bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 459: Targeting MUC1 mediated nucleotide metabolism sensitizes pancreatic tumors to radiation therapy
Authors: GUNDA VENUGOPAL , Souchek Joshua , Abrego Jaime , Goode Gennifer , Vernucci Enza , Shukla Surendra K. , DasGupta Aneesha , Chaika Nina V. , King Ryan , Yu Fang , Bessho Tadayoshi , Lin Chi , Shou Wang , Sicong Li , Singh Pankaj K.
Published: July 2017

Pancreatic cancer has the lowest survival rate of six perecent among all the cancers in the US and is projected to be the second leading cause of cancer related deaths in a decade. Radiation therapy provides only marginal increases in the survival rate in pancreatic cancer, due to poor responsiveness of pancreatic tumors. Clinical trials indicate a significant response to radiation only in twenty percent of primary pancreatic tumors. While multiple factors cause resistance to radiation therapy, the biological mechanisms mediating such innate resistance are currently being explored. Overexpression of MUC1 facilitates chemo and radiation resistance in pancreatic cancer. MUC1 overepxression also promotes pancreatic tumor growth through metabolic upregulation. We investigated the in vivo role of MUC1 mediated metabolic alterations in radiation response of pancreatic tumors. Our findings indicate that MUC1 expressing pancreatic tumors survive better upon radiation treatment. Metabolomic analysis through liquid chromatography coupled tandem mass spectrometry approach revealed that MUC1 expressing pancreatic tumors exhibit higher glycolytic and nucleotide metabolites upon irradiation. MUC1 expressing tumors also possess relatively higher PPP and nucleotide metabolites. Glycolytic inhibition using bromopyruvate revealed that MUC1 induced radiation resistance could be abrogated through inhibition of glucose carbon flux into nucleotide metabolism by BrPA. Treatment with BrPA effectively reduced glycolysis, pentose phosphate pathway and nucleotide levels in irradiated MUC1 expressing cells. Furthermore, a combination of BrPA and radiation reduced tumor growth in MUC1 expressing tumors. Metabolomic analysis showed a decrease in the glycolysis, PPP and nucleotide metabolites in MUC1 expressing tumors upon combination treatment with radiation and BrPA. Hence, our findings demonstrate that glycolytic inhibition could be used to effectively target MUC1 mediated radiation-resistance in pancreatic tumors.

Citation Format: VENUGOPAL GUNDA, Joshua Souchek, Jaime Abrego, Gennifer Goode, Enza Vernucci, Surendra K. Shukla, Aneesha DasGupta, Nina V. Chaika, Ryan King, Fang Yu, Tadayoshi Bessho, Chi Lin, Wang Shou, Li Sicong, Pankaj K. Singh. Targeting MUC1 mediated nucleotide metabolism sensitizes pancreatic tumors to radiation therapy [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 459. doi:10.1158/1538-7445.AM2017-459
Abstract 4768: A SHARP /Xist complex regulates breast cancer stem cells
Authors: Zhu Yongyou , Shang Li , Colacino Justin , Brooks Michael , Harouaka Ramdane , Lin Chang-Ching , Luo Ming , Wicha Max S.
Published: July 2017

Many cancers including breast cancer display hieratical organization and are driven by a cellular sub-population that displays stem cell properties. These cancer stem cells (CSC’s) mediate metastasis and contribute to treatment resistance highlighting the importance of elucidating CSC regulatory pathways. Nuclear proteins and lncRNAs involved in the maintenance of chromatin structure have been shown to play roles in CSC regulation. Among them the polycomb proteins bmi-1 and EZH2 are involved in epigenetic regulation of CSC’s in breast and other cancers. SHARP/SPEN, an RNA binding protein, has been previously been found to bind the lncRNA Xist which, by recruiting the PRC2 polycomb complex mediates transcriptional repression of the X chromosome. We utilized breast cancer cell lines and xenograft models to examine the role of the SHARP/Xist complex in the regulation of breast CSC’s. In a series of breast cancer cell lines including MCF7 (luminal )and SUM 159 (basal/claudin low) cells, SHARP mRNA and protein as well as the lncRNA Xist were more highly expressed in cells expressing aldehyde dehydrogenase as accessed by the Aldefluor assay than in ALDH -cells . Conditional knockdown of SHARP or Xist in SUM159 significantly decreased the ALDH+ CSC population without affecting cell growth in vitro. Furthermore, SHARP or Xist knockdown significantly reduced tumor initiating capacity and growth of SUM 159 cells in NOD/SCID mice. To determine the mechanism of breast CSC regulation by the SHARP complex we compared mRNA expression patterns of control and SHARP knockdown SUM 159 cells using RNAseq. There was significant overlap between the genes regulated by SHARP with genes regulated by the PCR1 and PRC2 polycomb complexes in addition to many histone genes. These studies suggest that the SHARP/Xist complex my regulate breast CSC’s through epigenetic regulatory histone and polycomb genes. Since SHARP expression is elevated in a number of cancers including breast cancer this pathway may represent a novel therapeutic target.

