MicroRNA-17∼92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways
2013; Springer Nature; Volume: 32; Issue: 17 Linguagem: Inglês
10.1038/emboj.2013.178
ISSN1460-2075
AutoresHyun Yong Jin, Hiroyo Oda, Maoyi Lai, Rebecca L. Skalsky, Kelly Bethel, Jovan Shepherd, Seung Goo Kang, Wen‐Hsien Liu, Mohsen Sabouri-Ghomi, Bryan R. Cullen, Klaus Rajewsky, Changchun Xiao,
Tópico(s)MicroRNA in disease regulation
ResumoArticle6 August 2013free access MicroRNA-17∼92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways Hyun Yong Jin Hyun Yong Jin Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Kellogg School of Science and Technology, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Hiroyo Oda Hiroyo Oda Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USAPresent address: Department of Immunology and Pathology, Research Institute, National Center for Global Health and Medicine, 1-7-1, Konodai, Ichikawa-shi, Chiba 272-8516, Japan Search for more papers by this author Maoyi Lai Maoyi Lai Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Rebecca L Skalsky Rebecca L Skalsky Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Kelly Bethel Kelly Bethel Department of Pathology, Scripps Clinic, La Jolla, CA, USA Search for more papers by this author Jovan Shepherd Jovan Shepherd Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Seung Goo Kang Seung Goo Kang Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Wen-Hsien Liu Wen-Hsien Liu Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Mohsen Sabouri-Ghomi Mohsen Sabouri-Ghomi Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Bryan R Cullen Bryan R Cullen Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Klaus Rajewsky Klaus Rajewsky Program of Cellular and Molecular Medicine, Children's Hospital, Immune Disease Institute, Harvard Medical School, Boston, MA, USAPresent address: Max Delbrück Center for Molecular Medicine, Berlin-Buch 13092, Germany Search for more papers by this author Changchun Xiao Corresponding Author Changchun Xiao Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Hyun Yong Jin Hyun Yong Jin Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Kellogg School of Science and Technology, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Hiroyo Oda Hiroyo Oda Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USAPresent address: Department of Immunology and Pathology, Research Institute, National Center for Global Health and Medicine, 1-7-1, Konodai, Ichikawa-shi, Chiba 272-8516, Japan Search for more papers by this author Maoyi Lai Maoyi Lai Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Rebecca L Skalsky Rebecca L Skalsky Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Kelly Bethel Kelly Bethel Department of Pathology, Scripps Clinic, La Jolla, CA, USA Search for more papers by this author Jovan Shepherd Jovan Shepherd Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Seung Goo Kang Seung Goo Kang Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Wen-Hsien Liu Wen-Hsien Liu Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Mohsen Sabouri-Ghomi Mohsen Sabouri-Ghomi Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Bryan R Cullen Bryan R Cullen Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Klaus Rajewsky Klaus Rajewsky Program of Cellular and Molecular Medicine, Children's Hospital, Immune Disease Institute, Harvard Medical School, Boston, MA, USAPresent address: Max Delbrück Center for Molecular Medicine, Berlin-Buch 13092, Germany Search for more papers by this author Changchun Xiao Corresponding Author Changchun Xiao Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Author Information Hyun Yong Jin1,2,‡, Hiroyo Oda1,‡, Maoyi Lai1,‡, Rebecca L Skalsky3, Kelly Bethel4, Jovan Shepherd1, Seung Goo Kang1, Wen-Hsien Liu1, Mohsen Sabouri-Ghomi1, Bryan R Cullen3, Klaus Rajewsky5 and Changchun Xiao 1 1Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA 2Kellogg School of Science and Technology, The Scripps Research Institute, La Jolla, CA, USA 3Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, NC, USA 4Department of Pathology, Scripps Clinic, La Jolla, CA, USA 5Program of Cellular and Molecular Medicine, Children's Hospital, Immune Disease Institute, Harvard Medical School, Boston, MA, USA ‡These authors contributed equally to this work. *Corresponding author. Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel.:+858 784 7640; Fax:+858 784 7643; E-mail: [email protected] The EMBO Journal (2013)32:2377-2391https://doi.org/10.1038/emboj.2013.178 Present address: Department of Immunology and Pathology, Research Institute, National Center for Global Health and Medicine, 1-7-1, Konodai, Ichikawa-shi, Chiba 272-8516, Japan PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR-17∼92 expression is frequently found in human cancers, mainly due to gene amplification and Myc-mediated transcriptional upregulation. Here we show that B cell-specific miR-17∼92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc-driven lymphomagenesis stringently requires two intact alleles of miR-17∼92. We experimentally identified miR-17∼92 target genes by PAR-CLIP and validated select target genes in miR-17∼92 transgenic mice. These analyses demonstrate that miR-17∼92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR-17∼92-driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma-bearing mice. These findings establish miR-17∼92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation. Introduction Studies of human cancer genomes have revealed numerous genetic alterations at the structural and sequence levels. By the time a cancer is diagnosed, it often comprises millions to billions of cells carrying genetic alterations that initiated malignant transformation and many others acquired along the way. Therefore, the contribution of each genetic alteration to cancer varies substantially. Some genetic alterations are strong and causal ‘drivers’ that confer selective growth advantages to cancer cells; others are weaker but important ‘contributors’ to the development of cancer; however, most are incidental ‘passengers’ that have been accumulated by chance during the cancer's life history (Chin and Gray, 2008). The key challenge in cancer research is to distinguish the drivers and contributors from the passengers. Assessing the contribution of individual alterations to cancer in genetically engineered mice has proven to be the gold standard for establishing their causality in cancer (Frese and Tuveson, 2007), and is essential for the development of cancer therapeutics. Studies during the past few years have demonstrated that microRNAs (miRNAs), a class of small non-coding RNAs, play important roles in human cancers (Croce, 2009). MiRNAs are endogenously encoded-single stranded RNAs of ∼22 nucleotides (nts) in length that bind to their target mRNAs and regulate their translation and stability (Ambros, 2004; Bartel, 2004; Bushati and Cohen, 2007). Despite the demonstrated importance of miRNAs, determining miRNA targets has been a major obstacle (Thomas et al, 2010). Many bioinformatic algorithms were developed to predict miRNA target genes, mainly based on seed pairing and evolutionary conservation (Rajewsky, 2006). However, these algorithms typically predict hundreds to thousands of target genes for each miRNA, and different algorithms often produce divergent results. Moreover, these algorithms sometimes fail to predict the most biologically important miRNA targets (Thomas et al, 2010). To overcome this problem, two groups recently developed methods to experimentally identify direct miRNA–target mRNA interactions: HITS-CLIP (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation) and PAR-CLIP (Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) (Chi et al, 2009; Hafner et al, 2010). These new methods hold the promise to identify miRNA binding sites in almost any cells and have the potential to revolutionize miRNA research, in the same way that chromatin immunoprecipitation (ChIP) did to transcription factor research, but their utility still awaits validation in animal models with gain- and loss-of-function mutations for individual miRNAs. Gene expression profiling of human cancer cells has revealed specific miRNA expression signatures in many human cancers (Croce, 2009). However, the development of miRNA-based therapeutics has lagged behind, largely due to the lack of in-depth understanding of function and molecular mechanism of miRNAs in carcinogenesis. We attempt to address this issue through the analysis of miR-17∼92 in the context of lymphomagenesis. MiR-17∼92 is the first miRNA gene implicated in cancer and encodes for six distinct miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92) (Mendell, 2008). These miRNAs fall into four miRNA families (miR-17, miR-18, miR-19, and miR-92 families), with members in each family sharing the same seed sequence. The genomic region encoding human miR-17∼92 is frequently amplified in lymphoma, leukemia, and solid tissue cancers, and the encoded miRNAs are highly