Leukemia (2012) 26, 1064 --1072 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu

ORIGINAL ARTICLE The miRNA-17B92 cluster mediates chemoresistance and enhances tumor growth in mantle cell lymphoma via PI3K/AKT pathway activation

ERao1,2,3,7, C Jiang1,7,MJi1, X Huang1, J Iqbal1, G Lenz4,5, G Wright6, LM Staudt5, Y Zhao2, TW McKeithan1, WC Chan1 and K Fu1

The median survival of patients with mantle cell lymphoma (MCL) ranges from 3 to 5 years with current chemotherapeutic regimens. A common secondary genomic alteration detected in MCL is chromosome 13q31-q32 gain/amplification, which targets a microRNA (miRNA) cluster, miR-17B92. On the basis of gene expression profiling, we found that high level expression of C13orf25, the primary transcript from which these miRNAs are processed, was associated with poorer survival in patients with MCL (P ¼ 0.021). We demonstrated that the protein phosphatase PHLPP2, an important negative regulator of the PI3K/AKT pathway, was a direct target of miR-17B92 miRNAs, in addition to PTEN and BIM. These proteins were down-modulated in MCL cells with overexpression of the miR-17B92 cluster. Overexpression of miR-17B92 activated the PI3K/AKT pathway and inhibited chemotherapy-induced apoptosis in MCL cell lines. Conversely, inhibition of miR-17B92 expression suppressed the PI3K/AKT pathway and inhibited tumor growth in a xenograft MCL mouse model. Targeting the miR-17B92 cluster may therefore provide a novel therapeutic approach for patients with MCL.

Leukemia (2012) 26, 1064--1072; doi:10.1038/leu.2011.305; published online 25 November 2011 Keywords: miR-17B92 cluster; mantle cell lymphoma; PHLPP2; PTEN; PI3K/AKT pathway

INTRODUCTION with higher expression of miR-17B92 develop a lymphoprolifera- Mantle cell lymphoma (MCL) is an aggressive hematological tive disorder and autoimmunity and die prematurely. Lympho- malignancy, comprising 5--10% of human B-cell lymphomas.1 The cytes from these mice show increased proliferation and median survival of patients with MCL ranges between 3 and 5 attenuated cell death. In these mice, miR-17B92 suppresses the 17 years.2 Cytogenetically, MCL is characterized by the chromosomal expression of PTEN and BIM. translocation t(11;14)(q13;q32), which results in aberrant expres- Several genes in the phosphatidylinositol 3-kinase (PI3K)/AKT sion of cyclin D1.3 However, studies in transgenic mice indicate signaling pathway, including PIK3CA, AKT1, PDK1 and PPP1R2, 18,19 that the t(11;14)(q13;q32) alone is insufficient to result in are overexpressed in primary MCL tumors and MCL cell lines; lymphoma and additional genetic alterations are necessary.4,5 and the PI3K/AKT pathway is constitutively activated in a 20 Secondary genomic alterations are frequently detected in MCL,6 of subset of MCL. Activation of PI3K/AKT signaling may be which chromosome 13q31-q32 gain/amplification is one of the secondary to overexpression of miRNAs via downregulation of 21,22 21 most common.7 Studies have shown that gain/amplification at several negative regulators such as PTEN, SHIP and 13 13q31-q32 targets a microRNA (miRNA) cluster, miR-17B92, PPP2R2A. However, the functional role of miRNAs in MCL which resides within intron 3 of C13orf25 (or MIRH1), a non- remains elusive. protein-coding gene at 13q31.3.8,9 The miR-17B92 cluster Herein, we show that high expression of miR-17B92 is consists of six miRNAs that are transcribed as a polycistronic correlated with poorer survival in patients with MCL. We found unit.9,10 Overexpression of miR-17B92 has been observed in that the protein phosphatase PHLPP2 (PH domain and Leucine- lymphomas and other solid tumors.9,10 The oncoprotein MYC rich repeat Protein Phosphatase 2), a negative regulator of the upregulates miR-17B92 expression, and overexpression of the PI3K/AKT pathway, is a direct target of the miR-17B92 cluster, in cluster accelerates MYC-induced lymphomagenesis in mice9,11 addition to PTEN and BIM. We demonstrated that overexpression and contributes to tumorigenesis.10,12 --14 of miR-17B92 in MCL cells activates the PI3K/AKT pathway and The miR-17B92 cluster is essential for B-cell development; in its impedes chemotherapy-induced apoptosis by collaboratively absence, the proapoptotic protein BIM is upregulated, and B-cell down-modulating multiple negative regulators in the PI3K/AKT development is blocked at the pro-B to pre-B transition.15 pathway and by decreasing BIM expression. We further showed Similarly, expression of miR-17B92 target genes, including BIM, that knockdown of miR-17B92 miRNAs inhibits tumor growth in a is upregulated in Dicer-deficient pro-B-cells.16 Transgenic mice xenograft MCL mouse model.

