bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1512: Functional characterization of novel transcription-regulating cancer drug targets, CDK8 and CDK19, using CRISPR/Cas9 knockout and a highly selective CDK8/19 kinase inhibitor
Authors: Chen Mengqian , Hu Bing , Ji Hao , Altilia Serena , Liang Jiaxin , McDermott Martina , Lim Chang-uk , Porter Donald C. , Broude Eugenia , Roninson Igor
Published: July 2017

The Mediator complex-associated cyclin-dependent kinase CDK8 is an oncogenic transcription-regulating serine/threonine kinase that mediates multiple cancer-associated transcriptional pathways. Despite recent high-profile attention to CDK8 as a novel cancer drug target, very little is known about the function of CDK8’s closely related paralog CDK19. Using CRISPR/Cas9n system we generated CDK8/CDK19 single-knockout (CDK8-KO and CDK19-KO) and double-knockout (CDK8/19-dKO) derivatives of HEK293 cells. RNA-Seq was used to characterize the effects of a highly selective small-molecule CDK8/19 kinase inhibitor Senexin B on gene expression in the parental, single-knockout and double-knockout cell lines. This analysis was conducted in the absence or in the presence of TNF-alpha, an inducer of transcription factor NF-kappa-B that we have previously shown to be potentiated by CDK8. The following results were obtained. (1) CDK8 and CDK19 have complementary functions in stabilization of their partner Cyclin C (independent of their kinase activity) and phosphorylation of transcription factor STAT1 at S727 (dependent on the kinase activity). (2) Senexin B treatment affected gene expression in wild-type 293 cells but had almost no effect in CDK8/19-dKO cells, indicating a very high degree of target selectivity. (3) In contrast to the results with CDK8/19-dKO, most of the genes affected by Senexin B in the wild-type cells were also affected in CDK8-KO and CDK19-KO cells, indicating complementary functions of CDK8 and CDK19. (4) Re-expression of either CDK8 or CDK19, but not of a CDK8 kinase-dead (D173A) mutant, in CDK8/19-dKO cells restored CDK8/19 kinase-dependent gene expression as well as the regulatory effects of Senexin B. (5) Many more genes were inhibited rather than induced by Senexin B, indicating that CDK8/19 act primarily as positive regulators of transcription. (6) The total number of genes affected by Senexin B was greatly increased in cells treated with TNF-alpha, suggesting that cooperation with other transcription factors (such as NF-kappa-B) is the primary role of CDK8/19. Our results indicate that complete suppression of cancer-relevant activities of CDK8 requires simultaneous inhibition of both CDK8 and CDK19.

Citation Format: Mengqian Chen, Bing Hu, Hao Ji, Serena Altilia, Jiaxin Liang, Martina McDermott, Chang-uk Lim, Donald C. Porter, Eugenia Broude, Igor Roninson. Functional characterization of novel transcription-regulating cancer drug targets, CDK8 and CDK19, using CRISPR/Cas9 knockout and a highly selective CDK8/19 kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1512. doi:10.1158/1538-7445.AM2017-1512
Abstract 1514: A novel approach for targeting androgen receptor signaling axis in prostate cancer
Authors: Kaochar Salma , Foley Christopher , Coarfa Cristian , Mitsiades Nicholas
Published: July 2017

Background and Objectives: The androgen receptor (AR) is a ligand-activated transcription factor (TF) that plays critical oncogenic roles. Medical castration therapy is the mainstay treatment for advanced PrCa, but the AR signaling axis frequently remains active in castration-resistant PrCa (CRPC). Despite advances in AR targeting with abiraterone and enzalutamide, disease progression re-occurs in the form of CRPCs that still express AR-dependent genes. This highlights the need for novel approaches to block AR signaling and CRPC growth.

The GATA family of TFs contains six members in mammals, all of which bind a consensus DNA sequence (A/T)GATA(A/G) to regulate gene expression. GATA2 is the predominant family member in prostate luminal epithelial cells. In a search for TFs that control AR expression, we found that GATA2 induces AR gene expression as well as promotes the recruitment of coactivators to the AR transcriptional complex. We found that GATA2 protein expression was strongly correlated with AR protein expression (Spearman's correlation coefficient: 0.323, p-value <0.0001) in our PrCa specimens (n=383). The intensity of GATA2 immunostaining in our BCM patient cohort positively correlated with Gleason score (R=0.37, p-value <0.01) and was a significant predictor of biochemical recurrence (p-value <0.001). We also demonstrated that GATA2 directly promotes expression of both full-length and AR splice-variant, resulting in a strong positive correlation between GATA2 and AR expression in both PrCa cell lines and patient specimens. Conversely, GATA2 expression is repressed by androgen and AR, suggesting a negative feedback regulatory loop that, upon androgen deprivation, derepresses GATA2 to contribute to AR overexpression in CRPC. Simultaneously, GATA2 is necessary for optimal transcriptional activity of both full-length and AR splice-variants. Using ChIP-Seq we found that GATA2 colocalizes with AR and Forkhead box protein A1 (FOXA1) on chromatin to enhance recruitment of steroid receptor coactivators (SRC1, SRC2, and SRC3) and formation of the transcriptional holocomplex. Lastly, we identified a GATA2 small molecule inhibitor (SMI) that can suppress the expression and transcriptional function of both full-length and splice-variant AR and exerted potent anticancer activity against PrCa cell lines. We propose that the inhibition of GATA2 is a ‘first-in-field’ approach to target AR expression and function, including ligand-independent AR, for the treatment of CRPC.

