bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1507: Fatty acid oxidation mediated autophagy regulation in triple negative breast cancer
Authors: Jung Kwang Hwa , Park Jun Hyoung , Sirupangi Tirupataiah , Vithayathil Sajna , Wong Lee-Jun , Kaipparettu Benny A.
Published: July 2017

Compared to hormone regulated/responsive (ER+) breast cancer (BC), triple negative BC (TNBC) patients have a worse overall survival, a significantly shorter disease-free survival, and a shorter post-recurrence survival. Using transmitochondrial cybrid (cybrid) (compares different mitochondria under a common defined nuclear background)-based discovery and validation in cell lines, patient derived xenografts (PDXs) and BC clinical tissues/data, we recently published that fatty acid oxidation (FAO) is a major energy pathway in TNBC. We also showed that FAO is a major regulator of Src activation by autophosphorylation at Src Y419 in metastatic TNBC. Our data suggest that inhibition of FAO induce cell growth inhibition. Short-term treatment of FAO inhibitors induces both cell cycle arrest and apoptosis. We observed cell cycle arrest in G1-S phase in FAO inhibitor treated TNBC cells. Apoptotic markers like cleaved Caspase-3 and cleaved PARP induced in FAO inhibited cells. Short-term treatment of FAO inhibitor also showed induction of AKT pathway. Further analysis on the role of FAO in TNBC suggests that FAO is a critical player in autophagy signaling in TNBC. Long-term treatment with FAO inhibitors or knockdown of FAO rate-limiting proteins carnitine palmitoyltransferase I (CPT1) induce autophagic cell death with beclin-1 and LC3-II induction in TNBC. However, no such phenomenon observed in estrogen receptor positive (ER+) breast cancer cell lines. Treatment with autophagy inhibitor 3-methyladenine (3-MA) abolished the FAO-induced cell growth inhibition and reduced beclin-1 and LC3-II induction. Ongoing studies using knock-down and over expression approaches focus on the role of specific pathways that are responsible for mitochondrial energy reprogramming regulated autophagy signaling in metastatic TNBC.

Citation Format: Kwang Hwa Jung, Jun Hyoung Park, Tirupataiah Sirupangi, Sajna Vithayathil, Lee-Jun Wong, Benny A. Kaipparettu. Fatty acid oxidation mediated autophagy regulation in triple negative breast 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 1507. doi:10.1158/1538-7445.AM2017-1507
Abstract 1522: Targeted gene therapy · c-MYC and hTERT in the scope
Authors: Rezzoug Francine , Thomas Shelia D. , Tella Segen , Miller Donald M.
Published: July 2017

In the past decade immunotherapy and kinase inhibitors have revolutionized the approach for cancer therapy, particularly for melanoma which has benefit from this new therapy in the last 5 years. Although, new treatment increase patient survival, there is no cure and treatments are often limited to a subtype of patients. Therefore there is still an urgent need for efficient therapy for melanoma. Among potential targets for cancer therapy, c-MYC and hTERT which are overexpressed in up-to 85% of all cancers are the most promising. The overexpression of hTERT provides indefinite division to cancer cells. Deregulation of c-MYC affect a large array of genes involved in cell cycle, proliferation/differentiation and apoptosis. Notably, both c-MYC and hTERT genes have in their promoter area a G-rich sequence that form secondary structure called G-quadruplex. These G-quadruplex structures are part of the silencers and are required for the repression of c-MYC or hTERT transcription. Mutations in the G-rich sequences of hTERT promoter destabilize the G-quadruplex and are associated with incidence of melanoma. We have shown that oligonucleotides encoding for the G-quadruples sequences of these genes promoter downregulate their respective gene expression and inhibit cell growth in different cell lines. We investigated the effect of targeting c-MYC or hTERT gene expression in melanoma cell lines using oligonucleotides targeted to the silencer G-quadruplex sequence. Four cell lines where investigated (SK-Mel-2, SK-Mel-3, SK-Mel-28 and A375) for the effect of Pu27 (targeting c-MYC) and Tert-FL (targeting hTERT) on cell proliferation using MTT assay. The treatment for 6 days resulted in 60% growth inhibition in the presence of Pu27 and 50% in the presence of Tert-FL for SK-Mel-2, SK-Mel-3, SK-Mel-28 while A375 was less sensitive. Gene expression evaluated by QRT-PCR showed down-regulation of c-MYC and hTERT in cell exposed to Pu27 and hTERT in cells exposed to Tert-FL. hTERT expression is regulated by c-MYC and in our experiment the down-regulation of c-MYC correlate with down-regulation of hTERT suggesting a cascade effect. In addition, SOX2 gene expression was also down-regulated by Pu27 suggesting an effect on the cancer stem cells (CSC). Our data demonstrate that oligonucleotides targeted to c-MYC and hTERT down-regulate gene expression associated with melanoma cell growth inhibition and possibly on metastasis.

