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

Supplementary Figure 4. Overlap of positive and negative correlations.

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

Ewing's sarcoma family of tumors (ESFT) refers to aggressive malignancies which frequently harbor characteristic EWS-FLI1 or EWS-ERG genomic fusions. Here, we report that these fusion products interact with the DNA damage response protein and transcriptional coregulator PARP-1. ESFT cells, primary tumor xenografts, and tumor metastases were all highly sensitive to PARP1 inhibition. Addition of a PARP1 inhibitor to the second-line chemotherapeutic agent temozolamide resulted in complete responses of all treated tumors in an EWS-FLI1–driven mouse xenograft model of ESFT. Mechanistic investigations revealed that DNA damage induced by expression of EWS-FLI1 or EWS-ERG fusion genes was potentiated by PARP1 inhibition in ESFT cell lines. Notably, EWS-FLI1 fusion genes acted in a positive feedback loop to maintain the expression of PARP1, which was required for EWS-FLI–mediated transcription, thereby enforcing oncogene-dependent sensitivity to PARP-1 inhibition. Together, our findings offer a strong preclinical rationale to target the EWS-FLI1:PARP1 intersection as a therapeutic strategy to improve the treatment of ESFTs. Cancer Res; 72(7); 1608–13. ©2012 AACR.

Recurrent gene fusions involving oncogenic ETS transcription factors (including ERG, ETV1, and ETV4) have been identified in a large fraction of prostate cancers. The most common fusions contain the 5′ untranslated region of TMPRSS2 fused to ERG. Recently, we identified additional 5′ partners in ETV1 fusions, including TMPRSS2, SLC45A3, HERV-K_22q11.23, C15ORF21, and HNRPA2B1. Here, we identify ETV5 as the fourth ETS family member involved in recurrent gene rearrangements in prostate cancer. Characterization of two cases with ETV5 outlier expression by RNA ligase–mediated rapid amplification of cDNA ends identified one case with a TMPRSS2:ETV5 fusion and one case with a SLC45A3:ETV5 fusion. We confirmed the presence of these fusions by quantitative PCR and fluorescence in situ hybridization. In vitro recapitulation of ETV5 overexpression induced invasion in RWPE cells, a benign immortalized prostatic epithelial cell line. Expression profiling and an integrative molecular concepts analysis of RWPE-ETV5 cells also revealed the induction of an invasive transcriptional program, consistent with ERG and ETV1 overexpression in RWPE cells, emphasizing the functional redundancy of ETS rearrangements. Together, our results suggest that the family of 5′ partners previously identified in ETV1 gene fusions can fuse with other ETS family members, suggesting numerous rare gene fusion permutations in prostate cancer. [Cancer Res 2008;68(1):73–80]

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Supplementary Figures 1-2, Tables 1-2 from Characterization of TMPRSS2:ETV5 and SLC45A3:ETV5 Gene Fusions in Prostate Cancer

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Impairment of double-stranded DNA break (DSB) repair is essential to many cancers. However, although mutations in DSB repair proteins are common in hereditary cancers, mechanisms of impaired DSB repair in sporadic cancers remain incompletely understood. Here, we describe the first role for a long noncoding RNA (lncRNA) in DSB repair in prostate cancer. We identify PCAT-1, a prostate cancer outlier lncRNA, which regulates cell response to genotoxic stress. PCAT-1 expression produces a functional deficiency in homologous recombination through its repression of the BRCA2 tumor suppressor, which, in turn, imparts a high sensitivity to small-molecule inhibitors of PARP1. These effects reflected a posttranscriptional repression of the BRCA2 3′UTR by PCAT-1. Our observations thus offer a novel mechanism of “BRCAness” in sporadic cancers. Cancer Res; 74(6); 1651–60. ©2014 AACR.

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In the 20 years since the completion of the Human Genome Project, cancer biology remains rooted in the assumption that the human genome encodes ~20,000 protein-coding genes. Yet, I and others have shown that thousands of “non-canonical” open reading frames (ncORFs) populate the human genome, potentially representing a dramatic expansion of the cancer proteome. Despite their abundance, little is known about the role of ncORFs as cancer driver genes. We developed functional genomics approaches to pursue this question across human cancers. To determine whether ncORFs represent biologically active proteins, we experimentally interrogated 553 candidates selected from ncORF datasets. Of these, 257 (46%) showed evidence of stable protein expression using multiple assays, and 401 (72%) induced gene expression changes when expressed in cancer cell lines. The bioactivity of ncORFs was dependent on their ability to translate a protein: mutation of the ORF start codon prevented induction of gene expression changes observed with the wild type ncORF in 48 of 51 (94%) cases. Using custom CRISPR/Cas9 knock-out screens targeting >2,000 ncORFs in 20 cancer cell lines, we found that genomic knock-out of approximately 10% of ncORFs induced viability defects in cancer cells. We focused on two candidates for functional studies. In breast cancer, we described G029442 - renamed glycine-rich extracellular protein-1 (GREP1) - as a secreted protein that is highly expressed and prognostic for poor patient outcomes. Knock-out of GREP1 in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. In medulloblastoma, we found that MYC-driven medulloblastoma cells are enriched for bioactive upstream ORFs (uORFs) that are encoded within the 5’ untranslated regions of mRNAs. We validated the ASNSD1 uORF as a top genetic vulnerability in multiple models of medulloblastoma, and its overexpression is sufficient to increase neural stem cell proliferation. Mechanistically, ASNSD1 uORF promotes a MYC-associated cellular program and interacts with the multiprotein prefoldin complex, which is required for tumors to maintain post-transcriptional regulation. Our work supports a generalizable principle that ncORFs commonly encode biologically-active proteins in diverse malignancies. Ongoing investigation of ncORFs therefore represents a new frontier in cancer research with the potential to define the next generation of therapeutic target genes. Citation Format: John R. Prensner, Ian Yannuzzi, Karl Clauser, Karsten Krug, Oana Enache, Adam Brown, Amy Goodale, David E. Root, Pratiti Bandopadhayay, Todd Golub. Non-canonical proteins are cancer cell vulnerabilities in diverse malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3624.

