Identification of Novel Therapeutic Targets in Atm-Deficient Lymphomas Using a Whole Genome CRISPR/CAS-9 Screen

ATM is a principal DNA damage response protein that synchronises a complex network of cellular responses to double stranded DNA breaks. ATM gene is recurrently mutated in a wide range of lymphoid malignancies, including B-cell chronic lymphocytic leukemia (CLL), T-prolymphocytic leukaemia (T-PLL), mantle cell lymphoma (MCL) and diffuse B cell lymphoma (DLBCL). ATM pathway is utilized by many DNA damaging agents and consequently inactivation of this pathway can lead to chemoresistance. Furthermore, in the absence of ATM tumour cells exhibit genomic instability that can lead to clonal selection and evolution even under current targeted treatments. Consequently there is clear need to understand dependency pathways in ATM-deficient tumours and apply tailored targeted therapies that will specifically eliminate those tumour cells. We have previously presented a novel murine model of ATM-deficiency that spontaneously generate lymphoid tumours, mostly DLBCL. These tumours have been successfully propagated both in recipient mice and in vitro, where several cell lines have been generated. Genome editing methods, such as CRISPR/CAS-9, permit the targeted disruption of specific genes. Protocols for genome wide screens have been developed based on this technology which can be used to identify genes that are essential for cellular survival. As such, these screens can be used to identify dependency pathways for tumours with specific genetic lesions. Using lentiviral transduction we established two cell lines that stably expressed CAS-9. We then performed a genome wide CRISPR screen using the GeCKO library to identify novel therapeutic targets in these Atm-deficient tumours. This library consists of 130,209 unique single guide RNA (sgRNAs), targetting 20,611 genes including 1176 miRNAs. A comparative analysis was performed of sgRNA drop-out following 15 cellular doublings. This revealed a number of pathways including those already known to be synthetically lethal with ATM deficiency, such as ATR and PARP. Pathway analysis of the top genes from this drop-out analysis identified oxidative phosphorylation, the spliceosome, ribosome biogenesis, N-glycan biosynthesis, pyrimidine metabolism and purine metabolism as the most significantly affected pathways. Furthermore, the drop-out screen revealed a number of miRNAs, including MiR-3470a, Mir-3971, MiR-669f and MiR-719. These data provide a unique molecular assessment of the dependency of ATM-deficient lymphomas and provide a number of novel putative therapeutic targets for treating such tumours. Disclosures No relevant conflicts of interest to declare.

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Recurrent DNA Mutations In Non-Hodgkin Lymphomas Reveal Candidate Therapeutic Targets

Blood ◽

10.1182/blood.v116.21.632.632 ◽

2010 ◽

Vol 116 (21) ◽

pp. 632-632

Author(s):

Andrew J Mungall ◽

Ryan D Morin ◽

Jianghong An ◽

Oleksandr Yakovenko ◽

Merrill Boyle ◽

...

Keyword(s):