Citation Format: Francine Rezzoug, Shelia D. Thomas, Segen Tella, Donald M. Miller. Targeted gene therapy · c-MYC and hTERT in the scope [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 1522. doi:10.1158/1538-7445.AM2017-1522
Abstract 1531: Therapeutic strategies for osteosarcoma stem cells by regulating adipocyte differentiation based on actin dynamics
Authors: Nobusue Hiroyuki , Takahashi Nobuhiro , Onishi Nobuyuki , Shimizu Takatsune , Sugihara Eiji , Oki Yoshinao , Akashi Koichi , Kano Koichiro , Saya Hideyuki
Published: July 2017

We generated induced cancer stem cells that initiate osteosarcoma (OS) by overexpression of c-MYC and loss of Ink4a/Arf in bone marrow-derived stromal cells, and found that the loss of adipogenic potential is an essential event for OS tumorigenesis. Therefore, our understanding of regulatory mechanisms of adipocyte differentiateon would greatly contribute to control OS tumorigenesis. Here, we show that regulation of the transcriptional coactivator MKL1 (megakaryoblastic leukemia 1) by actin cytoskeleton dynamics drives adipocyte differentiation mediated by PPARγ. Exposure of preadipocytes to an adipogenic cocktail results in the rapid disruption of actin stress fibres as a consequence of downregulation of RhoA-ROCK signaling, which regulates the formation of actin stress fibers and focal adhesions, and the consequent increase in monomeric G-actin levels. The resulting increase in the amount of G-actin leads to the interaction of G-actin with MKL1, which prevents nuclear translocation of MKL1 and allows expression of PPARγ followed by adipogenic differentiation. We further found that depletion of MKL1 can drive adipocyte differentiation in preadipocytes and even in nonadipogenic fibroblasts in the absence of an adipogenic cocktail, and these data revealed that MKL1 functions as a gatekeeper that controls adipocyte differentiation. In addition, we found that treatment with ROCK inhibitors induced terminal adipocyte differentiateon even in OS stem cells through regulation of MKL1 by actin cytoskeleton dynamics, which resulted in a significant suppression of tumorigenesis. Our findings provide new insight into the regulatory mechanism of adipogenesis, and trans-differentiation approach by controlling actin cytoskeleton dynamics may provide a novel therapeutic strategy for targeting cancer stem cells.

Citation Format: Hiroyuki Nobusue, Nobuhiro Takahashi, Nobuyuki Onishi, Takatsune Shimizu, Eiji Sugihara, Yoshinao Oki, Koichi Akashi, Koichiro Kano, Hideyuki Saya. Therapeutic strategies for osteosarcoma stem cells by regulating adipocyte differentiation based on actin dynamics [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 1531. doi:10.1158/1538-7445.AM2017-1531
Abstract 1538: A mouse model that mimics human MEN1 tumor is effective in preclinical neuroendocrine tumor therapeutics
Authors: Wong Chung , Clausen Richard , Tang Laura , Davidson Christian , Vosburgh Evan , Levine Arnold , Xu Eugenia
Published: July 2017

Conditional knockout of Men1 in β cells leads to development of pituitary adenomas and pancreatic neuroendocrine tumors (PanNETs) by the age of eight to twelve months. This suggests that other events, like loss of additional mutations, may be required for development of tumors in the absence of MEN1. Since it was reported that mutations in genes of PI3K/mTOR pathway are found in human neuroendocrine tumors, we hypothesized that double deletion of PTEN and MEN1 may accelerate neuroendocrine tumorigenesis. Mice with double deletion of PTEN and MEN1 in β cells were produced by crosses among mice with Cre gene under the control of Rat Insulin promoter (Rip-Cre), floxed allele Men1 (Men1fl/fl), and Pten (Ptenfl/fl). Then mice with the genotype of Men1fl/fl Ptenfl/fl Rip-Cre were monitored for development of tumors. Consistent with our hypothesis, mice with conditional deletion of PTEN and MEN1 leads to fully developed PanNETs and pituitary adenomas by three to four months old, much earlier than that with Men1 deletion alone. These tumors resemble human MEN1 tumors at a histological and molecular level. Activation of phospho-AKT was observed in these tumors and Rapamycin treatments lead to delayed PanNETs and pituitary adenomas for at least two months. This mouse model (Men1fl/fl Ptenfl/fl Rip-Cre) directly supports that PI3K/mTOR pathway plays a critical role in neuroendocrine tumorigenesis, and also provides a platform to study new therapeutic opportunities for PanNETs and neuroendocrine tumors of the pituitary through the targeting of PI3K/mTOR pathway and MEN1 pathway. This model represents an important tool for studying the biology and preclinical therapeutics of human neuroendocrine tumors.

Citation Format: Chung Wong, Richard Clausen, Laura Tang, Christian Davidson, Evan Vosburgh, Arnold Levine, Eugenia Xu. A mouse model that mimics human MEN1 tumor is effective in preclinical neuroendocrine tumor therapeutics [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 1538. doi:10.1158/1538-7445.AM2017-1538
Abstract 1542: Identifying genetic modifiers of PTEN using the Collaborative Cross mouse panel
Authors: Lanier Amanda , Barrington William , Threadgill David
Published: July 2017