1Departments of Pathology and Microbiology and Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA; 2Transplantation Biology Research Division, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Beijing, China; 3Graduate School, Chinese Academy of Sciences, Beijing, China; 4Department of Hematology, Oncology and Tumor Immunology, Charite´ - Universita¨tsmedizin Berlin, Berlin, Germany; 5Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Health Institutes, Bethesda, MD, USA and 6Biometrics Research Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Health Institutes, Bethesda, MD, USA. Correspondence: Dr K Fu, Departments of Pathology and Microbiology and Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198-3135, USA. E-mail: [email protected] 7These authors contributed equally to this work. Received 7 March 2011; revised 8 September 2011; accepted 15 September 2011; published online 25 November 2011 Chemoresistance and tumor growth in mantle cell lymphoma E Rao et al 1065 MATERIALS AND METHODS Jeko-pTRIPZ-control or Jeko-pTRIPZ-Sponge cells suspended in 100 ml PBS. Cell lines Tumor growth was assessed using the two largest perpendicular axes MCL cell lines Z138c, Granta-519 and Jeko-1 were maintained in RPMI measured with standard calipers. Tumor volume was calculated based on 1640, and HEK293T and NIH3T3 cells were maintained in DMEM. Both the formula V ¼ p  L  S  S/6 (L, the long axis; S, the short axis). Tumor size media were supplemented with 10% fetal bovine serum and 1% penicillin/ and body weight were measured every other day after inoculation of MCL streptomycin. cells. Animals were killed when the largest tumor diameter reached 20 mm or after the loss of 410% of body weight. All animal studies were conducted in accordance with the NIH guidelines for animal care. All Gene expression profiling experimental procedures and protocols were approved by the Institutional Gene expression profiling was performed on 92 patients with cyclin D1-positive Animal Care and Use Committee at the University of Nebraska Medical 23 MCL as previously described. A total of 82 cases were re-profiled using Center. Affymetrix HG-U133 plus 2.0 arrays (http://llmpp.nih.gov.). The expression level of the C13orf25 gene was determined using probe set 232291 (UG cluster ID: Statistical analysis Hs. 24115). The following MCL cell lines were also studied: HBL2, Jeko-1, Mino, Rec-1, SP53, UPN1 and Z138c. The study was approved by the Institutional Data were analyzed with the Student’s t-test using the SPSS 11.0. program Review Board at the University of Nebraska Medical Center. (IBM Corporation, Armonk, NY, USA). Po0.05 was considered statistically significant. Data are presented as mean ± s.e.m. Array-based comparative genomic hybridization (aCGH) aCGH was performed as previously described.24 Genomic DNA from MCL RESULTS cell lines was extracted using the DNeasy kit (Qiagen, Germantown, Expression of C13orf25 correlates with overall survival in patients MD, USA). Normal male genomic DNA was used as reference. The aCGH with MCL protocol from NimbleGen Systems (Madison, WI, USA) was followed. We previously profiled 92 primary samples of MCL23 and found Briefly, tumor and reference DNAs were fragmented by sonication and that the proliferation signature is the main predictor of clinical random-prime labeled with Cy3 and Cy5 dyes, respectively. Labeled outcome in these patients. Of these, 82 cases were reexamined material was cohybridized to microarrays consisting of 386 165 oligonu- using Affymetrix HG-U133 plus 2.0, which showed that c13orf25 B cleotide probes spaced at 5 kb intervals throughout the human genome, were highly expressed in these cases. The expression of individual washed and scanned (Axon Instruments, Union City, CA, USA). miRNAs within the miR-17B92 cluster was also evaluated in a subset of these cases by quantitative RT-PCR assay. Compared Plasmid constructions with normal naive B-cells (n ¼ 3), the tumor cells in MCL cases The vectors were constructed as described in the Supplementary (n ¼ 30) expressed two to three-fold higher levels of miR-17B92 Materials and methods. The primers