Purine-Rich Box-1–Mediated Reduced Expression of CD20

Research Article

Purine-Rich Box-1–Mediated Reduced Expression of CD20 Alters Rituximab-Induced Lysis of Chronic Lymphocytic Leukemia B Cells Amani Mankaı¨, 1 Anne Bordron,1 Yves Renaudineau,1 Christine Martins-Carvalho,1 Shinichiro Takahashi,2 Ibtissem Ghedira,3 Christian Berthou,1 and Pierre Youinou1

1‘‘Immunologie et Pathologie’’ Research Unit, Medical School, Brest, France; 2Department ofMolecular Hematology, University Graduate School ofMedical Science, Kitasato, Japan; and 3Immunology Research Unit, Faculty ofPharmacy, Monastir, Tunisia

Abstract sensitivity ofmalignant cells to CD5-induced (8) and IgM-induced apoptosis (9). The anti-CD20 monoclonal antibody rituximab has been less successful in treating chronic lymphocytic leukemia (CLL) In CLL, the B-cell marker CD20 is the ideal target for B-cell–depleting antibodies. Consequently, the first anti-B lym- than lymphoma, possibly due to the lower density of CD20 phocyte monoclonal antibody (mAb) approved by the Food and on B lymphocytes from CLL patients than on those from Drug Administration (10) to treat non–Hodgkin’s lymphoma lymphoma patients. This lowering may result from insuffi- (NHL) was an anti-CD20 mAb: rituximab. It was then tested in ciency of one of the transcription factors of cd20. Of these, CLL, but why the response rate to rituximab is lower in CLL purine-rich box-1 (PU.1) is poorly expressed in CLL. To than in NHL remains to be established (11). Theoretically, to kill estimate its weight in CD20 expression, pu.1 cDNA was the cells (12–14), rituximab combines apoptosis, complement- transfected into CLL B cells and shown to raise the mediated lysis (CML), and antibody-dependent cellular cytotox- membrane expression of CD20 and to improve the rituximab- icity (ADCC). One possibility is therefore that diverging response induced lysis of transfected cells. Granulocyte macrophage rates reflect different usages of effector mechanisms. If this colony-stimulating factor and all-trans-retinoic acids were interpretation is correct, variations in the treatment could be not involved in the defective expression of PU.1 or the due to low membrane expression ofCD20 in CLL compared excessive methylation of the pu.1 gene, because 6 of 14 with NHL (15). Briefly, the more rituximab molecules that bind CLL samples tested were normally methylated. This was to the cells increase the chances ofthe cells being killed and confirmed by the failure of DNA methyltransferase inhib- provide a rationale for increasing the density of CD20 on CLL B itors to restore pu.1 transcription in hypermethylated CLL, cells (16). and, in fact, the expression of PU.1 was down-regulated by One step further, the reduced expression of CD20 may result excessive expression of the FMS proto-oncogene–like tyro- from a defect in transcription factors (TF). Given interactions of sine kinase 3 (Flt3) receptor. This abnormality is consistent purine-rich box-1 (PU.1) with related binding sites in the with our finding of elevated levels of Flt3 ligand (FL) in promoter of cd20 (17), investigation ofthis TF is appropriate. 20 of 23 CLL sera tested. We propose that FL-dependent It belongs to a complex (18), containing also the PU.1-interacting increased Flt3 signaling prevents the expression of PU.1, partner (Pip), the octamer-binding protein 2 (Oct), and the B-cell which down-regulates that of CD20, and accounts for Oct-binding protein 1 coactivator (BOB). Transcription of cd20 is resistance of leukemic B cells to rituximab-induced lysis. initiated by the binding ofPU.1/Pip and Oct2/BOB.1 to its [Cancer Res 2008;68(18):7512–19] promoter. Combinatorial effects are then induced on granulocyte macrophage colony-stimulating factor (GM-CSF; ref. 19) and Introduction all-trans-retinoic acids (ATRA) by PU.1 (20), whereas BOB.1 Contrary to the long-held beliefthat chronic lymphocytic facilitates posttranslational modifications (21). In addition, the leukemia (CLL) results from accumulation of B cells (1), the FMS proto-oncogene–like tyrosine kinase 3 (Flt3) receptor current interpretation is that the increased growth rate ofthe represses PU.1 (22) and is in turn repressed by PU.1 (23). tumor has overtaken its decreased apoptosis (2). As CLL refers to Internal tandem duplication (ITD) in its juxtamembrane domain heterogeneous entities, delineation ofpatient groups with a given results in Flt3 ligand (FL)-independent activation ofFlt3 prognosis remains essential (3). For example, patients at risk ofa (reviewed in ref. 24), but this is normally activated by a cognate poor outcome are characterized by the expression ofCD38 and molecule termed FL. the absence ofVH gene mutations (4). This latter peculiarity The cd20 gene can be silenced by abnormal transcription, may be replaced (5) by the ratio oftranscripts forlipoprotein crippling mutations, methylation ofthe promoter ofone TF, or lipase (LPL) to those for a disintegrin and metalloproteinase methylation ofthe binding sites forone ofthem. Given the DNA 29 (ADAM29). Severity is also associated with expression of methyltransferase (DNMT)-induced widespread nonrandom CpG a full-length form of CD79b (6), presence of ZAP70 (7), and island methylation in CLL (25), the possibility exists that pu.1 transcription is repressed by such methylation. Transfection of pu.1 cDNA into CLL B cells up-regulates CD20 expression and improves rituximab-induced CML and ADCC. Rather than Note: A. Mankaı¨and A. Bordron contributed equally to this work. C. Berthou and P. Youinou contributed equally to this work. methylation-mediated blockade ofthe gene, excessive engagement Requests for reprints: Pierre Youinou, Laboratory ofImmunology, Brest ofFlt3 may preclude transcription of pu.1 and thereby diminish University Medical School Hospital, BP 824, F 29609 Brest, France. Phone: 33-2-98- transcription of cd20. We failed to detect ITP and observed that 22-33-84; Fax: 33-2-98-22-38-47; E-mail: [email protected]. I2008 American Association for Cancer Research. the level ofFL is elevated in the majority ofthe CLL sera. This doi:10.1158/0008-5472.CAN-07-6446 increase might engage Flt3 in leukemia.

