bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5170: Targeting FGFR signaling to disrupt cellular cross-talk in pancreatic cancer
Authors: Coetzee Abigail , Carter Edward , Heward James , Mardakheh Faraz , Grose Richard , Kocher Hemant
Published: August 2019

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with a 5 year survival rate of less than 5%. PDAC tumors consist of a desmoplastic stroma, which limits the effectiveness of chemotherapy. Pancreatic stellate cells (PSCs), which form a key part of this stroma, become activated in response to tumor development. Activated PSCs enter a cross-talk with cancer cells to induce tumor cell proliferation and invasion, leading to metastatic spread. Nuclear fibroblast growth factor receptor 1 (nFGFR1) has been found in PSCs at the invasive edge of PDAC tumors. Inhibition of FGFR1 prevents its nuclear translocation in PSCs, which results in decreased invasion in 3D in vitro PDAC models. Nuclear translocation of FGFR1 in PSCs appears to be a vital mechanism that triggers the transcription of key proteins involved in PDAC invasion.

We have used a powerful combination of chromatin immunoprecipitation (ChIP-seq) and sub-cellular mass spectrometry to determine the transcriptional targets of nFGFR1 and consequent sub-cellular protein flux. These techniques have allowed us to dissect the functional consequences of FGFR1 knockdown or inhibition in the PSCs. Candidate drivers of invasion are being validated in state-of-the-art 3D in vitro PDAC models. We have extended these functional studies to combination therapy with the clinical agent gemcitabine (targeting cancer cells) and all-trans retinoic acid (ATRA, modulating PSCs), providing translational relevance for our findings. We are embarking on validating this novel strategy using in vivo co-culture xenograft models with specific reference to FGFR1. Effectively disrupting the cross-talk between the tumor and stroma, either alone or in combination with other therapies, could translate to improved therapeutic responses in PDAC patients in the clinic.

Citation Format: Abigail Coetzee, Edward Carter, James Heward, Faraz Mardakheh, Richard Grose, Hemant Kocher. Targeting FGFR signaling to disrupt cellular cross-talk in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5170.
Abstract 5171: GRP78 as a potential modulator of cell adhesion markers in metastatic prostate cancer and multiple myeloma
Authors: Cultrara Christopher , Sabatino David , Zilberberg Jenny
Published: August 2019

Background: Glucose regulated protein 78 (GRP78) is a resident chaperone of the endoplasmic reticulum and a master regulator of the unfolded protein response under physiological and pathological cell stress conditions. GRP78 is overexpressed in many cancers, regulating a variety of signaling pathways associated with tumor initiation, proliferation, adhesion and invasion which contributes to metastatic spread. GRP78 can also regulate cell survival and apoptotic pathways to alter responsiveness to anticancer drugs. Tumors that reside in or metastasize to the bone and bone marrow (BM) space can develop pro-survival signals through their direct adhesive interactions with stromal elements of this niche thereby resisting the cytotoxic effects of drug treatment. In this study, we report a direct correlation between GRP78 and the adhesion molecule N-cadherin (N-cad), known to play a critical role in the adhesive interactions of multiple myeloma and metastatic prostate cancer with the bone microenvironment.

Methods: N-cad expression levels (transcription and protein) were evaluated upon siRNA mediated silencing of GRP78 in the MM.1S multiple myeloma and the PC3 metastatic prostate cancer cell lines. Furthermore, we evaluated the effects of GRP78 knockdown (KD) on epithelial-mesenchymal (EMT) transition markers, morphological changes and adhesion of PC3 cells.

Results: We demonstrate that GRP78 KD: 1) induced the concomitant downregulation of N-cad in both PC3 and MM.1S cells, 2) resulted in significant decreases of both N-cad and E-cad protein levels in PC3 cells, 3) induced TGF-β1 and Snail-2 expression, potentially accounting for the observed downregulation of E-cad. Our data also suggests that N-cad regulation via GRP78 KD supersedes the effects of TGF-β1; i.e., N-cad was downregulated in spite of the fact that TGF-β1 expression was significantly increased upon GRP78 KD.

Conclusion: This work implicates GRP78 as a modulator of cell adhesion markers in MM and PCa. Our results may have clinical implications underscoring GRP78 as a potential therapeutic target to reduce the adhesive nature of metastatic tumors to the bone niche.

Citation Format: Christopher Cultrara, David Sabatino, Jenny Zilberberg. GRP78 as a potential modulator of cell adhesion markers in metastatic prostate cancer and multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5171.
Abstract 5172: Deposition of platelets in sinusoids and its contribution to hepatocarcinogenesis
Authors: Tanaka Hiroki , Horioka Kie , Yamamoto Masahiro , Sawada Koji , Hasebe Takumu , Yamazaki Kosuke , Ogawa Katsuhiro
Published: August 2019

