Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis
Ryan A. Hlady, Slavomira Novakova, Jana Opavska, David Klinkebiel, Staci L. Peters, Juraj Bies, Jay Hannah, Javeed Iqbal, Kristi M. Anderson, Hollie M. Siebler, Lynette M. Smith, Timothy C. Greiner, Dhundy Bastola, Shantaram Joshi, Oksana Lockridge, Melanie A. Simpson, Dean W. Felsher, Kay-Uwe Wagner, Wing C. Chan, Judith K. Christman, Rene Opavsky
Ryan A. Hlady, Slavomira Novakova, Jana Opavska, David Klinkebiel, Staci L. Peters, Juraj Bies, Jay Hannah, Javeed Iqbal, Kristi M. Anderson, Hollie M. Siebler, Lynette M. Smith, Timothy C. Greiner, Dhundy Bastola, Shantaram Joshi, Oksana Lockridge, Melanie A. Simpson, Dean W. Felsher, Kay-Uwe Wagner, Wing C. Chan, Judith K. Christman, Rene Opavsky
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Research Article Hematology

Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis

Abstract

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b–/– lymphomas, but not in Dnmt3b–/– pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b–/– lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.

Authors

Ryan A. Hlady, Slavomira Novakova, Jana Opavska, David Klinkebiel, Staci L. Peters, Juraj Bies, Jay Hannah, Javeed Iqbal, Kristi M. Anderson, Hollie M. Siebler, Lynette M. Smith, Timothy C. Greiner, Dhundy Bastola, Shantaram Joshi, Oksana Lockridge, Melanie A. Simpson, Dean W. Felsher, Kay-Uwe Wagner, Wing C. Chan, Judith K. Christman, Rene Opavsky

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Figure 1

Accelerated lymphomagenesis and increased relapse rate in Dnmt3b-deficient mice.

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Accelerated lymphomagenesis and increased relapse rate in Dnmt3b-deficie...
(A) EμSRα-tTA, Teto-MYC, Teto-Cre, and ROSA26LOXPEGFP transgenes and the Dnmt3b conditional knockout allele (Dnmt3b2LoxP) used. Blue indicates that tTA directly regulates transcription from Teto promoters and indirectly recombines loxP sites (triangles). (B) Kaplan-Meier survival curves of MYC;Dnmt3bfl/fl (F/F), MYC;Dnmt3b+/– (+/–), and MYC;Dnmt3b–/– (–/–) mice. MS, n, and P values (log-rank test) are indicated. (C) Expression of Dnmt3b, MYC, Dnmt1, and Dnmt3a assessed by Western blot in normal thymocytes (N), control lymphomas (MYC;Dnmt3bfl/fl), and Dnmt3b-deficient (MYC;Dnmt3b–/–) lymphomas. β-Actin served as a loading control. (D) In vitro growth of unselected MYC;Dnmt3b–/– cells infected with the indicated retroviral constructs. Cells were plated at a concentration of 0.2 × 106 per ml (day 0) and replated every 2 days. Percent EGFP+ cells was determined by FACS. Data are mean ± SEM of 3 independent experiments. P < 0.01, 2-way ANOVA/Tukey test, for the last time point. (E) Representative examples of FACS in D. Percent EGFP+ cells is indicated. (F) Western blot analysis of Dnmt3b levels in MSCV-IRES-EGFP (control) and MSCV-Dnmt3b-IRES-EGFP (Dnmt3b) infected MYC;Dnmt3b–/– cells from D and E. (G) Kaplan-Meier relapse-free survival of MYC;Dnmt3bfl/fl and MYC;Dnmt3b–/– cohorts. Terminally ill mice were placed on doxycycline and monitored for tumor relapse. P < 0.01, log-rank test.