bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2281: Molecular mechanisms by which cFLIP overexpression regulates TRAIL-induced NF-κB activation and lymphoma survival
Authors: Habelhah Hasem , Workman Lauren , Zhang Laiqun
Published: May 2015

Although recombinant TRAIL and agonistic antibodies directed against TRAILR1/2 are considered potential anti-cancer agents, their anti-cancer response in clinical trials was poor with the vast majority of patients showing no remission. In addition, TRAIL has been found to act as tumor promoters in certain contexts, increasing metastasis in apoptosis-resistant cancer cells by activating NF-κB. Therefore, defining the mechanism that permits TRAIL to activate NF-κB is critical for the development of strategies that maximize the potential effectiveness of TRAIL in clinical applications. We found that cFLIP-mediated and caspase-8-dependent cleavage of RIP1 at two sites, D180 in the kinase domain (KD) and D324 in the intermediate domain (ID), determines the activation state of the IKK/NF-κB pathway in response to TRAIL treatment. In apoptosis sensitive cells, caspase-8 cleaves RIP1 at KD and ID domains immediately after its recruitment to the receptor complex, impairing IKK recruitment and activation. In cFLIP-overexpressing cells, cFLIP restricts caspase-8 activity and RIP1 cleavage, generating a KD-cleaved RIP1 fragment that is capable of activating the NF-κB but not the apoptotic pathway. Of note, cFLIP expression, caspase-8 activity and RIP1 cleavage in KD per se are not directly involved in IKK activation; instead, they suppress RIP1-dependent activation of the apoptotic and necrotic cascades, and thereby ensure the integrity of the NF-κB pathway. Moreover, inflammatory ligands such as CD40L deplete cytoplasmic pool of TRAF2 and cIAP1/2, and promote RIP1 cleavage in lymphomas. Inhibition of RIP1 cleavage in KD suppresses NF-κB activation, and significantly increases TRAIL-induced cell death even in cFLIP-overexpressing lymphomas. Importantly, a portion of RIP1 is constitutively cleaved in many types of human and mouse B-cell lymphomas. These data suggest that, in addition to TRAF2- and cIAP1/2-mediated ubiquitination, cFLIP-regulated and caspase-8-dependent limited cleavage of RIP1 is a new layer of mechanism that promotes NF-κB activation and cancer cell survival.

Citation Format: Hasem Habelhah, Lauren Workman, Laiqun Zhang. Molecular mechanisms by which cFLIP overexpression regulates TRAIL-induced NF-κB activation and lymphoma survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2281. doi:10.1158/1538-7445.AM2014-2281
Abstract 2297: 15-Deoxy-Δ12,14-prostaglandin J2 upregulates the expression of 15-hydroxyprostaglandin dehydrogenase through DNA methyltrasferase 1 inactivation
Authors: Jang Hye-Ok , Lee Ha-Na , Kim Areumnuri , Kim Do-Hee , Surh Young-Joon , Na Hye-Kyung
Published: May 2015

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme that catalyzes the conversion of prostaglandin E2 to a biologically inactive keto metabolite. The down regulation of 15-PGDH is ubiquitously observed in various human malignancies including breast cancer. Hypermethylation of CpG islands present in the gene promoter region is considered to silence the expression of 15-PGDH. However, the molecular mechanisms underlying regulation of 15-PGDH remain largely unknown. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of peroxisome proliferator-activated receptor γ, has been reported to have anti-inflammatory and anti-carcinogenic activities. In the present study, we have found that 15-PGJ2 upregulates expression and catalytic activity of 15-PGDH in human breast cancer (MDA-MB-231) cells. 15d-PGJ2 decreased the level of CpG methylation in the 15-PGDH promoter in MDA-MB-231 cells as determined by the bisulfite genome sequencing and methyl specific PCR. 15d-PGJ2 inhibited catalytic activity of methyltransferase 1 (DNMT1) but did not influence expression of either mRNA or protein of DNMT1. siRNA knockdown of DNMT1 induced expression of 15-PGDH. Biotinylated 15d-PGJ2 directly interacted with DNMT1 and reduced its catalytic activity. Chromatin-immunoprecipitation analysis revealed that 15d-PGJ2 significantly attenuated DNMT1 binding to the site for AP-1 transcription factor present in the 15-PGDH promoter regions. We also observed that 15-PGJ2 induced expression of c-Fos, but not c-Jun in the nuclear extract of MDA-MD-231 cells. Moreover, 15d-PGJ2 increased the c-Fos binding to the 15-PGDH promoter regions. A non-electrophilic analogue 9,10-dihydro-15d-PGJ2 failed to suppress the methylation of CpG islands present in 15-PGDH promoter and did not affect DNMT1 activity and 15-PGDH expression. This finding suggests that the α,β-unsaturated carbonyl group is essential for 15d-PGJ2-mediated inactivation of DNMT1 and expression of 15-PGDH. In conclusion, 15d-PGJ2 plays as a hypomethylating agent through direct interaction with DNMT1 and thereby suppresses DNMT1-mediated hypermethylation of 15-PGDH promoter, leading to upregulation of 15-PGDH expression.