Cell Proliferation ◽

B Cell ◽

Cell Lines ◽

Small Molecule ◽

Cell Lymphoma ◽

Three Dimensional ◽

Therapeutic Targets ◽

B Cell Lymphoma ◽

Wild Type ◽

Proliferation Assays

Abstract Abstract 632 Introduction: Non-Hodgkin lymphomas (NHL) are the most common type of lymphoma and can be broadly classified as indolent (slow-growing) diseases, progressing over many years; and aggressive (fast-growing) diseases, which progress rapidly. The latter class includes diffuse large B-cell lymphoma (DLBCL), which accounts for approximately 30% of all NHL diagnoses. Three DLBCL subtypes have been identified based on gene expression profiling, namely: germinal center B-cell (GCB), activated B-cell (ABC) and primary mediastinal B-cell lymphoma (PMBCL). These subtypes show substantial differences in response to treatment and ultimate disease outcome, suggesting that molecular subtyping is an important prognostic indicator and that each subtype may benefit from a distinct treatment regimen. Despite recent advances in cancer genomics revealing molecular and mutational differences between these subtypes, further studies focused on the common NHL subtypes are required to identify critical players in the pathogenesis of DLBCL that may be targeted by pharmacological intervention to improve patient outcome. Methods: Using ultra-high throughput whole genome shotgun sequencing (WGSS) and whole transcriptome shotgun sequencing (WTSS/RNA-seq) we have discovered protein-coding mutations in NHL genomes. With a focus on recurrent and likely gain-of-function mutations we have established procedures to model the three-dimensional structures of mutant proteins and using a computational “molecular docking” pipeline have identified candidate molecules with specificity for the mutant protein. These small molecule compounds are acquired and tested in cell proliferation assays against a suite of DLBCL cell lines characterized for target mutations. Results: Mutations affecting a single key tyrosine in the catalytic site of enhancer of zeste, homolog 2 (EZH2), a member of the Polycomb-group family involved in transcriptional repression were identified (Morin, R. et al. 2010 Nature Genetics 42(2):181-5). This mutation, in a gene previously unknown to be mutated in cancer, is restricted to the GCB subtype of lymphomas and is highly prevalent in patient samples and DLBCL cell lines. Mutations have also been observed in other proteins involved in epigenetic regulation and thus afford potentially novel therapeutic targets. In proof-of-principle experiments small molecule inhibitors were identified using molecular docking approaches to target the effect of EZH2 mutations in both mutant and wild-type DLBCL cell lines. We identified and imported 96 compounds from the Developmental Therapeutic Program NCI/NIH repository. These compounds were tested in alamarBlue cell proliferation assays revealing three with activity at 10uM concentration in EZH2 mutant but not wild-type cells. Computational optimization of these compounds is underway to identify related compounds with improved activities at reduced concentrations. Conclusions: High-throughput sequencing platforms have enabled the identification of recurrent, non-synonymous protein mutations in tumor genomes and transcriptomes. Such a catalogue of mutations provides new avenues of exploration for targeted therapy including small molecule inhibitors. Despite intensive efforts launched in recent years to determine the crystal structure for every human protein, many (including EZH2) do not currently have three dimensional structures. This poses a challenge to novel drug discovery but can be overcome using homology modeling and/or targeting other members of a pathway. Our observations also demonstrate the importance of epigenetic regulation in NHL tumorigenesis and thus provide potential new therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

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Novel therapeutic targets in diffuse large B-cell lymphoma

European Journal of Cancer Supplements ◽

10.1016/j.ejcsup.2013.07.041 ◽

2013 ◽

Vol 11 (2) ◽

pp. 262-263 ◽

Cited By ~ 4

Author(s):

Georg Lenz

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Therapeutic Targets ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Large B Cell ◽

Novel Therapeutic

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Essentiality of CREBBP in EP300 truncated B-cell lymphoma revealed by genome-wide CRISPR-Cas9 screen

10.1101/746594 ◽

2019 ◽

Author(s):

Man Nie ◽

Likun Du ◽

Bo Zhang ◽

Weicheng Ren ◽

Julia Joung ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Cell Lines ◽

Time Course ◽

Course Design ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Genetic Alterations ◽

Genome Wide ◽

A Genome

AbstractHistone acetyltransferases (HATs), including CREBBP and EP300, are frequently mutated in B-cell malignancies and usually play a tumor-suppressive role. In this study, we performed whole genome and transcriptome sequencing and a genome-wide CRISPR-Cas9 knockout screen to study a germinal center B-cell like diffuse large B-cell lymphoma (DLBCL) cell line (RC-K8). Using a summarizing method that is optimized to address the complexity introduced by the time-course design, we identified a distinct pattern of genetic essentialities in RC-K8, including a dependency on CREBBP and MDM2, shown already at early time points and a gradually increased dependency on oxidative phosphorylation related genes. The dependency on CREBBP is associated with the corresponding genetic alterations identified in this cell line, i.e. a balanced translocation involves EP300, which resulted in a truncated form of protein that lacks the critical bromodomain and HAT domain. We further evaluated the previously published CRISPR-Cas9 screens and identified a genetic essentiality of CREBBP or EP300 gene in a small set of cancer cell lines, including several DLBCL cell lines that are highly sensitive for EP300 knockout and with CREBBP mutations or copy number loss. The dependency of the remaining HAT function in CREBBP and/or EP300-deficient genotype was validated by testing the HAT-domain inhibitor A-485. Our study suggests that integration of the unbiased, time-course-based functional screen results with the genomic and transcriptomic data can identify druggable vulnerability in individual or subgroups of cell lines/patients, which may help to develop more effective therapeutic strategies for cancers that are genetically highly heterogeneous, like DLBCL.

