT levels in ODN-primed or unprimed B-CLL at varying intervals after IL-15. Statistical by guest on September 23, 2021 significance determined by two-sided, paired t test. Note: Values for baseline pAKT fluorescence in medium- and 20-h ODN-primed B-CLL with no IL-15 exposure were 259 6 131 and 266 6 126 (mean 6 SD); not significantly different by statistical analysis.

Consistent with the roles of CD122 (IL-2/15Rb) in mediat- was not uniformly observed. Together, the collective data within ing STAT5 activation within T/NK cells (19) and in promoting Fig. 2 show that IL-15 → CD122 signaling in ODN-primed IL-15–driven growth of ODN-primed B-CLL (17), we noted that B-CLL cells sustains STAT5 pathway activation throughout IL-15–induced STAT5 phosphorylation in B-CLL was blocked by clonal expansion. a neutralizing anti-CD122 mAb that abrogated IL-15–driven B-CLL growth (17) (Supplemental Fig. 1). Comparative tyrosine phosphorylation of STAT1, STAT3, and Levels of activated STAT5 remain elevated within cycling STAT5 in B-CLL clones with differing prior exposure to ODN B-CLL cells and IL-15 Chronic IL-15/CD122/ɣc signaling is necessary for sustained Because members of the STAT family of TFs can interact and B-CLL clonal expansion within ODN + IL-15–stimulated cultures cross-regulate one another (45–47), better insight into ODN and (17). The former, together with evidence that pSTAT5 levels IL-15 synergy might emerge from assessing their relative activa- remain elevated at 40 h post–IL-15 (Fig. 2C), prompted us to tion in unprimed and ODN-primed B-CLL 6 IL-15. Past reports investigate whether pSTAT5 levels remain high during ODN + indicated that B-CLL (but not normal B cells) show constitu- IL-15–driven cycling of CFSE-labeled B-CLL clones. Two-color tive serine (S727) phosphorylation of both STAT1 and STAT3 flow cytometric analysis of day 6 cultures revealed that not only (a modification that can influence TF activity) (reviewed in Ref. are pSTAT5 levels sustained, but in some clones, they are further 48), but tyrosine phosphorylation at residues Y701 (STAT1) and elevated during cycling (Fig. 2D, 2E). Data in Fig. 2E represent Y705 (STAT3) is negligible (49–51). The latter sites are of interest pSTAT5 expression of several ODN + IL-15–activated clones, because each can affect dimerization, nuclear translocation, and each normalized on the basis of levels in the major undivided TF activity (52, 53). Consequently, a cohort of six B-CLL (three fraction of parallel ODN-only cultures. This revealed that within U-CLL and three M-CLL) was examined for baseline and induced ODN + IL-15–stimulated B-CLL cultures, both the undivided and tyrosine phosphorylation of STAT1 and STAT3, as well as STAT5, divided fractions exhibit statistically heightened levels of pSTAT5 under these varying conditions (Fig. 3A–C). (p = 0.02 and p = 0.03 for undivided/divided cells, respectively). STAT1Y701. In agreement with a past B-CLL study (50), 20-h ODN The divided fraction of certain B-CLL clones (e.g., U-CLL1953 exposure triggered STAT1Y701 phosphorylation in some clones in Fig. 2D and M-2018 in Supplemental Fig. 2) manifest greater (e.g., U-2277) above the negligible levels in nonstimulated cells. pSTAT5 than the undivided fraction of the same culture, but this The low frequency of ODN-triggered clones with upregulated The Journal of Immunology 2929 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. IL-15 augments STAT5 activation in ODN-primed B-CLL. STAT5 activation was monitored by intracellular staining for pSTAT5Y694/699 and subsequent flow cytometry. (A) Histograms representing pSTAT5 levels in M-CLL1013 and U-CLL1158 cells treated as indicated (RMFI values inserted). (B) Fold increase in pSTAT5 levels (ratio: +IL-15/no IL-15) within medium-precultured cells (left) and ODN-primed cells (right) at varying intervals (1, 20, and 40 h) post–IL-15. Center legend shows individual B-CLL tested. (C) Box plots summarizing fold increase in pSTAT5 at varying intervals after IL-15 addition to medium-only cohorts (white bars) and parallel ODN-primed cohorts (gray bars). Statistically significant differences were reached by two-sided, paired t test. (D) Two-color flow cytometry of CFSE-labeled U-CLL1953 cells stained for pSTAT5 after 6 d of stimulation as indicated. Top-row dot plots show gating for viable cells. Bottom-row two-color dot plots of specific pSTAT5 staining (dark gray) or isotype-control staining (light gray) in viable cells of varying division status. Inserted values represent the proportion of divided cells. (E) Box plots of pooled data comparing pSTAT5 levels in viable-gated and division-gated B-CLL cells from 5- to 6-d cultures stimulated by ODN alone or by ODN + IL-15. pSTAT5 levels were normalized in each experiment on the basis of levels in undivided cells of ODN-only cultures. (The former gate was used for all comparisons because certain B-CLL clones yielded insufficient viable cells in the divided fraction of ODN-only cultures.) Box plots represent pooled data from seven CLL clones: U-770, U-791, U-1953, M-827, M-1013, M-1993, and M-2018. Figure presents a more comprehensive analysis of pSTAT5 expression in undivided/divided cells from the seven individual B-CLL clones. pSTAT1Y701 likely represents interclonal diversity in kinetics of STAT3Y705. Although STAT3Y705 phosphorylation was low in the response. In the past study, some clones failed to show ele- unstimulated B-CLL, levels uniformly rose in all B-CLL clones vated pSTAT1Y701 at 24 h after ODN exposure, but all did so following 20-h ODN exposure. The increase was quite notable in after 72 h (Fig. 3A). 4/6 B-CLL and less prominent in 2/6 clones (borderline p = 0.05 2930 MECHANISM FOR CpG DNA AND IL-15 SYNERGY IN B-CLL GROWTH

FIGURE 3. Priming of blood B-CLL cells with ODN for 20 h results in upregulated pSTAT3Y705. We further examined whether ODN priming and/or later IL-15 exposure influences STAT1 and STAT3 tyrosine phosphorylation at residues Y701 and Y705, respectively. Intracellular levels of (A) pSTAT1Y701 and (B) pSTAT3Y705 as well as (C) pSTAT5Y694 were monitored by immunofluorescence staining and flow cytometry of viable-gated cells in a new cohort of six B-CLL clones. Expression was assessed in both ODN-primed or unprimed cultures 6 subsequent 60-min exposure to IL-15. Statistical analyses were performed with a paired, two-sided t test when pooled data passed normality tests. In cases of nonparametric data, the Wilcoxon signed rank test was employed. The latter applied to pSTAT1: untreated versus ODN only; ODN only versus ODN + IL-15, and pSTAT3: untreated versus IL-15 only. Downloaded from statistical significance in two-sided, paired t test). Importantly, 4.2- to 6.6-fold above levels in respective nonsupplemented cultures. IL-15 did not further modulate pSTAT3Y705 levels at 15 min The IL-15–triggered CCND2 mRNA is consistent with past findings (Fig. 3B) or at 20 h after its pulse (data not shown). ODN-induced that IL-2 upregulates cyclin D2 protein in ODN-activated B-CLL upregulation of pSTAT3Y705 in B-CLL cells resembles that reported cells (62).

