Published OnlineFirst June 7, 2016; DOI: 10.1158/1078-0432.CCR-16-0609 Biology of Human Tumors Clinical Cancer Research Recurrent Mutations in the MTOR Regulator RRAGC in Follicular Lymphoma Zhang Xiao Ying1, Meiyan Jin2, Luke F. Peterson1, Denzil Bernard1, Kamlai Saiya-Cork1, Mehmet Yildiz1, Shaomeng Wang1, Mark S. Kaminski1, Alfred E. Chang3, Daniel J. Klionsky2, and Sami N. Malek1 Abstract Purpose: This study was performed to further our understand- Mutations in RRAGC distinctly clustered on one protein surface ing of the biological and genetic basis of follicular lymphoma and area surrounding the GTP/GDP–binding sites. Mutated RRAGC to identify potential novel therapy targets. proteins demonstrated increased binding to RPTOR (raptor) and Experimental Design: We analyzed previously generated substantially decreased interactions with the product of the tumor whole exome sequencing data of 23 follicular lymphoma cases suppressor gene FLCN (folliculin). In stable retrovirally trans- and one transformed follicular lymphoma case and expanded fected 293T cells, cultured in the presence or absence of leucine, findings to a combined total of 125 follicular lymphoma/3 multiple RRAGC mutations demonstrated elevated MTOR acti- transformed follicular lymphoma. We modeled the three- vation as evidenced by increased RPS6KB/S6-kinase phosphory- dimensional location of RRAGC-associated hotspot muta- lation. Similar activation phenotypes were uncovered in yeast tions. We performed functional studies on novel RRAGC engineered to express mutations in the RRAGC homolog Gtr2 and mutants in stable retrovirally transduced HEK293T cells, stable in multiple lymphoma cell lines expressing HA-tagged RRAGC- lentivirally transduced lymphoma cell lines, and in Saccharo- mutant proteins. myces cerevisiae. Conclusions: Our discovery of activating mutations in RRAGC Results: We report recurrent mutations, including multiple in approximately 10% of follicular lymphoma provides the amino acid hotspots, in the small G-protein RRAGC, which is mechanistic rationale to study mutational MTOR activation and part of a protein complex that signals intracellular amino acid MTOR inhibition as a potential novel actionable therapeutic concentrations to MTOR, in 9.4% of follicular lymphoma cases. target in follicular lymphoma. Clin Cancer Res; 1–11. Ó2016 AACR. Introduction Follicular lymphoma has a varied clinical course that is influ- enced by follicular lymphoma tumor cell–intrinsic and cell- Follicular lymphoma constitutes the most common indolent extrinsic deregulations (2, 5, 6, 8–12). Cell-intrinsic factors, B-cell lymphoma, with an incidence and prevalence of approx- including genomic aberrations and epigenetic deregulations, imately 14,000 and approximately 100,000 cases, respectively in prominently influence the follicular lymphoma phenotype. At the United States (1). Follicular lymphoma remains incurable the genomic level, follicular lymphoma is characterized by recur- with conventional therapies and most patients receive multiple rent acquired structural abnormalities, including translocation treatment regimens during the course of their illness. The devel- (14;18) deregulating BCL2 expression, acquired genomic copy opment of targeted follicular lymphoma–directed therapies is in number aberrations (aCNA), and frequent acquired uniparental- early stages (2–7) and lags behind progress made in other lym- disomy (aUPD)/copy neutral LOH (cnLOH; refs. 13–17). Fur- phoproliferative diseases such as chronic lymphocytic leukemia thermore, an expanding group of recurrently mutated genes has (CLL) or mantle cell lymphoma (MCL). recently been shown to underlie the pathogenesis of follicular lymphoma (KMT2D/MLL2, CREBBP, EZH2, EP300, ARID1A, HIST1H1 B-E, STAT6, and others) and some of these afford novel 1Division of Hematology and Oncology, Department of Internal Med- opportunities for targeted therapy developments (18–26). icine, University of Michigan, Ann Arbor, Michigan. 2Life Sciences To further our understanding of the biological and genetic basis 3 Institute, University of Michigan, Ann Arbor, Michigan. Department of follicular lymphoma and to identify targets for novel therapeutic of Surgery, University of Michigan, Ann Arbor, Michigan. approaches, we have further analyzed follicular lymphoma whole Note: Supplementary data for this article are available at Clinical Cancer exome sequencing (WES) data and have identified recurrent muta- Research Online (http://clincancerres.aacrjournals.org/). tions in the MTOR regulator RRAGC, a small G-protein. RRAGC Prior presentation: Presented as part of a podium presentation at the American forms heterodimers with either RRAGA or RRAGB and together Society of