Citation Format: Hye-Ok Jang, Ha-Na Lee, Areumnuri Kim, Do-Hee Kim, Young-Joon Surh, Hye-Kyung Na. 15-Deoxy-Δ12,14-prostaglandin J2 upregulates the expression of 15-hydroxyprostaglandin dehydrogenase through DNA methyltrasferase 1 inactivation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2297. doi:10.1158/1538-7445.AM2014-2297
Abstract 2307: DNA methylation as a reprogramming modulator: An epigenomic roadmap to induced pluripotency
Authors: Lee Dong-Sung
Published: May 2015

During cellular reprogramming to induced pluripotent stem cells (iPSCs), somatic cells rebuild their epigenetic architecture to acquire a steady self-renewing state. Here we systematically characterize the epigenomic roadmap to pluripotency by performing whole genome bisulfite sequencing of the “Project Grandiose” (PG) samples, along with analysis of histone modification and the transcriptome. The differentially methylated regions (DMRs) of the DNA samples revealed that hypermethylated-DMRs (Hyper-DMRs) gradually accumulated throughout reprogramming whereas there was a distinct gain in hypomethylated-DMRs (Hypo-DMRs) once cells reached the ESC-like state. ESC-associated transcription factor binding sites (ESC-TFBSs) were highly enriched in the Hypo-DMR group. During high transgene expression, leading to the pluripotent F-class state, cells exhibited focal DNA demethylation at the binding sites of the activated reprogramming factors, whereas ESC-like pluripotent cells exhibited widespread demethylation surrounding these sites. Genes associated with CpG rich promoters demonstrated a stable low DNA methylation state with strong engagement of H3K4me3 and H3K27me3 histone marks (histone modification-driven control). CpG poor promoter-related genes were repressed by DNA methylation, with transcriptional activity occurring upon promoter demethylation (DNA methylation-driven control). We further report that a proportion of genes proposed to be ESC-pluripotency predictors, including Dppa2, Eras, and Esrrb, are associated with CpG poor promoters and become transcriptionally activated upon DNA demethylation in ESC-like cells. Gene expression leading to ESC-like pluripotency is impacted and specifically orchestrated by dynamic changes of DNA methylation and histone modification. These results suggest that DNA methylation plays a crucial role within the epigenetic switch that drives somatic cells to pluripotency.

Citation Format: Dong-Sung Lee. DNA methylation as a reprogramming modulator: An epigenomic roadmap to induced pluripotency. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2307. doi:10.1158/1538-7445.AM2014-2307
Abstract 2311: NDN, an imprinted tumor suppressor gene inhibits ovarian cancer cell growth and motility and is downregulated by genetic and epigenetic mechanisms
Authors: Yang Hailing , Das Partha , Yu Yinhua , Baggerly Keith , Wang Ying , Mao Weiqun , Marquez Rebecca T. , Lu Zhen , Liu Jinsong , Bast Robert C.
Published: May 2015

NDN is a maternally imprinted gene on chromosome 15q11.2 that is consistently expressed in normal ovarian epithelium, but dramatically downregulated in the majority of ovarian cancer cell lines and surgical specimens, determined by gene expression array and RT-PCR analysis. On tissue microarrays, little or no NDN protein could be detected by immunohistochemical analysis in73% of 351 epithelial ovarian cancers. Loss of NDN expression could be attributed, in part, to LOH in 28% of 18 informative cases and to hypermethylation of CpG sites 1 and 2 in the NDN promoter region in 21% and 27% of 43 ovarian cancers, respectively. All hypermethylated cases were associated with NDN down-regulation. Promoter hypermethylation and decreased NDN expression were also found in 7 of 10 ovarian cancer cell lines. Treatment with the demethylating agent decitabine increased NDN expression in 4 of 7 hypermethylated cell lines. Treatment with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) increased NDN expression in 2 cell lines with normal or reduced methylation. However, when the two drugs were combined, they didn't show additive or synergistic effect. Re-expression of NDN in ovarian cancer cell lines such as SKOv3 and HEY significantly inhibited cell growth, associated with apoptosis, but not autophagy or necrosis. Re-expression of NDN also inhibited cell migration. These observations support the hypothesis that NDN is an imprinted tumor suppressor gene and its loss of function can be caused by multiple mechanisms in ovarian cancer.

Citation Format: Hailing Yang, Partha Das, Yinhua Yu, Keith Baggerly, Ying Wang, Weiqun Mao, Rebecca T. Marquez, Zhen Lu, Jinsong Liu, Robert C. Bast. NDN, an imprinted tumor suppressor gene inhibits ovarian cancer cell growth and motility and is downregulated by genetic and epigenetic mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2311. doi:10.1158/1538-7445.AM2014-2311
Abstract 2314: The combination of DNA methylation and H3K27me3 is a cancer-specific dual epigenetic modification
Authors: Takeshima Hideyuki , Wakabayashi Mika , Hattori Naoko , Yamashita Satoshi , Ushijima Toshikazu
Published: May 2015

Alterations of epigenetic modifications are promising targets of cancer therapy, and several epigenetic drugs are now used in the clinical field. At the same time, individual epigenetic modifications have physiological roles. Therefore, a cancer-specific combination of epigenetic modifications (dual modifications) is considered to be better as a therapeutic target. However, it is unclear whether or not such cancer-specific dual modification is present. In this study, we aimed to clarify whether cancer-specific dual modification is present by focusing on the combination of two major repressive modifications, DNA methylation and trimethylation of histone H3 lysine 27 (H3K27me3).

