bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 412: Application of a serial liquid biopsy ctDNA assay for monitoring efficacy of anti-angiogenic lung cancer therapy
Authors: Minarik Marek , Svaton Martin , Belsanova Barbora , Semyakina Anastasiya , Baxa Jan , Fiala Ondrej , Pesek Milos , Benesova Lucie
Published: August 2019

INTRODUCTION: Patients in advanced stages of non-small cell lung carcinomas (NSCLC), who and unsuited for targeted biological therapy because of lack of actionable molecular predictors are frequently treated by anti-angiogenic therapy. The effectiveness of such therapy is primarily relying on imaging techniques including CT or hybrid PET/CT evaluating a combination of morphological factors (dimensions and volume) and, more recently, also functional parameters including the metabolic activity, tumor vascularization etc. The ability to track the course of the disease at the same time all of these parameters is prerequisite to enable timely monitoring and therapy change in case of an early detection of resistance. This study was aimed at utility of circulating-tumor DNA (ctDNA) as an tool for monitoring of therapy response. and assessment of phenotypic parameters of the tumor.

PATIENTS AND METHODS: A total of 50 patients with confirmed Stage IV lung adenocarcinomas showing negativity on ALK, BRAF, EGFR, RET, ROS1 and MET predictors were prospectively enrolled into the study. Patients were treated under a standard protocol by a combination of paclitaxes/carboplatin/bevacizumab and followed by dual PET/CT. For each patient cytology tissue sample was subjected to test for a panel of somatic mutations. The found mutations were subsequently detected in ctDNA in plasma extracted from peripheral blood collected prior to therapy start and then in 1-month intervals during the therapy. The occurrence and quantity of ctDNA were correlated with the objective therapy response (RECIST) and to the functional imaging parameters of metabolic activity and vascularization.

RESULTS: ctDNA was initially positive in 29 patients. ctDNA levels closely reflected the response to the therapy with complete or partial remission expressed as reduction or absence of ctDNA, while disease stabilization or progression signaled by persisting or increasing ctDNA levels. There was a borderline correlation between ctDNA and vascularization evaluated by dual PET/CT (p=0.055).

CONCLUSION: In a subset of patients ctDNA can readily serve as a surrogate marker for semi-continuous monitoring of the effect of anti-angiogenic therapy. Work supported by Czech Ministry of Health project AZV 17-30748A.

Citation Format: Marek Minarik, Martin Svaton, Barbora Belsanova, Anastasiya Semyakina, Jan Baxa, Ondrej Fiala, Milos Pesek, Lucie Benesova. Application of a serial liquid biopsy ctDNA assay for monitoring efficacy of anti-angiogenic lung cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 412.
Abstract 4226: TNFRSF14 is a cell surface marker of MITF expression in melanoma
Authors: Dutton-Regester Ken , Hayward Nicholas K.
Published: August 2019

Background: Transcriptional ‘cell states’ (defined as the genes expressed in a cell in any given point in time) can determine response to targeted and immune-based therapies in late-stage melanoma treatment. MITF and AXL are common markers of drug-sensitive and drug-resistant ‘cell states’, respectively; and extensive heterogeneity of both cell populations have been observed in melanoma cell lines and tumors. Understanding the mechanisms controlling these ‘cell-states’ may lead to therapeutic strategies to overcome resistance and improve patient survival.

Problem: To date, certain experiments assessing cell population heterogeneity dynamics in melanoma (such as live cell sorting applications) has been limited by the lack of surface markers defining the MITF ‘cell-state’. Furthermore, existing antibodies against MITF commonly detect multiple expressed isoforms which can limit the accuracy of downstream experimental assays, such as in-cell western blot detection.

Methods and Results: To find a suitable cell surface marker defining the MITF ‘cell state’, we used the PARIS GenePattern module with data from the Cancer Cell Encyclopedia. TNFRSF14, our highest ranked gene, was highly correlated to MITF transcript expression in melanoma cell lines. We confirmed this at the protein level through western blot detection and flow cytometry. Using flow cytometry, we could accurately follow ‘cell-state’ population dynamics of AXL and MITF following shRNA knockdown of MITF and during acquired drug resistance in culture.

Outcomes: We have identified TNFRSF14 as a robust cell surface marker of MITF in melanoma. Our TNFRSF14 and AXL live cell flow protocol will be useful for those exploring ‘cell-state’ population dynamics and heterogeneity in melanoma and could contribute to new mechanistic insights of drug resistance.

