16 in Chronic Lymphocytic Leukemia

ORIGINAL ARTICLE Defective DROSHA processing contributes to downregulation of MiR-15/-16 in chronic lymphocytic leukemia

D Allegra1,2, V Bilan1, A Garding1,3,HDo¨ hner1, S Stilgenbauer1, F Kuchenbauer1 and D Mertens1,2

The MIR-15A/-16-1 tumor suppressor microRNAs (miRNAs) are deleted in leukemic cells from more than 50% of patients with chronic lymphocytic leukemia (CLL). As these miRNAs are also less abundant in patients without genomic deletion, their downregulation in CLL is likely to be caused by additional mechanisms. We found the primary transcripts (pri-miRNAs) of MIR-15a/- 16/-15b to be elevated and processing intermediates (precursor miRNAs) to be reduced in cells from CLL patients (22/38) compared with non-malignant B-cells (n ¼ 14), indicating a block of miRNA maturation at the DROSHA processing step. Using a luciferase reporter assay for pri-miR processing we validated the defect in primary CLL cells. The block of miRNA maturation is restricted to specific miRNAs and can be found in the cell line MEC-2, but not in MEC-1, even though both are derived from the same CLL patient. In these cells, the RNA-specific deaminase ADARB1 leads to reduced pri-miRNA processing, but full processing efficiency is recovered upon deletion of the RNA-binding domains or nuclear localization of ADARB1. Thus, we show that, apart from genomic deletion or transcriptional downregulation, aberrant processing of miRNA leads to specific reduction of miRNAs in leukemic cells. This represents a novel oncogenic mechanism in the pathogenesis of CLL.

Leukemia (2014) 28, 98–107; doi:10.1038/leu.2013.246 Keywords: miRNA; DROSHA; miR-15; miR-16; chronic lymphocytic leukemia; CLL; ADARB1

INTRODUCTION includes the MIR-15B/-16-2 genes localized in an intron of the MicroRNAs (miRNAs) are short non-coding RNAs which regulate SMC4 gene (Figure 1b). MIR-15B and MIR-16-2 are the only two post-transcriptional silencing and thereby modulate physiological other miRNA genes of the MIR-15/-16 family that are highly 32 and pathophysiological mechanisms such as leukemogenesis.1–3 expressed in B cells and produce a mature miR-16 and miR-15b, Their properties make them ideal technical tools4 and suited, which is closely related to miR-15a. Thus, the cluster MIR-15B/-16-2 for example, as biomarkers.5–7 MiRNAs are transcribed as long might be functionally redundant with MIR-15A/-16-1 and could primary precursors (pri-miRNAs), which are cleaved in the nucleus compensate for the genomic loss of MIR-15A/-16-1, especially as 18 by DROSHA in combination with cofactors like DGCR8. deletions in 3q25 are not frequent in CLL. The product of pri-miRNA cleavage is the precursor miRNA There is accumulating evidence that miR-15a and miR-16-1 act (pre-miRNA), which is exported to the cytoplasm and further as tumor suppressors in CLL: monoallelic deletion of the MIR-15A/- cleaved by DICER1 into a functioning miRNA:miRNA* complex.8 16-1cluster in mice results in the accumulation of clonal CD5 þ 33 MiRNA maturation is controlled by growth factors, hormones and B cells and in a CLL-like disease. The low penetrance of this other stimuli.9 pathologic cell accumulation is increased by either the deletion of 34 33 Chronic lymphocytic leukemia (CLL) is characterized by an a larger region or by the deletion of both copies of 13q14.3. accumulation of mature CD5 þ B cells that are dependent on This higher penetrance upon deletion of both copies suggests microenvironmental support10,11 and that often harbor chromosomal that in CLL cells with the deletion of one copy of 13q14.3 only, aberrations12,13 and genetic mutations.14–16 The most frequent additional mechanisms like post-transcriptional regulation that aberration occurs at chromosomal band 13q14.3, which is deleted reduce levels of 13q14.3 candidate tumor suppressor genes in 450% of CLL patients.17,18 In addition, in 13q14.3 a complex including MIR-15a/-16-1 would lead to a more aggressive cellular epigenetic regulatory mechanism has been identiﬁed that involves phenotype of CLL cells and a selective advantage. the two large non-coding RNA genes DLEU1 and DLEU219–21 and Several factors might inﬂuence expression of the MIR-15/-16 leads to downregulation of neighboring candidate tumor suppressor family miRNAs in CLL: transcription of SMC4 is activated by nuclear genes in-cis.22 The critical region in 13q14 is also recurrently lost in factor-kB35 and E2F1, which also stimulates DLEU2 expression.36 mantle cell lymphoma,23–25 prolymphocytic leukemia26 and also in a In contrast, c-myc represses the transcription of DLEU2.37 There is number of solid tumor entities listed in.27 Thus, 13q14.3 likely harbors evidence that also post-transcriptional regulation might contribute one or more tumor suppressor genes.28 to the decrease of miR-15/-16 levels in CLL: a mutation in pri-miR- The DLEU2 gene localized in the critical region is the host gene 16-1 found in two CLL cases has been postulated to have an impact of the miRNA cluster MIR-15A/16-1.29–31 Of interest, a cluster of on pri-miR processing.38,39 A corresponding mutation is present in genes homologous to the 13q14.3 candidate tumor suppressor New Zealand Black mice, which spontaneously develop CLL and genes exists that is localized in chromosomal band 3q25 and have reduced miR-16 levels in the spleen.40 Therefore, although