DNA methylation and H3K27me3 analyses in human colon, breast, and prostate cancer cell lines by Infinium HumanMethylation450 and ChIP-on-chip, respectively, revealed that 24.7±4.1% of DNA methylated genes were co-localized with H3K27me3 in cancer cells. In contrast, only 11.8±7.1% of DNA methylated genes were co-localized with H3K27me3 in their normal counterpart cells. 29.9±17.4% of genes with the dual modifications had neither DNA methylation nor H3K27me3 in normal counterpart cells. Expression levels of genes with the dual modifications were as low as those of genes with only DNA methylation in cancer cells. Analysis of re-activation of genes with the dual modifications by a DNA demethylating reagent, 5-aza-2′-deoxycytidine (5-aza-dC), and/or an EZH2 inhibitor, GSK126, revealed that genes with the dual modifications, such as IGFBP7, SFRP1, and SLC6A15, were re-activated more efficiently by the combination treatment than by a single treatment with 5-aza-dC. In contrast, such a combination effect on gene re-activation was not observed for genes with only DNA methylation. Furthermore, the combination treatment by 5-aza-dC and GSK126 had an additive inhibitory effect on growth of cancer cells.

These results showed that genes with the dual modifications, DNA methylation and H3K27me3, are increased in cancer cells, and the combination of a DNA demethylating reagent and an EZH2 inhibitor is useful for re-activation of these genes.

Citation Format: Hideyuki Takeshima, Mika Wakabayashi, Naoko Hattori, Satoshi Yamashita, Toshikazu Ushijima. The combination of DNA methylation and H3K27me3 is a cancer-specific dual epigenetic modification. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2014-2314
Abstract 2316: Multilayer omics analyses in CpG island methylator phenotype clear cell renal cell carcinomas
Authors: Arai Eri , Sakamoto Hiromi , Ono Masaya , Takahashi Yoriko , Miyata Sayaka , Fujimoto Hiroyuki , Gotoh Masahiro , Yamada Tesshi , Kanai Yae
Published: May 2015

[Background] We have reported that CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poorer patient outcome. We have established the criteria for CIMP diagnosis in clear cell RCCs by quantification of DNA methylation levels of RCC-specific CIMP marker genes.

[Purpose] The aim of this study was to reveal the molecular pathways which significantly participate in CIMP-positive renal carcinogenesis and become a therapeutic target of CIMP-positive clear cell RCCs.

[Methods] Our previous methylome analysis using Illumina Infinium HumanMethylation27 BeadArray identified CIMP-negative and -positive clear cell RCCs in the present cohort (n=101). In the present study, genome (whole-exome), transcriptome and proteome analyses using Agilent SureSelect All Exon capture followed by sequencing on an Illumina HiSeq 2000 platform, Agilent SurePrint Human Gene Expression microarray and two dimensional image converted analysis of liquid chromatography-mass spectrometry, respectively, were performed in tissue specimens of 87 CIMP-negative and 14 CIMP-positive clear cell RCCs and the corresponding non-cancerous renal cortex tissue specimens.

[Results] The number of genes showing genetic aberrations in CIMP-negative clear cell RCCs was quite low, indicating that CIMP-negative RCCs lacked distinct genetic characteristics. On the other hand, many genes, such as genes encoding hisotone modification enzymes and tumor-suppressor genes, showed 10% or more incidence of genetic aberrations in CIMP-positive clear cell RCCs. Based on MetaCore pathway analysis, although mRNA or protein expression alterations in CIMP-negative clear cell RCCs were not significantly accumulated in any molecular pathways, such alterations in CIMP-positive clear cell RCCs were accumulated in the 19 pathways (P<0.05), such as molecular pathways involved in cell cycle and cell adhesion. Quantitative RT-PCR analysis verified expression alterations of genes involved in the most frequently affected pathway in CIMP-positive clear cell RCCs.

[Conclusion] The most frequently affected pathway may be a novel therapeutic target of CIMP-positive clear cell RCCs. We are now examining the effectiveness of the inhibitor of the most frequently affected pathway in RCC cell lines showing CIMP.

Citation Format: Eri Arai, Hiromi Sakamoto, Masaya Ono, Yoriko Takahashi, Sayaka Miyata, Hiroyuki Fujimoto, Masahiro Gotoh, Tesshi Yamada, Yae Kanai. Multilayer omics analyses in CpG island methylator phenotype clear cell renal cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2014-2316
Abstract 2330: Transcription factor MAZ promotes cell growth and aggressive behavior of human pancreatic cancer cells
Authors: Maity Gargi , Sarkar Sandipto , Dhar Kakali , Dhar Gopal , Haque Inamul , Banerjee Sushanta K. , Banerjee Snigdha
Published: May 2015