expressed in these cancer cells (Mendell, 2008). Previous studies showed that miR-17∼92 overexpression accelerated carcinogenesis initiated by other oncogenic mutations, such as Myc and Notch activation or Rb family deletion (He et al, 2005; Mavrakis et al, 2010; Conkrite et al, 2011). These results established miR-17∼92 as an important contributor to cancer, but it remains unclear whether elevated miR-17∼92 expression per se is sufficient to drive carcinogenesis. The answer to this question will determine whether targeting miR-17∼92 miRNAs or their downstream pathways has any therapeutic value. In addition to gene amplification, miR-17∼92 expression can be deregulated by other mechanisms. Myc, one of the most common and potent oncogenes (Dang, 2012), activates miR-17∼92 expression by directly binding to its genomic locus (O'Donnell et al, 2005). Myc overexpression is the defining feature of Burkitt lymphoma, a disease state characterized by Myc translocation to the immunoglobulin (Ig) locus (Klapproth and Wirth, 2010). A recent study of Burkitt lymphoma patient biopsies found drastic miR-17∼92 overexpression in all the 28 cases examined (Schmitz et al, 2012), confirming that activation of the Myc→miR-17∼92 axis is a ubiquitous feature of this malignancy. Another study showed that deletion of miR-17∼92 in established Myc-driven lymphoma cell lines slowed down their growth in tissue culture and in immunodeficient hosts, suggesting that miR-17∼92 contributes to the optimal growth of those cancer cell lines (Mu et al, 2009). Established cancer cell lines differ from primary cancers in that the former can survive and proliferate in the absence of their natural tumour microenvironment, probably enabled by additional genetic alterations obtained during the tissue culture process. While the study of cancer cell lines is largely responsible for the early progress in cancer research, recent studies suggested that many of those initial observations need to be re-evaluated in autochthonous tumour models (Frese and Tuveson, 2007). Therefore, it remains unclear how critical miR-17∼92 is in the development of autochthonous lymphomas driven by Myc. Here we address these issues directly by generating (1) mice with B cell-specific transgenic miR-17∼92 expression, and (2) mice with deletion of the miR-17∼92 gene in a Myc transgenic Burkitt lymphoma model, and then monitoring lymphoma development in the resulting mice over their lifespan. Furthermore, we experimentally identified miR-17∼92 target genes in B cells by PAR-CLIP, validated select target genes in miR-17∼92 transgenic B cells, and explored the possibility of targeting miR-17∼92 downstream pathways to treat miR-17∼92-driven cancers. Results B cell-specific miR-17∼92 transgenic mice develop lymphomas We have previously created a miR-17∼92 transgenic allele (termed miR-17∼92 Tg) by homologous recombination into the Rosa26 locus. The expression of this transgene can be turned on conditionally by Cre recombinase (Xiao et al, 2008). To directly test the role of elevated miR-17∼92 expression in B cell lymphomagenesis, we generated miR-17∼92 Tg/Tg;CD19Cre mice (termed TG mice hereafter) in which the miR-17∼92 transgene is turned on specifically in the B-cell lineage. We detected a 3–4-fold increase in miR-17∼92 expression in TG B cells (Figure 1A). This level of overexpression is relatively modest compared to the drastic increase (5–30-fold) that is routinely seen for miR-17∼92 in human lymphomas (He et al, 2005; Schmitz et al, 2012). Analysis of B-cell development in the bone marrow did not reveal any significant alterations. In the spleen of TG mice, we observed an expansion of CD19+B220lowCD43+CD5+ B1-like cells, which are present mainly in the peritoneal cavity of wild-type mice, as well as a slight increase in the total B-cell number at the age of 2–4 months (Figure 1B and Supplementary Figure S1A). Figure 1.Mice with B-cell-specific transgenic miR-17∼92 expression develop lymphoma. (A) miR-17∼92 expression in control and TG B2 cells (TG) were determined by northern blot. miRNA/U6 ratios in control B2 cells was arbitrarily set as 1. (B) Flow cytometry analysis of splenocytes of 2-month-old mice. (C) Kaplan–Meier survival curves of 104 TG and 69 littermate control mice. The P-value was determined by Mantel–Cox log-rank test. (D) Splenomegaly, lymphadenopathy, tumour cell infiltration into liver (arrow), and subcutaneous tumour in TG mice. Spl, spleen; LN, lymph nodes. Scale bars, 1 cm. (E) Representative