bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 812: γ-tocotrienol induces mitophagy in breast cancer cells through activation of LKB1-AMPK pathway
Authors: Dronamraju Venkatesh , Sylvester Paul W.
Published: August 2019

The dysregulation of glucose metabolism in cancer cells manifests as a characteristic phenomenon, referred to as the Warburg effect. Warburg effect is an over-reliance on aerobic glycolysis by cancer cells to meet the energy demand of proliferation. Previous studies have shown that γ-tocotrienol, a natural vitamin E derivative, inhibits aerobic glycolysis in human breast cancer cells through its modulation of key regulatory enzymes protein kinase B (Akt) and 5’-AMP activated kinase (AMPK). In the present study, we investigated the effects of γ-tocotrienol-induced activation of AMPK on mitochondrial dynamics in human breast cancer cells. Western blotting and immunocytochemistry analysis revealed that in human breast cancer cells MDA-MB-231 and T-47D, γ-tocotrienol removed the negative regulation of Akt on liver kinase B1 (LKB1), an upstream promoter of AMPK by increasing active complex formation and cytosolic localization of LKB1. In addition, γ-tocotrienol-induced activation of AMPK improved mitochondrial membrane potential through inhibition of uncoupling protein 1 (UCP1) as revealed by western blot analysis and mitochondrial membrane potential assay. High performance liquid chromatography (HPLC) analysis of NAD+/NADH ratio showed that γ-tocotrienol-induced AMPK activation improved the NAD+ ratio in human breast cancer cells, which indicates improving mitochondrial oxidative phosphorylation. In addition, γ-tocotrienol induced AMPK activation in human breast cancer cells causes increased mitochondrial fission, an indicator of mitophagy, as revealed by Western blot analysis of mitochondrial fission factor (MFF). In summary, these results show that γ-tocotrienol-induced AMPK activation modulates metabolic reprogramming in part by influencing the mitochondrial dynamics in human breast cancer cells. This work was supported in part by funds provided by the Louisiana Cancer Foundation.

Citation Format: Venkatesh Dronamraju, Paul W. Sylvester. γ-tocotrienol induces mitophagy in breast cancer cells through activation of LKB1-AMPK pathway [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 812.
Abstract 827: Estimating breast tissue-specific epigenetic age using next-generation methylation sequencing data
Authors: Castle James R. , Lin Nan , Liu Jingpeng , Wang Chi , Liu Yunlong , He Chunyan
Published: August 2019

Epigenetic age captures both genetic and environmental influences across time on cellular functions and is an indicator of biological aging. Epigenetic age may deviate from chronological age substantially in individuals. Epigenetic age is also tissue-specific. Emerging evidence suggests that female breast tissue ages faster than other parts of the body according to epigenetic age estimation using the "Horvath Clock" model. The Horvath method is based on the DNA methylation of 353 CpG loci on the outdated Illumina microarray platforms. The increasing availability of next-generation sequencing data calls for method development that uses DNA methylation sequencing data to estimate tissue-specific epigenetic age. We developed a new method to estimate breast tissue-specific epigenetic aging using next-generation methylation sequencing data and assessed the difference between epigenetic and chronological ages, known as epigenetic age acceleration (EAA), in different breast tissue types. The Illumina TruSeq Methyl Capture EPIC Sequencing technology was used to obtain DNA methylation profiles of approximately 3.3 million CpG sites in 111 tumor, 48 matched adjacent normal, and 462 normal breast tissue samples. A total of approximately 1.4 million CpG sites remained after quality control. Following the Horvath approach, we used an elastic net penalized regression model to regress chronological age on CpG sites in normal breast tissue and defined a new set of 247 clock CpGs specific to breast tissue with randomly divided training (n = 370) and testing (n = 92) data sets. We estimated breast tissue-specific epigenetic age and EAA in tumor, adjacent, and normal breast tissue. We found that breast tissue-specific epigenetic age was positively correlated with chronological age (r=0.87; P<2.2X10-16). Neither normal nor adjacent normal breast tissue showed a significant EAA. However, tumor breast tissue had a significant and increased EAA (median = 8.0 years; P=6.9X10-9). While triple-negative breast tumors showed no significant EAA, hormone receptive-positive and Her2-positive breast tumors had a significant and increased EAA (median=9.5 and 13.1 years; P=1X10-5 and 0.02, respectively). Results of this new model were compared to similar results using the Horvath Clock model and were found to be qualitatively consistent. Further research is needed to determine whether epigenetic age acceleration in normal breast tissue is predictive of breast cancer risk and how breast cancer risk factors influence the rate of acceleration.