1Department of Internal Medicine III, Ulm University, Ulm, Germany; 2Cooperation Unit ‘Mechanisms of Leukemogenesis’, German Cancer Research Center DKFZ, Heidelberg, Germany and 3Signalling to Chromatin Laboratory, Institute of Molecular Biology, Mainz, Germany. Correspondence: Dr D Mertens, Department of Internal Medicine III, University of Ulm, Albert-Einstein-Allee 23, Ulm 89081, Germany. E-mail: [email protected] or [email protected] Received 8 March 2013; revised 7 August 2013; accepted 8 August 2013; accepted article preview online 26 August 2013; advance online publication, 24 September 2013 Defective DROSHA processing of MiR-15/-16 in CLL D Allegra et al 99 levels of mature miR-15a and miR-16 are reduced in CLL patients, numbers of cells were available for extraction (4107 cells) and (ii) the a number of processes have been shown to have an impact on selective loss does not occur with miR-16, miR-15b and only to lesser transcriptional and post-transcriptional regulation of the miRNAs extent with miR-15a.46 Reverse transcription and real-time PCR were 39 independently of genomic deletion of the critical region. performed as described in Allegra et al and in Supplementary We therefore asked whether post-transcriptional processing Experimental Methods. MiRNAs were profiled with the miRNA Microarray Release 14.0 (no. G4471A-029297; Agilent, Boeblingen, Germany) in 23 CLL defects of the MIR-15/-16 family might be a common feature in patients. Each miRNA was normalized to the average of six housekeeping CLL pathogenesis. Here we report for the first time impaired miRNA RNAs represented in the array (U1, U2, U4, U5, U6 and MRP). Results were maturation as a novel mechanism contributing to the down- comparable between microarray and quantitative reverse transcription- regulation of tumor suppressor miRNAs in leukemic cells. Expres- PCR data. MiRNAs whose levels differed significantly between the CLL high sion of the mature and precursor forms of MIR-15/-16 family ratio and normal ratio groups were identified and visualized using members was analyzed in CLL cells and compared with B cells from GenePattern (http://genepattern.broadinstitute.org/). Data are accessible at healthy individuals. We observed a dysregulation of the processing the gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo/, Series intermediates pointing toward defective pri-miRNA processing in a GSE30927). For full procedures refer to Supplementary Experimental subset of CLL patients. In these cells, defective processing leads to Methods. reduced levels of mature miR-15a, miR-15b and miR-16, but no reduction of most other miRNAs. This defect was also found in the ADARB1 overexpression and mutation cell line MEC-2butnotinMEC-1, even though these cell lines are The ADARB1 expression constructs ‘Wt’, ‘dN’ and ‘E397A’ contained the derived from the same CLL patient, albeit at different time points in wild-type (Wt) open reading frame of ADARB1 (gene ID 104)(‘Wt’), the the course of the disease. In the processing defective MEC-2cells, Wt open reading frame lacking the amino acids 4–72 that target the which originate from a later disease stage, we identified over- protein to the nucleus (‘dN’) and the Wt open reading frame with an expression of the RNA-editing gene ADARB1, a RNA-specific exchange A -4C at position 1621 that leads to an amino acids exchange E397A at the active site with abrogation of the enzymatic RNA-editing deaminase that has previously been reported to modify pre- 47 41 activity of ADARB1 protein (‘E397A’). Open reading frames were miRNAs and thereby interfere with miRNA processing. We could contained in the gateway pc3D plasmid that is derived from the human show that the RNA-binding domain and nuclear localization of expression backbone plasmid pcDNA3.1. All plasmids were kind gifts ADARB1 are essential for the observed tumor-specific miRNA from Mary A O’Connell and Bret SE Heale, MRC Human Genetics Unit, processing defect and thereby uncover the molecular cause of a Edinburgh, UK. novel leukemogenic tumor suppressor mechanism.