Introduction: Numerous transcripts annotated as long noncoding RNAs play a central role in cancer biology. For many, their noncoding status is merely a presumption. Genome-wide sequencing of ribosomal footprints has nominated thousands of unstudied open reading frames (ORFs) within lncRNAs, representing an expansion of the proteome. Here, we investigate previously unstudied proteins in cancer cell biology. Methods: Ribosome profiling data was analyzed with RibORF. 96 hours after infection with lentivirus for selected ORFs, L1000 expression profiling was performed on 4 cell lines. A CRISPR library was screened across 8 cells lines with sgRNA sequencing on days 0, 7 and 21 post-infection. Results: We analyzed ribosomal profiling data for 14 cell lines (~320 million sequencing reads). We nominated 28530 non-canonical ORFs within annotated protein-coding genes, 6697 ORFs in annotated lncRNAs, and 1252 ORFs in pseudogenes. For further study, we selected 553 candidate ORFs that exhibited compelling features, including DNA conservation, translational efficiency, protein domain, among others. We validated protein expression for 260 of 553 ORFs (47%): 89 (16%) had supporting peptides in deep-coverage proteomics datasets; 233 (42%) expressed protein after ectopic expression of individual V5-tagged cDNAs; 10 of 30 tested untagged ORFs expressed protein by biochemical in vitro translation. Ectopic overexpression followed by RNA profiling revealed 259 cDNAs that caused cellular transcriptional changes in at least one of four cancer cell lines (A549, HA1E, A375, MCF7). 137 of the 259 (49%) were validated proteins. As controls, we generated methionine-mutant constructs: 65 of 71 mutant cDNA experiments were unable to cause similar expression changes. We used a CRISPR library to identify novel ORF dependencies in 8 Cas9-derivatized cancer cell lines (MCF7, A549, A375, PC3, HEPG2, HELA, HA1E, HT29). For 42 ORFs, ≥ 2 targeting sgRNAs produced ≤ -1 log fold depletion in ≥ 1 cell lines. These ORFs were re-tested with a second sgRNA library. Next, we investigated compelling candidates more deeply with immunoprecipitation with mass spectrometry. For the cancer outlier transcript LINC01314, which encodes a highly conserved 59 amino acid protein harboring a cortexin domain (pfam domain cl12620), we found interactions with IMMT, SAMM50, and CHCHD3, members of a mitochondrial complex. Another example is LINC00116, which encodes a highly conserved 56 amino acid protein that binds the importin-nuclear pore complex. Conclusion: We establish a framework to discover, validate, and characterize unstudied proteins. About half of tested ORFs generated a detectable protein, and of these, half impacted cellular transcription. We discover novel gene dependencies, and are elucidating mechanisms for several ORFs. Together, our work is the first large-scale attempt to study the role of unannotated proteins in cancer cell biology. Citation Format: John R. Prensner, Oana Enache, Zhe Ji, Karsten Krug, Karl R. Clauser, Xiaoping Yang, Federica Piccioni, David E. Root, Todd R. Golub. Integrative functional proteogenomics for unannotated or uncharacterized proteins in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4344.

Ewing's sarcoma family of tumors (ESFT) refers to aggressive malignancies which frequently harbor characteristic EWS-FLI1 or EWS-ERG genomic fusions. Here, we report that these fusion products interact with the DNA damage response protein and transcriptional coregulator PARP-1. ESFT cells, primary tumor xenografts, and tumor metastases were all highly sensitive to PARP1 inhibition. Addition of a PARP1 inhibitor to the second-line chemotherapeutic agent temozolamide resulted in complete responses of all treated tumors in an EWS-FLI1–driven mouse xenograft model of ESFT. Mechanistic investigations revealed that DNA damage induced by expression of EWS-FLI1 or EWS-ERG fusion genes was potentiated by PARP1 inhibition in ESFT cell lines. Notably, EWS-FLI1 fusion genes acted in a positive feedback loop to maintain the expression of PARP1, which was required for EWS-FLI–mediated transcription, thereby enforcing oncogene-dependent sensitivity to PARP-1 inhibition. Together, our findings offer a strong preclinical rationale to target the EWS-FLI1:PARP1 intersection as a therapeutic strategy to improve the treatment of ESFTs. Cancer Res; 72(7); 1608–13. ©2012 AACR.