Citation Format: Salma Kaochar, Christopher Foley, Cristian Coarfa, Nicholas Mitsiades. A novel approach for targeting androgen receptor signaling axis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1514. doi:10.1158/1538-7445.AM2017-1514
Abstract 1523: The mechanism of action of BCL6 in glioblastoma
Authors: Jones Nicole M. , Fabre Marie-Sophie , Senanayake Dinindu Sachindra , Hatzi Katerina , Melnick Ari M. , McConnell Melanie J.
Published: July 2017

Glioblastoma (GBM) is the most common and most deadly brain tumor to occur in adults. Initially patients respond to radiation and chemotherapy, which primarily work by causing large amounts of DNA damage, causing apoptosis of the cells. However, this process does not happen effectively in GBM and understanding how these cells resist cell death in response to therapy is key to improving the efficacy of treatment. BCL6 is a transcription factor that stops cell death in response to DNA damage. Recent work in our lab has shown BCL6 to be present in untreated GBM tumors and up-regulated in treated GBM cells. This evidence indicates that BCL6 may be used as a mechanism of therapy resistance by GBM cells. In this study, the objective was to determine the mechanism of action of BCL6 in GBM cells using luciferase reporter assays, quantitative chromatin immunoprecipitation (qChIP) and RNA sequencing. We observed that BCL6 was transcriptionally active in GBM as shown by a reduction in luciferase activity when BCL6 was present. qChIP experiments revealed that BCL6 binding changed over time and was different with different types of DNA-damaging treatment. Preliminary analysis of our RNA sequencing data has identified a unique subset of genes which are upregulated when BCL6 is inhibited and downregulated in response to chemotherapy. These changes indicate that these genes may be regulated by BCL6 in chemotherapy treated cells. All of these results illustrate that BCL6 appears to have an active and relevant function in GBM cells, which demonstrates that BCL6 is an attractive therapeutic target in GBM.

Citation Format: Nicole M. Jones, Marie-Sophie Fabre, Dinindu Sachindra Senanayake, Katerina Hatzi, Ari M. Melnick, Melanie J. McConnell. The mechanism of action of BCL6 in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1523. doi:10.1158/1538-7445.AM2017-1523
Abstract 1525: Chemically induced degradation of the oncogenic transcription factor BCL6
Authors: Kerres Nina , Steurer Steffen , Schlager Stefanie , Bader Gerd , Berger Helmut , Blake Sophia , Caligiuri Maureen , Engen John , Ettmayer Peter , Gerstberger Thomas , Gerlach Daniel , Jacob Roxana , Lucas Simon , Mayer Moriz , Nikolai Mischerikow , Klaus Rumpel , Dirk Scharn , Renate Schnitzer , Voss Tilman , Andreas Zoephel , Kraut Norbert , McConnell Darryl , Mark Pearson , Koegl Manfred
Published: July 2017

The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B-cell lymphoma (DLBCL). It is a DNA-binding protein that represses gene transcription through the recruitment of co-repressor proteins via its BTB domain. Disruption of the interaction of BCL6 with co-repressor proteins interferes with the physiological and oncogenic functions of BCL6. We have used structure-based drug desgin to develop potent compounds that block this interaction with half maximal inhibitory concentration values (IC50s) below 3 nM. In addition to inhibiting co-repressor binding, a subset of the identified inhibitors also caused rapid disappearance of BCL6 protein in cells. This effect was mediated by compound-induced multi-ubiquitylation of BCL6 and degradation by the proteasome and was dependent on the presence of a functional DNA binding domain on BCL6. Compounds that induced BCL6 degradation displayed significantly stronger induction of expression of BCL6-repressed genes than compounds that merely inhibited co-repressor interaction. BCL6-degrading inhibitors had anti-proliferative effects in several DLBCL cell lines, while non-degrading BCL6 inhibitors only had minor effects on proliferation. The fact that the magnitude of effects elicited by this novel class of BCL6 degrading compounds greatly exceeds that of our equipotent classical co-repressor interaction inhibitors offers exciting new opportunities for the development of BCL6-based lymphoma therapeutics.

Citation Format: Nina Kerres, Steffen Steurer, Stefanie Schlager, Gerd Bader, Helmut Berger, Sophia Blake, Maureen Caligiuri, John Engen, Peter Ettmayer, Thomas Gerstberger, Daniel Gerlach, Roxana Jacob, Simon Lucas, Moriz Mayer, Mischerikow Nikolai, Rumpel Klaus, Scharn Dirk, Schnitzer Renate, Tilman Voss, Zoephel Andreas, Norbert Kraut, Darryl McConnell, Pearson Mark, Manfred Koegl. Chemically induced degradation of the oncogenic transcription factor BCL6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1525. doi:10.1158/1538-7445.AM2017-1525
Abstract 1528: Forecasting novel therapies by understanding the role BRD4 in regulating the Notch3 signaling in ovarian cancer
Authors: Villar-Prados Alejandro , Wu Sherry Y. , Roszik Jason , Sood Anil K. , Engelhardt Margaret I.
Published: July 2017