Hematology Meeting, Orlando FL, 2015. these dimers are part of a multiprotein complex that under con- Corresponding Author: Sami N. Malek, University of Michigan, 1500 East Medical ditions of amino acid sufficiency facilitates recruitment of MTOR to Center Drive, 4312 Cancer Center, Ann Arbor, MI 48109-0936. Phone: 734-763- lysosomal membranes resulting in MTOR activation through RHEB 2194; Fax: 734-647-9654; E-mail: [email protected] (27–31). Through sequencing of RRAGC in a combined 125 doi: 10.1158/1078-0432.CCR-16-0609 follicular lymphoma/3 transformed follicular lymphoma cases, Ó2016 American Association for Cancer Research. we identified novel RRAGC mutations, including multiple www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst June 7, 2016; DOI: 10.1158/1078-0432.CCR-16-0609 Ying et al. 3 program (http://primer3.ut.ee/) and sequence information Translational Relevance generated using direct sequencing as described. Mutations were Follicular lymphoma constitutes the second most common confirmed to be somatically acquired using unamplified lympho- non-Hodgkin lymphoma (NHL) in the United States, with ma cell–derived DNA and paired CD3 cell–derived DNA as over 100,000 patients living with the disease. Almost all templates isolated from highly pure flow-sorted cells. patients with follicular lymphoma need therapy within years from diagnosis, and most patients receive multiple chemo- or RRAGC cDNA mutagenesis, retroviral and lentiviral vector immunotherapies over their lifetimes. Despite recent progress, generation, cell transfection, and cell transduction to generate the development of targeted therapies in follicular lymphoma stable cell lines is in its early stages. Here, we report on recurrent activating Reagents and mutagenesis. A pCMV-SPORT plasmid containing mutations in the MTOR regulator RRAGC, a small G-protein the RRAGC cDNA (cat#: MHS6278-202757712; accession: and a component of the cellular amino acid sensing and BC016668) was purchased from Thermo Scientific, and used as sufficiency signaling pathway in approximately 10% of follic- a template to generate mutant RRAGC cDNAs using the Quik- ular lymphoma. Follicular lymphoma–associated RRAGC ChangeLightning Site-Directed Mutagenesis Kit (Stratagene/Agi- mutations activated MTOR as evidenced by elevated phos- lent). Full-length wild-type (WT) and mutant HA-tagged RRAGC phorylated RPS6KB/S6-kinase phosphorylation in multiple were constructed using PCR and cloned into the PacI/NotI sites of experimental systems. These data provide the mechanistic the lentiviral vector FG9 (a gift from Dr. Colin Duckett, University rationale to study mutational MTOR activation in follicular of Michigan; ref. 32). HA-tagged raptor (#8513) and RRAGB WT lymphoma and MTOR inhibition as a potential novel action- (#19301) was purchased from Addgene (27). Anti-HA antibody able therapeutic target in follicular lymphoma subsets. was purchased from Roche (clone 3F10, #11867423001). Anti- raptor (05-1470) was from Millipore. Anti-b-actin (#A544) was from Sigma Aldrich. Antibodies against RRAGC (#3360), anti- folliculin (#3697), anti-RRAGB (#8150), anti-RRAGA (#4357), mutation hotspots, in approximately 10% of follicular lymphoma. anti-S6Kinase (#9202) and anti–pS6-Kinase-Thr 389 (#9205) Detailed functional studies in multiple complementary experimen- were purchased from Cell Signaling Technology. tal systems demonstrated that follicular lymphoma–associated RRAGC mutations are activating MTOR as evidenced by elevated Transient transfection studies in HEK293T cells. HEK293T cells phosphorylated (p)-RPS6KB/S6-kinase (S6K) levels. In aggregate, were transfected in 10-cm dishes with 1 or 2 mg of plasmids these data provide novel insights into the properties of recurrent encoding either WT or mutant forms of RRAGC or various other follicular lymphoma–associated RRAGC mutations and have constructs as indicated using polyethylenimine (Polyscience Inc., implications for novel therapy developments targeting MTOR in #23966). genetically defined follicular lymphoma subsets. Generation of stable transduced HEK293T cell lines. RRAGC cDNAs Materials and Methods excised from pCR Blunt topo vectors using EcoR1 were ligated into Patient characteristics and study source material MSCV-IRES-PURO (MIP; ref. 33) previously dephosphorylated Twenty-three follicular lymphoma cases and one transformed with shrimp alkaline phosphatase (#78390; Affymetrix). Retro- follicular lymphoma provided the source material for massive viral production and infection of cells followed by puromycin parallel sequencing (WES) as described and characteristics of these selection were performed as described previously (33). The cases have been published (18, 22). Results