bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B21: A long noncoding RNA LINC00467 regulates autophagy in cancer
Authors: Pridham Kevin J. , Guo Sujuan , Sheng Zhi
Published: May 2016

Autophagy is a degradation pathway utilized by cells under stressful conditions such as nutrient deprivation or drug treatment to support extended survival. During this process, cellular components are broken down to macromolecules to be re-assimilated for consumption to sustain cell survival. With autophagy serving an important role between life and death for a cell, it has implications in not only maintaining homeostasis of normal cells but also the growth and survival of malignant cancer cells. Long noncoding RNAs (LncRNAs) are RNA transcripts longer than 200 nucleotides with no protein-coding potential and play an important role in the regulation of gene expression. Albeit certain LncRNAs are overexpressed in cancer with a strong correlation with cancer risk and prognosis, the relationship of LncRNAs and autophagy has not yet been extensively explored. Our previous work has identified LINC00467 (long intergenic noncoding RNA 00467 or C1orf97) as a potential autophagy regulator in chronic myeloid leukemia (CML) cells. We further hypothesized that LINC00467 is a common autophagy regulator in cancer. To test this hypothesis, we analyzed the gene expression pattern of this LncRNA using RT-PCR, monitored its expression in cancers by querying different gene expression databases, measured expression by RT-PCR in different cancer cell lines, and measured autophagy in cancer cells upon depletion of LINC00467. Our results show that (1) LINC00467 is expressed as two transcripts (733 and 797 nucleotides); (2) LINC00467 is ubiquitously expressed in different types of cancer; and (3) LINC00467 depletion activates autophagy in multiple cancer cell lines. This work will illuminate an otherwise unknown and new autophagy-regulatory pathway and will add a new dimension to our knowledge of the regulation of cancer cell survival and death. Of particular importance, this new autophagy regulatory pathway may provide new and effective therapeutic targets.

Citation Format: Kevin J. Pridham, Sujuan Guo, Zhi Sheng. A long noncoding RNA LINC00467 regulates autophagy in cancer. [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 B21.
Abstract B31: Bladder cancer patient urinary exosomes and tumors contain long noncoding RNA that may serve as therapeutic targets and biomarkers
Authors: Berrondo Claudia , Flax Jonathan , Siebert Aisha , Kucherov Victor , Rosenberg Alex , Fucile Christopher , Beckham Carla
Published: May 2016

While long non-coding RNA (lncRNA) play important roles in tumor biology including bladder cancer (BC), no previous studies have shown that lncRNAs in BC patient urinary exosomes (UEs) can function as biomarkers of disease. Exosomes are 30-150nm secreted membrane-bound vesicles that contain active biomolecules like: messenger(m)RNA, micro(mi)RNA, lncRNA and proteins generated from their cells of origin. In this study, we identified a panel of lncRNAs and mRNAs enriched in the UEs and tumors of BC patients, including the well-characterized lncRNA, HOTAIR. In order to elucidate if HOTAIR has a functional role in BC, we knocked it down in BC cell lines. Loss of HOTAIR resulted in reduced expression of known HOTAIR epithelial-to-mesenchyme transition (EMT) target genes, as well as reduced migration, and invasion using trans-well and 3-D culture assays. These data suggest that HOTAIR may serve as both a biomarker in UEs and as a potential therapeutic target. Critically, we RNA-Sequenced primary tumors, distal normal tissue (DNT) and UEs of BC patients and UEs from healthy volunteers (HV) to identify two novel lncRNAs, LINC00477 and LINC00940 enriched in BC patients' UEs and tumors. These candidate lncRNAs were confirmed with quantitative real time (qRT-PCR). Taken together, UE lncRNA content reflect the lncRNA content of BC tumors and may serve as biomarkers of disease. Further investigation is needed to validate the use of the lncRNAs presented in this study as biomarkers and putative therapeutic targets in relevant patient populations.

Citation Format: Claudia Berrondo, Jonathan Flax, Aisha Siebert, Victor Kucherov, Alex Rosenberg, Christopher Fucile, Carla Beckham. Bladder cancer patient urinary exosomes and tumors contain long noncoding RNA that may serve as therapeutic targets and biomarkers. [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 B31.
Abstract B38: Cancer-related circulating long noncoding RNAs in serous extracellular vesicles: Their characterization and potential application
Authors: Lei Dong , Wanrun Lin , Peng Qi , Midie Xu , Xiang Du
Published: May 2016

Background: Circulating nucleic acids (CNAs) are extracellular nucleic acids found in cell-free sera, plasma and other bodily fluids of healthy subjects and cancer patients. We have previously demonstrated that human serum or plasma contains microRNAs (miRNAs) and long non-coding RNA (lncRNAs) that are significantly up-regulated or down-regulated in various types of cancer and are of good diagnostic value for screening. The mechanisms underlying the stability of serum RNAs are unclear; lncRNAs may be protected by extracellular vesicles (EV) including apoptotic body (AB), microvesicle (MV) and exosome (EXO), as for circulating miRNAs. In order to find optimal method to evaluate the potential utility of circulating lncRNAs for cancer diagnosis or prognosis, more exploration should be undertook for the distribution of circulating lncRNAs.

