Somatic Superenhancer Duplications and Hotspot Mutations Lead to Oncogenic Activation of the KLF5 Transcription Factor.

The Krüppel-like family of transcription factors plays critical roles in human development and is associated with cancer pathogenesis. Krüppel-like factor 5 gene (KLF5) has been shown to promote cancer cell proliferation and tumorigenesis and to be genomically amplified in cancer cells. We recently reported that the KLF5 gene is also subject to other types of somatic coding and noncoding genomic alterations in diverse cancer types. Here, we show that these alterations activate KLF5 by three distinct mechanisms: (i) Focal amplification of superenhancers activates KLF5 expression in squamous cell carcinomas; (ii) Missense mutations disrupt KLF5-FBXW7 interactions to increase KLF5 protein stability in colorectal cancer; (iii) Cancer type-specific hotspot mutations within a zinc-finger DNA binding domain of KLF5 change its DNA binding specificity and reshape cellular transcription. Utilizing data from CRISPR/Cas9 gene knockout screening, we reveal that cancer cells with KLF5 overexpression are dependent on KLF5 for their proliferation, suggesting KLF5 as a putative therapeutic target.Significance: Our observations, together with previous studies that identified oncogenic properties of KLF5, establish the importance of KLF5 activation in human cancers, delineate the varied genomic mechanisms underlying this occurrence, and nominate KLF5 as a putative target for therapeutic intervention in cancer. Cancer Discov; 8(1); 108-25. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1.

[1]  C. Teng,et al.  Phosphorylation of Kruppel-like factor 5 (KLF5/IKLF) at the CBP interaction region enhances its transactivation function. , 2003, Nucleic acids research.

[2]  Yusuke Nakamura,et al.  Genome-wide association study identifies multiple susceptibility loci for pancreatic cancer , 2014, Nature Genetics.

[3]  Swneke D. Bailey,et al.  Laying a solid foundation for Manhattan--'setting the functional basis for the post-GWAS era'. , 2014, Trends in genetics : TIG.

[4]  Colin N. Dewey,et al.  RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome , 2011, BMC Bioinformatics.

[5]  Cheryl H. Arrowsmith,et al.  Prevalent p53 mutants co-opt chromatin pathways to drive cancer growth , 2015, Nature.

[6]  Swneke D. Bailey,et al.  Integrative functional genomics identifies an enhancer looping to the SOX9 gene disrupted by the 17q24.3 prostate cancer risk locus , 2012, Genome research.

[7]  Timothy E. Reddy,et al.  Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements , 2015, Nature Methods.

[8]  V. Yang,et al.  A Colon Cancer-derived Mutant of Krüppel-like Factor 5 (KLF5) Is Resistant to Degradation by Glycogen Synthase Kinase 3β (GSK3β) and the E3 Ubiquitin Ligase F-box and WD Repeat Domain-containing 7α (FBW7α)* , 2014, The Journal of Biological Chemistry.

[9]  L. Mirny,et al.  The 3D Genome as Moderator of Chromosomal Communication , 2016, Cell.

[10]  K. Soo,et al.  Epigenomic profiling of primary gastric adenocarcinoma reveals super-enhancer heterogeneity , 2016, Nature Communications.

[11]  L. Jia,et al.  KLF5 promotes breast cancer proliferation, migration and invasion in part by upregulating the transcription of TNFAIP2 , 2016, Oncogene.

[12]  Chandra Sekhar Pedamallu,et al.  Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas , 2016, Nature Genetics.

[13]  Jing Liang,et al.  Chromatin architecture reorganization during stem cell differentiation , 2015, Nature.

[14]  P. Leder,et al.  The human c-myc oncogene: Structural consequences of translocation into the igh locus in Burkitt lymphoma , 1983, Cell.

[15]  V. Chatterjee,et al.  Mutation of the gene encoding human TTF-2 associated with thyroid agenesis, cleft palate and choanal atresia , 1998, Nature Genetics.

[16]  Trevor J Pugh,et al.  Landscape of genomic alterations in cervical carcinomas , 2013, Nature.

[17]  P. Gregory,et al.  Controlling Long-Range Genomic Interactions at a Native Locus by Targeted Tethering of a Looping Factor , 2012, Cell.

[18]  Steven J. M. Jones,et al.  Comprehensive genomic characterization of head and neck squamous cell carcinomas , 2015, Nature.

[19]  J. Simons,et al.  KLF5 promotes cell proliferation and tumorigenesis through gene regulationin the TSU‐Pr1 human bladder cancer cell line , 2006, International journal of cancer.