Advances in cancer research have led to the rapid development of novel drug therapies designed for optimal personalized patient care. Despite this surge of treatments, there is still a challenge in accurately predicting the most effective targeted therapy for a specific cancer. Additionally, the potential of systematically identifying and re-purposing currently existing therapeutics designed for one specific cancer to treat other malignancies remains understudied. To address this problem, our group has developed an integrative computational therapy-forecasting algorithm. This algorithm incorporates data from The Cancer Genome Atlas (TCGA) as well as molecular and survival correlations to identify candidate anti-tumor drugs that can be re-purposed for other malignancies by targeting novel pathways. Our computational analysis predicted that bromodomain inhibitors, which inhibit bromodomain-containing proteins such as BRD4, would target the Notch3 pathway in high-grade serous ovarian carcinoma (HGSC). Upregulation of Notch3 plays a crucial role in HGSC tumorigenesis and is associated with worse patient survival. Current Notch3 targeted therapies are not effective, thus designing therapeutic strategies to target Notch3 are critical for HGSC. We hypothesize that inhibition of BRD4 is an effective therapeutic target in HGSC by downregulating the Notch3 pathway. Current in vitro results demonstrated that BRD4 inhibition either by using chemical inhibitors or siRNA results in a decrease in HGSC cell viability by both MTT and 2-D colony formation assays. Furthermore, inhibition of BRD4 resulted in a decrease in both Notch3 transcription and protein levels. Given that BRD4 regulates gene transcription, we performed chromatin immunoprecipitation (ChIP) and observed that BRD4 was present in the Notch3 gene promoter. These findings suggest that by inhibiting BRD4 in HGSC, we can target Notch3 and biologically validates our initial in sillico analysis. The successful completion of this project could have major implications for development of personalized therapies not only for HGSC but for other cancers as well.

Note: This abstract was not presented at the meeting.

Citation Format: Alejandro Villar-Prados, Sherry Y. Wu, Jason Roszik, Anil K. Sood, Margaret I. Engelhardt. Forecasting novel therapies by understanding the role BRD4 in regulating the Notch3 signaling in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1528. doi:10.1158/1538-7445.AM2017-1528
Abstract 1533: Transcriptional inhibition of brachyury in chordoma is associated with adoption of quiescent phenotype
Authors: Seldomridge Ashlee N. , Jung Jinkyu , Sonnemann Heather M. , Giles Amber , Meetze Kristan , Gilbert Mark R. , Palena Claudia M. , Park Deric M.
Published: July 2017

BACKGROUND: Brachyury is a mesoderm specification transcription factor involved in notochord development and overexpressed in a variety of cancers, including chordoma. High levels of brachyury protein expression in cancers is associated with a poor prognosis in part due to its role in mediating epithelial-mesenchymal transition (EMT). TG02 is a multikinase inhibitor that targets transcriptional regulation of cyclin-dependent kinases (CDKs). Because chordoma is characterized by a relative paucity of genomic mutations and largely driven by the super-enhancer activity of brachyury expression, we investigated the downstream effect of a transcriptional inhibitor, TG02, in chordomas.

METHODS: Established chordoma cell lines, UCH-1 and UM-Chor1, were exposed to increasing concentrations of TG02 to determine effect on brachyury protein expression. qPCR was used to analyze brachyury mRNA expression as well as the mesenchymal proteins, vimentin and fibronectin, typically associated with brachyury expression in tumor cells. Cell migration was

evaluated using wound healing assay. CellTiterGlo Luminescence Assay was used to quantify cell viability.

RESULTS: TG02 down-regulated protein expression of brachyury in a dose-dependent manner. Attenuation of brachyury expression was seen within 4 hours and persisted for 5 days upon single exposure to a clinically relevant concentration of TG02. Brachyury downregulation did not affect cell count or viability, and was associated with a quiescent phenotype, including impaired migration. Expression of vimentin and fibronectin, both associated with EMT, was also suppressed within 4 hours of TG02 treatment and persisted for 24 hours.

CONCLUSIONS: Inhibition of brachyury expression and its downstream signaling is associated with quiescent behavior of chordoma cells. Pharmacologically induced transcriptional targeting of brachyury using TG02, a potent CDK9 inhibitor, represents a potential therapeutic strategy.

Citation Format: Ashlee N. Seldomridge, Jinkyu Jung, Heather M. Sonnemann, Amber Giles, Kristan Meetze, Mark R. Gilbert, Claudia M. Palena, Deric M. Park. Transcriptional inhibition of brachyury in chordoma is associated with adoption of quiescent phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1533. doi:10.1158/1538-7445.AM2017-1533
Abstract 1534: The significance of a p53 gain-of-function mutation in the acquisition of metastatic traits in the SW13 cell line
Authors: Montgomery McKale , Hull Elizabeth
Published: July 2017

