Characterization of KRAS Rearrangements in Metastatic Prostate Cancer

Published OnlineFirst April 3, 2011; DOI: 10.1158/2159-8274.CD-10-0022

ReseARcH BRieF

characterization of KRAS rearrangements in Metastatic Prostate cancer

Xiao-Song Wang1– 3, * Sunita Shankar1, 3, * Saravana M. Dhanasekaran1, 3, * Bushra Ateeq1, 3, Atsuo T. Sasaki9, 10, Xiaojun Jing1, 3, Daniel Robinson1, 3, Qi Cao1, 3, John R. Prensner1, 3, Anastasia K. Yocum1, 3, Rui Wang,1, 3 Daniel F. Fries 1, 3, Bo Han1, 3, Irfan A. Asangani 1, 3, Xuhong Cao1, 3, Yong Li1, 3, Gilbert S. Omenn2 , Dorothee Pflueger7, 8, Anuradha Gopalan11 , Victor E. Reuter 11, Emily Rose Kahoud 9, Lewis C. Cantley9, 10, Mark A. Rubin7 , Nallasivam Palanisamy 1, 3, 6, Sooryanarayana Varambally1, 3, 6, and Arul M. Chinnaiyan1–6

ABstRAct Using an integrative genomics approach called amplification breakpoint ranking and assembly analysis, we nominated KRAS as a gene fusion with the ubiquitin- conjugating enzyme UBE2L3 in the DU145 cell line, originally derived from prostate cancer metastasis to the brain. Interestingly, analysis of tissues revealed that 2 of 62 metastatic prostate cancers harbored aberrations at the KRAS locus. In DU145 cells, UBE2L3-KRAS produces a fusion protein, a specific knockdown of which attenuates cell invasion and xenograft growth. Ectopic expression of the UBE2L3-KRAS fusion protein exhibits transforming activity in NIH 3T3 fibroblasts and RWPE prostate epithelial cells in vitro and in vivo. In NIH 3T3 cells, UBE2L3-KRAS attenuates MEK/ERK signaling, commonly engaged by oncogenic mutant KRAS, and instead signals via AKT and p38 mitogen-activated protein kinase (MAPK) pathways. This is the first report of a gene fusion involving the Ras family, suggesting that this aberration may drive metastatic progression in a rare subset of prostate cancers.

siGnificance: This is the first description of an oncogenic gene fusion of KRAS, one of the most studied proto-oncogenes. KRAS rearrangement may represent the driving mutation in a rare subset of metastatic prostate cancers, emphasizing the importance of RAS-RAF-MAPK signaling in this disease. Cancer Discovery; 1(1); 35–43. © 2011 AACR.

Authors’ Affiliations: 1 Michigan Center for Translational Pathology, 2 National intRoDuction Center for Integrative Biomedical Informatics, Center for Computational To understand the characteristic features of driving gene Medicine and Bioinformatics, and 3Departments of Pathology and 5Urology, 4Howard Hughes Medical Institute, and 6Comprehensive Cancer Center, fusions in cancer, we previously carried out a large-scale in- University of Michigan Medical School, Ann Arbor, Michigan; 7Departments of tegrative analysis of cancer genomic datasets matched with Pathology and Laboratory Medicine and 8Urology, Weill Cornell Medical gene rearrangement data (1). As part of this analysis, we ob- College, and 11Department of Pathology, Memorial Sloan-Kettering Cancer served that in many instances a small subset of tumors or 9 Center, New York, New York; Beth Israel Deaconess Medical Center, Division cancer cell lines harboring an oncogenic gene fusion displays of Signal Transduction, and Departments of Medicine and 10Systems Biology, Harvard Medical School, Boston, Massachusetts characteristic amplification at the site of genomic rearrange- * These authors contributed equally to this article. ment (refs. 2–6; Supplementary Fig. S1A and B). Such am- Note : Supplementary data for this article are available at Cancer plifications usually affect a portion of the fusion gene and Discovery Online ( http://www.aacrjournals.org ). are generally considered secondary genetic lesions associated Corresponding Author: Arul M. Chinnaiyan, Departments of Pathology and with disease progression, drug resistance, and poor progno- Urology, University of Michigan Medical School, 1400 E. Medical Center sis (2, 4–8). In contrast, high-level copy number changes that Drive, 5316 UMCCC, Ann Arbor, MI 48109. Phone: 1-734-615-4062; Fax: result in the marked overexpression of oncogenes usually en- 734-615-4498; E-mail: [email protected] compass the target genes at the center of overlapping ampli- doi: 10.1158/2159-8274.CD-10-0022 fications across a panel of tumor samples. Thus, a “partially” ©2011 American Association for Cancer Research. amplified cancer gene may suggest that this gene participates