Citation Format: Yongyou Zhu, Li Shang, Justin Colacino, Michael Brooks, Ramdane Harouaka, Chang-Ching Lin, Ming Luo, Max S. Wicha. A SHARP /Xist complex regulates breast cancer stem 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 4768. doi:10.1158/1538-7445.AM2017-4768
Abstract 482: miRNAs involved in LY6K and estrogen receptor-α contribute to tamoxifen susceptibility in breast cancer
Authors: Kim Yesol , Son Dasom , Yoo Kyung Hyun , Park Jong Hoon
Published: July 2017

Estrogen receptor-alpha is a clinically important therapeutic target for breast cancer. However, tumors that lose ERα are less responsive to anti-estrogens such as tamoxifen. MicroRNAs are small RNAs that regulate expression of their target gene and dysregulations of miRNA has been identified in many diseases including human cancer. However, only a few miRNAs associated with tamoxifen resistance has been reported. In this study, we found that lymphocyte antigen 6 complex, which is a member of the Ly-6/μPAR superfamily and related to breast cancer progression and metastasis, is inversely correlated with ERα expression. We, for the first time, found miRNAs involved in the regulatory molecular mechanism between ERα and LY6K and related to tamoxifen susceptibility in breast cancer. miR-192-5p, induced by LY6K, downregulates ERα directly and induced tamoxifen resistance in ERα-positive breast cancer cells. In addition, re-expression of ERα in ERα-negative breast cancer cells increased miR-500a-3p expression and directly inhibits LY6K expression. Ectopic expression of miR-500a-3p sensitized ERα-negative cells to tamoxifen by increasing apoptosis. Finally, we observed an inverse correlation between LY6K and ERα in primary breast cancer samples. We found that patients with recurrence showed high expression of miR-192-5p after tamoxifen treatments. In addition, expression of miR-500a-3p was significantly correlated to survival outcome. As miRNAs involved in the regulatory mechanism between LY6K and ERα can affect tamoxifen resistance, downregulating miR-192-5p or re-expressing miR-500a-3p could be a potential therapeutic approach for treating tamoxifen resistant patients.

Citation Format: Yesol Kim, Dasom Son, Kyung Hyun Yoo, Jong Hoon Park. miRNAs involved in LY6K and estrogen receptor-α contribute to tamoxifen susceptibility in 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 482. doi:10.1158/1538-7445.AM2017-482
Abstract 484: Epigenetic regulation of hsa-miR-3663 in colon cancer
Authors: Pique Laia , Ferrerira Humberto Jorge , Esteller Manel
Published: July 2017

Colon Cancer is the third most common cancer in the world and a major cause of morbidity and mortality. Molecular mechanisms of colon carcinoma have been deeply studied and current evidences indicate that microRNAs play a pivotal role in its tumorigenesis and progression. MicroRNAs (miRNAs) are small 19 to 22 nucleotides of RNA classified as non-coding RNAs that negatively regulate gene expression at the post-translational level controlling numerous biological processes including development, cell proliferation, apoptosis, differentiation and cell migration. Aberrant expression of miRNAs due to epigenetic alterations has been associated with carcinogenesis. Aimed to identify epigenetically regulated miRNAs involved in colon cancer, we analyzed DNA methylation profiles available in The Cancer Genome Atlas. One of the main hits identified was hsa-miR-3663, hypermethylated in 26% of colon cancer patients (n=286, p< 0.001) in comparison with complete lack of methylation in normal tissues (n=38), suggesting its potential function as a putative tumor suppressor. In order to study the role of DNA methylation controlling hsa-miR-3663, HumanMethylation450K methylation profiles were generated for a panel of colon cancer cell lines (n=10), including the HCT116-DNA methyltransferase knock-out model (DKO, double knockout of DNMT1 and DNMT3B). We found a significant correlation between methylation status of hsa-miR-3663 and expression in colon cancer cell lines. Furthermore, epigenetic regulation of hsa-miR-3663 was confirmed in HCT116 methylated cell line by restored expression upon treatment with DNA demethylating agent 5-aza-2′-deoxycytidine and in the HCT116-DKO model. In order to elucidate the function of hsa-miR-3663, we stably expressed hsa-miR-3663 in a panel of five methylated colorectal cancer cell lines (HCT116, RKO, DLD1, SW48 and SW480). Our initial functional assays revealed that hsa-miR-3663 is not involved in cell proliferation or migration. We are now performing additional assays to further study hsa-miR-3663 and its potential role in colon cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Laia Pique, Humberto Jorge Ferrerira, Manel Esteller. Epigenetic regulation of hsa-miR-3663 in colon 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 484. doi:10.1158/1538-7445.AM2017-484
Abstract 5011: Tumor associated macrophages promote malignant phenotypes of disseminated human gastric cancer cells in intraperitoneal cancer immune microenvironment
Authors: Sakamoto Shuichi , Kagawa Shunsuke , Kuwada Kazuya , Ito Atene , Kagawa Tetsuya , Kikuchi Satoru , Kuroda Shinji , Yoshida Ryuichi , Tazawa Hiroshi , Fujiwara Toshiyoshi
Published: July 2017

