Published OnlineFirst May 26, 2016; DOI: 10.1158/0008-5472.CAN-16-0058 Cancer Therapeutics, Targets, and Chemical Biology Research Diverse, Biologically Relevant, and Targetable Gene Rearrangements in Triple-Negative Breast Cancer and Other Malignancies Timothy M. Shaver1,2, Brian D. Lehmann1,2, J. Scott Beeler1,2, Chung-I Li3, Zhu Li1,2, Hailing Jin1,2, Thomas P. Stricker4, Yu Shyr5,6, and Jennifer A. Pietenpol1,2 Abstract Triple-negative breast cancer (TNBC) and other molecularly discovered a clinical occurrence and cell line model of the target- heterogeneous malignancies present a significant clinical chal- able FGFR3–TACC3 fusion in TNBC. Expanding our analysis to lenge due to a lack of high-frequency "driver" alterations amena- other malignancies, we identified a diverse array of novel and ble to therapeutic intervention. These cancers often exhibit geno- known hybrid transcripts, including rearrangements between mic instability, resulting in chromosomal rearrangements that noncoding regions and clinically relevant genes such as ALK, affect the structure and expression of protein-coding genes. How- CSF1R, and CD274/PD-L1. The over 1,000 genetic alterations ever, identification of these rearrangements remains technically we identified highlight the importance of considering noncod- challenging. Using a newly developed approach that quantita- ing gene rearrangement partners, and the targetable gene tively predicts gene rearrangements in tumor-derived genetic fusions identified in TNBC demonstrate the need to advance material, we identified and characterized a novel oncogenic fusion gene fusion detection for molecularly heterogeneous cancers. involving the MER proto-oncogene tyrosine kinase (MERTK) and Cancer Res; 76(16); 1–11. Ó2016 AACR. Introduction quency and widely varying clonality of therapeutically action- able alterations across subtypes, including mutations in Despite advances in precision medicine, the treatment of PIK3CA and BRAF,amplification of EGFR,lossofPTEN,and many molecularly heterogeneous cancers remains challenging expression of androgen receptor (AR;refs.3–5). However, a due to a lack of recurrent alterations amenable to therapeutic significant proportion of TNBC cases lack somatic alterations of intervention. To make significant clinical advances against any established "driver" gene, highlighting the importance of these recalcitrant cancers, integrative genomic and molecular continued genomic analysis and discovery integrated with analyses are required to understand their complexity and to molecular profiling of the tumor microenvironment (3). identify targetable features arising from lower-frequency genet- Adefining feature of TNBC and other clinically challenging, ic events. Our laboratory has identified six molecular subtypes molecularly heterogeneous cancers such as ovarian carcinoma of one such heterogeneous disease, triple-negative breast cancer and lung squamous cell carcinoma is copy number alteration (TNBC), with each subtype displaying unique ontologies and (CNA), which is frequently accompanied by mutation or inac- differential response to standard-of-care chemotherapy (1, 2). tivation of the p53 tumor suppressor (4, 6–8). The genomic Ongoing genomic analysis of TNBC has identified a low fre- instability that gives rise to CNA can also result in chromo- somal rearrangements and gene fusions, which have long been 1Department of Biochemistry,Vanderbilt University, Nashville,Tennes- recognized as oncogenic drivers and effective drug targets in a see. 2Vanderbilt-Ingram Cancer Center,Vanderbilt University Medical variety of hematological and solid malignancies (9). In recent Center, Nashville, Tennessee. 3Department of Statistics, National 4 years, the advent of next-generation sequencing (NGS) has Cheng Kung University, Tainan, Taiwan. Department of Pathology, fi Microbiology, and Immunology,Vanderbilt University Medical Center, enabled the identi cation of a number of gene fusion events Nashville, Tennessee. 5Center for Quantitative Sciences, Vanderbilt in epithelial cancers, with varying frequency and therapeutic 6 University Medical Center, Nashville, Tennessee. Department of Bio- relevance (10–14). statistics, Vanderbilt University Medical Center, Nashville, Tennessee. In order to broaden our understanding of the somatic altera- Note: Supplementary data for this article are available at Cancer Research tions underlying TNBC, we sought to identify known and novel Online (http://cancerres.aacrjournals.org/). gene rearrangements affecting the structure and/or expression T.M. Shaver and B.D. Lehmann contributed equally to this article. level of protein-coding transcripts. While a number of compu- Corresponding Authors: Jennifer