bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 809: Response of metabolic phenotype to chemotherapy agents in a simulated tumor microenvironment
Authors: Hoffman David L. , Rumble Julie M.
Published: August 2019

Chemotherapeutic agents inhibiting fatty acid oxidation can sensitize cells to the tumor microenvironment.

Chemotherapeutic agents (CA), while standard-of-care for treating numerous types of cancer are, by nature, poisonous. Studies have shown that treatment with certain CA, such as Irinotecan, leads to chemotherapy associated steatohepatitis (CASH) resulting from the inhibition of mitochondrial fatty acid oxidation (FAO). While inhibition of FAO may have harmful effects on the liver, it may have a different effect in the tumor microenvironment (TME). Within the TME exists a unique metabolic environment, consisting of low [O2], low pH and high concentrations of fatty acids. Occupying the TME are immunosuppressive myeloid derived suppressor cells (MDSCs), which aid in tumor growth by suppressing T cells, which would normally attack the tumor. It is thought that the MDSCs localized to the TME undergo an alteration in metabolic phenotype, allowing them to utilize the high concentrations of fatty acids found in the TME as their primary carbon source, becoming reliant on FAO. It is therefore hypothesized that normoxic MDSCs will alter their metabolic phenotype in response to a simulated TME to favor FAO. This altered metabolic phenotype will sensitize MDSCs to inhibition of FAO by SN-38, the active metabolite of Irinotecan. The response of metabolic phenotype to hypoxic conditions will also be examine in HCT116 colorectal carcinoma cells.

Note: This abstract was not presented at the meeting.

Citation Format: David L. Hoffman, Julie M. Rumble. Response of metabolic phenotype to chemotherapy agents in a simulated tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 809.
Abstract 815: Genome-wide DNA methylation signatures to predict pathologic complete response from combined neoadjuvant chemotherapy with bevacizumab in breast cancer
Authors: Hsu Ping-Ching , Kadlubar Susan , Siegel Eric , Rogers Lora J. , Todorova Valentina K. , Su Joseph , Makhoul Issam
Published: August 2019

Neoadjuvant chemotherapy is given before surgery to patients with primarily unresectable, advanced-stage cancers to render the tumor resectable, and to facilitate breast conservation. Previously, we have reported a prospective phase II trial for women with stage IIA-B/IIIA-B-C breast cancer with improved pathologic complete response (pCR) when using bevacizumab in the neoadjuvant setting. Chemotherapy agents are given orally or intravenously during multiple cycles of systemic treatments. However, the effect of the treatment can only be evaluated upon the completion of therapy. Here, we aim to identify epigenetic signatures in blood for patients at baseline as non-invasive markers to predict their pCR from the treatment, and to determine if treatment-related changes in epigenetic profiles reflect responsiveness to therapy. We performed genome-wide DNA methylation profiling using blood-derived DNA, and found that pre-treatment methylation status of BRD9 was predictive of responsiveness to therapy. Post-treatment global methylation differences were also observed between responders and non-responders. Most differentially methylated CpGs were located in promoter CpG island regions for responders, but in the open sea region for non-responders. DNMT3B was hypomethylated while most of the other genes were hypermethylated after 4 cycles of treatment. Hypomethylation of DNMT3B could potentially lead to the increased methylation of oncogenes and genes responsible for cell growth and proliferation, facilitating responsiveness to the therapy. These results support the development of BRD9 as a biomarker for treatment selection before neoadjuvant therapy with bevacizumab, and indicate DNMT3B as a potential target to improve clinical response.

Citation Format: Ping-Ching Hsu, Susan Kadlubar, Eric Siegel, Lora J. Rogers, Valentina K. Todorova, Joseph Su, Issam Makhoul. Genome-wide DNA methylation signatures to predict pathologic complete response from combined neoadjuvant chemotherapy with bevacizumab in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 815.
Abstract 817: Assessment and manipulation of DNA methylation in cancer cells using terahertz radiation
Authors: Yang Hee-Jin , Cheon Hwayeong , Paik Jin Ho , Son Joo-Hiuk
Published: August 2019

Carcinogenesis is closely associated with abnormal regulation of gene expression, among which DNA methylation in one of the key epigenetic modifications. Therefore, several studies have been directed towards the evaluation of DNA methylation. Here, we aimed to experimentally observe the specific signal of methylated DNA in various blood cancer cell lines using terahertz (THz) radiation, which is presented as a resonance in the spectrum. THz radiations are electromagnetic waves having frequencies of 0.1−10 THz, which falls between those of infrared radiation and microwaves. Molecular low-energy motions in THz region, such as collective vibrations, torsion of molecular structures, and hydrogen bonds, is correlated with biological characteristics, i.e., vibrational modes of DNA, structural modes in proteins, cell membrane vibrations, or enzyme activity. These features are usually presented as a characteristic resonance of the modes in the THz spectrum. In our study, the degree of global DNA methylation was obtained by the quantification of the resonance around 1.7 THz, and the results were in good agreement with those obtained by enzyme-linked immunosorbent assay (ELISA). Further, DNA demethylation was performed using high-power THz radiation around the resonance frequency of 1.7 THz, which lead to a 10% to 72% decrease in the degree of DNA methylation as shown in Table 1. Thus, our data shows the feasibility of using THz radiation for the assessment and manipulation of DNA methylation. This is the first study wherein the sensing and manipulation of DNA methylation could be achieved using a non-contact and non-invasive optical technique. Use of THz radiation for the assessment of DNA methylation might be helpful for its rapid processing, while THz manipulation of DNA methylation can avoid the toxicity of chemical demethylating agents. We propose that THz radiation may be useful for the diagnosis of cancer and we expect that THz demethylation might find potential application in cancer therapy.