Platelets can activate hepatocyte proliferation not only via releasing the platelet factors, but also promoting for liver sinusoidal endothelial cells (LSEC) and Kupffer cells to produce the factors such as IL-6 and TGF-α that are indispensable for hepatocyte proliferation. We previously reported that in the transgenic (TG) mice with liver specific human BRAFV600E mutation, which corresponds to the mouse BrafV637E that is highly prevalent in mouse DEN-induced hepatic tumors, exhibit increased liver weight five times larger than that of normal mice. Furthermore, hepatocytes in these mice overexpress thrombopoietin (TPO) in association with megakaryocytosis/thrombocytosis, and large numbers of platelets were deposited in the liver sinusoids. In the present study, we investigated whether platelets are deposited in liver sinusoids in hepatic tumors in human and mice. CD61 immunohistochemical staining revealed the increased numbers of platelets were deposited in the sinusoids in foci, adenomas and hepatocellular carcinomas (HCC) induced by neonatal treatment with DEN compared to surrounding normal hepatic tissues. Moreover, increased numbers of platelets were deposited in sinusoids of dysplastic nodules and well-differentiated HCC compared to normal liver tissue and cirrhotic nodules in human. In addition, we evaluated the correlation between the tumor diameters and peripheral platelets numbers in the patients who were primarily diagnosed as HCC in our institute. The result indicated that the numbers of peripheral platelets are positively correlated with the tumor diameters. These results indicate that platelets contribute to development of HCC via interacting sinusoidal cells.

Citation Format: Hiroki Tanaka, Kie Horioka, Masahiro Yamamoto, Koji Sawada, Takumu Hasebe, Kosuke Yamazaki, Katsuhiro Ogawa. Deposition of platelets in sinusoids and its contribution to hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5172.
Abstract 5178: Genomic and transcriptomic alterations of chromatin factors in head and neck cancer
Authors: Cheng Hui , Bao Riyue , Waes Carter Van , Saloura Vassiliki
Published: August 2019

Chromatin factors play central roles in the transcriptional regulation and maintenance of genomic stability and have been implicated in cancer initiation and progression in multiple cancer types. No study has systematically investigated the genomic and expression alterations of chromatin factors in head and neck squamous cell carcinoma (HNSCC). To this purpose, we utilized the molecular profiles from over 510 HNSCCs in the Cancer Genome Atlas (TCGA), and thoroughly characterized the mutational, copy number and transcriptional alterations of 457 chromatin factors and histones. Over 75% of all HNSCC samples have genomic alterations in protein methyltransferases and protein demethylases. Protein lysine methyltransferases MLL2 and NSD1 are the most frequently mutated chromatin factors in this disease. Actin Like 6A (ACTL6A), a component of the SWI/SNF chromatin remodeling complex, SUMO specific peptidase 5 (SENP5) and Nuclear receptor binding SET domain protein 3 (NSD3) are the most frequently copy-number amplified and overexpressed chromatin factors. Interestingly, hierarchical clustering of gene expression of a set of chromatin factors identified a molecular subtype with significantly better overall survival (OS), regardless of HPV-status, stage and NSD1 mutation status, a factor recently reported to render better OS in HNSCC. Systematic characterization of the deregulation of chromatin factors may elucidate novel epigenetic mechanisms and provide novel targets for cancer therapy in HNSCC. Supported by NIDCD intramural projects ZIA-DC-000016, 73 and 74.

Note: This abstract was not presented at the meeting.

Citation Format: Hui Cheng, Riyue Bao, Carter Van Waes, Vassiliki Saloura. Genomic and transcriptomic alterations of chromatin factors in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5178.
Abstract 518: Hypoxia and high interstitial pressure present within the tumor microenvironment can promote T cell suppression and exhaustion
Authors: Rancan Chiara , Marusinec Rachel , Lim James , Fong Lawrence
Published: August 2019

Introduction: The tumor microenvironment is thought to be hostile to the optimal functionality of immune cells. In addition to hypoxia, tumors have a higher interstitial pressure than normal tissue. We examined the impact of hypoxia and increased pressure on human T cell function in vitro. We hypothesize that hypoxia and high pressure induced a more profound and durable exhaustion of activated T cells.

Methods: We cultivated human healthy PBMCs stimulated with phytohemagglutinin (PHA) or CD3/CD28 beads under tumor hypoxic conditions (1%O2, 2PSI) and compared it to normoxic in vitro culture conditions (20% O2, 0 PSI). We utilized the AVATAR system (Xcellbio), a novel bioreactor with highly customable cultivation conditions that allows for the modulation of oxygen and pressure. Functionality and phenotypic changes induced by different cultivation conditions were assessed by flow cytometry and cytokine release assays.

Results: Upon stimulation of PBMCs for 3 days, we observed that hypoxia markedly reduced IFNγ and TNFα cytokine release. Additionally, hypoxia induced an overall decrease in surface expression of the activation and metabolic markers CD71 (transferrin) and CD98 (neutral amino acids transporter), while promoting an increased expression of the inhibitory receptor PD1. This was true for both CD4+ and CD8+ T cells. Following this initial 3-day “priming” stimulation, cells spent 2-days in a “resting period” and underwent a second stimulation under a different condition (cells under normoxia were cultured under hypoxia and vice versa). We found that cells initially primed under hypoxic conditions maintained higher levels of PD1 expression after the second stimulation, regardless of whether the latter condition was normoxic or hypoxic.

