Oncogene (2010) 29, 1421–1430 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Genomic and functional analysis identifies CRKL as an oncogene amplified in lung cancer YH Kim1,7, KA Kwei1,7, L Girard2, K Salari1,3, J Kao1, M Pacyna-Gengelbach4, P Wang5, T Hernandez-Boussard3, AF Gazdar2, I Petersen6, JD Minna2 and JR Pollack1 1Department of Pathology, Stanford University, Stanford, CA, USA; 2Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, USA; 3Department of Genetics, Stanford University, Stanford, CA, USA; 4Institute of Pathology, University Hospital Charite´, Berlin, Germany; 5Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA and 6Institute of Pathology, Universita¨tsklinikum Jena, Jena, Germany DNA amplifications, leading to the overexpression of related mortality (Jemal et al., 2008). Nearly 80% oncogenes, are a cardinal feature of lung cancer and of lung cancers diagnosed are non-small-cell lung directly contribute to its pathogenesis. To uncover such cancers (NSCLCs), which are classified into three main novel alterations, we performed an array-based compara- histological subtypes: adenocarcinoma, squamous cell tive genomic hybridization survey of 128 non-small-cell carcinoma and large cell carcinoma. Despite the lung cancer cell lines and tumors. Prominent among our advancement of surgical, cytotoxic and radiological findings, we identified recurrent high-level amplification at treatment options over the years, lung cancer therapy cytoband 22q11.21 in 3% of lung cancer specimens, with remains largely ineffective from a clinical standpoint, another 11% of specimens exhibiting low-level gain spanning as evidenced by a low 5-year survival rate (o15%), that locus. The 22q11.21 amplicon core contained eight and underscores the aggressive nature of the disease. named genes, only four of which were overexpressed (by Much effort has been directed toward elucidating transcript profiling) when amplified. Among these, CRKL the pathogenetic alterations underlying the initiation encodes an adapter protein functioning in signal transduc- and progression of NSCLC, with the hope of developing tion, best known as a substrate of the BCR-ABL kinase in novel therapeutics to selectively target those alterations chronic myelogenous leukemia. RNA-interference-mediated in vivo. Indeed, recent application of epidermal growth knockdown of CRKL in lung cancer cell lines with (but not factor receptor (EGFR) tyrosine kinase inhibitors without) amplification led to significantly decreased cell has been moderately successful in the treatment proliferation, cell-cycle progression, cell survival, and cell of NSCLCs harboring activating point mutations of motility and invasion. In addition, overexpression of CRKL EGFR (Lynch et al., 2004; Paez et al., 2004). It is in immortalized human bronchial epithelial cells led to likely that other genetic alterations in NSCLC await enhanced growth factor-independent cell growth. Our discovery, and once characterized might provide useful findings indicate that amplification and resultant overexpres- targets for therapy. sion of CRKL contribute to diverse oncogenic phenotypes in Genomic DNA amplifications are a frequent class lung cancer, with implications for targeted therapy, and of aberrations in NSCLC, where increased gene dosage highlight a role of adapter proteins as primary genetic leads to overexpression of key cancer genes. Genomic drivers of tumorigenesis. profiling studies of NSCLC, using cDNA (Tonon et al., Oncogene (2010) 29, 1421–1430; doi:10.1038/onc.2009.437; 2005; Kwei et al., 2008), BAC (Garnis et al., 2006), published online 7 December 2009 oligonucleotide (Tonon et al., 2005; Kendall et al., 2007) and single nucleotide polymorphism (Zhao et al., 2005; Keywords: CRKL; lung cancer; DNA amplification; Weir et al., 2007) arrays have revealed focal amplicons genomic profiling; adapter protein harboring known oncogenes, such as KRAS (12p12.1), EGFR (7p12.2), ERBB2 (17q12), MET (7q31.2), MYC (8q24.1), CDK4 (12q14.1) and CCND1 (11q13.2), and have led to the recent discovery of TITF1 (14q13) Introduction as a lineage-dependent oncogenic transcription factor amplified in lung cancer (Kendall et al., 2007; Weir Lung cancer is the leading cause of cancer death in the et al., 2007; Kwei et al., 2008). For other recurrent United States, accounting for almost 30% of all cancer- amplicons, the driver oncogene(s) have not yet been identified, and mapping such loci provides a starting Correspondence: Dr JR Pollack, Department of Pathology, Stanford point for cancer gene discovery and characterization. University, CCSR Building, Room 3245A, 269 Campus Drive, Here, from a cDNA microarray-based genomic profiling Stanford, CA 94305-5176, USA. analysis of 128 lung cancer specimens, we identify E-mail: [email protected] 7These authors contributed equally to this work. amplification of CRKL (22q11) as a recurrent genetic Received 17 February 2009; revised 4 September 2009; accepted 2 event promoting cell proliferation, survival and invasion November 2009; published online 