in normal B lymphocytes (54). Regarding MYC mRNA, IL-15–pulsed cultures showed a minor http://www.jimmunol.org/ STAT5Y694. Consistent with Fig. 2 data, experiments with this transient boost in this mRNA species at 4 h postexposure in 3 of 5 additional B-CLL cohort showed that low-level activation of clones tested (fold increase of 1.1, 1.7, and 2.4). This increase STAT5 (pSTAT5Y694) is manifest in both untreated and ODN- was statistically significant when DCt values were assessed primed B-CLL and that levels of activated STAT5 in ODN- (Supplemental Fig. 3) but not so when fold-increase values primed cells rise significantly upon IL-15 exposure (p = 0.02). were evaluated (Fig. 4A). Of note, the three B-CLL populations In this cohort, although some unprimed clones responded to IL-15 manifesting an early IL-15–driven rise in MYC mRNA all divided with elevated pSTAT5Y694, the increase was not statistically sig- extensively in response to ODN + IL-15 (data not shown). Of the nificant for the pool (Fig. 3C). 2/5 B-CLL clones showing no IL-15–induced elevation in MYC Thus, the present findings 1) that ODN priming triggers tyrosine mRNA, one responded poorly to ODN + IL-15, whereas the other by guest on September 23, 2021 phosphorylation of STAT1 and STAT3, but not STAT5, at func- mounted a vigorous proliferative response (data not shown). Thus, tionally relevant residues and 2) that IL-15 signals activate STAT5, within the clones tested, there was no absolute correlation between but have no notable effect on STAT1 and STAT3, suggests the IL-15–boosted MYC mRNA and B-CLL growth. possibility that dynamic relationships between these STATS (and In contrast to these minor effects of IL-15 on MYC mRNA levels their downstream pathways) contribute to ODN and IL-15 synergy within ODN-primed B-CLL, MYC expression is notably elevated (8, 10). Although these interactions were not actively investigated by IL-15/STAT5 signaling in T/NK cells (58, 59). A possible in this study, supplementary data to be presented later provide reason is that ODN priming itself heightens MYC transcription. some support. In fact, ODN elevates MYC message in normal B cells (63), and in B-CLL, ODN is a strong activator of NF-kB (17, 44) that influ- IL-15 signaling in ODN-primed B-CLL cells rapidly increases ences MYC transcription in other cells (64). Our study has not CCND2 mRNA encoding cyclin D2, with minimal effects on excluded the possibility that cMyc protein in B-CLL is boosted MYC mRNA by IL-15. In T cells, IL-2 → CD122/ɣc signaling is reported to

Cyclin D2 is important in G1 progression, essential for B-1 cell elevate cMyc protein via a posttranscriptional mechanism (65). development (55), elevated in blood-derived B-CLL (56), and IL-2/15Rb-inducible in T cells (57). Furthermore, the c-Myc TF is IL-15 signaling dampens mRNA for several mediators of upregulated following IL-15/CD122 signaling in T/NK cells (58, 59) the DDR and is overexpressed within B-CLL proliferation centers of patient Samples of total mRNA from the above (Fig. 4A) and additional lymphatic tissue (60). B-CLL clones were examined for IL-15–induced changes in These features prompted us to examine whether IL-15 signaling ATM, TP53BP1, and MDC1 messages by qRT-PCR. Fig. 4B augments CCND2 and MYC transcripts within ODN-primed B-CLL presents the calculated fold-change values for IL-15–altered ex- cells. Total mRNA was isolated from primed cultures at varying pression of ATM, TP53BP1, and MDC1 mRNA, and Supplemental intervals following IL-15 as well as from parallel cultures without Fig. 3 shows the DCt values from which these calculations were IL-15, and specific mRNA was measured by quantitative RT-PCR made. Importantly, IL-15 prompted a statistically significant de- (qRT-PCR). Supplemental Fig. 3 presents DCt values from indi- cline in ATM, TP53BP1, and MDC1 mRNA from 9 to 16 h fol- vidual B-CLL experiments obtained by comparing CCND2 to lowing its introduction. The decline in TP53BP1 and ATM GAPDH reference mRNA. Fig. 4A shows pooled data for fold- mRNA was sustained for at least 20–24 h following IL-15 increase values derived from the former by the 2^(2ΔΔCt) exposure, but the drop in MDC1 appeared more transient. In method (61). A pulse of IL-15 into ODN-primed cultures additional experiments with B-CLL clones U-515 and U-430, prompted a significant rise in CCND2 mRNA at 20–24 h fol- mRNA levels of these DDR genes were assessed both prior to lowing the cytokine; this began at ∼4 h and by 20–24 h ranged from culture (t = 0 h) and after 4 d of stimulation with ODN 6 IL-15 The Journal of Immunology 2931 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. In ODN-primed B-CLL, IL-15 signaling augments mRNA for cyclin D2 and represses mRNA for DDR proteins ATM, 53BP1, and MDC1. Total mRNA was isolated from B-CLL cells that received 20-h ODN priming and were subsequently pulsed with IL-15 or medium for varying intervals. qRT-PCR with specific primers was performed as detailed in Materials and Methods. IL-15–induced changes in specific mRNA are shown on bar graph ordinates as fold increase (ratio of mRNA within ODN + IL-15–treated versus ODN only–treated B-CLL) as calculated from ΔCt values by the 2^(2ΔΔCt) method (61). p values for statistical significance between IL-15–pulsed versus –nonpulsed cultures were determined by two-sided, paired t test. (A) IL-15 influence on MYC and CCND2 mRNA. A 4-h pulse = mean of five clones tested (CLL 693, 849, 1031, 1692, 1953); 9–16-h = mean of three clones tested (CLL 693, 887, 1953); 20–24-h = mean of five clones (693, 849, 1031, 1692, 1953). (B) IL-15 influence on ATM, P53BP1, and MDC1 mRNA. Levels were significantly reduced upon IL-15 exposure (p , 0.001 to 0.003), albeit the suppressive effect on MDC1 mRNA appeared to be transient. A 4-h pulse = mean of six clones tested (CLL 693, 849, 887, 1031, 1692, 1953); 9–16-h = mean of three clones tested (CLL 693, 887, 1953); 20–24-h = mean of seven clones tested (CLL 693, 849, 887, 1031, 1692, 1993, 2018). (C) Levels of ATM, TP53BP1, and MDC1 mRNA within U-CLL430 and U-CLL515 cells examined in unstimulated state (t = 0) or after 4 d of culture with ODN alone or ODN + IL-15. For each mRNA species, mean ΔCt values from triplicate qRT-PCR assays of t = 0 unstimulated cells or day 4 cultures stimulated with ODN + IL-15 are expressed as a ratio of the mean ΔCt values from day 4 cultures exposed to ODN alone, using the 2^(2ΔΔCt) method. Values for U-CLL430 mRNA in ODN and ODN + IL-15 cultures represents mean 6 SEM values from three separate experiments. Asterisks linked to the U-CLL430 experiments indicate that differences in mRNA levels between ODN + IL-15 versus ODN-only cultures reached statistical significance by two-sided, paired t test. Values for U-CLL515 are from one experiment with triplicate qRT-PCR determinations.