RESULTS EXPERIMENTAL PROCEDURES Processing intermediates of MIR-15/-16 family members are Primary samples, fluorescence in situ hybridization and cell dysregulated in CLL purification Expression of miR-16 in CLL patients is highly variable. A patient cohort was carefully selected for analysis that is representative of Unexpectedly, it is not significantly reduced in CLL cells with symptomatic disease in order to also identify aberrations associated with loss of one copy of 13q14.3 (13q þ / À ) compared with cells advanced stage.42,43 The cohort is thus enriched for cytogenetic with retention of both copies (13q þ / þ )(Supplementary aberrations associated with aggressive disease (del(17p) (5/38, 13%), Figures S1A–C). Thus, gene dosage is unlikely to influence del(11q) (7/38, 18%)), whereas the presence of del(13q) remains balanced miR-16 levels in CLL. Rather, transcriptional or post-transcrip- (19/38, 50%) (Supplementary Table S5). Peripheral blood was collected tional processes regulate levels of mature miR-15a/-16, espe- with informed written consent from CLL patients (Ethics Committee, University of Ulm, approval no. 96/08). Peripheral blood mononuclear cells cially in 13q þ / þ cells. Interestingly, also miR-15b is downregulated in CLL (Supplementary Figure S1D), even were purified from whole blood using density-gradient centrifugation with 18 Biocoll (Biochrom AG, Berlin, Germany). Fluorescence in situ hybridization though it is not frequently deleted. This downregulation analysis was performed as previously described.13 Patients with could be caused by a reduced processing of primary miRNA chromosomal deletions in at least 70% of peripheral blood mononuclear transcripts (pri-miRNAs; Figure 1a). In this case, elevated levels cells were selected (average 87.5%). Patients with normal karyotype had an of pri-miRNAs would correlate with reduced levels of mature average leukocyte count of 54 giga/l. For healthy donors, buffy coats were miRNA molecules. To investigate the transcriptional and post- collected from age-matched donors with informed oral consent (German transcriptional regulation of MIR-15/-16 family members, Red Cross, Ulm, Germany). CD19 þ peripheral blood mononuclear cells we therefore measured all processing intermediates of miR- were purified using CD19-microBeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) as described previously.44 Cells were viably frozen and stored in 15a, miR-16-1, miR-15b and miR-16-2 in cells from CLL patients nitrogen until use. and CD19 þ B cells from healthy probands. However, it has to be kept in mind that only a subset of non-malignant CD19 þ cells is best suited as counterpart to CLL cells.48 We used Cell culture, LiCl treatment, transfection and luciferase assays quantitative PCR-amplicons localized upstream of the miRNA All cell lines were cultured in 5% CO2 at 37 1C in RPMI-1640 supplemented clusters to measure the expression of the respective host genes with 10% FBS (PAN Biotech, Aidenbach, Germany) and 3 mM glutamine. For and quantitative PCR-amplicons spanning the miRNA hairpins b-catenin stimulation, MEC-1 and MEC-2 cells were seeded in medium to measure the unprocessed pri-miRNAs (Figure 1b). Detection containing 30 mM LiCl or NaCl and collected after 24 h. Control B cells and of pre-miRNAs by quantitative reverse transcription-PCR was CLL cells were transfected with 2.5 mg of either FF_pri-wt or ss plasmid and 2.5 mg of pRL-CMV, using AMAXA B-cell solution (Lonza, Cologne, Germany) optimized to avoid cross amplification with the respective pri- and progU-015. For cell lines, AMAXA solution V was used with progA-023. miRNAs (see Supplementary Experimental Procedures and After 24 h, cells were lysed in 50 ml of Passive Lysis Buffer (Promega, Supplementary Figure S2 for schematic on amplicon design). Mannheim, Germany) for 10 min at room temperature. Fifteen microlitres Values obtained for CLL cells from n ¼ 38 patients were of cleared lysate was used to measure luciferase activity in Firefly and normalized to the average of healthy controls (n ¼ 14; 45 Renilla buffers as described. Figure 1c–e, average represented by dashed line). We found that pri-miRNAs-15a, -16-1 and -16-2 were upregulated, RNA extraction, reverse transcription, quantitative reverse whereas pre- and mature miRNAs were downregulated in CLL transcription-PCR and microarray analysis cells compared with healthy control cells that showed the RNA was extracted using TRIzol reagent (Invitrogen, Darmstadt, Germany). expected differences in processing intermediates (Figures 1c, d Recent reports on the selective loss of short structured RNAs with low GC and Supplementary Figure S3). As there was no change in from small numbers of cells46 do not apply to our work as (i) sufficient the expression of the primary transcripts DLEU2 and SMC4

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Figure 1. The precursors of the pri-miR-15/-16 family are dysregulated in CLL cells. (a) Schematic of the putative processing defect. (b) Schematic of the miRNA clusters in 13q14.3 and 3q25. Black bars indicate the position of the amplicons used for quantitative reverse transcription-PCR (qRT-PCR) analysis. (c–e) Upstream sequences (white), pri-miR, pre-miR and mature miR levels (darker shades of blue) of the 13q14 miRs (c), the 3q25 miRs (d) and miR-26b (e) were quantiﬁed with qRT-PCR in peripheral blood mononuclear cells (PBMCs) from CLL patients (n ¼ 38) and CD19 þ PBMCs from healthy donors (n ¼ 14) as described previously39 and in Supplementary Experimental Procedures. Values obtained for CLL patient samples are shown, normalized to the average of healthy controls (dashed line). Although upstream transcripts are not deregulated, pri-miR levels are higher and pre- and mature miRs levels are lower in CLL cells for the miR-15/-16 families, whereas this is not the case for miR-26b. Boxes indicate the median and interquartile range, whiskers the 10–90 percentile. *Po0.05; **Po0.01; ***Po0.001. (f–i) In-line with a processing defect, levels of mature miRs are anticorrelated to levels of pri-miR for the MIR-15/-16 family, whereas this is not the case for miR-155. Pearson correlation coefﬁcients are given (‘R’), levels of transcripts and miRs are normalized to the respective average of healthy controls. Pri-miR-16 is the average of pri-miR-16-1 and pri-miR-16-2. Empty symbols represent del(13q) patients.