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Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst April 3, 2011; DOI: 10.1158/2159-8274.CD-10-0022 research brief Wang et al. in a genomic fusion event important in cancer progression. A potential 5' partners. In total, six 5' amplified genes were partially amplified gene could arise from several independent found in K-562, and 5 matched the level of ABL1 3' am- genetic accidents, including the formation of the gene fusion plification. After curation of the amplification breakpoints, and subsequent amplification, suggesting possible selective BCR and NUP214 were nominated as ABL1 fusion partner pressure in cancer cells for this aberration. Toward this end, candidates (Supplementary Fig. S1D, right; see Methods and we developed an integrative genomic approach called ampli- Supplementary Fig. S2 for criteria of candidate selection). fication breakpoint ranking and assembly (ABRA) to discover These findings demonstrated the feasibility of this method in causal gene fusions from cancer genomic datasets. nominating driver gene fusions from genomic datasets. To uncover driving gene fusions contributing to prostate To nominate novel gene fusions contributing to the progres- cancer progression, we applied ABRA analysis to genomic data sion of advanced prostate cancer, we applied this method to from 10 prostate cancer cell lines. Of most interest, this led an array comparative genomic hybridization (aCGH) dataset to the identification of a KRAS gene fusion in a rare subset of 10 prostate cancer cell lines. Interestingly, the top candidate of metastatic prostate cancer. RAS proteins play a critical role nominated in the ETS gene fusion-negative prostate cancer cell in cellular physiology, development, and tumorigenesis (9, 10). line, DU145, was the KRAS locus, which exhibited a clear break- Mutations in RAS have been identified in a wide spectrum point accompanied by a 3' amplification of KRAS (Fig. S1A, of cancers (9), but rarely in prostate cancer (11). To date, on- left; Supplementary Table S2). This result was particularly in- cogenic alterations in the Ras pathway have been exclusively triguing owing to the well-known role of KRAS as an onco- restricted to activating point mutations, including the most gene (9) and the observation that activating point mutations of commonly studied Gly-to-Val substitution at codon 12 and KRAS are found in a number of tumor types but are rarely seen substitutions at codons 13 and 61 of the different RAS isoforms in prostate cancer (11). Interestingly, the activation of down- (9, 12, 13). Gene fusions involving RAS genes have thus far not stream signaling intermediaries of the RAS–mitogen-activated been described as a class of cancer-related mutations. This is the protein kinase (MAPK) pathway has been observed in prostate first description of a mutant chimeric version of KRAS and thus cancer by a number of studies (15–17). may represent a new class of cancer-related alterations. To assemble amplification breakpoints in the KRAS gene with more confidence, we carried out replicate aCGHs for Results DU145 cells. Matching the amplification level of KRAS with the 5' amplified genes from DU145 cells, we identified 10 On the basis of the fusion breakpoint principle previously potential 5' partner candidates that were suggested by ei- described (1), amplifications associated with gene fusions ther of the 2 aCGHs. After curation, C14orf166, SOX5, and usually involve the 5' region of 5' partners and the 3' region UBE2L3 remained as the top 5' partner candidates for KRAS of 3' partners. Further, the amplification levels of 5' and 3' fu- (Fig. 1A, right; Supplementary Table S3), based on the crite- sion genes will be identical owing to their coamplification as ria detailed in Supplementary Fig. S2. a single fusion gene. This observation provided the rationale To experimentally validate the predicted fusions of to assemble putative gene fusions from amplification break- C14orf166-KRAS, SOX5-KRAS, and UBE2L3-KRAS, we designed points by matching the amplification levels of candidate 5' primer pairs from the first exons of candidate 5' partners and 3' partners. We therefore developed ABRA analysis, which and the last exon of KRAS, as well as the exons next to the leverages the in vivo amplification and breakpoint analysis in breakpoints (Supplementary Table S3). Reverse transcriptase cancer cells to assemble novel gene fusions and predict their (RT)–PCR analysis of DU145 cells identified a specific fusion tumorigenicity. Concept signature analysis (ConSig) was de- band for UBE2L3-KRAS, but not for the others. Subsequent se- veloped in a previous study (1) and provides a ConSig score, quencing of the RT-PCR product confirmed fusion of UBE2L3 which is helpful in ranking biologically relevant candidates exon 3 to KRAS exon 2, as schematically depicted in Fig. 1B. based on prior knowledge and has been incorporated into To assess the abundance of the UBE2L3-KRAS fusion tran- ABRA analysis. scripts, we analyzed a panel of prostate cell lines by SYBR The detailed methodology of ABRA analysis is depicted in green quantitative PCR (Fig. 1C) and RNase protection assay Supplementary Fig. S1C and discussed in the Supplementary (Supplementary Fig. S3). UBE2L3-KRAS was highly expressed Data. We initially focused this analysis on cancer cell lines be- in DU145 cells, but not in the other prostate cancer cell lines cause breakpoint analyses are more reliable in uniform cellular tested; this result was confirmed by subsequent paired-end populations, as opposed to tumors, which comprise multiple transcriptome sequencing of DU145 cells (Supplementary cell types, many of which are not malignant. Fig. S4). Moreover, mutation analysis of the fusion sequences Next, we tested the ABRA approach using a published from DU145 did not reveal canonical point mutations in the single-nucleotide polymorphism microarray (aSNP) data- fusion allele of KRAS (Supplementary Fig. S5). set (2) generated from 36 leukemia cell lines, including the To examine the chromosomal rearrangements involv- K-562 chronic myeloid leukemia cell line known to harbor ing UBE2L3 and KRAS loci in DU145 cells, we carried out the amplified BCR-ABL1 fusion (14). We inferred the rela- FISH analysis. By both KRAS split probe and UBE2L3-KRAS tive DNA copy number data and identified all 5' and 3' fusion probe FISH analysis, DU145 cells clearly showed a amplified genes from the 36 cell lines (≥2 copies). In this rearrangement at the KRAS genomic loci and fusion with dataset, ABL1 was the top-ranking gene, with a 3' copy num- UBE2L3 (Fig. 1D). In addition, we observed low-level ampli- ber increase (Supplementary Fig. S1D, left; Supplementary fication (3 copies) of the UBE2L3-KRAS fusion consistent Table S1). The amplification levels of all 5' amplified genes with its nomination by the ABRA approach. To extend our in K-562 cells were then matched with ABL1 to nominate studies to prostate tumors, we carried out a combination