(Fig. 4C). Interestingly, although culture with ODN alone MDC1 (ATM = TP53BP1 . MDC1) [molecules with critical roles notably elevated levels of each DDR mRNA above base- in 1) dampening cell growth and viability and 2) fostering DNA line levels (increases of 2- to 5-fold), the cultures receiving repair following DNA damage (66)]. both ODN and IL-15 stimuli maintained levels of these DDR mRNA species more comparable to baseline. In this study PI3K and STAT5 pathways contribute to IL-15–augmented we did not investigate the kinetics of the ODN-induced rise CCND2 mRNA and IL-15–reduced ATM and TP53BP1 mRNA in ATM, TP53BP1,andMDC1 mRNA nor the mechanisms The involvement of PI3K/AKT and/or STAT5 in IL-15–driven responsible. modulation of the above mRNA species was examined with Together, the above studies demonstrate that IL-15 significantly pharmacologic inhibitors of PI3K (LY294002) and STAT5 alters the B-CLL repertoire of mRNA molecules encoding certain (pimozide and STAT5 Inhibitor II [CAS-285986-31-4]). Using critical cell cycle regulatory proteins. Although IL-15 signaling in several ODN-primed B-CLL clones, we quantified specific mRNA primed B-CLL cells prompts a rapid (within 4 h) rise in mRNA for following 20-h exposure to IL-15 6 inhibitor (or vehicle alone), growth-promoting cyclin D2 ...c-Myc, by 9–16 h such sig- normalizing ΔCt values from all IL-15–pulsed cultures based on naling also fosters a decline in transcripts for ATM, 53BP1, and ΔCt values from parallel ODN-primed cultures pulsed only with 2932 MECHANISM FOR CpG DNA AND IL-15 SYNERGY IN B-CLL GROWTH vehicle (Fig. 5). MDC1 mRNA assessments are not indicated (p = 0.049). Furthermore, both pimozide (Fig. 5C, middle bar) because results were variable, with no consistent trend observed and STAT5 Inhibitor II (Fig. 5C, right bar) reversed IL-15– (data not shown). We suspect the latter variability reflects the fact driven repression of TP53BP1 mRNA in five of six B-CLL, that inhibitor studies were performed at 20 h post–IL-15 when albeit at levels that did not reach statistical significance IL-15–mediated MDC1 mRNA repression was not consistently (p =0.14andp = 0.09, respectively). The magnitude of the re- evidenced (Fig. 5B). versal varied between B-CLL clones and might represent the use of both STAT5 inhibitors at doses near their reported IC . CCND2 mRNA 50 U-CLL1239 was an exception; manifesting reduced rather an PI3K inhibitor ablated the IL-15–driven rise in CCND2 mRNA in augmented TP53BP1 mRNA when exposed to STAT5 inhibitors. half (two of four) of the B-CLL populations tested (Fig. 5A, left When this variant was removed from analysis, the TP53BP1- bar). Interestingly, both clones whose CCND2 mRNA was not boosting effect of pimozide and STAT5 Inhibitor II approached inhibited by PI3K inhibitor were of the M-CLL subset (M-2018 or reached statistical significance (p =0.09andp = 0.03, and M-1993; open symbols). These two were among the five of six respectively). clones whose CCND2 mRNA levels were inhibited by STAT5 Altogether, the above pharmacologic approaches revealed that inhibitor, pimozide (Fig. 5A, middle bar). Given the suggestion both IL-15–induced PI3K/AKT and STAT5 pathways can con- that PI3K → AKT activation may be reduced in M-CLL (Fig. 1C), tribute to the repression of ATM and TP53BP1 mRNA in ODN- these data suggest that the STAT5 pathway is a compensatory primed B-CLL. means for upregulating CCND2 mRNA. Nonetheless, larger co- IL-15–induced AKT and STAT5 phosphorylation in B-CLL are horts of both U-CLL and M-CLL clones will be needed to discern Downloaded from modulated by PI3K inhibitor, LY294002, and STAT5 whether this pattern is reproducible. Less potent STAT5 Inhibitor inhibitor, pimozide II (CAS-285986-31-40), with an EC50 ∼50 mM versus EC50 =5 mM for pimozide, was less effective at reducing CCND2 mRNA, As a means of confirming the upstream inhibitory activity of with only two of five B-CLL clones affected (Fig. 5A, right bar). these pharmacologic agents, we examined levels of pAKTS473 and U-CLL1058 was an anomaly in showing augmented CCND2 pSTAT5Y694 in ODN-primed B-CLL cells pre-exposed to either of mRNA upon IL-15 exposure with STAT5 inhibitor, pimozide. the above inhibitors (or vehicle alone) for 2 h before the pulse with http://www.jimmunol.org/ (As in other U-CLL clones, its CCND2 mRNA was suppressed IL-15 (60 min) (Fig. 5E, middle and bottom histograms). These with PI3K inhibitor). were compared with levels in parallel ODN-primed cultures with Together, these findings show that IL-15–induced PI3K/AKT vehicle alone (Fig. 5E, top histograms). PI3K inhibitor blocked and STAT5 pathways can each contribute to the IL-15–driven IL-15–induced pAKTS473 upregulation as expected (30% inhibi- rise in CCND2 mRNA but that B-CLL clones differ in the relative tion) (Fig. 5E; middle left histogram). Furthermore, pimozide participation of each pathway. Because the above experiments blocked the IL-15–induced rise in pSTAT5Y694 as expected were performed at a single time point (20 h after IL-15 exposure), (42% inhibition) (Fig. 5E, bottom right histogram). The STAT5 it is possible that the differences reflect clonal diversity in the inhibitor was also effective in reducing IL-15–triggered upregu- temporal use of these pathways. Our findings that both PI3K and lation of pAKTS473 (45% inhibition) (Fig. 5E, bottom left histo- by guest on September 23, 2021 STAT5 pathways contribute to IL-15 → CD122/ɣc-driven cyclin- gram). This latter observation is consistent with past reports that 2 in B-CLL cells resemble observations with IL-2–signaled activated STAT5 engages both the PI3K p85 regulatory subunit and T cells (67). the scaffolding protein Gab, thereby promoting AKT activation (68, 69). Additionally, STAT5 was reported essential for AKT/ ATM mRNA p70S6 activity during IL-2–induced T cell proliferation (21). Fig. 5B (left bar) shows that exposure to PI3K inhibitor imme- Thus, IL-15–induced STAT5 phosphorylation in ODN-primed diately prior to IL-15 uniformly augmented ATM mRNA (reversed B-CLL likely precipitates two events: 1) STAT5 dimerization and the IL-15–driven decline) in four of four B-CLL tested (U-1058, translocation to the nucleus for function as a TF and 2) activation U-1239, M-1993, and M-2018). Although the difference in ATM of a parallel AKT signaling pathway that can also influence gene expression (ratio of IL-15–pulsed versus ODN-only cultures) was expression. not significantly different in inhibitor- versus vehicle-pulsed cul- IL-15 signaling in ODN-primed B-CLL cells dampens protein tures (p = 0.16), this quite likely represents the wide variation of expression of ATM, 53BP1, and MDC1 values within n = 4 experiments. Of note, when fold change was assessed by direct comparisons of ATM mRNA in ODN + IL- We investigated whether downstream DDR proteins are corre- 15–stimulated cultures with/without inhibitor, the inhibitor- spondingly IL-15 attenuated through Western blotting of cell potentiated rise in ATM mRNA was statistically significant lysates and immunofluorescence staining. Results from such (p = 0.029) (by nonparametric Mann–Whitney rank sum test). experiments, employing U-CLL430 and U-CLL515, appear in STAT5 inhibitor, pimozide, also attenuated the IL-15–driven de- Fig. 6A, 6B. A strong band representing full-length 53BP1 protein cline in ATM mRNA (observed in five of six B-CLL) (Fig. 5B, (∼345 kDa) and a weak band representing MDC1 (∼225 kDa) middle bar) with p values approaching statistical significance were detectable in day 4 lysates from both CLL. Full-length ATM (p = 0.08) Less potent STAT5 Inhibitor II augmented ATM mRNA (∼350 kDa) was noted only in CLL-515, consistent with the ATM- but only in the clones most strongly affected by pimozide (U-1058 impaired status of the CLL430 clone (11q22 del and ATM muta- and M-2018) (Fig. 5B, right bar). Taken together, the above tion) (8). Notably, B-CLL cells stimulated with ODN + IL-15 findings suggest that both PI3K and STAT5 contribute to IL-15– expressed lesser levels of ATM and 53BP1 proteins as compared mediated repression of ATM mRNA. with parallel cultures with only ODN. Although full-length MDC1 protein appeared relatively unaf- TP53BP1 mRNA fected, ODN + IL-15–stimulated cultures of both B-CLL exhibited The inhibitor of PI3K reversed IL-15–mediated suppression of markedly lower levels of an MDC1 band with a molecular mass of TP53B1 mRNA in most B-CLL (three of four) (Fig. 5C, left ∼70 kDa (Fig. 6A, 6B). The latter was prominently expressed in bar), with the upturn in mRNA reaching statistical significance lysates of ODN-only cultures and likely represents a previously The Journal of Immunology 2933 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 5. Effect of PI3K and STAT5 inhibitors on IL-15–upregulated CCND2 and IL-15–repressed ATM and TP53BP1 transcripts. (A–C) To examine whether IL-15 modulation of mRNA levels involves PI3K and/or STAT5 activity, ODN-primed B-CLL (20 h) were exposed to PI3K inhibitor LY294002 (20 mM) or one of two STAT5 inhibitors: pimozide (5 mM) or STAT5 Inhibitor II (CAS-285986-31-4) (100 mM) for 15 min prior to cytokine addition. DMSO only was vehicle control. Following an additional 20 h of culture, total mRNA was harvested and qRT-PCR performed to quantify levels of CCND2, ATM, and TP53BP1 mRNA, with GAPDH