Results: The shapes and sizes of three subgroups of extracellular vesicles, including AB, MV and EXO, were evaluated. NanoSight particle tracking analysis and transmission electron microscopy (TEM) showed the MVs, pelleted at 12, 000×g, were with the size range of 75-465 nm; and EXOs, pelleted at 120,000×g, with the size range of 45-205 nm. In serous vesicles of both colorectal cancer and healthy subjects, 17 of the 39 cancer-related lncRNAs were detected, including TDRG1, MEG3, DSCAM-AS1, 7SK, MIR31HG, CBR3-AS1, TUG1, BCAR4, EPB41L4A, PCA3, FAS-AS1, SUMO1P3, uc338, PRNCR1, PTENP1 and CUDR. The amount of most lncRNAs were higher in EXO than those in AB and MV. In addition, AB contains higher amount of most lncRNAs than MV.

Results: The shapes and sizes of three subgroups of extracellular vesicles, including AB, MV and EXO, were evaluated. NanoSight particle tracking analysis and transmission electron microscopy (TEM) showed the MVs, pelleted at 12, 000×g, were with the size range of 75-465 nm; and EXOs, pelleted at 120,000×g, with the size range of 45-205 nm. In sera of both colorectal cancer and healthy subjects, 16 out of the 39 cancer-related lncRNAs were detected. The amount of most lncRNAs were higher in EXO than those in AB and MV. In addition, AB contains higher amount of most lncRNAs than MV.

Conclusions: Among three types of vesicles in sera, EXOs were the richest reservoir for almost all measured lncRNAs, while the MVs contained the least amount of lncRNAs. EXOs, as their intercellular origin, have reasons to contain higher amount of lncRNAs among serous vesicles and seem to be the most promising research materials in the field of circulating lncRNA.

Citation Format: Dong Lei, Lin Wanrun, Qi Peng, Xu Midie, Du Xiang. Cancer-related circulating long noncoding RNAs in serous extracellular vesicles: Their characterization and potential application. [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 B38.
Abstract IA01: Divergent transcription and long noncoding RNA in cancer
Authors: Sharp Phillip A.
Published: May 2016

The processes of divergent transcription and RNA splicing control the synthesis of many long noncoding RNAs in mammalian cells. These RNAs could facilitate the binding of transcription factors and other complexes thus enhancing transcription of genes. Their existence suggests that nuclear RNA forms connectivity for location of regulatory complexes in cells. These probably change during the transformation process, perhaps accounting for some of the concordant changes in gene expression.

Citation Format: Phillip A. Sharp. Divergent transcription and long noncoding RNA in cancer. [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 IA01.
Abstract IA10: microRNAs and lncRNAs in mammalian physiology and cancer
Authors: Mendell Joshua T.
Published: May 2016

Gain- and loss-of-function of noncoding RNAs can potently influence cellular behavior in normal physiologic states and in diseases such as cancer. In particular, dysregulation of microRNA (miRNA) expression has been clearly shown to result in dramatic phenotypic consequences. We previously demonstrated extensive control of miRNA expression by well-characterized oncogenic and tumor suppressor networks including the MYC, KRAS, and p53 pathways. More recently, we have uncovered germline and somatic mutations in core miRNA processing components such as DROSHA and DICER in tumors. The analysis of novel mouse models with gain- and loss- of miRNA function has revealed potent, and often unexpected, consequences of dysregulated miRNA expression and activity in vivo. More recently, we have applied these same approaches to dissect novel mechanisms of long noncoding RNA (lncRNA) regulation and function in mammalian physiology and cancer. I will present our latest results related to the regulation and function of noncoding RNAs and how these findings may be exploited for the development of novel therapeutic approaches.

Citation Format: Joshua T. Mendell. microRNAs and lncRNAs in mammalian physiology and cancer. [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 IA10.
Abstract IA14: Noncoding RNAs of viral and cellular origin: Links to oncogenesis
Authors: Steitz Joan A. , Lee Nara , Moss Walter N. , Yario Therese A. , Vilborg Anna , Passarelli Maria C. , Tycowski Kazimierz T.
Published: May 2016

EBER2 is a highly abundant nuclear noncoding RNA expressed by Epstein-Barr virus (EBV). Probing its possible chromatin localization by Capture Hybridization Analysis of RNA Targets (CHART) revealed EBER2's presence at the terminal repeats (TRs) of the latent EBV genome, overlapping previously identified binding sites for the important B-cell transcription factor PAX5. EBER2 interacts with and is required for PAX5 localization to the TRs. Knockdown of EBER2 or PAX5 decreases EBV lytic replication, underscoring the essential role of the TRs in viral replication, which is known to be linked to the onset of EBV-associated tumors. Recruitment of the EBER2-PAX5 complex is mediated by base pairing between EBER2 and nascent transcripts from the TR locus. The interaction is evolutionarily conserved in the related primate herpesvirus CeHV15 despite great sequence divergence in both EBER2 and the TRs. Using base pairing with nascent transcripts to guide an interacting transcription factor to its DNA target site in chromatin is a previously undescribed function for a trans-acting noncoding RNA.