36 | CANCER DISCOVERY JUNE 2011 www.aacrjournals.org

Published OnlineFirst April 3, 2011; DOI: 10.1158/2159-8274.CD-10-0022

KRAS Rearrangements in Prostate Cancer research brief

Aa 1.6 1.5 0 3.0 1.2 -1.5 UBE2L3

2.0 1.5 1.5 0.8 0 0

ConSig score -1.5 -1.5 1.0 - KRAS 0.4 SOX5

1.5 3’ copy number gain (log2) 0 0 DU145 NCI660 PC3 NCI660 NCI660 VCaP NCI660 PC3 CA-HPV-10 MDA-PCa-2b 0.5 -0.5 C14orf166 CBX2 KRAS CREB5 STRN3 NEK7 POLB ZNF605 MYO16ANXA11 CHAF1A Bb Cc

UBE2L3 KRAS 15 104 200 387 2907 170 292 471 631 5297 Primer q1 1 2 3 4 1 2 3 4 5 10 Primer q2 78 443 182 748 5 104 200 387 509 688 848 5514 (log 2) 1 2 3 2 3 4 5 UBE2L3-KRAS 0 78 965

A C C G A C C A A G G C C T G C T G A A A -5 UBE2L3-KRAS/GAPDH NPP DU145

3 D UBE2L3 /KRAS 4 KRAS split (5’ 1 3’ 2 ) d 1 1 22q11.21

19,839,192 20,551,225 DU145

UBE2L3 5’ 1 2 3 4 3’ 3 RP11-317J15 DU145 DU145 PCA0216 12p12.1 2.5 DU145 PCA0211 PCA0216

0 25,023,924 25,441,965 KRAS chr12: 24,256,437-26,292,645 -2.5 KRAS 3’ 5 4 3 2 1 5’ 2 1 Tumor tissues UM cohort (FISH, n = 103) MSKCC cohort (aCGH, n = 218) RP11-157L6 4 RP11-68I23 Localized prostate cancer 0/78 0/181 RP11-608F13 Metastatic prostate cancer 0/25 2*/37

Figure 1. Identification and characterization of a novel KRAS rearrangement in metastatic prostate cancer. A, left, amplification breakpoint analysis and ConSig scoring (yellow line) of 3' amplified genes from a panel of advanced prostate cancer cell lines nominating KRAS as a fusion gene candidate with 3' amplification (red columns) in the DU145 prostate cancer cell line. Right, matching the amplification level of 5' amplified genes in DU145 cells nominates SOX5, C14orf166, and UBE2L3 as 5' fusion partner candidates for KRAS. Relative quantification of DNA copy number data from the genomic regions 1 Mb apart from the candidate fusion genes is shown. The x-axis indicates the physical position of the genomic aberrations; fusion partners are indicated by gray arrows. B, sequencing results from RT-PCR, revealing fusion of UBE2L3 with KRAS in DU145. Structures for the UBE2L3 and KRAS genes have their basis in the Genbank reference sequences. Numbers above the exons (boxes) indicate the last base of each exon. Open reading frames are shown in darker shades. Exons of UBE2L3-KRAS fusion are numbered from the original reference sequences. Line graphs show the position and DNA sequencing of the fusion junction. C, panel of prostate cancer cell lines analyzed for UBE2L3- KRAS mRNA expression by SYBR assay with the fusion primers. NPP, normal prostate pool. D, left, genomic organizations of UBE2L3 and KRAS loci, with red and green bars indicating the location of bacterial artificial chromosome clones. For genes, the direction of transcription is indicated by arrows and exons, by bars. Right, FISH assay (top) and copy number data analysis (bottom) confirm the fusion of UBE2L3 to KRAS in DU145 cells and recurrent rearrangements at the KRAS locus. The left FISH image shows 3 copies of fusion signals (yellow arrows), using colocalizing probes for the fusion. The FISH image on the right shows triplicate KRAS 3' signals in DU145 and 3' deletion of KRAS in a metastatic prostate tumor, PCA0216, using probes that tightly encompass the KRAS locus. The middle image shows relative quantification of copy number aCGH data at the KRAS locus in DU145 and metastatic prostate tumors PCA0211 and PCA0216. At bottom is a summary of KRAS rearrangements revealed by FISH and copy number analysis of a series of prostate cancer tissues from the University of Michigan (UM) and Memorial Sloan-Kettering Cancer Center (MSKCC); table indicates number of positive cases divided by total number of cases evaluated. Positive cases: PCA0211 (spine metastasis), 3' amplification; PCA0216 (bladder metastasis), 3' deletion.

of KRAS split probe FISH (n = 103 total cases) and aCGH we identified 2 of 62 metastatic prostate cancers that har- breakpoint (n = 218 total cases) analysis of 259 clinically bored a rearrangement at the KRAS locus (Fig. 1D). One of localized prostate cancers and 62 metastatic prostate can- the index cases, PCA0216, which was a soft tissue metasta- cers from UM and MSKCC. Interestingly, although clinically sis, was validated by both aCGH and FISH. The other index localized cases did not show aberrations at the KRAS locus, case, PCA0211, was a bone metastasis and was validated only