Recurrent gene fusions involving oncogenic ETS transcription factors (including ERG, ETV1, and ETV4) have been identified in a large fraction of prostate cancers. The most common fusions contain the 5′ untranslated region of TMPRSS2 fused to ERG. Recently, we identified additional 5′ partners in ETV1 fusions, including TMPRSS2, SLC45A3, HERV-K_22q11.23, C15ORF21, and HNRPA2B1. Here, we identify ETV5 as the fourth ETS family member involved in recurrent gene rearrangements in prostate cancer. Characterization of two cases with ETV5 outlier expression by RNA ligase–mediated rapid amplification of cDNA ends identified one case with a TMPRSS2:ETV5 fusion and one case with a SLC45A3:ETV5 fusion. We confirmed the presence of these fusions by quantitative PCR and fluorescence in situ hybridization. In vitro recapitulation of ETV5 overexpression induced invasion in RWPE cells, a benign immortalized prostatic epithelial cell line. Expression profiling and an integrative molecular concepts analysis of RWPE-ETV5 cells also revealed the induction of an invasive transcriptional program, consistent with ERG and ETV1 overexpression in RWPE cells, emphasizing the functional redundancy of ETS rearrangements. Together, our results suggest that the family of 5′ partners previously identified in ETV1 gene fusions can fuse with other ETS family members, suggesting numerous rare gene fusion permutations in prostate cancer. [Cancer Res 2008;68(1):73–80]

Long non-coding RNA (lncRNAs) have been shown to play an important role is a variety of cellular processes including cancer initiation and progression. Recently, we described a landscape of lncRNA using RNA-Seq data from more than 5000 cancer samples from 18 tissue types. Using novel bioinformatics based approach we were able to discover almost 50,000 novel transcripts. This list includes a subset of lncRNAs that contain ultra-conserved elements (UCE) termed “Highly Conserved Long Intergenic Non-Coding RNAs (HICLINCs)”. This provides a new opportunity for exploring the functions of highly conserved lncRNAs using genomic approaches. Among them, we focus on an lncRNA which we have termed HiClinc-1 (Highly Conserved Intergenic long-noncoding RNA-1), that is specifically expressed only in testis in benign tissue, and re-expressed in a broad range of cancer types. We described that HiClinc-1 exists in the messenger Ribonucleoprotein (mRNP) complex and binds to Insulin-Like Growth Factor 2 mRNA Binding Protein 1 (IGF2BP1), one of the component of mRNP complex in both human and zebrafish. HiClinc-1 regulates cell proliferation both in vivo and in vitro through the regulation of IGF2BP1's downstream target Insulin-Like Growth Factor 2 (IGF2). HiClinc-1 also enhanced tumor growth in mouse xenograft model. Notably, Human-HiClinc-1 injection in zebrafish embryos accelerated the onset and growth of NRAS K61-induced melanoma. Similarly, HiClinc-1 knockout background showed delayed onset of NRAS K61-induced melanoma. Taken together, we identified a novel Cancer-Testis lncRNA, which is highly conserved through human to zebrafish that can be a potential therapeutic target for cancer treatment. Citation Format: Yasuyuki Hosono, John Prensner, Matthew Iyer, Yashar Niknafs, Rohit Malik, Anton Poliakov, Xuhong Cao, Jordan Shavit, Rohit Mehra, Weibin Zhou, Arul M. Chinnaiyan. HiClinc-1, a highly conserved Cancer-Testis lncRNA, regulates cell proliferation and tumor onset. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR10.

Introduction: Integrated clinical sequencing (RNA and whole-exome) has been used to assist in clinical decision making for pediatric and young adult cancer patients in the Peds-MiOncoSeq study. Here we present novel biological findings from this study that shed light onto disease pathogenesis. Actionable sequencing results affecting patient management have been presented previously by our group. Experimental Procedures: Peds-MiOncoSeq is an observational, consecutive case series (May 2012-October 2014) of 102 pediatric and young adult cancer patients (ages 0-22 years) at the University of Michigan who underwent exome (tumor and germline DNA) and transcriptome (tumor RNA) sequencing with genetic counseling. Sequencing was performed with standard protocols on an Illumina HiSeq 2000 or 2500. Sequence reads were aligned to the reference genome build hg19, GRCh37. Somatic and germline variant calling was performed with VarScan2 and Pindel for indels; gene fusion identification with Tophat2; and gene expression with Cufflinks. Recommendations were discussed in a Precision Medicine Tumor Board and reported to treating physicians and families. Results: In 91/102 patients (28 hematologic and 63 solid tumor patients), there was adequate tumor tissue for sequencing and analysis. Of these, 42 had potentially actionable molecular events (previously presented by our group), while another 44 patients yielded informative but non-actionable biology only. We uncovered both known and novel disease-associated mutations. In particular, sarcomas had frequent TP53 mutations (8/23 patients), FGF pathway activation (5/23), and PDGF pathway activation (3/23). Other recurrently mutated genes included beta-catenin in liver tumors (3/6), FLT3 in leukemias (3/20), and ATRX in neuroblastoma (3/7). Across disparate tumor types, we observed MAPK pathway activation (via NF1 loss (n=3), or RAS (n=5) or RAF mutations (n=3)), SWI/SNF complex inactivation (n=9), NOTCH1/2 aberrations (n=7), and STAG2 mutations (n=3). Surprisingly, we discovered mutations in the transcription factor HNF1A in both leukemia and osteosarcoma, which is not known in these cancers. 12 patients had significant germline variants including unexpected rare cancer risk genes (BAP1 in melanoma, BARD1 in neuroblastoma) which revealed potential cancer predisposition syndromes for several patients. Interestingly, an activating germline MITF E318K mutation was discovered in an omental mass of unclear etiology with hematolymphoid histology. Most notably, 32/91 patients had a driving gene fusion. Fusions were more common in hematologic compared to solid tumors (OR=4.0, 95% CI 1.6–10.4). However, sarcomas displayed a comparable rate to hematologic malignancies: 52% of sarcomas, 59% of liquid tumors, 12% of other solid tumors had a driving fusion. Sarcomas also harbored actionable kinase fusions such as LMNA-NTRK1 and ATIC-ALK. Recurrent BCOR-CCNB3 fusions were further found in sarcoma and in a spindle cell carcinoma, which is previously unknown in this tumor type. Other novel findings were a previously unknown YAP-MAML2 fusion in meningioma, a LRP6-ETV6 fusion in glioma, a EBF1-PDGFRB fusion in leukemia, and lastly a SFPQ-TFE3 fusion in a colon adenocarcinoma, which is the first description of this fusion in this tumor type. Conclusions: Here, we catalog the landscape of tumor biology in the Peds-MiOncoSeq study. Highlighted in our study are somatic HNF1A mutations and rare germline variants (MITF, BAP1, BARD1). Most importantly, our study demonstrates the importance of gene fusions across cancer subtypes, and we describe many novel fusions warranting further study. Our work therefore provides a framework for clinical sequencing programs as well as a rich collection of novel disease-associated molecular events. This abstract is also presented as Poster A44. Citation Format: John R. Prensner, Rajen J. Mody, Yi-Mi Wu, Robert J. Lonigro, Xuhong Cao, Sameek Roychowdhury, Pankaj Vats, Kevin M. Frank, Irfan Asangani, Nallasivam Palanisamy, Raja M. Rabah, Laxmi Priya Kunju, Jessica Everett, Elena M. Stoffel, Jeffrey W. Innis, Dan R. Robinson, Arul M. Chinnaiyan. The landscape of molecular aberrations in pediatric and young adult cancer patients undergoing clinical sequencing for disease management: Novel biological findings from the Peds-MiOncoSeq study. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR07.

Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration resistant prostate cancer (CRPC). Substantial prior work has focused on targeting AR directly; however, the identification and therapeutic targeting of co-activators of AR signaling remains an underexplored area of potential clinical significance. Here we demonstrate that the MLL (mixed-lineage leukemia) complex, a well-known contributor in MLL-fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via its critical subunit, menin. Small molecule inhibition of the menin-MLL interaction blocks AR signaling and inhibits the growth of castration resistant tumors in vivo. Furthermore, we find that menin is up-regulated in castration resistant prostate cancer and high expression correlates with poor overall survival. Taken together, our study identifies the MLL complex as a critical co-activator of AR that can be targeted in advanced prostate cancer. Citation Format: Rohit Malik, Amjad P. Khan, Irfan A. Asangani, Marcin Cieślik, John R. Prensner, Xiaoju Wang, Matthew K. Iyer, Xia Jiang, Dmitry Borkin, June Escara-Wilke, Rachell Stender, Yi-Mi Wu, Xuhong Cao, Felix Y. Feng, Jolanta Grembecka, Tomasz Cierpicki, Arul M. Chinnaiyan. Targeting the MLL complex in castration resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3636. doi:10.1158/1538-7445.AM2015-3636

Prostate cancer is the leading cancer diagnosis in men, but only a subset of patients die from the disease. Understanding the molecular basis of aggressive prostate cancer remains elusive and few genomic biomarkers exist to guide clinical management. Long non-coding RNAs (lncRNAs) have emerged as a class of regulatory genes that play a role in several biological and disease processes, including cancer. Recently, we identified SChLAP1, a novel, prognostic lncRNA expressed in a subset of prostate tumors. Mechanistically, SChLAP1 interacts with and inhibits genome-wide binding of the SWI/SNF nucleosome-remodeling complex. Additionally, SWI/SNF plays a key role in the epigenetic control of gene expression and has been shown to have tumor suppressive ability. Given the emerging role of SChLAP1 in the pathogenesis of aggressive prostate cancer and its interaction with the tumor suppressive SWI/SNF complex, exploring the detailed mechanism of action of SChLAP1 warranted further investigation. To identify the regions of SChLAP1 necessary for its function, we created 250 base pair deletion constructs of SChLAP1 and measured RNA-enrichment following SWI/SNF pull-down as well as cell invasion using a Boyden chamber matrigel assay. To investigate whether SChLAP1 preferentially interacts with specific components of the SWI/SNF complex, we performed RNA immunoprecipitation assays using antibodies targeting various enzymatic subunits of SWI/SNF. SChLAP1 enrichment was measured by qPCR. Our results indicate that there is a 250bp region of SChLAP1 that mediates its interaction with SWI/SNF and promotes an invasive phenotype. Furthermore, SChLAP1 shows binding specificity for certain SWI/SNF subunits. Future studies to elucidate the details of this interaction will provide a more comprehensive understanding of the biological and therapeutic consequences of SChLAP1 in prostate cancer progression. Citation Format: Udit Singhal, Anirban Sahu, John R. Prensner, Qi Cao, Arul M. Chinnaiyan. SChLAP1 mediated epigenetic modifications in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2869. doi:10.1158/1538-7445.AM2015-2869