We have uncovered a novel transcript type derived from human protein-coding genes that is represented by thousands of examples genome-wide. These RNAs, which we call DoGs for downstream of gene containing transcripts, are inducible by osmotic stress through an IP3 receptor signaling-dependent pathway, indicating active regulation. DoGs possess long non-coding regions (often >45 kb) and remain chromatin bound. Their levels are increased by decreased termination of the upstream transcript, a previously undescribed mechanism for rapid transcript induction. Relative depletion of polyA signals in DoG regions correlates with increased levels of DoGs after osmotic stress. DoGs may play a role in reinforcing the nuclear scaffold after stress.

1. Lee, N., Moss, W., Yario, T., Steitz, J.A. (2015). An EBV noncoding RNA binds nascent RNA to drive host PAX5 to viral DNA. Cell 160, 607-618.

2. Vilborg, A., Passarelli, M.C., Yario, T.A., Tycowski, K.T. and Steitz, J.A. (2014). Widespread inducible transcription downstream of human genes. Mol. Cell. 59, 449-461.

Citation Format: Joan A. Steitz, Nara Lee, Walter N. Moss, Therese A. Yario, Anna Vilborg, Maria C. Passarelli, Kazimierz T. Tycowski. Noncoding RNAs of viral and cellular origin: Links to oncogenesis. [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 IA14.
Abstract IA23: Targeting nuclear noncoding RNAs
Authors: Corey David
Published: May 2016

RNA interference (RNAi) is well known as a mechanism for controlling mammalian mRNA translation in the cytoplasm, but what would be the consequences if it also functioned in cell nuclei? Although RNAi has also been found in nuclei of plants, yeast, and other organisms, there has been relatively little progress towards understanding the potential involvement of mammalian RNAi factors in nuclear processes including transcription and splicing. When RNAi factors bind small RNAs, they form ribonucleoprotein complexes that can be selective for target sequences within different classes of nuclear RNA substrates. The versatility of nuclear RNAi may supply a previously underappreciated layer of regulation to transcription, splicing, and other nuclear processes. This talk focuses on recent work targeting noncoding RNA at the frataxin and C9orf72 loci as examples for how nuclear RNAi can be used to control gene expression.

Citation Format: David Corey. Targeting nuclear noncoding RNAs. [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 IA23.
Abstract IA25: Regulatory control of lincRNA function through formation of complex RNA structural motifs
Authors: Chillon Isabel , Somarowthu Srinivas , Liu Fei , Pyle Anna Marie
Published: May 2016

While the regulatory role of lincRNAs is becoming increasingly appreciated, the functional role of RNA structures within these molecules is not well understood. We have embarked on a study to understand the folding of lincRNA molecules, and to identify functional structural modules within large RNA transcripts. Using approaches to promote the native folding of large RNA molecules, we have been purifying lincRNAs, mapping their structures with chemical probes, and then using this information to construct structural maps of lincRNA architecture. This work is complemented by phylogenetic and genetic approaches for exploring the functional relevance of RNA structures identified in our study. In this way, we have mapped the structure of lincRNAs such as HOTAIR and lincRNA p21, identifying novel structural motifs, enabling evolutionary comparisons and facilitating targeted mutational studies of lincRNA function.

Citation Format: Isabel Chillon, Srinivas Somarowthu, Fei Liu, Anna Marie Pyle. Regulatory control of lincRNA function through formation of complex RNA structural motifs. [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 IA25.
Abstract PR01: Strand-specific in vivo screen of cancer-associated miRNAs unveils key drivers and oncogenic targets
Authors: Ge Yejing , Zhang Liang , Nikolova Maria , Reva Boris , Fuchs Elaine
Published: May 2016

MiRNAs are evolutionarily conserved small non-coding RNAs that orchestrate a wide spectrum of physiological and pathological processes in stem cells (SCs) and human cancers. Intimately wired into oncogenic and tumor suppressive networks, miRs are valuable as not only diagnostic biomarkers but also therapeutic targets. Squamous cell carcinomas (SCCs) are among the most life-threatening human cancers world-wide. Using skin as paradigm, we describe the first panoramic view of miRs during the development, homeostasis and malignant transformation of skin epithelium, whose SCs are the target of most common SCCs. Focusing on abundant and enriched miRs in SCC-SCs versus normal skin SCs, we devised lentiviral miR expression platform and conducted pooled in vivo functional screens for oncomiRs in mice. Deep sequencing of genomic DNA from basal progenitors of tumors or unaffected skin epithelium revealed preferential enrichment of 29/169 miRs within the tumors. We followed up with the top hits and showed candidate miRs are indeed aberrantly induced in both mouse and human SCCs. After functionally documenting their tumor-promoting roles in mouse SCCs in vivo and human xenografts, we applied CRISPR technology to ablate the miR loci in cells derived from these SCCs, and then used lentiviral delivery methods to carry out specific miR rescues of the tumorigenic phenotype. To look for candidate targets of newly identified oncomiRs, we analyzed TCGA database that are inversely correlated with our candidate miR as well as genes that are de-repressed upon miR deletion. We discovered potent tumor suppressors whose knockdown rescued the deficit in tumor formation from oncomiR-deficient cells and whose overexpression reversed miR-driven tumorigenesis. Furthermore, we demonstrated CRISPR-mediated deletion of the endogenous miRNA binding site renders the target cells no longer sensitive to miRNA regulation. Finally, we pinpointed the cellular mechanism of how miR-target regulates SCC progression. Empowered by mouse genetics and high throughput approaches, our study has unveiled a rich set of putative miRs that drive skin SCC malignancy, and discovered their oncogenic targets. These findings shed new light on increasingly important oncomiR loci and its action on a devastating cancer.