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Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst April 3, 2011; DOI: 10.1158/2159-8274.CD-10-0022 research brief Wang et al. by aCGH; FISH analysis was attempted, but because of decal- RNA interference against KRAS, UBE2L3, and the chimeric cification, adequate hybridization was not possible (Fig. 1D; junction of UBE2L3-KRAS (Supplementary Fig. S7A). The Supplementary Fig. 6A). Further investigation of the available UBE2L3-KRAS protein was found specifically in DU145 cells gene and exon expression data for case PCA0211 suggested and not in a panel of other prostate cell lines (Fig. 2C). Specific that this case did not overexpress ETS family genes but instead expression of the protein was independently confirmed by exhibited high expression of KRAS exons 2 to 6 (except exon 1), mass spectrometry assessment of DU145 cells, using a mul- similar to the DU145 cell line (Supplementary Fig. S6B and C). tiple reaction monitoring (MRM) assay, which does not re- We next examined expression of the UBE2L3-KRAS pro- quire antibody-based detection (Fig. 2D). Overexpression of tein. The predicted 296 amino acid fusion protein trims UBE2L3-KRAS in HEK 293 cells, however, did not result in 17 amino acids from the C-terminus of UBE2L3 (Fig. 2A). The detectable fusion protein. Interestingly, in the presence of the full length of the KRAS protein is preserved, with a 4–amino proteosomal inhibitor bortezomib, expression of the fusion acid insertion between UBE2L3 and KRAS. Using both a protein was clearly apparent, suggesting decreased stability monoclonal antibody raised against the Ras family and a of the fusion protein in the overexpression system (Fig. 2C). polyclonal antibody raised against KRAS specifically, we de- One area that needs to be explored and may explain tected a 33-kDa fusion protein in addition to the 21-kDa the protein stability issues is the function of the UBE2L3 band corresponding to wild-type KRAS in DU145 cells ubiquitin-conjugating (E2) enzyme portion of the fusion (Fig. 2B). Specificity of the band attributed to the Wang,UBE2L3- (18). We et therefore al. attempted Figure to detect ubiquitination 2 of KRAS protein was shown by knocking down expression using the UBE2L3-KRAS protein. We identified a rat anti-Ras

A a Bb

NontargetKRAS siRNA 1 150 200 295 aa Untreated UBE2L3 siRNA UBE2L3-KRAS 50 100 250 RAS siRNA monoclonal UBE2L3-KRAS UBCc domain UBE2L3 isoform1 UBE2L3-KRAS UBCc domain UBE2L3 isoform 2 KRAS UBCc domain H-N-K KRas–like domain polyclonal UBE2L3-KRAS KRAS H-N-K KRas–like domain KRAS ␤-Actin

DU145

C c D d MAASRRLMKELEEIRKCGMKNFRNIQVDEANLLTWQGLIVPDNPPYD KGAFRIEINFPAEYPFKPPKITFKTKIYHPNIDEKGQVCLPVISAENWKP PrEC DU145 RWPE VCaP 22RV1 PC3 HEK293 ATKTDQGLLKMTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIED －＋－＋－＋－＋－＋－＋－＋ +UBE2L3-KRASBort SYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINN TKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDL RAS UBE2L3-KRAS monoclonal ARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSK TKCVIM KRAS UBE2L3-KRAS polyclonal KRAS ␤-Actin SYGIPFIETSAKSFEDIHHYR

UBE2L3:KRAS: IYHPNIDEKKRAS:Fusion TDQ breakpoint:GLLK

DU145 LnCaP 33–36 kDa (UBE2L3-KRAS) VCaP DU145 LnCaP 20–23 kDa (wt KRAS) VCaP DU145 LnCaP 17–20 kDa (wt UBE2L3) VCaP

Figure 2. Characterization of the UBE2L3-KRAS fusion protein. A, UBE2L3, KRAS, and the predicted UBE2L3-KRAS fusion protein. B, expression of the UBE2L3-KRAS fusion protein in DU145 cells. Immunoblot analysis of DU145 cells using an anti-RAS mouse monoclonal antibody and an anti- KRAS rabbit polyclonal antibody detects a 33-kDa fusion protein specific to DU145 cells. Small interfering RNA (siRNA) duplexes are indicated. β-Actin was used to demonstrate equal loading. C, survey of the UBE2L3-KRAS fusion protein in a panel of prostate cancer cell lines and stabilization of protein expression with a proteosome inhibitor, bortezomib (Bort). Cell lines are indicated and treated in the presence or absence of 500 nM bortezomib for 24 hours. HEK293 cells were transfected with an expression construct encoding UBE2L3-KRAS. Immunoblot analysis was carried out using KRAS polyclonal and RAS monoclonal antibodies. D, MS assay for detection of UBE2L3-KRAS protein in DU145 cells. An MRM-MS assay was developed to detect the UBE2L3-KRAS fusion protein. Top, sequence of the UBE2L3-KRAS fusion protein with amino acids from UBE2L3 (red) and from KRAS (green). Tryptic peptides used for MRM-MS analysis are underlined. Bottom, matrix represents positive measurement (red) of peptides from corresponding gel fractions of DU145, LNCaP, and VCaP whole-cell lysates.