Conclusions: Taken together these observations suggest that T cell priming in hypoxic and high pressure conditions present in tumors induce an irreversible state of T cell exhaustion and point out a dominant effect of hypoxia over normoxia. Our data suggest that environmental culture conditions greatly affect T cell responses and should be taken into account when studying T cell functionality, especially in the investigation of novel therapeutic agents targeting the immune system.

Citation Format: Chiara Rancan, Rachel Marusinec, James Lim, Lawrence Fong. Hypoxia and high interstitial pressure present within the tumor microenvironment can promote T cell suppression and exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 518.
Abstract 5180: Enhanced and controlled chromatin extraction for chromatin-based epigenetic assays in FFPE tissues
Authors: Zhong Jian , Ye Zhenqing , Clark Chad , Lenz Samuel , Nguyen Justin , Yan Huihuang , Robertson Keith , Farrugia Gianrico , Zhang Zhiguo , Ordog Tamas , Lee Jeong-Heon
Published: August 2019

Background: Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples and are hampered by low chromatin yield in FFPE samples due to the lack of reliable and efficient chromatin preparation methods. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays.

Results: During rehydration of FFPE tissues, applying tissue-level cross-link reversal to the deparaffinized tissue at 65°C dramatically increased chromatin yield in the soluble fraction. We found the resulting chromatin to be compatible with ChIP-qPCR and ChIP-seq analysis of histone marks (H3K4me3, H3K27me3, and H3K27Ac) and RNA polymerase II. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37°C, the majority of soluble chromatin was over 1 kb in size. The soluble chromatin prepared at 45-60°C showed a typical nucleosomal pattern shown in cell lines and frozen tissues. The majority of the chromatin prepared at 65°C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays.

Conclusions: Our method achieves efficient extraction of high-quality chromatin from FFPE samples suitable for targeted and genome-wide epigenetic assays while minimizing degradation and damages. This approach provides a new platform for developing chromatin-based assays applicable to archived FFPE samples to circumvent the over-fixed nature of FFPE tissues.

This work was supported, in part, by National Institutes of Health grant P01DK068055 and the Mayo Clinic Center for Individualized Medicine Epigenomics Program.

Citation Format: Jian Zhong, Zhenqing Ye, Chad Clark, Samuel Lenz, Justin Nguyen, Huihuang Yan, Keith Robertson, Gianrico Farrugia, Zhiguo Zhang, Tamas Ordog, Jeong-Heon Lee. Enhanced and controlled chromatin extraction for chromatin-based epigenetic assays in FFPE tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5180.
Abstract 5183: ONC201 induces acetylation of histone binding within the p21 (CDKN1A) gene promoter
Authors: Zhang Yiqun , Zhou Lanlan , Zhang Shengliang , Ralff Marie D. , Zhao Shuai , Abbosh Philip H. , Sidker Rahmat , El-Deiry Wafik S.
Published: August 2019

ONC201/TIC10 is a promising anticancer agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. Preliminary clinical data indicates ONC201 induces clinical benefit in a subset of patients with histone H3 K27M glioma, among other tumors. Since H3 K27M mutation reduces levels of H3K27 di- and tri-methylation and in turn alters the expression of genes by epigenetic modulation, we investigated ONC201 effects on epigenetic regulation in cancer. We treated the colorectal cancer cell line HCT116 with the histone deacetylase inhibitor entinostat or ONC201. Chromatin immunoprecipitation (ChIP) was performed with anti-histone H3 (acetyl K9) antibody using lysates from drug-treated tumor cells. Immunoprecipitated DNA was probed by PCR using primers across the promoter of p21 (WAF1; CDKN1A). The results indicate that both ONC201 and entinostat induce the acetylation of histone H3 binding within the p21 promoter. We further observed that ONC201 plus entinostat have synergistic effects in this p21 promoter acetylation activity. Activation of p21 gene expression by enhancing histone acetylation may be relevant in cancer suppression by ONC201.

Citation Format: Yiqun Zhang, Lanlan Zhou, Shengliang Zhang, Marie D. Ralff, Shuai Zhao, Philip H. Abbosh, Rahmat Sidker, Wafik S. El-Deiry. ONC201 induces acetylation of histone binding within the p21 (CDKN1A) gene promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5183.
Abstract 5189: Activity of polycomb repressive complex 2 determines sensitivity to epigenetic therapy
Authors: Chen Fan , Brainson Christine F.
Published: August 2019