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Materials and Methods compensate for inconsistency. In experiments on PU.1-induced expression ofCD20, CD72 (stained with PE-anti-CD72 mAb fromAbcam) and CD5 Patients and controls. Thirty-six untreated patients fulfilling the criteria (stained with PC5-anti-CD5 mAb from Beckman Coulter) were taken as for the diagnosis of CLL (26) participated in the study. At disease stage A, positive and negative controls, respectively. there were 16 men plus 9 women ranging in age from 54 to 82 years, and at Functional B lymphocyte subsets can be distinguished using combina- disease stage B, there were 4 men plus 7 women ranging in age from 55 to tions ofmarkers. In particular, the relative expression ofIgD and CD38 83 years (3). Circulating B cells from two patients with mantle cell has led to a model for mature B-cell homeostasis from naive Bm1 cells lymphoma and another two with marginal zone lymphoma served as + À + + (IgD , CD38 ) to activated Bm2 cells (IgD , CD38 ), germinal center founder disease controls (27). Venous blood was also collected from 14 healthy Bm2’ cells (IgD+, CD38++), centroblast Bm3 and centrocyte Bm4 cells volunteers, and tonsils from 7 children at elective surgery. Informed consent À À À (IgD , CD38++), and early memory and memory (IgD , CD38 ) Bm5 cells was obtained from CLL patients, normal volunteers, and the guardians of (28). Quadruple combinations ofmarkers were devised to phenotype the children. The protocol was approved by the Institutional Review Board tonsillar B-cell–enriched suspensions. They were stained with PC7-anti- at Brest University. CD5, FITC-anti-CD20, PE-anti-IgD (BD PharMingen), and PC5-anti-CD38 Cell preparation. Peripheral blood mononuclear cells (PBMC) were mAb. CD5+ B cells were gated and distributed into CD20low and CD20high separated by density gradient centrifugation, and B lymphocytes were populations. Each ofthem was subdivided according to the expression of purified using a B-cell isolation kit (Miltenyi). Tonsil single-cell suspensions IgD and CD38. To serve as an additional control, normal blood was were filtered through a 70-mm nylon mesh strainer, and B lymphocytes stained with PE-anti-CD3, PC5-anti-CD19, and PC7-anti-CD5. The MFI of were subsequently purified as above. B-cell–enriched populations were then À FITC-anti-CD20 was then measured in CD3 CD19+CD5+ cells. incubated with unconjugated anti-CD5 mAb (BD PharMingen). For further Cell culture. B cells were seeded at 5 Â 106 per well and maintained studies, goat anti-mouse IgG antibody-coated magnetic beads selected À in 200 AL ofRPMI 1640 with 10% FCS (LifeTechnologies), 2 mmol/L distinguished negatively the CD5 B cells and positively the CD5+ B cells. L-glutamine, and antibiotics. The cells were incubated for 24 h alone, with Natural killer (NK) cells for ADCC were isolated from normal PBMC 5,000 to 10,000 units ofGM-CSF (R&D Systems) or with 1 to 100 Amol/L of by removing non-NK cells using the Miltenyi NK cell isolation kit. Their ATRA. Methylation was inhibited by 10 to 50 Amol/L of5-azacytidine or purity was verified by fluorescence-activated cell sorting (FACS) analysis procainamide (both from Sigma). using three mAbs: FITC-anti-CD3, phycoerythrin (PE)-anti-CD56, and Western blot analysis. For Western blot analysis, 2 Â 107 cells were PE-cyanin 7 (PC7)-anti-CD16 (Beckman Coulter). lysed by incubation for 30 min at 4jC with Triton X-100 in a buffer made Staining with FITC-anti-CD4, PC7-anti-CD8, and PE-anti-CD19 of140 mmol/L NaCl, 1 mmol/L EDTA, and Tris-HCl and supplemented mAb established that isolated B cells were 98% pure, and staining with with 1 mmol/L phenylmethylsulfonyl fluoride, 10 Ag/mL aprotinin, and FITC-labeled Annexin V (Beckman Coulter) that they were 98% viable. 1 mmol/L sodium orthovanadate. FACS analyses. Forward and side scatter gates were set to acquire 5,000 The lysates were centrifuged at 10,000 Â g at 4jC for 10 min, run on 10% events per experiment. Unstained cells and isotype-matched FITC-, PE-, SDS-PAGE, and blotted to nitrocellulose membrane (Bio-Rad). Unbound PC7-, and PC5-labeled irrelevant mAbs were used as internal controls. The sites were quenched with 5% nonfat dry milk in 10 mmol/L Tris, 0.1% mean fluorescence intensity (MFI) of CD20 on B cells was measured by Tween 20 for 1 h, washed thrice in the same buffer, and probed with mouse FITC- or PC5-conjugated anti-CD20, and that ofFlt3 by PE-anti-CD135 anti-CD20 (BD PharMingen), anti-PU.1 (Santa Cruz Biotechnology), anti-Pip (BD PharMingen). The results were validated by calibration beads to (Ozyme), and anti-actin or anti-BOB.1 (Sigma) mAb or with rabbit anti-Oct2 (Abcam) antibody for 1 h. After three more washes, membranes were incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse Table 1. Primers used in RT-PCR or goat anti-rabbit immunoglobulin (both from Jackson ImmunoResearch Laboratories)for1h.Boundanti-CD20mAbwasrevealedwith 5-chloronaphtol, and other bound antibody by enhanced chemilumines- ¶ ¶ 18S sense 5 -GGCTACCACATCCAAGGAAGG-3 cence (ECL). Absorbances ofspecificbands were normalized to actin and ¶ ¶ 18S antisense 5 -CCAATTACAGGGCCTCGAAAG-3 quantified using the Biocapt Express software. ¶ ¶ CD20 sense 5 -CCAATTACAGGGCCTCGAAAG-3 The lysates ofleukemic B cells were subjected to immunoprecipitation ¶ ¶ CD20 antisense 5 -CCAATTACAGGGCCTCGAAAG-3 with rabbit anti-Flt3 antibody (Ozyme), resolved on 10% SDS-PAGE, ¶ ¶ PU.1 sense 5 -GTGCCCTATGACACGGATCT-3 transferred to polyvinylidene difluoride membrane (Amersham), ¶ ¶ PU.1 antisense 5 -GTAGAGGACCTGGTGGCC-3 and probed with antibody to Flt3 and tyrosine phosphorylated Flt3 ¶ ¶ Pip sense 5 -CCCAGCTTGTGAAAATGGTT-3 (BD Biosciences; ref. 29) and revealed as above. The negative and ¶ ¶ Pip antisense 5 -TCAGCTCCTTCACGAGGATT-3 the positive controls were, respectively, tonsillar B cells and acute ¶ ¶ Oct2 sense 5 -AGCCCTCAAGGCAGCCACT-3 lymphoblastic cell line cells, referred to as SEM cells and kindly donated ¶ ¶ Oct2 antisense 5 -TTCAAGAAGAGCGGCGAGGT-3 by Dr. O. Heidenreich (University ofTu¨bingen, Tu¨bingen, Germany). ¶ ¶ BOB.1 sense 5 -TGTGAAGCCAGTGAAGG-3 Quantitative reverse transcription-PCR. The RNeasy Mini kit (Qiagen) ¶ ¶ BOB.1 antisense 5 -AACACTGAGGAGGGCCCCA-3 was used to extract mRNA. Its integrity was verified by ethidium bromide ¶ ¶ Flt3 sense 5 -CACGGGAAAGTGGTGAAGAT-3 staining ofthe 18S and 28S species on agarose gel electrophoresis. mRNA ¶ ¶ Flt3 antisense 5 -GGAATGCCAGGGTAAGGATT-3 (1 Ag) was reverse transcribed into cDNA using a High-Capacity cDNA ¶ ¶ Flt3 ligand sense 5 -AGCCCAACAACCTATCTCCT-3 Archive kit with random hexamers according to the manufacturer’s ¶ ¶ Flt3 ligand antisense 5 -GTCTGGACGAAGCGAAGACA-3 instructions (Applied Biosystems). ¶ ¶ DNMT1 sense 5 -CGGTTCTTCCTCCTGGAGAATGTCA-3 Quantitative reverse transcription-PCR (RT-PCR) was performed with the ¶ ¶ DNMT1 antisense 5 -CACTGATAGCCCATGCGGACCA-3 ABI PRISM 7000 Sequence Detector where the 18S rRNA gene enabled to ¶ ¶ DNMT3A sense 5 -CTCCTGTGGGAGCCTCAATGTTACC-3 normalize mRNA. Some primers have been described (30), and the ¶ ¶ DNMT3A antisense 5 -CAGTTCTTGCAGTTTTGGCACATTCC-3 remainder were designed using Primer Express software version 2.0 ¶ ¶ DNMT3B sense 5 -CTCGAAGACGCACAGCTGACGAC-3 after Genbank sequences (Table 1): NM_001379 for DNMT1, BC043617 ¶ ¶ DNMT3B antisense 5 -CCTATAACAACGGCAAAGACCGAGC-3 for DNMT3A, AF331857 for DNMT3B, NR_003286 for 18S, NM_152866 ¶ ¶ LPL sense 5 -GGGCATGTTGACATTTACCC-3 for CD20, X52056 for PU.1, NM_002460 for Pip/IRF4, NM_002698 for ¶ ¶ LPL antisense 5 -AGCCCTTTCTCAAAGGCTTC-3 Oct2, and NM_004119 for Flt3. Temperatures were 50jC for 2 min, 95jC ¶ ¶ ADAM29 sense 5 -GACCAGGGTGCTATCCTTGA-3 for 10 min, 95jC for 15 s, and 60jC for 1 min for 40 cycles. Numbers ¶ ¶ ADAM29 antisense 5 -GGGGCTTGATTTCATAAGCA-3 oftranscripts were determined fromthreshold cycle numbers, the standard curves were normalized to 18S, and the levels were www.aacrjournals.org 7513 Cancer Res 2008; 68: (18). September 15, 2008