Gastric cancer is the third leading cause of cancer-related death, and peritoneal dissemination is one of the most frequent site of recurrence. Although the role of tumor microenvironment is recognized, it is still unclear how intraperitoneal cancer-immune microenvironment contributes to peritoneal metastasis. Thus, we investigated the interaction between intraperitoneal cancer and immune cells, especially focusing on tumor-associated macrophages (TAM) in the peritoneal cavity. The peritoneal wash from gastric cancer patients was subjected to conventional cytology, flow cytometry, and immuno-fluorescent assay using antibodies against CD45 (leukocytes), CD14 (monocytes), CD80/163 (M1/M2 macrophages, respectively), in combination with genetically engineered cancer-imaging viral agent ‘TelomeScan’ which specifically expresses GFP in telomerase-positive cells. In cytology-positive samples, CD163-positive macrophages could be detected in approximately 70% of intraperitoneal macrophages with TelomeScan-positive cancer cells, while cytology negative samples contained few CD163-positive macrophages. In addition, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were induced to differentiate into CD163-positive M2 macrophages (TAM-like macrophages), and co-cultured with gastric cancer cells to examine the effect on their malignant phenotype such as migration and invasion. We found that PBMCs-derived CD163-positive M2 macrophages significantly potentiated the ability of migration and invasion in gastric cancer cells. In case of existence of free cancer cells in peritoneal cavity, intraperitoneal macrophages appeared to differentiate into TAM and they potentially accelerate malignant phenotypes of gastric cancer cells. These results suggested that intraperitoneal cancer-immune microenvironment may play an important role to promote peritoneal disseminations in gastric cancer.

Citation Format: Shuichi Sakamoto, Shunsuke Kagawa, Kazuya Kuwada, Atene Ito, Tetsuya Kagawa, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Toshiyoshi Fujiwara. Tumor associated macrophages promote malignant phenotypes of disseminated human gastric cancer cells in intraperitoneal cancer immune microenvironment [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 5011. doi:10.1158/1538-7445.AM2017-5011
Abstract 5186: Vemurafenib selectively radiosensitizes BRAF V600E mutant papillary and anaplastic thyroid carcinoma cells
Authors: Robb Ryan N. , Yang Linlin , Williams Terence
Published: July 2017

BRAF activating mutations act as oncogenic drivers and are highly prevalent in thyroid cancer, occurring in about 60% of papillary thyroid cancer (PTC) as well as 30-40% anaplastic thyroid cancer (ATC). MAPK signaling and treatment resistance is driven by BRAF mutations in thyroid cancer. Among the most common of these mutations is BRAFV600E, which can be selectively inhibited by vemurafenib.

Using a panel of PTC and ATC cell lines we assessed how the presence/absence of BRAFV600E impacts radiation sensitivity. We used radiation clonogenics, comet assays, nuclear foci formation, immunoblotting, and mouse xenografts models to determine the effect of vemurafenib on PTC and ATC cells. Analysis of radiation clonogenics implicated higher radioresistance in cell lines containing the BRAFV600E mutation as compared to wild-type BRAF. Additionally, forced expression of BRAFV600E in a wild-type thyroid cancer cell line induced radioresistance. Vemurafenib inhibited MAPK signaling in V600E mutant cell lines, but showed no effect in BRAF wild-type cell lines. Vemurafenib pretreatment selectively radiosensitized BRAFV600E mutants in vitro, as assessed by radiation clonogenic assays. Comet assays revealed impairment of DNA repair in BRAFV600E lines when treated with vemurafenib and radiation. Additionally, γ-H2A.x westerns and nuclear foci staining indicated that vemurafenib pretreatment decreases the tumor cell’s ability to repair DNA double-strand breaks (DSB) in BRAFV600E cell lines. Vemurafenib also appeared to alter the kinetics of formation and resolution of 53BP1 and Rad51 nuclear foci in these cell lines. Evaluation of DSB functional repair using GFP reporter constructs, suggests that BRAFV600E induces NHEJ repair, which can be attenuated by vemurafenib treatment. Finally, vemurafenib effectively increases radiosensitivity of BRAF V600E tumors in vivo.

From our results, BRAF activating mutation appears to be associated with radioresistance. Vemurafenib selectively radiosensitizes both PTC and ATC cells through inhibition of DNA repair mechanisms. Combining vemurafenib and radiation may improve therapeutic control for BRAFV600E mutant thyroid cancers.

Citation Format: Ryan N. Robb, Linlin Yang, Terence Williams. Vemurafenib selectively radiosensitizes BRAF V600E mutant papillary and anaplastic thyroid carcinoma 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 5186. doi:10.1158/1538-7445.AM2017-5186
Abstract 5233: Pancreatic cancer specific glycosylations survay by a panel of lectin staining; Tn antigen exposure as a result of o-glycan truncation
Authors: Ozawa Yusuke , Oda Tatsuya , Shimomura Osamu , Tateno Hiroaki , Hirabayashi Jun , Ohkohchi Nobuhiro
Published: July 2017