southern blot analysis of tumour clonality using a JH4 probe. Arrowheads indicate clonal bands corresponding to VDJ or DJ rearrangements. Numbers indicate different lymphoma-bearing TG mice. Download figure Download PowerPoint We monitored a cohort of 104 TG and 69 littermate control mice for 2 years for lymphoma development. As shown in Figure 1C, all TG mice died during this period, with an average lifespan of 40 weeks. Macroscopic examination of TG mice, sacrificed when they were sick, revealed splenomegaly and lymphadenopathy, in some cases accompanied by tumours in extranodal tissue compartments such as liver and skin (Figure 1D). Clonal expansion of B cells was observed in 24 of 30 sick TG mice analysed (Figure 1E and Supplementary Table S1). We performed histological and immunohistochemical analyses of involved organs from the 24 mice with clonal B cell expansion (Figure 2 and Supplementary Figure S2, Supplementary Table S1). Of these 24, 12 mice developed diffuse large B-cell lymphoma (DLBCL), characterized by sheets of large-to-intermediate-sized, transformed-appearing lymphoid cells with numerous mitotic figures and a moderate-to-high proliferative rate as assessed by Ki-67 staining. Six mice showed lymphoid proliferations characterized by admixed centrocytic (cleaved)- and centroblastic (non-cleaved)-appearing lymphocytes, with a low-to-moderate proliferative rate as assessed by Ki-67 staining, forming a variably nodular pattern; these mice were diagnosed with follicular lymphoma (FL). One mouse showed proliferation characterized by CD138+ plasmablastic-appearing cells with numerous mitoses and a high Ki-67 positivity rate; these features are characteristic of anaplastic plasmacytoma (AP). Two mice had lymphoid proliferations that were categorized as small B-cell lymphoma (SBL). Among the other four mice, one had clonal B cell lymphoid hyperplasia in the peritoneal cavity, one had aggressive B-cell lymphoma similar to Burkitt or high-grade mucosa-associated lymphoid tissue (MALT) lymphoma, and the other two mice had lymphomas whose histologic types were not determined (Figure 2, Supplementary Figure S2, and Supplementary Table S1). Mouse lymphoma cells differ from wild-type B cells in their capacity to transfer disease upon transplantation into healthy mice. To determine whether TG lymphoma cells are able to transfer disease, we transplanted primary lymphoma cells into Rag1−/− (immunodeficient) and wild type-C57BL/6 (immunosufficient) mice. Most TG lymphomas were able to establish secondary lymphomas in Rag1−/− recipients, while lymphoma cells from 6 out of 23 TG mice were able to establish secondary lymphomas in C57BL/6 recipients (Supplementary Table S1). In all transplantable cases, the primary and secondary lymphoma cells exhibited identical immunophenotypes and clonal bands of the IgH locus (Supplementary Figure S1B and C). Figure 2.Histological and molecular analyses of lymphomas developed in TG mice. (A) Representative immunohistochemical analyses of tumours in TG mice. DLBCL, diffuse large B cell lymphoma; AP, anaplastic plasmacytoma; FL, follicular lymphoma. Scale bars, 20 μm. (B) Western blot analysis of Bcl6 and IRF4 expression in purified lymphoma cells. Naïve and activated (by anti-CD3/CD28 for 48 h) CD4 T cells were used as negative and positive controls, respectively. (C) Clonality and lymphoma subtypes of TG lymphomas. Note that mouse no. 14 had FL in mesenteric lymph node and DLBCL on small intestine, and was counted in both the FL and DLBCL categories.Source data for this figure is available on the online supplementary information page. Source data for Figure 2b [embj2013178-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint DLBCL, FL, and AP are thought to originate from germinal centre (GC) or post-GC B cells. For the 18 cases falling into these three categories, we performed immunohistochemical (IHC) analyses of Bcl6 and IRF4 expression, as well as PNA-binding ability (Supplementary Figure S2B and Supplementary Table S1). In addition, we expanded 6 cases of lymphomas (5 DLBCL and 1 AP) by transplanting into Rag1−/− mice and purifying lymphomas cells from the recipient mice, and analysed their expression of Bcl6 and IRF4 protein, as well as V gene mutation status (Figure 2B and Supplementary Table S2). Consistent with their classification as DLBCL, FL, and AP, 13 of these 18 cases are positive in IHC for Bcl6, a signature transcription factor of GC B cells. Among the 5 IHC Bcl6-negative cases, 2 cases (no. 22 and