(Figures 1c, d, white bars), we could exclude that reduced Only a subset of CLL patients exhibits defective pri-miR-16-1 transcription contributed to the downregulation of the mature processing miRNAs. This processing defect could not be observed in miR- In order to address the heterogeneity of miR-15/-16 family 26b that was used as a negative control (Figure 1e). The pattern processing in different CLL patients, we introduced the pri-/pre- of deregulation we observed indicated a block of miRNA miR-16 ratio as an indirect measure of DROSHA processing maturation at the level of DROSHA cleavage: reduced DROSHA activity. To this end, CLL patients were stratiﬁed into two activity would result in accumulation of its substrates (pri- subgroups (Figure 2a): (1) CLL samples with a pri-/pre-miR-16 miRNAs) and reduction of its products (pre- and mature ratio within one standard deviation (s.d.) from the average of miRNAs) (Figure 1a). If the increase in pri-miRNAs and the healthy controls and (2) a second patient subgroup with a decrease in mature miRNAs were caused by the same process, putative processing defect displaying a high pri-/pre-miR-16 their levels should be inversely correlated. Indeed, we found a ratio, which indicates defective DROSHA activity. In line with our negative correlation between pri- and mature miRNAs of the model, the high ratio group had reduced levels of mature miR-16, miR-15/-16 family (Figures 1f–h) that could not be detected for miR-15a and miR-15b compared to both the normal ratio group miR-155, which is transcriptionally upregulated in CLL cells and to healthy controls (Figures 2b–d). This difference in the two (Supplementary Figures S1E, F). Levels of miR-155 are positively patient subgroups could not be observed for miR-155 or miR-26b correlated with pri-miR-155 as you would expect if no that are not affected by the observed processing defect regulation occurs during miRNA processing (Figure 1i). (Figures 2e, f).

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Figure 2. A subset of CLL patients with defective MIR-15/-16 family processing. (a) CLL patients were grouped based on the pri-/pre-miR-16 ratio. The shaded area represents one s.d. from the average of healthy controls, empty symbols represent del(13q) patients that are evenly distributed among the two subgroups. (b–f) Levels of miR-16, miR-15a, miR-15b, miR-155 and miR-26b were measured by qRT-PCR in normal B cells and PBMCs from CLL patients of the two groups deﬁned in (a). Open symbols represent del(13q) patients. Although no difference can be seen between the CLL subgroups for miR-155 and miR-26b, the miR-15/-16 family mature miRs show signiﬁcantly lower levels in cells from patients with a putative processing defect (‘CLL high ratio’).

If an aberration at 13q14.3 is causative for CLL as has been suggested previously,22,33 it should be affected in all patients so that no correlation of 13q14.3 aberrations with specific prognostic or genetic subtypes of patients would be expected. In line with this hypothesis, we could not find a consistent correlation of defective processing of the miR-15/16 genes localized at 13q14.3 or at 3q25.3 with overall survival (Supplementary Figure S4A), cytogenetic aberrations (Supplementary Figure S4B), immunoglobulin mutational status (immunoglobulin heavy variable) (Supplementary Figure S4C; for P-values see Supplementary Figure 3. MiRNome profiling uncovers additional miRs involved in Table S1) or time since diagnosis (Supplementary Table S4, mean the processing defect. The miRome of cells from n ¼ 23 CLL patients time since diagnosis 48.2 and 36.4 months for processing classified as having a processing defect of the miR-15/-16 family was defective and intact group, P ¼ 0.46 Student’s t-test). profiled using miRNA Microarrays Release 14.0 (Agilent) and normalized to six housekeeping RNAs (U1, U2, U4, U5, U6 and MRP). Seven miRs that differed significantly (Po0.05) between the MiRNome profiling uncovers additional miRNAs involved in CLL ‘high ratio’ and ‘normal ratio’ group are depicted in a heatmap, defective processing in CLL where shades of green and red indicate miR levels above and below the mean of the array, respectively. To test which proportion of miRNAs are deregulated as consequence of defective processing, we performed whole miRNome analysis on cells from 23 patients of both CLL of a vault RNA49 and is not processed by DROSHA. Thus, the subgroups with intact and with defective processing (Figure 3). processing defect we describe here is restricted to a small subset Similar to the quantification with quantitative reverse transcrip- of miRNAs. Intriguingly, target genes of these seven potentially tion-PCR, miR-15b, miR-16 and miR-16-2* were significantly differentially processed miRNAs (Figure 3) are significantly reduced in the CLL high ratio group (Figure 3, Supplementary enriched in the T-cell receptor signaling pathway, several Figure S5). Levels of miR-15a were not significantly different on components of which are deregulated in CLL cells the microarray. However, as miR-15a is only transcribed from (Supplementary Tables S3, S7). 13q14.3, the influence of the 13q deletion status on its levels is more pronounced than for miR-16. Accordingly, when only 13q þ / þ patients were considered, miR-15a was also reduced in high Reduced DROSHA processing activity in CLL cells with a pri-miRNA ratio CLL patients, although due to low sample number processing defect significance was not reached (n ¼ 12, P ¼ 0.054, Supplementary In order to functionally show that a subgroup of CLL samples Figure S6). Only four other miRNAs were significantly down- displays defective processing, we directly measured DROSHA regulated in the high ratio group (miR-886-3p, miR-7, miR-181d enzymatic activity in living primary CLL cells using a recently and miR-338-3p, Supplementary Table S2), whereas no miRNA was developed method for the intracellular quantification of pri-miR- upregulated, further underlining specificity of the observed 16-1 processing (schematic shown in Figure 4a).39 This assay is processing defect. Reduction of miR-886-3p is probably not based on two reporter constructs: (i) FF_pri-16-1wt that allows the directly related to the DROSHA defect, as this miRNA is a fragment expression of firefly luciferase with pri-miR-16-1 inserted into the