Citation Format: Ken Dutton-Regester, Nicholas K. Hayward. TNFRSF14 is a cell surface marker of MITF expression in melanoma [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 4226.
Abstract 425: Development and validation of liquid biopsy assay for copy number loss in cancers
Authors: Zhang Simo , Xie Feng , Chang Amy , Zhao Zhixin , Montesinos Carlos , Wang Xiaohong , Zhou Kemin , Jia Shidong , Yu Jianjun , Du Pan
Published: August 2019

Introduction: Copy number loss (CNL) of tumor suppressor genes, such as PTEN, RB1 and BRCA1/2, are common and key characteristics of many cancers. However, non-invasive detection of CNL has represented a strong demand and practical challenge in the clinic. Here we report a high-sensitive liquid biopsy NGS assay coupled with a proprietary bioinformatics pipeline to profile and detect CNL using cell-free circulating DNA (cfDNA).

Method: We developed PrediSeq, a proprietary liquid biopsy NGS assay to detect CNL, in addition to SNV, indels, rearrangement, and copy number gain. We leveraged unique molecular identifier (UMI) and proprietary bioinformatics algorithm to suppress background noise caused by PCR pre-amplification and sequencing steps. In-house developed bioinformatics pipeline, DeepSea, is used for ultra-sensitive detection of CNL and other genomic alterations. Reference materials, matched tissue and plasma samples were used for assay validation.

Results: PrediSeq CNL assay was validated using serially diluted reference materials with known copy number status. The method was evaluated with the orthogonal whole-genome sequencing using a set of clinical plasma samples with matched tissues. About 82% of CNL calls (ATM, BRCA1, BRCA2, CDKN2A, RB1) in were confirmed between plasma PrediSeq and tissue WGS. In alignment with literature, CNL of ATM, CDKN2A, BRCA1, BRCA2, was observed in plasma samples of a cohort of Her2+, HR+, and triple-negative breast cancer patients. Similarly, we observed the dynamic changes of plasma-based CNL of BRCA2, PTEN, RB1, CDKN2A, ATM genes in patients of metastatic castration-resistant prostate cancer (mCRPC) and metastatic hormone sensitive prostate cancer (mHSPC).

Conclusion: PrediSeq CNL assay has been successfully developed and validated to detect copy number loss in circulation, providing valuable molecular biomarkers to guide patient selection and treatment monitoring in precision oncology and clinical drug development.

Citation Format: Simo Zhang, Feng Xie, Amy Chang, Zhixin Zhao, Carlos Montesinos, Xiaohong Wang, Kemin Zhou, Shidong Jia, Jianjun Yu, Pan Du. Development and validation of liquid biopsy assay for copy number loss in cancers [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 425.
Abstract 4273: Inhibition of autophagy increases HNSCC sensitivity to cancer therapies
Authors: Lee Yong-Syu , Skiba Justin , Kubatzke Jaimee , Nickel Kwangok P. , Kimple Randall
Published: August 2019

Background: Radiation and EGFR-targeted therapies are commonly used in the treatment of head and neck squamous cell carcinoma (HNSCC). These treatments fail to control a significant number of cancers resulting in a 5-year survival rate that remains around 40-50%. We have shown that both cetuximab and radiation induce autophagy, a pro-survival cellular stress response, in head and neck cancer. In this study, we examine the consequence of autophagy inhibition and investigate the molecular mechanism underlying therapy-induced autophagy.

Methods: Autophagy was assessed using a nano-Luc LC3 reporter (Promega), immunofluorescence for LC3, p62, and acridine orange in HNSCC cell lines. RNAi knockdown of EGFR and LAPTM4B were used to test the involved signaling molecules. Radiation was delivered using a RS225 cabinet irradiator at a dose rate of approximately 3 Gy/min with dose validation by TLD using custom phantoms. Cetuximab was delivered via intraperitoneal injection. Vps34 inhibitor SAR405 and ULK1 inhibitor SBI-0206965 were used to determine whether inhibition of autophagy reduces cell survival or represses cancer cell growth in the clonogenic assay. A flank xenograft model using A253 cells was used to test the combination of autophagy inhibitors and current therapies in vivo.

Results: As previously shown, both cetuximab and radiation induced autophagy by two times. Knockdown of EGFR and LAPTM4B decreased autophagy (62.5% and 65%, respectively) when assessed using the nano-Luc reporter assay. Similar results were seen using IF. Using a clonogenic survival assay, the combination of SAR405 and radiation resulted in complete loss of cell survival suggesting a radiosensitizing effect. In vivo, SAR405 treatment improved tumor control when combined with radiation or cetuximab when compared to either treatment alone.

Conclusions: Therapy induced autophagy is dependent upon expression of both EGFR and LAPTM4B. Inhibition of autophagy resulted decreased cell survival in vitro and resulted in decreased in vivo tumor growth. These results suggest that inhibition of autophagy may be a viable approach to sensitize HNSCC to anti-cancer treatments.