used are listed in Supplementary miRNAs (Supplementary Figure S2). To further study the Table S1 and S2. prognostic significance of c13orf25 overexpression, we identified five cases which had significantly higher levels of c13orf25 Establishment of MCL cell lines with ectopic expression or (4 mean þ 1.5 s.d.) compared with the other 77 MCL cases. All conditional knockdown of miR-17B92 five cases showed a high proliferation signature (Figure 1a), and four had 13q31-32 gain/amplification by aCGH analysis (data not Z138c and Granta-519 cell lines were stably transduced with pRevTet-On shown). Gene set enrichment analysis25 revealed the enrichment (Clontech, Palo Alto, CA, USA) to generate stable cell lines (Z138c-Tet-On of gene signatures that were highly associated with cell and Granta-Tet-On) according to the manufacturer’s instructions. The proliferation in these cases (Supplementary Table S3). Kaplan-- Z138c-Tet-On and Granta-Tet-On cell lines was then transduced with Meier analysis showed that patients with high levels of C13orf25 the TMP2-miR-17B92 vector (Supplementary Figure S1A). These cells had poorer overall survival (P ¼ 0.021) with median survival of only were selected with puromycin, and GFP-expressing cells were isolated 1.06 years, compared with median survival of 2.75 years for the by fluorescence-activated cell sorting. The cell lines were grown in the rest of the cohort (Figure 1b). presence of doxycycline (1 mg/ml) to maintain the overexpression of miR-17B92. For knockdown of miR-17B92, Jeko-1 cells were trans- duced with lentivirus packaged with the pTRIPZ-Sponge construct MiR-17B92 overexpression inhibits chemotherapy-induced (Supplementary Figure S1B), pMD2G envelope vector and psPAX2 apoptosis in MCL cells packaging plasmid in HEK293T cells. Transduced cells were selected with To study the effects of miR-17B92 overexpression, we established puromycin, and doxycycline-induced RFP-expressing cells were isolated by virally transduced MCL cell lines, Z138c-miR-17B92 (Z138c-miR) fluorescence-activated cell sorting. and Granta-519-miR-17B92 (Granta-miR). Compared with their respective control cells, Z138c-miR and Granta-miR cells expressed B Luciferase assays 3.6-fold and 2.9-fold higher levels of miR-20a, determined by 5 real time RT-PCR. Similarly higher levels of miR-17 were also HEK 293T or NIH3T3 cells were plated at 1  10 cells per well in a 24-well demonstrated in these cells (Figure 2a). plate 24 h before transfection. pGL3-promoter plasmids containing the 0 Topotecan is a potent topoisomerase I inhibitor, which induces wild-type or mutated 3 -UTRs of various potential targets were co- DNA damage and leads to apoptosis in cells through the intrinsic transfected with pRL-SV40 using Exgen 500 (Fermentas, Hanover, MD, pathway in a caspase 3-dependent manner.26 Overexpression of USA). For miRNA inhibition studies, sponge plasmids targeting particular B 0 miR-17 92 significantly decreased cell death in Z138c cells and miRNAs were co-transfected into HEK293T cells with 3 -UTR reporter Granta-519 cells treated with topotecan, as determined by a plasmids and pRL-SV40. Luciferase assays were performed 24 h after proliferation assay (Figure 2b) or annexin V-staining (Figure 2c). transfection using the Dual Luciferase Reporter Assay System (Promega, Similar results were also seen when these cell lines were treated Madison, WI, USA). Firefly luciferase activity was normalized to Renilla with doxorubicin or etoposide, a topoisomerase II inhibitor luciferase activity for each reaction. Transfected wells were analysed in (Supplementary Figure S3A and B). In addition, significantly triplicate for each group. smaller fractions of cleaved poly-(ADP-ribose) polymerase and cleaved caspase 3 were present in Z138c-miR compared with Xenograft experiments control cells (Figure 2d). Conversely, knockdown of miR-17B92 Six- to eight-week-old female CB-17/SCID mice (The Jackson Lab, Bar activity using a mixture of antagomirs against individual miRNAs Harbor, ME, USA) were subcutaneously inoculated in the flank with 5  106 within the miR-17B92 cluster (Applied Biosystems/Ambion,