This abstract is also presented as Poster A02.

Citation Format: Yejing Ge, Liang Zhang, Maria Nikolova, Boris Reva, Elaine Fuchs. Strand-specific in vivo screen of cancer-associated miRNAs unveils key drivers and oncogenic targets. [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 PR01.
Abstract PR02: The diverse roles of long noncoding RNAs in the p53 tumor suppressor pathway
Authors: Bendor Jordan , Zamudio Jesse , Jacks Tyler , Dimitrova Nadva
Published: May 2016

The p53 tumor suppressor is a transcription factor, which plays a central role in the cellular response to DNA damage and oncogenic stress. Using genetic approaches in the mouse as well as antisense technology, we have previously shown that the p53-regulated lncRNA, lincRNA-p21 activates the expression of its neighboring gene, the critical G1/S checkpoint mediator, p21. Mechanistically, we found that lincRNA-p21 localizes at its site of transcription, where it acts in cis to stabilize the binding of p53 and its transcriptional co-activator hnRNP-K at the promoter of p21. Importantly, using CRISPR-based approaches to modify the endogenous lincRNA-p21 locus in primary human fibroblasts, we show that the role of lincRNA-p21 in promoting p21 levels is conserved between mouse and human.

To expand our understanding of the contribution of lncRNAs to the p53 tumor suppressor pathway, we have performed p53 ChIPseq and RNAseq under conditions of oncogenic stress and identified multiple previously uncharacterized lncRNAs that are regulated in a p53-dependent manner. One of them, lincRNA-Dhx15 is a novel lncRNA expressed from a bidirectional promoter shared with the RNA helicase Dhx15. LincRNA-Dhx15 is a direct target of p53 by virtue of multiple p53 response elements located in its first intron and appears to be preferentially expressed under conditions of senescence. We have generated a conditional knockout of lincRNA-Dhx15 in the mouse and found that absence of lincRNA-Dhx15 leads to impaired induction of senescence following prolonged exposure to DNA damage. Moreover, by gene expression profiling, we observed a downregulation of a subset of p53 target genes, suggesting that lincRNA-Dhx15 plays a global role in the p53 pathway. Surprisingly, we found that lincRNA-Dhx15 is exported to the cytoplasm and appears to promote the translational of p53 itself. The mechanistic basis for lincRNA-Dhx15 function is under active investigation. Altogether, these studies reveal that p53-regulated lncRNAs act through diverse mechanisms to add important layers of regulation to the p53 transcriptional network.

Citation Format: Jordan Bendor, Jesse Zamudio, Tyler Jacks, Nadva Dimitrova. The diverse roles of long noncoding RNAs in the p53 tumor suppressor pathway. [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 PR02.
Abstract PR05: Dicer1 deletion: Sufficient for angiosarcoma development and a haploinsufficient tumor suppressor in rhabdomyosarcoma
Authors: Hanna Jason A. , Garcia Matthew R. , Rehg Jerold , Hatley Mark E.
Published: May 2016

In this study, the role of Dicer and microRNAs (miRNAs) is investigated in pediatric and adult soft tissue sarcomas with the hypothesis that understanding miRNAs and their biogenesis may provide biological insights into the pathogenesis of these diseases and identify novel therapeutic targets. In several mouse models of cancer, Dicer1 is thought to function as a haploinsufficient tumor suppressor where loss of a single allele promotes tumorigenesis yet loss of both alleles is detrimental to tumor formation. Heterozygous germline mutations in DICER1 in humans is associated with rare malignant and benign neoplasias including embryonal rhabdomyosarcoma (ERMS).

With DICER1 syndrome mutations found in ERMS, a pediatric soft tissue sarcoma resembling skeletal muscle precursors that fail to differentiate, we sought to further understand the role of Dicer and miRNAs utilizing a transgenic mouse model of ERMS combined with conditional Dicer1Flox/Flox null mice. The ERMS mouse model results from activation of a constitutively active SmoM2 allele with Cre recombinase expressed from the adipose protein 2 (aP2) promoter resulting in unrestrained hedgehog signaling in the aP2 lineage. Heterozygous Dicer1 loss led to increased penetrance of and a decrease in latency to onset of tumors. In agreement with the haploinsufficient tumor suppressor model, homozygous Dicer1Flox/Flox mice rarely developed tumors in stark contrast to wild type and heterozygous littermates. Furthermore, tumors that did develop in Dicer1Flox/Flox mice escaped Cre recombination and retained a flox allele indicating functional Dicer1 activity.