Citation Format: James R. Castle, Nan Lin, Jingpeng Liu, Chi Wang, Yunlong Liu, Chunyan He. Estimating breast tissue-specific epigenetic age using next-generation methylation sequencing data [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 827.
Abstract 833: Integrative analysis of TCGA pancreatic ductal adenocarcinoma data
Authors: Mishra Nitish K. , Southekal Siddesh , Guda Chittibabu
Published: August 2019

Pancreatic cancer is a heterogeneous type of cancer that exhibits different histopathological, molecular and genomic phenomena. Pancreatic ductal adenocarcinoma (PDAC) is the deadliest type of pancreatic cancer with a five-year survival of only 8%. Prognostic biomarkers for early detection of PDAC are not available. In this study, we used clinical, DNA methylation, and gene expression profiles of 146 TCGA PDAC patients. We analyzed the global patterns of DNA methylation and correlated the promoter DNA methylation with corresponding gene expression. CpG sites with a difference of 0.2 in the mean β value of the primary tumor and normal samples were considered as differentially methylated. We observed differential methylation of about 12-thousand CpG sites between tumor and normal samples, the majority of which are hypermethylated. Chromosome 1, 20, 18 and 19 are predominately hypermethylated and chromosome 9 is hypomethylated. CpG islands and regions that are in close proximity to CpG islands (shores) have more hypermethylated sites compared to the far away regions (shelf). Pathway enrichment analysis of differentially methylated genes enabled us to understand how changes in methylation affect biological pathways involved in the progression of PDAC. Our analysis reveals enrichment of MAPK signaling, Rap1 signaling, calcium signaling pathway, etc. Promoter DNA methylation is highly associated with its corresponding gene expression. A significant correlation between methylated CpG sites within 1.5kb from TSS and corresponding gene expression was observed between 4,971 CpGs and 1,744 corresponding genes, out of these about 92% of the genes showed a negative correlation. Survival analysis showed a statistically significant correlation between the expression levels of genes, NEK2, and ASPM with overall survival of the patients. In the future, we will further analyze the long non-coding RNA (lncRNA) promoter methylation, and role of lncRNA and miRNA in PDAC patients’ survival.

Citation Format: Nitish K. Mishra, Siddesh Southekal, Chittibabu Guda. Integrative analysis of TCGA pancreatic ductal adenocarcinoma data [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 833.
Abstract 853: Essential role of autocrine FGF19-FGFR4 signaling in head and neck tumorigenesis
Authors: Lang Liwei , Gao Lixia , Shay Chloe , Zhao Xiangdong , Shull Austin , Teng Yong
Published: August 2019

The fibroblast growth factor 19 gene FGF19 has been reported to be amplified in several cancer types and encodes for a key autocrine signaler known to promote tumorigenic growth. Thus, it is imperative to understand which cancers are oncogenically addicted to FGF19 amplification as well as the role it serves in these cancer types. We report for the first time that high FGF19 amplification in head and neck squamous cell carcinomas (HNSCC) is associated with increased autocrine secretion of FGF19 and poor patient outcome in HNSCC. FGF19 amplification corresponded with constitutive activation of FGFR4-dependent ERK/AKT-p70S6K-S6 signaling, and addition of human recombinant FGF19 could promote proliferation and soft agar colony formation in HNSCC cells with low FGF19 expression through activating FGFR4 and downstream signaling cascades. Consistently, FGF19 knockout counteracted the observed effects in HNSCC cells carrying high endogenous FGF19, and significantly suppressed tumor growth in an orthotopic mouse model of HNSCC. This study demonstrates that FGF19 gene amplification corresponds with an increased dependency upon FGF19/FGFR4 autocrine signaling in HNSCC, revealing a therapeutic target for this cancer type.

Note: This abstract was not presented at the meeting.

Citation Format: Liwei Lang, Lixia Gao, Chloe Shay, Xiangdong Zhao, Austin Shull, Yong Teng. Essential role of autocrine FGF19-FGFR4 signaling in head and neck tumorigenesis [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 853.
Abstract 864: Mutant IDH1 is essential for chondrosarcoma growth
Authors: Li Luyuan , Hu Xiaoyu , Eid Josiane E. , DeSalvo Joanna , Trent Jonathan C.
Published: August 2019

Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers, including chondrosarcomas. IDH mutations detected in human cancers invariably are heterozygous missense substitutions. These mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into 2-hydroxyglutarate (D-2HG), an oncometabolite. Due to the structural similarity between D-2HG and α-KG, it has been reported that high levels of D-2HG competitively inhibit α-KG-dependent dioxygenases such as TET, JHDM and PHD enzymes, thus contributing to tumorigenesis. We sought to determine the role of IDH1 mutations in the tumorigenesis of human chondrosarcomas by inactivating mutant IDH1 using pharmacological and genetic approaches. In our study, we employed two human chondrosarcoma cell lines, JJ012 and HT1080, that carry endogenous IDH1 mutations. IDH mutation analysis was performed by PCR-based DNA sequencing, and D-2HG levels were measured by tandem mass spectrometry. Mutant IDH1 was knocked down via siRNA and knocked out via CRISPR/Cas9. We analyzed the effect of mutant IDH1 on chondrosarcoma growth in murine xenograft models. We found that knockdown of mutant IDH1 via siRNA significantly reduced D-2HG production in chondrosarcoma cells. In addition, mutant IDH1 knockdown dramatically inhibited colony formation in the sarcoma cells. Consistently, genetic knockout of mutant IDH1 almost completely depleted D-2HG production and significantly inhibited colony formation in the chondrosarcoma cells. To assess the significance of these results in vivo, we implanted the mutant IDH1- knockout cells in nude mice and studied their capacity for tumor initiation and growth. In these models, we observed that loss of mutant IDH1 led to a marked attenuation of chondrosarcoma formation. Our findings clearly demonstrate that mutant IDH1 plays a vital role in chondrosarcoma tumor formation. By investigating the role of IDH mutations in the pathogenesis of chondrosarcomas, we aim to uncover the potential therapeutic targets against this aggressive cancer.