Figure 1. Expression of CD20, PU.1 (plus Pip), and Oct2 (plus BOB.1) in CD5+ B cells from CLL patients compared with CD5+ B cells from normal blood, control tonsils, and non-CLL B-cell lymphomas. A, blotted B-cell extracts were probed with anti-CD20 mAb, developed with HRP-anti-mouse antibody, and measured by densitometry. Oct2, BOB.1, PU.1, and Pip were probed with mouse or rabbit antibody, developed with HRP-labeled goat anti-mouse or goat anti-rabbit antibody, and quantitated by ECL. B, left, B-cell–enriched suspensions were stained with PC5-anti-CD5 and FITC-anti-CD20. The MFI of CD20 was then measured in gated CD5+ B cells. From the left to the right, CD5+ B cells from CLL (black histogram), normal blood and control tonsil (white histograms), and non-CLL malignantB cells (gray histograms). Right, tonsillar B-cell–enriched populations were stained with PC7-anti-CD5, FITC-anti-CD20, PE-anti-IgD, and PC5-anti-CD38. The CD5+ B cells were gated and separated into CD20low and CD20high subpopulations. Each subpopulation was distributed into Bm1 through Bm5 cells according to their relative expression of IgD and CD38. C, left, low expression of CD20 in CD5+ (white histogram) and bimodal expression of CD20 in CD5À B cells (black histogram) from control tonsils; right, similar expression of CD20 in CD5+ (white histogram) and CD5À B cells (black histogram) from normal blood. D, correlation between CD20 mRNA and PU.1 mRNA in CLL B cells.