EZH2 is a histone methyltransferase that tri-methylates histone H3 at lysine 27 (H3K27me) in the context of the Polycomb Repressive Complex 2, and is highly correlated with poor prognosis of many cancers. However, recent data suggest a de-coupling of EZH2 expression from its enzymatic activity, and many poor prognosis tumors also have low H3K27me3. Therefore, we hypothesize that deletion of Ezh2 would drive more aggressive tumors which may have distinct epigenetic vulnerabilities. To test this hypothesis, we generated the LSL:KrasG12D/+; p53 flox/flox (LSL: lox-stop-lox) mice (Kras/p53 mice) with either zero, one or two floxed alleles of Ezh2 and induced lung tumors with intranasal adeno-Cre virus administration. Supporting our hypothesis, we observed mice with two floxed alleles of Ezh2 (Ezh2 null) had higher tumor burden, higher tumor grade and lower survival than Ezh2 wild-type or heterozygous Kras/p53 mice. Ezh2 null tumors also had a higher propensity to metastasize than Ezh2 wild-type tumors. Consistent with these results, Ezh2 null Kras/p53 tumors and cell lines had increased expression of genes involved in epithelial-mesenchymal transition (EMT), KRAS-driven tumorigenesis, metastasis, and cell cycle regulation. In order to examine the therapeutic vulnerabilities of Ezh2 depleted lung cancers, we built a panel of 2-dimensional cell lines and 3-dimensional organoid cultures of the Kras/p53 tumors that were Ezh2 null, heterozygous and wild-type. We reasoned that using the H3K27me3 demethylase inhibitor might be able to stabilize the residual H3K27me3 in Ezh2 null cells and decrease their aggressive properties. Intriguingly, while we observed no difference in sensitivity to H3K27me3 demethylase inhibitor in the various Ezh2 genotypes of 2-dimensional cultures, both 3-dimensional cultures and in vivo allografts demonstrated Ezh2 null tumors but not Ezh2 WT or heterozygous are sensitive to the H3K27me3 demethylase inhibitor. These data are interesting because they suggest that assays of 3-dimensional organoids may more faithfully recapitulate responses to epigenetic drugs than standard cell lines, and that aggressive tumors with low H3K27me3 might be selectively killed by demethylase inhibitor. We are now expanding our studies to include other targeting agents, including inhibitors of the kinase WEE1 and inhibitor of the chromatin reader BRD4.

Citation Format: Fan Chen, Christine F. Brainson. Activity of polycomb repressive complex 2 determines sensitivity to epigenetic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5189.
Abstract 519: EVOEXS21546 is a novel non-brain penetrant A2AR inhibitor for cancer immunotherapy with accelerated drug development
Authors: Fons Pierre , Bell Andy , Esquerre Michael , Versluys Stephanie , Bertrand Florie , Hopkins-Navratilova Iva , Robinson Sean , Visentin Virgile , Schreyer Adrian , Cox Richard , Mirey Emilie , Pelissier Emilie , bergeaud Celia , Culurgioni Simone , Besnard Jeremy , Payne Andrew , Menegotto Jerome , Machet Frederic , Poussereau-Pomie Celine , Cogo Eric , Paillasse Michael , Ferigo Federica , Pagliarusco Sabrina , Lisztwan Joanna , Whittaker Mark , Johnstone Craig , Hopkins Andrew
Published: August 2019

Evotec and Exscientia are collaborating to develop an innovative drug discovery platform for accelerating small molecule development in Immuno-Oncology. The primary focus is to target the immunosuppressive adenosine pathway. We have firstly sought to generate a novel and selective, non-brain penetrant A2AR antagonist. EVOEX21546 has been selected as our lead candidate with in vitro potency on primary human CD3+ T-lymphocytes for inducing the recovery of IL-2 production after CADO-mediated inhibition of T-cell activation. In addition, we have demonstrated the compound’s effect on other key biological features of T-cell activation including IFN-gamma production and T-cell proliferation. ADME/DMPK data show that EVOEX21546 has a favourable pharmacological profile consistent with its evaluation in syngeneic tumour models. Furthermore, we are pursuing efforts to understand the Mechanism-of-Action of EVOEXS21546 in order to identify the most relevant biomarker of activity for clinical translation. Finally, from the perspective to accelerate the IND submission, EVOEXS21546 entered an INDiGO campaign, an integrated and rapid process to reach IND complemented by high-end integrated CMC. These results, generated in the frame of the A2AR inhibitor pathway, have paved the way to an optimized process for identifying, improving and accelerating the path from drug discovery to the entering into the clinic for Immuno-Oncology drugs.

Citation Format: Pierre Fons, Andy Bell, Michael Esquerre, Stephanie Versluys, Florie Bertrand, Iva Hopkins-Navratilova, Sean Robinson, Virgile Visentin, Adrian Schreyer, Richard Cox, Emilie Mirey, Emilie Pelissier, Celia bergeaud, Simone Culurgioni, Jeremy Besnard, Andrew Payne, Jerome Menegotto, Frederic Machet, Celine Poussereau-Pomie, Eric Cogo, Michael Paillasse, Federica Ferigo, Sabrina Pagliarusco, Joanna Lisztwan, Mark Whittaker, Craig Johnstone, Andrew Hopkins. EVOEXS21546 is a novel non-brain penetrant A2AR inhibitor for cancer immunotherapy with accelerated drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 519.
Abstract 5190: EZH2 epigenetically silences anti-tumor immunity in melanoma via dual histone and DNA methylation
Authors: Tiffen Jessamy , Gunatilake Dilini , Gallagher Stuart , Hersey Peter
Published: August 2019

Despite major advances in immuno- and targeted therapies for metastatic melanoma, not all patients respond to such treatments and the development of therapy resistance remain key obstacles. Novel targets are required and the epigenetic modifier EZH2 may represent such a solution.

