Kalpana Kannan

Recurrent chimeric RNAs enriched in human prostate cancer identified by deep sequencing

Transcription-induced chimeric RNAs, possessing sequences from different genes, are expected to increase the proteomic diversity through chimeric proteins or altered regulation. Despite their importance, few studies have focused on chimeric RNAs especially regarding their presence/roles in human cancers. By deep sequencing the transcriptome of 20 human prostate cancer and 10 matched benign prostate tissues, we obtained 1.3 billion sequence reads, which led to the identification of 2,369 chimeric RNA candidates. Chimeric RNAs occurred in significantly higher frequency in cancer than in matched benign samples. Experimental investigation of a selected 46 set led to the confirmation of 32 chimeric RNAs, of which 27 were highly recurrent and previously undescribed in prostate cancer. Importantly, a subset of these chimeras was present in prostate cancer cell lines, but not detectable in primary human prostate epithelium cells, implying their associations with cancer. These chimeras contain discernable 5′ and 3′ splice sites at the RNA junction, indicating that their formation is mediated by splicing. Their presence is also largely independent of the expression of parental genes, suggesting that other factors are involved in their production and regulation. One chimera, TMEM79-SMG5, is highly differentially expressed in human cancer samples and therefore a potential biomarker. The prevalence of chimeric RNAs may allow the limited number of human genes to encode a substantially larger number of RNAs and proteins, forming an additional layer of cellular complexity. Together, our results suggest that chimeric RNAs are widespread, and increased chimeric RNA events could represent a unique class of molecular alteration in cancer.

Two RNA subunits and POT1a are components of Arabidopsis telomerase.

Telomerase is a ribonucleoprotein (RNP) reverse transcriptase whose essential RNA subunit (TER) functions as a template for telomere repeat synthesis. Here we report the identification of two divergent TER moieties in the flowering plant Arabidopsis thaliana. Although both TER1 and TER2 copurify with telomerase activity and serve as templates for telomerase in vitro, depletion of TER1, but not TER2, leads to decreased telomerase activity and progressive telomere shortening in vivo. Moreover, mutation of the templating domain in TER1 results in the incorporation of mutant telomere repeats on chromosome ends. Thus, TER1 provides the major template for telomerase in vivo. We also show that POT1a binds TER1 with a Kd of 2 × 10-7 M and the two components assemble into an enzymatically active RNP in vivo. In contrast, TER1-POT1b and TER2-POT1a associations were not observed. In other organisms POT1 proteins bind telomeric DNA and provide chromosome end protection. We propose that duplication of TER and POT1 in Arabidopsis fueled the evolution of novel protein–nucleic acid interactions and the migration of POT1 from the telomere to the telomerase RNP.

Dyskerin Is a Component of the Arabidopsis Telomerase RNP Required for Telomere Maintenance

ABSTRACT Dyskerin binds the H/ACA box of human telomerase RNA and is a core telomerase subunit required for RNP biogenesis and enzyme function in vivo. Missense mutations in dyskerin result in dyskeratosis congenita, a complex syndrome characterized by bone marrow failure, telomerase enzyme deficiency, and progressive telomere shortening. Here we demonstrate that dyskerin also contributes to telomere maintenance in Arabidopsis thaliana. We report that both AtNAP57, the Arabidopsis dyskerin homolog, and AtTERT, the telomerase catalytic subunit, accumulate in the plant nucleolus, and AtNAP57 associates with active telomerase RNP particles in an RNA-dependent manner. Furthermore, AtNAP57 interacts in vitro with AtPOT1a, a novel component of Arabidopsis telomerase. Although a null mutation in AtNAP57 is lethal, AtNAP57, like AtTERT, is not haploinsufficient for telomere maintenance in Arabidopsis. However, introduction of an AtNAP57 allele containing a T66A mutation decreased telomerase activity in vitro, disrupted telomere length regulation on individual chromosome ends in vivo, and established a new, shorter telomere length set point. These results imply that T66A NAP57 behaves as a dominant-negative inhibitor of telomerase. We conclude that dyskerin is a conserved component of the telomerase RNP complex in higher eukaryotes that is required for maximal enzyme activity in vivo.

Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma

Significance High-grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer and is typically detected only at advanced stages due to lack of effective early screening tools. Fusion genes are among the most cancer-specific signatures known and, when highly recurrent, they have the potential to serve as screening tools. Here we identified BCAM-AKT2 as a cancer-specific fusion gene present in 7% of HGSC tumors, a significant frequency in this highly heterogeneous disease. This fusion results in an aberrant kinase whose constant activity contributes to cancer formation. Thus, the BCAM-AKT2 fusion gene could be important for understanding and identifying clinically relevant subtypes of HGSC, and could be a novel therapeutic target for developing small-molecule drugs. High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. Excessive genomic rearrangements, which are expected to create fusion oncogenes, are the hallmark of this cancer. Here we report a cancer-specific gene fusion between BCAM, a membrane adhesion molecule, and AKT2, a key kinase in the PI3K signaling pathway. This fusion is present in 7% of the 60 patient cancers tested, a significant frequency considering the highly heterogeneous nature of this malignancy. Further, we provide direct evidence that BCAM-AKT2 is translated into an in-frame fusion protein in the patient’s tumor. The resulting AKT2 fusion kinase is membrane-associated, constitutively phosphorylated, and activated as a functional kinase in cells. Unlike endogenous AKT2, whose activity is tightly regulated by external stimuli, BCAM-AKT2 escapes the regulation from external stimuli. Moreover, a BCAM-AKT2 fusion gene generated via chromosomal translocation using the CRISPR/Cas9 system leads to focus formation in both OVCAR8 and HEK-293T cell lines, suggesting that BCAM-AKT2 is oncogenic. Together, the results indicate that BCAM-AKT2 expression is a new mechanism of AKT2 kinase activation in HGSC. BCAM-AKT2 is the only fusion gene in HGSC that is proven to translate an aberrant yet functional kinase fusion protein with oncogenic properties. This recurrent genomic alteration is a potential therapeutic target and marker of a clinically relevant subtype for tailored therapy of HGSC.

CDKN2D-WDFY2 Is a Cancer-Specific Fusion Gene Recurrent in High-Grade Serous Ovarian Carcinoma

Ovarian cancer is the fifth leading cause of cancer death in women. Almost 70% of ovarian cancer deaths are due to the high-grade serous subtype, which is typically detected only after it has metastasized. Characterization of high-grade serous cancer is further complicated by the significant heterogeneity and genome instability displayed by this cancer. Other than mutations in TP53, which is common to many cancers, highly recurrent recombinant events specific to this cancer have yet to be identified. Using high-throughput transcriptome sequencing of seven patient samples combined with experimental validation at DNA, RNA and protein levels, we identified a cancer-specific and inter-chromosomal fusion gene CDKN2D-WDFY2 that occurs at a frequency of 20% among sixty high-grade serous cancer samples but is absent in non-cancerous ovary and fallopian tube samples. This is the most frequent recombinant event identified so far in high-grade serous cancer implying a major cellular lineage in this highly heterogeneous cancer. In addition, the same fusion transcript was also detected in OV-90, an established high-grade serous type cell line. The genomic breakpoint was identified in intron 1 of CDKN2D and intron 2 of WDFY2 in patient tumor, providing direct evidence that this is a fusion gene. The parental gene, CDKN2D, is a cell-cycle modulator that is also involved in DNA repair, while WDFY2 is known to modulate AKT interactions with its substrates. Transfection of cloned fusion construct led to loss of wildtype CDKN2D and wildtype WDFY2 protein expression, and a gain of a short WDFY2 protein isoform that is presumably under the control of the CDKN2D promoter. The expression of short WDFY2 protein in transfected cells appears to alter the PI3K/AKT pathway that is known to play a role in oncogenesis. CDKN2D-WDFY2 fusion could be an important molecular signature for understanding and classifying sub-lineages among heterogeneous high-grade serous ovarian carcinomas.

Identification of cancer fusion drivers using network fusion centrality

SUMMARY Gene fusions are being discovered at an increasing rate using massively parallel sequencing technologies. Prioritization of cancer fusion drivers for validation cannot be performed using traditional single-gene based methods because fusions involve portions of two partner genes. To address this problem, we propose a novel network analysis method called fusion centrality that is specifically tailored for prioritizing gene fusions. We first propose a domain-based fusion model built on the theory of exon/domain shuffling. The model leads to a hypothesis that a fusion is more likely to be an oncogenic driver if its partner genes act like hubs in a network because the fusion mutation can deregulate normal functions of many other genes and their pathways. The hypothesis is supported by the observation that for most known cancer fusion genes, at least one of the fusion partners appears to be a hub in a network, and even for many fusions both partners appear to be hubs. Based on this model, we construct fusion centrality, a multi-gene-based network metric, and use it to score fusion drivers. We show that the fusion centrality outperforms other single gene-based methods. Specifically, the method successfully predicts most of 38 newly discovered fusions that had validated oncogenic importance. To our best knowledge, this is the first network-based approach for identifying fusion drivers. AVAILABILITY Matlab code implementing the fusion centrality method is available upon request from the corresponding authors.