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Targeting MYC-Driven B-Cell Lymphoma By Inhibition of the Histone Methyltransferase DOT1L

Blood ◽

10.1182/blood-2018-99-115475 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2839-2839 ◽

Cited By ~ 2

Author(s):

Anagha Deshpande ◽

Benson Chen ◽

Parham Ramezani-Rad ◽

Alessandro Pastore ◽

Luyi Zhao ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Cell Lines ◽

Target Genes ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Advisory Committees ◽

Genome Wide

Abstract Aberrant activation of the MYC proto-oncogene is a recurrent feature in human B-cell lymphomas of diverse sub-types, correlating with adverse prognosis and therapy resistance. Direct pharmacological MYC-targeting has proved difficult, but recent studies have shown that targeting chromatin regulators critical for MYC-driven oncogenesis may provide alternative avenues for therapeutic intervention. Recently, it has been demonstrated that MYC-driven oncogenesis in certain solid tumors is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L. We hypothesized that B-cell lymphomas with hyperactive MYC-signaling might be responsive to DOT1L inhibition. In order to test this hypothesis, we tested the effect of the DOT1L inhibitor Pinometostat (EPZ-5676) on a panel of human B-cell lymphoma cell lines featuring elevated MYC. Pinometostat treatment reduced global H3K79 methylation levels, accompanied by a time and dose-dependent decrease in proliferation of several Burkitt's lymphoma cell lines including P493-6, Daudi and Raji. We observed that key MYC-target genes including CDK4, PPAT and NPM1 were significantly downregulated upon Pinometostat treatment, suggesting that DOT1L is required for the transcriptional activation of MYC-target genes in these cells. Pinometostat-treated B-lymphoma cells showed a significant decrease of cells in S-phase compared to controls as assessed by BrdU-labeling assays. Similar results were also obtained in a panel of B-cell lymphoma cell lines with MYC-rearrangements including mantle cell lymphoma (MCL) cell lines Jeko-1, JVM2, Mino-1 and Maver-1 and the diffused large B-cell lymphoma (DLBCL) cell line Karpas 422. Next, we sought to investigate whether the DOT1L-dependence of MYC-driven B-cell lymphoma could be reproduced in a well-defined model of MYC-driven B-cell lymphoma. Towards this end, we utilized a mouse model in which expression of the Cre recombinase from a B cell specific promoter leads to ectopic expression of a transgenic human MYC allele and concomitant deletion of the tumor suppressor Pten in B cells. Similar to our in vitro studies, Pinometostat treatment led to a significant reduction in proliferation of B-cell lymphoma cells from these mice with an IC50 of 0.5 µM. Furthermore, we sought to ascertain whether these findings reflected on-target effects related to DOT1L inhibition. Therefore, we deleted DOT1L using CRISPR/Cas9 in B-cell lymphoma cell lines and assessed the effect on proliferation using competitive-proliferation assays. We observed that DOT1L-deletion progressively diminished the relative growth of anti-DOT1L sgRNA-expressing P493-6 and Jeko1 cells compared to non-targeted cells invitro. In order to test the requirement for DOT1L in lymphoma propagation in vivo, we performed intravenous injections of equal number of Jeko-1 cells with either anti-DOT1L or anti-Renilla control sgRNAs into sub-lethally irradiated non-obese diabetic/severe combined immunodeficiency mice (NOD/SCID) mice. Mice injected with control anti-Renilla sgRNAs succumbed to disease with a median latency of 34 days while the latency of disease in the anti-DOT1L sgRNA cohort was 45 days. In summary, DOT1L depletion significantly delayed disease latency in this invivo disseminated model of B-cell lymphoma (P=0.02). We then performed transcriptomic analyses of Pinometostat-treated B-cell lymphoma cell lines compared to DMSO-treated counterparts using RNA-seq. Gene-set enrichment analysis (GSEA) of RNA-seq data from three different B-cell lymphoma cell lines demonstrated that Pinometostat treatment significantly decreased the expression of MYC-target genes. In order to investigate the intriguing role of DOT1L in regulating MYC-target gene expression, we used ChIP-seq to assess the genome-wide occupancy of MYC following DOT1L inhibitor treatment. Strikingly, our studies demonstrated that DOT1L inhibition significantly reduced the chromatin occupancy of MYC. Taken together, our experiments demonstrate the role of DOT1L in MYC-driven B-cell lymphoma pathogenesis invitro and invivo. Furthermore, our genome-wide studies demonstrate the importance of DOT1L for genomic MYC occupancy. Based on these findings, we propose that therapeutic DOT1L targeting may be a viable strategy in MYC-driven B-cell lymphoma. Disclosures Weigert: Roche: Research Funding; Novartis: Research Funding. Rickert:Pfizer: Employment. Ren:Elli Lilly: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arima Genomics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deshpande:Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.