Background: Long noncoding RNAs (lncRNAs) have recently been associated with the development and progression of a variety of human cancers. To date, the interplay between known oncogenic drivers, such as estrogen receptor (ER), and lncRNAs has not been well described. In this study, we identify M41 as the top outlier lncRNA in ER-positive vs ER-negative breast cancer and investigate its role in preclinical cancer phenotypes and clinical outcomes. Methods and Materials: RNA sequencing was performed on 89 breast cancer samples and cell lines, including 42 ER+ cases, and a modified cancer outlier analysis was used to identify lncRNAs enriched in ER-positive disease. To assess ER regulation of the top enriched lncRNA (M41), ChIP-Seq and ChIP-PCR was used to detect binding of ER to M41 promoter and qPCR was used to determine changes in M41 expression following 10 nM estradiol treatment in MCF7 and T47D cells. Following knockdown via siRNA, the impact of M41 expression was assessed on cell invasion, migration, proliferation, and anchorage-independent growth. The impact of M41 knockdown on tamoxifen sensitivity was assessed by cell proliferation studies in MCF7 cells with acquired tamoxifen resistance. Lastly, clinical associations between M41 expression and grade/node status, as well as event-free survival (EFS), was determined using ANOVA and Kaplan-Meier analyses of TCGA samples. Results: M41, an uncharacterized lncRNA located on chr21q22.2, was identified as the top outlier lncRNA in ER-positive vs ER-negative breast cancer. M41 demonstrated outlier expression (RPKM values>50) in 15% of ER-positive cancers, and was not significantly expressed in normal breast tissue. ChIP studies show that ER robustly binds to the M41 promoter. Estradiol stimulation significantly increased M41 expression in a time-dependent manner. Knockdown of M41 significantly inhibited all assessed oncogenic phenotypes in the ER-positive MCF7 and T47D cells, with a 60-80% decrease in both invasion and anchorage-independent growth, but had no effect in the ER-negative MDA-MB-231 cell line (which has minimal M41 expression). M41 expression was greater than 10-fold higher in tamoxifen-resistant MCF7 cells compared to parental controls (p Conclusion: We have identified M41 as an ER-associated oncogenic lncRNA that contributes to preclinical cancer phenotype, promotes tamoxifen resistance in cell line models, and associates with poor outcomes in clinical samples. We suggest that M41 represents a novel biomarker candidate for the prognosis of ER-positive breast cancers and provides new insight into the biological complexity of breast tumor biology. Citation Format: Felix Y Feng, Teng Ma, Corey Speers, Matthew K Iyer, Shuang Zhao, John R Prensner, James M Rae, Lori J Pierce, Arul M Chinnaiyan. The long noncoding RNA M41 promotes aggressiveness and tamoxifen resistance in ER-positive breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-1.

Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castrate resistant prostate cancer (CRPC), an advanced and frequently lethal form of this disease. Substantial prior work has focused on targeting AR directly; however, the identification and therapeutic targeting of co-activators of AR signaling remains an underexplored area of potential clinical significance. Here we demonstrate that the MLL complex acts as a co-activator of AR signaling. AR directly interacts with the sub-unit menin to recruit MLL and its complex to AR target genes. Inhibition of the menin-MLL interaction can block AR signaling and inhibit the formation of castration resistant tumors in vivo. Furthermore, we find that menin is up-regulated in localized and metastatic prostate cancer and high menin expression correlates with poor overall survival. Taken together our study identifies a novel co-activator complex of AR that can be targeted in CRPCs. Citation Format: Rohit Malik, Amjad P. Khan, John R. Prensner, Matthew K. Iyer, Dmitry Borkin, Xiaoju Wang, Xia Jiang, Shruthi Subramaniam, Yang Shi, Rachell Stender, Yi-Mi Wu, Xuhong Cao, Jolanta Grembecka, Tomasz Cierpicki, Arul Chinnaiyan. Targeting novel co-activators of androgen receptor in castration resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1398. doi:10.1158/1538-7445.AM2014-1398

Prostate cancer is the second most common epithelial cancer and second leading cause of cancer death in men. Prostate cancers remain indolent in the majority of individuals but behave aggressively in a minority of patients. The molecular basis for this clinical heterogeneity remains incompletely understood. Long noncoding RNAs (lncRNAs) have emerged as a prominent layer of transcriptional regulation implicated in various biological and disease processes, including several types of cancer. Therefore, we hypothesized that lncRNAs may play a role in mediating aggressive prostate cancer. Previously, our lab utilized RNA sequencing across a cohort of prostate tissues to discover differentially expressed lncRNAs in cancer versus benign samples. We identifed a novel transcript termed SChLAP1 (Second Chromosome Locus Associated with Prostate-1) overexpressed in a subset of localized and metastatic cancers. Clinical analyses showed that SChLAP1 levels independently predict poor outcomes, including metastasis and prostate cancer-specific mortality. In vitro and in vivo experiments indicated that SChLAP1 is critical for cancer cell invasiveness and metastasis. Mechanistically, we found that SChLAP1 binds to and antagonizes the genome-wide localization of the tumor-suppressive SWI/SNF nucleosome-remodeling complex. Given the scaffolding capabilities of lncRNAs, previous studies describing an inhibitory role of SWI/SNF on PRC2 function, and the known oncogenic role of PRC2 in prostate cancer, we hypothesized that SChLAP1 may mediate PRC2 activity in conjunction with SWI/SNF to promote aggressive disease. Here, we show that a SChLAP1-associated gene signature nominates PRC2-related concepts, and in vitro studies indicate an interaction between SChLAP1 and PRC2 that enhances PRC2 genome-wide localization as well as its histone methyltransferase activity. These results suggest a mechanistic model in which SChLAP1 simultaneously engages multiple epigenetic complexes, inhibiting tumor-suppressive functions of SWI/SNF while enhancing oncogenic activity of PRC2, which leads to aggressive prostate cancer. Citation Format: Anirban Sahu, Matthew K. Iyer, John R. Prensner, Arul M. Chinnaiyan. The role of long noncoding RNA SChLAP1 in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 541. doi:10.1158/1538-7445.AM2014-541