The therapeutic potential of histone deacetylase inhibitors (HDACi), shown to epigenetically regulate a variety of genes, has yet to be fully realized due to their documented pleiotropic effects in vivo. Currently, four HDACi are FDA-approved for the treatment of hematological malignancies, but their effectiveness against other types of cancers have been shown to be limited at best and, as a result, most of these clinical trials have failed. A comprehensive review of the recent literature has identified that approximately 50% of all cell lines treated with an HDACi demonstrate an increase in metastatic potential as measured by increases in migration and invasion. Of these cell lines that respond with increased metastatic potential to HDACi treatment, 60% have a gain-of-function mutation within p53 (p=0.03). As HDACi have been shown to destabilize mutant p53, they have been used in studies to examine the effects of HDACi treatment on cancers with an identified p53 mutation. This is concerning as the specific p53 mutation in these cancers is not always known, and thus treatment could potentially result in an increase in cancer cell growth. Previous work in our lab demonstrates that the human SW13 adrenal adenocarcinoma cell line, which harbors a homozygous H193Y gain-of-function mutation, does have an HDACi-induced metastatic phenotype; treated cells demonstrate significant increases in MMP activity and ~50% reduction in p53 protein expression compared to untreated cells. To investigate the role that a decrease in mutant p53 expression plays in the acquisition of metastatic characteristics we utilized stable shRNA plasmids to knock-down p53 expression in the SW13 cell line. HeLa cells, which possess a wild-type p53 gene, were used as a positive control. We found that a decrease in p53 expression alone was not sufficient to induce changes in SW13 growth or invasiveness. In fact, even though p53 expression was knocked down by ~70% in the shRNA lines, HDAC inhibition still induced the acquisition of metastatic properties in these cells. Intriguingly, while the DNA binding activity of mutant p53 was correlated with protein expression and was significantly decreased following HDAC inhibition, the same response was not observed in HeLa cells. These findings suggest that p53 status may be an important determinant of therapeutic response to HDAC inhibition.

Note: This abstract was not presented at the meeting.

Citation Format: McKale Montgomery, Elizabeth Hull. The significance of a p53 gain-of-function mutation in the acquisition of metastatic traits in the SW13 cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1534. doi:10.1158/1538-7445.AM2017-1534
Abstract 155: Addition of RP6530, a dual PI3Kδ/γ inhibitor, accentuates Romidepsin activity in NHL cells in vitro
Authors: Viswanadha Srikant , Eleswarapu Satyanarayana , Nyayapathy Seeta , Vakkalanka Swaroop
Published: July 2017

Background: Relapsed or refractory Non-Hodgkin’s Lymphoma (NHL) represents a significant unmet medical need. Novel treatment options currently being explored include Romidepsin, a histone deacetylase inhibitor, which has demonstrated significant preclinical and clinical activity. Because the PI3K pathway is often activated in NHL, combining Romidepsin with a PI3K δ/γ inhibitor may be a viable alternative to chemotherapy. RP6530 is a novel, potent, and selective PI3K δ/γ inhibitor that demonstrated high potency against PI3Kδ (IC50=25 nM) and γ (IC50=33 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Dose-escalation trials to evaluate the safety and efficacy of RP6530 in patients with hematological malignancies are currently underway at several sites in US and Europe. The objective of this study was to evaluate the effect of a combination of Romidepsin and RP6530 in NHL cells.

Methods: Combination of Romidepsin and RPRP6530 was evaluated in the following NHL cell lines: OCI-LY-1, OCI-LY-10, RAJI, DAUDI, TOLEDO, JEKO, MAVER, and SU-DHL-1. Synergism or additivity between Romidepsin and RP6530 was determined using different concentrations of the compounds in a 5 x 5 grid and calculated based on the BLISS score. Effect on apoptosis and cell cycle were estimated by flow cytometry. Phosphorylated AKT and PARP were determined by Western Blotting.

Results: Synergism or additivity between Romidepsin and RP6530 for anti-proliferative activity was noticed in all the cell lines tested with effect being most pronounced in OCI-LY-1, OCI-LY-10, JEKO, and SU-DHL-1. Incubating cells with 3-5 nM Romidepsin and 3 or 5 μM RP6530 resulted in a 2 to 5-fold increase in the total apoptotic population. Cell cycle analysis indicated that the combination of Romidepsin (1-5 nM) and RP6530 (1-5 μM) caused a G0/G1 arrest (2 to 3-fold) with a corresponding increase in percent of apoptotic cells. While Romidepsin had minimal effect of pAKT, treatment with RP6530 reduced expression by 40-90% across cell lines. Further, Romidepsin + RP6530 enhanced expression of cleaved PARP (8 to 60-fold) compared to either agent alone

Conclusions: Addition of RP6530, a potent and selective dual PI3Kδ/γ inhibitor, potentiated Romidepsin activity in NHL cells. Findings provide a rationale for use of the combination in future clinical trials involving naïve or relapsed NHL patients thereby providing a safer alternative to currently available therapy.

Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Seeta Nyayapathy, Swaroop Vakkalanka. Addition of RP6530, a dual PI3Kδ/γ inhibitor, accentuates Romidepsin activity in NHL cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 155. doi:10.1158/1538-7445.AM2017-155
Abstract 1550: Epigenetic downregulation and growth inhibition of IGFBP7 in gastric cancer
Authors: Kim Jin , Kim Min A , Byeon Sun-Ju , Kim Woo Ho
Published: July 2017

Insulin-like growth factor-binding protein 7 (IGFBP7) has been found to be a tumor suppressor in several human cancers, but the role of IGFBP7 in gastric cancer has not yet been fully investigated. In this study, we examined the epigenetic downregulation of IGFBP7 expression in gastric cancer. Analysis by qRT-PCR, western blot, and methylation specific-PCR revealed that DNA methylation was inversely correlated with IGFBP7 expression, and the expression of IGFBP7 was restored after treatment with 5-aza-2'-deoxycytidine. Immunohistochemistry showed that IGFBP7 expression was downregulated in 47.5% of advanced gastric cancer patients. Univariate and multivariate analysis showed that IGFBP7 expression and tumor stage were independent prognostic factors. In cell culture, IGFBP7 knockdown increased gastric cancer cell growth, invasion, and migration, whereas IGFBP7 overexpression in gastric cancer cells showed cell growth inhibition and apoptosis. Our data suggest that IGFBP7 functions as a tumor suppressor in gastric cancer via an epigenetic pathway.