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Human BCL11B Is Expressed in Normal T Cells and Differentially Expressed in T-Cell Malignancies.

Blood ◽

10.1182/blood.v106.11.4393.4393 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4393-4393

Author(s):

Karen Pulford ◽

Linden Lyne ◽

Giovanna Roncador ◽

Ryo Kominami ◽

Alison H. Banham

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Cell Lines ◽

Zinc Finger ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Expression Data ◽

Wide Range ◽

T Cell Malignancies

Abstract B-cell lymphomaleukaemia 11B (BCL11B), the human homologue of murine Bcl11b/Rit-1/CTIP2, was originally identified as a novel tumor suppressor gene in a study of gamma-radiation induced thymic lymphomas in mice. Later studies, however, showed Bcl11b to have a vital role in T cell development and survival, with BCL11B translocations involving a variety of partner genes being reported in both T-cell and myeloid leukemias. The human BCL11B gene, located at 14q32.2, encodes an 832aa Kruppel C2H2 zinc finger protein that is functionally uncharacterized but likely to act as a transcriptional regulator. Analysis of publically available normal tissue Affymetrix microarray expression data indicates an expression pattern restricted to hematopoietic cells, with high levels of BCL11B transcripts being present only in peripheral blood T cells, NK cells, thymus and tonsil. We have used two rabbit polyclonal anti-BCL11B antibodies to study the distribution of BCL11B protein in both normal and neoplastic human cells. These reagents, raised against two distinct regions of the murine Bcl11b protein (zinc finger and C-terminus), recognized the human BCL11B protein. While neither antibody stained B-cells in tonsil, one was crossreactive with the highly homologous BCL11AXL protein by Western blotting. In normal tissues, BCL11B protein expression was confined to the nuclei of the vast majority of T cells in thymus (foetal and adult) and tonsil. High levels of BCL11B were detected in T-cell lines, including the Molt-4, CCRF-CEM and Jurkat T-cell acute lymphoblastic leukaemia (T-ALL) derived cell lines. No expression was detected in any B-cell derived (pre-B to plasma cell stage) or myeloid cell lines studied. These results are consistent with the microarray gene expression data. In T-cell malignancies, BCL11B protein was only detected in a proportion of tumors, including 5/6 T-ALLs (one being weakly stained) and 2/8 peripheral T-cell lymphomas (weak cytoplasmic staining only). Interestingly, no expression was detected in ALK-positive anaplastic large cell lymphoma lines or tumors. Further studies of a larger series of T-cell malignancies are in progress. All other tumors studied, including B-ALL, chronic lymphocytic leukaemia, diffuse large B-cell lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, follicular lymphoma, myeloma and Hodgkin’s lymphoma, were unlabelled. In conclusion, the distribution pattern of the BCL11B protein in a wide range of both normal and neoplastic tissues is described for the first time. The study of BCL11B expression is an invaluable first step towards elucidating the role of this protein in T-cell biology and the significance of its differential expression in T-cell malignancies.