Citation Format: Luyuan Li, Xiaoyu Hu, Josiane E. Eid, Joanna DeSalvo, Jonathan C. Trent. Mutant IDH1 is essential for chondrosarcoma growth [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 864.
Abstract 873: Molecular mechanisms of non-small cell lung cancer growth and drug resistance
Authors: Alam SK Kayum , Astone Matteo , Liu Ping , Wang Li , Coyle Abbygail M. , Dankert Erin N. , Hoffman Dane K. , Hall Stephanie R. , Zhang Wei , Kuang Rui , Roden Anja C. , Mansfield Aaron S. , Hoeppner Luke H.
Published: August 2019

Lung cancer is the deadliest and most frequently diagnosed type of tumor worldwide, with 1.6 million deaths reported annually. Non-small cell lung cancer (NSCLC) represents 85% of all lung cancer cases and carries a poor 5-year survival rate below 15%. Prognoses remain dismal due to the large number of patients diagnosed with advanced stage disease and the development of resistance to current therapies. A better understanding of acquired drug resistance will help to circumvent the progression of lung cancer and make significant strides in improving NSCLC patient treatment. Our recent work demonstrates that dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) and its amino-terminally truncated splice variant (t-DARPP) promote lung tumor growth in orthotopic mouse models. IHC staining of 62 human lung adenocarcinoma tissues showed that t-DARPP expression is elevated with increasing tumor (T) staging score, which represents the size of the primary tumor and whether it has grown into nearby areas, as a metric of tumor progression and growth. Correspondingly, a computational biology analysis of 513 lung adenocarcinoma patients revealed upregulation of t-DARPP isoform expression correlates with advanced T stage and is associated with poor overall survival. We identified a novel physical interaction between DARPP-32 and inhibitory kappa B kinase-α (IKKα) that promotes NSCLC cell migration through activation of non-canonical NF-κB2 signaling. It has been shown that DARPP-32 overexpression inhibits gefitinib-induced apoptosis in gastric cancer and t-DARPP contributes to the trastuzumab resistance phenotype in breast cancer. Our recent findings suggest DARPP-32 and t-DARPP overexpression in NSCLC promotes resistance to specific molecular targeted inhibitors by enabling tumor cells to evade apoptosis via Akt- and Erk-dependent cell survival mechanisms. Current ongoing studies are focused on manipulating expression of DARPP-32 isoforms in lung tumor cells to prevent resistance to targeted therapies in NSCLC patients.

Citation Format: SK Kayum Alam, Matteo Astone, Ping Liu, Li Wang, Abbygail M. Coyle, Erin N. Dankert, Dane K. Hoffman, Stephanie R. Hall, Wei Zhang, Rui Kuang, Anja C. Roden, Aaron S. Mansfield, Luke H. Hoeppner. Molecular mechanisms of non-small cell lung cancer growth and drug resistance [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 873.
Abstract 874: The CRTC1/MAML2 oncogene induces a PGC-1a splice variant that sensitizes mucoepidermoid carcinoma cells to PPARg inhibition
Authors: Musicant Adele M. , Sharma Kshitij P. , Henry Erin C. , Tasoulas Jason , Padilla Ricardo , Sengupta Monideepa , Flaveny Colin , Amelio Antonio L.
Published: August 2019