as housekeeping control. In each experiment, parallel cultures primed with ODN alone + vehicle served as baseline for calculating fold changes in mRNA (as in Fig. 4). Values for fold change in specific mRNA within ODN + IL-15 + vehicle cultures versus ODN + vehicle cultures are shown above each box plot (mean 6 SD of the multiple B-CLL tested). Box plots represent the level of IL-15–altered mRNA in inhibitor-treated cultures expressed as a percentage of that in respective IL-15–pulsed, vehicle-only cultures. Inserted symbols show responses of individual B-CLL (open symbols = U-CLL; closed symbols = M-CLL). (A) Effect of PI3K and STAT5 inhibitors on IL-15–augmented expression of CCND2 mRNA. Box plot showing relative CCND2 mRNA in IL-15–pulsed B-CLL cells exposed either to PI3K inhibitor (four CLL) or to STAT5 inhibitors (six CLL). A value of 100% indicates equivalence with mRNA levels in IL-15–pulsed, vehicle-only cultures. The table insert shows statistical values for the represented data. p values with asterisk were determined by excluding the single variant B-CLL. (B) Effect of PI3K and STAT5 inhibitors on IL-15–mediated repression of ATM mRNA. (C) Effect of PI3K and STAT5 inhibitors on IL-15–mediated repression of TP53BP1 mRNA. (D and E) Effects of PI3K inhibitor, LY294002, and STAT5 inhibitor, pimozide, on IL-15–induced AKT and STAT5 phosphorylation in B-CLL. ODN-primed U-CLL2245 cells were pretreated with either inhibitor or vehicle alone for 2 h prior to a 60-min pulse with IL-15 (or medium). (Figure legend continues) 2934 MECHANISM FOR CpG DNA AND IL-15 SYNERGY IN B-CLL GROWTH reported 70-kDa MDC1 cleavage fragment generated by action viable-gated populations but were represented in the whole culture of caspase-3 at position 173 of MDC1 (70). In support of this lysates evaluated by Western blotting. possibility, the anti-MDC1 mAb used for detection binds the Taken together, the above experiments demonstrate that IL-15 N-terminal forkhead-associated (FHA) domain preserved in cas- signaling within ODN-primed B-CLL cells significantly attenuates pase-3–cleaved MDC1 (71). Furthermore, B-CLL cells stimulated the expression of DDR molecules with roles in the recognition of with ODN alone undergo caspase-mediated death (8). Of note, DNA damage, induction of cell cycle blocks, and DNA repair (66). when total MDC1 expression is taken as the sum of full-length Both ATM/ATM and TP53BP1/53BP1 mRNA/protein are repro- protein and cleaved fragment, it becomes quite apparent that ducibly repressed in B-CLL receiving ancillary IL-15 signals. MDC1 protein levels decline in IL-15–supplemented cultures. Furthermore, IL-15 signals also dampened MDC1 mRNA in a time- This corresponds with the lesser MDC1 mRNA levels in day 4 sensitive manner and, as demonstrated by immunoblotting, reduced ODN + IL-15–stimulated cultures versus cultures exposed to ODN levels of total MDC1 protein (full-length + 70-kDa fragment). alone (Fig. 4C). Bioinformatics analysis of STAT TF binding to TP53BP1, Flow cytometry results, following immunofluorescent staining to ATM, and MDC1 gene loci monitor DDR protein levels, are presented in Fig. 6D, 6E. In ODN- primed B-CLL cultures, a 48–72-h pulse with IL-15 led to notably Following their phosphorylation and dimerization, STAT TF are lower 53BP1 protein expression than seen in primed B-CLL cells translocated to the nucleus where they bind gene promoters and not exposed to IL-15; furthermore, 53BP1 protein remained sup- regulate gene expression. To gain insight into whether STAT1, pressed at 114 h post–IL-15 (Fig. 6C, 6D). ATM protein was STAT3, and STAT5 can engage sites within ATM, TP53BP1, and similarly reduced, albeit with slightly slower kinetics (Fig. 6C, 6D). MDC1 gene promoters, we employed publicly available ENCODE Downloaded from Exposure to IL-15 alone, without ODN priming, did not reduce ChIP-seq data derived from several human non–B-CLL cell lines. baseline levels of these DDR proteins (data not shown), consistent We sought evidence for STAT TF binding as well as TBP binding with the negligible expression of its major signaling receptor, (other TFs were not evaluated). Importantly, evidence of STAT CD122, in unprimed B-CLL (17). A statistical analysis of pooled TF binding near the TSS of these genes would suggest STAT- data from six to seven B-CLL clones (Fig. 6E) shows that IL-15– regulated expression. Such a conclusion would be further strengthened by the following: presence of TBP at the TSS, driven repression of ATM and 53BP1 proteins is highly significant http://www.jimmunol.org/ (p , 0.0001). presence of flanking CTCF insulators, evidence of chromatin IL-15–driven STAT5 activation has a role in precipitating this openness indicated by DNase hypersensitivity clusters, and by decline in ATM and 53BP1 proteins. This is indicated from presence of a peak of H3K27ac histone marks, often indicative of findings that 1) STAT5 contributes to the acute repression of ATM regulatory activities (72, 73). Because STAT5 repression can re- and TP53BP1 mRNA shortly following IL-15 exposure (Fig. 5B, flect the latter’s competition with other STATs that bind GAS 5C), 2) reduced levels of ATM and 53BP1 proteins within repli- motifs (47), we also looked for signs that STAT5 binding sites cating blasts of ODN + IL-15–stimulated cultures are accompa- overlapped with binding sites for other STAT TFs. The schematic nied by elevated levels of pSTAT5 (Fig. 6E), and 3) there is a in Fig. 7 highlights major pertinent results from this bioinformatics statistically significant correlation between IL-15–driven repres- study. Supplemental Fig. 4 displays the Genome Browser tracks by guest on September 23, 2021 sion of ATM and TP53BP1 mRNA and later ATM and 53BP1 from promoter and promoter-proximal regions of genes for ATM, protein levels (regression curves in Fig. 6F). TP53BP1 (reflecting TSS regions for transcript variants 1 and 2 Of note, although both Western blotting and flow cytometry and distinctly variant 3), and MDC1. These contain additional assays yielded concordant conclusions regarding IL-15 repression information. Note that in these tracks, darkness of the gray boxes is of ATM and 53BP1 proteins, this was not the case for MDC1 proportional to maximal signal strength. protein. Rather, by flow cytometrytherewasnoindicationthat ATM. For the ATM promoter region, there are TF binding sig- MDC1 levels were reduced in IL-15–supplemented cultures nals for various STATs. Within the ATM promoter of the (Δ median fluorescence intensity [MFI] of 970 and 959 for B-lymphoblastoid cell line, GM12878, STAT5A potentially U-CLL1953 cells stimulated with ODN + IL-15 and ODN alone, competes with STAT1 and STAT3 (STAT5A has a high signal respectively). This contrasts with diminished MDC1 protein (sum strength, with the signal strength for STAT1 and STAT3 being of full-length and 70-kDa fragment) when IL-15–supplemented much weaker). Within the K562 cell line, STAT5A within the first cultures are compared with ODN-only cultures by Western blot- ATM intron has a strong signal (Fig. 7A, Supplemental Fig. 4A). ting (Fig. 6A, 6B). The differing conclusions from these two TP53BP1. Top tracks: In the HeLa-S3 cell line, which expresses assays may well reflect the fact that caspase-3+ apoptotic cells alternative TP53BP1 transcripts 1 and 2, a strong signal for STAT1 were excluded from flow cytometric analysis. Consistent with binding was seen in the promoter near the TSS but no evidence this interpretation, blotting data in Fig. 6A shows that most for STAT3 or STAT5 binding. Bottom tracks: In the K562 mye- MDC-1 protein within ODN-only cultures is present as a loblastoid and MCF 10A-Er-Src mammary gland lines, which 70-kDa caspase-3 cleavage fragment. Thus, cells expressing high express TP53BP1 transcript variant 3, our bioinformatics analysis levels of caspase-3–fragmented MDC1 were likely not within the revealed STAT3 binding near the TSS (in MCF 10A-Er-Src