Citation Format: Jin Kim, Min A Kim, Sun-Ju Byeon, Woo Ho Kim. Epigenetic downregulation and growth inhibition of IGFBP7 in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1550. doi:10.1158/1538-7445.AM2017-1550
Abstract 1553: Delineating the role of multiple copies of RNA binding domains in human nucleolin and its homologs using a computational approach
Authors: Begum Kamrun , Steinberg Ruchama Chaya , Saxena Anjana , Singh Shaneen
Published: July 2017

All eukaryotes have nucleoli, the sub-nuclear compartments where ribosomes synthesis occur. Nucleolin (NCL), an abundant RNA binding phosphoprotein,constitutes 5-10% of total nucleolar proteins. This multifunctional protein plays defined role/s in many critical cellular processes e.g. chromatin remodeling, ribosome biogenesis, transcriptional and translational regulation of various non-coding as well as coding RNAs. NCL binds to its target RNAs via two or multiple RNA binding domains (RBDs) to control gene expression during normal cell cycle as well as during cellular response to stress. A variety of tumors express elevated levels of NCL where NCL plays a direct role/s in increasing expressions of genes involved in cell survival, angiogenesis and metastasis. A comparison of NCL in various organisms shows that it is highly conserved, especially in its RNA binding domains that can vary in number (2 to 4 consecutive domains). Interestingly, the individual NCL-RBDs are more conserved across different species rather than within the same protein. RBDs are known to interact in pairs with RNA via RNA recognition motifs called RNP motifs, and complementary nucleic acid motifs or elements in RNA allow interaction with NCL. Vertebrate NCL proteins contains four RBDs, however most studies have focused only on the role/s of RDB 1,2; none probing the possible functional redundancy of the additional RBDs nor identifying any target-specificity for the RBD 3,4 in human NCL and other homologs. Earlier data from our lab suggested RBD 3,4 can bind RNA in a structurally analogous manner to RBD1,2 albeit with altered affinity. Here, we continue this research by investigating various homologs of human NCL to better understand the conserved mechanism of its interaction with RNA and whether the additional RBDs, are in fact, redundant in function. To test this hypothesis, RBDs from various homologs were modeled using template based methods and analyzed by a combination of sequence and structure analysis tools. We show that there are key residue differences in individual RBDs from various homologs that potentially manifest as differences in binding affinities. This study reveals new insights into NCL RBD function and describes the evolution of RBD function with respect to nucleic acid binding.

Citation Format: Kamrun Begum, Ruchama Chaya Steinberg, Anjana Saxena, Shaneen Singh. Delineating the role of multiple copies of RNA binding domains in human nucleolin and its homologs using a computational approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1553. doi:10.1158/1538-7445.AM2017-1553
Abstract 1555: Pan-cancer patterns of synthetic lethality: statistical modeling of gene dependency profiles
Authors: Yeerna Huwate , Rangan Ramya , Aguirre Andrew , Kim William , Vazquez Francisca , Weir Barbara , Ghandi Mahmoud , Tsherniak Aviad , Boehm Jesse , Hahn William , Mesirov Jill , Tamayo Pablo
Published: July 2017

We present a methodology to fit statistical models of cell-viability profiles from RNAi-gene knockdowns. This method allows us to classify genes according to the degree of skewness in their viability distributions. The set of genes with the highest degree of skewness is highly enriched with many known oncogenes and tumor suppressors. We characterize many of these genes, compare them against the results of large sequencing efforts, and use them as inputs to a matrix-decomposition procedure that identifies the most salient cell viabilities shared by different cancer types. We catalog these pan-cancer patterns of synthetic lethality and characterize them by the genomic, transcriptional, and phenotypic features. This analysis provides a rich catalog of the most salient Achilles’ Heels of Pan-Cancer that can be helpful to identify new therapeutic strategies across cancers.

Citation Format: Huwate Yeerna, Ramya Rangan, Andrew Aguirre, William Kim, Francisca Vazquez, Barbara Weir, Mahmoud Ghandi, Aviad Tsherniak, Jesse Boehm, William Hahn, Jill Mesirov, Pablo Tamayo. Pan-cancer patterns of synthetic lethality: statistical modeling of gene dependency profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1555. doi:10.1158/1538-7445.AM2017-1555
Abstract 1563: A machine learning approach to predict platform specific gene essentiality in cancer
Authors: Gilvary Coryandar M. , Madhukar Neel S. , Gayvert Kaitlyn M. , Rickman David S. , Elemento Olivier
Published: July 2017