[20]  Clifford A. Meyer,et al.  Cistrome: an integrative platform for transcriptional regulation studies , 2011, Genome Biology.

[21]  B. Clurman,et al.  The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Chris Sander,et al.  Emerging landscape of oncogenic signatures across human cancers , 2013, Nature Genetics.

[23]  A. Services,et al.  Integrated genomic and molecular characterization of cervical cancer. , 2017 .

[24]  R. Blumenthal,et al.  Structural basis for Klf4 recognition of methylated DNA , 2014, Nucleic acids research.

[25]  A. Ferrando,et al.  The Ubiquitin Ligase FBXW7 Modulates Leukemia-Initiating Cell Activity by Regulating MYC Stability , 2013, Cell.

[26]  E. Lander,et al.  Comprehensive assessment of cancer missense mutation clustering in protein structures , 2015, Proceedings of the National Academy of Sciences.

[27]  Shuangxi Li,et al.  The Fbw7/Human CDC4 Tumor Suppressor Targets Proproliferative Factor KLF5 for Ubiquitination and Degradation through Multiple Phosphodegron Motifs* , 2010, The Journal of Biological Chemistry.

[28]  V. Yang,et al.  Krüppel-like factor 5 mediates cellular transformation during oncogenic KRAS-induced intestinal tumorigenesis. , 2008, Gastroenterology.

[29]  Benjamin J. Raphael,et al.  Integrated genomic characterization of oesophageal carcinoma , 2017, Nature.

[30]  Joshua M. Stuart,et al.  The Cancer Genome Atlas Pan-Cancer analysis project , 2013, Nature Genetics.

[31]  Mark D. Robinson,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[32]  Zhongmei Zhou,et al.  BAP1 promotes breast cancer cell proliferation and metastasis by deubiquitinating KLF5 , 2015, Nature Communications.

[33]  S. Elledge,et al.  Phosphorylation-Dependent Ubiquitination of Cyclin E by the SCFFbw7 Ubiquitin Ligase , 2001, Science.

[34]  J. M. Shields,et al.  Identification and Characterization of a Gene Encoding a Gut-enriched Krüppel-like Factor Expressed during Growth Arrest* , 1996, The Journal of Biological Chemistry.

[35]  A. Sivachenko,et al.  Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer , 2012, Nature Genetics.

[36]  Ryan M. Layer,et al.  LUMPY: a probabilistic framework for structural variant discovery , 2012, Genome Biology.

[37]  Derek Y. Chiang,et al.  The landscape of somatic copy-number alteration across human cancers , 2010, Nature.

[38]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[39]  G. Heinze,et al.  Expression of KLF5 is a Prognostic Factor for Disease-Free Survival and Overall Survival in Patients with Breast Cancer , 2006, Clinical Cancer Research.

[40]  Marcin Imielinski,et al.  Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers , 2015, Nature Genetics.

[41]  David A. Orlando,et al.  Mediator and Cohesin Connect Gene Expression and Chromatin Architecture , 2010, Nature.

[42]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[43]  M. Crossley,et al.  Mammalian Krüppel-like transcription factors: more than just a pretty finger. , 1999, Trends in biochemical sciences.

[44]  Wen Tan,et al.  Genome-wide association study identifies five loci associated with susceptibility to pancreatic cancer in Chinese populations , 2011, Nature Genetics.

[45]  Benjamin E. Gross,et al.  The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. , 2012, Cancer discovery.

[46]  T. Golub,et al.  Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma , 2005, Nature.

[47]  T. Golub,et al.  Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting. , 2016, Cancer discovery.

[48]  R. Young,et al.  Histone H3K27ac separates active from poised enhancers and predicts developmental state , 2010, Proceedings of the National Academy of Sciences.

[49]  M. Rydzanicz,et al.  Chromosomal gains and losses indicate oncogene and tumor suppressor gene candidates in salivary gland tumors. , 2008, Neoplasma.

[50]  Zhongmei Zhou,et al.  TAZ antagonizes the WWP1-mediated KLF5 degradation and promotes breast cell proliferation and tumorigenesis. , 2012, Carcinogenesis.

[51]  R. Young,et al.  Super-Enhancers in the Control of Cell Identity and Disease , 2013, Cell.

[52]  Max A. Horlbeck,et al.  Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation , 2014, Cell.

[53]  H. Aburatani,et al.  CDX1 confers intestinal phenotype on gastric epithelial cells via induction of stemness-associated reprogramming factors SALL4 and KLF5 , 2012, Proceedings of the National Academy of Sciences.

[54]  Steven J. M. Jones,et al.  Integrated genomic and molecular characterization of cervical cancer , 2017, Nature.