The outermost coating of every cell type is predominantly composed of cell-specific glycans, and glycosylation changes in cancer has been used in clinical field as a tumor markers, such including AFP and CA19-9. We focused pancreatic cancer, the most intractable cancer with difficulty in early diagnosis, and insisted to glasp glycosylation patterns by using a panel of lectin histochemical stainings. Lectins are subgroup of proteins that specifically recognize and bind glycans, and thus helpful indication of glycosylation patterns. Lectin microarry is one of the advanced technologies which inables glycosylation patters of target materials at onece, however, obtained data with homogenized protein matelials applied lacks histological location and information. Instead, we used a panel of 20 types of lectins histochemical staining for cancer and normal tissues. The staining patterns were firstlly classified according to the ratio between cancer cells and stromal cells. a) Cancer high (cancer cell ++ and stromal cells -) b) equal (cancer cells ++ or + and stromal cells ++ or +) c) Cancer Low (cancer cells - or + and stromal cells ++ or +). Further more, the stainig in cancer cell were compaired with that of non cancerous pancreatic ductal epitherial cells. Among 20 lectins, CNL, Clitocybe nebularis Lectin binds motifs containing α/βGalNAc, Tn antigen, and blood group A, showed cancer cell staining higher than normal tissue in pancreatic cancer. Pancreatic cancer cell (31 of 58 patients,71%) were positive for CNL, but normal pancreatic duct cells (2 of 42 patients,4%) were positive in patients of pancreatic cancer. CNL is known to recognize Tn Antigen in O-glycosylation. Previously, it reported that during malignancy, glycosylation displays abnormal expression of shortened or truncated glycans, such as a Tn antigen, T antigen and their sialylated forms, ST, STn antigen. CNL staining pattern we showed in this report is considered to matching Tn staining. Our result indicated that speccific affinity of CNL lectin in pancreatic cancer and counterpart glycoprotein are STn antigen. We may insist that STn glycoprotein antigen could be used as a diagnostic marker of Pancreatic cancer and also it would be a target of new tumor-targetting strategies.

Citation Format: Yusuke Ozawa, Tatsuya Oda, Osamu Shimomura, Hiroaki Tateno, Jun Hirabayashi, Nobuhiro Ohkohchi. Pancreatic cancer specific glycosylations survay by a panel of lectin staining; Tn antigen exposure as a result of o-glycan truncation [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 5233. doi:10.1158/1538-7445.AM2017-5233
Abstract 5281: Komen Tissue Bank donors: Genetically determined ethnicity and race
Authors: McCarty Julia R. , Jiang Guanlong , Johnson Mariah L. , Mahin Teresa , Scherer Nicholas , Dunn Jonathon , Storniolo Anna Maria , Marino Natascia
Published: July 2017

Background: Several evidences indicate that different racial and ethnic backgrounds affect the incidence and severity of diseases such as breast cancer and diabetes, and the response to therapy. For example White and African American women are more likely to develop breast cancer than Hispanic and Asian women. Moreover, African American women are more likely to develop more aggressive (Triple Negative Breast Cancer), more advanced-stage breast cancer at a young age. Therefore, given the health disparity and with the advance of personalized medicine, it is becoming critical to address population stratification. The Komen Tissue Bank at IU Simon Cancer Center (KTB), the only biobank of normal breast tissue from healthy women, is employing a more accurate approach to detect population stratification through the use of Ancestry Informative Markers (AIMs).

Methodology: A total of 2,973 DNA samples were obtained from the KTB. Genotyping was performed using the KASP technology (LGC Genomics) and a 41-SNP panel (labeled 41-AIM panel) selected from Nievergelt et al, 2013. Genotype analysis using the 41-AIM panel along with a Bayesian clustering method (STRUCTURE) was able to discern continental origins including European/ Middle East (Caucasian), East Asia, Central/South Asia, Africa, Americas, and Oceania. A reference set was obtained from the HeatMap. The results were compared with the self-reported data.

Results/Conclusion: Our genetic ancestry analysis indicates that out of 2,973 KTB tissue donors, 2,132 are primarily Caucasian, 532 African, 234 of Asian, 72 Latino, 3 Oceanic origins. High mixture of racial markers was observed in several samples. Moreover, when we compare these findings with the self-reported data we find a 10.9% of discrepancies indicating the imprecision of the self-reported information. The implementation of the genetic ancestry data into the KTB creates the potential for a more accurate study of health disparities.

Citation Format: Julia R. McCarty, Guanlong Jiang, Mariah L. Johnson, Teresa Mahin, Nicholas Scherer, Jonathon Dunn, Anna Maria Storniolo, Natascia Marino. Komen Tissue Bank donors: Genetically determined ethnicity and race [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 5281. doi:10.1158/1538-7445.AM2017-5281
Abstract 5296: Evaluation of p16/Ki-67 dual stain, cytology, and HPV16/18 genotyping for triage of HPV-positive women in a large screening population
Authors: Wentzensen Nicolas A. , Fetterman Barbara , Bremer Renee , Castle Philip , Tokugawa Diane , Poitras Nancy , Hosfield Elizabeth , Lorey Thomas , Schiffman Mark , Kinney Walter
Published: July 2017

Objectives: Primary HPV testing has been approved in the United States. Screening intervals can be safely extended for HPV-negative women, but the challenge lies in discriminating transient HPV infections from precancers among HPV-positives. In a large study at Kaiser Permanente Northern California (KPNC), candidate strategies for triage of HPV-positive women are being evaluated, including cytology, p16/Ki-67 dual stain (DS, CINtec PLUS) and HPV16/18 genotyping (cobas).

Methods: Over 13,000 HPV-positive women participating in cervical cancer screening at KPNC were enrolled. The dual stain and HPV genotyping assays were implemented and conducted at KPNC. Baseline results for 7,124 women are available and detection of CIN3 is currently evaluated only in cytology-positive women.