& 2012 Macmillan Publishers Limited Leukemia (2012) 1064 --1072 Chemoresistance and tumor growth in mantle cell lymphoma E Rao et al 1066

Figure 1. Overexpression of C13orf25 is associated with poor clinical outcome in MCL patients. (a) MCL cases were divided into quartiles on the basis of the proliferation signature and, within each quartile; the data are shown in order of C13orf25 expression. The corresponding proliferation signatures are shown at the top, with red indicating the highest levels and green the lowest. The five cases (marked in red) with highest C13orf25 (4 mean þ 1.5 Â s.d.) showed high proliferative signatures. (b) Kaplan--Meier plot showing that high C13orf25 expression is associated with poorer overall survival (P ¼ 0.021).

Carlsbad, CA, USA) decreased cell survival when treated with containing a segment of the PTEN 30-UTR that includes predicted etoposide in Mino and Z-138c cells, but only minimally in Jeko-1 binding sites for miR-20a/-17-5p and miR-19. We also constructed cells (Supplementary Figure S3C and D). Furthermore, treatment a luciferase reporter plasmid containing point mutations in the with antagomirs increased the number of apoptotic cells and predicted miRNA-binding sites within the PTEN 30-UTR (PTENmut) increased caspase 3/7 activity in Z138c (Supplementary Figure S3E (Supplementary Figure S4A). The luciferase activity of the reporter and F) and Mino cells (data not shown). Our observations suggest gene containing the wild-type PTEN 30-UTR was decreased 40% that overexpression of miR-17B92 protects the MCL cells from compared with the control construct in HEK293T cells, in which apoptosis when challenged with chemotherapeutic agents and, the miR-17B92 cluster is highly expressed,17 but the decrease was conversely, that knock down of the activity of miR17-92 promotes not seen with the reporter gene PTENmut (Supplementary Figure chemotherapy-induced apoptosis in MCL cells. S4B). To further demonstrate that the effects seen were directly mediated by miR-17B92, we co-transfected the 30-UTR reporter plasmid PTEN or PTENmut along with TMP2 or TMP2-miR-17B92 Overexpression of miR-17B92 miRNAs downregulates PTEN plasmid into NIH3T3 cells, which have much lower levels of and BIM endogenous miR-17B92.17 We found that enforced miR-17B92 To gain insight into the molecular mechanisms by which expression in NIH3T3 cells decreased the luciferase activity of the miR-17B92 overexpression enhances tumor cell resistance to reporter gene with a wild-type PTEN 30-UTR, but not the reporter chemotherapy-induced apoptosis, we searched for putative miR- gene PTENmut (Supplementary Figure S4C). Furthermore, we 17B92 target genes predicted by TargetScan, Pictar and miRbase studied the effect on targets upon knocking down these miRNAs programs. Several genes with known roles in proliferation and in MCL cells. The miR-20a ‘sponge’ construct expresses a mRNA apoptosis, such as PTEN and BIM, are among the top predicted with seven tandem miR-20a binding sites in the 30-UTR and acts targets of miR-17B92 based on a series of established criteria to sequester miR-20a miRNA.28 Coexpression of the miR-20a (Supplementary Table S4).27 ‘sponge’ construct in HEK293T cells upregulated PTEN protein As PTEN has been previously reported as direct target of the expression by 72% (Supplementary Figure S4D). Moreover, the miR-17B92, we examined its protein level in Z138c-miR cells by luciferase activity in the PTEN 30-UTR reporter gene was restored in immunoblot analysis. Compared with the control cells, the protein HEK293T cells by coexpression of the miR-20a ‘sponge’ plasmid level of PTEN were significantly reduced in cells with miR-17B92 (Supplementary Figure S4E). However, there was no significant effect overexpression (Figure 3a). To demonstrate that downregulation on the luciferase activity in reporter genes in which these binding of PTEN was mediated through miR-17B92 binding to the 30-UTR sites were mutated (Supplementary Figure S4E). The findings of PTEN mRNA, we constructed a luciferase reporter plasmid confirmed that PTEN was a direct target of the miR-17B92 cluster.

Leukemia (2012) 1064 --1072 & 2012 Macmillan Publishers Limited Chemoresistance and tumor growth in mantle cell lymphoma E Rao et al 1067

Figure 2. Overexpression of miR-17B92 enhances the survival of MCL cells treated with topotecan (TPT). (a) MiR-20a and miR-17 expressions in Z138c and Granta-519 cells transduced with TMP2 (vector) or TMP2-miR-17B92 retrovirus, as quantified by Taqman microRNA assays. Relative miRNAs expression was estimated from the CT value using b-actin for normalization. (b) Retrovirus-transduced Z138c or Granta-519 cells were incubated with the indicated concentrations of TPT for 48 h, and cell survival was determined by the MTS assay, as indicated by absorbance at 490 nm. (c) Retrovirus-transduced Z138c or Granta-519 cells were treated with 5 nM TPT for the indicated times, stained with annexin V-PE and 7-AAD, and analyzed by flow cytometry. (d) Z138c-control and Z138c-miR cells, treated with 5 nM TPT for the indicated durations, were lysed and immunoblotted with anti-caspase 3, anti-poly-(ADP-ribose) polymerase (PARP) and anti-a-tubulin antibodies.