Tumor suppressor genes code for proteins that limit cellular proliferation and cancer progression, and are often exploited in cancer therapies. One such tumor suppressor gene, Phosphatase and Tensin homolog deleted from Chromosome Ten (PTEN), mediates cell growth through negative regulation of phosphatidylinositol 3-kinase (PI3K) dependent pathways. PTEN is frequently deleted or mutated in a variety of human cancers. Mice carrying a PTEN transgene (Super-PTEN mice) were found to be resistant to tumor formation, and even small changes in PTEN expression and activity have been shown to influence cancer susceptibility and progression. We hypothesized that PTEN activity can be altered by modifier genes. To determine whether PTEN modifiers are influencing its activity, we are using the Collaborative Cross (CC), a panel of inbred mouse strains that has a similar amount of genetic variation as human populations. In CC-Super-PTEN crosses, we are utilizing an easily measured phenotype of Super-PTEN mice, reduced body weight, to identify CC strains with altered PTEN function. On average, weanlings carrying the PTEN transgene weigh 16% less than their wild type littermates, but this weight difference varies significantly across strains, from no weight reduction to as much as a 29% difference. This distribution of phenotypes has allowed us to use quantitative trait locus (QTL) mapping to identify potential modifiers of PTEN. Through association mapping, we have determined that the loci with the highest LOD scores reside on chromosomes 2, 12, 16, and X, and we are now reviewing genes within those loci to identify candidate modifiers of PTEN. Additionally, we are using several cancer models to determine the effects of said candidate genes and confirm that an altered PTEN weight phenotype correlates with changes in cancer susceptibility and progression. Ultimately, this research will demonstrate the use of the CC mouse panel to identify novel genetic modifiers and improve our understanding of PTEN regulation.

Citation Format: Amanda Lanier, William Barrington, David Threadgill. Identifying genetic modifiers of PTEN using the Collaborative Cross mouse panel [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 1542. doi:10.1158/1538-7445.AM2017-1542
Abstract 1544: MTAP insufficiency promotes metastasis in lung cancer via diminishing protein dimethylation
Authors: Chang Wen-Hsin , Ho Bing-Ching , Su Kang-Yi , Chen Jian-Wei , Wu Reen , Yang Pan-Chyr , Yu Sung-Liang
Published: July 2017

Genome instability, metabolic switch, and activating metastasis are three of the cancer hallmarks, which always collectively contribute to cancer malignancy. We identified methylthioadenosine phosphorylase (MTAP) as a metastasis suppressor in a series isogenic lung cell lines with different invasion ability by array CGH. Using siRNA silencing and ectopic expression of MTAP, we found that MTAP not only inhibited invasion and colony forming in vitro, but also reduced metastasis and tumorigenesis in vivo. Clinically, patients with low MTAP expression associated with poor overall survival and progression-free survival. Furthermore, the metabolite analysis of clinical specimens showed that low MTAP expression leads to MTA substrate accumulation and product decrease. MTA accumulation in MTAP knockout cells reduced the level of symmetric arginine dimethylation of proteins. By using LC/MS, we identified a novel methyl-protein, whose level is elevated in MTAP knockout cells with the depletion of symmetric arginine dimethylation. Functionally, the unmethylated protein would be protected from proteolysis via caspase 3 cleavage. Moreover, the unmethylated mutant protein promoted migration and invasion in vitro strongly than the wild-type one did. In conclusion, we identify a novel marker in low MTAP expression or MTAP knockout cells, providing a potential therapeutic candidate in lung adenocarcinoma.

Citation Format: Wen-Hsin Chang, Bing-Ching Ho, Kang-Yi Su, Jian-Wei Chen, Reen Wu, Pan-Chyr Yang, Sung-Liang Yu. MTAP insufficiency promotes metastasis in lung cancer via diminishing protein dimethylation [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 1544. doi:10.1158/1538-7445.AM2017-1544
Abstract 1552: Integrative bioinformatics analyses of ChIP seq and RNA seq data reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer
Authors: Ramos Jairo , Roy Deodutta
Published: July 2017

The expression of redox sensitive transcription factor -nuclear respiratory factor 1 (NRF1) expression significantly correlates with histological grades and prognosis of breast cancer. However, the molecular mechanism by which NRF1 may contribute in the development of breast cancer is not clear. In this study we examined whether NRF1 is a molecular risk factor of breast cancer. We also identified regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer. Steps followed for the investigation included the selection of mRNA microarray dataset from TCGA and Metabric, selection and analysis of ChIP-Seq datasets from different breast cancer cell lines to identify NRF1 target genes possibly involved in breast cancer through Molecular Pathway, Gene Ontology and statistical analysis. Analysis of mRNA expression in breast cancer patients showed that NRF1 was significantly overexpressed in breast cancer tissues compared to normal tissues. ChIP-Seq analysis of two different breast cancer cell line - MCF7 and T7D showed that there are more than 8,000 genes, which contain NRF1 binding DNA motif(s). The NRF1 binding to ChIP DNA sequences of target gene is cell context dependent. High percentage of known breast cancer (~50%) susceptibility genes are transcriptional targets of NRF1. Network analysis revealed that NRF1 regulates genes, which are key regulators of epithelial-mesenchymal transition (EMT), stemness, cell apoptosis, cell cycle regulation, chromosomal integrity, and DNA damage and repair. Furthermore, we observed that NRF1 regulates target genes of both breast cancer and cancer KEGG pathways. Many of the MicroRNAs involved in cancer and genes of signaling pathways involved in cancer initiation and progression such as MAPK, PI3K/AKT and Notch signaling are also transcriptional targets of NRF1. In summary, this study reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast cancer. Dysregulation of NRF1 signaling pathways may contribute in the development of breast cancer. Clinical confirmation of our study will have significant impact on our understanding of the role of NRF1 as a valuable biomarker for breast cancer diagnosis and prognosis and will provide strong rationale for the future studies to further develop NRF1 for breast cancer therapeutic target. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR.