Mucoepidermoid carcinoma (MEC) is the most common type of malignant salivary gland tumor. MECs frequently exhibit a recurrent t(11;19)(q21;p13) chromosomal translocation that fuses the transcriptional coactivators CRTC1 (CREB-Regulated Transcription Coactivator 1; C1) and MAML2 (Mastermind-Like 2; M2). Though many C1/M2-positive tumors are easily resected, some do progress to advanced, metastatic disease accompanied by poor 5-year survival rates. Thus, it is critical to understand the mechanisms by which C1/M2 drives tumor growth and progression in order to identify therapeutic vulnerabilities. Our lab recently identified a correlation between C1/M2 expression and increased levels of the growth hormone IGF-1. This hormone plays critical roles in the development and homeostasis of normal salivary glands, and its dysregulation has been implicated in several cancers including colon and lung cancers. Interestingly, we demonstrate that IGF-1 is not directly upregulated by C1/M2. Rather, we have discovered a novel C1/M2-driven signaling circuit whereby constitutive CREB activation drives the increased production of a minor PGC-1α (PPARγ Coactivator 1α) splice variant, PGC-1α4, which is responsible for the high IGF-1 levels observed in C1/M2-positive MEC cells. Notably, the PGC-1α4 splice variant is minimally expressed in normal tissues and C1/M2-negative cell lines but is selectively upregulated in C1/M2-positive cell lines. We show that C1/M2 directly binds CREB at the PGC-1α4 promoter to induce expression of this variant, resulting in increased IGF-1 expression. IGF-1 promoter analysis revealed a significant enrichment of PPARγ binding motifs and functional studies confirm that this promoter is regulated by PPARγ. Consequently, we demonstrate that a novel PPARγ inverse agonist is capable of repressing IGF-1 promoter activity and reducing growth and clonogenic potential of C1/M2-positive cell lines that exhibit high PGC-1α4 expression. Collectively, our results reveal a novel therapeutic vulnerability in salivary MEC cells that may extend more broadly to other cancers that exhibit dysregulated CREB activity.

Citation Format: Adele M. Musicant, Kshitij P. Sharma, Erin C. Henry, Jason Tasoulas, Ricardo Padilla, Monideepa Sengupta, Colin Flaveny, Antonio L. Amelio. The CRTC1/MAML2 oncogene induces a PGC-1a splice variant that sensitizes mucoepidermoid carcinoma cells to PPARg inhibition [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 874.
Abstract 877: Novel role of truncated HMGA2 in regulating reactive oxygen species in prostate cancer cells
Authors: Hawsawi Ohuod , Henderson Veronica , Burton Liza , Dougan Jodi , Millena Ana Cecillia , Adams Vanessa , White Elshaddai Z. , Wang Guang Di , Zhang Qiang , Hinton Cimona , Khan Shafiq , Odero-Marah Valerie
Published: August 2019

High mobility group A (HMGA2), a non-histone protein, is up-regulated in several cancers. Due to chromosomal rearrangement of HMGA2 gene, full-length or wild-type HMGA2 can be truncated leading to loss of the C-terminus and the 3’UTR. Both wild-type and truncated isoforms have been detected in uterine leiomyoma patients. However, the functional role of truncated HMGA2 has not been investigated. We hypothesize that truncated HMGA2 plays a role in prostate cancer progression. We analysed expression of wild-type vs truncated HMGA2 in a panel of prostate cancer cell lines using real-time PCR and western blot analyses. We utilized LNCaP cells stably overexpressing wild-type HMGA2 or truncated HMGA2 in LNCaP cells to detect the basal reactive oxygen species (ROS) levels using DCFDA dye. We analysed Jun-D expression, a putative downstream effector of HMGA2, by western blot analysis, and utilized siRNA to knockdown Jun-D. We performed migration and cell viability assays following Jun-D knockdown. Additionally, we performed proteomic analysis following immunoprecipitation with HMGA2 antibody in nuclear extracts from LNCaP cells overexpressing wild-type HMGA2 WT or cytoplasmic extracts from cells overexpressing truncated HMGA2. Finally, we utilized tissue microarray to analyse expression of HMGA2 by immunohistochemisty. Our results showed that prostate cancer cell lines expressed varying amounts of wild-type and truncated HMGA2. LNCaP cells overexpressing truncated HMGA2 exhibited increased nuclear expression of Jun-D, as well as increased ROS compared to LNCaP cells overexpressing wild-type HMGA2 or empty vector Neo control. Knockdown Jun-D in LNCaP cells overexpressing truncated HMGA2 abrogated ROS induction and cell migration with no effect on cell proliferation. Additionally, truncated HMGA2 interacted with a number of proteins, including Ras GTPase-activating protein-binding protein 1(G3BP1), a cytoplasmic stress granule protein that responds to oxidative stress. Truncated HMGA2 is mainly localized to the cytoplasm, and interestingly we also observed cytoplasmic HMGA expression in prostatic tissue with chronic inflammation. Therefore, truncated HMGA2 may promote prostate cancer progression via Jun-D-mediated ROS. GRANT SUPPORT: NIH 1P20MD002285 and NIH/NCRR/RCMI G12RR003062-22

Citation Format: Ohuod Hawsawi, Veronica Henderson, Liza Burton, Jodi Dougan, Ana Cecillia Millena, Vanessa Adams, Elshaddai Z. White, Guang Di Wang, Qiang Zhang, Cimona Hinton, Shafiq Khan, Valerie Odero-Marah. Novel role of truncated HMGA2 in regulating reactive oxygen species in prostate 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 877.
Abstract 878: IGFBP2 is required to activate EGFR-DNA-PKcs pathway to protect esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage
Authors: Zhou Zhangjian , Lu Heng , Zhu Shoumin , Gomaa Ahmed , Chen Zheng , Yan Jin , El-Rifai Wael , Dang Chengxue , Peng Dunfa
Published: August 2019

The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett’s esophagus (BE) and its progression to EAC have been linked to gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks (DSBs). Immunohistochemistry analysis demonstrated frequent overexpression of IGFBP2 in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA DSBs and apoptosis, following transient exposure (20 min) to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against DSBs and apoptosis. Using western blotting and immunofluorescence assays, we found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are required for DNA damage repair activity. Using co-immunoprecipitation assay, we detected IGFBP2 forms complex with EGFR and DNA-PKcs and co-localizes with EGFR and DNA-PKcs in nucleus, following acidic bile salts treatment. We also demonstrated, using cycloheximide (CHX) chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure. In summary, our data indicate that IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.