& 2012 Macmillan Publishers Limited Leukemia (2012) 1064 --1072 Chemoresistance and tumor growth in mantle cell lymphoma E Rao et al 1068 PHLPP2 contains sequences matching the seed sequences for miR-18, miR-20a/-17-5p and miR-92 (Figure 4a). Immunoblotting analysis demonstrated that PHLPP2 protein was also down- regulated in Z138c-miR cells compared with the control cells (Figure 3a). The luciferase activities of report plasmids P1, which contains putative binding sites for miR-18 and miR-20a/-17-5p, and P2, which does not contain any known binding sites for the miR-17B92 miRNAs, were comparable to the control plasmid (Figure 4b). However, the luciferase activity of reporter plasmid P3, which contains two putative binding sites for miR-92, was decreased B40% compared with the control, but the decrease was not seen when both binding sites for miR-92 were mutated (P3-92-2M) (Figure 4b). The finding was further confirmed using a miR-92 sponge plasmid that restored B80% of the luciferase activity of P3 (Figure 4c). However, sponge plasmids for miR-18 or miR-20 failed to restore the luciferase activity of their respective reporter plasmids. Furthermore, we showed that the sponge plasmids for miR-92 or the entire miR-17B92 cluster, but not the sponge plasmids for miR-18, -19 or -20, upregulated PHLPP2 protein levels in HEK293T cells (Figure 4d). The findings indicated that PHLPP2 is a direct target of the miR-17B92 cluster and more specifically, that miR-92 has an important role in PHLPP2 regulation. We performed gene expression profiling analysis in MCL cell B lines including HBL2, Jeko-1, Mino, Rec-1, SP53, UPN1 and Z138c Figure 3. MiR-17 92 modulates the PI3K/AKT pathway. (a) Z138c- along with 82 primary MCL tumor samples. Expression levels of control and Z138c-miR samples were immunoblotted with PTEN and PHLPP2 antibodies and quantified by the Odyssey Infrared PHLPP2 in Mino, Rec-1 and SP53 were significantly decreased and Imaging System (Lincoln, NE, USA). The ratios of PTEN and PHLPP2 in reached only 50% of the average expression level in primary MCL miR-17B92-overexpressing cells compared with the control cells are tumors and cell lines studied. We also performed aCGH in these shown. (b) Granta-control and Granta-miR cells were immunoblotted cell lines and found that Rec-1 contained a single-copy B7Mb with antibody against BIM. The ratio of BIM in miR-17B92- deletion in chromosome 16 that includes PHLPP2 (Figure 4e). overexpressing cells compared with the control cells is shown. (c) Z138c-control and Z138c-miR samples were immunoblotted with the indicated antibodies. (d) AKT phosphorylated at Serine-473 was B S473 Knockdown of the miR-17 92 cluster suppresses tumor cell stained with Alexa Fluor 647 Rabbit anti-pAKT antibody or IgG proliferation in vitro and tumor growth in a xenograft isotype control using the intracellular immunofluorescent staining MCL mouse model method. The fluorescence histograms of stained cells are shown. We constructed a doxycycline-inducible sponge lentiviral con- struct targeting all the miRNAs within the cluster (Supplementary Figure S1B). By transducing it into Jeko-1 cell line, which expresses BIM, a BH3-only proapoptotic protein, is also markedly down- a high level of miR-17B92,29 we generated a stable cell line (Jeko- regulated in Granta-miR cells (Figure 3b). Among the three cloned Sponge (Jeko-S)) in which miR-17B92 could be conditionally 0 fragments of the BIM 3 -UTR (Supplementary Figure S5A), B2 knocked down. As the miRNAs were sequestered instead of being contains binding sites for miR-17-5p/-20a and -92, whereas B3 degraded, we used a functional assay such as measuring the levels contains binding sites for miR-19 and -92. The luciferase activity of of target proteins or the activity of AKT pathway to confirm that a reporter plasmid containing the wild-type B2 or B3 was the miRNAs were knocked down in our system. Specifically, upon decreased 40% compared with the control construct (Supplemen- doxycycline administration, the protein levels of PHLPP2 and PTEN tary Figure S5B). Luciferase activity was partially restored by in Jeko-S cells increased B72% and 32%, respectively, compared co-transfection with sponge plasmids against miR-20a and -92, with the control cells (Jeko-Control) with or without doxycycline; and to a lesser extent miR-19 (Supplementary Figure S5C). These and Jeko-S cells without doxycycline); and p-AKT decreased to findings suggest that miR-20/-17-5p and miR-92 have an B75% of the control levels (0.62/0.83) (Figure 5a). As a important role in BIM downregulation. consequence, Jeko-S cells showed decreased cell proliferation upon doxycycline administration (Figure 5b). MiR-17B92 cluster modulates the PI3K/AKT pathway in MCL cells Next, we developed a xenograft MCL mouse model by We then examined the activity of the PI3K/AKT pathway in MCL inoculating Jeko-1 cells into the flanks of NOD-SCID mice. After cells. Immunoblot analysis showed higher levels of phosphory- the inoculation of 3 weeks, all mice developed measurable tumors. lated AKT (p-AKT) and its targets, GSK-3b and p70S6K, in Z138c- There was no difference in tumor size among mice inoculated with Jeko-Control or Jeko-S cells without doxycycline treatment. miR than in control cells (Figure 3c). Similarly, an intracellular B immunofluorescence assay (Figure 3d), showing that the level of To study the effect of miR-17 92 inhibition on tumor growth, half p-AKTSer473 in Z138c-miR, was significantly higher than that in of the mice were administered doxycycline. Whereas there was no control cells. Our findings demonstrate that miR-17B92 over- difference in tumor growth in mice inoculated with Jeko-Control expression activates the PI3K/AKT pathway, presumably at least in cells with or without doxycycline, growth of Jeko-S tumors treated part through downregulation of PTEN. with doxycycline was markedly suppressed compared with untreated Jeko-S tumors (1.96±0.89 g versus 3.36±0.74 g, Po0.001) (Figures 5c and d). PHLPP2 and PTEN protein levels PHLPP2, a negative regulator of the AKT pathway, is a direct target were upregulated in doxycycline-treated Jeko-S tumors but not in of the miR-17B92 cluster controls. Conversely, the levels of p-AKT and RPS6 were decreased We have also analyzed the 30-UTR of other known negative 20% and 50%, respectively (Figure 5e). Cell cycle analysis showed regulators of the PI3K/AKT pathway and found that the 30-UTR of that knockdown of miR-17B92 expression blocked the G1--S