A. Pietenpol, Vanderbilt-Ingram Cancer tational approaches have been developed to identify hybrid Center, 652 Preston Research Building, 2200 Pierce Avenue, Nashville, RNA and DNA sequences using NGS data, numerous technical TN 37232-6838. Phone: 615-936-1512; Fax: 615-936-1790; E-mail: hurdles complicate accurate and efficient gene rearrangement [email protected]; and Brian D. Lehmann, detection (15). For instance, widespread regions of sequence [email protected] homology and current limitations in read length result in doi: 10.1158/0008-5472.CAN-16-0058 sometimes-ambiguous alignment and a large number of false Ó2016 American Association for Cancer Research. positives. To combat this, many detection methodologies use www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst May 26, 2016; DOI: 10.1158/0008-5472.CAN-16-0058 Shaver et al. filters designed to enrich for biologically relevant gene rearran- Ba/F3 IL3 withdrawal gements, such as restricting both potential fusion partners to Stably transfected Ba/F3 cells were seeded in 6-well plates protein-coding loci (16). While these filters enrich for currently (40,000 cells/well) in growth medium Æ 1 ng/mL IL3. Live-cell known gene fusions, they are not effective for the discovery of counts were manually determined at three sequential 24-hour less canonical events, such as rearrangements between coding timepoints by visual inspection using a hemocytometer and and noncoding regions of the genome. We developed a new Trypan blue (Bio-Rad). algorithm, Segmental Transcript Analysis (STA), which uses exon-level expression estimates to rank a population of sam- Cloning and generation of stable cell lines ples based on their likelihood of harboring a rearrangement in The TMEM87B-MERTK expression construct, corresponding a given gene. We identified a number of known and novel to amino acids 1-55 of TMEM87B (NM_032824) and amino rearrangements involving functionally diverse gene partners in acids 433-1000 of MERTK (NM_006343), was synthesized in a TNBC, and expanded our analysis and discovery to a wider, pMK-RQ-Bb vector using GeneOptimizer and GeneArt (Life multicancer cohort from The Cancer Genome Atlas (TCGA). Technologies) and cloned into pBABE-puro (19) (Addgene) The DNA-validated rearrangements that we identified include by EcoRI/SalI restriction digest and ligation (New England noncoding portions of the genome acting as both 50 and 30 BioLabs). The complete pBABE-puro-TMEM87B-MERTK ex- partners in clinically relevant hybrid transcripts. pression construct sequence is available upon request. To generate stably transfected cell lines, pBABE-puro-TMEM87B- Materials and Methods MERTK or pBABE-puro empty vector retroviruses were pack- aged in Phoenix cells (Orbigen) and Ba/F3 and MCF10A Cell culture cells were transduced with virus for two 24-hour intervals with SUM185PE (Asterand, March 2010) and MCF10A cells (ATCC, 8 mg/mL polybrene (Sigma), then selected and maintained June 2012) were cultured as previously described (1). Ba/F3 cells with 1.5 mg/mL (Ba/F3) or 0.5 mg/mL (MCF10A) puromycin (provided by Dr. Christine Lovly, Vanderbilt University, Novem- (Sigma). ber 2014) were cultured in RPMI þ GlutaMAX (Gibco 61870) with 1 ng/mL IL3 (Life Technologies) and 5% (v/v) FBS (Gemini). All cell lines were maintained in 100 U/mL peni- Immunoblotting cillin and 100 mg/mL streptomycin (Gemini) and tested neg- SUM185PE cells were lysed 72 hours after siRNA transfection. ative for mycoplasma (Lonza). Cell Line Genetics performed Ba/F3 cells were grown in suspension and incubated for 90 positive short tandem repeat DNA fingerprinting analysis on minutes in the indicated media before lysis. MCF10A cells seeded SUM185PE (March 2011); cells were cultured for fewer than in 10-cm plates were incubated for 180 minutes in the indicated 2 months after identification. We also verified SUM185PE by growth media before in-well lysis. All cells were lysed in RIPA manual identification of unique variants in NGS data. DNA buffer supplemented with protease and phosphatase inhibitors. fingerprinting analysis was not performed on MCF10A or Ba/ Cell lysates [40 mg (SUM185PE) or 30 mg (Ba/F3 and MCF10A)] F3 cells, but the Ba/F3 cell line displayed the previously were separated on polyacrylamide gels and transferred to poly- published IL3 dependence phenotype (17). vinyl difluoride membranes (Millipore). Immunoblotting was performed using FGFR3 B-9 (1:200, Santa Cruz), GAPDH FGFR3/TACC3 siRNA transfection MAB374 (1:1000, Millipore), MERTK D21F11 (1:1000, Cell SUM185PE cells (8,000 cells/well) were reverse transfected Signaling Technology), phospho-Akt Ser473 D9E (1:2000, Cell in a 96-well format with RNAiMax (0.1 mL, Life Technologies) Signaling Technology),