Two hours prior to cytokine pulse, cultures were pretreated with vehicle 6 LY294002 (20 mM) or pimozide (5 mM). By flow cytometry, cells were assessed for (D) viability and (E)pAKTS473 (left) and pSTAT5Y694 (right) fluorescence. Top-row histograms in (E): ODN-primed cultures with vehicle but no cytokine. Middle-row histograms in (E): ODN-primed cultures with IL-15 6 PI3K inhibitor. Bottom-row histograms in (E): ODN-primed cultures with IL-15 6 STAT5 inhibitor. Solid-line and dotted-line histograms represent cultures with vehicle alone or with inhibitor, respectively. Filled histograms represent isotype-control staining. Values for RMFI-specific staining/MFI isotype control are shown in each plot; when two values are provided, the top and bottom values represent RMFI without and with inhibitor. Percentage of inhibition was calculated as follows: (1 2 [RMFI with no INH – RMFI with INH]/RMFI with no INH) 3 100. Viability dot plots in (D): Top row = ODN-only cultures; bottom row (left to right) = ODN + IL-15 with vehicle; ODN + IL-15 with PI3K inhibitor; ODN + IL-15 with STAT5 inhibitor. Similar observations were made with another B-CLL clone (U-2258) with lower levels of IL-15–induced pAKT and pSTAT5 (data not shown). INH, inhibitor. The Journal of Immunology 2935 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 6. DDR protein levels are reduced in ODN-primed B-CLL receiving IL-15 signals. (A) ATM, 53BP1, and MDC1 protein levels were examined in day 4 lysates of ODN 6 IL-15–stimulated B-CLL cultures by electrophoretic separation and Western blotting. The asterisk by CLL430 denotes this clone’s lack of ATM protein due to del11q22 and a coding region mutation (8). MDC1 protein is manifest both as full-length MDC1 protein (∼225 kDa) and a MDC1 cleavage fragment (∼70 kDa) (70). Values below each lane represent relative densitometric levels adjusted on the basis of b-actin loading control. (B) Calculated values for CLL430 and CLL515 expression of ATM, 53BP1, and MDC1 (full-length or cleavage fragment) (Figure legend continues) 2936 MECHANISM FOR CpG DNA AND IL-15 SYNERGY IN B-CLL GROWTH mammary gland line) and more distal STAT5A binding in such experiments with diverse B-CLL clones are shown in the K562 cell line. All other relevant tracks (DNase cluster, Fig. 8C. Data are normalized by expressing values for total yield H3K27ac, TBP binding, bracketing of CTCF bindings) are in (Fig. 8C, left) and viability (Fig. 8C, right) as a percentage of support of an active TSS in this latter region. Altogether, these respective values in parallel ODN + IL-15–stimulated cultures latter observations show the involvement of STAT3 and STAT5A with vehicle alone. Both yield and viability are significantly in regulation of TP53BP1 transcript variant 3 (Fig. 7B, Supplemental reduced by day 0 and day 3 exposure to pimozide (day 0 . day 3). Fig. 4B). On average, a day 3 pulse with this STAT5 inhibitor precipitates a MDC1. In a region occupied by TBP and CTCP insulators, STAT3 60% decline in yield and a 20% decline in viability of progeny, binding was seen in two cell lines (HeLa-S3 and the mammary cell indicating that growth is more affected than viability. Additional line): STAT1 binding in HeLa-S3 and K562 cell lines and STAT5A pooled data in Fig. 8D, 8E show that both STAT5 inhibitors binding in the B cell line, GM12878, and K562. In K562 cells, (pimozide or STAT5 Inhibitor II [CAS-285986-31-4]) block STAT5A competes with STAT1 (and STAT2) at the same site. growth when added late after B-CLL activation; a day 4 pulse with Together, the above bioinformatics analyses support the con- either inhibitor reduced the day 6 yield of highly divided B-CLL cept that IL-15–driven DDR gene repression in B-CLL might in a dose-related manner. The dose responses for pimozide and involve direct binding of pSTAT5 to the relevant DDR gene STAT5 Inhibitor II inhibition were consistent with the published promoters or to a promoter-proximal region in the TP53BP1 IC50 for each: 5 and 47 mM, respectively (39, 40). A higher dose of locus. Future ChIP-seq studies of activated B-CLL clones should pimozide (10 mM) significantly compromised yield of viable clarify whether these regions within TP53BP1, ATM,andMDC1 B-CLL in all division subsets (Fig. 8D). At least in part, the latter loci of B-CLL cells display differences in their occupancy of the reflects the inhibitor’s more pronounced antiviability effects at this Downloaded from STAT1, STAT3, and STAT5 following ODN priming 6 IL-15 dose (data not shown). Disparate STAT5 functions are reported to (Fig. 7C, Supplemental Fig. 4C). require differing levels of STAT5 (21, 77, 78), which might explain why STAT5 inhibitors at low doses compromise growth . viability. Pharmacologic inhibitors of PI3K and JAK/STAT5 signaling To test whether growth inhibition by delayed pimozide treat- pathways reduce B-CLL blastogenesis and clonal expansion ment was linked to reduced STAT5 activation, we monitored Y694 Because PI3K and STAT5 are involved in IL-15–driven tran- pSTAT5 levels within day 4 cultures of ODN + IL-15– http://www.jimmunol.org/ scriptional changes that should foster B-CLL growth, we exam- stimulated B-CLL 6 a 12-h pulse with 5 mMpimozide(n =3 ined whether PI3K and STAT5 inhibitors blocked IL-15–driven B-CLL). Pimozide-treated cultures exhibited a 17 6 12% de- B-CLL clonal expansion (Fig. 8). Photomicrographs in Fig. 8A cline in pSTAT5 (mean 6 SD) when compared with parallel show the PI3K inhibitor, Ly294002, aborted IL-15–driven B-CLL vehicle-treated cultures (p = 0.04 by unpaired, one-sided t test). blastogenesis and clustering in ODN-primed cultures and, fur- Thus, blocked B-CLL cycling following the delayed addition of thermore, increased the appearance of shrunken cells with apo- pimozide is correlated with diminished, albeit not fully ablated, ptotic morphology. These observations are consistent with our STAT5 activation. above findings, past reports that PI3K is critical for B-CLL via- Although all ODN + IL-15–stimulated B-CLL clones (six of six) bility and growth (74), and evidence that PI3K inhibitor blocks were subject to the growth-inhibitory effects of pimozide, clonal by guest on September 23, 2021 IL-15/IL-2/CD122/ɣ signaling in T cells through a process differences in the extent of inhibition were apparent. To illuminate involving cyclin D2 repression and arrested G0/G1 transition the basis for this diversity, we focused on five clones that had been (67, 75, 76). monitored both for 1) ATM and 53BP1 protein expression and The effects of abrogated STAT5 function on B-CLL viability/ 2) growth inhibition by pimozide treatment at day 3 or day 4 after growth have never been examined, to our knowledge, and were ODN + IL-15 activation. Regression analysis revealed a statisti- of particular interest. Hence, CFSE-labeled B-CLL cells from cally significant, direct relationship between the extent of growth several patients were used to test for STAT5 inhibitor–blocked inhibition by pimozide and the effectiveness of IL-15 at repressing B-CLL clonal expansion. Results are summarized in Fig. 8B–F. A ATM ( p = 0.016) and 53BP1 (p = 0.013) protein levels (Fig. 8F). representative experiment with U-CLL996 (Fig. 8B) shows un- This provides further support for the conclusion that the IL-15 → equivocally that early (t = 0 h) exposure to the STAT5 inhibitor, STAT5 signaling axis contributes to ATM and 53BP1 repression. pimozide, at 5 mM, abrogates ODN + IL-15–induced B-CLL CD122/ɣc-associated JAK1 and JAK3 are upstream media- cycling and impairs the day 5 recovery of viable cells. Consis- tors of IL-15–induced STAT5 phosphorylation in T/NK cells tent with importance of sustained IL-15/CD122/ɣc signaling for (20). Thus, inhibitors of these upstream kinases (e.g., JAK1/2 B-CLL growth (10, 17), clonal expansion is curtailed even if this inhibitor, ruxolitinib, and preferential JAK3 inhibitor, tofacitinib STAT5 inhibitor is delayed to day 3. Pooled results from three [JAK3 . JAK1 . JAK2]) (79) should be effective at blocking

proteins in ODN + IL-15 lysates as a percentage of that seen in ODN-only lysates. (C) Fluorescence histograms representing ATM and 53BP1 protein expression in viable-gated U-CLL1953 cells stimulated with ODN (t = 0) and IL-15 (t = 20) and harvested at t = 68, 92, or 134 h. Inserted values represent ratio of MFI (RMFI) in test mAb-stained cells (solid line)/MFI in isotype control cells (filled gray). (D) Bar graph of results from the U-CLL1953 ex- periment showing ATM and 53BP1 protein expression at the intervals following IL-15 pulse to cultures prestimulated with ODN for 20 h. Data are plotted as a ratio of RMFI in ODN + IL-15–treated cells/RMFI in ODN-treated cells (mean 6 SEM of staining replicates). (E) Pooled results from CFSE-labeled B-CLL experiments monitoring pSTAT5, ATM protein, and 53BP1 protein levels as a function of division status within 5–6-d cultures stimulated with ODN + IL-15 or ODN alone. (Experiments involved n = 7 CLL [770, 791, 827, 1031, 1953, 1993, 2018], except for ATM analyses [n = 6 CLL]). Data expressed as ratio of specific fluorescence in ODN + IL-15 cultures versus ODN-only cultures. Dotted line represents normalized fluorescence in ODN-only cultures. Statistical analyses by two-sided, unpaired t test: *p = 0.02 when compared with ODN-only cells; **p , 0.0001. (F) Linear regression analysis of mRNA versus protein expression of ATM (left) and 53BP1 (right) within five B-CLL clones. Specific mRNA was assessed in 20-h ODN-primed B-CLL 6 ad- ditional 20 h of culture with medium or IL-15; specific protein was assessed by staining and flow cytometry of 5–6-d cultures stimulated by ODN 6 IL-15 (M-1031, M-2018, M-1993, U-1953, and U-1692). mRNA and protein levels are expressed as a ratio of assessed levels in ODN + IL-15 cultures versus ODN-only cultures. The Journal of Immunology 2937 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. Schematic summary of STAT1, STAT3, and STAT5 binding to promoter regions of ATM, TP53BP1, and MDC1 from ENCODE-derived ChIP-seq data derived from non–B-CLL lines. ENCODE ChIP-seq data (Materials and Methods) was used for our bioinformatics evaluation of (A) ATM, (B) TP53BP1, and (C) MDC1 loci. This database provides information on DNA binding of 161 TFs, including STATs, TBP, and CTCF (insulator) within several established cell lines, but we focused in this study on STAT1, STAT3, STAT5, TBP, and CTCF binding to promoter regions. Note that ENCODE provides data only for STAT5A binding; binding of closely related STAT5B that shares the same GAS specificity is not indicated. Browser tracks from which this schematic was derived are shown in Supplemental Fig. 4.