38 | CANCER DISCOVERY JUNE 2011 www.aacrjournals.org

KRAS Rearrangements in Prostate Cancer research brief monoclonal antibody that precipitated the 33-kDa UBE2L3- (Supplementary Fig. S7B), a system classically used to study KRAS protein as well as additional bands in the 40- to 55-kDa RAS biology (12, 19). Of note, enforced expression of UBE2L3- region, which were specific to HEK 293T cells expressing the KRAS induced loss of fibroblast morphology and increased fusion (Supplementary Fig. S8A). These shifted bands are cell proliferation and focus formation (Supplementary recognized by both anti-Ras and anti-HA tagged ubiquitin Fig. S9A; Fig. 3A and B). Cell-cycle analysis revealed an increase antibodies, and their molecular weights match the predic- in the S-phase fraction of cells (Supplementary Fig. S9B). To tion for ubiquitinated fusion proteins. We further validated determine the effects of UBE2L3-KRAS expression on tumor these ubiquitinated UBE2L3-KRAS proteins in DU145 growth in vivo, we implanted nude mice with stable NIH cells (Supplementary Fig. S8B). These data suggest that the 3T3 vector control cells or NIH 3T3 UBE2L3-KRAS fusion– UBE2L3-KRAS protein is ubiquitinated, which may contribute expressing cells. We observed robust tumor formation by the to its decreased stability. Wang,UBE2L3-KRAS–expressing et al. Figure cells, but not 3 the vector-trans- To determine the function of the UBE2L3-KRASWang, fu- Wang,fected et cells (Fig.al. et 3C; Figure al. Supplementary Figure Fig.3 S10). 3 To interro- sion, we overexpressed the fusion protein in NIH 3T3 cellsWang, gate the potential et RAS-relatedal. Figure signaling pathways 3 engaged

60 b a pDEST40 UBE2L3-KRAS pBABE KRAS G12V A 60 bB 60a b pDEST40 UBE2L3-KRAS pBABE KRAS G12V a 50 pDEST40 UBE2L3-KRAS pBABE KRAS G12V 4 60 a 50 b pDEST40 UBE2L3-KRAS pBABE 5040 4 KRAS G12V 4 5040 4030 4 4030 1000 3020 800 1000 30 1000 Cell number (x 10 ) 20 2010 Vector 600 800 Cell number (x 10 ) 800 1000 Cell number (x 10 ) 2010 UBE2L3-KRASVector 400 600 10 Vector 600 800 Cell number (x 10 ) UBE2L3-KRAS 400 0 Number of foci 200 101 3 5 UBE2L3-KRAS Vector 7 400 600

0 Number of foci 200 0 UBE2L3-KRAS 0 Time (days) Number of foci 200 400 1 3 5 7 pDEST400 UBE2L3-KRAS pBABE KRAS G12V 1 3 5 7 0 0 Time (days) Number of foci 200 pDEST40 UBE2L3-KRAS pBABE KRAS G12V Time (days) pDEST40 UBE2L3-KRAS pBABE KRAS G12V 1 3 5 7 0 Time (days) c d pDEST40 UBE2L3-KRAS pBABE KRAS G12V C D BRAF V600EKRAS G12V 3000c pDEST40d UBE2L3-KRAS vector c pDEST40 UBE2L3-KRAS d pDEST40 UBE2L3-KRAS vector BRAFp-MEK V600EKRAS G12V 3000- - Bort BRAF V600EKRAS G12V + pDEST40+ UBE2L3-KRAS pDEST40 UBE2L3-KRAS vector 3000 c pDEST40 UBE2L3-KRAS d p-MEK 3 2500 - -UBE2L3-KRASBort + + pDEST40 UBE2L3-KRAS vector p-MEKBRAFTotal V600EKRAS MEK G12V 3000- + - + Bortwt KRAS

3 2500 pDEST40 UBE2L3-KRAS p44 UBE2L3-KRAS p-MEKTotal MEK 3 2500 UBE2L3-KRAS 2000 - + - + Bortwt KRAS p42 p44 Totalp-ERK MEK wt KRAS p44

3 2500 2000 UBE2L3-KRAS p42 Totalp-ERK MEK 2000 p42 p-ERKTotal ERK 1500 wt KRAS p44 2000 p42 p-ERKTotal ERK 1500 Total ERK 1500 p-AKT 1000 Totalp-AKT ERK 1500 1000 p-AKTTotal AKT 1000 Tumor volume (mm ) 500 p-AKTTotal AKT NIH 3T3- pDEST40 Totalp-p38MAPK AKT 1000 Tumor volume (mm ) 500 NIH 3T3- UBE2L3-KRASNIH 3T3- pDEST40 Tumor volume (mm ) 500 NIH 3T3- pDEST40 Totalp-p38MAPK AKT 0 NIH 3T3- UBE2L3-KRAS p-p38MAPKTotal p38MAPK

Tumor volume (mm ) 500 NIH 3T3- UBE2L3-KRASNIH 3T3- pDEST40 7 80 9 10 11 12 13 14 15 p-p38MAPKTotal p38MAPK 0 NIH 3T3- UBE2L3-KRAS Total␤-Actin p38MAPK 7 8Time 9 (days) 10 11 12 13 14 15 7 80 9 10 11 12 13 14 15 Total␤-Actin p38MAPK Time (days) ␤-Actin 7 8Time 9 (days) 10 11 12 13 14 15 ␤-Actin Time (days)

Figure 3. Transforming activities of the UBE2L3-KRAS fusion in NIH 3T3 cells. A, overexpression of UBE2L3-KRAS in NIH 3T3 cells, increasing cellular proliferation. pDEST40 represents an empty vector. B, focus formation from overexpression of UBE2L3-KRAS in NIH 3T3 cells. Oncogenic KRAS G12V was used as a positive control with respective empty vectors as negative controls (pDEST40 and pBABE). Top, photographs of representative plates. Bottom, quantification of focus formation shown by bar graph. C, UBE2L3-KRAS–transfected NIH 3T3 cells form tumors in nude mice. Stable polyclonal populations of NIH 3T3 cells expressing either the vector or the UBE2L3-KRAS fusion gene were injected s.c. into nude mice. Tumor growth was monitored from day 7 to 15, as indicated. The inset shows fusion protein in the stably transfected NIH 3T3 cells, which is further stabilized upon bortezomib treatment. D, downstream signaling pathways engaged by UBE2L3-KRAS fusion in NIH 3T3 cells. Lysates prepared from stably transfected NIH 3T3 polyclonal populations and vector controls were subject to immunoblot analysis for phospho- and total MEK, ERK, AKT, and p38 MAPK. Oncogenic BRAFV600E and KRASG12V were controls, with β-actin used as a loading control.