Serendipitously, aP2-Cre; SmoM2; Dicer1Flox/Flox mice that did not develop ERMS all developed multifocal angiosarcomas. This finding prompted the analysis of Dicer loss alone in the aP2 lineage and remarkably Dicer1Flox/Flox mice similarly developed angiosarcoma whereas Dicer1Flox/+ do not, providing evidence for Dicer loss independent of a cooperating oncogene driving tumorigenesis in a mouse model.

This study provides evidence for the role of Dicer and miRNAs in rhabdomyosarcoma and angiosarcomas. Further understanding the molecular mechanisms and genetic drivers of soft tissue sarcomas is critical for the development of novel targeted therapeutics. This is true especially for soft tissue sarcomas where treatment strategies and survival outcomes have not changed in decades despite numerous clinical trials.

This abstract is also presented as Poster B06.

Citation Format: Jason A. Hanna, Matthew R. Garcia, Jerold Rehg, Mark E. Hatley. Dicer1 deletion: Sufficient for angiosarcoma development and a haploinsufficient tumor suppressor in rhabdomyosarcoma. [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 PR05.
Abstract PR10: HiClinc-1, a highly conserved Cancer-Testis lncRNA, regulates cell proliferation and tumor onset
Authors: Hosono Yasuyuki , Prensner John , Iyer Matthew , Niknafs Yashar , Malik Rohit , Poliakov Anton , Cao Xuhong , Shavit Jordan , Mehra Rohit , Zhou Weibin , Chinnaiyan Arul M.
Published: May 2016

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.
Abstract PR14: Potent knock down of lncRNAs in vitro and in vivo with antisense LNA™ GapmeRs
Authors: Lai Johnathan , Ozen Asli , Mouritzen Peter , Tolstrup Niels , Frandsen Niels Montano
Published: May 2016

Exiqon develops strategies for the optimal design of single stranded LNA™-enhanced antisense oligonucleotides (ASOs, also known as LNA™ GapmeRs) that catalyze RNaseH dependent degradation of target mRNAs and long non-coding RNAs (lncRNA). We have developed an empirically derived design algorithm to provide ASOs that achieve potent target knockdown with a high hit-rate. We describe the latest improvements to the design algorithm, including the incorporation of alignments to both spliced and unspliced transcriptomes in the Ensembl database, to provide maximal target specificity.

Many lncRNAs are nuclear retained or have long residence time in the nucleus. This makes them hard to target by RNAi based methods. However since RNaseH is almost exclusively present in the nucleus nuclear RNAs are expected to be particularly sensitive LNA™ GapmeR targets. We have tested LNA™ GapmeRs to knockdown multiple different RNA targets in vitro – including mRNA and lncRNA targets residing in either cytoplasmic or nuclear compartments. Our results demonstrate that all of these targets were equally efficiently silenced irrespective of the type of RNA target and its subcellular localization. We also report highly efficient and long lasting knockdown of a nuclear retained lncRNA in a broad range of tissues in mice subjected to systemic adminstration of a LNA™ GapmeR. LNA GapmeRs are therefore excellent tools for lncRNA loss of function analysis in vitro and in vivo and provide an excellent platform for therapeutic targeting of lncRNA.

Risk of hybridization based off-target activity with gapmers has traditionally been considered to be low. However in a recent publication1 it was shown that unless properly designed antisense off-target activity with gapmers can be quite significant. Surprisingly, particularly sensitive off-targets were frequently localized in introns of primary transcripts causing significant reduction of the amount of mature spliced mRNAs. Since RNaseH is confined to the nucleus we therefore speculated that the true target of gapmers might be unspliced primary transcripts in the nucleus rather than the spliced mature transcripts that are exported to the cytoplasm. We present data showing that intron targeting LNA GapmeRs often provide highly potent knockdown of the mature spliced RNA. These results have profound implications on therapeutic gapmer design.

1. Kamola PJ, Nucl. Acids Res. 2015, pii: gkv857

This abstract is also presented as Poster B45.

Citation Format: Johnathan Lai, Asli Ozen, Peter Mouritzen, Niels Tolstrup, Niels Montano Frandsen. Potent knock down of lncRNAs in vitro and in vivo with antisense LNA™ GapmeRs. [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 PR14.
Abstract A02: Neuroblastoma patient-derived orthotopic xenografts: Clinically relevant models for drug testing
Authors: Braekeveldt Noémie , Wigerup Caroline , Gisselsson David , Mohlin Sofie , Merselius My , Beckman Siv , Tadeo Irene , Berbegall Anna P. , Öra Ingrid , Navarro Samuel , Noguera Rosa , Påhlman Sven , Bexell Daniel
Published: May 2016

Widespread metastasis is a major problem for the treatment of high-risk neuroblastoma. Relevant neuroblastoma animal models are hence needed to study and target high-risk metastatic neuroblastoma.