EZH2 methylates histones causing chromatin compaction and silencing of gene expression. Additionally, EZH2 has been shown to interact with DNA methyltransferases (DNMTs). Aberrant EZH2 in cancer results in the silencing of hundreds of tumor suppressor genes that would normally constrain cancer growth, and 26% of patients in the Australian Melanoma Genome Project (AMGP) display such abnormal EZH2.

Using ChIP-seq, microarrays and DNA methylation sequencing, we identified a subset of EZH2 target genes that are silenced by both histone and DNA methylation in melanoma. Interestingly many of these genes play a role in the anti-tumor immune response, including RASSF5 and ITGB2 that facilitate lymphocyte infiltration into tumors and correlate with better survival outcomes in melanoma patients when their expression is high. We show that such genes can be significantly up-regulated by combined treatment with inhibitors of both EZH2 and DNMT in melanoma cells in vitro.

Our studies suggest that aberrant EZH2 in melanoma drives aggressive disease via suppression of genes that would normally trigger the anti-tumor immune response. The progression of several preclinical, small molecule inhibitors of EZH2 and DNMTs remains promising.

Citation Format: Jessamy Tiffen, Dilini Gunatilake, Stuart Gallagher, Peter Hersey. EZH2 epigenetically silences anti-tumor immunity in melanoma via dual histone and DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5190.
Abstract 5195: Efficacy of histone deacetylase inhibitors in a 3D cell culture model
Authors: Irigoyen Macarena , Castillo Gonzalo
Published: August 2019

Epigenetic dysregulation, changes in gene expression not due to changes in the DNA sequence, is the underlying cause of many diseases including cancer. Identification of enzymatic chromatin-complex modulators that can restore chromatin homeostasis is critical to developing better therapeutic agents for both, hematologic and solid tumors. 3D cell culture models have been developed to overcome the limitations of monolayer in vitro assays, to include the complexity of the tumor microenvironment known to modulate epigenetic factors, such as cell-cell interaction and allowing for formations of metabolic gradients, factors known to play a role in epigenetic drug efficacy and resistance. In this study, we evaluated the efficacy of class I and II Inhibitors of histone deacetylases, SAHA and Trichostatin A, in a 3D cell culture in vitro assay across several human tumor carcinoma cell lines. The cell lines were grown in 3D cell cultures, and efficacy of the drugs was evaluated at 72 hours and ten days. All the cell lines grown as spheroids proved to be highly sensitive to SAHA and Trichostation A. AGS, a stomach adenocarcinoma cell line, showed the highest sensitivity to SAHA with an IC50 of 270 nM. Trichostatin A, on the other hand, displayed higher sensitivity to the ovarian carcinoma cell line, SKOV3, with an IC50 of 190 nM. In all cases, maximum potency was seen at the 10-day incubations with IC50s below 1 µM for all the cell lines in the panel. Drug-drug combinations with the epigenetic drugs were also studied, the results are discussed.

Citation Format: Macarena Irigoyen, Gonzalo Castillo. Efficacy of histone deacetylase inhibitors in a 3D cell culture model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5195.
Abstract 5197: Histone deacetylase inhibitors promote breast cancer metastasis by elevating NEDD9 expression
Authors: Hu Zong-long , Wang Ya-fang , Su Yi , Shen Yan-yan , Fang Yan-fen , Ding Jian , Chen Yi
Published: August 2019

The approved pan-histone deacetylase (HDAC) inhibitors have brought impressive therapeutic benefits for patients with a few subtypes of hematologic malignancies. Favorable outcomes in breast cancer have not been observed, although multiple HDAC inhibitors have been therapeutically validated in preclinical breast cancer models. Here, we report that pan-HDAC inhibitors provoke breast cancer metastasis in vitro and in vivo, which will severely impede their clinical benefits. Given the role of HDAC inhibitors in the epigenetic control of transcription, we utilized RNA sequencing (RNA-seq) analysis to analyze global gene-expression changes following LBH589 treatment of MCF-7 cells. Pathway enrichment analysis of the 1591 identified genes based on GO analysis showed that 146 GO were significantly altered. Interestingly, most of the changes were related to cytoskeletal, cell junction, and biological adhesion pathways. Genes in the above mentioned GO were intersected to obtain seven genes by Venn analysis. NEDD9 was among the most significantly upregulated transcripts by LBH589, for which was suggested as a metastasis marker in the last decade. We substantiated this result by detecting the mRNA and protein levels of NEDD9 in HDAC inhibitors treated cells. As with the RNA-seq results, a notable upregulation of NEDD9 mRNA and protein were induced by HDAC inhibitors in breast cancer in vitro and in vivo. Mechanistically, pan-HDAC inhibitors enhance acetylation of H3K9 at the NEDD9 gene promoter to upregulate NEDD9 expression via inhibition of HDAC4, and activate FAK phosphorylation. Notably, we demonstrated that FAK inhibitor can reverse pan-HDAC inhibitor-induced breast cancer metastasis, which may improve HDAC inhibitors efficacy for breast cancer. In summary, we demonstrated an NEDD9-FAK signaling pathway, which was induced by pan-HDAC inhibitors, leading to breast cancer metastasis, and thereby caused limitation of the clinical benefits of HDAC inhibitors on breast cancer. We propose that HDAC4 has an upstream regulatory role in NEDD9 expression, leading to FAK phosphorylation and subsequent stimulation of breast cancer metastasis through epigenetic regulation. Our results suggest a need for increased isoform selective HDAC inhibitor research and development.

