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Supplementary Methods, Figures, Tables, and References from Characterization of KRAS Rearrangements in Metastatic Prostate Cancer

Supplementary Methods and Data (posted 7/5/2011) from Characterization of KRAS Rearrangements in Metastatic Prostate Cancer

PDF file, 138K, High-throughput profiling of survival following exposure to �-radiation.

PDF file, 109K, Clonogenic survival of lung SqCC after radiation (S1); High-throughput profiling of lung SqCC after radiation (S2); Mutations in NFE2L2 and KEAP1 in lung SqCC cell lines (S3); MSigDB Gene Sets from Fig S5 (S4); TP53 alterations, LOH, and mRNA levels in lung SqCC cell lines (S5).

PDF file, 282K, Clonogenic survival following exposure to �-radiation.

PDF file, 103K, NFE2L2 is frequently activated in human cancers.

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PDF file, 86K, ssGSEA identifies gene sets that correlate with radiation resistance (left) and sensitivity (right). Top 15 gene sets are listed.

PDF file, 20K, High-throughput profiling of lung cancer cell lines.

PDF file, 1002K, ssGSEA identifies gene sets that correlate with radiation resistance (left) and sensitivity (right). Top 50 gene sets are listed.

Radiotherapy is one of the mainstays of anticancer treatment, but the relationship between the radiosensitivity of cancer cells and their genomic characteristics is still not well defined. Here, we report the development of a high-throughput platform for measuring radiation survival in vitro and its validation in comparison with conventional clonogenic radiation survival analysis. We combined results from this high-throughput assay with genomic parameters in cell lines from squamous cell lung carcinoma, which is standardly treated by radiotherapy, to identify parameters that predict radiation sensitivity. We showed that activation of NFE2L2, a frequent event in lung squamous cancers, confers radiation resistance. An expression-based, in silico screen nominated inhibitors of phosphoinositide 3-kinase (PI3K) as NFE2L2 antagonists. We showed that the selective PI3K inhibitor, NVP-BKM120, both decreased NRF2 protein levels and sensitized NFE2L2 or KEAP1-mutant cells to radiation. We then combined results from this high-throughput assay with single-sample gene set enrichment analysis of gene expression data. The resulting analysis identified pathways implicated in cell survival, genotoxic stress, detoxification, and innate and adaptive immunity as key correlates of radiation sensitivity. The integrative and high-throughput methods shown here for large-scale profiling of radiation survival and genomic features of solid-tumor–derived cell lines should facilitate tumor radiogenomics and the discovery of genotype-selective radiation sensitizers and protective agents. Cancer Res; 73(20); 6289–98. ©2013 AACR.

The NKX2-1 transcription factor, a regulator of normal lung development, is the most significantly amplified gene in human lung adenocarcinoma. To study the transcriptional impact of NKX2-1 amplification, we generated an expression signature associated with NKX2-1 amplification in human lung adenocarcinoma and analyzed DNA-binding sites of NKX2-1 by genome-wide chromatin immunoprecipitation. Integration of these expression and cistromic analyses identified LMO3, itself encoding a transcription regulator, as a candidate direct transcriptional target of NKX2-1. Further cistromic and overexpression analyses indicated that NKX2-1 can cooperate with the forkhead box transcription factor FOXA1 to regulate LMO3 gene expression. RNAi analysis of NKX2-1-amplified cells compared with nonamplified cells demonstrated that LMO3 mediates cell survival downstream from NKX2-1. Our findings provide new insight into the transcriptional regulatory network of NKX2-1 and suggest that LMO3 is a transcriptional signal transducer in NKX2-1-amplified lung adenocarcinomas., National Institutes of Health (U.S.) (NIH/NCI T32 Institutional Training Program fellowship (5T32CA009361-28)), National Cancer Institute (U.S.) (grant 5R01CA109038), National Cancer Institute (U.S.) (grant 5P20CA90578)

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. Read the Commentary on this article by Edgren et al., p. 12 This article is highlighted in the In This Issue feature, p. 4

The NKX2-1 transcription factor, a regulator of normal lung development, is the most significantly amplified gene in human lung adenocarcinoma. To better understand how genomic alterations of NKX2-1 drive tumorigenesis, we generated an expression signature associated with NKX2-1 amplification in human lung adenocarcinoma, and analyzed DNA binding sites of NKX2-1 by genome-wide chromatin immunoprecipitation (ChIP-seq) from three NKX2-1-amplified human lung adenocarcinoma cell lines. Integration of these expression and cistromic analyses suggested that aberrant over-expression of NKX2-1 primarily activates the expression of transcriptional target genes whose expression is not trans-activated by NKX2-1 at physiological expression levels, and identified LMO3, itself encoding a transcription regulator, as a candidate direct transcriptional target of NKX2-1. NKX2-1 amplification is most significantly associated with over-expression of the LMO3 gene, a member of the LMO family of oncogenes that are translocated in T-ALL, the same disorder in which NKX2-1 translocation has been observed. We further show that NKX2-1 interacts physically with the LMO3 locus, and activates its expression. RNA interference analysis of NKX2-1-amplified cells compared to non-amplified cells demonstrated that LMO3 mediates cell proliferation downstream of NKX2-1. Further cistromic analyses found that consensus binding motifs including a nuclear hormone receptor signature and a Forkhead box motif, indicating that NKX2-1 might cooperate with Forkhead box FOXA1 and nuclear hormones to regulate gene expression. Genome-wide analysis of FOXA1 occupancy by ChIP-seq in the NCI-H3122 cell line revealed that FOXA1 binds not only to the LMO3 locus upstream of NKX2-1 binding site but also to many NKX2-1 occupied regions (47% of total NKX2-1-bound sites). Our findings provide new insight into the transcriptional regulatory network of NKX2-1 and suggest that LMO3 is a transducer of lineage specific cell survival of NKX2-1-amplified lung adenocarcinomas. Citation Format: Hideo Watanabe, Joshua M. Francis, Michele S. Woo, Banafsheh Etemad, Wenchu Lin, Daniel F. Fries, Shouyong Peng, Eric L. Snyder, Purushothama Rao Tata, Anna C. Schinzel, Jeonghee Cho, Peter S. Hammerman, Roel G. Verhaak, William C. Hahn, Jayaraj Rajagopal, Tyler Jacks, Matthew Meyerson. Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3127. doi:10.1158/1538-7445.AM2013-3127