We developed neuroblastoma patient-derived orthotopic xenografts (PDXs) using viably cryopreserved or fresh patient neuroblastoma fragments which were implanted orthotopically into immunodeficient NSG mice. Immunohistochemistry showed that PDXs retain neuroblastoma markers and a highly infiltrative growth pattern. Importantly, we found distant metastasis to lungs, liver and bone marrow. Single nucleotide polymorphism array analysis confirmed that PDXs maintain patient-specific chromosomal aberrations 1p del, MYCN amp and 17q gain. In vitro cultures established from the PDXs express neuroblastoma markers and retain their tumorigenic and metastatic ability in vivo after orthotopic injection. PDXs were highly vascularized with mouse endothelial cells and one PDX model formed tumor vasculature through co-engrafted human tumor endothelial cells. Tumors also contained pericytes, cancer-associated fibroblasts, macrophages and extracellular components resembling the patient disease. PDXs established in athymic nude mice additionally developed intratumoral lymphatic vessels and contained mouse-derived CD45+ lymphoid cells. Thus, PDXs maintain important tumor microenvironment hallmarks for high stage neuroblastoma. Furthermore, we demonstrate the feasibility of using short-term in vitro cultured PDX-derived cells as a drug-testing model, and show that the HIF2A oncogene is transcriptionally regulated at hypoxia via the PI3K/mTORC2 pathway in these cells. The results establish the PI3K and mTORC2 pathways as potential therapeutic targets of aggressive neuroblastoma.

In conclusion, neuroblastoma orthotopic PDXs resemble many clinical aspects of the patient disease, e.g., distant metastases, and PDXs are valuable models for preclinical drug testing.

Citation Format: Noémie Braekeveldt, Caroline Wigerup, David Gisselsson, Sofie Mohlin, My Merselius, Siv Beckman, Irene Tadeo, Anna P. Berbegall, Ingrid Öra, Samuel Navarro, Rosa Noguera, Sven Påhlman, Daniel Bexell. Neuroblastoma patient-derived orthotopic xenografts: Clinically relevant models for drug testing. [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 A02.
Abstract A14: Checkpoinblockade enhances oncolytic herpes virotherapy in immunosuppressive sarcoma models
Authors: Chun Chun-yu , Wang Pin-Yi , Hutzen Brian , Haworth Kellie , Conner Joe , Cripe Timothy
Published: May 2016

Most solid tumors are characterized by an immunosuppressive microenvironment, limiting the effectiveness of antitumor immunotherapeutics. Programmed cell death protein (PD)-1-mediated T cell suppression via engagement of its ligand, PD-L1, is of particular interest due to recent successes in selected adult cancers. The utility of PD1-directed therapy for pediatric cancers is unknown, especially given the paucity of mutations and thus infrequent neoantigens in many types of childhood tumors. Oncolytic virotherapy induces tumor shrinkage via a multistep process including direct tumor cell lysis, induction of cytotoxic or apoptosis-sensitizing cytokines, and induction of anti-tumor T cell responses. We recently demonstrated that intratumoral injection of an oncolytic herpes virus induced growth delays and in some cases durable remissions in several mouse models of rhabdomyosarcoma. The effects were T cell mediated, rendering surviving mice resistant to tumor rechallenge and being absent in athymic nude hosts (Leddon et al., Mol Ther-Oncolytics 1, Article number: 14010, 2015). We found these tumor models express PD-L1, suggesting that T cell checkpoints may in part limit virus-induced antitumor immunity. Here we show the implantable C57Bl/6 rhabdomyosarcoma model, M3-9-M, showed a moderate response to single-agent Seprehvir (HSV1716), a virus currently in pediatric clinical trials (NCT00931931, NCT02031965). Single-agent PD-1 blockade also showed moderate but significant tumor growth delay with no complete responses. Strikingly, combining these two therapies together substantially prolonged overall survival with several complete responses post 40 days treatment. We hypothesize that virotherapy-induced chemokine secretion increases the recruitment of antitumor T cells, while PD-1 blockade increases their effectiveness. Overall, our data suggest the combination of PD-1 and oncolytic herpes virotherapy may be an effective treatment strategy for some cancers.

Citation Format: Chun-yu Chun, Pin-Yi Wang, Brian Hutzen, Kellie Haworth, Joe Conner, Timothy Cripe. Checkpoinblockade enhances oncolytic herpes virotherapy in immunosuppressive sarcoma models. [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 A14.
Abstract A34: EWS/FLI regulates transcriptional activation in Ewing sarcoma via length dependent GGAA microsatellites
Authors: Johnson Kirsten M. , Gangwal Kunal , Robertson James , Cheatham Thomas E. , Lessnick Stephen L.
Published: May 2016