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Figure 4. Reduced DROSHA processing activity in CLL cells from patients with a pri-miR processing defect. (a) Schematic of firefly luciferase reporter for measurement of DROSHA processing activity (adapted with permission from Allegra et al39). The upper construct contains a hairpin in the 30 untranslated region (30UTR) of the luciferase transcript that is a DROSHA substrate, while the second construct contains four mutations abrogating the hairpin. Thus, the upper transcript is degraded depending on the DROSHA activity levels, the transcript from the lower construct is not processed and serves as internal control. The processing activity is given by the ratio of luciferase activity of reactions with the non-mutated vs the mutated construct.39 (b, c) Pri-miR-16-1-processing activity was assessed in viable B cells from healthy probands and CLL patients from both groups either with processing defect or without. As expected, the processing of miR-155 is not different between the two patient groups. In contrast, a significant difference in the DROSHA processing activity of pri-miR-16-1 can be observed in living cells from the ‘CLL high ratio’ group of patients.

0 3 untranslated region and (ii) as an internal control FF_pri-16-1ss cells and therefore are unlikely to be causative for the DROSHA that contains in the 30untranslated region a mutated version of processing defect in CLL. pri-miR-16-1 that does not fold normally (‘ss’, single stranded) and is therefore not processed by DROSHA. In normal B cells and in CLL cells with normal pri-/pre-miR-16 ratio, FF_pri-16-1wt mRNA is MEC-1 and MEC-2 cells represent a cellular model of the DROSHA cleaved by DROSHA and produces less luciferase than FF_pri-16-1ss processing defect in CLL mRNA that is not processed by DROSHA. In cells with normal To identify a cellular model system of the DROSHA processing processing, the ratio of luciferase activity of the two plasmids wt/ss defect in CLL, we measured the processing intermediates of MIR- is therefore lower (Figure 4b). In contrast, in cells from patients with 15/-16 family members and of miR-26b in cell lines obtained from a putative processing defect (‘high-ratio’), cleavage of the RNA CLL, mantle cell lymphoma, prolymphocytic leukemia patients, derived from FF_pri-16-1wt by DROSHA is reduced and more and in LCL-WEI, a cell line derived from a non-malignant B cell luciferase is produced, so that the ratio of luciferase activity is (Supplementary Table S4). Of note, all of the CLL-derived lines higher (Figure 4b), validating the observed defect in living cells. were obtained using transformation with Epstein-Barr virus, which In contrast, processing of miR-155 that was measured as a negative is likely to add phenotypic changes not present in the original CLL control with specific dedicated wt/ss plasmids was not different in cells. However, to our knowledge these CLL cell lines are the best the two patient subgroups, underlining specificity of the observed model to investigate CLL in vitro apart from directly using primary defect. In summary, the quantification of DROSHA processing cells, whereas primary CLL cells are difficult to transfect.52 The CLL- activity in live CLL cells confirms the impairment of DROSHA derived cell line MEC-2 showed deregulation of miRNA processing processing activity in CLL cells with a high pri-/pre-miR-16 ratio. intermediates resembling the pattern we observed in CLL patients (Figure 6). When compared to the other cell lines, MEC-2 cells had higher levels of pri-miR-15b, pri-miR-16-1 and pri-miR-16-2, Reduced processing by DROSHA does not correlate with whereas levels of the corresponding pre- and mature miRNAs downregulation of DROSHA or DROSHA cofactors and of the miRNA host genes were not increased. Only for miR- We next wanted to determine whether the DROSHA processing 15a, the levels of pri-miRNA were not enhanced compared with defect was due to reduced expression of components of the pri- pre-miRNA but were similar to miR-26b which was used as miRNA processing machinery. First, we measured the mRNA levels a control. To confirm this correlative finding, we tested whether a of DROSHA and DGCR8 in CLL patients and observed no reduction reduced capacity to process pri-miR-16-1 could also be detected in the high ratio group (Figures 5a, b). Similarly, the levels of in viable cells using our luciferase reporter system (Figure 7a). Five additional factors required for efficient processing of pri-miRNAs of the cell lines (LCL-WEI, Granta, EHEB, JVM-3 and MEC-1) had a were also not downregulated: KHSRP50 was similarly expressed in processing efficiency similar to primary non-malignant B cells both CLL subgroups, whereas p68 and p7251 were even increased (CD19 þ ), whereas processing was more efficient in JVM-2 cells. in the high ratio group compared with the normal ratio group In contrast, MEC-2 and I83-E95 cells had significantly impaired (Figures 5c–e), which could be speculated to be caused by pri-miR-16-1 cleavage, similar to what we observed in the CLL high feedback mechanisms owing to defective processing. Thus, none ratio group (Figure 7a). Interestingly, pri-miR-16-1 processing was of these factors are downregulated in processing defective CLL significantly different in MEC-1 compared with MEC-2 even