Results: Among 7,124 HPV-positive women, 4,107 (57.7%) were cytology-positive (ASC-US+), 3,056 (42.9%) were DS-positive, and 1,406 (19.7%) were positive for HPV16 or HPV18. Among all 3,017 HPV-positive, cytology-negative women, 911 (30.2%) were DS- positive, and 508 (16.8%) were positive for HPV16 or HPV18. Of 315 CIN3 detected so far, 280 (88.9%) were DS-positive and 176 (55.9%) were positive for HPV16 or HPV18. Major triage strategies including cytology alone, DS alone, as well as combinations of cytology and genotyping, and DS and genotyping will be presented at the meeting.

Discussion: In one of the largest clinical implementation studies of triage strategies, we showed that primary HPV screening followed by DS can reduce colposcopy referral compared to HPV-cytology co-testing while achieving high sensitivity. Additional follow-up is underway to evaluate the programmatic performance of several major candidate strategies.

Citation Format: Nicolas A. Wentzensen, Barbara Fetterman, Renee Bremer, Philip Castle, Diane Tokugawa, Nancy Poitras, Elizabeth Hosfield, Thomas Lorey, Mark Schiffman, Walter Kinney. Evaluation of p16/Ki-67 dual stain, cytology, and HPV16/18 genotyping for triage of HPV-positive women in a large screening population [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 5296. doi:10.1158/1538-7445.AM2017-5296
Abstract 5327: Glioma derived cancer stem cells are hypersensitive to proteasomal inhibition
Authors: Lee Dae-Hee , Oh Sang Cheul , Kwon Yong Tae , Lee Yong J. , Yoo Young Dong
Published: July 2017

Although proteasome inhibitors (PIs) are used as anticancer drugs to treat various cancers, their relative therapeutic efficacy on stem cells vs. bulk cancers remains unknown. Here, we show that stem cells derived from gliomas, GSCs, are up to 1,000-fold more sensitive to PIs (IC50, 27-70 nM) compared with their differentiated controls (IC50, 47->>100 µM). The stemness of GSCs correlates to increased ubiquitination, whose misregulation readily triggers apoptosis. PI-induced apoptosis of GSCs is independent of NFκB but involves the phosphorylation of JNK as well as the transcriptional activation of endoplasmic reticulum (ER) stress-associated proapoptotic mediators. In contrast to the general notion that ER stress-associated apoptosis is signaled by prolonged unfolded protein response (UPR), GSC-selective apoptosis is instead counteracted by the UPR. ATF3 is a key mediator in GSC-selective apoptosis. Pharmaceutical uncoupling of the UPR from its downstream apoptosis sensitizes GSCs to PIs in vitro and during tumorigenesis in mice. Thus, a combinational treatment of a PI with an inhibitor of UPR-coupled apoptosis may enhance targeting of stem cells in gliomas.

Key words: glioma; glioma stem cells; proteasome inhibitors; ubiquitin proteasome system; apoptosis; c-Jun N-terminal kinase (JNK)

Citation Format: Dae-Hee Lee, Sang Cheul Oh, Yong Tae Kwon, Yong J. Lee, Young Dong Yoo. Glioma derived cancer stem cells are hypersensitive to proteasomal inhibition [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 5327. doi:10.1158/1538-7445.AM2017-5327
Abstract 5358: Comprehensive evaluation of Illumina’s TruSight® Tumor 170 Panel to estimate tumor mutational burden
Authors: Zhang Shile , So Alex S. , Kaplan Shannon , Kruglyak Kristina M.
Published: July 2017

Tumor mutational burden (TMB), or the density of variants in a tumor, is typically assessed by whole exome sequencing (WES) and has been shown to correlate with efficacy of immunotherapy treatment. Targeted cancer gene panels are broadly used to assess mutational status in cancer related genes but have not historically been used to estimate TMB. Recently, two studies have demonstrated that TMB can be accurately estimated using these cancer gene panelsi,ii. Illumina’s TruSight® Tumor 170 (TST170, research use only, RUO) panel is a comprehensive next-generation sequencing (NGS) assay that covers the coding regions of 170 genes associated with solid tumors. TST170 targets DNA and RNA variants from the same FFPE tumor sample in a single sequencing run. Here we evaluate the performance of TMB estimation with TST170.

TST170 is an enrichment-based targeted panel designed to capture mutational changes, including single nucleotide variant, indels, amplifications, splice variants and fusions. The TST170 analysis pipeline is able to call variants with frequencies down to 5%. In the current study, TMB was calculated as the number of reported variants per megabase after germline polymorphism filtering. First, we evaluated the performance of TST170 for TMB estimation using 6,000 TCGA samples that had been analyzed by WES. TMB estimated from the TST170 targeted regions showed a high correlation to TMB estimated from WES (R2=0.91). Next, we evaluated the prognostic value of TMB estimated from TST170 by overlapping the TST170 targeted regions with WES data for 202 subjects treated with CTLA-4 or PD-1 from three clinical studies. Higher TMB estimated from the TST170 targeted regions was observed in subjects who responded to checkpoint inhibitors. Finally, we assessed the correlation of TMB estimation using matched samples profiled with both WES and TST170 and again saw high correlation between the two methods. In summary, our analysis indicates that the panel content of TST170 can be used to accurately estimate TMB from tumor samples.

i Garofalo A, Sholl L, Reardon B, et al. The impact of tumor profiling approaches and genomic data strategies for cancer precision medicine. Genome Med. 2016;8(1):79. doi:10.1186/s13073-016-0333-9.

ii Campesato, L., Barroso-Sousa, R., Jimenez, L., et al. Comprehensive cancer-gene panels can be used to estimate mutational load and predict clinical benefit to PD-1 blockade in clinical practice. Oncotarget, 2015;6(33), 34221-34227.