Change of methylation status of blood cancer DNA using high-power THz radiation.SamplesNo ExposureHigh-power THz exxpsoureChange (%) THzChange (%) ELISATHzELISARatio1THzELISARatio2119.621.8510.6111.661.0611.0040.5742.70211.341.388.2210.201.198.5710.0513.77315.151.4310.596.240.738.5558.8148.95417.731.4712.063.510.447.9880.2070.07511.271.437.8810.001.367.3511.274.90STDEV1.58STDEV1.24THz, the amplitude of terahertz resonance; ELISA, optical density of Enzyme linked immunosorbent assay; STDEV, standard deviation1 THz/ELISA of no exposure2 THz/ELISA of high-power THz exposure

Citation Format: Hee-Jin Yang, Hwayeong Cheon, Jin Ho Paik, Joo-Hiuk Son. Assessment and manipulation of DNA methylation in cancer cells using terahertz radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 817.
Abstract 820: Enzymatic Methyl-Seq: methylome analysis of challenging DNA samples
Authors: Williams Louise , Ponnaluri V K Chaithanya , Sexton Brittany S. , Saleh Lana , Marks Katherine , Samaranayake Mala , Ettwiller Laurence , Guan Shengxi , Church Heidi E. , Dai Nan , Tamanaha Esta , Yigit Erbay , Langhorst Bradley , Sun Zhiyi , Evans Thomas C. , Vaisvila Romualdas , Dimalanta Eileen , Davis Theodore B.
Published: August 2019

DNA isolated from blood draws (cell-free DNA (cfDNA)) or from archival material like formalin fixed paraffin embedded (FFPE) tissues have advanced the field of cancer genetics. DNA methylation (5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC)) is a key epigenetic factor that plays an important role in cellular processes and it’s misregulation results in diseased states like cancer. Advances in the field of sample preparation from biological matrices and genomics have enabled cancer biomarker identification based on methylation profiling.

Bisulfite sequencing is the standard method to detect methylation and has been employed for both targeted and whole genome methylation analysis. However, the chemical based bisulfite conversion of cytosines to uracils also results in DNA damage which subsequently results in shorter DNA insert sizes as well as introducing bias into the data. Robust biomarker detection relies primarily on the ability to profile methylation accurately. Analysis of DNA methylation from cfDNA and FFPE DNA is challenging as the DNA is typically of low quality and quantity. To overcome the drawbacks of bisulfite sequencing, we developed an enzyme based methylation detection technology, called NEBNext Enzymatic Methyl-Seq (EM-Seq). DNA damage is minimized enabling longer insert sizes, lower duplication rates and minimal GC bias resulting in more accurate quantification of methylation in the sample DNA.

Using EM-Seq, we profiled cfDNA and FFPE DNA from multiple tissue types. Results for these challenging DNA types showed that the EM-Seq libraries had longer inserts, lower duplication rates, higher percentages of mapped reads and less GC bias compared to WGBS libraries. These libraries also identified a higher number of CpG’s and the estimated global methylation levels were in good agreement with the absolute levels quantified using LC/MS. In conclusion, EM-Seq libraries have superior sequencing metrics resulting in robust methylation profiling for these types of challenging DNA samples.

Citation Format: Louise Williams, V K Chaithanya Ponnaluri, Brittany S. Sexton, Lana Saleh, Katherine Marks, Mala Samaranayake, Laurence Ettwiller, Shengxi Guan, Heidi E. Church, Nan Dai, Esta Tamanaha, Erbay Yigit, Bradley Langhorst, Zhiyi Sun, Thomas C. Evans, Romualdas Vaisvila, Eileen Dimalanta, Theodore B. Davis. Enzymatic Methyl-Seq: methylome analysis of challenging DNA samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 820.
Abstract 821: DNA hypermethylation of IRX4 is a frequent event that may confer growth advantage to pancreatic cancer cells
Authors: Chakma Kanchan , Gu Zhaodi , Motoi Fuyuhiko , Unno Michiaki , Horii Akira , Fukushige Shinichi
Published: August 2019