Activating mutations in the Ras oncogenes characterize 20-40% of all non-small cell lung cancer (NSCLC), the leading cause of cancer mortality in the United States, which establishes Ras genes as the most commonly mutated oncogenes in this malignancy. Mutations in the KRAS oncogene characterize more than 20% of all NSCLC malignancies and recent studies suggest that those tumors can be divided into KRAS dependent (KRAS-Dep) and KRAS independent (KRAS-Ind), according to their dependency on KRAS mutant for cell survival and proliferation. Although, the KRAS signaling pathway has been considerably studied using high throughput transcriptomic or proteomic technologies in isolation, the specific network of effector genes and proteins that drives carcinogenesis in NSCLC is still far from being understood. Integrative profiling with different omics technologies harbors the potential for characterizing the specific network of effector proteins associated with specific oncogenes. We developed a bioinformatics approach to reconstruct active and targetable networks associated with KRAS dependency in non-small cell lung cancer (NSCLC). By integrating matched global transcriptomics, proteomics and phosphoproteomics datasets, we stratified NSCLCs into those dependent on, or independent of, KRAS signaling by defining the S score that combines transcript, protein and phosphoprotein abundance in order to identify differential expressed proteins. We demonstrate that the S score outperforms naïve integration methods and improves the functional specificity of the proteins identified as differential expressed. Next, we reconstruct targetable network modules associated with KRAS dependency by employing the Prize Collecting Steiner Tree (PCST) algorithm to formulate the module reconstruction. This reconstruction identified three conspicuous modules centered on KRAS and MET, LCK and PAK1, and B-Catenin. We validated activation of those proteins in KRAS dependent cell lines and performed functional studies defining LCK as a critical gene to the cell proliferation in KRAS-dependent, but not KRAS-independent, NSCLCs. We further define a functional module of LCK signaling in NSCLC, in which LCK activates downstream proteins such as PAK1 in order to regulate cell death. Finally, we find evidence of LCK signaling in human NSCLCs tissues. These results are the first evidence that suggest LCK as potential targetable protein in the context of KRAS dependency. In summary, our integrative analysis establishes a novel mechanistic basis for KRAS dependency in NSCLC and nominates KRAS-LCK-PAK1 network module as a potentially druggable pathway in KRAS-dependent lung cancers. Citation Format: Alejandro O. Balbin, John Prensner, Anirban Sahu, Anastasia Yocum, Sunita Shankar, Mohan Dhanasekaran, Xuhong Cao, Alexey Nesvizhskii, Arul Chinnaiyan. Reconstructing targetable pathways in KRAS dependent lung cancer by integrating transcriptome, proteome and phosphoproteome. [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 5214. doi:10.1158/1538-7445.AM2013-5214

The Ewing's sarcoma family of tumors (ESFTs) are aggressive malignancies which frequently harbor EWS-FLI1 or EWS-ERG genomic fusions. Here, we demonstrate that these fusion products interact with, and depend on, poly (ADP-ribose) polymerase 1 (PARP1), a DNA damage response protein and transcriptional co-regulator. This interaction occurs in a DNA-independent manner. ESFT cell lines and xenografts are preferentially sensitive to PARP1 inhibition, and the addition of a PARP1 inhibitor to the second-line chemotherapeutic agent temozolamide resulted in complete response of all treated tumors in an EWS-FLI1 ESFT xenograft model. PARP inhibition blocked Ewing's cell line, but not control osteosarcoma or rhabdomyosarcoma cell line invasion, as well as the formation of lung metastasis in a xenograft model of ESFT. Mechanistically, the EWS-FLI1 and EWS-ERG fusions induce DNA damage, which is potentiated by PARP1 inhibition in ESFT cell lines. In a positive feedback loop, EWS-FLI1 fusions maintain the expression of PARP1 by direct regulation of the PARP1 promoter. This regulation is independently supported by a gene expression array data set of 20 ESFT patient tumors (Spearman r = 0.8165). Finally, because front-line therapy for this disease includes an intense regimen of five cytotoxic chemotherapy agents from which patients can quickly relapse with even more aggressive disease, we studied the effect of PARP inhibition in two Ewing's sarcoma cell line models derived after patient relapse. Importantly, these two cell lines maintained a preferential sensitivity to PARP1 inhibition despite their general chemoresistance. These findings suggest that targeting the EWS-FLI1: PARP1 interaction axis is a promising therapeutic strategy for the Ewing's sarcoma family of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4681. doi:1538-7445.AM2012-4681

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic histone methyltransferase subunit of the Polycomb Repressive Complex 2 (PRC2), that trimethylates histone H3 at lysine 27 (H3K27me3) resulting in the silencing of target genes. PRC2 plays a critical role in many basic cellular processes including cell proliferation, differentiation, early embryogenesis, and X chromosome inactivation. In cancer, EZH2 upregulation is implicated in metastasis and tumor aggressiveness of prostate and breast cancer and several other solid tumors. Recently our lab reported the genomic loss of miR-101 microRNA accompanying EZH2 overexpression in tumor cells. Here we identified several microRNAs that were downregulated by EZH2 and their levels were restored upon EZH2 depletion in cancer cell lines, and expression levels of these microRNAs were negatively correlated with EZH2 in human prostate tumors. Additionally, H3K27me3 modification was observed in the upstream regions of the miRNAs, suggesting a direct role for EZH2 in their regulation. Ectopic overexpression of the miRNAs suppressed cell proliferation, invasion, anchorage-independent growth, sphere formation and xenograft tumor growth of aggressive prostate and breast cancer cell lines. Finally, our investigations showed that the miRNAs also repress the expression of Polycomb Repressive Complex 1 (PRC1) members BMI1 and RING2, leading to a global decrease in the epigenetic marker, ubiquityl-H2A-K119 (uH2A) in cells, a key step in PRC1-mediated silencing. Our findings provide compelling argument for a regulatory axis joining PRC2 and PRC1 through miRNAs. This novel link between PRC2 and PRC1 indicates a coordinated mechanism by polycomb group proteins to promote an aggressive cancer phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2795. doi:10.1158/1538-7445.AM2011-2795