expressed relative to those oftranscripts in normal B cells, which were Transfection of malignant B cells. The method for isolating the pu.1 assigned a value of1. probe has been described elsewhere (32). It was inserted into a pcDNA-3.1 PCR analyses of pu.1 promoter methylation and ITD of flt3 and FL. expression plasmid, and two suspensions ofCLL B lymphocytes were The methylation status ofthe pu.1 promoter was determined essentially electroporated at 40% efficiency using the B-cell nucleofactor kit (Amaxa). as described by Ushmorov and colleagues (31). Briefly, genomic DNA was The first suspension was transfected with pu.1-containing pcDNA-3.1 purified using a DNA blood kit (Qiagen), and 50 ng were digested with constructs, and the second with empty pcDNA-3.1 vectors. Both were 20 units of MspIorHpaII (Invitrogen) for 3 h at 37jC. Then, 2 AL ofthe cotransfected with green fluorescent protein (GFP)-containing pMax vector. digest were amplified with 5¶-TTAGCCCCCAAAGTCATCCCTCTCA-3¶, plus After a 24-h culture, CD20 and PU.1 were evaluated at the mRNA level by 5¶-ACCCTTCCATTTTCGACTCCTGTAAC-3¶, to flank the 5¶-CCGG-3¶ bind- quantitative RT-PCR, and at the protein level by FACS. ing sites ofthe pu.1 promoter. PCR comprised denaturation at 94jCfor Killing assays. B lymphocytes from patients and controls were 5 min, 40 cycles at 94jC for 30 s, 62jC for 1 min, 72jC for 1 min, and a distributed into three groups ofaliquots of10 6 B cells each. The first 10-min final extension at 72jC. The products were resolved in 1% agarose group served for apoptosis, the second for CML, and the third for ADCC. In gel and stained with ethidium bromide. each group, aliquots underwent a 4-h incubation with rituximab (Roche) at For analysis ofITD ofthe flt3 gene, 50 ng ofgenomic DNA were amplified the doses of0, 1, 10, 25, and 50 Ag/mL, respectively, and lysis ofaliquots with 5¶-CAATTTAGGTATGAAAGCC-3¶ plus 5¶-CAAACTCTAAATTTTCTC-3¶ without rituximab was subtracted from that of the rituximab-containing in 50 AL reaction volume by 10 cycles at 95jC for 1 min, 50jC for 1 min, and aliquots with rituximab. 72jC for 1 min followed by 35 cycles at 95jC for 1 min, 47jC for 30 s, and In the CML assay, B cells were incubated with 15% human AB serum for 72jC for 1 min, ending with a final extension at 72jC for 10 min. The final 6 h. Propidium iodide (PI) was added 10 min before harvest for FACS products were checked as above. The wild-type allele was f129 bp, and the analysis. In the ADCC assay, B cells were incubated with 106 NK cells for ITD allele ranged from 149 to 240 bp. 6 h. In the apoptosis assay, B cells were incubated in medium without To validate our search for mutations in the flt gene, the promyelocytic serum and evaluated by FACS analysis through a combination ofPI and leukemia cell line HL-60 (American Type Culture Collection) and the acute PE-Annexin V. monocytic leukemia cell line MV4-11 (gift of G. Kromer, Institut National de ELISA for FL. The level ofFL was measured in 23 sera fromCLL patients la Sante et de la Recherche Medicale, Paris, France) were taken as positive and 10 sera from normal volunteers. The sandwich ELISA kit from R&D and negative controls, respectively. Primer pairs specific for FL were also Systems was used according to the manufacturer’s instructions. Values used to amplify a 352-bp sequence in B cells from two CLL samples and two greater than 75 pg/mL (i.e., above 3 SDs ofthe mean of30 normal sera) were normal tonsils. considered to be increased.