Citation Format: Jairo Ramos, Deodutta Roy. Integrative bioinformatics analyses of ChIP seq and RNA seq data reveals a complex regulatory landscape of NRF1 network involved in the pathogenesis of breast 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 1552. doi:10.1158/1538-7445.AM2017-1552
Abstract 1564: A computational framework for removing mouse contamination in tumors sequenced from patient-derived xenografts
Authors: Amin-Mansour Ali , Jané-Valbuena Judit , Mu Xinmeng Jasmine , Garraway Levi
Published: July 2017

Introduction: Using patient derived xenograft (PDX) models has become an effective way for investigating response to standard or new therapeutics in cancer. Human cancer cells injected in mice are allowed to establish tumors and subjected to desired treatments. The PDX tumors are later harvested and characterized, often by massive parallel sequencing. However, a major challenge with analyzing these data is the presence of stromal mouse genomic material, frequently resulting in artifacts in downstream variant detection. We present a computational method to eliminate mouse contamination in PDX.

Method: We used the Burrows-Wheeler Aligner to map reads obtained from sequencing the PDX samples to a combined human and mouse reference genome . We remove reads that are mapped to the mouse reference. The remaining reads are then processed through standard mutation calling pipelines for somatic mutation detection. To test the efficacy of our method, we created in silico mixtures of human and mouse whole-exome sequencing reads from a melanoma patient’s tumor and an immortalized mouse cell line captured with human exome baits. We then carried out a sensitivity analysis to examine how changing the mean target coverage of sequencing, or mouse contamination levels affects our results. For each of the computational experiments, we evaluated somatic mutations detected from the synthetic samples in comparison to the original human sample.

Results: We calculated the sensitivity and specificity of detecting somatic mutations to determine our algorithm’s performance. In all instances, we found greater than 99% for both sensitivity and specificity.

Conclusions: Our results demonstrate that our method works accurately towards removing mouse reads in PDX samples. This task could also be applied to separating sequence reads from other species.

Citation Format: Ali Amin-Mansour, Judit Jané-Valbuena, Xinmeng Jasmine Mu, Levi Garraway. A computational framework for removing mouse contamination in tumors sequenced from patient-derived xenografts [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 1564. doi:10.1158/1538-7445.AM2017-1564
Abstract 1566: Differential expression of Epstein Barr virus miRNAs in triple-negative breast cancer
Authors: Blanchard Alexander , Walls Tiffany , Vidal Ramon , Bonn Stefan , Harrison Scott , Muganda Perpetua
Published: July 2017

Triple-negative breast cancers (TNBC) accounts for up to 20% of breast cancer cases. These tumors are negative for estrogen, progesterone, and HER2 receptors, which are markers used for diagnosis and treatment of other types of breast cancer. Clinically, TNBCs are notoriously aggressive, respond poorly to targeted therapies, have high rates of relapse, as well as poor prognosis. The molecular basis of TNBC oncogenesis is currently unknown, but it is possible that viral factors, such as Epstein-Barr Virus (EBV) miRNAs, play a role. EBV miRNAs have been implicated in the oncogenesis of several forms of cancer, including Burkitt’s lymphoma and nasopharyngeal carcinoma. The objective of this study, therefore, was to determine the differential expression of EBV miRNAs in TNBC tumors as compared to normal breast tissue. We conducted a comprehensive profiling of viral miRNAs in 48 TNBC tumors as compared to 15 control normal breast tissues, utilizing deep sequencing analysis software and publically available deep sequencing data. Four EBV miRNAs (BART18-3p, BART 8-5p, BART15, BART22) were significantly expressed in 11-17% of the TNBC tumors, and not in control normal breast tissue. Four novel putative EBV miRNAs were found to be differentially expressed in TNBC tumors as compared to control normal cells. Two of the novel putative EBV miRNAs were differentially expressed above 125 reads per million (RPM) in 20% and 27% of the TNBC tumors as compared to 0% and 6.6% of normal breast tissues, respectively. One novel putative EBV miRNA was expressed above 125 RPM in 83% of TNBC tumors as compared to 13% of normal breast tissue. One novel putative EBV miRNA was expressed above 125 RPM in 66% of the normal breast tissue as compared to 2% of the TNBCs. These putative EBV miRNAs localize within genomic regions of EBV, including the LMP-2 region. Ongoing work has so far computationally validated one of these miRNAs as a novel EBV miRNA; in vitro validation studies are in progress. This is the first report on the differential expression of EBV miRNAs in TNBC tumors. Since TNBC tumors are extremely heterogeneous, it is intriguing that 20% of TNBC tumors specifically express the same EBV miRNA. Our findings suggest that these differentially expressed EBV miRNAs may potentially play a role in the pathogenesis of TNBC.