Aberrant MUC1-TRIM46-KRTCAP2 Chimeric RNAs in High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. By analyzing the mRNA-seq reads from The Cancer Genome Atlas (TCGA), we uncovered a novel cancer-enriched chimeric RNA as the result of splicing between MUC1, a highly glycosylated transmembrane mucin, TRIM46, a tripartite motif containing protein, and KRTCAP2, a keratinocyte associated protein. Experimental analyses by RT-PCR (reverse transcription PCR) and Sanger sequencing using an in-house cohort of 59 HGSC patient tumors revealed a total of six MUC1-TRIM46-KRTCAP2 isoforms joined by different annotated splice sites between these genes. These chimeric isoforms are not detected in non-cancerous ovaries, yet are present in three out of every four HGSC patient tumors, a significant frequency given the exceedingly heterogeneous nature of this disease. Transfection of the cDNA of MUC1-TRIM46-KRTCAP2 isoforms in mammalian cells led to the translation of mutant MUC1 fusion proteins that are unglycosylated and cytoplasmically localized as opposed to the cell membrane, a feature resembling the tumor-associated MUC1. Because the parental MUC1 is overexpressed in 90% of HGSC tumors and has been proposed as a clinical biomarker and therapeutic target, the chimeric MUC1-TRIM46-KRTCAP2 isoforms identified in this report could represent significantly better MUC1 variants for the same clinical utilities.

Abstract MIP-076: A RECURRENT BCAM-AKT2 FUSION GENE LEADS TO A CONSTITUTIVELY ACTIVATED AKT2 FUSION KINASE IN HIGH-GRADE SEROUS OVARIAN CARCINOMA

High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. Excessive genomic rearrangements, which are expected to create fusion oncogenes, are the hallmark of this cancer. Using high-throughput RNAseq, we identify a cancer-specific gene fusion between BCAM, a membrane adhesion molecule, and AKT2, a key kinase in the PI3K signaling pathway. This fusion is present in 7% of the 60 patient cancers tested, a significant frequency considering the highly heterogeneous nature of this malignancy. Further, we provide direct evidence that BCAM-AKT2 is translated into an in-frame fusion protein in the patient9s tumor. The resulting AKT2 fusion kinase is membrane-associated, constitutively phosphorylated, and activated as a functional kinase in cells. Unlike endogenous AKT2, whose activity is tightly regulated by external stimuli, BCAM-AKT2 escapes the regulation from external stimuli. Moreover, a BCAM-AKT2 fusion gene generated via chromosomal translocation using the CRISPR/Cas9 system leads to focus formation in both OVCAR8 and HEK-293T cell lines, suggesting that BCAM-AKT2 is oncogenic. Together, the results indicate that BCAM-AKT2 expression is a new mechanism of AKT2 kinase activation in HGSC. BCAM-AKT2 is the only fusion gene in HGSC that is proven to translate an aberrant yet functional kinase fusion protein with oncogenic properties. This recurrent genomic alteration is a potential therapeutic target and marker of a clinically relevant subtype for tailored therapy of HGSC. Citation Format: Kalpana Kannan, Cristian Coarfa, Pei-Wen Chao, Liming Luo, Yan Wang, Amy E. Brinegar, Shannon M. Hawkins, Aleksandar Milosavljevic, Martin M. Matzuk, and Laising Yen. A RECURRENT BCAM-AKT2 FUSION GENE LEADS TO A CONSTITUTIVELY ACTIVATED AKT2 FUSION KINASE IN HIGH-GRADE SEROUS OVARIAN CARCINOMA [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-076.

Abstract 4973: Recurrent chimeric RNAs enriched in human prostate cancer identified by deep-sequencing

Chimeric transcripts unrelated to chromosomal rearrangement are primarily generated by two transcription-induced mechanisms: read-through/splicing and trans-splicing. Both mechanisms result in fused transcripts that possess sequences from both genes. Although the exact cause and regulation of chimeric transcripts are unknown, chimeric RNAs, like fusion genes, are expected to increase the proteomic diversity in cells through chimeric proteins or altered regulation of participating mRNAs. Despite its importance, few studies have reported on chimeric RNAs, especially regarding their presence and roles in human cancers. In this study, we took advantage of the analytical power of paired-end high throughput sequencing to characterize chimeric transcripts enriched in human prostate cancer. By deep-sequencing the transcribed mRNA (transcriptome) of 20 clinical prostate cancer tissues, in addition to 10 matched benign prostate tissues from the same patients, we obtained greater than 500 million useful sequence reads. This strategy combined with stringent bioinformatic criteria enabled the identification of 27 novel and highly recurrent chimeric RNAs that have never been described before. The novel chimeric transcripts were experimentally validated to be present in human prostate cancer samples in a recurrent manner. Further experimental analysis identify several chimeric transcripts are present in the human prostate cancer cell lines, but not in primary human prostate epithelium cells, implying their associations with cancer mechanisms. Importantly, one of these chimeras appeared to be highly cancer-enriched, as it is confirmed to be expressed at significantly higher levels in human prostate cancers but present at very low levels in non-cancer prostates. Our results suggest that chimeric RNAs are more abundant than previously estimated. The RNA fusion junctions of these novel recurrent chimeras, as determined experimentally, suggest that many of them would generate altered or aberrant protein sequences, potentially leading to abnormal functions in cancer. 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 4973. doi:10.1158/1538-7445.AM2011-4973