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Statistical analyses. Results are expressed as mean F SD. Comparisons circles). These data suggest that PU.1 might affect CD20 were made using the Mann-Whitney U test for unpaired data and the expression in CLL, although its expression did not correlate Wilcoxon test for paired data. Correlations were established using the with the disease stage or the VH mutational status (Table 2). Spearman’s test. We found less PU.1 protein (Fig. 1A) in CLL than in normal blood and tonsil B cells [0.02 F 0.01 versus 0.30 F 0.05 (P < 0.05) Results and versus 0.25 F 0.04 (P < 0.05)]. Compared with CLL, in Expression of CD20 at the mRNA and protein levels. There non-CLL B-cell lymphoproliferative disorders, there were was less CD20 mRNA in CD5+ B cells ofCLL blood than in CD5 + more transcripts for CD20 (1.28 F 0.29 versus 0.17 F 0.14; B cells ofnormal blood and control tonsils. To compensate P < 0.05) as well as for PU.1 (0.66 F 0.11 versus 0.08 F 0.01; for inconsistency, a value of 1 was assigned to the mean of P < 0.05). 14 normal samples ofblood B cells, and other samples were rated In contrast, Oct2, BOB.1, and Pip had comparable levels of at 1.8 F 0.09 in 7 control tonsils and 0.17 F 0.14 in 36 CLL patients mRNA and protein in CLL and normal blood CD5+ B cells. They (P < 0.01, compared with normal blood and tonsils). were also unrelated to disease stage and VH mutational status Western blot analysis detected fewer CD20 molecules (Fig. 1A) (Table 2). The absence ofdifferenceindicates that the reduction in in CLL than in normal blood and tonsil B cells [0.02 F 0.01 the density ofCD20 could not be ascribed to any ofthese three versus 0.22 F 0.05 (P < 0.01) and versus 0.29 F 0.04 (P < 0.01)]. factors. Defective expression of CD20 was confirmed by FACS analysis PU.1-induced expression of CD20. Next, we explored (Fig. 1B, left), where the CD20 MFI ofgated CD5 + B cells was 10.8 F the possible role ofPU.1 in the decrease in the expression of 1.1 in CLL, 41.2 F 7.2 in normal blood (P < 0.01, compared with CD20 in CLL B lymphocytes. To this end, B cells from eight patients CLL), and 55.5 F 7.2 in control tonsils (P < 0.01, compared with were transfected with GFP-pu.1–containing pcDNA-3.1 constructs CLL). Contrary to CLL B cells, CD20 was overexpressed in B cells or with empty vectors. Transfection of pu.1 enhanced the from lymphoma. transcription of pu.1 (P < 0.05, between pu.1-containing constructs Clearly, the CD20 MFI distinguished two populations ofton- and empty vectors in Fig. 2A). This promoted an increase in sillar B cells. The presence ofCD38 and the absence ofIgD the expression ofCD20 at the mRNA ( P < 0.05 between pu.1- (Fig. 1B, right) localized the CD20high B cells within the Bm3 containing constructs and empty vectors in Fig. 2A) and the centroblast/Bm4 centrocyte population (28). Interestingly, there protein levels (P < 0.05, between pu.1-containing constructs and À were more CD20 molecules in CD5 (Fig. 1C, black histograms) empty vectors in Fig. 2B). Consequently, increases in PU.1 mRNA than in CD5+ B cells (Fig. 1C, white histograms) from normal blood correlated to increases in CD20 mRNA (P < 0.05 in Fig. 2A). An and tonsils. example is shown in Fig. 2C (left), where the specificity of PU.1 Reductions in the number ofmRNA copies forCD20 were not was confirmedby raised expression ofCD72, the gene ofwhich is different in the two disease stages studied in our patient group, controlled by PU.1 (33), in contrast to CD5, the gene ofwhich is neither were they in patients with a different VH mutational status, not (34). as defined by the LPL/ADAM29 ratios (Table 2). A high density of CD20 improves the rituximab-induced Expression of the PU.1/Pip and the Oct2/BOB.1 complexes. lysis. A standing question is whether raised expression ofCD20 The role ofsome TFs in the reduced expression ofCD20 in CLL ameliorates the rituximab-mediated cell lysis. CML (Fig. 3A, left) was then determined. There were fewer transcripts for PU.1 in and ADCC (Fig. 3B, left) were significantly increased in a dose- CLL than in control B cells (Fig. 1A), and therefrom a correlation dependent manner [Fig. 3A (right) and B (right)]. Changes in (P < 0.01) between PU.1 mRNA and CD20 mRNA (Fig. 1C) in CLL surface expression of CD20 (7.5 F 5.4%) correlated with improve- (Fig. 1D, closed circles) but not in lymphoma (Fig. 1D, open ments ofCML (6.1 F 3.1%; P < 0.05) as well as with those of

