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Oncogenes and Tumor Suppressors
Sleeping Beauty Screen Identifies RREB1 and Other Genetic Drivers in Human B-cell Lymphoma Eric P. Rahrmann1,2, Natalie K.Wolf1, George M. Otto2, Lynn Heltemes-Harris2,3,4, Laura B. Ramsey4, Jingmin Shu2, Rebecca S. LaRue2, Michael A. Linden2,5, Susan K. Rathe2, Timothy K. Starr2,6, Michael A. Farrar2,3,4, Branden S. Moriarity2,7,8, and David A. Largaespada1,2,7,8
Follicular lymphoma and diffuse large B-cell lymphoma (DLBCL) are the most common non-Hodgkin lymphomas distinguishable by unique mutations, chromosomal rear- rangements, and gene expression patterns. Here, it is dem- onstrated that early B-cell progenitors express 20,30-cyclic- nucleotide 30 phosphodiesterase (CNP) and that when targeted with Sleeping Beauty (SB) mutagenesis, Trp53R270H
mutation or Pten loss gave rise to highly penetrant lymphoid diseases, predominantly follicular lymphoma and DLBCL. In efforts to identify the genetic drivers and signaling path- ways that are functionally important in lymphomagenesis, SB transposon insertions were analyzed from splenomegaly specimens of SB-mutagenized mice (n ¼ 23) and SB-muta- genized mice on a Trp53R270H background (n ¼ 7) and identified 48 and 12 sites with statistically recurrent trans- poson insertion events, respectively. Comparison with
human data sets revealed novel and known driver genes for B-cell development, disease, and signaling pathways: PI3K–AKT–mTOR, MAPK, NFkB, and B-cell receptor (BCR). Finally, functional data indicate that modulating Ras- responsive element-binding protein 1 (RREB1) expression in human DLBCL cell lines in vitro alters KRAS expression, signaling, and proliferation; thus, suggesting that this proto- oncogene is a common mechanism of RAS/MAPK hyper- activation in human DLBCL.
Implications: A forward genetic screen identified new genetic drivers of human B-cell lymphoma and uncovered a RAS/ MAPK–activating mechanism not previously appreciated in human lymphoid disease. Overall, these data support target- ing the RAS/MAPK pathway as a viable therapeutic target in a subset of human patients with DLBCL.
Introduction B-cell malignancies comprise a large family of diseases ranging
fromhighly curableHodgkin lymphoma to themore diverse non- Hodgkin lymphoma subtypes including the indolent follicular lymphoma and the aggressive, genetically heterogeneous diffuse large B-cell lymphoma (DLBCL; ref. 1). Molecular profiling of B-cell malignancies has identified defining genetic features for many of the subtypes leading to new therapeutic targets and increased survival-rates for some diseases (1). DLBCL, which
occur predominantly in older adults, diagnosis and treatment have greatly been impacted by the genetic profiling efforts. DLBCL is categorized into two unique molecular subtypes based on gene expression profiling: activated B-cell–like (ABC) and germinal center B-cell–like (GCB; ref. 1). Transcriptomic and genomic analyses identified recurrent genomic aberrations and signaling pathway alterations unique to each subtype and common to both (2, 3). Mutations in genes altering B-cell receptor (BCR) signaling andNFkB activation (e.g.,CD79A,MALT1, andMYD88) aremore common in ABC DLBCL, whereas mutations in genes altering histone modifications and B-cell homing (e.g., EZH2, CREBBP, andMLL2) are more common in GCB DLBC (4–6). Mutations in TP53, immunosurveillance genes (e.g., B2M, CD58), epigenetic modifiers (e.g., CREBBP), and MYC copy number alteration (CNA) gains occur in both subtypes (2). Whole-genome and -exome sequencing efforts have identified over 300 recurrently mutated genes in primary DLBCL samples (3, 5, 7, 8). However, there is still limited knowledge on functional impact of many of these mutations and genetic alterations on disease initiation and progression; genetically engineered mouse models (GEMM) pro- vide a platform to begin evaluating these putative targets.
The Sleeping Beauty (SB) somatic cell mutagenesis system has successfully identified genetic drivers of various cancers including hepatic, intestinal, pancreatic, osteosarcoma, and T-cell (9–14). We previously reported the identification of novel genetic drivers of peripheral nerve–related cancers targeting SB mutagenesis to 20,30-cyclic-nucelotide 30 phosphodiesterase (Cnp)-expressing cells in mice in the context of EGFR overexpression with
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota. 2Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota. 3Lab Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota. 5Center for Immunology, University of Minnesota, Minneapolis, Minnesota. 4Department of Laboratory Medicine and Pathology, Division of Hematopathology, University of Minnesota, Minneapolis, Minnesota. 6Department of Ob-Gyn and Women's Health, University of Minnesota, Minneapolis, Minnesota. 7Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota. 8Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota.
Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).
Corresponding Author: Eric P. Rahrmann, University of Cambridge, Robinson Way, Cambridge CB2 0RE, United Kingdom. Phone: 012-2373-0854; Fax: 012-2376-9881; E-mail: firstname.lastname@example.org
�2018 American Association for Cancer Research.
Molecular Cancer Research
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Published OnlineFirst October 24, 2018; DOI: 10.1158/1541-7786.MCR-18-0582
Trp53R270Hmutation (12). Mutagenesis alone or in the context of onlyTrp53R270Hmutationwas inefficient at developing peripheral nervous system tumors (12). We describe here how these animals developed highly penetrant (65%) lymphoid disease (follicular lymphoma and DLBCL). Analysis of SB-induced lymphomas identified 59 common insertion sites (CIS), of which several were associated with signaling pathways altered in human DLBCL formation: PI3K–AKT–mTOR, NFkB, and BCR signaling. We also identified several novel proto-oncogenes and tumor suppressor genes (TSG) for B-cell lymphoma, for example, Ras-responsive element binding protein 1 (Rreb1) and Ambra1, respectively. Furthermore, we described new roles for Rreb1, a MAPK pathway effector, inDLBCLmaintenance and its impact onKras expression, revealing an unknown mechanism for RAS activation in DLBCL.
Materials and Methods Transgenic animals
Three transgenes were used to induce SB mutagenesis: Condi- tionally expressed SB (R26SB11LSL; ref. 15), Cnp promoter–driven cre recombinase (Cnp-Cre; ref. 16) and oncogenic transposon, concatemer (T2/Onc15). Cnp-Cre;R26SB11LSL;T2/Onc15 (SB- mutagenized) mice underwent insertional mutagenesis in Cnpþ
cells. Genotyping PCR was performed on phenol-chloroform– extracted mouse tail DNA (10, 16, 17). Conditionally expressed Pten (Ptenf/f) and Trp53 (Trp53R270H) allele mice were utilized (17, 18). B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J reporter mice (The Jackson Laboratory) were utilized for lineage tracing studies. Allmicewerebredand cared forunder the guidelines of the University of Minnesota Animal Care and Use Committee.
V(D)J PCR One-hundred nanograms of DNA from control and SB-
mutagenized spleens underwent PCR to assess V(D)J clonality for VHJ558/JH3, VHQ52/JH3, VH7183/JH3, and DHL/JH3 recombi- nation (19). PCR for Actb served as the loading control.
Flow cytometry Single-cell suspensions from bone marrow (femur and tibia),
spleen, and lymph nodes were stained with the following anti- bodies: a-IgM (Jackson ImmunoResearch), a-IgD (11–26), a-BP-1 (FG35.4), a-CD5 (53-7.3), a-CD19 (1D3), a-CD21/35 (7E9), a-CD23 (B3B4), a-CD24 (M1/69), a-CD25 (PC61.5), a-CD38 (90), a-CD43 (S7, BD Biosciences), a-CD45R (RA3- 6B2) for Hardy fractionation (20). Antibodies were obtained from eBioscience unless otherwise indicated. SA-PerCP-Cy5.5 (eBioscience) was used to detect biotinylated antibodies. Cells were assayed on a LSRII flow cytometer (BD Biosciences); data were analyzed using FlowJo software (Treestar).
Transposon insertion site analysis DNA-T2/Onc junctions were amplified by linker-mediated
PCR (LM-PCR), purified using MinElute 96 UF Plates (Qiagen), and submitted for high-throughput HiSeq 2500 sequencing (Illumina) or 454 pyrosequencing (12). A total of 4 � 107 100-bp reads (Illumina) and 384,919 100-bp reads (454 pyro- sequencing) were processed and analyzed using Transposon Annotation Poisson Distribution Association Network Connec- tivity Environment (TAPDANCE) software and gene-centric CIS analysis software (21, 22). Mouse build NCBI37/mm9 was used to map insertion cites and subsequent analyses.
IHC TheM.O.M. kit (Vector Laboratories Inc.) was used for blocking
and antibody incubations. Primary antibodies: Ki67 (1:100; Leica Biosystems), RREB1 (1:100; Sigma-Aldrich), pErk (1:100; Cell Signaling Technology), pAkt (1:100; Cell Signaling Technology), Kras (1:100, Santa Cruz Biotechnology), and SB (1:100; R&D Systems). Corresponding biotinylated secondary antibodies (1:250; Vector Laboratories Inc.) were used followed by incuba- tion with Vectastain ABC Kit (Vector Laboratories Inc.) and developed using peroxidase substrate kit DAB (Vector Laborato- ries Inc.). Slides were counterstained with hematoxylin, dehy- drated, cleared with xylene, and mounted with permount (Ther