Loss-of-function (LOF) screenings across a set of diverse cancer cell lines has the potential to reveal novel synthetic lethal interactions, cancer-specific vulnerabilities, and guide treatment options. These were traditionally done using shRNAs, but with the recent emergence of CRISPR technology there has been a shift in methodology. The Achilles project is to date the largest cancer LOF screening effort undertaken, however we found a large amount of inconsistency between their shRNA and CRISPR-Cas9 essentiality results for the same set of cell lines. Here we characterize the differences between genes found to be essential in either CRISPR or shRNA screens. We found that certain features such as gene expression, network connectivity and conservation could accurately separate out essential genes that were found exclusively in either one of these screens. This information could be tremendously useful in understanding the differences in the CRISPR and shRNA screening results. Furthermore, one limitation with Project Achilles was that they conducted shRNA screens on 216 cell lines, but only 33 cell lines in CRISPR. Therefore we developed a model that integrates these genetic, network, and population features to predict CRISPR results from shRNA screenings, and found that our model can accurately identify CRISPR essential genes better than approaches just based on the shRNA results (p-value < 10-5, d-statistic =~0.5 ). This potentially eliminates the need for a costly CRISPR screen, predicts essential genes that would be missed in the shRNA screen, and provides new data on thousands of genes in almost 200 cell lines. Additionally we integrated prior screening results to build a second set of models to predict gene essentiality for untested genes with no LOF screening needed. We found this accurately predicted whether a gene would be marked as essential as well as what type of platform (CRISPR or shRNA) was more likely to accurately identify essentiality. When predicting genes which were exclusively essential in CRISPR we observed an area under the receiver operating characteristic curve (AUC) of 0.82. Overall, these methods allow for a more comprehensive essentiality analysis of genes; which is not possible by single screening platforms.

Citation Format: Coryandar M. Gilvary, Neel S. Madhukar, Kaitlyn M. Gayvert, David S. Rickman, Olivier Elemento. A machine learning approach to predict platform specific gene essentiality in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1563. doi:10.1158/1538-7445.AM2017-1563
Abstract 1567: Oncogenesis may result from multiple different combinations of a small number of tumorigenic mutations
Authors: Anandakrishnan Ramu , Varghese Robin , Kinney Nick , Garner Harold R.
Published: July 2017

Cancer is known to result from genetic mutations, both inherited and somatic. Yet, decades of investigation and the availability of extensive genomic data, have failed to reveal the specific mutations that directly result in tumorigenesis for most cancers. One possible reason is that tumorigenesis may result from one or more of multiple different combinations of mutations, while investigators generally search for a single set of mutations. A mathematical model of the probability of “hitting” one of many possible combinations of a small number of mutations was compared to the actual distribution of accumulated mutations at diagnosis as reported on the cancer genome atlas (TCGA). This “multi-combination multi-hit” model reproduces the distribution of accumulated mutations from the TCGA database with surprising accuracy (RMSD = 1.5%) for a model where one or more of 100 different possible combinations of three mutations result in tumorigenesis. We speculate that each of the three mutations affects one of the several genes in three distinct cell proliferation control pathways, such as senescence, apoptosis, autophagy and necrosis, and different combinations of mutations affecting three of these pathways may result in tumorigenesis. We have identified several such combinations for further investigation.

Citation Format: Ramu Anandakrishnan, Robin Varghese, Nick Kinney, Harold R. Garner. Oncogenesis may result from multiple different combinations of a small number of tumorigenic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1567. doi:10.1158/1538-7445.AM2017-1567
Abstract 1576: UGT2B17 promotes castration-resistant prostate cancer progression through enhancing ligand-independent AR signaling
Authors: Li Haolong , Xie Ning , Chen Ruiqi , Verreault Mélanie , Fazli Ladan , Gleave Martin E. , Barbier Olivier , Dong Xuesen
Published: July 2017

Background: Castration resistant prostate cancer (CRPC) is characterized by a shift of androgen receptor (AR) signaling from ligand-dependent to ligand-independent. Defining mechanisms that control AR signaling transformation is important to develop therapies for disease control. UDP-glucuronosyltransferase 2B17 (UGT2B17) is a key enzyme that maintains androgen homeostasis by catabolizing AR agonists into inactive forms. Although enhanced UGT2B17 expression by antiandrogens was reported in androgen-independent prostate cancers, its role in regulating AR signaling transformation and CRPC progression remain unknown.

Method and Results: We first evaluated the UGT2B17 protein expression levels by immunohistochemistry (IHC) on Vancouver Prostate Centre tissue microarrays. We show that higher UGT2B17 protein expression in prostate tumors is associated with higher Gleason score, metastasis and CRPC progression. The expression and activity of UGT2B17 are also higher in androgen-independent compared to androgen-dependent cell lines. Interestingly, enzalutamide-resistant MR49F cells expresses low UGT2B17 mRNA but strong protein expression, suggesting that posttranslational mechanisms may enhance UGT2B17 protein stability in these cells. We then constructed PCa cell lines with gain-of-function UGT2B17 by lentivirus. Functional analyses indicate that UGT2B17 stimulates cancer cell proliferation, invasion, and xenograft progression to CRPC after prolonged androgen deprivation. To further decipher the molecular mechanisms by which UGT2B17 enhance PCa cell growth independent of androgen, we performed gene microarray using LNCaP(mock) and LNCaP(UGT2B17) cells cultured under the regular serum condition or the prolonged androgen deprivation condition. Microarray analyses reveal that UGT2B17 suppresses androgen-dependent AR transcriptional activity, while enhancing androgen (ligand)-independent AR transcriptional activity. The latter targets genes associated with cell mitosis. These UGT2B17 actions in reprogramming androgen signaling are mainly mediated by activating the c-Src kinase. We confirmed that, in CRPC tumors, UGT2B17 expression is positively associated with c-Src activation.