Long intervening non-coding RNAs (lincRNAs) have been implicated in diverse biological processes including p53 signaling and chromatin remodeling, but have not been thoroughly profiled in human cancers. Here, we have developed an approach for ab initio reconstruction of poly-A+ transcriptome sequencing (RNA-seq) data for the unbiased discovery of novel transcripts. To accomplish this, we developed AssemblyLine, a method that clusters and filters large collections of transcripts to produce a consensus transcriptome. To demonstrate AssemblyLine, we sequenced a cohort comprised of 81 prostatic tissues (20 benign, 47 localized tumors, and 14 metastases) and 21 prostatic cell lines using the Illumina Genome Analyzer II and generated 1.723 billion sequence fragments. We successfully aligned 1.42 billion reads with Tophat – a program capable of ab initio splice junction discovery – and then used Cufflinks to model sample-specific transcriptomes totaling 8.25 million transcripts. AssemblyLine condensed the 8.25 million original transcripts into 35,415 distinct transcriptional loci, of which 1,859 (5.2%) represented candidate lincRNAs that lacked genomic overlap with known gene annotations. These putative RNAs lacked robust open reading frames suggesting that the vast majority were non-coding. Further, they exhibited evolutionary conservation and were enriched with histone modifications supporting independent transcriptional start sites and active transcription. Together, these results add confidence to AssemblyLine's nomination process and suggest that these novel lincRNAs may be transcriptionally active in prostate cancer. We then selected 106 transcripts that were differentially expressed in localized prostate cancer when compared to benign adjacent tissue (False Discovery Rate < 0.05), and 15 transcripts with profound cancer outlier expression profiles for further study. These 121 Prostate Cancer Associated Transcripts (PCATs) accurately classified benign, localized, and metastatic prostate cancer tissues by unsupervised hierarchical clustering. Consistent with AssemblyLine's nominations, PCR-based experiments on selected transcripts in an independent tissue cohort showed high validation rates for the transcript structure and expression level predictions. Furthermore, in vitro studies of PCAT-1, a novel lincRNA observed in our dataset as highly upregulated in prostate cancer, revealed direct regulation by the histone methyltransferase EZH2. siRNA knockdown of PCAT-1 in LNCaP, a prostate cancer cell line, caused a 25-50% decrease in cell proliferation. Thus, this study establishes a paradigm for ab initio transcriptome annotation and discovery of novel lincRNAs in cancer tissues. Further, these results provide intriguing evidence that lincRNAs are aberrantly expressed and may play a role in prostate cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 929. doi:10.1158/1538-7445.AM2011-929

In cancer, the genome undergoes global DNA methylation changes, which contributes to disease progression. We mapped the global DNA methylation patterns in prostate tissues and cells (n=19) from fifty nanograms of genomic DNA. The methylated regions enriched by Methylplex technology, were subjected -Next Generation Sequencing (M-NGS). A total of 28 lanes of next generation sequencing yielded approximately 530 million sequence reads. The reads uniquely mapped to human genome were analyzed by H-Peak program to identify 1.8 million methylated regions from the 28 runs. While twenty percent of all CpG islands (CGIs) (68,508) were methylated in tissues, the promoter CGI methylation gradually increased from ∼12.6% in benign samples to 19.3% and 21.8% in localized and metastatic cancer tissues. However surprisingly, the total methylation events in intergenic/intronic regions between benign adjacent and cancer tissues were largely comparable. We found distinct patterns in promoter methylation around transcription start sites where methylation occurred directly on the CGIs, flanking regions and on CGI sparse promoters. Among the 6,691 methylated promoters in prostate tissues, 2481 differentially methylated regions (DMRs) were cancer specific and several previously studied targets were among them. A novel cancer specific DMR in WFDC2 promoter showed 77% methylation in cancer (17/22), 100% methylation in transformed prostate cell lines (6/6), none in the benign tissues (0/10) and normal PrEC cells. Integration of LNCaP DNA methylation and H3K4me3 data suggested a role for DNA methylation in alternate transcription start site utilization. Specifically, we detail the regulation of RASSF1, a gene previously reported to be silenced through DNA methylation in PCa and NDRG2. We show that variant-1 of RASSF1, but not variant-2 or -3, is specifically silenced in LNCaP through promoter methylation, and normal cells do not exhibit this phenomenon. Treatment with 5-Aza-2-deoxycytidine preferentially induced re-expression of variant-1 of RASSF1 in LNCaP, confirming transcriptional regulation of this isoform through DNA methylation. The methylated promoters lacking CGIs showed sparse H3K4me3 modification, interestingly methylated promoters containing CGIs showed a mutually exclusive H3K4me3/DNA methylation marks. Finally, we observed a difference in the methylation of LINE-1 elements between transcription factor ERG positive and negative cancers. The comprehensive methylome map presented here will further our understanding of epigenetic regulation of the prostate cancer genome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4840. doi:10.1158/1538-7445.AM2011-4840