Citation Format: Zhangjian Zhou, Heng Lu, Shoumin Zhu, Ahmed Gomaa, Zheng Chen, Jin Yan, Wael El-Rifai, Chengxue Dang, Dunfa Peng. IGFBP2 is required to activate EGFR-DNA-PKcs pathway to protect esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage [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 878.
Abstract 888: Exploring the signaling and activity interactome between oncogenic RAS and the nucleotide pool-detoxifying enzyme MTH1
Authors: Samaranayake Govindi J. , Troccoli Clara I. , Jayaraj Christina , Durden Brittany C. , Nagathihalli Nagaraj , Merchant Nipun , Rai Priyamvada
Published: August 2019

MutT Homolog 1 (MTH1) is a NUDIX pyrophosphorylase that hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, thus preventing their incorporation into DNA. Our prior work has shown that MTH1 is critical for the maintenance of multiple pro-tumorigenic phenotypes in oncogenic RAS-driven cancer cells, with its depletion leading to decreased tumor formation in vivo. Our subsequent analyses of TCGA patient datasets showed elevated MTH1 expression to be significantly associated with poorer disease-free survival in RAS-mutated cancers, such as that of the lung and pancreas. We found that MTH1 mRNA levels were positively correlated with KRAS levels even in early-stage non-small cell lung cancer patient tissues, and that the introduction of oncogenic KRAS was sufficient to upregulate MTH1 mRNA and protein levels. The aim of this study is to identify RAS-effector signaling intermediates affecting MTH1 expression and activity.

Chemical inhibitors of the MAPK/ERK, PI3K/AKT and NOX pathways, plus oncogenic RASV12-effector domain mutants (RASV12- S35/ E38/ G37/ C40), were used to identify key signaling molecular mediators of MTH1 expression in the distinct RAS isoforms (H- and K-RAS). The dependencies of the different KRASG12-mutant polymorphisms (KRASG12- C/ D/ V) on MTH1 expression and activity, as well as candidate transcription factors regulating MTH1 expression, were evaluated.

Our work shows MTH1 at the nexus of crosstalk between different effector pathways activated downstream of RAS. Dissecting these signaling intermediates are important in identifying alternate pathways of MTH1 regulation, which may manifest as resistance mechanisms to standard-of-care cancer treatments. Our work will also help understand how to best leverage MTH1 as a therapeutic target in oncogenic RAS-driven cancers driven by the different isoforms, and their respective mutant polymorphisms.

Citation Format: Govindi J. Samaranayake, Clara I. Troccoli, Christina Jayaraj, Brittany C. Durden, Nagaraj Nagathihalli, Nipun Merchant, Priyamvada Rai. Exploring the signaling and activity interactome between oncogenic RAS and the nucleotide pool-detoxifying enzyme MTH1 [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 888.
Abstract 899: Selective clearance of cells undergoing therapy-induced senescence in a model of triple negative breast cancer
Authors: Tyutyunyk Liliya , Saleh Tareq , Gewirtz David A.
Published: August 2019

Despite the ability of chemotherapy (and/or radiation) to eliminate the majority of tumor cells, some residual surviving cells may be able to escape cell death by entering into a prolonged senescence-based growth arrest. In addition to apoptosis, the majority of anti-cancer therapies induce senescence that has long been considered to be a favorable outcome of treatment due to its perceived irreversibility. However, accumulating studies in the literature demonstrate that therapy induced senescence may reflect one form of tumor dormancy and disease recurrence, when senescent cells are able to regain proliferative capacity. Tumor cells that escape dormancy often demonstrate an aggressive phenotype and are refractory to further treatment. Senescent cells are characterized by a specific (though often heterogeneous) secretory profile (Senescence Associated Secretory Phenotype or SASP), some components of which have been shown to promote tumorigenesis and may assist in neoplastic escape after therapy. In the current work, we evaluated the capacity of the BCL protein family inhibitor, ABT 263, to selectively clear breast tumor cells induced into senescence by doxorubicin. MDA-MB231-231 human breast cancer cells (a model of triple negative breast cancer) were induced into senescence upon exposure to a clinical concentration of the chemotherapeutic agent, doxorubicin (Dox). While a fraction of the MDA-MB-231 cell population underwent cell death, a large subpopulation entered into a state of prolonged growth arrest that lasted for several days, after which time the cells recovered proliferative capacity and formed colonies. At the time points when cells were arrested, prominent Senescence Associated β-Galactosidase staining marker indicative of senescence. In addition, tumor tissue collected from mice that received injections of Dox also were positive for β-Galactosidase staining. Cells induced into senescence and treated with ABT263 for 18 hours showed a significant increase in apoptosis and did not regain colony forming capabilities. We therefore postulate that senescence and escape from senescence may represent a form of tumor dormancy and disease recurrence, respectively. Consequently, if Therapy Induced Senescence is a deleterious outcome of treatment, it is of importance to identify strategies that might prevent senescent cells from neoplastic escape and induce cell death in order to completely eliminate residual tumors and thereby interfere with disease recurrence.