Epigenetic gene silencing by aberrant DNA methylation is one of the important mechanisms leading to the loss of key cellular pathways in tumorigenesis. Methyl-CpG targeted transcriptional activation (MeTA) reactivates hypermethylation-mediated silenced genes in a different way from DNA demethylating agents. Previous study using microarray coupled with MeTA (MeTA-array) identified seven commonly hypermethylation-mediated silenced genes in pancreatic cancer cell lines. In this study, IRX4 (Iroquois homeobox 4) was focused on because IRX4 was located at chromosome 5p15.33 where one of pancreatic cancer susceptibility loci has been identified through genome-wide association study (GWAS). IRX4 has also been identified as a candidate tumor suppressor gene in prostate cancer. IRX4 was greatly downregulated in all of the analyzed 12 pancreatic cancer cell lines by promoter hypermethylation. In addition, IRX4 promoter region was found to be frequently and specifically hypermethylated in primary resected pancreatic cancers (15/22: 68%); corresponding normal pancreatic tissues were unmethylated. To examine whether IRX4 has the growth inhibitory effect on pancreatic cancer cell lines, colony formation assay using PK-1 and PK-9 cell lines has been performed; compared with empty vector-transfected cells, IRX4 expression vector-transfected cells formed significantly reduced number of colonies. Furthermore, tetracycline-inducible IRX4 expressing vector system was constructed and a pancreatic cancer cell line PK-1 was analyzed for further functional investigation; induction of IRX4 suppressed cell growth in PK-1. Our results suggest that DNA methylation-mediated silencing of IRX4 is a frequent event that may confer growth advantage to pancreatic cancer cells and MeTA may be a powerful tool to search critical hypermethylated genes in tumorigenesis.

Citation Format: Kanchan Chakma, Zhaodi Gu, Fuyuhiko Motoi, Michiaki Unno, Akira Horii, Shinichi Fukushige. DNA hypermethylation of IRX4 is a frequent event that may confer growth advantage to pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 821.
Abstract 835: Altered DNA modifications in Barrett’s oesophagus and oesophageal adenocarcinoma
Authors: Kriaucionis Skirmantas , Tomova Marketa , Kirschner Sophie , Brazauskas Pijus , Kgatle Mankgopo , Owen Richard , White Michael , Schuster-Boeckler Benjamin , Lu Xin
Published: August 2019

Cytosine in human DNA can be modified by DNA methyltransferases (DNMTs) and the Ten-Eleven Translocation (TET) family of oxygenases. Activity of these enzymes results in four modifications, among which 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the major ones. Different patterns of localisation of both these modifications relate to gene transcription and the developmental origin of different cell types. Cancers have been shown to have globally altered DNA modifications, however it is poorly understood when in carcinogenesis the shift in DNA modifications happens and how it influences tumour growth. Here we mapped 5mC and 5hmC genome-wide at single nucleotide resolution in Barrett’s oesophagus, a well-known pre-malignant precursor of oesophageal adenocarcinoma (OAC). To get better insight into alterations in Barrett’s oesophagus (BO), we also analysed samples from OAC, adjacent normal oesophagus and gastric and duodenal epithelia. We found substantial global hypomethylation outside genic regions in Barrett’s oesophagus. Specific hypermethylation was evident in gene regulatory regions as defined by DNAseI hypersensitive sites. Moreover, we observed elevated methylation at CpG islands in a fraction of genes, which have reduced expression in Barrett’s oesophagus. While we find 5hmC depleted both in BO and OAC, distribution of the modification in CpG islands is different. Overall our results demonstrate that cancer-specific epigenetic alterations can already be found in benign Barrett’s oesophagus, suggesting early redistribution of DNA modifications in the process of transformation.

Citation Format: Skirmantas Kriaucionis, Marketa Tomova, Sophie Kirschner, Pijus Brazauskas, Mankgopo Kgatle, Richard Owen, Michael White, Benjamin Schuster-Boeckler, Xin Lu. Altered DNA modifications in Barrett’s oesophagus and oesophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 835.
Abstract 838: Epigenetic reprogramming of different cancer-related pathways at the single cell level after low-dose DNA demethylation therapy
Authors: Takeshima Hideyuki , Yoda Yukie , Watanabe Naoko , Wakabayashi Mika , Ushijima Toshikazu
Published: August 2019

The efficacy of DNA demethylation therapy is highly dependent on the dosing regimen. Administration of a drug at a dose much lower than the maximum tolerated dose (MTD) for a prolonged period shows a high therapeutic efficacy, despite its small demethylating effect. However, the mechanisms underlying the high therapeutic efficacy of low-dose treatment are still unclear. In this study, we aimed to reveal the mechanisms by analyzing decitabine (DAC)-treated individual cancer cells. To reveal the epigenetic reprogramming at the single cell level and its functional consequences, individual DAC-treated HCT116 cells (H3 clones) were cloned from DAC-treated bulk HCT116 cells. Genome-wide DNA methylation was analyzed using InfiniumHuman450 beadChip. In bulk cells, only partial reduction of DNA methylation levels was observed, and no genes were completely demethylated. In contrast, in cloned cells, a much larger reduction of DNA methylation levels was observed, depending upon genes. Promoter CpG islands of 233-470 genes of 1,188 hypermethylated genes in HCT116 were completely demethylated (Δβ ≥ 0.6) in some of the clones analyzed. Demethylated genes were highly variable among individual clones, and different cancer-related genes, such as those involved in the p53 pathway, WNT pathway, and cell cycle regulation, were completely demethylated, depending upon clones. For example, CDKN2A (p16) was completely demethylated only in a H3-32 clone, and this clone showed slower cell growth rate (14.6% of untreated cells), and aneuploid cells were detected possibly due to cellular senescence. These results showed that epigenetic reprogramming at the single cell level involves diverse cancer-related pathways, and this mechanism is likely to underlie the high efficacy of low-dose DNA demethylation therapy.