Citation Format: Zong-long Hu, Ya-fang Wang, Yi Su, Yan-yan Shen, Yan-fen Fang, Jian Ding, Yi Chen. Histone deacetylase inhibitors promote breast cancer metastasis by elevating NEDD9 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5197.
Abstract 5199: A novel role for BAP1 in development and tumor suppression
Authors: Owens Dawn A. , Kuznetsov Jeffim N. , Kurtenbach Stefan , Agüero Tristan , King Mary Lou , Harbour J. William
Published: August 2019

Uveal melanoma (UM) is the second most common form of melanoma and one of the most deadly types of cancer. Recent discoveries in our laboratory and others have led to a deeper understanding of the driver mutations UM. The initiating mutation that is present in virtually all UM occurs in one of four genes in the Gaq signaling pathway (the “Gaq” mutations) and result in sustained activation of proliferative signals. A second mutation occurs later in one of three genes – BAP1, SF3B1 or EIF1AX (the “BSE” mutations), which determines metastatic risk – BAP1 (high risk), SF3B1 (intermediate risk) or EIF1AX (low risk). Surprisingly, we recently discovered that the Gaq mutation and the subsequent BSE mutation occur very close in molecular time. This work suggests that Gaq mutations may cause epigenetic changes that predispose to BSE mutations. We have shown that BAP1-mutant UMs exhibit a de-differentiation and stem-cell like phenotype, suggesting that BAP1 may normally regulate differentiation programs during embryonic development. Consistent with this possibility, BAP1 loss in mouse is embryonic lethal. To study the role of BAP1 in early vertebrate development, therefore, we chose Xenopus laevis (African clawed frog), a widely used model for studying gene function during development. We found that targeted depletion of the Xenopus BAP1 protein (xBap1) causes a striking developmental phenotype affecting multiple germ layers, and that this phenotype is associated with disruption of super enhancers around differentiation genes that are normally maintained by BAP1. Taken together, these studies suggest that loss of BAP1 promotes tumor progression and metastasis, at least in part, by disrupting tightly controlled differentiation programs that govern cell identity. These findings may nominate new therapeutic targets in BAP1-mutant cancers.

Citation Format: Dawn A. Owens, Jeffim N. Kuznetsov, Stefan Kurtenbach, Tristan Agüero, Mary Lou King, J. William Harbour. A novel role for BAP1 in development and tumor suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5199.
Abstract 5203: Segregating activities of the estrogen receptor in gene regulation
Authors: Yang Mei , Zhang Zhao , Xu Kexin
Published: August 2019

Upon stimulation with its ligand estradiol (E2), the estrogen receptor (ER) obviously can induce both transcriptional activation and repression in breast cancer cells. Although the mechanism of gene activation has been extensively scrutinized, how ER targets are turned off is generally overlooked. Some theories have been proposed to explain the trans-suppression process, such as engagement of corepressors, physiological squelch of coactivators, or decommission of RNA Polymerase II, etc. However, none of these models really elucidate the intrinsic and external factors that segregate ER activities in transcriptional regulation. In this study, we identified a functional domain of ER, which is specifically required for E2-mediated gene repression. While the DNA-binding domain (DBD) was essential for the overall response to E2, deletion of AF2 fragment at C-terminus of ER specifically reverted the E2-repressed transcriptional program into E2-activated one. We further demonstrated that RNAs transcribed from these E2-repressed enhancers (eRNAs) act as the exterior determinants of ER activity in gene repression. In addition to stabilizing promoter-enhancer looping structure, these eRNAs recruit ER to particular chromatin loci, and facilitate the hierarchical formation of a functional complex responsible for transcriptional repression. Thus, our findings suggest that by targeting distinct functional domains of ER, we may be able to reprogram expression of a specific subset of ER targets, which play critical roles in conferring malignant phenotypes of breast cancer cells. Meanwhile, as a distinctive class of cis-acting elements besides chromatin regulatory regions, eRNAs help to refine ER-dependent transcriptional program that contributes to the development and progression of breast cancer.