Conclusion: These results indicate that UGT2B17 expedites CRPC progression by enhancing ligand-independent AR signaling that activates predominantly cell mitosis in cancer cells.

Citation Format: Haolong Li, Ning Xie, Ruiqi Chen, Mélanie Verreault, Ladan Fazli, Martin E. Gleave, Olivier Barbier, Xuesen Dong. UGT2B17 promotes castration-resistant prostate cancer progression through enhancing ligand-independent AR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1576. doi:10.1158/1538-7445.AM2017-1576
Abstract 1580: Combined neuroendocrine differentiation and androgen receptor hyperactivity in MAP37 and CHD1 null prostate cancer
Authors: Rider Leah C. , Rodrigues Lindsey U. , Karimpour-Fard Anis , Romero Lina , Gillette Claire , Costello James C. , Cramer Scott D.
Published: July 2017

We recently functionally validated a clinically aggressive subtype of prostate cancer characterized by dual deletion of MAP3K7 and CHD1. This subtype accounts for up to 25% of prostate cancer deaths. Androgen signaling is a critical component of prostate tumorigenesis and disease progression, which is exploited in treatment of prostate cancer with androgen deprivation and anti-androgen therapies. Neuroendocrine differentiation is thought to provide a selective growth advantage of prostate cancer in androgen-depleted conditions such as surgical or chemical castration. REST is a transcriptional repressor of neuronal gene expression and has been implicated in neuroendocrine differentiation of prostate cancer. The impact of combined MAP3K7 and CHD1 loss on androgen signaling and neuroendocrine differentiation has not been determined. Here, we demonstrate that dual loss of MAP3K7 and CHD1 simultaneously stimulates enhanced androgen signaling and enhanced neuronal differentiation correlated with enhanced growth in both androgen-depleted and androgen-replete conditions. These changes occur via increased androgen receptor chromatin binding, loss of CHD1 chromatin binding, loss of REST expression, and reduced chromatin binding by REST. Suppression of CHD1 and MAP3K7 attenuates the antiproliferative activity of the anti-androgen Enzalutamide. Furthermore, we translate these findings to clinical samples and show that decreased expression of CHD1, MAP3K7, and REST is associated with previous therapeutic intervention, castrate-resistant disease, and progression to a neuroendocrine phenotype. Overall, patients with low expression of all three have poorer disease-free survival. Together these data support the lineage plasticity of prostate tumors with loss of CHD1 and MAP3K7 exhibiting both androgen-dependent and -independent phenotypes leading to poor clinical outcome, which poses challenges to conventional therapeutic approaches.

Citation Format: Leah C. Rider, Lindsey U. Rodrigues, Anis Karimpour-Fard, Lina Romero, Claire Gillette, James C. Costello, Scott D. Cramer. Combined neuroendocrine differentiation and androgen receptor hyperactivity in MAP37 and CHD1 null prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1580. doi:10.1158/1538-7445.AM2017-1580
Abstract 1601: Anti-tumor effect of GAGomer-mediated intra-tumoral IL-2 expression following systemic administration
Authors: Alpert Genia , Altreuter David , Anamandla Sunil , Birt Arlyssa , Cinamon Guy , Merimi Keren Cohen , Or Orli Even , Gefen Nir , Gurvich Nadia , Gyuris Jeno , Lerner Lorena , Mondshine Adi , Wang Hong
Published: July 2017

Cancer immunotherapies are revolutionizing cancer treatment. Unfortunately, a large proportion of patients with solid tumors do not respond to currently available immune-therapeutics. The lack of response is due to a variety of mechanisms tumors adopt to avoid immune mediated clearance. The multiplicity of immunosuppressive mechanisms operational in the tumor microenvironment may not be overcome by single agents and require interventions at multiple control points. However, systemic exposure to combinations of immunoregulators may result in severe, dose limiting, acute and chronic toxicities that might be prevented if the effect of these agents is focused to the tumor microenvironment.

We are engaged in the discovery of a novel class of immuno-oncology drugs aimed at maximizing the effect of immunoregulatory molecules in the tumor microenvironment and minimizing systemic adverse effects. These drugs incorporate plasmids, engineered to program tumor cells to produce and secrete immune-regulatory proteins, within hyaluronic acid (HA) coated lipid nanoparticles, called GAGomers, which specifically target tumor cells that overexpress activated HA receptors (GAG-pDNA). GAG-pDNA based therapeutics promise highly potent but localized activation of the immune system exclusively in the tumor microenvironment following systemic administration, leading to the destruction of tumor cells by activated immune cells without debilitating toxic side effects.

To demonstrate the feasibility of the GAG-pDNA approach we have incorporated a plasmid directing the expression of murine IL-2 into GAGomers (GAG-pIL2) and assessed the anti-tumor activity of the construct after systemic delivery into tumor bearing mice. GAG-pIL2 administration resulted in statistically significant inhibition of tumor growth, which correlated with elevated IL-2 levels in the tumor and increased infiltration of T-cells into the tumor microenvironment.

These experiments demonstrate the feasibility of programming tumor cells using GAG-pDNA to produce and secrete immunoregulatory molecules into the tumor microenvironment and trigger robust anti-tumor immune responses.