Citation Format: Liliya Tyutyunyk, Tareq Saleh, David A. Gewirtz. Selective clearance of cells undergoing therapy-induced senescence in a model of triple negative 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 899.
Abstract 90: The “reproducibility crisis” of animal studies in oncology - How did we get here, and how can we resolve it
Authors: Hentze Hannes , Ibsen Eric
Published: August 2019

Historically, a large majority of clinical phase III trials fail to gain regulatory approval due primarily to a lack of demonstrated efficacy, demonstrating the challenge of conducting clinically relevant efficacy studies in laboratory animals. In 2012, Amgen researchers published their finding that an astoundingly-high proportion (90%) of more than 50 republished in vivo studies in oncology was not reproducible. This sparked an intense debate about data validity in preclinical sciences, and several reviews have been published in the last two years analysing this issue. A number of best-practice initiatives and resources have since been created along with the development of animal study workflow software applications and databases to address the causes of this significant, global problem in cancer research. Major sources of irreproducibility in animal research such as bias, suboptimal disease models, lack of controls, and most importantly bad documentation practices will be discussed. Several initiatives have resulted in practically-applicable concepts and publicly-available resources to address this issue, such as ARRIVE guidelines, PHISPS protocols and the N3R Design Assistant, will be described. Electronic spreadsheets have been the most prevalently-used tool to plan and conduct preclinical efficacy studies in oncology. The advent of animal study workflow software and database technology, designed specifically for in vivo oncology research, has seen increasingly widespread adoption in recent years in both academia and industry. By employing standardized and more detailed approaches to the processes documenting highly detailed study design, data collection, method and procedure descriptions and adherence, real-time monitoring of animal welfare parameters, standardized graphing, data analyses and report generation, measurement and task scheduling, as well as data preservation approaches, will be explored. Using some real-life examples, the advantages and disadvantages of preclinical study automation and the use of electronic spreadsheets will be discussed in the context of helping to resolve the animal study “Reproducibility Crisis”.

Citation Format: Hannes Hentze, Eric Ibsen. The “reproducibility crisis” of animal studies in oncology - How did we get here, and how can we resolve it [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 90.
Abstract 950: In vivo efficacy and pharmacodynamic modulation of JNJ-64619178, a selective PRMT5 inhibitor, in human lung and hematologic preclinical models
Authors: Millar Hillary J. , Brehmer Dirk , Verhulst Tinne , Haddish-Berhane Nahor , Greway Tony , Gaffney Dana , Boeckx An , Heerde Erika Van , Nys Thomas , Portale Joseph , Philippar Ulrike , Wu Tongfei , Laquerre Sylvie , Packman Kathryn
Published: August 2019

Protein arginine methyltransferase 5 (PRMT5), a type II methyltransferase, is responsible for symmetric arginine di-methylation of multiple cellular proteins involved in the regulation of cellular transcription. PRMT5 is involved in cellular processes such as survival, proliferation, and apoptosis, and an elevated tumor PRMT5 protein level has recently been correlated with poor survival of cancer patients.

JNJ-64619178, a selective PRMT5 inhibitor, showed inhibition of cellular growth in several cell lines representing multiple cancer histologies in vitro. From this, a broad selection of xenograft models was chosen to demonstrate potent anti-tumor efficacy.

Xenograft models representing small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), acute myeloid leukemia (AML), and non-Hodgkin lymphoma were chosen to demonstrate anti-tumor efficacy. Biologically significant tumor growth inhibition up to 99% was observed in both solid and hematological xenograft models, including an aggressive disseminated model of AML, with oral doses of 1 to 10 mg/kg, once daily. Importantly, continued inhibition of tumor growth was observed for several weeks following dosing cessation.

Dosing of JNJ-64619178 results in inhibition of Sym-Arg di-methylation of SMD1/3 proteins, core components of the spliceosome in the tumor, and general Sym-Arg di-methylation of serum proteins. These serve as pharmacodynamic markers of PRMT5 inhibition in xenograft models. Potent and prolonged inhibition of SMD1/3 di-methylation was observed in the SCLC model, during and after the dosing period. This has led to the exploration of alternative dosing regimens preclinically.