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Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst April 3, 2011; DOI: 10.1158/2159-8274.CD-10-0022 research brief Wang et al. by UBE2L3-KRAS in NIH 3T3 cells, we carried out a series oncogenes involved in gene rearrangements in large ge- of immunoblot analyses on key signaling intermediaries. nomic datasets. As reported in the literature for NIH 3T3 cells, KRAS is a UBE2L3-KRAS encodes a protein encompassing most of the stronger inducer of the MEK/ERK cascade, whereas HRAS UBE2L3 protein and the full length of KRAS, which is ubiqui- is a stronger activator of the PI3K/AKT pathway (20). Of tinated and exhibits a high turnover rate. Importantly, recur- note, UBE2L3-KRAS overexpression resulted in attenuated rent genomic rearrangements at the KRAS locus were found in endogenous MEK and ERK phosphorylation (Fig. 3D) in 2 of 62 metastatic prostate tumors in addition to the DU145 NIH 3T3 cells; the signaling was instead directed to AKT and metastatic prostate cancer cell line. Although a number of on- p38 MAPK cascades, both of which have been implicated in cogenic activating point mutations of KRAS have been identi- prostate cancer (15, 17). fied, this is the first description of a mutant chimeric version As activation of the MEK/ERK pathway is dependent on of KRAS that is oncogenic and thus may represent a new class membrane attachment of Ras proteins, we investigated Ras of cancer-related alterations. Consistent with this finding, we subcellular localization, using immunofluorescence assays. recently described gene fusions of BRAF and RAF1 in 1% to 2% Interestingly, Ras proteins, which are normally distributed of prostate tumors, further implicating the RAS-RAF-MAPK in the cytoplasm, were found to be highly enriched in the late signaling pathway in subsets of prostate cancer (21). A sum- endosome after ectopic expression of UBE2L3-KRAS fusion mary of gene fusions involving this pathway that have been in NIH 3T3 cells (Supplementary Fig. S11). We speculate that discovered in prostate cancer is shown in Fig. 4D. The rarity of this relocation of Ras proteins may decrease their association KRAS rearrangement will limit its clinical relevance to a small with the cellular membrane and possibly enhance growth fac- subset of patients with metastatic prostate cancer. tor receptor signaling in the endosome. Although both KRAS G12V and UBE2L3-KRAS exhibit an To investigate the role of UBE2L3-KRAS fusion in a pros- oncogenic phenotype in vitro and in vivo, UBE2L3-KRAS tate background, we overexpressed the fusion in RWPE overexpression leads to attenuation, rather than activation, prostate epithelial cells (Supplementary Fig. S7C). The of the MEK/ERK pathway in NIH 3T3 cells. Instead, it ap- expression of the fusion protein was enhanced by incuba- pears that the KRAS fusion enriches Ras proteins in the en- tion with bortezomib (Fig. 4A, inset). Overexpression of the dosome and switches signaling to the AKT and p38 MAPK UBE2L3-KRAS fusion in RWPE cells led to increased cell inva- cascades. This observation may have important implica- sion, proliferation, and a transient increase in tumor growth tions in understanding the biological nature of this most in nude mice (Fig. 4A; Supplementary Fig. S12). Of note, in studied proto-oncogene. These signaling switches were not the RWPE model, signaling pathway analysis did not reveal observed in RWPE cells expressing the fusion, which by- inhibition of the MEK/ERK pathway or activation of AKT/ passes the ERK pathway (unlike the KRAS G12V mutant), p38 MAPK (data not shown). Although the MEK inhibitor indicating that other downstream effectors are being en- U0126 inhibits the invasion of RWPE cells overexpressing gaged in the prostate background. KRAS rearrangements, either wild-type or mutant KRAS, treated RWPE cells overex- although rare in the prostate cancer setting, could modu- pressing the fusion continued to exhibit invasive properties late metastasis, using signaling pathways that have thus in the presence of U0126 (Supplementary Fig. S13), suggest- far not been associated with KRAS. Future studies will be ing that downstream effectors other than MEK/ERK may be needed to elucidate the details of how chimeric KRAS en- engaged by the fusion in the prostate context. gages endogenous RAS-related signaling pathways in the To further confirm the function of endogenous UBE2L3- context of prostate cancer. KRAS in DU145 cells, we performed stable knockdown of UBE2L3-KRAS fusion and generated chicken embryo cho- Methods rioallantoic membrane and mouse xenograft models. This Amplification Breakpoint Ranking and Assembly resulted in decreased cell invasion and proliferation in vitro, Cell lines used for aCGH analysis were obtained from either as well as inhibition of tumor formation in the in vivo models American Type Culture Collection or collaborators and authenti- (Fig. 4B and C; Supplementary Fig. S14). cated by providers (detailed in the Supplementary Data). The aCGH data from prostate cancer cell lines were segmented by the circular binary segmentation algorithm (22), and the genomic position of each Discussion amplification breakpoint was mapped with the genomic regions of The addiction of cancer cells to causal gene fusions often all human genes. The 3' amplified genes were rated by their ConSig results in amplification in vivo, which may be exploited score (1), which identified KRAS as the top candidate. Matching to reveal unidentified recurrent gene rearrangements. On the amplification level of 3' KRAS with 5' amplified genes from the basis of this observation, we developed an integrative DU145 cells nominated UBE2L3, SOX5, and C14orf166 as 5' partner genomic-based approach called ABRA to explore driving candidates. The aCGH data used in this study have been deposited in the National Center for Biotechnology Information Gene Expression gene fusions contributing to the progression of prostate Omnibus with the accession number GSE26447. cancer. This investigation led to the nomination of an oncogenic UBE2L3-KRAS fusion in DU145 prostate cancer RT- PCR, Nuclease Protection Assay, and FISH cells, which was delineated by amplification breakpoints RT-PCR with the fusion primers UBE2L3-S2 and KRAS-R2 at KRAS and UBE2L3 loci, validating the ABRA analysis (Supplementary Table S3) confirmed the UBE2L3-KRAS fusion in approach. Although the ConSig score was not necessary DU145 cells. Fusion qPCR was performed on a panel of prostate to nominate KRAS as an oncogenic partner, it remains a cancer cell lines using primers UBE2L3-q1 and KRAS-q2 (StepOne useful approach for the identification of potential novel Real-Time PCR System; Applied Biosystems). Ribonuclease protection