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Figure 5. DROSHA and cofactors of DROSHA are not downregulated in processing defective CLL cells. (a–e) Genes involved in pri-miR processing are not downregulated in CLL cells with a processing defect, suggesting a different underlying molecular cause. Shown are qRT-PCR measurements in cells from the patient cohort (n ¼ 38, see Supplementary Table S5) with normal ratio (n ¼ 16) and from patients with a processing defect (n ¼ 22) that are normalized to two housekeeping genes (ACTB and LMNB1). though these cell lines originate from the same CLL patient albeit thus be used as a negative control not harboring the processing at different time points of disease progression.53 In contrast, the defect. Subsequently, pri-miR-16-1-processing activity was processing of pri-miR-34a and pri-miR-155 was comparable in measured (Figure 8c). Underlining its role in miR-16-1 processing, these cell lines, underlining that the defect is restricted only to overexpression of the wild-type (Wt) form of ADARB1 leads to specific miRNAs (Figures 7b, c). Thus, MEC-2 cells can be used as a reduced processing of pri-miR-16-1. In contrast, the processing of model of the DROSHA processing defect. We therefore used MEC- pri-miR-155 used as a control was not altered (Figure 8d). ADARB1 2 cells in comparison with MEC-1 cells that do not harbor this protein has two copies of highly conserved double-stranded-RNA- defect in order to identify the underlying molecular cause. binding domains, which are important for the editing activity of common RNAs and for sequence-dependent editing59 (Figure 8b). As the impact of RNA-modifying enzymes on the processing on -catenin stimulation by LiCl reduces processing of pri-miR-16-1 60 but not pri-miR-16-2 pri-miRNAs occurs mostly via RNA editing, we tested whether the binding of ADARB1 alone is sufficient to block processing. To The Wnt/-catenin pathway has been reported to be deregulated this end we used ADARB1 variants, where the enzymatic activity of in CLL cells54 and its inhibition leads to the induction of apoptosis ADARB1 has been deleted by separately mutating aspartic acid at of CLL cells.55 Intriguingly, Wnt/b-catenin activation has also been position 397 (‘A397E’), the RNA-binding potential (RNA-binding reported to inhibit miR-16 processing, but the details of this domain) and the nuclear localization signal (‘dN’).47 Intriguingly, regulation are not known.56 We therefore stimulated b-catenin in ablation of the RNA-editing activity did not change the capacity of MEC-1 and MEC-2 cells by LiCl, which led to reduced processing of ADARB1 to reduce pri-miR-16-1 processing (‘A397’, Figure 8c). pri-miR-16-1 but not of pri-miR-16-2 in MEC-1 and MEC-2 cell lines In contrast, removal of either the RNA-binding potential or the (Supplementary Figure S8). However, maturation of other miRNAs nuclear localization domains (‘RNA-binding domain’ and ‘dN’) was also impaired and the transcription of SMC4 in MEC-1 cells leads to a loss of the capacity of ADARB1 to interfere with pri-miR- was reduced, suggesting that b-catenin does not very likely have a 16-1 processing (Figure 8c), suggesting that these properties of role in the processing defect we identified in CLL. ADARB1 protein are essentially involved in the miRNA processing defect detected in the MEC-2 CLL cells. Overexpression of ADARB1 leads to reduced processing of pri-miR-16-1 in CLL cells In order to identify the molecular mechanism causing the DISCUSSION observed processing defect in CLL cells, we compared the MiRNAs have been implicated as central players in the develop- transcriptomes of MEC-2 cells that harbor the processing defect ment of cancer, and miR-15a/-16-1 were the first shown to be to transcriptomes of MEC-1, but also JVM-2 and I83-E95 to deregulated in malignant cells.38,61 As their processing to the characterize the processing defect specific to MEC-2 cells effective mature forms is intricately regulated,62 it is very likely (Figure 8a). The most deregulated genes were ADARB1 (upregu- and has been proposed before that miRNA processing could lated) and SLC15A4 (downregulated). SLC15A4 (‘solute carrier be affected in malignant cells.63 The enzymatic activities 15A4’ alias PHT1) is involved in peptide shuttling across the involved in miRNA processing like DICER1 and TARBP2 can act cellular membrane,57 which cannot intuitively be linked to miRNA as haploinsufficient tumor suppressors in different cancer processing. The ADARB1 gene product (schematic in Figure 8b) is entities,64,65 whereas the upregulation of DROSHA has been involved in RNA processing.58 In order to test whether the described in cervical squamous cell carcinoma.66 observed upregulation of ADARB1 also has functional impact on Our work shows that the miR-15/-16 family of tumor suppressor miRNA processing, we overexpressed recombinant ADARB1 in miRNAs is post-transcriptionally downregulated in CLL, owing to a HEK-293T cells that are of non-hematopoietic origin and could specific defect of DROSHA processing. Patients with defective

