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Next-Generation Sequencing of Translocation Renal Cell Carcinoma Reveals Novel RNA Splicing Partners and Frequent Mutations of Chromatin-Remodeling Genes

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Next-Generation Sequencing of Translocation Renal Cell Carcinoma Reveals Novel RNA Splicing Partners and Frequent Mutations of Chromatin-Remodeling Genes Published OnlineFirst June 4, 2014; DOI: 10.1158/1078-0432.CCR-13-3036 Clinical Cancer Biology of Human Tumors Research Next-Generation Sequencing of Translocation Renal Cell Carcinoma Reveals Novel RNA Splicing Partners and Frequent Mutations of Chromatin-Remodeling Genes Gabriel G. Malouf1, Xiaoping Su7, Hui Yao7, Jianjun Gao8, Liangwen Xiong8, Qiuming He8, Eva Comperat 2, Jer ome^ Couturier3, Vincent Molinie4, Bernard Escudier5, Philippe Camparo6, Denaha J. Doss9, Erika J. Thompson9, David Khayat1, Christopher G. Wood10, Willie Yu11, Bin T. Teh11, John Weinstein7, and Nizar M. Tannir8 Abstract Purpose: MITF/TFE translocation renal cell carcinoma (TRCC) is a rare subtype of kidney cancer. Its incidence and the genome-wide characterization of its genetic origin have not been fully elucidated. Experimental Design: We performed RNA and exome sequencing on an exploratory set of TRCC (n ¼ 7), and validated our findings using The Cancer Genome Atlas (TCGA) clear-cell RCC (ccRCC) dataset (n ¼ 460). Results: Using the TCGA dataset, we identified seven TRCC (1.5%) cases and determined their genomic profile. We discovered three novel partners of MITF/TFE (LUC7L3, KHSRP, and KHDRBS2) that are involved in RNA splicing. TRCC displayed a unique gene expression signature as compared with other RCC types, and showed activation of MITF, the transforming growth factor b1 and the PI3K complex targets. Genes differentially spliced between TRCC and other RCC types were enriched for MITF and ID2 targets. Exome sequencing of TRCC revealed a distinct mutational spectrum as compared with ccRCC, with frequent mutations in chromatin-remodeling genes (six of eight cases, three of which were from the TCGA). In two cases, we identified mutations in INO80D, an ATP-dependent chromatin-remodeling gene, previously shown to control the amplitude of the S phase. Knockdown of INO80D decreased cell proliferation in a novel cell line bearing LUC7L3–TFE3 translocation. Conclusions: This genome-wide study defines the incidence of TRCC within a ccRCC-directed project and expands the genomic spectrum of TRCC by identifying novel MITF/TFE partners involved in RNA splicing and frequent mutations in chromatin-remodeling genes. Clin Cancer Res; 20(15); 1– 12. Ó2014 AACR. Introduction Authors' Affiliations: Departments of 1Medical Oncology and 2Pathology, Groupe Hospitalier Pitie-Salp etri^ ere, Assistance Publique Hopitaux de Translocation renal cell carcinoma (TRCC) is a rare Paris, Faculty of Medicine Pierre et Marie Curie, Institut Universitaire de Cancerologie GRC5, University Paris 6; 3Department of Genetics, Institut subtype of kidney cancer that was added to the World Curie; 4Department of Pathology, Hopital^ Saint Joseph, Paris; 5Department Health Organization (WHO) classification in 2004 (1) and of Medical Oncology, Institut Gustave Roussy, Villejuif; 6Centre de Patho- is histologically and genomically a heterogeneous disease logie, Amiens, Picardie, France; Departments of 7Bioinformatics and Computational Biology and 8Genitourinary Medical Oncology, 9DNA Anal- (2, 3). TRCC is characterized by translocations involving the ysis Facility, Department of Genetics, 10Division of Surgery, Department of genes for transcription factors E3 (TFE3) and EB (TFEB; Urology, The University of Texas MD Anderson Cancer Center, Houston, refs. 1–4). TFE3 and TFEB belong to the microphthalmia Texas; and 11Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore, Singapore transcription factor/transcription factor E (MITF/TFE) fam- Note: Supplementary data for this article are available at Clinical Cancer ily of basic helix-loop-helix leucine zipper (bHLH-LZ), and Research Online (http://clincancerres.aacrjournals.org/). are often called MITF TRCC. Although the TFEB gene has been reported to fuse exclusively with the Alpha gene, G.G. Malouf, X. Su, and H. Yao contributed equally to the work and share the role of first author as first coauthors. leading to t(6;11)(p21;q21) translocation, the TFE3 gene Corresponding Authors: Nizar M. Tannir, University of Texas MD Ander- (Xp11.2) has been found to rearrange with at least five son Cancer Center, 1155 Pressler Street, Unit 1374, Houston, TX 77030- different partners: PRCC (1q21), ASPSCR1 (17q25), SFPQ 3721. Phone: 713-563-7265; Fax: 713-745-0422; E-mail: (1p34), NONO (Xq12), and CLTC (17q23; refs. 1–6). The [email protected]; and Gabriel G. Malouf, Department of Medical Oncology, Hopital^ de la Pitie-salp etriere,^ 43, Boulevard de l'Hopital,^ 75013, breakpoints of those translocations