Table 2. Transcripts for CD20, PU.1, Oct2, Pip, and Flt3 in B cells from 36 CLL patients classified according to stage and mutational status

mRNA Stage Mutational status

A B Mutated Unmutated

n =25 n =11 n =26 n =10

CD20 0.22 F 0.48 0.23 F 0.30 0.24 F 0.25 0.20 F 0.20 PU.1 0.49 F 0.30 0.68 F 0.53 0.52 F 0.87 0.62 F 0.44 Oct2 1.76 F 1.72 1.48 F 1.29 1.54 F 1.53 1.70 F 1.58 Pip 1.34 F 0.98 1.87 F 1.56 1.37 F 1.03 1.53 F 0.9 BOB.1 1.28 F 0.54 1.23 F 0.48 1.32 F 0.63 1.26 F 0.31 Flt3 19.8 F 34.8 6.85 F 4.7 19.05 F 34.06 7.52 F 8.13

NOTE: No significant differences were seen between stage A and stage B, and between VH mutated and VH unmutated B cells. cDNAs were subjected to quantitative RT-PCR (for the primers, see Table 1). Their relative expression was adjusted to 18S levels and compared with the transcript levels in B cells from normal blood (assigned an arbitrary value of 1).

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Figure 2. CD20 in CLL B cells following transfection with pu.1-containing construct or empty vector. A, transfection of CLL B cells with pu.1 cDNA augments the transcription of pu.1 and cd20, as shown by fold increases of the ratios of cd20 to 18S following transcription with pu.1-containing constructs, relative to the ratios of cd20 to 18S following transcription with empty vectors (left), and fold increases of the ratios of pu.1 to 18S following transcription with pu.1-containing constructs, relative to the ratios of pu.1 to 18S following transcription with empty vectors (right). B, the MFI of CD20 was increased in transfected (black columns) compared with untransfected (white columns) B cells. C, the effect of the PU.1 TF on cd20 was confirmed by unchanged CD5 butincreased CD72 ( dotted lines for histogram before transfection and solid lines for histograms after transfection).

ADCC (21.4 F 8.7%; P < 0.05). Not surprisingly, apoptosis was Is pu.1 methylation regulated? Because both GM-CSF (19) and not influenced by the expression level of CD20, as efficient ATRA (20) increase the expression ofPU.1 in myeloid cells, we rituximab-induced apoptosis requires cross-linking ofrituximab looked for similar mechanisms in B cells. Although GM-CSF and with a second-layer antibody (35). ATRA were added at doses as high as 10,000 units and 100 Amol/L,

Figure 3. Increase in the density of CD20 improves the efficacy of rituximab (RTX). A, left, individual values of CML with 25 Ag/mL rituximab; right, dose-dependent effects of rituximab on CML. B, left, individual values of ADCC with 25 Ag/mL; right, dose-dependent effects of rituximab on ADCC. Black columns, pu.1-containing constructs; white columns, empty vectors.