PRMT5 inhibitor JNJ-64619178 is currently being investigated in a Phase I clinical trial, based on its high selectivity and potency, favorable pharmacokinetics and safety properties, and strong preclinical efficacy and pharmacodynamic data.

Citation Format: Hillary J. Millar, Dirk Brehmer, Tinne Verhulst, Nahor Haddish-Berhane, Tony Greway, Dana Gaffney, An Boeckx, Erika Van Heerde, Thomas Nys, Joseph Portale, Ulrike Philippar, Tongfei Wu, Sylvie Laquerre, Kathryn Packman. In vivo efficacy and pharmacodynamic modulation of JNJ-64619178, a selective PRMT5 inhibitor, in human lung and hematologic preclinical 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 950.
Abstract 954: SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS mutant tumors treated with MEK inhibitors
Authors: Lu Hengyu , Liu Chen , Velazquez Roberto , Wang Hongyun , Dunkl Lukas M. , Kazic-Legueux Malika , Haberkorn Anne , Billy Eric , Manchado Eusebio , Brachmann Saskia M. , Moody Susan , Engelman Jeffrey A. , Hammerman Peter S. , Caponigro Giordano , Mohseni Morvarid , Hao Huaixiang
Published: August 2019

Introduction: FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with MEK inhibitor (MEKi) such as trametinib in several KRAS mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTKs) are also feedback activated in KRAS mutant cell lines after MEKi treatment.

Experimental procedures: We profiled a large panel (n>32) of KRAS mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using a SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We then performed in vitro and in vivo combination efficacy studies and pathway analysis in various KRAS mutant cancer models.

Results: We find that RTK-driven feedback activation widely exists in KRAS mutant cancer cells and involves several RTKs including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS in KRAS G12C or G12D models. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting MAPK pathway and KRAS mutant cancer cell proliferation in vitro and in vivo.

Conclusions: Our findings suggest that MAPK inhibition in KRAS mutant cancer provokes feedback re-activation of the pathway that often involves RTK activity and SHP2 inhibition may enhance the efficacy of MEKi in KRAS mutant tumors. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS mutant cancers in the clinic.

Citation Format: Hengyu Lu, Chen Liu, Roberto Velazquez, Hongyun Wang, Lukas M. Dunkl, Malika Kazic-Legueux, Anne Haberkorn, Eric Billy, Eusebio Manchado, Saskia M. Brachmann, Susan Moody, Jeffrey A. Engelman, Peter S. Hammerman, Giordano Caponigro, Morvarid Mohseni, Huaixiang Hao. SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS mutant tumors treated with MEK inhibitors [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 954.
Abstract 958: Tumor-vascular interactions promote STING-driven inflammation in the tumor microenvironment
Authors: Campisi Marco , Sundararaman Shriram K. , Kitajima Shunsuke , Chiono Valeria , Kamm Roger D. , Barbie David A.
Published: August 2019

The recruitment of T cells following intratumoral administration of Stimulation of Interferon Genes (STING) agonists in the tumor microenvironment (TME) is a critical event in the STING-driven antitumor immune response, a pathway with great relevance in the context of cancer immunotherapy. We have previously demonstrated that LKB1 mutation is associated with suppression of tumor cell STING levels and reduced production of T-cell chemoattractants such as CXCL10 in KRAS-driven non-small cell lung cancer (NSCLC). Consistent with this, immunohistochemical staining of patient samples showed poor infiltration of CD3, CD4, and CD8 T cells into LKB1 negative versus LKB1 intact cancer epithelium, and instead, retention of T-cells in stroma. To examine how LKB1 alters immune cell recruitment in a STING-dependent manner, we used a 3-D microfluidic co-culture system to study interactions between vasculature and tumor spheroids derived from a KRAS/LKB1 mutated (KL) cell line with LKB1 reconstitution +/- STING deletion. To form the vasculature, we co-cultured tumor spheroids with fibroblasts and endothelial cells for 7 days, and identified changes in morphology, cytokine production, and gene expression that occur in co-culture. We first observed that co-culture induced synergistic production of multiple immune cell chemo-attractants such as CXCL10, CCL2, CCL5, and G-CSF. Interestingly, this more physiologic ex vivo tumor model of LKB1 reconstitution revealed particularly strong cooperative production of STING-dependent cytokines such as CXCL10 in the vasculature. Moreover, STING depletion in LKB1 reconstituted tumor cells did not significantly attenuate production of CXCL10 and other cytokines in co-culture, suggesting that tumor/vessel interaction may promote STING activation in the vasculature regardless of cancer cell-intrinsic STING function. Furthermore, although there was no appreciable response after treatment of KL cancer cells with cGAMP based STING agonists, treatment of isolated 3-D vascular networks with cGAMP enhanced vascular permeability and increased production of CXCL10 and CCL5, possibly contributing to defective chemokine gradients that retain T cells near the vasculature. Thus, developing these more complex models that incorporate the vasculature may elucidate important aspects of STING biology and may ultimately aid further development of effective immunotherapies targeting this signaling axis.