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Ofatumumab demonstrates activity against rituximab-sensitive and -resistant cell lines, lymphoma xenografts and primary tumour cells from patients with B-cell lymphoma

British Journal of Haematology ◽

10.1111/j.1365-2141.2011.08966.x ◽

2011 ◽

Vol 156 (4) ◽

pp. 490-498 ◽

Cited By ~ 49

Author(s):

Matthew J. Barth ◽

Francisco J. Hernandez-Ilizaliturri ◽

Cory Mavis ◽

Ping-Chiao Tsai ◽

John F. Gibbs ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Primary Tumour ◽

B Cell Lymphoma ◽

Resistant Cell ◽

Tumour Cells

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Deletions of the cyclin-dependent kinase inhibitor genes p16INK4A and p15INK4B in non-Hodgkin's lymphomas

Blood ◽

10.1182/blood.v86.4.1534.bloodjournal8641534 ◽

1995 ◽

Vol 86 (4) ◽

pp. 1534-1539 ◽

Cited By ~ 57

Author(s):

AF Gombart ◽

R Morosetti ◽

CW Miller ◽

JW Said ◽

HP Koeffler

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Point Mutations ◽

B Cell Lymphoma ◽

Chromosome 9 ◽

Wide Range ◽

Hodgkin's Lymphomas ◽

Homozygous Deletions ◽

Large B Cell

The tumor suppressor genes p16INK4A and p15INK4B map to the 9p21 chromosomal locus and are either homozygously deleted or mutated in a wide range of human cancer cell lines and tumors. Although chromosome 9 abnormalities have been described in non-Hodgkin's lymphomas (NHLs), to date, the mutational status of these genes has not been determined for these malignancies. A total of five cell lines and 75 NHLs were examined for homozygous deletions or point mutations in the coding regions of both the p15 and p16 genes using Southern blot and/or polymerase chain reaction-single-strand conformation polymorphism analyses. Homozygous deletions of either the p16 gene or both the p15 and p16 genes were observed in one diffuse large B-cell lymphoma cell line and two uncultured lymphomas consisting of one large B-cell and one mixed T-cell lymphoma. In contrast, point mutations were not detected in either the cell lines or lymphomas. These results indicate that the rate of alterations in the p15 and p16 genes is low for lymphomas, but loss of p16 and/or p15 may be involved in the development of some lymphomas.

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Targeted Therapy of B Cell Lymphoma with a Direct Inhibitor of the NF-κB Subunit c-Rel

Blood ◽

10.1182/blood.v124.21.507.507 ◽

2014 ◽

Vol 124 (21) ◽

pp. 507-507

Author(s):

Yusuke Shono ◽

Andrea Z. Tuckett ◽

Hsiou-Chi Liou ◽

Samedy Ouk ◽

Ekaterina Doubrovina ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Tnf Α ◽