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cancer in the United States and the eighth worldwide. PDAC is most aggressive type of cancer that spreads rapidly and is seldom detected in its early stages as signs and symptoms may not appear until pancreatic cancer is quite advanced. MAZ (Myc-associated zinc-finger protein) or SAF1 (Serum amyloid A activating factor 1) gene is a member of multiple Cys2-His2-type zinc finger proteins that are activated in response to various inflammatory signals and may act as a transcription factor with dual roles in transcription initiation and termination. Deregulation of MAZ expression has been shown to be related to the development and progression of various cancers like prostate cancer, breast cancer and pancreatic cancer. However, mode of action of MAZ in carcinogenesis is unclear. We found that MAZ mRNA and protein levels were markedly elevated in pancreatic adenocarcinoma tissue samples as compared to adjacent normal tissues and the expression profile was increased as the disease progressed. Similarly, MAZ mRNA and protein levels were increased several folds in aggressive pancreatic cancer cell lines as compared to non-transformed pancreatic cancer cell lines. Functional analysis demonstrates that siRNA-based depletion of MAZ significantly inhibits in vitro cellular growth, colony formation, anchorage independent growth and invasion in pancreatic cancer cells. Mechanistically, MAZ regulates BIRC5, a pancreatic cancer promoting gene, to possibly exert its pathobiological function. Moreover, we found that CCN1/Cyr61 is an upstream regulator of MAZ. Collectively, these findings indicate that MAZ is a downstream target of CCN1 and plays an important role in pancreatic cancer progression and differentiation. Thus, MAZ could be an ideal target for PDAC management. [This work is supported by VA Merit Award Grant (SB/SKB)]

Citation Format: Gargi Maity, Sandipto Sarkar, Kakali Dhar, Gopal Dhar, Inamul Haque, Sushanta K. Banerjee, Snigdha Banerjee. Transcription factor MAZ promotes cell growth and aggressive behavior of human pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2330. doi:10.1158/1538-7445.AM2014-2330
Abstract 2342: Related study on the fat mass and obesity associated FTO gene and melanoma
Authors: Niu Tianhui , Xiong Min , Tian Yan
Published: May 2015

FTO is a Fe(II)/2-oxoglutarate-dependent oxidative DNA/RNA demethylase and plays a key role in the fat metabolism and several chronic illnesses including cancer development. Several independent SNPs in the intron 8 of the FTO gene, showed nominally significant association with risk of melanoma. But the functional regulatory mechanism of FTO protein is poorly understood. Thus, the research of downstream factors and interaction proteins of FTO may identification of the mechanism FTO participated in and discovery of the FTO pathway may provide a noval evidence to the diagnosis and put forward new targets for the treatment of melanoma. Recent reports show that there is an important link between FTO and melanoma cancer. Our recent studies find that there are differences of FTO expression between normal skin and melanoma cancer. First of all, higher FTO protein expression was detected by western blot in melanoma cancer cells, compared with primary cultured normal skin cells. However, the mRNA expression of both normal tissue and melanoma cancer are at the similar level. Secondly, FTO expression was also investigated in 80 clinical samples. Compared with normal skin tissues, malignant melanoma tissues had stronger positive immunohistochemical (IHC) staining of FTO in nucleus, and IHC scoring confirmed the significantly higher FTO protein expression in carcinoma tissues. In the normal skin tissues, FTO only expressed in basal nuclei; but in the melanoma cancer, FTO expressed in the dermal cells both at the nucleus and membrane. Especially, the expression of FTO is closely related with the pathological staging of melanoma. The increased FTO expression in melanoma carcinoma suggested that aberrant FTO expression may be an early molecular event in cancer development. Simultaneously, we focus on identifying the specific regulation function of FTO in the melanoma cancer cell, using the cDNA microarray and Two-dimensional fluorescence difference gel electrophoresis combining with MALDI-TOF-MS technique to screen the interaction proteins and their mutual regulation, determine the molecules interaction with FTO in the melanoma cancer development. Taken together, these data demonstrated that there is an important link between FTO and melanoma cancer, FTO gene plays an important role in melanoma pathogenesis.

Note: This abstract was not presented at the meeting.

Citation Format: Tianhui Niu, Min Xiong, Yan Tian. Related study on the fat mass and obesity associated FTO gene and melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2342. doi:10.1158/1538-7445.AM2014-2342
Abstract 2344: 3D extracellular stiffness cues drive localized changes in gene expression
Authors: Bainer Russell , Yui Yoshihiro , Mumenthaler Shannon , Mallick Parag , Podlaha Ondrej , Michor Franziska , Liphardt Jan , Licht Jonathan , Weaver Valerie
Published: May 2015

The stiffness of the extracellular matrix (ECM) stimulates mechanotransduction pathways that regulate tissue development and tumor progression. We previously showed that a stiff ECM potentiates cell growth and survival, enhances cell migration to drive tumor cell invasion, and drives malignant progression of the mammary gland in culture and in vivo, but the specific transcriptional and molecular events that occur as cells acquire these phenotypic changes are not well understood. To clarify this process, we used expression microarrays, tandem mass spectrometry, and RNA sequencing to identify changes in gene expression levels, isoform usage, and protein abundance that occur as intact ascini respond to distinct stiffness environments. We found that in stiffer ECM conditions ascini acquire consistent changes in gene expression related to cell adhesion and mRNA splicing, and specifically induce the expression of a set of genes involved in epithelial cell differentiation that includes multiple SPRR and S100 proteins. Remarkably, these genes map to an apparent stiffness-mediated transcriptional hotspot on chromosome 1q21, a region containing elevated transcription in many cancers but whose activity has not been related to mechanotransduction. We then used a heuristic approach to identify additional candidate force-mediated transcriptional hotspots throughout the genome that contain multiple genes that are coordinately activated or silenced in response to elevated ECM stiffness. Provocatively, we find that genes whose expression levels are responsive to ECM stiffness cues are disproportionately located within chromosomal regions that associate with the nuclear lamina, suggesting that these transcriptional changes may be due in part to force-dependent alteration of genomic contacts with the nuclear envelope. These studies provide biological insight into the divergent cellular responses to distinct stiffness environments and suggest that genome regulatory responses to the force environment may specifically target distinct chromosomal regions via a mechanism that remains to be elucidated.