Citation Format: Shile Zhang, Alex S. So, Shannon Kaplan, Kristina M. Kruglyak. Comprehensive evaluation of Illumina’s TruSight® Tumor 170 Panel to estimate tumor mutational burden [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 5358. doi:10.1158/1538-7445.AM2017-5358
Abstract 5464: Catalase mediates the survival of anchorage-independent ovarian cancer cells
Authors: Libbing Cassandra L. , Versagli Calli A.
Published: July 2017

Among United States women, ovarian cancer is the fifth leading cause of cancer-related death and the deadliest of the female reproductive system cancers. This is due to a lack of early detection, in effect, leading to primary diagnosis in later, metastasized stages for which there are few effective treatment options. In order to metastasize, ovarian cancer cells must be able to survive detachment from the extracellular matrix (ECM). When a non-tumorigenic mammary epithelial cell detaches from the ECM, research has shown that levels of Reactive Oxygen Species (ROS) rise in the cell and inhibit cell metabolism resulting in decreased cell survival. Previous research has also shown that ECM-detached breast cancer cells are able to utilize antioxidant enzymes to neutralize ROS thereby rescuing ATP levels and promoting cell survival. This antioxidant activity has also been shown to bolster cancer cells’ survival in anchorage independence. Although studied in breast cancer cells, no work has been done in ovarian cancer, which exhibits a very unique mode of metastasis through the peritoneal fluid. To begin to understand the role of antioxidant enzymes in metastatic ovarian cancer cells, we have worked to characterize the role of catalase, a prominent antioxidant enzyme, in the survival of ECM-detached SKOV3 ovarian cancer cells. In order to do so, we utilized short hairpin RNA (shRNA) to engineer SKOV3 cells deficient in catalase. The catalase-deficient cells were then tested for colony formation in detachment and cell viability and proliferation in both attachment and detachment. The results show that elimination of catalase expression does not have a significant effect on proliferation in attachment or detachment, but significantly decreases both cell viability and colony formation of SKOV3 cells grown in ECM-detachment. This suggests that elimination of catalase expression may hold promise for targeted treatment in late stage ovarian cancer, calling for further investigation of the mechanisms behind these results and the role that other antioxidant enzymes play in ovarian cancer. Future studies include looking at this phenomenon in other ovarian cancer cell lines, and using mouse xenograft studies to examine the effects of antioxidant compounds on survival of metastatic ovarian cancer cells.

Citation Format: Cassandra L. Libbing, Calli A. Versagli. Catalase mediates the survival of anchorage-independent ovarian 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 5464. doi:10.1158/1538-7445.AM2017-5464
Abstract 5481: Role and regulation of Peroxiredoxin antioxidant proteins in K562 leukemia cells
Authors: Szabo Elizabeth , Phelan Shelley A.
Published: July 2017

Peroxiredoxins are a family of antioxidant proteins that have found to play a key role in many diseases, including cancer. A wide number of studies have found that Peroxiredoxin levels are elevated in several cancers, and reducing these levels can aid in inhibiting cell growth of cancer cells, suggesting that Peroxiredoxins confer resistance to reactive oxygen species (ROS)-induced cellular toxicity in cancer cells. Specific research has demonstrated that Peroxiredoxin levels are elevated in leukemia cells, and that targeting and inhibiting Peroxiredoxins in these cells can induce differentiation. Studies have also shown the use of doxorubicin as an effective chemotherapy treatment to significantly decrease cell proliferation and viability in leukemia. The purpose of this study is to examine Peroxiredoxin levels in K562 cells, a human chronic myelogenous leukemia cell line, and determine if high levels of Peroxiredoxins have the ability to protect cells from chemotherapy-induced death and possibly aid in chemoresistance. We first demonstrated that serum-deprived K562 cells exhibited significant growth inhibition over a 72 hour time period, but had no significant effect on viability. We also showed that treating K562 cells with 50 nM doxorubicin significantly decreased proliferation and viability. Corresponding changes in Peroxiredoxin protein levels are currently being analyzed to determine the effect of both growth-inhibition and chemotherapy-induced cytotoxicity on the Peroxiredoxin family in K562 cells, and siRNA experiments are being conducted to examine the protective role of Peroxiredoxins in these cells. Based on previous studies on MCF-7 cells conducted in our lab, we predict that Peroxiredoxin levels will be modified under both conditions, and that suppression of Prdxs by siRNA will increase chemotherapy susceptibility.