7 December 2009 in lung cancer. CRKL amplification in lung cancer YH Kim et al 1422 Results frequently amplified loci not associated with a known oncogene occurred at cytoband 22q11.21 (Figure 1a), Recurrent 22q11 amplicon in NSCLC spans CRKL where high-level amplification (fluorescence ratios 43, To identify recurrent DNA amplifications pinpointing corresponding to 45-fold amplification; Pollack et al., novel oncogenes in NSCLC, we analyzed cDNA array 1999) was found in 4 of 128 samples analyzed (3%), with comparative genomic hybridization (CGH) data gener- low-level gain spanning 22q11.21 present in an addi- ated on 52 NSCLC cell lines and 76 NSCLC tumors, tional 14 of 128 samples (11%). There was no significant the latter comprising 36 adenocarcinomas (including difference in the frequency of 22q11.21 gain/amplifi- 2 metastases) and 40 squamous cell carcinomas (with cation between adenocarcinoma and other histologies 1 metastasis), totaling 128 samples. One of the most (both for cell lines and for tumors), nor in the NSCLC 8q24.21 14q13.3 (MYC) 12p12.1 12 (TITF1) (KRAS) 12q14.1 10 11q13.2 (CDK4) 1q21.3 19q13.2 8 7p11.2 7q31.2 (CCND1) 17q12 (MET) 22q11.21 6 (EGFR) (ERBB2) 4 3p24.3 Frequency (%) 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2021 22 WBCs SAEC HBEC3-UI HBEC5-UI HBEC2-KT HBEC3-KT HBEC4-KT HBEC5-KT HBEC2-E BEAS-2B A549 CaLu-1 CaLu-3 CaLu-6 H23 H157 H322 H358 H441 H460 H661 H1155 H1299 H1355 H1395 H1437 H1648 H1650 H1666 H1770 H1781 H1792 H1819 H1975 H2009 H2073 H2087 H2122 H2126 H2347 H2882 H2887 H3122 H3255 HCC15 HCC44 HCC78 HCC95 HCC193 HCC366 HCC461 HCC515 HCC827 HCC1171 HCC1195 HCC1359 HCC1833 HCC2429 HCC2279 HCC2450 HCC2935 HCC4006 adeno tumors squamous tumors MM M Chr22 13 p 11.2 11.21 SNAP29 CRKL 11.23 AIFM3* q 12.1 LZTR1 12.3 THAP7 FLJ39582 P2RXL1 13.1 SLC7A4 13.2 13.31 13.33 Primary cells Adeno Adenosquamous Non-tumorigenic Bronchioloalveolar Neuroendocrine <1/2-fold 1 >2-fold Squamous Large cell NSCLC (unspecified) HCC515 H1819 CRKL Tel22q Figure 1 Recurrent 22q11 amplicon in non-small-cell lung cancer (NSCLC) spans CRKL. (a) Frequency plot of cytobands harboring high-level DNA amplification in NSCLC cell lines. Cytobands consisting known oncogenes in lung cancer are indicated, with 22q11.21 highlighted in red. (b) Heat map representation of array comparative genomic hybridization (CGH) profiles of NSCLC cell lines and tumors representing a segment of 22q11.21. Each column represents a different sample (histologies indicated, M ¼ metastasis) and each row represents a different gene ordered by chromosome position. Red indicates positive tumor/normal array CGH ratios (scale shown), and samples called gained (by cghFLasso) or highly amplified at 22q11.21 are marked below by closed black or red circles. Genes residing within the smallest common region of gain (amplicon core) are indicated; those with increased expression when amplified (see Figure 2a) are highlighted in red. AIFM3 (asterisked) was not present on the array but resides where shown. (c) fluorescence in situ hybridization (FISH) validation of CRKL amplification in NSCLC cell lines HCC515 and H1819. DNA amplification is indicated by increased CRKL (green) to telomere-22q (red) signals or signal clusters. Oncogene CRKL amplification in lung cancer YH Kim et al 1423 lines was there a significant association between homology with the CRK proto-oncogene (ten Hoeve 22q11.21 gain/amplification and mutation of KRAS, et al., 1993). Best known as a substrate of the BCR-ABL EGFR or TP53 (data not shown). Conversely, we oncogenic kinase in chronic myelogenous leukemia observed significant (false discovery rate o0.01) associa- (Sattler and Salgia, 1998), a role of CRKL in other cancer tion of 22q11.21 gain with concomitant gains elsewhere types remains largely unexplored. We therefore sought in the genome, namely at 9q34.3, 11q13.2–q13.3 to characterize a possible role of CRKL amplification (CCND1), 15q24.1 and 21q22.3 (Supplementary Table 2), in lung cancer. suggesting possible cooperative interactions. The smallest region of recurrent amplification (or amp- licon core) (Figure 1b), spanned approximately 170 kb CRKL amplification promotes cell proliferation within 22q11.21, and contained eight known RefSeq genes, and survival which included SNAP29 (synaptosomal-associated protein To assess the functional significance of CRKL amplifi- 29), CRKL (v-crk avian sarcoma virus CT10 oncogene cation and overexpression in NSCLC, we used small- homologue-like), AIFM3 (apoptosis-inducing factor, mito- interfering RNAs (siRNAs) directed against CRKL chondrion-associated 3), LZTR1 (leucine-zipper-like in two cell lines, HCC515 and H1819, for which CRKL transcriptional regulator 1), THAP7 (THAP domain- amplification had been validated by fluorescence in situ containing protein 7), FLJ39582 (hypothetical protein hybridization (Figure 1c), and with increased levels LOC439931), P2RXL1 (purinergic receptor P2X-like 1, of total and phosphorylated (activated) CRKL protein orphan receptor) and SLC7A4 (solute carrier family 7 (Figure 2b).