Citation Format: Russell Bainer, Yoshihiro Yui, Shannon Mumenthaler, Parag Mallick, Ondrej Podlaha, Franziska Michor, Jan Liphardt, Jonathan Licht, Valerie Weaver. 3D extracellular stiffness cues drive localized changes in gene expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2344. doi:10.1158/1538-7445.AM2014-2344
Abstract 2348: Low PU.1 expression not only attenuates neutrophil differentiation of AML cells but also increases resistance to cytotoxic therapies
Authors: Tschan Mario P. , Haimovici Aladin , Brigger Daniel , Schläfli Anna M. , Shan Deborah , Fey Martin F.
Published: May 2015

Inactivation of PU.1 results in a myeloid differentiation block and contributes to the pathogenesis of acute myeloid leukemia (AML). A long list of PU.1 transcriptional targets with a direct function in myeloid development exits and clearly supports its role in cellular hematopoietic differentiation processes. Furthermore, we and others showed that PU.1 positively affects cell survival, for example by directly activating the anti-apoptotic genes BCL2A1 and BCL-XL. Recently, we demonstrated that the novel PU.1 target Hexokinase 3 supports acute promyelocytic leukemia (APL) cell survival. Surprisingly, our current data indicate that knocking down PU.1 in NB4 APL significantly increased resistance in apoptosis responses to anthracyclins. These findings were confirmed in MOLM-3 AML cells. Both cell lines showed an induction of the anti-apoptotic gene CFLAR (c-FLIP). c-FLIP blocks the activation of procaspase-8 at the death-inducing signalling complex (DISC) and is involved in AML chemotherapy resistance. Using PU.1 and c-FLIP double knockdown APL cells, we could significantly restore the sensitivity to anthracyclin treatment. This indicates that the PU.1 knockdown-dependent increase of c-FLIP is responsible for the resistance towards anthracyclin treatment. Moreover, we identified the pro-apoptotic kinase DAPK2 as novel transcriptional target of PU.1 in APL cells. Interestingly, we found that inhibiting DAPK2 in APL cells significantly attenuater arsenic trioxide (ATO)-induced cell death. Together, we link PU.1 to apoptosis and resistance against antracyclins and ATO via regulating c-FLIP and DAPK2. Our data indicate that low PU.1 level found in AML patients not only contribute to a block in differentiation but also increase resistance to cytotoxic therapies in AML.

Citation Format: Mario P. Tschan, Aladin Haimovici, Daniel Brigger, Anna M. Schläfli, Deborah Shan, Martin F. Fey. Low PU.1 expression not only attenuates neutrophil differentiation of AML cells but also increases resistance to cytotoxic therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2348. doi:10.1158/1538-7445.AM2014-2348
Abstract 2363: Large scale gene expression analysis identifies key oncogenic pathways across hematological malignancies
Authors: Liuksiala Thomas , Teittinen Kaisa , Granberg Kirsi , Heinäniemi Merja , Annala Matti , Ylipää Antti , Mäki Markku , Nykter Matti , Lohi Olli
Published: May 2015

Hematological malignancies, consisting of all the subtypes of leukemia and lymphoma together with multiple myeloma, constitute a diverse assembly of cancers manifesting primarily in the circulatory or immune systems. Common to all of them is their developmental origin in the hematopoietic cell lineage, which normally produces mature blood cells. For this reason, numerous transcriptomic aberrations, along with their underlying mutations, are shared by different subtypes of hematological malignancies.

To enable concurrent gene expression analysis across a comprehensive range of leukemias, lymphomas and multiple myeloma, we compiled a hematological dataset from gene expression microarray data publicly available in the Gene Expression Omnibus data repository. This manually curated collection includes over six thousand neoplastic and nearly nine hundred healthy samples of multiple lineages descending from the hematopoietic stem cell. The cancer samples were manually re-annotated in a congruent manner into categories with three hierarchical levels so as to allow analysis at various levels of specificity. For instance, an early B cell acute lymphocytic leukemia (ALL) sample is classified first as leukemia, secondly as ALL, and in the third, most specific level, as early B-ALL.

The power of the dataset lays not only in its extensiveness in terms of subtypes of hematological malignancies, but also in the sheer number of samples within these categories. This enables the discovery of expression signatures with high statistical significance, as well as identification of key oncogenic pathways and regulatory networks within different subtypes. To demonstrate these analyses, we used our database to search for any specific expression patterns of small nucleolar RNAs (snoRNAs) in acute leukemias. Among thousands of leukemia patients, we identified a specific signature of SNORD114 in acute promyelocytic leukemia, previously revealed in a very restricted cohort of patients.