no. 16) are positive in western blot. This discrepancy between IHC and western blot is probably due to the limited sensitivity of Bcl6 IHC (Cattoretti et al, 2005). The expression of IRF4, which denotes B-cell maturation toward plasma cells during late GC B-cell differentiation (De Silva et al, 2012), ranged from negative, dim-positive, to positive (Figure 2B and Supplementary Figure S2B, Supplementary Table S1), suggesting that lymphomas developed in TG mice are heterogeneous in nature, and that some of them originated from late-stage GC B cells or plasmablasts. Only two cases are positive for PNA binding, which is characteristic of GC B cells. The inability of other lymphomas to bind to PNA may be due to constitutive PI3K signalling in these cells (Figure 8D), similar to that reported in a recent study of a new Burkitt lymphoma mouse model that combines constitutive Myc expression and PI3K signalling, in which GC B cell-derived lymphoma cells are also PNA-negative (Sander et al, 2012). Among all the six cases analysed, only one case (no. 1962) contains significant amount of mutations in their V genes (Supplementary Table S2). While most human DLBCL, FL and AP contain somatic mutations in their V genes, our result may reflect the difference between human lymphomas and lymphomas developed in genetically engineered mice, as reported in a recent study of a mouse model of DLBCL lymphomas driven by constitutive NFκB activation and Blimp1 deletion (Calado et al, 2010). Taken together, these results showed that elevated miR-17∼92 expression alone is sufficient to drive lymphomagenesis in mice with a marked preponderance of GC and post-GC B-cell-derived lymphomas. Myc-driven lymphomagenesis stringently requires two intact alleles of miR-17∼92 To evaluate the role of miR-17∼92 during the development of autochthonous lymphomas driven by Myc, we generated λ-Myc mice carrying conditional miR-17∼92 knockout alleles (miRfl/fl) under the control of CD19Cre (termed Myc;miRfl/fl; CD19Cre mice hereafter) (Rickert et al, 1997; Kovalchuk et al, 2000; Ventura et al, 2008). λ-Myc mice express a c-Myc transgene under the control of the B cell-specific λ light chain regulatory sequence. These mice develop monoclonal tumours recapitulating features of human Burkitt lymphoma (Kovalchuk et al, 2000). MiR-17∼92 miRNAs were significantly induced at the lymphoma stage, and this correlated with Myc expression (Figure 3A and B). The deletion of miR-17∼92 was confirmed in B cells of young Myc;miRfl/fl;CD19Cre mice (Figure 3B), which exhibited largely normal B-cell development in the bone marrow and spleen (Figure 3C). We monitored a cohort of 31 Myc;miRfl/fl; CD19Cre and 27 Myc;miRfl/fl mice over one and a half years for tumour development and survival (Figure 3D). Lymphoma-free survival of Myc;miRfl/fl;CD19Cre mice was significantly extended. Figure 3.Genetic ablation of the miR-17∼92 gene delays Myc-mediated lymphomagenesis. (A, B) The c-Myc protein (A) and miR-17∼92 (B) expression levels in B cells purified from 2–3-month-old mice of indicated genotypes and lymphoma cells purified from λ-Myc mice were determined by western (A) and northern (B) blot analysis, respectively. Left panels show representative blots, and right panels summarize quantification results from three independent experiments (n=5–8 in each group for western blot and n=6∼14 in each group for northern blot). The c-Myc/β-actin and miR/U6 ratios in WT B cells were arbitrarily set as 1. The residual bands in Myc;miRfl/fl;CD19Cre B cells in (B) are likely from cross-hybridization with miRNAs encoded in the two homologous clusters, miR-106a∼363 and miR-106b∼25. (C) Flow cytometry analysis of splenocytes of 2-month-old mice of indicated genotypes (n=5–10 in each group). Splenic B cell (B220+CD19+) numbers are summarized in the right panel. (D) Kaplan–Meier survival curves of 31 Myc;miRfl/fl;CD19Cre and 27 littermate control Myc;miRfl/fl mice. The P-value was determined by Mantel–Cox logrank test.Source data for this figure is available on the online supplementary information page. Source data for Figure 3a [embj2013178-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint We next performed phenotypic and molecular analyses of those lymphomas to gain insights into mechanisms underlying the delayed lymphoma development in Myc;miRfl/fl; CD19Cre mice. Tumours in the control Myc;miRfl/fl group were predominantly mature and immature B-cell lymphomas, which are typical for λ-Myc mice (Kovalchuk et al, 2000) (Figure 4A