differ according to the Paris, France. Phone: þ33 1 42 16 04 61; Fax: þ33142160423; TFE3 partner, and all TFE3 fusion proteins contain the [email protected] bHLH-LZ and transcriptional activation domains of TFE3 doi: 10.1158/1078-0432.CCR-13-3036 (7). Unlike TFE3, the TFEB gene rearranges with Alpha,an Ó2014 American Association for Cancer Research. intronless gene, leading to a translocation that preserves the www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst June 4, 2014; DOI: 10.1158/1078-0432.CCR-13-3036 Malouf et al. Cancer Center (MDACC) and Institut Curie, fresh tissue Translational Relevance specimens were obtained at the time of nephrectomy and We performed exome and RNA sequencing on seven stored at À80C until DNA and RNA extraction were MITF/TFE translocation renal cell carcinoma (TRCC) carried out. The clinicopathologic characteristics of these tumors and validated our findings in seven of 460 cases are summarized in Supplementary Table S1. Geno- (1.5%) clear-cell RCC cases from The Cancer Genome mic DNA with matched normal DNA (adjacent normal Atlas (TCGA). We discovered three novel partners of kidney tissue) was available from three cases (Supple- MITF/TFE (LUC7L3, KHSRP, and KHDRBS2) that are mentary Table S1). RNA from seven controls (three pap- involved in RNA splicing. TRCC displayed a unique gene illary RCC, one ccRCC, and three normal kidney samples) expression signature with activation of MITF, the trans- was included. RNA was available for seven cases (Sup- forming growth factor b1, and the PI3K complexes. plementary Table S1). DNA extraction was performed Genes differentially spliced between TRCC and using the DNeasy Blood & Tissue Kit (Qiagen) according other RCC types were enriched for MITF targets, sug- to the manufacturer’s instructions. RNA extraction was gesting a putative role for RNA splicing in kidney performed using the RNeasy Kit (Qiagen) according to carcinogenesis. Exome sequencing revealed mutations the manufacturer’s instructions. in the chromatin-remodeling gene INO80D.Ourstudy expands the spectrum of TRCC and raises potential RNA sequencing therapeutic implications. Total RNA for each sample was converted into a library of template molecules for sequencing on the Illumina HiSeq 2000 according to the NuGen Ovation RNA-Seq System V2 protocol. The details are reported in Supplementary Mate- TFEB full-length coding region, which becomes dysregu- rials and Methods. lated by the Alpha gene promoter (8). TRCC represents 15% of RCC in patients younger than Mapping/alignment 40 years (9, 10). The incidence of TRCC varies between 1% We checked the quality of the sequencing data by using and 6% according to previously published studies, several HTSeq package. The raw paired-end reads in FASTQ format TFE3 of which used morphology and expression alone to were then aligned to the human reference genome, screen for RCC cases with translocation (10–12). However, GRCh37/hg19, using MOSAIK alignment software (20). the true incidence of TRCC in an unselected cohort of MOSAIK works with paired-end reads from Illumina HiSeq pathologically confirmed clear-cell RCC (ccRCC) remains 2000 and uses both a hashing scheme and the Smith– to be determined. Next-generation sequencing highly Waterman algorithm to produce gapped optimal align- improved our understanding of ccRCC, known to bear ments and to map exon junction-spanning reads with a inactivation of the von Hippel-Lindau (VHL) tumor-sup- local alignment option for RNA-Seq. The resulting pair-wise pressor gene, located on chromosome 3p arm (13). Large- alignments were then consolidated into a multiple scale screening has identified several new cancer genes that sequence alignment (assembly) and saved as a standard PBRM1 include mutations in the SWI/SNF family gene (14) bam file. and BAP1 (15), as well as mutations in chromatin remo- KDM6A KDM5C SETD2 ( ) delers such as (16), , and 17 .To Identification of differentially expressed genes from date, the genetic basis and origins of TRCC remain poorly RNA-Seq understood on a genome-wide scale. The details are presented in Supplementary Materials and Compared with ccRCC, which displays gene expression Methods. profiles composed of two main transcriptomic subsets named ccA and ccB (18), the transcriptomic signature of Fusion detection from RNA-Seq TRCC remains obscure. Although in a previous study A modified version of VirusSeq that implements a MITF/TFE tumors of the family were shown to display a greedy algorithm with a robust statistical model was unique gene expression signature (19), their full transcrip- implemented and used in gene fusion discovery for tomic signature remains unknown. RNA-Seq data (21). The TRCC cases were confirmed by In this study, we report the incidence and describe the another bioinformatic pipeline called FusionSeq (22). MITF/TFE genomic profile of translocations of the family
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