Citation Format: Yong-Syu Lee, Justin Skiba, Jaimee Kubatzke, Kwangok P. Nickel, Randall Kimple. Inhibition of autophagy increases HNSCC sensitivity to cancer therapies [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 4273.
Abstract 4275: Gastric cancer stem cells under starvation stress might sustain their stemness via autophagy system
Authors: Togano Shingo
Published: August 2019

Background: Cancer stem cells (CSCs) play an important role for the progression of carcinoma. And, CSCs have a potential of resistance to various stresses such as starvation and chemotherapy. Autophagy is an intracellular degradation system which is induced various kinds of cells under various stress. In cancer cells, autophagy is activated and has involved in cancer survival. However, the significance of autophagy in CSCs has still remained to be unknown, especially in starvation and chemotherapy stress. The aim of this study is to clarify the role of autophagy in CSCs under starvation condition.

Materials and Methods: Two scirrhousgastric cancer cell lines, OCUM-2MD3 and OCUM-12 (parent cells) were used. Side population cells (SP cells) which include many CSCs, was known to the fraction of low dyeability of Hoechst 33342 identified by FACScan. Stemness of SP cells, OCUM-2MD3/SP and OCUM-12/SP, were examined by sphere colony assay and RT-PCR using stemness markers, Nanog, Oct3/4, CD44. Then we examined the effect of autophagy in CSCs under starvation condition, as follows.Parents cells and SP cells were evaluated about autophagy. Parents cells and SP cells were incubated in the presence and absence of glucose for various time. The expression level of autophagy molecule, LC3 was examined by western blotting. The effect of an autophagy inhibitor, Chloroquine, to SP frequency was examined by FACScan.

Results: The expression of Nanogand Oct3/4, were significantly higher in OCUM-2MD3/SP rather than parent cells OCUM-2MD3. Also, these stemness markers were significantly higher in OCUM-12/SP cells rather than parent cells. The number of sphere colony was greater in both SP cells rather than that in parent cells. The expression level of LC3 was high in both SP cells, in comparison to that in parent cells under glucose deficient condition.The autophagy inhibitor,Chloroquine, significantly decreased the SP fraction in both cells.

Conclusion: CSCs might have high autophagy activity under starvation stress and sustain their stemness by autophagy system. The autophagy inhibitor,Chloroquine, might be a promising agent for gastric cancer.

Citation Format: Shingo Togano. Gastric cancer stem cells under starvation stress might sustain their stemness via autophagy system [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 4275.
Abstract 4289: ERK3 negatively regulates IL-6/STAT3 signaling via SOCS3 in cancer cells
Authors: Shakya Astha , Chen Minyi , Markey Michael , Long Weiwen
Published: August 2019

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen activated protein kinase (MAPK) which is dysregulated in several cancers including lung cancer, breast cancer and melanoma. It has a single Ser-Glu-Gly (SEG) phospho-acceptor motif in its activation loop, instead of the Thr-Xaa-Tyr (TXY) motif conserved in the classical MAPKs. Moreover, compared to the conventional MAPKs, much less is known about the targets of ERK3 signaling. Hence, to elucidate the ERK3-regulated genes and pathways, we performed gene microarray analyses of both A549 lung cancer cells and A375 melanoma cells after treatment with either a siRNA specifically targeting ERK3 or a non-targeting control siRNA. Interestingly, IL-6 target genes were found to be highly upregulated upon ERK3 knockdown. Interleukin-6 (IL-6) is a pleiotropic cytokine which signals via the JAK/STAT3 pathway. Phosphorylation of STAT3 at Tyrosine 705 (Y705) by JAK activates STAT3 in response to IL-6 stimulation. IL-6 signaling is negatively regulated by a feedback inhibitor SOCS3 (Suppressor of cytokine signaling 3), which binds to and inhibits JAK kinase activity. Interestingly, we noted that ERK3 interacts with SOCS3 from a published Yeast-two-hybrid screening. We validated the interaction between ERK3 and SOCS3 by co-immunoprecipitation assay. In addition, we found that ERK3 downregulates the phosphorylation of STAT3 at Y705. In line with its negative regulation of oncogenic STAT3 activity, ERK3 inhibits melanoma cell growth, migration/invasion and colony formation in soft agar. Taken together, we have revealed a novel role for ERK3 in suppressing IL-6/STAT3 signaling via SOCS3 in cancer cells.