ODN + IL-15–driven B-CLL growth. Experiments with CFSE-labeled access of recirculating leukemic cells to 1) stromal cells providing cultures (Fig. 9) confirm this; both JAK inhibitors and the STAT5 viability/growth-sustaining stimuli (adhesion molecules, chemo- inhibitor, pimozide, curtailed cycling and compromised viabil- kines, BAFF, APRIL), 2) activated T cells expressing CD40L, and 3) ity upon their day 3 pulse into ODN + IL-15–stimulated B-CLL ligands engaging the unique BCRs of B-CLL clones (7, 80). The cultures. Although the histograms in Fig. 9A suggest that JAK in- latter include microbes as well as molecules on surfaces of apo- hibitors may be more effective than pimozide at impairing survival ptotic and stressed body cells (14, 80). As recently reviewed (8), a of progeny, further studies are needed. The time-course experiment major role for TLRs in fostering B-CLL growth was also sug- in Fig. 9B shows that both JAK inhibitors and pimozide compro- gested by elevated TLR9 (and other TLR) in B-CLL, by gene mise the day 6 yield of viable cells when pulsed into cultures as late expression arrays evidencing activated TLR and BCR pathways as day 4. Together, this study’s functional experiments support the in lymph node B-CLL, and by the emergence of function- conclusion that upstream JAK kinases and downstream PI3K/AKT enhancing MYD88 mutations in certain leukemic clones. None- and STAT5 are important mediators in sustaining the IL-15–driven theless, in vitro exposure of quiescent B-CLL to TLR ligands clonal expansion of ODN-primed B-CLL cells. resulted in only meager B-CLL cycling, and in a major subset of The Fig. 10 schematic summarizes insights obtained concerning B-CLL, significant apoptosis (8). An integrative explanation is that ODN + IL-15–driven cycling from this and earlier reports (8, 10) B-CLL clonal growth as seen in normal B cells requires several in the context of B-CLL growth within pseudofollicles of patient diverse stimuli acting in a coordinated manner to foster the lymphoid tissues. In particular, it emphasizes potential new ave- appropriate panoply of molecules controlling cell cycle and nues for blocking this growth in patients. viability. Consistent with the latter view, certain cytokines exhibit sig- Discussion nificant synergy with the TLR9 ligand CpG DNA (ODN) in During the past decade of intense B-CLL research, lymphatic driving B-CLL growth in vitro (8, 62, 81). IL-2, a T cell cytokine, tissue–restricted growth of B-CLL was attributed to a greater was first implicated (81), and augmented DNA synthesis was 2938 MECHANISM FOR CpG DNA AND IL-15 SYNERGY IN B-CLL GROWTH Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 8. PI3K and STAT5 inhibitor effects on ODN + IL-15–stimulated B-CLL growth. (A) Twenty-hour ODN-primed M-CLL1993 cultures were pulsed with IL-15 (following 15-min preincubation with PI3K inhibitor (LY294002 at 20 mM) or vehicle alone). After 20 h further culture, B-CLL cells were viewed by phase microscopy (2003) and photographed. Inhibitor-treated cultures showed a decline in enlarged cell clusters and a rise in cells with shrunken, apoptotic features. Similar findings were obtained in a parallel experiment with M-CLL2018 (data not shown). (Figure legend continues) The Journal of Immunology 2939 Downloaded from

FIGURE 9. Inhibitors of JAK kinases block ODN + IL-15–driven B-CLL growth. (A) CFSE-labeled M-CLL1031 cells were stimulated with ODN + IL-15 on day 0 and pulsed on day 3 with either STAT5 inhibitor, pimozide (5 mM), or a maximal inhibitory dose (50 nM) of JAK1/2 inhibitor, ruxolitinib, or http://www.jimmunol.org/ JAK1/3 (JAK3 . JAK1) inhibitor, tofacitinib. Cultures were harvested on day 6 and analyzed by flow cytometry, with CFSE fluorescence shown in gated viable cells (solid line) and dead cells (gray fill). (B) Effect of day 3, day 4, or day 5 pulse of the above inhibitors on yield of viable, CFSE-labeled M-CLL1031 in day 6 ODN + IL-15–stimulated cultures. Yield is expressed as percentage of that in ODN + IL-15–stimulated cultures pulsed with vehicle alone at the indicated intervals (mean 6 SEM of triplicate cultures). *Indicates that treated cultures are significantly different from respective cultures with vehicle alone (p , 0.05 by one-sided, unpaired t test). Similar results obtained in a replicate experiment. attributed to enhanced expression of cyclin D2 and cyclin D3 by and macrophages, in the spleens and lymph nodes from B-CLL IL-2 and ODN, respectively (62). To date, IL-2+ T cells have not patients (8, 10). These IL-15+ cells were found proximal to and been described in B-CLL pseudofollicles, but CD40L+ CD4+ sometimes within B-CLL proliferative foci (8) and also noted in by guest on September 23, 2021 T cells have been reported (82, 83). IL-15, a cytokine produced normal human spleens (8). It warrants noting that mean and me- by multiple stromal cells, was more recently shown to exhibit dian levels of soluble IL-15 within blood of B-CLL patients are synergy with ODN in promoting B cell growth (8, 10). IL-15 higher than those in controls, albeit both exhibit a considerable signals significantly extended in vitro cycling of ODN-primed range (84). Gene expression data from the ImmGen database B-CLL clones and blocked ODN-triggered apoptosis (8, 17, 44, (https://www.immgen.org) (85) show elevated IL15 mRNA as well 50). as IL15RA needed for in vivo trans-presentation of IL-15 (18, 86) Importantly, multiple lines of evidence show that IL-15 mRNA/ within several dendritic cell and macrophage subpopulations of protein is amply expressed in lymphoid tissues where B-CLL humans and mice. Granulocytes were also identified as high growth occurs. Using immunohistochemistry, we recently found producers of IL15 but not IL15RA mRNA. Although not excluding IL-15–producing cells, resembling both follicular dendritic cells a role for the latter in B-CLL, trans-presentation of IL-15 by

(B) CFSE-labeled M-CLL2018 cells were stimulated with ODN + IL-15 with the STAT5 inhibitor, pimozide (5 mM), or vehicle alone, pulsed into cultures at times indicated. At day 6, cultures were harvested and analyzed by flow cytometry. Viable and dead cell subpopulations (gated on the basis of V450– Pacific Blue staining and side scatter) were analyzed for past division history as described (8). Filled gray histograms = dead cells; solid line = viable cells. Dotted line represents viable cells from cultures with IL-15 alone (indicator of undivided cells). (C) Pooled experiments comparing effects of early (day 0) or late (day 3) pimozide pulses on day 6 B-CLL yield and viability in ODN + IL-15–stimulated cultures (CLL 996, 1993, and 2018). Left panel, Relative impact of day 0 or day 3 pimozide (5 mM) on total recovery of gated viable undivided and divided B-CLL fractions. Recovery is expressed as percentage of the absolute viable cell yield in stimulated cultures with vehicle alone. Right panel, Relative impact of day 0 or day 3 pimozide treatment on cell viability (expressed as % of vehicle control). *Indicates that cell yield or viability in the indicated fractions was statistically different from respective cultures treated with vehicle alone (two-sided, unpaired t test). (D and E) Extensively divided cells are preferentially compromised by delayed (day 4) exposure to STAT5 inhibitors. CFSE-labeled B-CLL (n = 3; CLL 321, 1692, and 1953) were stimulated with ODN + IL-15 on day 0, pulsed