Citation Format: Mei Yang, Zhao Zhang, Kexin Xu. Segregating activities of the estrogen receptor in gene regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5203.
Abstract 5213: A modified L-penetratin peptide targeting e2f-1, 2 and 3 is effective in combination with cisplatin or pemetrexed against prostate and lung cancer cell lines
Authors: Rather Gulam M. , Anyanwu Michael , Kerrigan John E. , Scotto Kathleen W. , Garbuzenko Olga , Minko Tamara , Szekely Zoltan , Bertino Joseph R.
Published: August 2019

Overexpression of the “activating” transcription factors, E2F1,2 and-3a induces genes involved in DNA synthesis and leads to cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more activating E2Fs is a recognized target in cancer therapeutics. Dysregulation/overexpression of E2Fs is also controlled by deletion or mutations in the retinoblastoma protein in tumors. In our previous studies we showed that a novel penetratin conjugated 7-mer peptide (PEP) inhibited transcription of the E2F1, 2 and 3a by bounding tightly to E2F promoters. The PEP was cytotoxic at low micro molar concentrations to several malignant cell lines. As the PEP was unstable in vivo, the PEP was encapsulated in PEGylated liposomes (PL-PEP). Treatment of xenografts of the pRB negative small cell lung cancer H-69 and castrate resistant prostate cancer DU145 tumors propagated in mice with the PL- PEP, caused tumor regression. To increase stability and potency of the PEP, L-Arg in the PEP was replaced by D-Arg. Molecular simulation studies showed that the D-Arg PEP secondary structure is more stable than the L- Arg peptide structure in water. In vitro studies showed that the D-Arg PEP was more potent and more resistant to degradation by serum proteases than the L- form. As E2F is important for DNA repair, and for transcription of thymidylate synthase, we tested the effects of the PEP in combination with cisplatin, a DNA damaging drug and pemetrexed, a potent thymidylate synthase inhibitor. Combination studies of the D-ARG pep with cisplatin (in DU145, PC3, LnCap and 4T1) cells and with pemetrexed (in non-small cell lung cancer, H2009, H441, H1975 and H2228) resulted in synergistic cytotoxicity as determined by Chou-Talalay analysis.

Citation Format: Gulam M. Rather, Michael Anyanwu, John E. Kerrigan, Kathleen W. Scotto, Olga Garbuzenko, Tamara Minko, Zoltan Szekely, Joseph R. Bertino. A modified L-penetratin peptide targeting e2f-1, 2 and 3 is effective in combination with cisplatin or pemetrexed against prostate and lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5213.
Abstract 5215: Targeting of HOX-PBX binding in glioblastoma multiforme as a novel therapeutic treatment
Authors: Morgan Richard , Primon Monika , Shnyder Steven , Short Susan , Kaur Balveen , Hong Bangxing , Bagwan Izhar , Rogers William , Pandha Hardev S.
Published: August 2019

The HOX genes encode a family of transcription factors that play an essential role in embryonic patterning. They are also aberrantly expressed in numerous cancers, including glioblastoma (GBM). Three Amino Acid Loop Extension Homeobox (TALE) proteins act as important co-factors for HOX proteins, modulating their binding affinities to genomic targets. TALE proteins include the Pre-B-cell leukemia homeobox (PBX) proteins 1-4, which bind anterior HOX proteins, facilitating their nuclear entry and limiting their degradation. HTL00-1 is a 2nd generation hexapeptide drug that inhibits HOX-PBX dimer formation, and has been shown to induce rapid apoptosis in cancer cells, but not normal cells, through the rapid upregulation of genes including cFos, DUSP1, and EGR1. We have found that both HOX and TALE genes are markedly dysregulated in primary GBM tumors as well as in murine (GL261), adult (LN18, U87-MG, U251-MG) and pediatric (KNS42, SF188) GBM cell lines, all of which are sensitive to HTL-001. These genes were even more highly expressed in experimentally induced GBM cancer stem cells (CSCs) compared with parental lines, with a corresponding increase in sensitivity to HTL-001. We also investigated the in vivo activity of HTL-001 in Black 6 mice carrying GL-261 subcutaneous and orthotropic tumors with twice weekly intraperitoneal delivery. HTL-001 was shown to cross the blood brain barrier using Alexa660 labelled peptide, and significant anti-tumor activity was observed in both subcutaneous and orthotropic models with increased survival (p<0.02 and p<0.0078, respectively). Resected tumors from HTL-001 treated mice showed marked evidence of apoptosis, tumor vasculature disruption and focal necrosis compared to untreated tumors. Taken together, our findings indicate that HOX-PBX inhibition is a potential therapeutic target for adult and pediatric GBM patients.

Citation Format: Richard Morgan, Monika Primon, Steven Shnyder, Susan Short, Balveen Kaur, Bangxing Hong, Izhar Bagwan, William Rogers, Hardev S. Pandha. Targeting of HOX-PBX binding in glioblastoma multiforme as a novel therapeutic treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5215.
Abstract 5220: Transcription regulatory networks associated with luminal master regulator expression and breast cancer survival
Authors: Byun Jung S. , Singhal Sandeep , Park Samson , Yi Dae IK , Caban Ambar , Vohra Nasreen , Perez-Stable Eliseo J. , Napoles Anna , Gardner Kevin L.
Published: August 2019