Citation Format: Thomas Liuksiala, Kaisa Teittinen, Kirsi Granberg, Merja Heinäniemi, Matti Annala, Antti Ylipää, Markku Mäki, Matti Nykter, Olli Lohi. Large scale gene expression analysis identifies key oncogenic pathways across hematological malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2363. doi:10.1158/1538-7445.AM2014-2363
Abstract 2364: Pancreatic cancer stem-like cells display aggressive behavior mediated through the activation of FoxQ1
Authors: Bao Bin , Azmi Asfar , Ahmad Aamir , Ali Shadan , Li Yiwei , Sarkar Fazlul H.
Published: May 2015

Pancreatic cancer (PC) remains as one of the most aggressive and deadly malignant diseases with poorest clinical outcome, and it is the 4th leading cause of cancer-related death in the USA. Higher mortality of patients diagnosed with PC is in part attributed to treatment resistance and early metastatic disease. Sub-populations of cancer cells commonly known as cancer stem cells (CSCs) or cancer stem-like cells (CSLCs) have been identified from most tumors including PC, and the existence of these cells are being recognized to be clinically relevant. Emerging evidence suggests that CSLCs are responsible for many cellular functions including their propensity of increased cell growth/proliferation, migration/invasion, metastasis, and chemo-radio-therapy resistance, all of these attributes lead to poor clinical outcome. However, the pathogenesis and biological significance of CSLCs in PC has not been well characterized. In the present study, we found that triple-marker positive cells (CD44+/CD133+/EpCAM+ cells) cells isolated from human PC cell lines behave like CSCs. These CSLCs exhibit aggressive behavior such as increased cell growth, migration, clonogenicity, and self-renewal capacity. The mRNA expression profiling analysis showed that CSLCs (triple-marker positive cells) exhibit differential expression of more than 1600 mRNAs including Bmi1, BMP4, BST2, BTG1, FOLR1, FoxQ1, PRKAR1A, Sox2, TACSTD2 and Wnt3a, compared to the cells that are triple-marker negative (CD44-/CD133-/EpCAM- cells). The knock-down of FoxQ1 by its siRNA in CSLCs resulted in the inhibition of aggressive behavior, consistent with reduced expression of EpCAM and Snail. These data clearly suggest the role of differentially expressed genes in the regulation of CSLC characteristics, which further suggest that targeting some of these genes in order to eliminate CSLCs could be important for the development of novel therapies for achieving better treatment outcome of patients diagnosed with PC.

Citation Format: Bin Bao, Asfar Azmi, Aamir Ahmad, Shadan Ali, Yiwei Li, Fazlul H. Sarkar. Pancreatic cancer stem-like cells display aggressive behavior mediated through the activation of FoxQ1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2364. doi:10.1158/1538-7445.AM2014-2364
Abstract 237: Gene expression profiling identifies Sulindac-resistant colon tumors
Authors: Hanley Matthew P. , Miyamoto Shingo , Rosenberg Daniel
Published: May 2015

Sulindac is a non-steroidal anti-inflammatory drug (NSAID) with established chemopreventive efficacy for colorectal cancer (CRC). Despite its tumor suppressive effects, occasional resistance to this drug has been observed in clinical trials. To establish underlying mechanisms that may contribute to sulindac resistance, we have identified differentially expressed gene sets in two groups of colon tumors - those that respond to sulindac treatment and those that do not. Strain A mice were injected (ip) with azoxymethane (AOM) once per week for six weeks, followed by sulindac (180 ppm) treatment for an additional 15 weeks. Throughout the sulindac treatment period, tumor response was monitored endoscopically using a 3-mm fiber optic camera; tumor size and multiplicity were measured in situ every 3 weeks, allowing a real-time evaluation of tumor response to sulindac. At sacrifice, colons were removed and tumors were stratified according to size; large tumors (≥3 mm) were considered to be sulindac-resistant, whereas tumors ≤2 mm were classified as sulindac-responsive. Endoscopic measurements showed that presumed resistant tumors had an average doubling time of 0.9 ± 0.16 weeks, while sulindac-responsive tumors had an average doubling time of 4.0 ± 2.0 weeks. Whole genome expression analysis was conducted using an Illumina Mouse WG-6 Expression Microarray. Using Gene Ontology analysis, genes associated with inflammation, survival and proliferation were found to be differentially expressed between the two groups. Of particular interest was an increased expression of IL-1β and CXCL2, which have previously been implicated as part of a paracrine drug-resistance network in breast and lung cancers. Additionally, the growth factor MDK, which is involved in drug-resistant neuroblastomas, was found to be up-regulated in the largest colon tumors. These findings provide new insights into potential signaling networks that may contribute to the inconsistent response to sulindac chemoprevention and provide potential new targets for cancer protection.

Citation Format: Matthew P. Hanley, Shingo Miyamoto, Daniel Rosenberg. Gene expression profiling identifies Sulindac-resistant colon tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 237. doi:10.1158/1538-7445.AM2014-237
Abstract 2371: Heterogeneity of resistance mechanisms in lung adenocarcinoma patients with acquired resistance to EGFR inhibitors
Authors: Mu Xinmeng Jasmine , Allen Eli Van , Wagle Nikhil , Chong Curtis , Butaney Mohit , Farlow Deborah , Getz Gad , Jänne Pasi A. , Garraway Levi A.
Published: May 2015