on day 4 with indicated doses of either pimozide (D) or STAT5 Inhibitor II (E), and harvested on day 6. Parallel cultures were pulsed with vehicle alone (0.03% final DMSO for pimozide experiments and 0.12% final DMSO for STAT5 Inhibitor II experiments). Values for absolute yield of viable cells per division and percent viability within each division were determined and expressed as a percentage of that in vehicle-only cultures. *Indicates that values were significantly different from vehicle control (two-sided, unpaired t test). (F) Linear regression analysis comparing growth inhibition by pimozide STAT5 inhibitor with relative IL-15–mediated inhibition of ATM and 53BP1 protein in five B-CLL clones (M-1031, M-2018, M-1993, U-1953, and U-1692), based on RMFI levels in ODN + IL-15 versus ODN-only cultures on day 4–5. Values for growth inhibition in day 6 ODN + IL-15–stimulated cultures were determined as follows: (1 2 [absolute recovery viable blasts in last three divisions of pimozide-treated cultures/absolute recovery of viable blasts in same three divisions of vehicle-treated cultures] 3 100). M-1031, M-2018, M-1993, and U-1953 cultures were pulsed with 5 mM pimozide on day 3 or day 4; U-1692 was pulsed with 10 mM pimozide on day 4. A significant correlation was noted between extent of IL-15–reduced ATM and 53BP1 and pimozide inhibition of growth. 2940 MECHANISM FOR CpG DNA AND IL-15 SYNERGY IN B-CLL GROWTH Downloaded from http://www.jimmunol.org/ FIGURE 10. Schematic illustrating hypothetical mechanisms for ODN + IL-15–driven B-CLL growth in patient lymphatic tissues and potential ther- apeutic interventions. B-CLL receiving TLR-9 signals, subsequent to BCR internalization of CpG DNA-bearing microbes and/or apoptotic cell debris, upregulate CD122 mRNA/protein and thereby more effectively receive signals from IL-15/IL-15Ra complexes present on adjacent IL-15–producing stromal cells (8, 11, 17) or, alternatively, from soluble IL-15/IL-15Ra complexes cleaved from the latter (127). Earlier studies showed that ODN + IL-15– driven in vitro B-CLL growth is positively influenced by del11q22 + del13q14, ATM mutations, and Trisomy 12 (8) and negatively influenced by CD122- or IL-15–specific neutralizing mAbs (17). The present study reveals that inhibitors of JAK1/3, PI3K, and STAT5 can each block ODN + IL-15–driven B-CLL growth, at least in part due to interference with IL-15–driven upregulation of cyclin D2 and IL-15–mediated repression of ATM and 53BP1. (Although less clear, it seems likely that pSTAT5 also participates in IL-15–mediated repression of MDC1). Thus, treatment of B-CLL patients with agents

that block IL-15 access to its signaling receptor and/or block early activation of JAK, PI3K, or STAT5 following IL-15/CD122/gc engagement could be by guest on September 23, 2021 effective new approaches to curtailing B-CLL growth within lymphatic tissue of patients.

IL-15Ra–positive stromal cells appears to be a dominant means of cytokine-driven growth not only depends upon sustained CD122 IL-15 signaling in vivo in mice (18, 86). → JAK → STAT5 activation but also upon activation of a IL-2 and IL-15 bind to specific IL-2Ra and IL-15Ra,re- CD122→ PI3K/AKT pathway, which is, at least in part, influenced spectively, yet employ the same major signaling complex CD122 by STAT5 activation. Importantly, both of the above pathways (IL2/15Rb)/cg (18). The latter is strongly linked to in vitro and contribute to IL-15–augmented CCND2 mRNA for cyclin D2 and in vivo B-CLL growth. Of note, neutralizing anti-CD122 mAb IL-15 repression of mRNA/protein for DDR mediators ATM, fully blocks IL-15–driven in vitro cycling of ODN-primed 53BP1, and MDC. Together, our findings suggest that therapeutic B-CLL (17). Furthermore, within B-CLL patients, blood leuke- molecules able to block IL-15 → CD122/gc → JAK → AKT and mic cells with recent division history exhibit significantly higher STAT5 signaling pathways within B-CLL cells may be particu- IL2RB mRNA than their more quiescent counterparts (17). Fi- larly effective at curtailing B-CLL growth within patient lymphoid nally, a recent clinical study reported that IL2RB mRNA was a tissues. major predictor of worse patient outcome (87). The discovery that PI3K/AKT is an intermediary in the above The ample evidence suggesting that TLR9 and CD122/gc sig- IL-15 functions is consistent with evidence that the PI3K/AKT/ naling pathways drive in vivo B-CLL clonal expansion and re- mTOR pathway is critical for this leukemia (89, 90). Although lated mutagenesis (88) emphasizes the relevance of our findings IL-15–driven AKT activation appears unreported in B-CLL, this regarding mechanisms. Together, findings from our present and pathway was noted in T/NK cells (23, 25). Interestingly, a past recent (17) studies indicate that synergy between CpG DNA study reported that degree of ODN-triggered AKT activation was and IL-15 involves a dynamic relationship between TLR-9– and the best discriminator of B-CLL subsets that undergo limited CD122/gc-triggered events. IL-15 is dispensable during the first growth (U-CLL) or, alternatively, apoptosis (M-CLL) upon culture 20 h following ODN exposure, during which acutely activated NF- with ODN alone (8, 9, 44). Even so, ODN-induced AKT activation kB promotes increased mRNA/protein for IL-15 receptors CD122 is transient (44) (this study). Thus, together these findings strongly (IL-2/15Rb) and IL-15Ra (17). During ODN priming, STAT1 and suggest that IL-15→Akt signaling through TLR9-upregulated STAT3 TF are phosphorylated at tyrosine residues (50) (this pa- IL-15 receptors compensates for weaker and/or transient ODN- per) that promote nuclear translocation and function. Within driven AKT activation, thereby permitting optimal survival/growth 24–36 h following ODN exposure, IL-15/CD122/gc signaling of both M-CLL and U-CLL clones. becomes critical for survival [particularly in M-CLL cells prone to Unlike for B cell acute lymphocytic leukemia (91) and acute ODN-induced apoptosis (8, 50)] and for continued cycling of both myelogenous leukemia (92), the importance of STAT5 for B-CLL M-CLL and U-CLL clones (17). The present study reveals that growth is little appreciated. Nonetheless, de Totero et al. (29) had The Journal of Immunology 2941 earlier suggested that STAT5 might have a role based on evi- recent B-CLL proliferation (8, 106). A separate B-CLL study dence that IL-15 promoted both STAT5 phosphorylation and correlated less effective translation of ATM mRNA to protein with DNA synthesis within CD40-activated B-CLL cells. The present greater CD40L + IL-4–driven viability/growth and reduced pa- mechanistic study, including evidence that IL-15–driven cycling is tient survival (107). Although mechanisms were undefined, it is blocked by STAT5 inhibitors, provides strong support for STAT5’s of interest that ATM protein was found highly expressed in relevance in B-CLL growth and suggests new treatments for this nonreplicating mantle-zone B lymphocytes and plasma cells but leukemia. weakly expressed in germinal center B cells (108). STAT5 is also important during periods of extended proliferation We found in this study that IL-15–mediated repression of ATM in normal B cell development. Mice with conditional STAT5 de- mRNA involves activation of both PI3K/AKT and STAT5 in letion in CD19+ B cells were blocked in pro-B to pre-B devel- B-CLL cells. Possibly, IL-15 → PI3K/AKT-mediated