The purpose of this study is to investigate how EWS/FLI transcriptionally activates gene targets via polymorphic GGAA microsatellites. Ewing Sarcoma is a pediatric bone malignancy initiated by a t(11;22) chromosomal translocation that produces the EWS/FLI oncoprotein. EWS/FLI transcriptionally activates and represses its target genes to mediate oncogenic reprogramming. Expression of its up-regulated targets correlates with EWS/FLI binding to associated GGAA microsatellites. These microsatellites show length polymorphisms, suggesting that microsatellite polymorphisms may have critical effects on EWS/FLI-responsiveness of key gene targets. For example, NR0B1 is necessary for EWS/FLI mediated oncogenic transformation, and we found a “sweet-spot” of 24-25 repeat length as optimal for EWS/FLI mediated transcriptional activity at NR0B1. The mechanism underlying this optimal length is unknown. We therefore explored the stoichiometry and binding affinity of EWS/FLI for different repeat lengths through biochemical studies combined with molecular modeling simulation. Our data demonstrate a complex relationship between microsatellite length and transcriptional activity. Fluorescence anisotropy studies demonstrate that FLI binding affinity is independent of GGAA microsatellite length. In contrast, the stoichiometry of protein to DNA binding increases in specific incremental patterns with increasing microsatellite repeats. Gel shift binding assays elucidate the minimal microsatellite length critical for binding. Overall our data suggests a model in which the DNA binding domain of multiple monomers of FLI function as independent binding units to facilitate transcriptional activity in a length-dependent fashion. We propose that GGAA microsatellites are necessary and sufficient for EWS/FLI-mediated oncogenic transformation and that repeat length affects optimal DNA binding stoichiometry and transcriptional activity.

Citation Format: Kirsten M. Johnson, Kunal Gangwal, James Robertson, Thomas E. Cheatham, Stephen L. Lessnick. EWS/FLI regulates transcriptional activation in Ewing sarcoma via length dependent GGAA microsatellites. [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 A34.
Abstract A47: Unusual suspects identified in neuroblastoma: An unexpected tumor suppression pathway
Authors: Choo Zhang'e , Koh Rachel Yu Lin , Wallis Karin , Koh Timothy Jia Wei , Kuick Chik Hong , Sobrado Veronica , Loh Amos Hong Pheng , Soh Shui Yen , Schlisio Susanne , Chang Kenneth Tou En , Chen Zhi Xiong
Published: May 2016

Developmental apoptosis of neural crest precursors is crucial in determining the final number of terminally differentiated cells such as sympathetic neurons. During neural development, cells undergo apoptosis as NGF becomes limiting. Aberrant developmental apoptosis is implicated in pediatric sympathetic nervous system tumors. When NGF becomes limiting, a developmental apoptotic pathway is activated which requires KIF1Bbeta. KIF1Bbeta is necessary and sufficient for apoptosis during NGF withdrawal. KIF1Bbeta maps to 1p36.2, a frequently deleted region in neuroblastomas, and a neural crest-derived cancer. We identified that KIF1Bbeta-induced apoptosis requires RNA/DNA helicase DHX9. KIF1Bbeta interacts with DHX9 to enhance translocation and accumulation of cytoplasmic DHX9 in the nucleus, resulting in transcription of apoptotic XAF1. Transcription-incompetent DHX9 is unable to potentiate KIF1Bbeta-induced cell death. Knockdown of DHX9 also protects from KIF1Bbeta-induced cell death whereas KIF1Bbeta loss-of-function domains or patient-associated point mutants are unable to translocate and accumulate cytoplasmic DHX9 in the nucleus, impairing XAF1 expression. Furthermore, XAF1 silencing protects from KIF1Bbeta-induced and NGF withdrawal-mediated apoptosis in vitro as well as promotes tumor growth in vivo whereas XAF1 overexpression is necessary and sufficient to induce apoptosis in vitro and delays tumor growth in vivo. Conditional knockout of KIF1Bbeta in the superior cervical ganglia neurons of mouse pups also specifically ablates XAF1 expression in vivo and ex vivo, suggesting that KIF1Bbeta and XAF1 are tightly regulated and act along the same pathway. Clinically, tissue microarray analysis of pre-treatment or post-treatment neuroblastoma patients who are 1p-intact or 1p-deleted, revealed strong prognostic significance of XAF1 expression in disease stratification. Our findings provide a mechanistic understanding on the development of neuroblastoma through unexpected candidates of tumorigenesis.

Citation Format: Zhang'e Choo, Rachel Yu Lin Koh, Karin Wallis, Timothy Jia Wei Koh, Chik Hong Kuick, Veronica Sobrado, Amos Hong Pheng Loh, Shui Yen Soh, Susanne Schlisio, Kenneth Tou En Chang, Zhi Xiong Chen. Unusual suspects identified in neuroblastoma: An unexpected tumor suppression pathway. [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 A47.
Abstract A52: Investigation of p53 restoration therapy in a mouse model of pineoblastoma
Authors: Harajly Mohammad , Zalzali Hassan , Nawaz Zafar , Saab Raya
Published: May 2016

Background: Pineoblastoma is a malignant childhood tumor with poor outcomes. We have previously characterized a mouse model of pineoblastoma induced by transgenic expression of Cyclin D1 in a p53 null background. We showed that Cyclin D1 expression in the setting of intact p53 results in pineal hyperplasia, but no onset of invasive tumors, reminiscent of pineal enlargement seen in a subset of children with RB haploinsufficiency. In the setting of p53 loss, pineoblastoma occurs with 100% penetrance. We now investigated the role of p53 restoration therapy in prevention and treatment of pineoblastoma using this mouse model.