Drug resistance is a major obstacle in overcoming cancer. Understanding the origin, extent and heterogeneity of resistance mechanisms within a patient is crucial to guide treatment and developing drugs/combinations to circumvent resistance. To this end, we studied autopsy samples collected from four EGFR-mutant lung adenocarcinoma patients who developed resistance to EGFR inhibitors. We used whole-exome sequencing to characterize genomic profiles of multiple metastasis tumors within each patient. To address the challenge of non-uniform variant detection power at sequenced genetic loci across multiple samples, we developed a computational framework that tackles variant detection from pooled tumor followed by a genotyping module. After the identification of somatic alterations, we determined the extent of heterogeneity of resistance mechanisms between multiple tumor samples. Our results revealed that multiple metastasis samples in a single patient can potentially use one common resistance mechanism. For example, one patient harbored a resistant EGFR T790M mutation in all 11 metastasis samples with a correlated allele frequency to the original L858R mutation. However, in some other cases, we found heterogeneous resistance mechanisms coexisting in multiple metastasis samples with various allele frequencies. For instance, one patient harbored both the resistance EGFR T790M mutation and an activating PIK3CA E542K mutation that appear with a range of allelic frequencies in multiple metastatic samples. Furthermore, branched evolutionary patterns of tumors were constructed using a phylogenetic approach, which inform resistance in the context of tumor evolution. Our findings are consistent with the model that cells harboring a variety of resistance mechanisms coexist in each metastasis tumor, and those subclones with higher selective advantage outgrow the others.

Citation Format: Xinmeng Jasmine Mu, Eli Van Allen, Nikhil Wagle, Curtis Chong, Mohit Butaney, Deborah Farlow, Gad Getz, Pasi A. Jänne, Levi A. Garraway. Heterogeneity of resistance mechanisms in lung adenocarcinoma patients with acquired resistance to EGFR inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2371. doi:10.1158/1538-7445.AM2014-2371
Abstract 2378: Harmonization of next generation sequencing data within consortia for gene discovery in familial breast cancer
Authors: Vijai Joseph , Hart Steven , Thomas Tinu , Wubbenhorst Bradley , Guidugli Lucia , Schrader Kasmintan , Maxwell Kara , Jacobs Lauren , Villano Danylo , Klein Robert , Lipkin Steven , Neuhausen Susan , Weitzel Jeffrey , Altshuler David , Couch Fergus , Offit Kenneth , Nathanson Katherine
Published: May 2015

Massively parallel next generation sequencing data (NGS) has proved to be useful in identification of rare Mendelian disorders. Efforts are on-going to use NGS for discovery of novel rare variants in common disease etiology. Distinguishing rare private mutations from causal variants remains a major challenge in complex disorders. With decreasing cost, investigators are generating NGS data for common cancers. However, power and other considerations suggest that a consortia approach of pooling data is more likely to succeed where individual efforts fail. Combining data within such consortia brings its own level of challenges. We enumerate these challenges and suggest recommendations based on our experience of combining and harmonizing NGS data on 358 breast cancer samples from four centers; (City of Hope n=8, Mayo Clinic n=221, MSKCC n=96 and University of Pennsylvania n=33). Major concerns were ability of individual datasets to discover known and novel variations, depth of coverage across the exome, and quality of variants. Using pre- and post-processed data, we demonstrate an increased power for detecting variants when using a joint-calling method, quality control (QC) and quality assurance pre- and post-merge, as well as the advantages of combined annotations and filtering procedures. To generate a filtered list of high confident calls, we subset the data on Depth per Allele (DP), Genotyping Quality (GQ), Allele balance, Phred likelihood score (PL) as well as using frequency filters on the alternate allele both within the data and comparing against convenience control summaries. We describe general principles and QC measures to be adopted when NGS data is used from diverse sources, specifically applicable to large consortia.

Note: This abstract was not presented at the meeting.

Citation Format: Joseph Vijai, Steven Hart, Tinu Thomas, Bradley Wubbenhorst, Lucia Guidugli, Kasmintan Schrader, Kara Maxwell, Lauren Jacobs, Danylo Villano, Robert Klein, Steven Lipkin, Susan Neuhausen, Jeffrey Weitzel, David Altshuler, Fergus Couch, Kenneth Offit, Katherine Nathanson. Harmonization of next generation sequencing data within consortia for gene discovery in familial breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2378. doi:10.1158/1538-7445.AM2014-2378
Abstract 2406: Epigenetics of chromosomal breakage sites and translocations
Authors: Burman Bharat , Zhang Zhuzhu , Burgess Rebecca , Roukos Vassilis , Lieb Jason , Misteli Tom
Published: May 2015

Chromosomal translocations are genetic hallmarks of most cancer cells. Translocations require the formation of DNA double-strand breaks (DSBs) at two or more genomic loci, followed by the illegitimate joining of broken chromosomal ends through DNA repair. There is increasing evidence that translocations occur at non-random sites in the genome, suggesting that certain regions of the genome are more susceptible to DNA breakage than others. We hypothesize that altered chromatin structure predisposes genomic sites to DNA breakage and translocations. To identify chromatin features that facilitate translocations, we have mapped histone modifications and DNase I hypersensitive sites (DHSs) at translocation-prone regions in anaplastic large cell lymphoma (ALCL) precursor cells. We find enrichment of active histone marks and a decrease in repressive marks near frequent translocation breakpoints. In a complementary approach using genome-wide histone modification mapping we have identified altered chromatin features at common leukemia and lymphoma breakpoints in CD34+ hematopoietic stem cells. In order to directly test the role of chromatin features in DNA breakage susceptibility, we have developed a protein-DNA tethering system that allows us to create local chromatin domains at pre-defined sites in the genome containing inducible DSB sites in vivo. By measuring the amount of DSBs using ligation-mediated PCR, we find that histone modifying enzymes that create active chromatin marks generally increase breakage susceptibility; however, repressive marks do not necessarily decrease breakage susceptibility. This suggests the involvement of other factors in breakage susceptibility. Taken together, these experiments provide first insights into the role of chromatin structure in the formation of nonrandom chromosomal breaks and the mechanisms that lead to clinically relevant translocations.