repression opment due to impaired IL-7–driven proliferation (93). Although involves regulatory microRNA. Importantly, studies of other this deletion appeared to have little, if any, impact on later B cell cell lineages show ATM mRNA levels regulated by miR-18a and development or on in vitro responses of mature B cells to anti- miR-421, both downstream of PI3K/AKT/mTOR (109–111). Our IgM + IL-4 (93), a more recent study found that germinal center studies with STAT5 inhibitor implied an additional role for B cells express elevated pSTAT5 (94). Furthermore, STAT5 STAT5. This might reflect 1) STAT5’s role in facilitating PI3K/ availability augmented B cell proliferation in response to AKT activation or 2) STAT5’s displacement of more transcrip- CD40L, IL-4, and IL-2 (94). IL-15 is reported to trigger STAT5 tionally permissive STAT molecules from a shared GAS site. Such activationinnaiveB1acellsfrom mouse peritoneum (31), but it a mechanism (47, 112) is one of several means whereby STAT5 remains unclear whether STAT5 influences B1 cell self-renewal. represses transcription of other genes (46, 47, 78, 112, 113) and Downloaded from Our present efforts to examine downstream targets of IL-15– was an implied mechanism for ATM repression in IL-15–driven triggered PI3K/AKT and STAT5 activation provide mechanistic CD8 T cells (34). Our bioinformatics analysis of ENCODE TF insights into how IL-15 fosters B-CLL cycling. First, CCND2 ChIP-seq data supports this mechanism; STAT5A, STAT3, and transcription appears to be enhanced given the rapid rise in STAT1 were found to occupy the same, or very proximal, GAS CCDN2 mRNA upon IL-15 exposure. Our evidence that both site within the ATM promoter in other diverse cell lines.

PI3K and STAT5 participate in this enhancement is consistent Although IL-15 repression of ATM mRNA was more effectively http://www.jimmunol.org/ with presence of a STAT5 binding site within the CCND2 pro- reversed by the PI3K inhibitor than inhibitors of STAT5, the op- moter (57). The latter is functionally relevant in early B cell posite was true for IL-15 repression of TP53BP1. This suggests a development (95) and in pre–B cell acute lymphoblastic leuke- different mechanism(s). Available ENCODE-based ChIP-seq data mia (91). Furthermore, T cell studies indicate that PI3K has a were not consistent with the above “displacement model,” given role in optimizing the binding of RNA polymerase II to the no evidence that STAT5 shares a GAS site with other STATs within CCND2 promoter (67). Thus, it appears that synergy between the TP53BP1 promoter of the lines investigated. Nonetheless, ODN and IL-15 [and related IL-2 (62)] involves CD122/cg- STAT1 did bind a TP53BP promoter in HeLa-S3 cells. ODN signals triggered activation of PI3K and STAT5 and cooperation of each in B-CLL activate STAT1 (Ref. 50 and this paper). Thus, a role for of the later in promoting CCND2 gene transcription for enhanced IL-15–upregulated pSTAT5 in displacing transcription-enhancing by guest on September 23, 2021 passage through the G1 → S phase transition. pSTAT1 cannot be excluded. The second insight regarding IL-15–fostered cycling was that A more intriguing possibility for STAT5-driven repression both PI3K/AKT and STAT5 contribute to repressed mRNA and emerged upon discovering a potential regulatory region in the protein for three critical DDR molecules: ATM, 53BP1, and TP53BP1 locus, located 3.7 kb from a TF start site for TP53BP1 MDC1 (96, 97). These findings resemble those from a recent study variant 3, one of three reported transcripts for this gene (114). with CD8+ T cell lines (34). In the latter, a greater effectiveness of Several attributes of this latter region suggest that it might be a IL-15 versus IL-2 at preventing senescence was linked to lower TP53BP1 enhancer under the influence of STAT5; 1) the region is mRNA levels of these DDR molecules with IL-15 present. Im- distal to the TP53BP1 promoter, 2) ChIP-seq data on this region portantly, their repressed expression within cycling B-CLL cells showed a unique STAT3 binding site located at some distance has significant implications for both growth and genomic stability from a unique STAT5 binding site, and 3) the region is charac- of the malignant clone. Within normal body cells, the ATM kinase terized by DNase I hypersensitivity clusters, by CTCF sites, and is promptly recruited to sites of DNA damage, where it undergoes by layered H3K27Ac marks (often found near active regulatory autophosphorylation (activation) (98). Activated ATM phosphor- elements) (72). Although far from definitive, these observations ylates local histone H2AX, yielding ɣH2AX (99), which enhances present the possibility that STAT5 repression of TP53BP1 might the recruitment of MDC1 scaffold protein through the latter’s involve chromatin alterations [e.g., STAT5 tetramer recruitment of BRCT domain (100). MDC1, in turn, recruits 53BP1, an im- the histone methyltransferase Ezh2 (115) and/or recruitment of a portant regulator of an intra-S phase checkpoint and mediator of NCoR repressor complex (45, 113, 116)]. Further study is needed DNA repair, to the site of DNA damage (100, 101). Additionally, concerning the variants of TP53BP1 expressed in B-CLL clones through its FHA domain, MDC1 recruits further ATM molecules and the means by which they are regulated. to the site of DNA damage, thereby augmenting the DDR re- The cyclic pattern of IL-15–driven MDC1 mRNA repression in sponse (97). Thus, collaborative function of the above DDR B-CLL cells made it difficult to study mechanism. Nonetheless, molecules is needed for optimal checkpoint regulation and DNA STAT5 activation is linked to MDC1 repression in IL-15–driven repair (66, 100). CD8+ T cell lines (34). Furthermore, our analysis of ENCODE- Although B-CLL clones harboring ATM gene deletion or derived ChIP-seq data from the MDC1 promoter in non–B-CLL inactivating mutations are strongly linked to lymphadenopathy hematopoietic cell lines is consistent with STAT5 displacement of and poor patient outcome in B-CLL (102–104), somewhat sur- other STATs as a possible mechanism. Namely, we found evidence prisingly, little is known regarding transcriptional regulation of that a single region of the MDC1 promoter could be bound by ATM in either malignant or normal B cells. Notwithstanding, a multiple STATs (STAT3, STAT5A, STAT1, and STAT2). Further previous study with B-CLL clones found that ATM mRNA levels supporting this hypothesis are findings that STAT3 facilitates were inversely related to expression of CD38 (105), a marker of MDC1 transcription in other cell types (117) and STAT3 is 2942 MECHANISM FOR CpG DNA AND IL-15 SYNERGY IN B-CLL GROWTH activated by ODN priming in B-CLL clones (50) (this paper). proliferation and disease progression in patients with early stage chronic → lymphocytic leukemia. Leukemia 31: 1348–1354. Thus, an abrupt rise in nuclear pSTAT5 following IL-15 CD122/ 7. Burger, J. A., and J. G. Gribben. 2014. The microenvironment in chronic gc signaling may reduce occupancy of more transcriptionally fa- lymphocytic leukemia (CLL) and other B cell malignancies: insight into dis- vorable pSTAT TFs at the MDC1 promoter. This hypothesis needs ease biology and new targeted therapies. Semin. Cancer Biol. 24: 71–81. 8. Mongini, P. K., R. Gupta, E. Boyle, J. Nieto, H. Lee, J. Stein, J. Bandovic, testing. T. 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