Citation Format: Hideyuki Takeshima, Yukie Yoda, Naoko Watanabe, Mika Wakabayashi, Toshikazu Ushijima. Epigenetic reprogramming of different cancer-related pathways at the single cell level after low-dose DNA demethylation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 838.
Abstract 841: Meningioma subgroups associated with functional genomic elements defined by DNA methylation
Authors: Malta Tathiane M. , Snyder James , Wells Michael , deCarvalho Ana , Poisson Laila , Souza Camila , Zadeh Gelareh , Aldape Kenneth , Tirapelli Daniela , Carlotti Carlos , Lee Yan , Kalkanis Steven , Walbert Tobias , Noushmehr Houtan
Published: August 2019

Although most meningioma are non-malignant, there is a high recurrence rate among atypical and anaplastic (malignant) meningiomas (grades II/III). In addition, malignant meningioma usually progresses after treatment. Recently, based on DNA methylation, two subgroups of meningioma were described with recurrence-free survival differences. Epigenetic deregulation at distinct genomic elements can affect changes in gene expression and alter the transcriptional profile of the cancer cells. We seek to understand the mechanisms of meningioma recurrence and progression after initial treatment. In order to address this, we will use DNA methylation data to identify candidate noncoding elements and their connection with genes that might explain differences in meningioma prognostic subgroups. Using published DNA methylation data we compared favorable and unfavorable meningioma subgroups and identified 3,045 differentially methylated probes (p< 0.0001, difference mean-methylation beta-value> 0.2). Focusing on probes within known functional genomics, we identified 18 highly conserved genomic enhancers known to be activated in cancer that can potentially drive meningioma recurrence. We next investigated links between these enhancers and their targeted genes by incorporating GeneHancer annotation. We found that the unfavorable subgroup of meningiomas presented hypomethylation within enhancer regions that have the potential to target PARK7, ARID4B, and FBH1. ARID4B was previously shown to be highly active in high-grade meningiomas. We also identified 16 enhancer regions that overlap known prognostic cancer enhancer, previously identified in other tumor types. Our findings were validated in independent cohort comprised by public and unpublished DNA methylation datasets. Our preliminary results are the first to suggest that DNA methylation changes can be used to identify noncoding regions associated with meningioma prognosis. Identification of noncoding regions associated with meningioma recurrence will provide knowledge of the role of epigenomics in the development of malignant meningioma and of opportunities for targeted therapy.

Citation Format: Tathiane M. Malta, James Snyder, Michael Wells, Ana deCarvalho, Laila Poisson, Camila Souza, Gelareh Zadeh, Kenneth Aldape, Daniela Tirapelli, Carlos Carlotti, Yan Lee, Steven Kalkanis, Tobias Walbert, Houtan Noushmehr. Meningioma subgroups associated with functional genomic elements defined by DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 841.
Abstract 842: Racial disparity in breast cancer: Tissue-specific epigenetic mechanisms
Authors: Lin Nan , Castle James , Liu Jinpeng , Shendre Aditi , Wang Chi , Liu Yunlong , He Chunyan
Published: August 2019

Racial disparity in breast cancer is well recognized. Compared with European American (EA) women, African American (AA) women have higher mortality rate despite low incidence rate, and they are more likely being diagnosed at younger age and with features that are more aggressive. Many risk factors of breast cancer, including social, culture, behavioral, genetics, and environmental factors, are disproportionally distributed across two racial groups. These factors could influence DNA methylation differently, as suggested by recent reports that describe biological differences of breast tumor in different racial group. However, no studies have systematically evaluate tissue-specific epigenetic mechanisms underlying the observed racial disparity in breast cancer development. We conducted a molecular epidemiological study of 276 EA and 186 AA healthy women. Genome-wide DNA methylation profiling was performed in normal breast tissue using the Illumina Methyl Captured EPIC-sequencing. Genome-wide association between race and DNA methylation identified differentially methylated sites near genes implicated in metastatic potential and aggressiveness of tumor, tamoxifen-resistance, immune response, and cancer-related metabolic pathways. Many of these genes have not been reported previously to be associated with breast cancer or racial disparities. Further analyses showed that differentially methylated sites across two racial groups were significantly overlapped with differentially methylated sites in tumor and normal breast tissue, suggesting tissue- and racial-specific epigenetic mechanisms underlying breast cancer development. Furthermore, meQTL analyses between known breast cancer risk loci and DNA methylation also suggested AA-specific risk loci were associated with DNA methylation sites implicated in metastatic potential and aggressiveness of tumor. Our study sheds light on tissue-specific genetic and epigenetic mechanisms underlying racial disparity in breast cancer. Further research is warranted to validate the findings in larger studies and to understand gene regulation network by linking with RNA-seq data.