Citation Format: Alexander Blanchard, Tiffany Walls, Ramon Vidal, Stefan Bonn, Scott Harrison, Perpetua Muganda. Differential expression of Epstein Barr virus miRNAs in triple-negative breast 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 1566. doi:10.1158/1538-7445.AM2017-1566
Abstract 157: Targeting distinct nodes of the PI3K/AKT/mTOR cascade in prostate cancer cells: impact on cell proliferation, apoptosis and pathway signaling
Authors: Marques Rute B. , Aghai Ashraf , Stam Wendy , Weerden Wytske M. van
Published: July 2017

Inactivation of the PTEN gene is a common event in PCa, leading to activation of the PI3K/AKT/mTOR pathway and promoting PCa progression. Inhibitors targeting this pathway are currently being developed as anticancer agents. While recent clinical trials with mTORC1 inhibitors have shown limited single-agent efficacy in castration-resistant prostate cancer (CRPC), it is still not known whether targeting alternative nodes of the PI3K/AKT/mTOR cascade may improve response. The current study aimed to characterize the response of PCa cell lines to multiple inhibitors targeting the PI3K, AKT and mTOR nodes of the cascade and identify possible mechanisms of resistance.

The impact of the different PI3K/AKT/mTOR inhibitors on cell proliferation and apoptosis was measured by WST-1 and ApoTox-Glo assays, respectively, in a broad panel of established PCa cell lines and primary cell cultures generated from patient-derived xenografts. PTEN-negative PCa cell lines showed strong response to PI3Kβ/δ, AKT and mTOR inhibition, whereas PTEN-positive cells were resistant to PI3Kβ/δ and showed variable sensitivity to mTOR inhibitors. MTORC1 inhibitor showed low IC50 but reached plateau at about 70-80% growth inhibition. In contrast, mTORC1/2 inhibitor could reach complete growth inhibition at ~1μM concentration, in sensitive cell lines. Furthermore, treatment with PI3K or AKT inhibitor in combination with androgen depletion induced apoptosis in various PTEN-negative models, which is consistent with previously reported synergistic effect of PI3K/AKT and androgen receptor co-targeting. The effect of the inhibitors on phosphorylation of PI3K downstream targets (AKT, PRAS40, GSK3, S6K1 and 4EBP1) was analyzed by phospho-specific western blotting. Perturbation of different nodes of the PI3K/AKT/mTOR cascade resulted in differential phosphorylation of downstream targets, but there was no evident correlation with in vitro growth inhibition.

In conclusion, the current study validates PI3Kβ and AKT as alternative targets in PTEN-negative PCa, whereas PTEN-positive models showed preferential yet variable responses towards mTOR inhibitors.

Citation Format: Rute B. Marques, Ashraf Aghai, Wendy Stam, Wytske M. van Weerden. Targeting distinct nodes of the PI3K/AKT/mTOR cascade in prostate cancer cells: impact on cell proliferation, apoptosis and pathway 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 157. doi:10.1158/1538-7445.AM2017-157
Abstract 1583: Inhibition of proline isomerase Pin1 interrupts the function of the androgen receptor N-terminal domain and suppresses androgen-independent growth of prostate cancer cells
Authors: Leung Jacky K. , Imamura Yusuke , Kato Minoru , Mawji Nasrin R. , Sadar Marianne D.
Published: July 2017

Patients with advanced prostate cancer continue to develop lethal castration-resistant prostate cancer (CRPC) despite hormone therapy and maintaining castrate levels of serum androgen. Most CRPC appears to be dependent on the androgen receptor (AR), but instead of the C-terminal ligand-binding domain, it is the N-terminal domain (NTD) harboring a powerful transactivation domain that drives AR transcriptional activity. This was supported by the discovery of AR splice variants (ARv567es and V7) which are constitutively active, do not encode a functional ligand-binding domain, and correlate with poor patient outcome. The AR NTD is intrinsically disordered, but it contains several putative binding sites for Pin1, a proline isomerase specific for phosphorylated-Ser/Thr-Pro motifs. Since the innate ability of the AR NTD to adopt multiple transient structures is important for transactivation of AR, we aimed to determine whether Pin1 regulates motifs within the AR NTD.

We tested several known inhibitors of Pin1 in cell-based assays that measure proliferation or transcription mediated by AR. Our results demonstrated that inhibition of Pin1 interrupted the function of the AR NTD. The Pin1 inhibitor juglone effectively and specifically blocked transcription mediated by AR induced by androgen, as well as transactivation of the AR NTD in the presence of IL-6. We found that Pin1 predominantly interacted with a specific region of the AR NTD containing two Pin1 binding sites, and by inhibiting Pin1 the interactions between endogenous AR and STAT3 became attenuated. Furthermore, Pin1 inhibitors were more effective than second-generation anti-androgens in blocking androgen-independent proliferation of LNCaP95 cells driven by AR variants.

Here we describe that Pin1 is a critical factor for transcription mediated by AR, regardless of ligand, by regulating the AR NTD. Understanding the molecular mechanisms that may promote AR signaling in the absence of androgen will aid the development of more effective therapies for CRPC.

Citation Format: Jacky K. Leung, Yusuke Imamura, Minoru Kato, Nasrin R. Mawji, Marianne D. Sadar. Inhibition of proline isomerase Pin1 interrupts the function of the androgen receptor N-terminal domain and suppresses androgen-independent growth of prostate cancer cells [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 1583. doi:10.1158/1538-7445.AM2017-1583
Abstract 1589: Zinc inhibits androgen receptor expression to inhibit prostate cancer cell growth
Authors: To Phuong Kim , Cho Young-Suk , Kwon Se-Young , Kang Taek Won , Kim Kyung Keun , Jung Chaeyong
Published: July 2017

Background: Prostate gland contains high level of intracellular zinc which is dramatically diminished during cancer development. Due to the obscure role of zinc in this process, therapeutic application using zinc and its supplement is very limited. This study aims to clarify the role(s) of zinc and its intervening mechanism.