High-throughput sequencing of polyA+ RNA (RNA-Seq) in human cancer shows remarkable potential to identify both novel disease-specific markers for clinical uses and uncharacterized aspects of tumor biology, particularly non-coding RNA (ncRNA) species. To illustrate this approach, we employed RNA-Seq on a cohort of 102 prostate tissues and cells lines. We found that aberrant expression profiles of novel tissue-specific ncRNAs distinguished benign, cancerous, and metastatic tumors, and we defined a core set of 121 novel ncRNAs whose dysregulation characterizes prostate cancer. Among these, a novel prostate-cancer specific ncRNA (termed PCAT-1) defined a subset of aggressive cancers with low expression of the epigenetic regulator EZH2, a component of the Polycomb Repressive Complex 2 (PRC2) commonly upregulated in metastatic cancers. In vitro chromatin immunoprecipitation, RNA immunoprecipitation, and drug treatment assays for core PRC2 genes indicated that the PRC2 complex directly binds and represses PCAT-1, and that PCAT-1 transcript reciprocally binds PRC2. By contrast, in vitro models with high levels of endogenous PCAT-1 transcript did not recapitulate PRC2-mediated repression, and in these cells siRNA-mediated knockdown of PCAT-1 showed a 25 – 50% decrease in cell proliferation. Using gene expression arrays, we determined that PCAT-1 contributes to the transcriptional regulation of genes in several key biological processes, including cell cycle. These data suggest that PCAT-1 exhibits two biological states: a PRC2-repressed state and an active state that promotes proliferation. Next, we showed that novel ncRNAs may serve a clinical purpose for the non-invasive detection and stratification of prostate cancer patients. We performed qPCR on patient urine samples (n=108) and found that a custom ncRNA expression signature, which includes PCAT-1, both diagnosed prostate cancer effectively and yielded prognostic information. Indeed, a high ncRNA expression signature value correlated with high-grade histology (Gleason score >=7 vs. Gleason score =6; p = 0.01). Taken together, the findings presented herein establish the utility of RNA-Seq to comprehensively identify unannotated ncRNAs, such as PCAT-1, implicated in cancer. Our data suggest that PCAT-1 promotes cell proliferation, that in its inactive state PCAT-1 is mechanistically repressed by PRC2, and that PCAT-1 may serve as a candidate biomarker for non-invasive clinical tests. We further speculate that applying these methodologies to other diseases may reveal key aspects of disease biology and clinically important biomarkers, particularly for diseases that currently lack good non-invasive tests in fluids such as blood serum or urine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4707. doi:10.1158/1538-7445.AM2011-4707

With a flood of cancer genome sequences expected soon, distinguishing ‘driver’ from ‘passenger’ mutations will be an important task. To discover the key characteristics of driver gene fusions in cancer, we performed a systematic analysis of known fusion genes using a compendium of ‘molecular concepts', inclusive of functional domains, pathways, gene ontologies and molecular interactions. Although analysis of domain architectures and shared pathways was less informative, we found that cancer-related fusion genes tend to share common gene ontologies or engage distinct interaction networks. We hypothesized that such ‘signatures’ of molecular concepts may be used to distinguish biologically meaningful gene fusions underlying cancer, similar to signature genes defining certain phenotypes. We therefore designed an algorithm, called concept signature score (ConSig score), to quantitatively rank human genes underlying cancer by the strength of their association with the molecular concepts characteristics of cancer genes. To integrate the use of high-throughput genomic data, we characterized the chromosomal imbalances associated with gene fusions, finding that recurrent gene fusions exhibit distinctive patterns of copy number alteration corresponding to differential portions of fusion partners. We named this pattern as the fusion breakpoint principle, and confirmed this principle by large-scale meta-analysis of recurrent gene fusions using high-resolution array CGH/SNP datasets. Next, we applied the ConSig technology to paired-end transcriptome sequencing data to benchmark fusion candidates, which were then assessed for chromosomal aberrations complying with the fusion breakpoint principle by integrating high-quality copy number data. We found that the ConSig score was able to identify the known EML4-ALK fusion as the top-ranked candidate in the H2228 lung cancer cell line, and, in addition, we found further evidence of a R3HDM2-NFE2 fusion in H1792 cell line. We show that the R3HDM2-NFE2 fusion, which results in overexpression of wild-type NFE2, promotes cell proliferation and invasion. Moreover, through analysis of SNP arrays and lung TMAs, we find that chromosomal rearrangements at the NFE2 locus are recurrent in a small subset of patient tumors, suggesting that NFE2 may contribute to a new class of lung cancer molecular biology. Together, the ConSig technology suggests the functional importance of putative fusions in cancer, whereas the breakpoint principle helps interpret large-scale cancer genomic data sets to explore potential recurrence. Thus the methodology described here can filter the large number of fusion candidates generated by paired-end sequencing data and preferentially identify driver gene fusions in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2214.

Genomic regions undergoing frequent alteration in human cancers often point to genes that play causal roles in oncogenesis. We have performed high-resolution analyses of copy-number alterations from 3131 cancer specimens across multiple histological types. We identified 158 regions of that are significantly altered by focal copy-number changes. Among these, 122 regions cannot be explained by the presence of a known cancer gene. Several gene families are enriched among these regions, including the BCL2 family of apoptosis regulators and the NF-κB pathway. Cell lines with amplifications of the anti-apoptotic genes MCL1 and BCL2L1 depend upon these genes for survival. Finally, we find that the majority of copy-number changes identified in individual cancer types are present across multiple cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5759.