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Figure 6. A cellular model for the processing defect of miR-15/-16 family. Pri-, pre- and mature forms of the MIR-15/-16 family were measured together with upstream sequences in six CLL cell lines (EHEB, JVM-2, JCM-3, MEC-1, MEC-2, Granta) and one non-leukemic cell line (LCL-WEI) and normalized to the average of all cell lines (solid horizontal line). Only MEC-2 cells showed high levels of pri-miR and low levels of mature miRNAs and therefore probably harbor a processing defect similar to CLL patients from the ‘high ratio’ group. This defect was not observed for miR-26b that was used as a negative control.

Figure 7. miR-16-1 is processed differently in B-cell- and CLL cell lines. (a) Pri-miR-16-1 is processed less efﬁciently in MEC-2 and I83-E95 cells as quantiﬁed using the intracellular processing assay39 (see also Figure 4). Interestingly, MEC-1 cells derived from the same patient but at an earlier time point do not display defective processing. (b) This defective processing is not seen with pri-miR-34a (c) or pri-miR-155.

pri-miR-16 processing also have signiﬁcantly lower levels of miR-7, to MIR-15/-16 family members. Processing of miR-7, miR-181d and miR-181d and miR-338-3p. These miRNAs and their respective miR-338-3p might also be blocked in high ratio CLL patients, or pri-miRNAs bear no obvious resemblance in sequence or structure their downregulation could be a secondary effect to the reduction

Leukemia (2014) 98 – 107 & 2014 Macmillan Publishers Limited Defective DROSHA processing of MiR-15/-16 in CLL D Allegra et al 105

Figure 8. Overexpression of ADARB1 leads to reduced processing of pri-miR-16-1 in CLL cells. (a) Transcriptome profiling identified ADARB1, a RNA-specific adenine deaminase involved in RNA-processing, and SLC15A4 to be the most deregulated genes in MEC-2 cells compared with cell lines without a processing defect (MEC-1, JVM-2) and I83-E95. Depicted are normalized log signal intensity ratios compared with MEC-2, top row the average. (b) Schematic of protein domains of ADARB1 and changes introduced into recombinant constructs (red arrows) used in c, d (modified from47). (c, d) Overexpression of wild-type (‘Wt’) and an enzymatically inactive form of ADARB1 (‘A397B’) in HEK-293T cells leads to significantly reduced processing of pri-miR-16-1 but not of pri-miR-155 quantified by an in vivo processing assay39 (see also Figure 4). In addition, removal of the RNA-binding domains (‘2xRBD‘) and the nuclear localization domain (’dN’) of ADARB1 reestablished full processing efficiency, suggesting that these protein domains of ADARB1 are essential for its involvement in pri-miRNA processing. of miR-15/-16. The latter explanation is also likely in the case for by DICER.68 Together with our data, this suggests that both miRNA miR-886-3p, which is not produced by DROSHA cleavage. processing steps are regulated by members from the same gene The only other report of a specific defect in pri-miRNA family. processing in cancer focused on miR-7.67 In glioblastoma ADARB1 is upregulated in lymphocytes of patients suffering patients, levels of miR-7 and its precursors pre-miR-7-1, -7-2 from lupus erythematosus58 and in inflammation,69 which is and -7-3 are strongly reduced compared with matched interesting as CLL cells are characterized by auto-reactivity and non-malignant tissues, whereas the corresponding pri-miRNAs immunological activation.70 Intriguingly, ADAR is also affected in a are not reduced. Of note, levels of miR-7 are also reduced in CLL syndrome called Aicardi Guitieres Syndrome.71 Cells of patients patients with defective pri-miR-16-1 processing, so that it would suffering from this syndrome also harbor mutations in SAMHD1,71 be interesting to test whether both processes have similar a gene that is recurrently mutated in CLL,16 where it is possibly a underlying causes. causative driver.72 These findings could point to a synergism In order to identify the mechanism of the defective DROSHA between ADAR and SAMHD1 in the pathogenesis of CLL and processing in CLL, we sequenced 33 patients from our cohort and further underscore the role of miRNA processing in the disease. found no genomic mutations in the two miRNA clusters in 13q14 What is the underlying molecular mechanism of ADARB1 and 3q25 (Supplementary Table S5). In addition, the mRNA levels leading to reduced processing of pri-miR-15/-16 in CLL? Genetic of DROSHA, DGCR8 and other pri-miRNA processing cofactors were evidence from Caenorhabditis elegans suggests that already the not decreased in processing-deficient CLL patients, making it binding of ADAR proteins to RNA impacts on the phenotype,60 unlikely that these genes are involved in the defect. Processing and ADARB1 has already been shown to inhibit the DROSHA defective CLL cells retained the ability to cleave pri-miR-155 that is processing of pri-376a independently of its enzymatic activity.47 not related to the miR-15/-16 family, indicating that DROSHA The homolog ADAR has also been shown to block RNA processing processing is not globally defective in CLL but rather specifically both by editing and by binding to RNA.60 In the present study we impaired for the MIR-15/-16 family members and possibly several could show that for the pri-miR-15/-16 family, blockage of the other miRNAs. adenosine deaminase enzymatic activity of ADARB1 does not We could also exclude that deregulation of the Wnt/b-catenin affect the processing defect induced by overexpression of the pathway is causative for the processing defect observed in CLL gene (Figure 8c). In contrast, deletion of the RNA-binding domains cells, even though it impacts on processing of miR-16-1. and the nuclear localization signal (‘dN’) that is required for In contrast, in MEC-1 cells that have a defective pri-miR-15/-16 shuttling of ADARB1 from the cytoplasm to the nucleus73 processing, we found the RNA-specific Adenosine Desaminase abrogated the potential of ADARB1 to inhibit processing of the ADARB1 specifically upregulated, and overexpression of ADARB1 in pri-miR molecules (Figure 8c). On the basis of the structure of the human embryonic kidney cells lead to defective processing of RNA-binding domains, it has been suggested that ADARB1 could pri-miRs. This is especially interesting as the homolog ADAR1 has interact with DROSHA (and DICER) proteins and thereby compete been shown during the preparation of this manuscript to with their RNA-processing activity.74 However, further work is modulate the successive processing step of pre-miRNA transcripts required to uncover the details of this novel molecular mechanism.