Leukemia (2012) 1064 --1072 & 2012 Macmillan Publishers Limited Chemoresistance and tumor growth in mantle cell lymphoma E Rao et al 1069

Figure 4. PHLPP2 is a direct target of the miR-17B92 cluster. (a) A map of the PHLPP2 30-UTR. The cloned segments that contain putative binding sites for miRNA are indicated, as are introduced point mutations. (b)30-UTR reporter plasmids were transfected into HEK293T cells. P1--P3: the various segments cloned from the 30-UTR of PHLPP2 as shown in (a). P1(18M): P1 segment mutated in the putative miR-18 binding site. P1(20M): P1 segment mutated in the putative miR-20-binding site. P3(92-2M): P3 segment mutated in both putative miR-92-binding sites. (c) HEK293T cells with or without P3 segment were transfected with control vector or sponge plasmids targeting miR-18 (S18), miR-20 (S20) or miR-92 (S92). (d) HEK293T cells were transfected with control vector or sponge plasmids targeting miR-18, miR-19, miR-20, miR-92 or the entire miR-17-92 cluster. PHLPP2 levels were determined by immunoblotting. (e) aCGH shows a single copy deletion involving PHLPP2 in Rec-1 cells.

phase transition without significantly affecting apoptosis cellular receptors. Nevertheless, our recent study of diffuse large (Figure 5f). Similarly, using the TUNEL assay, we did not detect B-cell lymphoma showed that the gain/amplification of the any significant changes in apoptosis between Jeko-S-tumors chromosomal region containing miR-17-92 occurs only in the treated with or without doxycycline (data not shown). germinal center-B-like subtype and is mutually exclusive of PTEN deletion,24 suggesting that PI3K/AKT activation may be mediated through the miR-17B92/PTEN pathway. DISCUSSION PI3K/AKT signaling can be terminated through at least two In the current study, we describe a novel oncogenic pathway different mechanisms: removal of the activating lipid second underlying the pathogenesis of MCL: that is, overexpression of messenger, catalyzed by PTEN,30 or dephosphorylation of miR-17B92 targets the protein phosphatase PHLPP2, in addition activated AKT. Dephosphorylation of AKT is mediated by PP2A- to PTEN and BIM, and thereby impedes chemotherapy-induced type phosphatases31 and members of a novel phosphatase family, apoptosis in MCL cells. We showed that high expression of the PHLPP, which includes PHLPP1 and PHLPP2 (also known as miR-17B92 cluster was associated with poorer survival in MCL PHLPPL).32 In the current study, we demonstrated that PHLPP2 is a patients. Inhibition of miR-17B92 augmented the protein level of direct target of miR-17B92, suggesting that miR-17B92 may PTEN and PHLPP2, and inhibited tumor growth in vivo. target both PHLPP2 and PTEN to activate the PI3K/AKT pathway in Constitutive activation of the PI3K/AKT pathway has been MCL. Although PHLPP1 and PHLPP2 both dephosphorylate the shown to contribute to survival in a subset of MCL. Rudelius same residue within the hydrophobic phosphorylation motif on et al.,20 found loss of PTEN expression in 5 of the 17 cases with AKT, they differentially terminate AKT signaling by regulating activated PI3K/AKT. Although loss of PTEN expression may be distinct AKT isoforms. It has been suggested that PHLPP1 has a secondary to gene mutation, gene deletion or epigenetic role in glucose homeostasis, whereas PHLPP2 has a role in cell mechanisms, our data demonstrate that miRNAs, specifically survival. Notably, whereas PHLPP1 modulates the phosphorylation miR-17B92, have an important role and provide a novel of MDM2 and GSK-3a through AKT2, PHLPP2 specially modulates mechanism for PTEN downregulation in MCL. p27 phosphorylation through AKT3 and the phosphorylation of There are clearly many potential causes for PI3K/AKT activation GSK-3b and the FOXO family of forkhead transcription factors in MCL other than PTEN alterations, such as amplification of one of through AKT1.32,33 Therefore, downregulation of PHLPP2 may lead the AKT genes, activation or mutation of one of the three RAS to a constitutive activation of AKT, especially AKT1 and AKT3, proto-oncogenes and activation or mutation of a wide range of resulting in inactivation of FOXO family members, GSK-3b and