The disparity in breast cancer burden of African Americans compared with European Americans is one of the most revealing instances in oncology associated with ethnicity. Recent studies demonstrated that there were highly intratumoral genetic heterogeneity and increased the frequency of basal subtypes among African Americans than European Americans. Because significant race-based disparities in breast cancer patients with hormone-receptor positive still persistent even after reflecting socio-economic status, there may be substantial roles playing by intrinsic biological factors contributing to the disparities. Transcriptional regulation governed by estrogen receptor plays critical roles in major changes in chromatin landscape that facilitate the assembly of other transcriptional complexes affecting mammary tumor initiation and proliferation. We investigated the estrogen receptor and its essential pioneer transcription factors how their expressions are associated with racial disparity with their prognostic significance. Co-expression analysis of estrogen receptor and its pioneer transcription factors suggest that ER-positive patients of women of European ancestry show much more favorable survival than women of African ancestry. Differential comparison of protein expression integrated with network-level gene expression analysis reveals significant differences in the predictive ability of the luminal master regulators based on race and survival. Moreover, we identified genes in the downstream of these master regulators that are highly correlated with race and survival. The comparative analysis of the predictive value of race-based cutoffs suggests that these master regulators of luminal differentiation have reduced transcriptional activity in the downstream regulatory network pathways in African ancestry. Functional characterization of the luminal master regulators and their downstream pathways may provide a better understanding of the intrinsic mechanisms that drive racial disparities in breast cancer survival.

Citation Format: Jung S. Byun, Sandeep Singhal, Samson Park, Dae IK Yi, Ambar Caban, Nasreen Vohra, Eliseo J. Perez-Stable, Anna Napoles, Kevin L. Gardner. Transcription regulatory networks associated with luminal master regulator expression and breast cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5220.
Abstract 5225: Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer
Authors: Tiwari Ritika , Manzar Nishat , Bhatia Vipul , Yadav Anjali , Carskadon Shannon , Gupta Nilesh , Zoubeidi Amina , Palanisamy Nallasivam , Ateeq Bushra
Published: August 2019

The Serine Peptidase Inhibitor, Kazal type 1 (SPINK1) overexpression represents the second-largest prostate cancer (PCa) subtype associated with increased risk of recurrence and poor prognosis. Regardless of molecular subtype, androgen-deprivation therapy (ADT) remains the mainstay treatment for locally advanced and metastatic PCa patients. However, majority of the treated individuals eventually progress to castration-resistant stage and a subset of these patients develop ADT-induced neuroendocrine prostate cancer. Despite evidences of detrimental effects of ADT on PCa, possible role of androgen signaling in SPINK1-mediated prostate oncogenesis remains unexplored. Here, we show that androgen receptor (AR) functions as a direct transcriptional repressor of SPINK1, and blocking AR signaling relieves its repression, leading to upregulation of SPINK1. In agreement, we observe an inverse association between SPINK1 levels and AR expression across multiple patient cohorts, and in neuroendocrine differentiated LNCaP cells. We show that AR and its corepressor, the RE1-silencing transcription factor (REST), occupy SPINK1 promoter and inhibits its transcription. On the other hand, in the absence of AR, lineage reprogramming factor SOX2 in turn binds to SPINK1 promoter leading to its positive transcriptional regulation in androgen-deprived conditions with concomitant increase in neuroendocrine markers. Additionally, stable knockdown of SPINK1 results in reduced epithelial-mesenchymal transition, decreased stemness and drug resistance. Collectively, our findings provide a plausible explanation to the paradoxical clinical outcomes of ADT, arising due to increase in SPINK1 levels. Finally, we emphasize the need to take a well-informed decision prior to ADT and develop alternative therapeutic strategies for castrate-resistant PCa patients.

Citation Format: Ritika Tiwari, Nishat Manzar, Vipul Bhatia, Anjali Yadav, Shannon Carskadon, Nilesh Gupta, Amina Zoubeidi, Nallasivam Palanisamy, Bushra Ateeq. Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5225.
Abstract 5228: Acute depletion of CTCF disrupts enhancer-promoter regulation ofMYCin B-cell acute lymphoblastic leukemia
Authors: Li Chunliang , Zhang Yang
Published: August 2019

Acute lymphoblastic leukemia (ALL), including B-ALL and T-ALL, is the most common childhood cancer and the leading cause of cancer death in children and young adults. Comprehensively genomic sequencing strategies have successfully sub-classified ALL based on genetic alterations including chromosomal rearrangement and aberrant gene mutations, of which frequent coding mutations of CTCF were discovered. However, the function of CTCF in ALL was not clearly defined yet. In a cohort of 264 human pediatric B-ALL patients, particularly in those carrying CTCF loss-of-function mutations, we identified a positive correlation of mRNA expression between CTCF and MYC. Then we integrated the miniAID-mClover3 cassette to the both alleles of endogenous human CTCF locus, which allows acute auxin-mediated degradation of the CTCF protein in a human pediatric B-ALL cell line. By employing combination strategies including whole transcriptome analysis and Capture-C, we discovered that only small number of genes were differentially expressed in CTCF depleted cells, among which MYC and it's downstream genes, not other B-ALL associated oncogenes, were specifically enriched. Mechanically, chromatin interaction capture assay reveals that acute depletion of CTCF primarily disrupted direct interaction between MYC promoter and its distal enhancer residing ~1.8 Mb downstream, subsequently down-regulating target genes in human B-ALL cancer cells. Thus, CTCF/MYC regulation axis is essential in B-ALL development.

Citation Format: Chunliang Li, Yang Zhang. Acute depletion of CTCF disrupts enhancer-promoter regulation ofMYCin B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5228.