[55]  G. Getz,et al.  GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers , 2011, Genome Biology.

[56]  Raffaele Pezzilli,et al.  Common variation at 2 p 13 . 3 , 3 q 29 , 7 p 13 and 17 q 25 . 1 associated with susceptibility to pancreatic cancer , 2022 .

[57]  A. Ferrando,et al.  The SCFFBW7 ubiquitin ligase complex as a tumor suppressor in T cell leukemia , 2007, The Journal of experimental medicine.

[58]  Aaron R. Quinlan,et al.  Bioinformatics Applications Note Genome Analysis Bedtools: a Flexible Suite of Utilities for Comparing Genomic Features , 2022 .

[59]  Daniel S. Day,et al.  Activation of proto-oncogenes by disruption of chromosome neighborhoods , 2015, Science.

[60]  Kari Stefansson,et al.  Common variants on 9q22.33 and 14q13.3 predispose to thyroid cancer in European populations , 2009, Nature Genetics.

[61]  Jesse R. Dixon,et al.  Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions , 2012, Nature.

[62]  M. Freedman,et al.  Chromosome 8q24-Associated Cancers and MYC. , 2010, Genes & cancer.

[63]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[64]  Steven J. M. Jones,et al.  Comprehensive genomic characterization of squamous cell lung cancers , 2012, Nature.

[65]  Steven A. Roberts,et al.  Mutational heterogeneity in cancer and the search for new cancer-associated genes , 2013 .

[66]  Wei Zheng,et al.  A genome-wide association study identifies pancreatic cancer susceptibility loci on chromosomes 13q22.1, 1q32.1 and 5p15.33 , 2010, Nature Genetics.

[67]  Hanfei Sun,et al.  Target analysis by integration of transcriptome and ChIP-seq data with BETA , 2013, Nature Protocols.

[68]  L. Hennighausen,et al.  Hierarchy within the mammary STAT5-driven Wap super-enhancer , 2016, Nature Genetics.

[69]  Mark W. Youngblood,et al.  Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas , 2016, Nature Genetics.

[70]  M. Gurney,et al.  The Notch Intracellular Domain Is Ubiquitinated and Negatively Regulated by the Mammalian Sel-10 Homolog* , 2001, The Journal of Biological Chemistry.

[71]  Adam A. Margolin,et al.  Addendum: The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity , 2012, Nature.

[72]  Hui Zhao,et al.  Five endometrial cancer risk loci identified through genome-wide association analysis , 2016, Nature Genetics.

[73]  Takafumi N. Yamaguchi,et al.  TMPRSS2–ERG fusion co-opts master transcription factors and activates NOTCH signaling in primary prostate cancer , 2017, Nature Genetics.

[74]  Neville E. Sanjana,et al.  Improved vectors and genome-wide libraries for CRISPR screening , 2014, Nature Methods.

[75]  David A. Orlando,et al.  Master Transcription Factors and Mediator Establish Super-Enhancers at Key Cell Identity Genes , 2013, Cell.

[76]  Teresa Palomero,et al.  A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia , 2014, Nature Medicine.

[77]  Yusuke Nakamura,et al.  Genome-wide association study identifies five new susceptibility loci for prostate cancer in the Japanese population , 2010, Nature Genetics.

[78]  Pablo Tamayo,et al.  ATARiS: Computational quantification of gene suppression phenotypes from multisample RNAi screens , 2013, Genome research.

[79]  Shawn M. Gillespie,et al.  An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma , 2016, Nature Genetics.

[80]  Nathan C. Sheffield,et al.  The accessible chromatin landscape of the human genome , 2012, Nature.

[81]  Rob Pieters,et al.  FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to γ-secretase inhibitors , 2007, The Journal of experimental medicine.

[82]  Swneke D. Bailey,et al.  Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression , 2012, Nature Genetics.

[83]  H. Clevers,et al.  KLF5 regulates the integrity and oncogenicity of intestinal stem cells. , 2014, Cancer research.

[84]  R. Young,et al.  An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element , 2014, Science.

[85]  Meagan E. Sullender,et al.  Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 , 2015, Nature Biotechnology.

[86]  H. Ng,et al.  Regulatory crosstalk between lineage-survival oncogenes KLF5, GATA4 and GATA6 cooperatively promotes gastric cancer development , 2014, Gut.

[87]  S. Imai,et al.  The NAD Biosynthesis Pathway Mediated by Nicotinamide Phosphoribosyltransferase Regulates Sir2 Activity in Mammalian Cells* , 2004, Journal of Biological Chemistry.

[88]  Tsutomu Ohta,et al.  Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy , 2008, Proceedings of the National Academy of Sciences.