Material and methods: Treated by zinc chloride (15-150 µM), several prostate cancer cell lines were applied to confocal microscopy for intracellular trafficking of exogenous zinc, in vitro proliferation assays for their growth, prostate specific antigen (PSA)-based reporter-mediated transactivation, and Western blot for detection of androgen receptor (AR), PSA and ubiquitination. Further in vivo studies were performed to demonstrate the effect of zinc (10-20 mg/kg) on xenograft cancer growth using syngeneic animals followed by tumor analyses.

Results: Zinc chloride suppressed androgen-dependent proliferation of human prostate cancer cells and accordingly zinc chloride dramatically inhibited androgen-mediated transactivation and several androgen target protein expressions, including PSA and p21. Further investigation showed that addition of zinc chloride strikingly downregulated AR protein levels after 4 hours up to 24 hours in both human LNCaP and murine TRAMP C2 prostate cancer cell lines. AR downregulation resulted from facilitated protein degradation instead of transcriptional control. Further in vivo study was carried out using syngeneic mice bearing C2 subcutaneous tumors. Peritoneal injection of zinc chloride significantly reduced tumor size. Analysis of these tumors revealed that there were reduced expression of AR and increased cell death.

Conclusions: Zinc has been shown to inhibit incumbent oncogenic NF-κB pathway. These results also suggest that intracellular zinc inhibits cell growth via downregulation of AR to inhibit growth of prostate cancer. Considering that AR functions as a major effector in prostate cancer development and progression into castration resistant prostate cancer, loss of zinc may be a critical step for this devastating disease and further studies can be performed to develop zinc-based cancer therapeutics.

Citation Format: Phuong Kim To, Young-Suk Cho, Se-Young Kwon, Taek Won Kang, Kyung Keun Kim, Chaeyong Jung. Zinc inhibits androgen receptor expression to inhibit prostate cancer cell growth [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 1589. doi:10.1158/1538-7445.AM2017-1589
Abstract 1618: Immune-modulating effect of bevacizumab in EGFR mutated lung adenocarcinoma
Authors: Feng Po-Hao , Lee Kang-Yun
Published: July 2017

Background: Host immunity affect treatment effect of lung cancer. Peripheral blood S100A9+ monocytic myeloid derived suppressor cells (MDSCs) is a predictive factor for treatment response of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) of lung adenocarcinoma patients. Bevacizumab have immune-modulating effect, including decreasing MDSCs, and improve treatment effect in combination with immunotherapy. However, the effect of immune modulation in combination of bevacizumab and EGFR-TKI in EGFR mutated lung adenocarcinoma patients is not clear.

Methods: Stage IV lung adenocarcinoma harboring sensitive EGFR mutation patients receiving first line EGFR-TKI or combination EGFR-TKI and bevacizumab were enrolled. The peripheral blood mononuclear cell (PBMCs) were collected, and S100A9+ MDSCs percentage was calculated by flow cytometry from CD14+S100A9+ in PBMC. Clinical data was collected.

Results: Eight patients receiving EGFR-TKI and bevacizumab, as combination group, and twenty patients received EGFR-TKI alone, as control group, were enrolled. PBMC S100A9+ MDSC decreased in combination group (decrease 39±18% from baseline), but not in control group. Combination group had longer progress free survival (PFS) comparing with control group. (Median PFS combination vs control group: 15.2 vs 9.9 months, Log Rank test, p=0.05) Increased of peripheral blood cytotoxic T cells also had trend in combination group but not in control group.

Conclusion: Besides the anti-angiogenesis effect, bevacizumab had immune modulated effect, especially in decreasing circulating S100A9+ MDSC in EGFR mutated lung adenocarcinoma patients. This might partially explain the longer PFS in combination of EGFR-TKI of anti-angiogenesis agent treatment.

Citation Format: Po-Hao Feng, Kang-Yun Lee. Immune-modulating effect of bevacizumab in EGFR mutated lung adenocarcinoma [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 1618. doi:10.1158/1538-7445.AM2017-1618
Abstract 1631: Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TM
Authors: Lowman Geoffrey , Linch Elizabeth , Miller Lauren , Topacio-Hall Denise , Looney Timothy , Pankov Alex , Sun Yongming , Peng Xinzhan , Andersen Mark , Hyland Fiona , Mongan Ann
Published: July 2017

T-cell repertoire (TCR) sequencing by next-generation sequencing (NGS) is a valuable tool for building a deeper understanding of the adaptive immune system. As immunotherapies, particularly T-cell dependent therapies, show increasing potential in treating cancer, the ability to gain a detailed, unbiased view of the TCR becomes imperative for biomarker discovery, immune response to treatment, and study of tumor microenvironments. A key question the field seeks to understand is the relationship between circulating T-cells and infiltrating T-cells at the tumor site. Here, we present a novel approach for TCR sequencing using the Ion S5 ™ sequencer which leverages simplified library construction workflows and offers a more complete characterization of the entire V(D)J region of TCRB. This method can leverage mRNA as input, minimizing requirements in starting materials and focusing sequencing to productive TCRB arrangements. This approach targets the constant (C) and the FR1 regions, minimizing the potential for primer bias and greatly increasing the phylogenetic information content compared to techniques that exclusively characterize the CDR3 domain. Our results show that the observed circulating T-cell repertoire size is approximately 2 orders of magnitude higher than the infiltrating T-cell repertoire. Accordingly, while it is difficult to fully capture the complete repertoire of circulating T-cells due to its vast diversity, we show that it is possible to reliably capture the complete infiltrating T-cell repertoire with as high as 10 samples on the Ion 530 ™ chip. Replicate sequencing runs of infiltrating T-cells offers correlation of ~0.9, indicating that the results were reproducible, and the samples were sequenced to appropriate depth. In summary, we believe that this workflow will allow researchers to more routinely characterize the infiltrating T-cell repertoire and offers the field a better understanding of the impact of repertoire diversity on tumor elimination.