& 2014 Macmillan Publishers Limited Leukemia (2014) 98 – 107 Defective DROSHA processing of MiR-15/-16 in CLL D Allegra et al 106 In conclusion, miR-15/16 are pivotal players in the pathogenesis 18 Haferlach C, Dicker F, Schnittger S, Kern W, Haferlach T. Comprehensive genetic of CLL. Here, we show for the first time that a defect in the characterization of CLL: a study on 506 cases analysed with chromosome banding post-transcriptional processing of tumor suppressor pri-miRNA analysis, interphase FISH, IgV(H) status and immunophenotyping. Leukemia 2007; transcripts is a novel miRNA-related tumor escape mechanism. 21: 2442–2451. In the future, defects in processing will need to be considered as 19 Mertens D, Philippen A, Ruppel M, Allegra D, Bhattacharya N, Tschuch C et al. the underlying cause whenever levels of mature miRNAs are Chronic lymphocytic leukemia and 13q14: miRs and more. Leuk Lymphoma 2009; reduced. 50: 502–505. 20 Mertens D, Wolf S, Schroeter P, Schaffner C, Dohner H, Stilgenbauer S et al. Down-regulation of candidate tumor suppressor genes within chromosome band CONFLICT OF INTEREST 13q14.3 is independent of the DNA methylation pattern in B-cell chronic lymphocytic leukemia. Blood 2002; 99: 4116–4121. The authors declare no conflict of interest. 21 Mertens D, Wolf S, Tschuch C, Mund C, Kienle D, Ohl S et al. Allelic silencing at the tumor-suppressor locus 13q14.3 suggests an epigenetic tumor-suppressor mechanism. Proc Natl Acad Sci USA 2006; 103: 7741–7746. ACKNOWLEDGEMENTS 22 Garding A, Bhattacharya N, Claus R, Ruppel M, Tschuch C, Filarsky K et al. We are thankful to the CLL patients and healthy probands for the generous donation Epigenetic upregulation of lncRNAs at 13q14.3 in leukemia Is linked to the of their primary tissue. We would also like to thank Stefan Fro¨hling and Claudia Scholl oitalic4In Ciso/italic4 downregulation of a gene cluster that targets NF-kB. for their comments and to Doris Winter for her excellent technical support. This work PLoS Genet 2013; 9: e1003373. was supported by the DKFZ intramural funding scheme (K109), by the Sander 23 Allen JE, Hough RE, Goepel JR, Bottomley S, Wilson GA, Alcock HE et al. Identifi- Foundation (no. 2010.036.1), by the Deutsche Jose´ Carreras Leuka¨mie-Stiftung (R06/ cation of novel regions of amplification and deletion within mantle cell 13v) and by the Deutsche Krebshilfe (Max-Eder Programme, no. 109321). lymphoma DNA by comparative genomic hybridization. Br J Haematol 2002; 116: 291–298. 24 Sander S, Bullinger L, Leupolt E, Benner A, Kienle D, Katzenberger T et al. Genomic REFERENCES aberrations in mantle cell lymphoma detected by interphase fluorescence in situ 1 Bartel DP. MicroRNAs: target recognition and regulatory functions. 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