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Figure 5. Knockdown of miR-17B92 resulted in decreased cell proliferation in vitro and significant tumor suppression in MCL xenografts. (a) Protein samples from Jeko-Control (Jeko-C) or Jeko-Sponge (Jeko-S) cells, which were treated with or without doxycycline (Dox), were immunoblotted with the indicated antibodies. (b) The proliferation of Jeko-C and Jeko-S cells, which were treated with or without doxycycline, was determined by the MTS assay. The experiments were repeated three times with similar results, and an average ratio to that of Jeko-C cells without doxycycline treatment is shown. (c) Relative tumor sizes over time in NOD/SCID mice. Relative tumor size is the ratio of tumor size at the indicated time compared with that at initiation of doxycycline treatment. (d) Representive tumors samples from the Jeko-S mice treated with or without doxycycline. The average tumor weight is shown on the right. (e) Samples from xenograft tumors were immunoblotted with the indicated antibodies. The average of all xenograft tumor samples is shown. (f) Cells isolated from xenograft tumors were fixed and subjected to cell cycle analysis by fluorescence-activated cell sorting (FACS). The percentage of cells in different cell cycle stages was determined. *Po0.05; **Po0.005; ***Po0.001.

p27kip1 through phosphorylation. Further investigation of the role downregulation in MCL. Repression of BIM by the miR-17B92 of PHLPP2 in MCL is warranted, particularly the potential cluster could also explain the observation that miR-17B92 synergistic effect with the down-modulation of PTEN expression overexpression synergizes with MYC in a mouse model of B-cell on PI3K/AKT signaling. The notion of PHLPP2 as a tumor lymphomagenesis, as a similar synergy in lymphomagenesis has suppressor is further supported by our finding that it is deleted been observed by combining MYC overexpression with BIM in one of the MCL cell lines, Rec-1 and the levels of PHLPP2 mRNA deficiency.38 Activation of the PI3K/AKT pathway may inactivate are decreased in several MCL cell lines. the FOXO family of transcription factors, leading to decreased BIM is one of the most potent pro-apoptotic BH3-only proteins, transcription of BIM.39 --41 The marked BIM down-modulation which binds to all pro-survival BCL2 family members with high therefore could be attributed to a combined effect of both direct affinity,34,35 thereby releasing BAX and BAK proteins, the critical targeting of BIM by miR-17B92 and indirect transcriptional downstream effectors of the BCL2-regulated pathway of apopto- suppression through FOXO inactivation.39 --41 The miR-17B92 sis.36 Homozygous deletions of BIM occur in several MCL cell lines cluster may therefore control signaling networks regulating cell including Jeko-1, SP53 and Z138c, and BIM expression is reduced survival at multiple levels, in a cooperative fashion, in MCL; thus, in Rec-1, but not in Granta-519 or JVM2 cells.37 Our study showed modest effects on multiple factors in the regulatory pathway may that overexpression of the miR-17B92 cluster markedly down- result in marked changes in BIM protein levels and in AKT modulated BIM and provided a novel mechanism for BIM activation.