Citation Format: Geoffrey Lowman, Elizabeth Linch, Lauren Miller, Denise Topacio-Hall, Timothy Looney, Alex Pankov, Yongming Sun, Xinzhan Peng, Mark Andersen, Fiona Hyland, Ann Mongan. Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TM [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 1631. doi:10.1158/1538-7445.AM2017-1631
Abstract 1643: Cytotoxic T lymphocyte CD3+, CD8+, and immunoscore as prognostic marker in patients after radical cystectomy
Authors: Mansure Jose J. , Yu Alice , Solanki Shraddha , Dias Ana B. , Burnier Miguel M. , Brimo Fadi , Kassouf Wassim
Published: July 2017

Purpose: Prognosis after radical cystectomy for bladder cancer can be unpredictable. There is increasing evidence that the presence of cytotoxic T lymphocytes correlates with survival. The Immunoscore, a new approach to classification of cancer using the number, type and distribution of immune cells, is an independent predictor for survival in colon cancer. Our objective was to evaluate the prognostic impact of lymphocyte distribution and the Immnoscore in bladder cancer.

Methods: Cystectomy sections with core tumor (CT) involvement and identifiable invasive margin (IM) were selected and stained for CD3+ and CD8+ lymphocytes. Three non-contiguous areas of highest lymphocyte density were selected from both CT and IM. The number of lymphocytes in each area was used to calculate the Immunoscore based on previously defined criteria. Kaplan-Meier curves were used to determine differences between Disease-free Survival (DFS) and Overall Survival (OS). Multivariate Cox proportional hazards model was used to determine hazard ratios.

Results: Total of 67 patients who had cystectomy for invasive bladder cancer were included in the study. High concentration of CD8+ lymphocytes in the tumour margin was associated with better DFS (P=0.005) and OS (P=0.03). Similar results were found for CD3+ lymphocytes with regards to DFS (P=0.05). A higher Immunoscore was also associated with better DFS (P=0.04). On multivariate analysis, CD8+ in the invasive margin was independently associated with better prognosis.

Conclusions: The host’s own immune system plays a valuable role in clinical outcome. Our data suggests that a strong immune response in the tumor margin is independently associated with better prognosis.

Citation Format: Jose J. Mansure, Alice Yu, Shraddha Solanki, Ana B. Dias, Miguel M. Burnier, Fadi Brimo, Wassim Kassouf. Cytotoxic T lymphocyte CD3+, CD8+, and immunoscore as prognostic marker in patients after radical cystectomy [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 1643. doi:10.1158/1538-7445.AM2017-1643
Abstract 1644: Combination of MT-3724 with sirolimus reduces anti-drug antibody response and prolongs drug exposure
Authors: Higgins Jack P. , Robinson Garrett L. , Rajagopalan Sangeetha , Brieschke Brigitte , Liu Jensing , Willert Erin K.
Published: July 2017

Molecular Templates is developing engineered toxin bodies (ETBs), potent recombinant immunotoxins that combine the specificity of an antibody fragment with the powerful direct cytotoxicity of the Shiga-like toxin A subunit. ETBs can induce their own internalization, route through the cell in a predictable manner, enzymatically and irreversibly destroy ribosomes to shutdown protein synthesis and induce apoptosis of tumor cells. This mechanism of action is distinct from that of other therapeutics, making ETBs an attractive treatment for patients who have become resistant to chemotherapy and other treatment modalities.

MT-3724 is Molecular Templates’ first-generation ETB targeting CD20, a surface receptor that is highly expressed on malignant B cells in hematological malignancies. Pre-clinical and clinical studies have shown promising results for non-Hodgkin’s lymphoma cell lines and patients; however anti-drug antibodies are formed in experimental animals and patients after repeat dosing. Sirolimus (rapamycin) is a macrolide compound approved to prevent organ rejection and in LAM, and has been used in tolerization protocols with replacement enzymes. In pre-clinical studies, we have co-administered sirolimus in combination with MT-3724 in rodents as well as in a non-human primate model in order to reduce the anti-drug antibody response to MT-3724. These studies demonstrated that the combination of sirolimus with MT-3724 decreased anti-drug antibody response, prolonged serum exposure of MT-3724, and improved B-cell depletion as compared to MT-3724 alone.

Molecular Template’s ETB technology has resulted in potent and targeted therapeutic agents that have a unique mechanism of action in the field of oncology. Our first-generation, CD20-targeted therapeutic, MT-3724, has promising clinical results in the refractory setting for non-Hodgkin’s lymphoma. The combination of sirolimus with MT-3724 is an attractive and feasible regimen which will be further explored in clinical studies.