Wide Range ◽

In Vitro Treatment ◽

Human B Cell

Abstract NF-kB plays important roles in immunity and oncogenesis, indicating that therapeutic targeting of this pathway could be beneficial in various clinical settings; however,an NF-kB-specific inhibitor does not exist in clinical practice to date. One approach toward development of such a compound is small-molecule-mediated direct inhibition of one or several members of the NF-kB family of transcription factors, a network that comprises five structurally related proteins including p50, p52, RelA, RelB and c-Rel. After screening of a library of 15,000 small molecules with a biochemical assay, we identified two scaffolds with inhibitory activity specific for the NF-kB subunit c-Rel. These scaffolds act as direct c-Rel inhibitors by modifying the conformation of the c-Rel protein, thus preventing DNA binding. We previously reported that in vitro treatment of T cells with the thiohydantoin IT-603 induces c-Rel deficiency, resulting in suppression of T cell alloactivation without compromising T cell activation triggered by recognition of tumor-associated or viral antigens (Shono et al., Cancer Discovery, 2014). Here, we for the first time demonstrate in vivo efficacy of a c-Rel inhibitor treatment regimen in mouse models of graft-versus-host disease (GVHD) and graft-versus-lymphoma (GVL), as well as xenograft models of human B cell lymphomas, revealing that inhibition of c-Rel activity allows not only for suppression of GVHD while retaining GVL activity, but it also mediates promising anti-lymphoma effects. We first show that the novel small molecule IT-901 is a more potent c-Rel inhibitor than IT-603 and has a superior pharmacokinetic profile. IT-901 displayed significantly improved in vivo efficacy, ameliorating GVHD while preserving the anti-lymphoma activity of T cells (Figure 1a,b). Recent genetic evidence has established a pathogenetic role for NF-kB signaling in lymphoid malignancies. We therefore sought to explore the potential of IT-901 for targeted therapy of human lymphomas. We analyzed six representative diffuse large B cell lymphoma (DLBCL) cell lines including activated B-like (ABC; HBL1, TMD8, U2932) and germinal center B-like (GCB; Ly19, SU-DHL4, SU-DHL8) cell lines for nuclear translocation of c-Rel and found that c-Rel was constitutively active in all cell lines. To examine if c-Rel inhibition with IT-901 alters cytokine production by DLBCL cells, we analyzed cytokine levels in the supernatant after in vitro incubation with IT-901. IT-901 treatment resulted in decreased levels of a wide range of cytokines in TMD8 cells, with the notable exceptions of interleukin 8 (IL-8), tumor necrosis factor (TNF)-α, and TNF-β (P<0.05, Figure 2a). We next investigated if IT-901 treatment affected growth of DLBCL cells in vitro. We found that IT-901 dose-dependently inhibited cell growth of both ABC and GCB cell lines with IC50 values between 3µM to 4µM. Interestingly, IT-901 at a concentration of 3µM did not have an anti-proliferative effect on TMD8 cells, suggesting that cytokines such as IL-8 and TNF-α may be upregulated as a mechanism of resistance to c-Rel inhibition by activating alternative survival pathways. Indeed, in vitro treatment of TMD8 cells with a TNF-α neutralizing antibody inhibited cell growth, and this effect was enhanced when combining TNF-α blockade with c-Rel inhibition (P<0.01, Figure 2b). Furthermore, we detected high HMOX1 protein levels in DLBCL cells treated with IT-901, suggesting that HMOX1 expression was induced, which is a hallmark of oxidative stress. Indeed IT-901 induced production of high levels of reactive oxygen species in lymphoma cells. This suggests that induction of oxidative stress may be a second mechanism contributing to the anti-lymphoma activity of IT-901. We next analyzed primary lymphoma cells and found that the c-Rel gene is widely expressed in human B cell malignancies and frequently amplified in DLBCL and EBV-transformed B cells. Importantly, intranuclear analysis of the c-Rel protein demonstrated that this transcription factor can be constitutively active in a wide range of human lymphomas. IT-901 efficiently inhibited growth of EBV-transformed B cells in vitro, and mediated significant anti-lymphoma activity in a xenograft model of EBV-induced lymphoma (P<0.01, Figure 2c). In summary, our findings underscore multiple therapeutic benefits and great potential for clinical translation of a novel c-Rel inhibitor. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11

Journal of Oncology ◽

10.1155/2019/9832956 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Danxia Zhu ◽

Cheng Fang ◽

Wenting He ◽

Chen Wu ◽

Xiaodong Li ◽

...

Keyword(s):

B Cell ◽

Binding Site ◽

Cell Lines ◽

Expression Vector ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Wild Type ◽

Protein Levels ◽

Large B Cell Lymphoma ◽

Large B Cell

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.

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