Citation Format: Nan Lin, James Castle, Jinpeng Liu, Aditi Shendre, Chi Wang, Yunlong Liu, Chunyan He. Racial disparity in breast cancer: Tissue-specific epigenetic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 842.
Abstract 843: Epigenomic disruption of adipogenic regulators in dedifferentiated liposarcoma
Authors: Takamatsu Hironori , Hattori Naoko , Asano Naofumi , Iida Naoko , Yoshida Akihiko , Kobayashi Eisuke , Nakayama Robert , Matsumoto Morio , Nakamura Masaya , Kawai Akira , Ushijima Toshikazu
Published: August 2019

Well-differentiated liposarcomas (WDLPS) and dedifferentiated liposarcomas (DDLPS) are the most common soft-tissue sarcomas in adults. They develop as a result of a disturbance in adipogenic differentiation however, the epigenomic changes underlying liposarcomagenesis are unknown. Here, we aimed to elucidate the role of epigenomic alterations in the initiation of DDLPS. Genome-wide DNA methylation profiles of 15 liposarcoma (6 WDLPS and 9 DDLPS) samples and 6 normal adipose tissue samples were obtained using the Infinium MethylationEPIC BeadChip. After grouping neighboring probes into genomic blocks (GBs, < 500 bp), principal component analysis of all the CpG GBs (n = 535,684) was conducted. Each of normal adipose tissue and WDLPS grouped closely while DDLPS distributed broadly, indicating heterogeneous methylation profiles among the DDLPS samples. Unsupervised hierarchical cluster analysis of enhancers (2,000 GBs with the high SD of 23,478 GBs) classified the samples into three tissue types, whereas that of promoters (1,000 GBs with the high SD of 8,668 GBs) could not classify the DDLPS samples distinctly. The Jonckheere-Terpstra trend test revealed 9,945 hypermethylated (p < 0.01, Δβ > 0.2) GBs in DDLPS compared to those in normal adipose tissues and WDLPS samples. Based upon a report on enhancers in adipocytes (Mikkelsen et al. Cell 143, 156-169. 2010), the hypermethylated GBs in DDLPS were enriched with typical- (2.2%: whole genome 1.2%) and super- (13.5%: whole genome 7.0%) enhancers. Genes involved in adipogenesis, including PPARG2, the master regulator of adipogenesis, and its target genes (FABP4 and PLIN1), were aberrantly methylated at the enhancers and repressed in DDLPS. Conversely, promoters were not enriched among hypermethylated GBs in DDLPS (1.2%: whole genome 4.1%). Taken together, these data indicate that aberrant methylation at the enhancers of adipogenic genes might be crucial for the generation of DDLPS. Evaluation of a demethylating agent in combination with a PPARγ agonist as a potential therapeutic is in progress.

Citation Format: Hironori Takamatsu, Naoko Hattori, Naofumi Asano, Naoko Iida, Akihiko Yoshida, Eisuke Kobayashi, Robert Nakayama, Morio Matsumoto, Masaya Nakamura, Akira Kawai, Toshikazu Ushijima. Epigenomic disruption of adipogenic regulators in dedifferentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 843.
Abstract 850: Evaluating preclinical efficacy of anti-HER2 drug combinations using ER+/HER2 mutant models
Authors: Devarakonda Vaishnavi , Williams LaTerrica , Seker Sinem , Lei Jonathan T. , Singh Purba , Han Airi , Anurag Meenakshi , Holloway Kimberly R. , Welm Alana L. , Ellis Matthew J. , Kavuri Shyam M.
Published: August 2019

Targeting HER2 is one of the greatest successes in oncology, and has resulted in the generation of a wide array of HER2-targeting agents. Our genomic approaches are revealing other mechanisms of HER2 activation, such as our discovery of activating HER2 mutations in different cancer types. From initial breast cancer and SUMMIT trial data, the pan-HER drug neratinib as monotherapy showed initial clinical response in ER+ breast cancer, but with frequent early relapse. This study investigates the preclinical efficacy of anti HER2 agents alone or in combination with endocrine therapy agents or in combination with CDK4/6 inhibitors using ER+/HER2 mutant cell lines and ex vivo HER2 mutant patient derived xenograft (PDX) model. ER+ breast cancer cell lines (T47D and MCF7) stably expressing HER2V777L, and ER+/HER2 mutant PDX model (HER2G778_P780 dup) were used to examine HER2 signaling. We found that MCF7/T47D cells expressing HER2V777L and HER2G778_P780 dup PDX tumors showed strongly activated autophosphorylation of HER2 and increased expression of CDK4, CDK6, phospho-Rb, and cyclin D1 as compared to MCF7/T47D cells expressing HER2WT or ER+/non-HER2mut PDX model, suggesting that HER2 mutations preferentially depend on CDK4/6 signaling for cell growth. Additionally, we showed that activating MCF7 HER2V777L cause resistance to endocrine therapy treatment (fulvestrant IC50 >5μM). Further, we show that neratinib alone is effective at higher concentrations (IC50 < 2μM) in MCF7/HER2V777L cells. Next we showed that abemaciclib alone exhibited moderate activity against MCF7 HER2V777L cells (IC50 < 0.4μM) and additional activity in combination with neratinib (IC50 < 0.06μM) was seen. Moreover, ex vivo HER2G778_P780 dup cells are relatively resistant to fulvestrant alone (IC50 < 0.2μM), neratinib alone (IC50 < 0.006μM), abemaciclib alone (IC50 < 0.04μM), and neratinib in combination with abemaciclib (IC50 < 0.005μM), suggesting that patients harboring ER+/HER2-mutant tumors may benefit from neratinib in combination with abemaciclib. Therefore, we propose that simultaneous targeting of both HER2 and the CDK4/6 axis is required for maximal inhibition of ER+ breast cancers harboring HER2 activating mutations.