Citation Format: Bharat Burman, Zhuzhu Zhang, Rebecca Burgess, Vassilis Roukos, Jason Lieb, Tom Misteli. Epigenetics of chromosomal breakage sites and translocations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2406. doi:10.1158/1538-7445.AM2014-2406
Abstract 241: CHOP-deficiency promotes chronic inflammation-induced pancreatic fibrosis
Authors: Taki Katsunobu , Ohmuraya Masaki , Sakata Kazuya , Hashimoto Daisuke , Abe Shinya , Nitta Hidetoshi , Hayashi Hiromitsu , Chikamoto Akira , Beppu Toru , Baba Hideo
Published: May 2015

Background and Aims: Endoplasmic reticulum (ER) stress is involved in the pathogenesis of various diseases, including neurodegenerative disease, diabetes mellitus, atherosclerosis, heart disease, and pancreatic disease. CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153 (GADD153) is a transcriptional regulator induced by ER stress and a key factor in the ER stress-mediated apoptosis pathway. Chronic inflammation in the pancreas causes progressive pancreatic fibrosis that eventually leads to pancreatic ductal adenocarcinoma. The aim of our study is to prove the role of the ER stress-CHOP pathway in chronic pancreatitis. Methods: We generated gene engineered chronic pancreatitis model (Spink3 SPINK1/-) mice, and crossed them with CHOP-deficient (Chop-/-) mice. We assessed the pancreatic fibrosis by hematoxylin eosin and Sirius red staining, and performed Western blotting to measure α-SMA, which is a maker of the activated pancreatic stellate cells, transforming growth factor β, amylase, and trypsinogen, which are makers of exocrine function, in wild-type (Chop+/+) and CHOP-deficient chronic pancreatitis model (Chop-/-; Spink3 SPINK1/-) mice at 2-weeks- and 4-weeks-old. Results: There were no significant differences between the pancreases of Chop+/+ and Chop-/- mice. The body weight of Chop-/-; Spink3 SPINK1/- mice was not so different from that of Spink3 SPINK1/- mice at 2-weeks-old, but the body weight of Chop-/-; Spink3 SPINK1/- mice was lighter than that of Spink3 SPINK1/- mice at 4-weeks-old (p=0.001). However, there was no significant difference between the survival of Spink3 SPINK1/- mice than that of Chop-/-; Spink3 SPINK1/- mice. We found the progression of pancreatic fibrosis and the activated pancreatic stellate cells in Chop-/-; Spink3 SPINK1/- mice compared with Spink3 SPINK1/- mice at 2-weeks-old. In addition, the expression levels of α-SMA and Desmin increased in Chop-/-; Spink3 SPINK1/- mice at 2-weeks-old. Conclusions: These results indicated that the ER stress-CHOP pathway may have an essential role in the suppression of pancreatic fibrosis through the inactivation of pancreatic stellate cells in chronic pancreatitis. Therefore, anti-inflammatory treatment against chronic pancreatitis through the ER stress-CHOP pathway may reduce the risk of pancreatic cancer.

Citation Format: Katsunobu Taki, Masaki Ohmuraya, Kazuya Sakata, Daisuke Hashimoto, Shinya Abe, Hidetoshi Nitta, Hiromitsu Hayashi, Akira Chikamoto, Toru Beppu, Hideo Baba. CHOP-deficiency promotes chronic inflammation-induced pancreatic fibrosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 241. doi:10.1158/1538-7445.AM2014-241
Abstract 2410: Crucial role of the non-BRCT automodification domain of PARP1 in either DNA binding and PARP inhibitor-mediated cell toxicity
Authors: Nakamura Jun , Tian Xu
Published: May 2015

Poly(ADP-ribose)polymerase (PARP) inhibitor (PARPi)-mediated synthetic lethality is an attractive new paradigm for cancer therapy. While it has been recently proposed some of PARPi may cause DNA single strand break (SSB)-PARP1 or -PARP2 complexes, leading to cell toxicity, the mode of action of PARPi-mediated synthetic lethality is not well understood. We analyzed DNA damage response to PARPi using an isogenic set of DT40 cells. Both PARP-1 ko mutants and PARP-1 ko mutants expressing DNA bindin domain-defficient PARP1 showed markedly more resistance to olaparib than parental DT40 cells, indicating that PARP1 binding to DNA strand break through DNA binding domain is critical in olaparib-mediated cell toxicity. While non-BRCT automodomain domain deletion mutant showed a resistant phenotype, olaparib causes similar toxicity in BRCT domain deletion mutant and parental DT40 cells. Furthermore, PARP1 localized in chromatin binding fraction in BRCT1 deletion mutant, but not in non-BRCT automodomain domain deletion mutant. These results indicate that non-BRCT automodification domain plays crucial role for the formation of stable DNA SSB-PARP1 complex and olaparib-mediated synthetic lethality.

Citation Format: Jun Nakamura, Xu Tian. Crucial role of the non-BRCT automodification domain of PARP1 in either DNA binding and PARP inhibitor-mediated cell toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2410. doi:10.1158/1538-7445.AM2014-2410