Citation Format: Elizabeth Szabo, Shelley A. Phelan. Role and regulation of Peroxiredoxin antioxidant proteins in K562 leukemia 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 5481. doi:10.1158/1538-7445.AM2017-5481
Abstract 5507: Investigation of the effects of alterations in the glutamate receptor, GRIK2 on osteosarcoma tumorigenesis
Authors: Mayers Justin G. , Gokgoz Nalan , Wunder Jay S. , Andrulis Irene L.
Published: July 2017

Osteosarcoma is a primary, malignant bone tumour mainly affecting young adults with a five-year survival rate of 60-70% depending on presentation of metastasis at diagnosis. Tumours are marked by genetic instability and heterogeneity and for this reason, molecular subtypes have yet to be characterized. Survival rates have remained consistent over the past few decades and novel molecular targets for treatment are needed. Current genomic strategies have identified recurrent copy number alterations. In a study of 44 flash frozen osteosarcoma tumours which were obtained at the time of biopsy and 25 blood-matched samples we identified recurrent copy number losses including a region at 6q16.3 in 14% of tumours. Focal deletions were observed in the 5’ intergenic space and first intron of the GRIK2 gene and do not overlap with any coding sequence. The relationship between alterations in this region and osteosarcoma tumourigenesis has yet to be explored in depth. GRIK2 encodes an ionotropic glutamate receptor that has been implicated as a tumour suppressor in gastric cancer. An interesting feature of the tumours was the overlap of focal deletions with a SETDB1 histone methyltransferase binding site indicating a potential connection with epigenetic regulation of GRIK2. The goal of this study is to examine the effects of non-coding, focal deletions on GRIK2 gene expression, function and regulation in osteosarcoma cells. GRIK2 transcript levels were measured in osteosarcoma tumours and cell lines using qRT-PCR. Functional assays were performed to characterize the role of GRIK2 in osteosarcoma cell proliferation, migration and apoptosis in vitro. Gene editing with CRISPR/cas9 was performed to examine the link between non-coding deletions and GRIK2 expression. GRIK2 transcript levels varied in tumours; several tumours with focal deletions had increased transcript levels relative to cell lines which demonstrated low expression levels. Due to the observation of low transcript levels, a gain of function approach was employed to examine GRIK2 function in vitro. Overexpression of GRIK2 decreased proliferation, migration and increased apoptosis in osteosarcoma cells. Gene editing of the SETDB1 binding site resulted in increased GRIK2 transcript levels relative to untreated cells. This study suggests that non-coding focal deletions are linked to high expression levels of GRIK2 which may confer a less aggressive phenotype and are involved in epigenetic regulation of the gene.

Citation Format: Justin G. Mayers, Nalan Gokgoz, Jay S. Wunder, Irene L. Andrulis. Investigation of the effects of alterations in the glutamate receptor, GRIK2 on osteosarcoma tumorigenesis [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 5507. doi:10.1158/1538-7445.AM2017-5507
Abstract 5597: Genomic profiling of syngeneic mouse cell lines and in vitro screen of the models against checkpoint inhibitors and target agents for preclinical application
Authors: Xing Frank , Qian Wubin , Dong Chunlan , Xu Xiaoxi , Guo Sheng , Shi Qian
Published: July 2017

Cancer immunotherapy has provided substantial clinical benefit in a significant number of patients with advanced diseases. In pre-clinical studies, CrownBio has developed a large collection of syngeneic models to facilitate in vivo efficacy testing of immune-oncology agents in mice (MuScreenTM). To combine the ostensibly separate therapeutic strategies of activating immune cells against and targeting the unique genetic characteristics of a tumor model, we sought to thoroughly characterize the mutation profiles of these syngeneic mouse cell lines and examine drug response profiles of these cell line models. The goal of this work was to provide an in vitro system in evaluating combination effectiveness when targeting both immune checkpoint markers and oncogenic targets in preclinical studies.

To this end, we investigated mutation and gene expression profiles of 18 mouse cancer cell lines out of the panel of 23 syngeneic mouse models included in the MuScreenTM for 50 well defined cancer-related mutations among 30,690 variants in exonic regions called by RNAseq (Illumina HiSeq X10). The somatic mutations revealed through the bioinformatics analysis include ALK (3 - frequency, same for the rest), BRAF (4), BRCA1 (7), BRCA2 (12), EGFR (3), ERBB2 (6), EGFR3 (2), FBXW7 (10), FLT3 (12), HRAS (1), KRAS (8), NRAS (1), PDGFRA (11), PTCH1 (9), PIK3CA (2), PTEN (6), RET (3), SETD2 (5), SMAD4 (3), SMO (13), TRP53 (13), TSC1 (3), and TSC2 (10). All of these oncogenic alterations are clinically actionable (Cancer Discovery 2, 82-93, 2012). The same set of genes was also subject to mRNA expression change analysis.

We screened the 18 mouse cell lines with a few selected target therapy agents, and performed combination assays of these agents together with check point inhibitors such as aPD1, aPDL1, in a co-culture system with mouse T cells. An IncuCyte real-time imaging platform was used to distinguish activities on T cells and tumor cells. The results and the implications will be discussed.