Citation Format: Vaishnavi Devarakonda, LaTerrica Williams, Sinem Seker, Jonathan T. Lei, Purba Singh, Airi Han, Meenakshi Anurag, Kimberly R. Holloway, Alana L. Welm, Matthew J. Ellis, Shyam M. Kavuri. Evaluating preclinical efficacy of anti-HER2 drug combinations using ER+/HER2 mutant models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 850.
Abstract 865: Isolation of circulating tumors cells from genetically engineered mouse models of lung adenocarcinoma
Authors: Burton Liza J. , Koo Junghui , Tucker-Burden Carol , Zhou Wei , Gilbert-Ross Melissa , Huang Chunzi , Sica Gabriel , Marcus Adam
Published: August 2019

The major cause of cancer-associated mortality is tumor metastasis, but our understanding of this process is far from complete. During successful dissemination, tumor cells invade the surrounding tissue of the primary tumor, intravasate into blood and lymphatic vessels, translocate to distant tissues, extravasate, adapt to the new microenvironment, and eventually seed, proliferate, and colonize to form metastases. Because dissemination mostly occurs through the blood, circulating tumor cells (CTCs) that have been shed into the vasculature and may be on their way to potential metastatic sites are of obvious interest (1). KRAS mutations are the most frequent oncogenic drivers of non-small cell lung cancer and when associated with co-mutations lead to a decrease in overall survival. We have previously established a lenti-Cre-induced Kras and Lkb1 mutant and Kras and p53 mutant genetically engineered mouse modes (KLLenti) and (KPLenti) that develop 100% lung adenocarcinoma to conduct the first study to isolate and maintain primary, CTC and metastatic cells that are KLLenti or KPLenti. We are able to isolate TTF-1+ and pan-cytokeratin+ CTCs from the KLlenti and KPlenti mice and validated their mutational status by genotyping, western blot, and immunofluorescence. Moreover, lkb1-mutant or p53-mutant primary tumor cells have different 3-D invasive properties and patterns, as well as the respective CTCs and metastatic cells when compared across different genetic sub-types. To further study gene expression patterns between primary, CTC, and metastatic sites RNAseq will be employed to identify pathways that drive metastasis within the genetic sub-types.

Citation Format: Liza J. Burton, Junghui Koo, Carol Tucker-Burden, Wei Zhou, Melissa Gilbert-Ross, Chunzi Huang, Gabriel Sica, Adam Marcus. Isolation of circulating tumors cells from genetically engineered mouse models of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 865.
Abstract 868: Epigenomic profiling of primary hepatocellular carcinoma reveals super-enhancer-associated chromatin regulator network
Authors: XU Liangliang , WU Feng , CHEUNG Otto K.W. , SZETO Lemuel L.M. , MOK Myth T.S. , Yip Kevin Y.L. , To Ka F. , CHENG Alfred S.L.
Published: August 2019

Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality worldwide. Parallel with the growing epidemics of obesity and diabetes, non-alcoholic fatty liver disease (NAFLD) has become the predominant cause leading to HCC in a dysregulated metabolic background. Recent HCC genomics studies present only a paucity of recurrent gene mutations, which lead to a sparkling interest in the epigenetic regulation of hepatic carcinogenesis. Chromatin modifications convert the metabolic insults from NAFLD to transcriptional program that contributes to HCC development. Super-enhancers (SEs) are subclass of regulatory elements with unusually strong enrichment for the binding of transcriptional coactivators to fulfill cell identity. Currently, it has been proposed that dysregulation of SEs underlies the development of diseases including cancers, but the role of SEs in NAFLD-associated HCC is still unknown.

In order to investigate the alterations of histone modifications in NAFLD-associated HCC development, nanoscale chromatin immunoprecipitation sequencing of multiple histone marks were performed. By integrating multiple epigenomic profiles from primary NAFLD-associated HCCs and matched non-tumor tissues, we identified recurrent SEs enriched that contribute to fatty liver, lipid metabolism and metabolic syndrome. Interestingly, we observed significant enrichment of recurrent active SEs in a number of target genes. Notably, through CRISPR/Cas9-induced knockout liver cell lines, we showed that deletion of two distinct SE regions significantly reduced the expression of a candidate target gene and attenuated the tumorigenic potential of HCC cells. In summary, our results show that the dysregulation of SEs contributes to human hepatic carcinogenesis, thus representing novel therapeutic targets for NAFLD-associated HCCs.