Citation Format: Marco Campisi, Shriram K. Sundararaman, Shunsuke Kitajima, Valeria Chiono, Roger D. Kamm, David A. Barbie. Tumor-vascular interactions promote STING-driven inflammation in the 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 958.
Abstract 963: The interplay between lifestyle-related factors and genetics with risk of invasive breast cancer among postmenopausal women from the UK Biobank
Authors: Arthur Rhonda S. , Wang Tao , Rohan Thomas
Published: August 2019

Background: Many genetic variants have been associated with increased risk of breast cancer. In contrast, an overall healthy lifestyle has been associated with a reduced risk. However, the degree to which an overall healthy lifestyle may attenuate the impact of genetic variants on risk of breast cancer remains equivocal. In this study, we examined the association of a healthy lifestyle index (HLI) with risk of breast cancer by genetic risk groups.

Methods: The study included 106,814 postmenopausal women of white descent aged 40 to 70 years who were enrolled in the UK Biobank cohort between 2006 and 2010. The HLI was based on a combination of diet, physical activity, smoking, alcohol consumption and anthropometry (ranging from 0 to 20, with the higher scores representing more favorable lifestyle). A polygenic risk score (categorized as low, intermediate and high) was generated as the sum of risk alleles weighted by the logOR of 117 breast cancer associated single nucleotide polymorphisms. A total of 1,949 incident invasive breast cancer cases were ascertained after a median follow-up of 5.7 years. Cox proportional hazards regression models were used to estimate the hazard ratios (HR) and 95% confidence intervals (CI) for the association of a healthy lifestyle risk score with risk of breast cancer by genetic risk groups.

Results: Compared to women with an unfavorable lifestyle (lowest quartile), women with a favorable lifestyle (highest quartile) had a 32% reduced risk of invasive breast cancer (HR: 0.68: 0.59, 0.79). Women with high genetic risk (highest tertile) had an 86% increased risk of invasive breast cancer (1.86: 1.66, 2.08) compared to those with low genetic risk (lowest tertile). Compared with women who had an unfavorable lifestyle, a favorable lifestyle did not significantly reduce risk of invasive breast cancer among women with low genetic risk (0.80: 0.63-1.02), while women with a favorable lifestyle had 32% and 43% reduced risk of invasive breast cancer in the intermediate and high genetic risk categories,

respectively (0.68: 0.53, 0.86 and 0.57; 0.44, 0.74, respectively). However, interaction between the genetic score and the lifestyle score did not achieve statistical significance (pinteraction=0.162).

Conclusion: This study suggests that an overall healthy lifestyle may attenuate the impact of genetics on risk of breast cancer, particularly among women with intermediate and high genetic risk.

Citation Format: Rhonda S. Arthur, Tao Wang, Thomas Rohan. The interplay between lifestyle-related factors and genetics with risk of invasive breast cancer among postmenopausal women from the UK Biobank [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 963.
Abstract 972: Direct observation in living tumors shows macrophage-dependent induction and dissemination of cancer stem cells in breast cancer
Authors: Sharma Ved P. , Wang Yarong , Tang Binwu , Karagiannis George S. , Xue Emily A. , Entenberg David , Borriello Lucia , Coste Anouchka , Surve Chinmay R. , Esposito Dominic , Oktay Maja H. , Wakefield Lalage M. , Condeelis John S.
Published: August 2019

Cancer stem cells (CSCs) play an important role during metastatic progression of breast cancer. However, the in vivo properties and dynamic behavior of CSCs are not well understood. Here, we employed high-resolution intravital multiphoton microscopy using a SOX2/OCT4 responsive fluorescent stem cell biosensor to directly observe CSC dynamics in the living animal using an orthotopic breast cancer xenograft model. We report that CSCs constitute a minority population (1-3%) in the primary tumors, and display the slow-migratory, invasive phenotype that is specifically associated with disseminating tumor cell population. We also report, for the first time, that CSCs are preferentially localized in direct contact with macrophages near and in tumor microenvironment of metastasis (TMEM) sites, the macrophage-containing intravasation doorway for tumor cells and that CSCs metastasize to lung and are strikingly enriched in early lung metastatic colonies. This is explained by our observation that, in vitro and in vivo, direct physical contact with macrophages induces stemness in non-stem cancer cells via juxtacrine Notch-Jagged1 signaling. These data indicate for the first time that macrophages play an actively inductive role in the CSC niche and promote TMEM-mediated CSC intravasation and early metastatic seeding.

Citation Format: Ved P. Sharma, Yarong Wang, Binwu Tang, George S. Karagiannis, Emily A. Xue, David Entenberg, Lucia Borriello, Anouchka Coste, Chinmay R. Surve, Dominic Esposito, Maja H. Oktay, Lalage M. Wakefield, John S. Condeelis. Direct observation in living tumors shows macrophage-dependent induction and dissemination of cancer stem cells 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 972.