[89]  Khay Guan Yeoh,et al.  A comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co-occurrence among distinct therapeutic targets , 2012, Gut.

[90]  J. Tchinda,et al.  Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. , 2006, Science.

[91]  J. Trent,et al.  Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour , 1983, Nature.

[92]  Mark D Robinson,et al.  edgeR for differential RNA-seq and ChIP-seq analysis: an application to stem cell biology. , 2014, Methods in molecular biology.

[93]  D. Coppola,et al.  Nicotinamide phosphoribosyltransferase in malignancy: a review. , 2013, Genes & cancer.

[94]  H. Wu,et al.  NAMPT overexpression in prostate cancer and its contribution to tumor cell survival and stress response , 2011, Oncogene.

[95]  J. Minna,et al.  Amplification and expression of the c-myc oncogene in human lung cancer cell lines , 1983, Nature.

[96]  Nicholas A. Sinnott-Armstrong,et al.  Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer , 2016, Nature Genetics.

[97]  Vincent W. Yang,et al.  Intestinal-enriched Krüppel-like Factor (Krüppel-like Factor 5) Is a Positive Regulator of Cellular Proliferation* , 2001, The Journal of Biological Chemistry.

[98]  M. Tyers,et al.  Structural Basis for Phosphodependent Substrate Selection and Orientation by the SCFCdc4 Ubiquitin Ligase , 2003, Cell.

[99]  Donna D. Zhang,et al.  The emerging role of the Nrf2–Keap1 signaling pathway in cancer , 2013, Genes & development.

[100]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[101]  S. Gabriel,et al.  Discovery and saturation analysis of cancer genes across 21 tumor types , 2014, Nature.

[102]  M. Meyerson,et al.  Copy number alterations unmasked as enhancer hijackers , 2016, Nature Genetics.

[103]  Daniel S. Day,et al.  Insulated Neighborhoods: Structural and Functional Units of Mammalian Gene Control , 2016, Cell.

[104]  A. Visel,et al.  ChIP-seq accurately predicts tissue-specific activity of enhancers , 2009, Nature.

[105]  Liliana Goumnerova,et al.  MYB-QKI rearrangements in Angiocentric Glioma drive tumorigenicity through a tripartite mechanism , 2016, Nature Genetics.

[106]  Matthew E. Ritchie,et al.  limma powers differential expression analyses for RNA-sequencing and microarray studies , 2015, Nucleic acids research.

[107]  Jaie C. Woodard,et al.  Survey of variation in human transcription factors reveals prevalent DNA binding changes , 2016, Science.

[108]  Benjamin E. Gross,et al.  Integrative Analysis of Complex Cancer Genomics and Clinical Profiles Using the cBioPortal , 2013, Science Signaling.

[109]  Ming Yu,et al.  Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation , 2013, Genes & development.

[110]  Peng Guo,et al.  Beyond proliferation: KLF5 promotes angiogenesis of bladder cancer through directly regulating VEGFA transcription , 2015, Oncotarget.

[111]  R. Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[112]  Michael D. Cole,et al.  Upregulation of c-MYC in cis through a Large Chromatin Loop Linked to a Cancer Risk-Associated Single-Nucleotide Polymorphism in Colorectal Cancer Cells , 2010, Molecular and Cellular Biology.

[113]  J Wade Harper,et al.  Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. , 2007, Molecular cell.

[114]  Jorma Isola,et al.  In vivo amplification of the androgen receptor gene and progression of human prostate cancer , 1995, Nature Genetics.

[115]  Eric S. Lander,et al.  Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma , 2016, Cell reports.

[116]  Neva C. Durand,et al.  A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping , 2014, Cell.

[117]  S. Philipsen,et al.  A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. , 1999, Nucleic acids research.

[118]  Clifford A. Meyer,et al.  Model-based Analysis of ChIP-Seq (MACS) , 2008, Genome Biology.

[119]  Jill M Dowen,et al.  Control of Cell Identity Genes Occurs in Insulated Neighborhoods in Mammalian Chromosomes , 2014, Cell.

[120]  Swneke D. Bailey,et al.  ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters , 2015, Nature Communications.

[121]  S. Gabriel,et al.  Pan-cancer patterns of somatic copy-number alteration , 2013, Nature Genetics.

[122]  David A. Orlando,et al.  Selective Inhibition of Tumor Oncogenes by Disruption of Super-Enhancers , 2013, Cell.

[123]  A. Regev,et al.  SOX2 Is an Amplified Lineage Survival Oncogene in Lung and Esophageal Squamous Cell Carcinomas , 2009, Nature Genetics.