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Figure 4. DNMT-induced methylation of the pu.1 promoter in CLL and normal blood B cells, and absence of effects of the DNMT inhibitors azathioprine (aza) and procainamide acid (PcA). A, left, the methylation status was determined by PCR of genomic DNA submitted to 10 to 50 Amol/L of methylation-sensitive HpaII or methylation-insensitive MspI; right, Pu.1 promoter methylation after treatment with azathioprine or procainamide acid. B, left, increased expression mRNA for Flt3 in CLL patients compared with normal controls; right, B-cell–enriched preparations from CLL patients (black histogram) and normal donors (white histogram with solid line) were stained with PE-anti-CD135 to determine the membrane expression level of FL relative to the control isotype (white histogram with dotted line). C, left, phosphorylation of Flt3 immunoprecipitated from CLL B cells, resolved by SDS-PAGE, and Western blotted for a phosphorylated-specific antibody. B cells from control tonsils and SEM line cells derived from a patient with acute myeloblastic leukemia, served as negative and positive controls, respectively. Right, Flt3 ITD is absent in CLL. The HL-60 line is the positive control, and the HV4-II line is the negative control. D, the serum levels of FL were found by ELISA to be higher than 3 SD above the mean of 30 normal sera (horizontal line denotes the cutoff level) in 20 of 23 CLL patients and none of 10 healthy volunteers.

respectively, the levels ofmRNA forPU.1 and CD20 were Flt3 kinase receptor is overexpressed and activated. unchanged. In other words, these agents failed to modify We found 15.8-fold more mRNA for Flt3 in CLL than in normal B membrane expression ofCD20. cells (Fig. 4B, left), and protein in excess was revealed by FACS Alternatively, undue activity ofDNMTs could methylate and analysis ofCLL B cells (Fig. 4 B, right). This is consistent with the thereby silence the pu.1 gene in CLL B cells. Supporting this view, restriction in pu.1 expression by Flt3 signaling (36) and the low À À DNMT1 and DNMT3A were 2.0 F 0.9–fold and 1.8 F 0.7–fold levels ofFlt3 transcripts in PU.1 / fetal liver hematopoietic increased, respectively, in 10 CLL patients relative to 4 healthy progenitors (37). Ofintriguing note, the amount ofmRNA forFlt3 controls. We investigated this further by evaluating DNMT-induced was higher in stage A than stage B patients (P < 0.01) and in VH methylation ofthe pu.1 promoter. The assay was based on the mutated than VH unmutated cases (P < 0.01). inability ofsome enzymes to digest a methylated 5 ¶-CCmGG-3¶ site. In addition to its overexpression, Flt3 was activated. As expected, Genomic DNA ofB cells from14 CLL patients and 4 healthy Western blot analysis ofimmunoprecipitate (Fig. 4 C, left) detected controls was digested with the methylation-sensitive enzyme HpaII a 130-kDa nonglycosylated and a 160-kDa glycosylated bands of or the methylation-insensitive enzyme MspI and subsequently Flt3. The anti-phosphorylated antibody showed that both forms amplified (Fig. 4A, left). were activated, although normal tonsillar B cells were negative and The group of14 patients was distributed into a first subgroup of SEM cells were positive. 8 individuals whose CpG included in the restriction sites were Two explanations deserve special attention. The first is the methylated, and a second subgroup of6 individuals whose CpG were FL-independent activation where ITD activates Flt3 and, by doing not methylated. To confirm such a dichotomy, five methylated CLL so, prevents the expression ofPU.1 (38). This option was samples were incubated with DNMT inhibitors after pilot experiments invalidated by the absence ofITD on genomic DNA (Fig. 4 C, had ensured that they did not alter cell viability (Fig. 4A, right). Three right). The second option is the FL-dependent phosphorylation. nonmethylated CLL samples served as negative controls. Although This seemed to be likely, as the serum levels ofFL (reviewed in ref. favoring the transcription of pu.1, DNMT blockade did not restore 24) were elevated (Fig. 4D) in 20 of23 CLL sera compared with expression of CD20. Furthermore, there was hardly any difference in none of10 sera fromhealthy volunteers. Our pilot experiments the MFI ofCD20 comparable in methylated and unmethylated groups reveal more transcripts for FL in CLL than in normal B cells (6.66 F 3.86 versus 5.83 F 1.85). That is, aberrant methylation ofthe (data not shown). Further experiments are currently in progress pu.1 gene does not explain its repression in CLL. to gain insight into this mechanism. www.aacrjournals.org 7517 Cancer Res 2008; 68: (18). September 15, 2008