Results: We found that restoration of p53 in mouse pre-malignant proliferating pineal tumors results in tumor prevention, shown by effective cell cycle exit and induction of cellular senescence, a tumor suppressor mechanism. Conversely, p53 reactivation in established malignant pineal tumors had no effect cell proliferation, unless paired with DNA damaging therapy. The response to p53 restoration did not correlate with levels of p19Arf expression. Interestingly, in premalignant lesions, p53 restoration was associated with secondary amplification of MAPK signaling and induction of p19Arf expression. Subsequent inactivation of p53 after senescence resulted in diffuse re-entry into the cell cycle and rapid tumor progression. Evaluation of a panel of human supratentorial primitive neuroectodermal tumors showed low activity of the p53 pathway, while only one of 6 tumors had p53 deletion or mutation.

Conclusion: Our data shows that, in a mouse model of Cyclin D1-driven pineoblastoma, restoration of p53 has different effects in premalignant versus invasive pineal tumors, where it may need to be paired with DNA damaging agents for effective tumor suppression. Furthermore, p53 activation may need to be continually sustained in such lesions undergoing senescence. Finally, p53 restoration approaches may be worth exploring in sPNET, where the p53 gene seems to be intact but the pathway inactive in the majority of examined tumors.

Citation Format: Mohammad Harajly, Hassan Zalzali, Zafar Nawaz, Raya Saab. Investigation of p53 restoration therapy in a mouse model of pineoblastoma. [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 A52.
Abstract B01: MicroRNAs as novel therapeutic adjuvants to treat high-risk medulloblastoma.
Authors: Abdelfattah Nourhan , Subbarayalu Panneerdoss , Onyeagucha Benjamin , Rajamanickam Subapriya , Chen Hung-I Harry , Rao Manjeet
Published: May 2016

Medulloblastoma is the most common malignant brain tumor in children, accounting for 18% of newly diagnosed brain tumors and 10% of all children cancer-related deaths. Despite improvement in the 5-year survival rate of medulloblastoma in recent years, only a small percentage of patients survive high-risk metastatic disease. The quality of life for those who do survive is often substantially reduced due to the toxicity associated with radiation and chemotherapy. Vincristine is a microtubule-destabilizing antimitotic drug, which is routinely administered in higher dosages to both high and average risk medulloblastoma patients. As a result, these patients suffer from devastating neurotoxic effects that include but not limited to: sensorimotor and autonomic neuropathy, hearing loss, mononeuropathy, and seizures. Using high-throughput microRNA mimic library screens, we identified a group of microRNAs that may improve the efficacy of vincristine against c-MYC amplified medulloblastoma as well as re-sensitize vincristine-resistant medulloblastoma. Our findings revealed that these microRNAs may act as tumor suppressors since their overexpression inhibited colony formation, migration and invasion ability of medulloblastoma cells. Furthermore, these microRNAs suppressed stem cell renewal/proliferation of c-MYC amplified medulloblastoma cells. Expression analysis, gene enrichment analysis and target prediction algorithms revealed that these microRNAs exert their vincristine sensitizing and tumor suppressor effect by targeting genes involved in microtubule organization, cell cycle regulation, DNA damage repair and mRNA translation. One of our most interesting targets is EIF4E3, which is a translation initiation factor. Our preliminary findings indicate that EIF4E3 may regulate medulloblastoma cell growth, progression and vincristine sensitivity by modulating c-MYC translation. Further experiments are underway to test the potential of candidate miRNA and EIF4E3 as vincristine sensitizer in vivo. In conclusion, this study may identify novel factors that have potential not only to decrease the current therapeutic dose of vincristine and therefore eliminate its side effects, but also have potential to multiply the efficacy of lower doses in order to overcome hard to treat high-risk tumors.

Citation Format: Nourhan Abdelfattah, Panneerdoss Subbarayalu, Benjamin Onyeagucha, Subapriya Rajamanickam, Hung-I Harry Chen, Manjeet Rao. MicroRNAs as novel therapeutic adjuvants to treat high-risk medulloblastoma.. [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 B01.
Abstract B26: Therapeutic targeting of sarcomas driven by EWSR1 fusion oncogenes by modulation of the fusion oncogene pre-mRNA
Authors: Slotkin Emily , Stanchina Elisa de , Cartegni Luca , Ladanyi Marc , Spraggon Lee
Published: May 2016

Many predominantly pediatric sarcomas are driven by chromosomal translocations that generate oncogenic fusion transcription factors. The Ewing sarcoma breakpoint region 1 (EWSR1) is one of the most commonly involved genes in sarcoma translocations. This gene has a large number of fusion partners, primarily associated with the pathogenesis of Ewing sarcoma, but also with other soft tissue sarcomas. Targeting the resulting aberrant oncogenic chimeric transcription factors would provide an ideal therapy, but this has approach has proved elusive with conventional small molecule approaches. We have developed an approach to employ splice-switching oligonucleotides to redirect the pre-mRNA splicing of EWSR1 fusion transcription factors. Through targeted exon skipping, and/or activation of intronic polyadenylation, these new compounds provide a targeted therapeutic approach for EWSR1 translocation positive sarcomas and other malignancies.

Citation Format: Emily Slotkin, Elisa de Stanchina, Luca Cartegni, Marc Ladanyi, Lee Spraggon. Therapeutic targeting of sarcomas driven by EWSR1 fusion oncogenes by modulation of the fusion oncogene pre-mRNA. [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 B26.