Citation Format: Genia Alpert, David Altreuter, Sunil Anamandla, Arlyssa Birt, Guy Cinamon, Keren Cohen Merimi, Orli Even Or, Nir Gefen, Nadia Gurvich, Jeno Gyuris, Lorena Lerner, Adi Mondshine, Hong Wang. Anti-tumor effect of GAGomer-mediated intra-tumoral IL-2 expression following systemic administration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1601. doi:10.1158/1538-7445.AM2017-1601
Abstract 1611: Intravesical BCG induces CD4 T Cell expansion in an immune competent model of bladder cancer
Authors: Kates Max , Nirschl Thomas , Sopko Nikolai , Hahn Noah , McConkey David , Baras Alex , Drake Charles , Bivalacqua Trintiy
Published: July 2017

Intravesical BCG Immunotherapy is the standard of care in treating non-muscle invasive bladder cancer (NMIBC), yet its mechanism of action remains elusive. Both innate and adaptive immune responses have been implicated in BCG activity. While prior research has indirectly demonstrated the importance of T cells and shown a rise in CD4+ T cells in bladder tissue after BCG, T cell subpopulations have not been fully characterized. We investigated the relationship between effector and regulatory T cells in an immune competent, clinically relevant rodent model of bladder cancer. Fischer 344 rats aged 7 weeks received 1.5mg/kg N-Nitroso-N-methylurea (MNU) every other week for 6 weeks (4 doses). Bladder dysplasia began by week 8 and by week 16 the majority of rats had a NMIBC phenotype. Beginning week 8 following the first MNU dose, rats were intravesically administered 0.3ml of BCG (Tice), cisplatin (1mg/ml), Mitomycin C (2mg/ml), MMC+ BCG, or saline (n=10 for all groups) weekly for 6 total doses. Animals were sacrificed at week 16, and bladders were processed for histopathology and digested into single cell suspensions for flow cytometry. Whole transcriptome expression profiling was then performed on sorted CD4 and CD8 cells of post-BCG tumors vs untreated tumors to assess T cell differentiation after BCG. Our data demonstrate that cancer progression in the MNU rat model of bladder cancer is characterized by a decline in the CD8/FoxP3 ratio, consistent with decreased adaptive immunity. By contrast, treatment with intravesical BCG leads to a large, transient rise in the CD4+ T cell population in the urothelium, and is both more effective and immunogenic compared to intravesical chemotherapy. Interestingly, whole transcriptome expression profiling of post-treatment intravesical CD4+ and CD8+ T cells revealed minimal differences in gene expression after BCG treatment. Together, our results suggest that while BCG induces T cell recruitment to the bladder, the T cell phenotype does not markedly change, implying that combining T cell activating agents with BCG might improve clinical activity.

Citation Format: Max Kates, Thomas Nirschl, Nikolai Sopko, Noah Hahn, David McConkey, Alex Baras, Charles Drake, Trintiy Bivalacqua. Intravesical BCG induces CD4 T Cell expansion in an immune competent model of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1611. doi:10.1158/1538-7445.AM2017-1611
Abstract 1613: Genetically modified neuronal stem cells expressing IL-12 and TNFSF14 attenuate highly metastatic mammary tumors in a mouse model
Authors: Shrestha Tej B. , Lu Dezhang , Basel Matthew T. , Pyle Marla , Bossmann Stefan H. , Troyer Deryl L.
Published: July 2017

Interleukin 12 (IL-12) is a heterodimeric cytokine encoded by two genes, p35 and p40. IL-12, also known as T cell-stimulating factor, induces proliferation of T-lymphocytes and natural killer (NK) cells, while also demonstrating an anti-angiogenic effect and inhibition of regulatory T cells. Similarly, LIGHT (TNFSF14) is a transmembrane protein which acts as a costimulatory factor for the activation lymphoid cells and stimulates the proliferation of T cells, leading to increased apoptosis of tumor cells. Expression of IL-12 and LIGHT specifically in the tumor region could activate T cells and stimulate the immune system to attenuate the tumor. Tumor homing neuronal stem cells C17.2 (NSC) were genetically engineered to express the mouse-LIGHT gene and IL-12 using a retrovirus. The expression of LIGHT and IL-12 genes in NSC was confirmed by PCR, ELISA and Western blot. To test the antitumor effects of the engineered cells, Balb/C mice bearing 4T1 mammary tumors were treated with NSC-LIGHT, NSC-IL-12 and NSC-LIGHT/IL-12 cells intratumorally (I.T.) at day nine. Repeated tumor measurement over time showed attenuation of mammary tumor growth in the both NSC/IL-12 and NSC/LIGHT groups in comparison with control groups PBS and NSC. Tumor growth was further reduced by treating the mice with NSC expressing both cytokines LIGHT and IL-12. Tumor metastasis was also evaluated by removing the primary tumor surgically and observing mice for any sign of metastatic burden. Treatment groups had less metastatic tumor burden and survived longer than the control group. Therefore, tumor homing NPC-LIGHT/IL-12 cells could be effective for the treatment of highly metastatic mammary tumors.

Citation Format: Tej B. Shrestha, Dezhang Lu, Matthew T. Basel, Marla Pyle, Stefan H. Bossmann, Deryl L. Troyer. Genetically modified neuronal stem cells expressing IL-12 and TNFSF14 attenuate highly metastatic mammary tumors in a mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1613. doi:10.1158/1538-7445.AM2017-1613