Citation Format: Jack P. Higgins, Garrett L. Robinson, Sangeetha Rajagopalan, Brigitte Brieschke, Jensing Liu, Erin K. Willert. Combination of MT-3724 with sirolimus reduces anti-drug antibody response and prolongs drug exposure [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 1644. doi:10.1158/1538-7445.AM2017-1644
Abstract 1646: Highly cytotoxic, completely human constructs targeting HER2 and containing the immuno-oncology payload granzyme B
Authors: Cheung Lawrence H. , Zhao Yunli , Mohamedali Khalid A. , Alvarez-Cienfuegos Ana , Hittelman Walter N. , Rosenblum Michael G.
Published: July 2017

Recent immunotherapeutic approaches designed to augment T- and B-cell mediated killing of tumor cells has met with clinical success suggesting that immuno-oncology (IO) approaches have tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade. We designed a recombinant fusion construct composed of a human anti-Her2 scFv fused to active GrB for recognition and delivery of GrB to tumor cells simulating T and B-cell therapy. The GrB-Fc-4D5 dimeric construct (mw 160 kDa) was generated and expressed in stably-transfected CHO-S cells at ~60 mg/L and purified to homogeneity. The enzymatic activity of the fusion construct was similar to commercially-available GrB and the affinity of the construct for purified Her2 extracellular domain (ECD) was 0.328 nM, comparable to that of Herceptin (0.150 nM). The GrB-Fc-4D5 construct was highly cytotoxic to Her2-positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC50 values of 56, 99 and 27 nM respectively. Using immunofluorescence, the fusion construct internalized rapidly into target (SKBR3 or SKOV3) cells within 1 h of exposure, rapidly delivering GrB to the cytoplasmic compartment in a similar manner to that of immune T and B-cell targeting but without the action of the transmembrane pore-forming agent perforin. Against a large panel of various tumor types, GrB-Fc-4D5 was highly cytotoxic to virtually all cells regardless of natural expression levels of the nominal endogenous GrB inhibitor PI-9. Contemporaneous studies with Kadcyla demonstrate similar levels of in vitro activity against virtually all HER2-positive cells tested. GrB-Fc-4D5 demonstrated activity against both log-phase and confluent tumor cells. In keeping with its relatively high molecular weight (~160 kDa), the construct demonstrated a terminal-phase half-life of 13.6 hrs. In vivo efficacy studies are currently ongoing in several orthotopic xenograft models. Also ongoing are pre-IND toxicology, histopathology and clinical chemistry studies. Research conducted, in part, by the Clayton Foundation for Research.

Citation Format: Lawrence H. Cheung, Yunli Zhao, Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Highly cytotoxic, completely human constructs targeting HER2 and containing the immuno-oncology payload granzyme B [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 1646. doi:10.1158/1538-7445.AM2017-1646
Abstract 1651: Targeting phosphatidylserine in combination with adoptive T cell transfer eliminates advanced tumors without off-target toxicities in a melanoma preclinical model
Authors: Hirschhorn-Cymerman Daniel , Schad Sara Sara , Budhu Sadna , Hong Zhong , Yang Xia , Jeff Hutchins T. , Freimark Bruce D. , Gray Michael J. , Wolchok Jedd , Merghoub Taha
Published: July 2017

A viable strategy to treat advanced cancers includes transferring of tumor-specific T cells. T cells that recognize tumor antigens can be expanded and reinvigorated ex-vivo. Furthermore, autologous T cells can be genetically modified to express anti-tumor T cell receptors or chimeric antigen receptors. Although the potency and specificity of tumor-specific T cells can be manipulated ex-vivo, once re-infused into patients, the T cells are subjected to immunosuppressive mechanisms established by the tumor. An important immune checkpoint regulator within tumors is phosphatidylserine (PS). Innate immune cells exposed to PS secrete suppressive cytokines and chemokines that can significantly impair the function and activation of anti-tumor T cells. Therefore, monoclonal antibodies that block PS activity can increase the anti-tumor potency of transferred T cells to treat aggressive cancers. Here we show that a PS targeting monoclonal antibody in combination with CD4+ T cells that recognize the melanoma antigen Trp1 can regress very advanced melanomas in all treated mice. Combination of anti-Trp1 CD4+ T cells with other immunomodulatory modalities such as anti-OX40 antibodies, can achieve equivalent treatment rates but these are typically accompanied by severe immune related adverse events. In contrast, in this setting, PS blockade did not show any off-target toxicities. Flow cytometry analysis revealed lower levels of CD206 expression concomitant with higher activation markers in macrophages and neutrophils in tumors from anti-PS treated mice. These results suggest that diminishing suppressive mechanisms locally in adoptive transfer protocols is a highly desirable strategy that can eliminate tumors while minimizing related adverse events.

Citation Format: Daniel Hirschhorn-Cymerman, Sara Sara Schad, Sadna Budhu, Zhong Hong, Xia Yang, Hutchins T. Jeff, Bruce D. Freimark, Michael J. Gray, Jedd Wolchok, Taha Merghoub. Targeting phosphatidylserine in combination with adoptive T cell transfer eliminates advanced tumors without off-target toxicities in a melanoma preclinical 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 1651. doi:10.1158/1538-7445.AM2017-1651