Discussion presence ofVH gene mutations (4). However, this comparing of Controversy persists over the relationship between efficacy of facts is contradicted by the absence of correlation between PU.1 the anti-CD20 mAb treatment and expression level ofits CD20 expression and the stage or the mutational status ofCLL B cells, target. A correlation has indeed been reported by some (39) but not suggesting that additional agents interfere with the relationship all groups (40). In view ofthe scarce expression ofCD20 by CLL B between pu.1 and Flt3. Indeed, PU.1 functions as a transcription cells (15), we postulated that enhancing its expression might repressor for the flt3 gene (23), and, in turn, its deficiency improve their susceptibility to rituximab. To assess this possibility, up-regulates the expression ofFlt3. In either case, defectivePU.1 is we transfected malignant cells with pu.1 cDNA. The density of an independent prognostic factor for CLL. Flt3 in excess might be CD20 was enhanced, and indeed, rituximab-induced CML and due to overproduction ofFL, in an autocrine loop forthe Flt3/FL ADCC were improved accordingly. system, as put forward by Brasel and colleagues (37). Expectedly, We thus wanted to decipher the mechanisms ofthe lowered Flt3 inhibition would release PU.1 and thereby increase the expression ofCD20 in CLL. Because one way to modulate a protein expression ofCD20. Unlike acute myeloblastic leukemia, Flt3 was synthesis is to interfere with its TF, we first evaluated the internalized and degraded by 5 min in leukemic B lymphocytes as importance ofsome TFs previously shown to act on cd20. Whereas well as normal B lymphocytes. Similar results were obtained in the Pip cofactor of PU.1, Oct2, and its cofactor BOB.1 were normal, three separate experiments so that the Flt3 blockade experiment PU.1 was defective, and the following expression of CD20 was rendered impossible. However, our pilot experiments showed diminished. Consistent with our interpretation, the transcription that the expression ofmRNA forFL was higher in leukemic cells ofPU.1 mRNA was elevated in other non-CLL malignant. compared with normal B cells. Transfection confirmed the role of PU.1 in the regulation of More effective strategies are required in the treatment of CLL CD20 by the presence ofeffecton the positive control CD72 (33) with rituximab. Recent findings have indicated that rituximab- and its absence ofeffectson the negative control CD5 (34). opsonized B cells are sequestered by the reticuloendothelial system Because our goal was to normalize CD20 expression on CLL B and released back into the circulation after the rituximab-CD20 lymphocytes, the results ofthe firstset ofexperiments focused complexes have been taken up by the phagocytes (44). To prevent our attention on PU.1 up-regulating agents. Like several groups the CD20 loss, and enhance targeting, the use oflow doses of (see ref. 41 for example), we failed to modify the expression of rituximab has been advised (45). Additional insight comes from the PU.1 and CD20 using GM-CSF or ATRA. Thus, normalization demonstration in a mouse transgenic for the human cd20 gene, ofPU.1 by ATRA in myeloid leukemic blasts (20), and the absence that resistance to CD20 involves circulatory dynamics, as access of ofnormalization in CLL B lymphocytes, reflects differences B cells to the circulation is required (46, 47). Finally, aberrant between the mechanisms activating the pu.1 promoter in the expression ofB-cell activating factorofthe tumor necrosis factor former and the latter cells (42). family (BAFF) by CLL B cells might be an additional mechanism for In addition, our data raise the issue ofthe mechanisms survival (48). This excess accounts for B-cell resistance to rituximab restricting the transcription of pu.1. It is unclear as to what TF is (49) and suggests to associate inhibitors ofBAFF with rituximab. involved because the relative contribution ofthe growth factorsto In conclusion, our beliefis that these results, although this transcription is not fully understood. Gene regulation was preliminary, are robust enough to provide a strong basis for future therefore considered, and it made sense to compare transcripts for work. Another area that requires further understanding is the role DNMT in leukemic and normal B cells because one ofthe ofFlt3 in the down-regulation ofPU.1. These findingsmay provide hallmarks ofmalignant B cells is hypermethylation ofDNA (24). new opportunities in the treatment ofB-cell malignancies. Despite the high expression level ofDNMT, the CpG islands ofthe pu.1 promoter were hypermethylated in only 8 ofthe 14 CLL cases Disclosure of Potential Conflicts of Interest tested. Such a variability renders methylation an unlikely mechanism for pu.1 repression. Not only may demethylation of No potential conflicts of interest were disclosed. pu.1 be assumed to be unnecessary to normalize CD20 expression, simply because some CLLs are not methylated, but it is also Acknowledgments insufficient because blockade of DNMT did not restore CD20 Received 11/30/2007; revised 5/2/2008; accepted 6/2/2008. expression. Grant support: ‘‘La Ligue Contre le Cancer’’ and ‘‘Association Ce´lineet Ste´phane.’’ Instead ofmethylation, CD20 expression might be inhibited by The costs ofpublication ofthis article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement in accordance Flt3. Consistent with this option is our finding of more Flt3 in stage with 18 U.S.C. Section 1734 solely to indicate this fact. A than in stage B patients and in VH mutated than VH unmutated We thank Drs. G. Kromer and O. Heidenreich for the generous gift of cell lines, Dr. E. Hardy for technical help, C. Se´ne´ and S. Forest for expert secretarial assis- cases. The influence of Flt3 on CD20 is reminiscent of the tance, and R. Budd (Birmingham, United Kingdom) for help with the writing of the association ofa low CD20 expression (43) with stage A and with the article.

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