Citation Format: Liangliang XU, Feng WU, Otto K.W. CHEUNG, Lemuel L.M. SZETO, Myth T.S. MOK, Kevin Y.L. Yip, Ka F. To, Alfred S.L. CHENG. Epigenomic profiling of primary hepatocellular carcinoma reveals super-enhancer-associated chromatin regulator network [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 868.
Abstract 870: The role of the intermediate prolactin receptor in breast cancer oncogenesis
Authors: Grible Jacqueline M. , Clevenger Charles V.
Published: August 2019

Epidemiological, cellular, and genetic analyses indicate the hormone prolactin (PRL) and its cognate receptor in humans (hPRLr) are significantly involved in breast cancer pathogenesis. Recent evidence demonstrated that a truncated mouse PRLr (mPRLrT) is oncogenic when expressed alongside its canonical long form counterpart (mPRLrL) [Cell Reports 17, 249-260]. The mPRLrT shares significant sequence homology with a naturally-occurring and widely-expressed hPRLr splice variant, the intermediate hPRLr (hPRLrI) isoform. Given this similarity, we hypothesized hPRLrI may also induce transformation, when expressed alongside wild-type long hPRLr (hPRLrL). Like the mPRLrT, hPRLrI co-expression with hPRLrL in the immortalized but not transformed human breast cell line MCF10A resulted in a significant increase in proliferation, migration, viability, and anchorage-independent growth. These results were not observed following overexpression of either isoform alone, demonstrating that hPRLrL and hPRLrI co-expression is necessary to induce transformation of normal mammary epithelia. To further characterize this transformation, we established MCF10A xenografts using female NOD scid gamma (NSG) mice. Following intraductal injection, we observed rapid tumor growth in the hPRLrL/I co-expression cohort, significantly over that of the cohorts harboring either isoform alone, validating our in vitro findings in vivo. To determine mechanisms of transformation, we examined both differential protein stability and altered signaling events. In analyzing receptor degradation, a cycloheximide assay revealed hPRLrL stability is increased when heterodimerized with hPRLrI. hPRLrL turnover has been reported to be impaired in human breast cancer, indicating this phenomenon may be involved in the observed hPRLrI-mediated transformation. In regards to differential signaling, we examined the Jak2/Stat5a pathway. Jak2 is a promiscuous kinase whose significant oncogenic actions are well-characterized, while Stat5a is a transcription factor whose activities are critical in attenuating the actions of Jak2. Following PRL stimulation, it was observed that hPRLrL/I co-expression induced approximately two-fold greater Jak2-Y1007/1008 phosphorylation (pJak2) compared to that induced by hPRLrL expression alone. Further, it was observed that hPRLrL/I co-expression induced ten-fold less Stat5a-Y694 phosphorylation (pY-Stat5a) than hPRLrL expression alone. These data indicate unchecked pJak2 activity may also be a contributing mechanism in the observed transformation. Overall, these results demonstrate that hPRLrI, alongside hPRLrL, is sufficient for transformation of normal breast tissue.

Citation Format: Jacqueline M. Grible, Charles V. Clevenger. The role of the intermediate prolactin receptor in breast cancer oncogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 870.
Abstract 880: Genetic ablation of the cystine transporter xCT in pancreatic ductal adenocarcinoma inhibits mTORC1, growth, survival and tumor formation: Implications for potentiating chemosensitivity via erastin
Authors: Vučetić Milica , Daher Boutaina , Durivault Jerome , Parks Scott K. , Pouyssegur Jacques
Published: August 2019

The lethality of pancreatic ductal adenocarcinomas (PDAC) calls for improved therapeutic strategies. Chemoresistance remains a primary challenge in PDAC treatment, and exploiting oxidative stress might offer novel therapeutic clues. In this regard, we explored the cystine/glutamate exchanger (SLC7A11/xCT) that contributes to the maintenance of the intracellular glutathione (GSH). We deleted xCT via CRISPR-Cas9 in two PDAC cell lines (MiaPaCa-2 and Capan-2) and cultivated xCT-KO clones in the presence of N-acetylcysteine (NAC). In both cell lines, xCT-deletion abolished >90% of 14C-cystine uptake and induced a rapid depletion of GSH following NAC removal. Although several cystine/cysteine transporters have been identified in human cells, our finding demonstrates that, in vitro, xCT is the major actor for GSH synthesis. Consequently, both xCT-KO cell lines exhibited amino-acid stress with ATF4 and GCN2 kinase activation, mTORC1 inhibition, and proliferation arrest followed by ferroptotic cell death. Importantly, tumor growth was also abolished in both KO cell lines indicating the key role of xCT in cellular cysteine availability in vivo. Moreover, the rapid depletion of intracellular GSH in xCT-KO cells led to accumulation of lipid peroxides and cell swelling, both being prevented by vitamin E or iron chelation, two hallmarks of cell death by ferroptosis. Finally, in vitro pharmacological inhibition of xCT by erastin (1μM) phenocopied xCT-KO and potentiated the cytotoxic effects of both gemcitabine and cisplatin in these PDAC cell lines. In conclusion, our findings strongly support the concept that xCT inhibition, by its dual induction of nutritional and oxidative cellular stresses, has the great potential of a successful anticancer strategy.