40 | CANCER DISCOVERY JUNE 2011 www.aacrjournals.org

Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst April 3, Wang,2011; DOI: 10.1158/2159-8274.CD-10-0022 et al. Figure 4 KRAS Rearrangements in Prostate Cancer Wang, et al. Figure research4 brief a Wang, et al. Figure 4 a A 20 pLenti-6 UBE2L3-KRAS a 1820 -pLenti-6+ -UBE2L3-KRAS+ Bort 0.9 UBE2L3-KRAS 20 - + - + Bort 0.80.9 1618 pLenti-6 UBE2L3-KRASwt KRAS UBE2L3-KRAS 0.7 - 0.8 4 - + Bort 18 16 + wt KRAS 0.9 14 UBE2L3-KRAS 0.60.7

4 0.8 16 14 wt KRAS 0.5 12 0.7 0.6 4 0.40.5 141012 0.6 0.30.4 12 0.5 810 0.20.3 Vector 0.4Absorbance (560 nm) 0.1 10 Cell number (x 10 ) 8 0.3 0.2 6 UBE2L3-KRASVector Clone 1 Absorbance (560 nm) 00.1 8 Cell number (x 10 ) 6 UBE2L3-KRASUBE2L3-KRAS Clone Clone 2 1 0.2 4 Vector Absorbance (560 nm) pLenti-6 UBE2L3-KRAS 0.1 0 Cell number (x 10 Cell number (x 10 ) UBE2L3-KRASUBE2L3-KRAS Clone Clone 3 2 6 4 UBE2L3-KRAS Clone 1 pLenti-6RWPEUBE2L3-KRAS DU145 2 0 UBE2L3-KRASUBE2L3-KRAS Clone Clone 4 3 UBE2L3-KRAS Clone 2 RWPE DU145 4 0 2 pLenti-6 UBE2L3-KRAS UBE2L3-KRASUBE2L3-KRAS Clone Clone 3 4 2 1 3 5 RWPE DU145 0 UBE2L3-KRAS Clone 4 1 Time (days) 3 5 0 b 1 Time 3 (days) 5 1 B b 4 Time (days) 1 0.8 b UBE2L3-KRAS 1 3.54 KRAS 0.8 4 UBE2L3-KRAS 3.5 0.6 3 UBE2L3-KRASKRAS 0.8 3.5 3 DU145 0.6 2.5 Scramble KRAS shRNA pool 0.4 DU145 0.6 3 2.5 ScrambleUBE2L3-KRASUBE2L3-KRAS 2 shRNA shRNA pool shRNAclone 1 pool 1 0.4 DU145 Absorbance (560 nm) 2.5 Scramble UBE2L3-KRASUBE2L3-KRAS 0.2 2 shRNA pool shRNA shRNAclone 1 pool 1 0.4 1.5 DU145 Absorbance (560 nm) UBE2L3-KRASUBE2L3-KRAS 0.2 2 shRNA shRNAclone 1 pool 1 Scramble 1.5 shRNADU145 pool Absorbance (560 nm) 0 Absorbance (450 nm) 1 Scramble 0.2 UBE2L3-KRAS 0 1.5 DU145 shRNA shRNA pool pool 1 Absorbance (450 nm) 1 0.5 ScrambleUBE2L3-KRASUBE2L3-KRAS shRNA shRNA shRNA pool clone pool 11 0 Scramble

Absorbance (450 nm) 1 0.5 UBE2L3-KRAS shRNAUBE2L3-KRAS pool UBE2L3-KRAS 0 UBE2L3-KRAS shRNA pool shRNA1 clone1 shRNA shRNA pool 1clone 1 Scramble 0.5 1 2 3 4 5 6 Days shRNAUBE2L3-KRAS pool UBE2L3-KRAS 0 UBE2L3-KRAS shRNA pool shRNA1 clone1 shRNA clone 1 Scramble 1 2 3 4 5 6 Days shRNAUBE2L3-KRAS pool UBE2L3-KRAS 0 shRNA pool RAS shRNA1 clone1 c 1 2 3 4 5 6 Days d c d (1% Kras aberrations)RAS (1% Kras aberrations) cC 400 Scramble d D RAS shRNA pool P MEKK1 PI3K 3 400 * < 0.05 (1% Kras aberrations)RAF 350 UBE2L3-KRAS Scramble **P < 0.001 shRNA pool pool 1 P MEKK1 PI3K 3 shRNA * < 0.05 (1-2% RAF fusions 400 350 RAF 300 ScrambleUBE2L3-KRASUBE2L3-KRAS **P < 0.001 involving BRAF, RAF1) shRNA shRNA shRNA pool clone pool 1 1P MEKK1 (1-2% RAF fusions PI3K 3 * < 0.05 * RAF 350250300 UBE2L3-KRASUBE2L3-KRAS**P < 0.001 involving BRAF, RAF1) shRNA shRNA pool 1clone 1 MKK3,4,6 (1-2% RAF fusions PDK 250 * 300200 UBE2L3-KRAS involving BRAF, RAF1) shRNA clone 1 MKK3,4,6 MEK PDK 250150200 * * MKK3,4,6 MEK PDK