and Supplementary Figure S3A). In contrast, the vast majority of Myc;miRfl/fl;CD19Cre lymphomas were IgM− and expressed markers characteristic of early precursor B cells (B220+CD19−CD4+CD43+IgM−) (Welner et al, 2008). These precursor B-cell lymphomas were found in only a small fraction of Myc;miRfl/fl mice (Figure 4A and Supplementary Figure S3A). The drastic shift in lymphoma types was further confirmed by Southern blot analysis of the IgH locus. Mature B and immature B-cell lymphomas in Myc;miRfl/fl mice exhibited clonal bands corresponding to VDJ or DJ rearrangements, whereas the IgH locus of all precursor B cell lymphomas in Myc;miRfl/fl; CD19Cre and Myc;miRfl/fl mice retained the germline configuration (Supplementary Figure S3B). Figure 4.Intact miR-17∼92 alleles are required for Myc-driven lymphomagenesis. (A) Distribution of lymphomas in Myc;miRfl/fl and Myc;miRfl/fl;CD19Cre mice based on developmental markers and IgH rearrangement status. Mature B-cell lymphoma, B220+CD19+IgM+AA4.1−; immature B-cell lymphoma, B220+CD19+IgM+AA4.1+; pre-B-cell lymphoma, B220+CD19+IgM−; and early B-lymphocyte precursor-derived lymphoma, B220+CD19−IgM−CD4+. The IgH locus of early B-lymphocyte precursor-derived lymphomas retain germline configuration, whereas other lymphomas display clonal VDJ or DJ rearrangements. Case numbers of each category are indicated. The dash line indicates opportunistic or low Myc expression in early B cell precursors. (B) Western blot analysis of Cre recombinase and Cd19 locus genotyping PCR of lymphoma cells of indicated genotypes. WT and CD19Cre+ B cells were used as controls. Note that the CD19Cre allele was generated by knocking in the Cre recombinase gene into the Cd19 locus. CD19Cre mice used in this study were heterozygous. (C) Genotyping PCR to detect deleted (‘Δ’) and floxed (‘flox’) miR-17∼92 alleles. Upper panel: B cells from miRfl/fl;CD19Cre and miRfl/fl mice were mixed at indicated ratios, and genomic DNA was extracted and used as PCR templates to evaluate the power of the assay. Lower panel: genomic DNA extracted from lymphomas of indicated genotypes were used as PCR templates, in comparison with lymph node samples of young Myc;miRfl/fl;CD19Cre (lane 1) and Myc;miRfl/fl (lane 2) mice, and purified B cells from one 19-month-old lymphoma-free survivor in the Myc;miRfl/fl;CD19Cre group (lane ‘S’). (D, E) Expression levels of miR-17∼92 (D) and c-Myc protein (E) in lymphoma cells of indicated genotypes were determined by northern (D) and western (E) blot analysis, respectively. Left panels show representative blots, and right panels summarize quantification results (n=5–6 in lymphoma groups and n=10 in the WT group for northern blot, and n=5–6 in each group for western blot). The miR/U6 and c-Myc/β-actin ratios in WT B cells were arbitrarily set as 1.Source data for this figure is available on the online supplementary information page. Source data for Figure 4 [embj2013178-sup-0003-SourceData-S3.pdf] Download figure Download PowerPoint The preponderance of CD19-negative precursor B cell lymphomas in Myc;miRfl/fl;CD19Cre mice suggests that those lymphomas lack Cre expression and hence escape CD19Cre-mediated deletion of the floxed miR-17∼92 alleles. Supporting this hypothesis, none of those lymphomas expressed Cre protein (Figure 4B), and they all retained intact miR-17∼92 alleles, a pattern identical to Myc;miRfl/fl lymphomas (Figure 4C, lower panel). In the few cases of CD19-positive pre/immature/mature B-cell lymphomas developed in Myc;miRfl/fl;CD19Cre mice, the complete absence of miR-17∼92 deletion was also observed. It is likely that these tumours were derived from the small fraction of B cells with insufficient amounts of Cre expression (Rickert et al, 1997). Consistent with the presence of intact miR-17∼92 alleles, lymphomas that ultimately developed in Myc;miRfl/fl; CD19Cre mice displayed drastically induced miR-17∼92 miRNAs expression, to the same degree as that in Myc;miRfl/fl lymphomas (Figure 4D). In addition, the c-Myc protein level was comparable between Myc;miRfl/fl;CD19Cre and Myc;miRfl/fl lymphomas (Figure 4E). Thus, the delayed lymphoma development in Myc;miRfl/fl;CD19Cre mice reflects that malignant transformation is restricted to CD19-negative early B-cell precursors, in which the activity of λ-light chain regulatory elements and activation of the Myc transgene might be opportunistic or low, and to a small fraction of CD19-positive B-lineage cells wit
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