Note: This abstract was not presented at the meeting.

Citation Format: Milica Vučetić, Boutaina Daher, Jerome Durivault, Scott K. Parks, Jacques Pouyssegur. Genetic ablation of the cystine transporter xCT in pancreatic ductal adenocarcinoma inhibits mTORC1, growth, survival and tumor formation: Implications for potentiating chemosensitivity via erastin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 880.
Abstract 892: Alterations of vitamin B1 homeostasis following oxidative stress in breast cancer and impact of supplementation on cellular redox homeostasis
Authors: Jonus Hunter C. , Ray Ashley E. , Zastre Jason A.
Published: August 2019

Vitamin B1, or thiamine, has been demonstrated to enhance the development and growth of breast tumors in vivo. In addition, alterations in the expression of critical thiamine homeostasis genes have been extensively described in pre-clinical models of breast cancer as well as in clinical breast tumor tissue. Despite the effort that has been placed on delineating alterations to thiamine homeostasis that occur during breast cancer, there remains little evidence demonstrating a molecular advantage for supplemental thiamine in breast cancer cells. Our previous work in colorectal cancer suggests that the proliferative benefits of thiamine may be related to the maintenance of oxidative stress. This appears to be facilitated through the adaptive regulation of the thiamine activating enzyme thiamine pyrophosphokinase-1 (TPK1) for thiamine pyrophosphate production. Here, using MCF7 cells as a model of breast cancer, we also identified a relationship between thiamine homeostasis and oxidative stress. Similar to previous findings in colorectal cancer, Western blot analysis revealed that TPK1 expression was up-regulated in response to enhanced levels of ROS induced by hypoxia and chemotherapeutic treatment. TPK1 knockdown mediated by shRNA reduced the proliferation of MCF7 cells, while the presence of supplemental thiamine promoted proliferation as determined by cell counting. The effect of supplemental thiamine on MCF7 proliferation corresponded with a direct impact to the antioxidant status of tumor cells measured by reduced nuclear accumulation of the transcription factor nuclear factor- erythroid 2-related factor 2 by Western blot analysis. Molecular probes detecting intracellular ROS revealed that supplemental thiamine did not reduce the basal level of ROS in MCF7 cells. However, enhanced thiamine reduced intracellular superoxide levels following stimulation with the electron transport chain inhibitor Antimycin A. These findings support that during supplemental thiamine conditions thiamine homeostasis may be exploited in breast cancer for a redox advantage contributing to tumor progression.

Citation Format: Hunter C. Jonus, Ashley E. Ray, Jason A. Zastre. Alterations of vitamin B1 homeostasis following oxidative stress in breast cancer and impact of supplementation on cellular redox homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 892.
Abstract 893: The cofilin-TEAD1-p27Kip1 pathway ablates cell senescence
Authors: Lee Yi-Jang , Tsai Cheng-Han , Chang Chun-Yuan
Published: August 2019

Morphological change is an explicit characteristic of cell senescence, but the underlying mechanisms remain to be addressed. Here we demonstrated, after a survey of various actin-binding proteins, that up-regulation of cofilin-1 was essential for the reduced rate of actin depolymerization in senescent cells, as well as their morphological enlargement . Additionally, the up-regulated cofilin-1 mainly existed in the serine-3 phosphorylated form according to the 2D gel immunoblotting assay. Accumulation of cofilin-1 in senescent cells depended on the post-translational mechanism, as the ubiquitin-proteasomal degradation of cofilin-1 was reduced without any change in mRNA level. Up-regulation of cofilin-1 was not only found in replicative senescence but also in oxidative stress- and K-Ras oncogene-induced senescence. Over-expression of wild-type cofilin-1 and constitutively phosphorylated cofilin-1 promoted cell senescence with increased cell size. Additionally, senescent phenotypes were reduced by knockdown of total cofilin-1. The senescence induced by over-expression of cofilin-1 was dependent on p27Kip1 but not p53 and p16INK4 expression. Knockdown of p27Kip1 alleviated cell senescence induced by oxidative stress or replicative stress. We also found that over-expression of cofilin-1 induced expression of p27Kip1 through transcriptional suppression of the TEAD1 transcription factor. The TEAD1 transcription factor played a transrepressive role on the p27Kip1 gene promoter as determined by the promoter deletion reporter gene assay. Interestingly, TEAD1 down-regulation was accompanied by up-regulation of cofilin-1 in senescence. Knockdown and restoration of TEAD1 in young cells and old cells could induce and inhibit p27Kip1 and cell senescence, respectively. Furthermore, senescence induced by the over-expression of cofilin-1 could be alleviated by ectopic expression of TEAD1. Taken together, current data suggest that cofilin-1/TEAD1/p27Kip1 signaling is involved in senescence-related morphological change and growth arrest.

Keywords: Cofilin-1, TEAD1, p27Kip1, morphology, growth arrest, senescence

Citation Format: Yi-Jang Lee, Cheng-Han Tsai, Chun-Yuan Chang. The cofilin-TEAD1-p27Kip1 pathway ablates cell senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 893.