200) Tumor volume (mm 100150 * ** JNK p38 ERK AKT * MEK 150) Tumor volume (mm 50100 ** ** * ** JNK p38 ERK AKT * Tumor volume (mm ) Tumor volume (mm 100 050 ** ** ** JNK p38 ERK Ets* AKT Week 2 Week* 3 Week 4 Week 5 50 0 ** (>50% ETS Ets* fusions) Week 2 **Week 3 Week 4 Week 5 0 (>50% Ets* ETS fusions) Week 2 Week 3 Week 4 Week 5 (>50% ETS fusions)

Figure 4. The oncogenicity of UBE2L3-KRAS fusion in the prostate context. A, expression of the UBE2L3-KRAS fusion in RWPE benign prostate epithelial cells, leading to increased cellular proliferation and invasion. Left, results of cell proliferation assays using stable RWPE cell clones infected with either pLenti-6 vector or UBE2L3-KRAS. Inset shows the 33-kDa fusion protein detected only in the fusion-transfected cells treated with bortezomib to enhance protein stability [data from a representative clone are shown (Clone 2)]. Right, modified Boyden chamber–Matrigel assays using pLenti-6 vector and fusion-expressing cells (Clone 2). Invading cells were stained with crystal violet and quantified. DU145 prostate cancer cells were used as a positive control. B, knockdown of the UBE2L3-KRAS fusion, reducing cell proliferation and invasion in DU145 cells. Left, cell growth relative to the control shRNA was monitored using WST-1 assay for 6 days. Inset shows immunoblot analysis for the 33-kDa fusion protein detected using Ras monoclonal antibody. Right, results of Matrigel invasion assay for DU145 pool and clone with UBE2L3-KRAS knockdown. Scrambled short hairpin RNA (shRNA) duplexes are used as control. C, knockdown of the UBE2L3-KRAS fusion, attenuating prostate tumor growth in mouse xenograft models; plot shows mean tumor volume trajectories over time for mice inoculated with DU145 pool (red) or single clone (green) after UBE2L3-KRAS stable knock-down. Error bars represent SEM at each time point. D, summary of RAS-RAF signaling pathways in relation to recurrent gene fusions characterized in prostate cancer. Genes that participate in fusion events are denoted in red. Within parentheses are percentages of prostate cancers harboring aberrations in the ETS family, RAF family, and KRAS gene locus. *ETS family members involved in gene fusions include ERG, ETV1, 4, and 5. Adapted from Gioeli et al. (16).

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Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst April 3, 2011; DOI: 10.1158/2159-8274.CD-10-0022 research brief Wang et al. assays were performed using a 230-bp fragment spanning the UBE2L3- Grant Support KRAS fusion junction. Interphase FISH was done on cell lines, This work was supported in part by the Early Detection Research paraffin-embedded tissue sections, and tissue microarrays, using bac- Network UO1 CA111275, Prostate SPORE P50CA69568, National terial artificial chromosome probes. The GenBank accession number Center for Integrative Bioinformatics (U54 DA21519-01A1), for UBE2L3-KRAS fusion sequence is JF343812. NIH (R01CA132874), the National Functional Genomics Center (W81XWH-09-2-0014), R01 CA125612-01, and R01 CA132874. A.M. Western Blotting and MRM-MS Chinnaiyan is supported by the Prostate Cancer Foundation, the Doris Lysates from DU145, PrEC, RWPE, 22RV1, VCaP, and PC3 cells, Duke Charitable Foundation Clinical Scientist Award and a Burroughs either untreated or treated with 500 nM of bortezomib for 12 hours, Wellcome Foundation Award in Clinical Translational Research and is were probed with anti-RAS monoclonal (Millipore) and anti-KRAS an American Cancer Society Research Professor. L.C. Canley is sup- rabbit polyclonal antibodies (Proteintech Group Inc.). Cell lysates ported by P01 CA089021. A.T. Sasaki is supported, in part, by Kanae from DU145 and LnCaP cells treated with bortezomib were sepa- Foundation for Research Abroad and a Genentech Fellowship. rated by sodium dodecyl sulfate-polyacrylamide gel electrophore- sis and subject to trypsin digestion. Transitions of tryptic digested Received November 9, 2010; revised January 6, 2011; accepted peptides were compared with those of labeled internal standard pep- January 20, 2011; published OnlineFirst April 3, 2011. tides (spanning the fusion junction) by MRM-MS to identify the fusion peptides; see the Supplementary Data for more details.

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KRAS Rearrangements in Prostate Cancer research brief

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Characterization of KRAS Rearrangements in Metastatic Prostate Cancer

Xiao-Song Wang, Sunita Shankar, Saravana M. Dhanasekaran, et al.

Cancer Discovery 2011;1:35-43. Published OnlineFirst April 3, 2011.

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