Richard Smith, Jeffrey D. Hancock, Michelle Kinsey, Leah A. Owen, and Stephen L. Lessnick

1. Knockdown EWS/FLI and measure transcript levels of all 33 genes via qRT-PCR Knockdown each validated gene and test for transformation 14 14 A673-iLuc/PQXIN A673-iLuc-lacz 12 12 A673-iluc-PPP1R1A A673-iLuc/NKX2.2 cDNA 10 10 A673-iPPP1R1A-2-lacz A673-iNKX2.2-1/PQXIN Rescue knockdown via cDNA 8 A673-iPPP1R1A-2-PPP1R1A 8 Cumulative population doubling A673-iNKX2.2-1/NKX2.2 cDNA 6 6 4 4 2 2 0 0 0 3 6 9 12 15 0 3 6 9 12 15 Days Days Functional Analysis of EWS/FLI Target Genes Via an RNAi Mini-Library Richard Smith, Jeffrey D. Hancock, Michelle Kinsey, Leah A. Owen, and Stephen L. Lessnick Center for Children at Huntsman Cancer Institute, Department of Oncological Sciences, and Division of Pediatric Hematology/Oncology University of Utah, Salt Lake City, UT Summary: NKX2.2 PPP1R1A Background: Ewing’s sarcoma is a highly malignant small round cell tumor of childhood. Most cases of Ewing’s sarcoma are associated with a recurrent chromosomal rearrangement, t(11;22)(q24;q12). This translocation encodes the EWS/FLI protein. Most prior work supports the hypothesis that EWS/FLI functions as an aberrant transcription factor to dysregulate genes involved in oncogenic transformation. The biggest questions in the field are (1) which genes are dysregulated by EWS/FLI, and (2) what are their roles in the oncogenic phenotype of Ewing’s sarcoma? To address these questions, we developed a system to study the effects of EWS/FLI in Ewing’s sarcoma cells. We used retroviral-mediated RNA interference (RNAi) to “knock-down” endogenous EWS/FLI expression in A673 Ewing’s sarcoma cells. We compared control cells to cells with EWS/FLI knocked-down using oligonucleotide microarray to perform gene expression analysis. To determine which genes were the most reproducibly dysregulated, we used a 2.5 fold change cutoff value and identified those that passed this filter in each of four experimental replicates. Using this approach, we identified 33 genes that were upregulated by EWS/FLI, and 180 genes that were downregulated. This result was somewhat surprising, since most prior data suggested that EWS/FLI functions as a transcriptional activator, and not as a repressor. Genes that are dysregulated by EWS/FLI are likely to include those that are involved in the oncogenic phenotype of Ewing’s sarcoma, and those that are not. To identify which of the upregulated genes are important for Ewing’s sarcoma transformation, we took a stepwise approach that culminated in functional analysis using a “mini-RNAi library” approach. 33 genes upregulated by EWS/FLI NKX2.2 ELN HOOK1 SSX3 NR0B1 EPHB3 SH2D1A CRLF PPP1R1A FCGRT CNTNAP2 GYG2 CDH12 GLI1 GSTM4 NPY1R ARHN SLC15A2 SCGB1A RET PEG3 C7 SSX2 WWOX qRT-PCR reveals knock-down of target transcript qRT-PCR reveals knock-down of target transcript A673 cells with PPP1R1A cDNA rescue A673 cells with NKX2.2 cDNA rescue Cumulative population doubling NKX2.2 ARHN ELN NR0B1 C7 SH2D1A PPP1R1A CNTNAP2 WWOX CDH12GSTM4 Growth curve shows little effect of PPP1R1A knock-down Growth curve shows little effect of NKX2.2 knock-down NKX2.2 NR0B1 Approach and Results: We first validated the microarray data using independently-derived biologic replicates. Of the 33 transcripts analyzed, 24 could be confirmed as upregulated by EWS/FLI in Ewing’s sarcoma cells using quantitative RT-PCR (qRT-PCR). We next developed a “mini-RNAi library” consisting of 3 different retroviral-RNAi constructs targeting the 3’ UTR of each of the 24 confirmed EWS/FLI upregulated transcripts. Each retroviral-RNAi construct was introduced into A673 Ewing’s sarcoma cells, and an analysis was performed, including qRT-PCR to evaluate knock-down of the target transcript, growth assays, and soft agar assays to evaluate oncogenic transformation. Of the 24 targets evaluated, 10 of these appeared to be necessary for the transformed phenotype, because knockdown of these caused alterations in growth in tissue culture or soft agar. To confirm that the loss in growth or transformation was specific to the targeted transcript, and not an “off-target effect,” we performed rescue experiments. Following knock-down of the particular transcript, a cDNA for that transcript was introduced that was resistant to the RNAi effect (because the endogenous 3’ UTR was absent from the construct). Phenotypic analysis was again performed. Transformation was rescued with two targets: NR0B1 and NKX2.2 (see next column for NKX2.2 data). In some instances, we observed that knockdown of a given target resulted in a loss of transformation, but that the effect could not be reversed by introduction of a cDNA expressing that protein. In some of these cases, this was true even though two different RNAi constructs gave a similar loss-of-transformation phenotype. Indeed, further analysis of some of these (such as PPP1R1A) revealed that the endogenous protein was not expressed at detectable levels (e.g., see column 3 for PPP1R1A data). This suggests that the RNAi effect observed was an “off-target” or other nonspecific effect. Discussion: -Two EWS/FLI target genes were identified that play a critical role in the transformed phenotype of Ewing’s sarcoma: NR0B1 and NKX2.2. -The “RNAi mini-library” screening approach can be used to identify functionally-relevant gene targets of oncogenic transcription factors. -The “RNAi mini-library” screening approach does suffer from a high rate of “off-target” or other nonspecific growth effects. -Two RNAi constructs targeting the same transcript is an inadequate control in these experiments. -Rescue experiments (in which the targeted protein is re-expressed from a cDNA that is immune to the RNAi effect) are critical to unveil nonspecific effects. Soft Agar Colonies Soft Agar Colonies iluc/empty iluc/empty iluc/NKX2.2 iPPP1R1A-3/ PPP1R1A iluc/PPP1R1A iNKX2.2/empty iPPP1R1A/empty iNKX2.2/NKX2.2 Soft agars show loss and rescue of transformation Soft agars show loss of transformation but no rescue iPPP1R1A-3/empty iPPP1R1A-3/ PPP1R1A iNKX2.2/NKX2.2 iNKX2.2/empty iluc/PPP1R1A iluc/NKX2.2 iluc/empty Acknowledgments: S.L.L. is supported by grant K08 CA96755, American Cancer Society grant MGO-111812, the Terri Anna Perine Sarcoma Fund, a Primary Children’s Medical Center Foundation Innovative Research Grant, a Hope Street Kids grant, and a Catalyst Grant from the University of Utah School of Medicine. J.D.H. is supported by the Children’s Health Research Center at the University of Utah, and an Amgen Fellow’s grant. iluc/empty Western shows knockdown and rescue of protein levels Western shows no endogenous PPP1R1A in A673 cells!