Citation Format: Frank Xing, Wubin Qian, Chunlan Dong, Xiaoxi Xu, Sheng Guo, Qian Shi. Genomic profiling of syngeneic mouse cell lines and in vitro screen of the models against checkpoint inhibitors and target agents for preclinical application [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 5597. doi:10.1158/1538-7445.AM2017-5597
Abstract 56: Antibody-drug conjugates containing glucuronide-tubulysin payloads display activity in MDR+ and heterogeneous tumor models
Authors: Burke Patrick J. , Hamilton Joseph Z. , Hunter Joshua H. , Cochran Julia H. , Pires Thomas A. , Leiske Christopher I. , Emmerton Kim K. , Senter Peter D. , Lyon Robert P. , Jeffrey Scott C.
Published: July 2017

While antibody-drug conjugates (ADCs) find increasing application in cancer treatment regimens, de novo or treatment-emergent resistance mechanisms could impair clinical benefit. Two resistance mechanisms that emerge under continuous ADC exposure in vitro include upregulation of transporters that confer multidrug resistance (MDR+) and loss of cognate antigen expression. New technologies that circumvent these resistance mechanisms may serve to extend the utility of next generation ADCs. Recently, we developed the quaternary ammonium linker system to expand the scope of conjugatable payloads to include tertiary amine-containing compounds and applied the linker to tubulysins, a highly potent class of microtubule disrupting agents that maintain activity in MDR+ cell lines. Quaternary ammonium-linked glucuronide-tubulysin drug-linkers were synthesized and evaluated as ADCs. The resulting conjugates were potent and immunologically specific across a panel of cancer cell lines, including those displaying the MDR phenotype. The ADCs also demonstrate potent bystander activity in a co-culture model containing a mixture of antigen-positive and -negative cell lines. Incorporation of a PEG12 side chain in the linker enabled loading at 8-drugs/Ab for increased in vivo potency while maintaining suitable ADC pharmacokinetic properties. In vivo, the glucuronide-tubulysin conjugates displayed activity in MDR+ xenograft models and bystander activity in an admixed Ag+/Ag- heterogeneous tumor model. Thus, the glucuronide-tubulysin drug-linkers represent a promising new payload for ADCs, combining conjugate potency in the presence of the MDR phenotype with robust activity in models of tumor antigen heterogeneity.

Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Joshua H. Hunter, Julia H. Cochran, Thomas A. Pires, Christopher I. Leiske, Kim K. Emmerton, Peter D. Senter, Robert P. Lyon, Scott C. Jeffrey. Antibody-drug conjugates containing glucuronide-tubulysin payloads display activity in MDR+ and heterogeneous tumor models [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 56. doi:10.1158/1538-7445.AM2017-56
Abstract 5637: An oral Androgen Receptor PROTAC degrader for prostate cancer
Authors: Neklesa Taavi K. , Snyder Lawrence B. , Bookbinder Mark , Chen Xin , Crew Andrew P. , Crews Craig M. , Dong Hanqing , Gordon Deborah , Macaluso Jennifer , Raina Kanak , Rossi AnnMarie , Taylor Ian , Vitale Nicholas , Wang Jing , Willard Ryan R. , Zimmermann Kurt
Published: July 2017

The Androgen Receptor (AR) remains the principal driver of castration-resistant prostate cancer during the transition from a localized to metastatic disease. Most patients initially respond to inhibitors of the AR pathway, but the response is often short-lived. The majority of patients progressing on enzalutamide or abiraterone exhibit genetic alterations in the AR locus, either in the form of amplifications or point mutations in the AR gene. Given these mechanisms of resistance, our goal is to eliminate the AR protein using the PROteolysis TArgeting Chimera (PROTAC) technology. Further, we sought to make an orally bioavailable AR PROTAC. Medicinal chemistry efforts yielded a small molecule AR PROTAC that simultaneously binds E3-ubiquitin ligase and AR, thus leading to ubiquitination and degradation of AR. This molecule has been characterized in in vitro and in vivo preclinical studies. Our lead oral AR PROTAC degrades approximately 98% of total AR in all cell lines tested, with 50% degradation concentration (DC50) < 1 nM. AR degradation suppresses the expression of AR-target gene PSA, inhibits cell proliferation, and induces potent apoptosis in VCaP cells. No activity is observed in AR-null cell lines, such as PC-3. While enzalutamide loses its activity in the presence of elevated androgens, AR PROTAC maintains its antiproliferative activity. Further, AR PROTAC is able to degrade all clinically relevant mutant AR proteins. A robust oral bioavailability is observed across multiple species and overall ADME properties are encouraging. Approximately 95% AR degradation is observed in AR-amplified VCaP xenografts at doses as low as 10 mg/kg. Congruent with AR degradation, a dose responsive tumor growth inhibition is observed in AR-dependent xenograft studies. In summary, we report the first orally bioavailable AR PROTAC that robustly degrades AR in vitro and in vivo.

Citation Format: Taavi K. Neklesa, Lawrence B. Snyder, Mark Bookbinder, Xin Chen, Andrew P. Crew, Craig M. Crews, Hanqing Dong, Deborah Gordon, Jennifer Macaluso, Kanak Raina, AnnMarie Rossi, Ian Taylor, Nicholas Vitale, Jing Wang, Ryan R. Willard, Kurt Zimmermann. An oral Androgen Receptor PROTAC degrader for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5637. doi:10.1158/1538-7445.AM2017-5637