Leukemia (2012) 1064 --1072 & 2012 Macmillan Publishers Limited Chemoresistance and tumor growth in mantle cell lymphoma E Rao et al 1071 High expression of miR-17B92 facilitates cell proliferation and 3 Tsujimoto Y, Jaffe E, Cossman J, Gorham J, Nowell PC, Croce CM. Clustering of 15 --17 inhibits apoptosis in lymphocytes. MCL patients with high breakpoints on chromosome 11 in human B-cell neoplasms with the t(11;14) miR-17B92 expression also showed the enrichment of gene chromosome translocation. Nature 1985; 315: 340 --343. signatures that were highly associated with cell proliferation. 4 Bodrug SE, Warner BJ, Bath ML, Lindeman GJ, Harris AW, Adams JM. Cyclin D1 Inhibition of miR-17B92 in Jeko-1 cells modestly decreased transgene impedes lymphocyte maturation and collaborates in lymphomagenesis proliferation in vitro but induced a dramatic G1/S arrest in vivo. with the myc gene. EMBO J 1994; 13: 2124 --2130. These results suggest that the miR-17B92 cluster has an 5 Lovec H, Grzeschiczek A, Kowalski MB, Moroy T. Cyclin D1/bcl-1 cooperates with important role in tumor proliferation. In addition to PTEN and myc genes in the generation of B-cell lymphoma in transgenic mice. EMBO J 1994; 13: 3487 --3495. PHLPP2, other targets of miR-17B92 may also participate in 42 43 6 Wlodarska I, Pittaluga S, Hagemeijer A, De Wolf-Peeters C, Van Den Berghe H. regulating proliferation, such as p21 and p57. Secondary chromosome changes in mantle cell lymphoma. Haematologica 1999; The much greater effect of inhibition of miR-17B92 in vivo than 84: 594 --599. in vitro is intriguing and suggests that miR-17B92 may modulate 7 Salaverria I, Zettl A, Bea S, Moreno V, Valls J, Hartmann E et al. Specific secondary tumor/microenvironment interactions to facilitate tumor prolifera- genetic alterations in mantle cell lymphoma provide prognostic information tion in addition to its effects on tumor cells per se. In particular, independent of the gene expression-based proliferation signature. J Clin Oncol miR-17B92 has been shown to promote angiogenesis through 2007; 25: 1216 --1222. several pathways. In RAS-expressing cells, miR-17B92 promotes 8 Ota A, Tagawa H, Karnan S, Tsuzuki S, Karpas A, Kira S et al. Identification and tumor angiogenesis in vivo by targeting the anti-angiogenic characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Res 2004; 64: 3087 --3095. proteins thrombospondin-1 and connective tissue growth factor.12 B 9 He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S et al. Mir-17 92 may also inhibit the TGFb pathway and attenuate its A microRNA polycistron as a potential human oncogene. Nature 2005; 435: 44 antiangiogenic effects. Furthermore, it has been shown that 828 --833. miR-92 may target the von Hippel--Lindau gene product, and 10 Hayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, Tomida S et al. increases VEGF expression.45 A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung The miR-17B92 cluster is known to be upregulated by the E2F cancers and enhances cell proliferation. Cancer Res 2005; 65: 9628 --9632. family.46 In addition, not only is it upregulated by MYC, but we 11 O’Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated also recently reported evidence that inhibitory members of the microRNAs modulate E2F1 expression. Nature 2005; 435: 839 --843. MYC family repress transcription of the locus.47 Indirect activation 12 Dews M, Homayouni A, Yu D, Murphy D, Sevignani C, Wentzel E et al. B Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster. of AKT by miR-17 92 is expected to provide positive feedback, as Nat Genet 2006; 38: 1060 --1065. AKT, by inhibiting GSK3b, decreases phosphorylation of MYC at 48 13 Lu Y, Thomson JM, Wong HY, Hammond SM, Hogan BL. Transgenic over- threonine-58 and thereby decreases its degradation. Similarly, expression of the microRNA miR-17-92 cluster promotes proliferation and activation of AKT is expected to result in inhibition of p27 and p21, inhibits differentiation of lung epithelial progenitor cells. Dev Biol 2007; 310: with activation of CDKs, inhibition of the RB family, and activation 442 --453. of E2F.49 Thus, the miR-17B92 miRNAs may promote their own 14 Navarro A, Bea S, Fernandez V, Prieto M, Salaverria I, Jares P et al. MicroRNA transcription through AKT activation. expression, chromosomal alterations, and immunoglobulin variable heavy chain The finding that inhibition of miR-17B92 activity significantly hypermutations in Mantle cell lymphomas. Cancer Res 2009; 69: 7071 --7078. reduced tumor growth in xenograft mice indicates that miR- 15 Ventura A, Young AG, Winslow MM, Lintault L, Meissner A, Erkeland SJ et al. 17B92 may serve as a target for tumor therapy. Inhibition of Targeted deletion reveals essential and overlapping functions of the miR-17 B through 92 family of miRNA clusters. Cell 2008; 132: 875 --886. individual miRNAs in the miR-17 92 cluster has previously been 16 Koralov SB, Muljo SA, Galler GR, Krek A, Chakraborty T, Kanellopoulou C et al. Dicer shown to inhibit tumorigenesis and tumor growth in different ablation affects antibody diversity and cell survival in the B lymphocyte lineage. 50 51 cancers, including multiple myeloma and neuroblastoma. Cell 2008; 132: 860 --874. Targeting miR-17B92 cluster in MCL may therefore provide a 17 Xiao C, Srinivasan L, Calado DP, Patterson HC, Zhang B, Wang J et al. new therapeutic approach for this incurable disease. Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nat Immunol 2008; 9: 405 --414. 18 Rizzatti EG, Falcao RP, Panepucci RA, Proto-Siqueira R, Anselmo-Lima WT, CONFLICT OF INTEREST Okamoto OK et al. Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT, WNT and TGFbeta The authors declare no conflict of interest. signalling pathways. Br J Haematol 2005; 130: 516 --526. 19 Ghobrial IM, McCormick DJ, Kaufmann SH, Leontovich AA, Loegering DA, Dai NT et al. Proteomic analysis of mantle-cell lymphoma by protein microarray. Blood ACKNOWLEDGEMENTS 2005; 105: 3722 --3730. 20 Rudelius M, Pittaluga S, Nishizuka S, Pham TH, Fend F, Jaffe ES et al. 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