Citation Format: Astha Shakya, Minyi Chen, Michael Markey, Weiwen Long. ERK3 negatively regulates IL-6/STAT3 signaling via SOCS3 in 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 4289.
Abstract 430: Prostate specific antigen (PSA) as a diagnostic and prognostic marker of prostate cancer
Authors: Simon Ronald , Kluth Martina , Fischer Kristine , Bonk Sarah , Hube-Magg Claudia , Höflmayer Doris , Lennartz Maximilian , Lutz Florian , Sauter Guido , Büscheck Franziska , Uhlig Ria , Dum David , Luebke Andreas M. , Wittmer Corinna , Jacobsen Frank , Burandt Eike , Steurer Stefan , Wilczak Waldemar , Minner Sarah
Published: August 2019

Since several decades, prostate specific antigen (PSA) is regarded as a specific marker for healthy and neoplastic prostate tissues, and PSA level changes in blood and tissue are discussed as prognostic markers of prostate cancer. Using a custom-made mouse monoclonal anti-PSA antibody, we studied expression of PSA in a tissue microarray (TMAs) comprising 17,456 prostate cancers with long-term follow-up data. PSA staining was typically present in the cytoplasm and pronounced at the apical cell pole. PSA expression was often reduced (15%) or even completely lost (1%). Reduction of PSA expression was significantly linked to adverse features of prostate cancer, including advanced stage, high classical and quantitative Gleason grade, presence of lymph node metastasis, a positive surgical margin, increased cell proliferation, and reduced time to recurrence (p<0.0001 each). Complete lack identified a small subset of patients with the worst prognosis. There was also in impact of the subcellular localization of PSA expression: Loss of the apical staining pattern worsened the prognosis in cancers with otherwise identical levels of cytoplasmic staining. In a multivariable analysis including pT stage, Gleason grade, nodal stage, and resection margin status, loss of PSA expression proved to predict outcome independently from these parameters. Of note, PSA even stratified the prognosis of 971 cancers harboring PTEN deletions in our cancer set. In conclusion, gradual reduction of PSA staining, including a loss of the apical staining pattern, is a strong and independent prognostic marker in prostate cancer.

Citation Format: Ronald Simon, Martina Kluth, Kristine Fischer, Sarah Bonk, Claudia Hube-Magg, Doris Höflmayer, Maximilian Lennartz, Florian Lutz, Guido Sauter, Franziska Büscheck, Ria Uhlig, David Dum, Andreas M. Luebke, Corinna Wittmer, Frank Jacobsen, Eike Burandt, Stefan Steurer, Waldemar Wilczak, Sarah Minner. Prostate specific antigen (PSA) as a diagnostic and prognostic marker of prostate 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 430.
Abstract 4313: Identifying and characterizing epigenetic «driver» genes («epidrivers») in regulatory pathways involved in tumorigenesis and tumour cell plasticity
Authors: Halaburkova Andrea , Cahais Vincent , Cuenin Cyrille , Khoueiry Rita , Ouzounova Maria , Ghantous Akram , Herceg Zdenko
Published: August 2019

One of the ground-breaking discoveries of the international cancer genome sequencing endeavours is the high frequency of mutational and non-mutational changes in epigenetic regulator genes (ERGs), which constitute a “genetic smoking gun” that epigenetic mechanisms lie in the very heart of cancer biology. However, functional importance of disruption of ERGs in tumorigenesis and cancer phenotype is poorly understood. We hypothesise that these genes are candidates to be drivers («epidrivers») of cancer onset and progression, thus regulating mechanisms underpinning cancer development. Moreover, deregulation of epidrivers may play a role in epithelial-to-mesenchymal transition (EMT) and emergence of cancer resilience. The overarching aim of this study was to identify and functionally characterize “Epidrivers” in tumorigenesis and cancer cell plasticity. To this end, we have set up novel epigenome-wide functional screens in human cultured cells (and organoids) combined with state-of-the-art genome-editing approach (based on CRISPR/Cas9 screen) and multiparametric phenotyping. We designed a custom-made lentiviral CRISPR library that consists of 1,649 gRNAs targeting all known ERGs (426 genes). Lentiviral CRISPR library was used to deliver the ERG gRNAs to target cells expressing the RNA-guided DNA endonuclease Cas9. Independent clones (derived from transduced breast and lung cancer cell lines constitutively expressing Cas9) are screened for acquisition of distinct features of transformed cells. The results of the identification of Epidriver genes based on the analysis of deregulation of core cellular processes, epigenome (ChIP-seq) as well as EMT and multiparametric phenotyping, will be presented.

Citation Format: Andrea Halaburkova, Vincent Cahais, Cyrille Cuenin, Rita Khoueiry, Maria Ouzounova, Akram Ghantous, Zdenko Herceg. Identifying and characterizing epigenetic «driver» genes («epidrivers») in regulatory pathways involved in tumorigenesis and tumour cell plasticity [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 4313.
Abstract 4315: UNC0642 inhibits G9a and induces apoptosis of human bladder cancer cells
Authors: Huang Ruimin , Sun Jingya , Li Meiqian , Dong Yu , Zhang Yuanheng , Yan Jun
Published: August 2019

Urinary bladder cancer (UBC) is one of the most common malignancies in the world, with the characteristics of frequent recurrence and metastasis despite the standard chemotherapy with gemcitabine and cisplatin combination. Hence, it is needed to develop new drugs to tackle down UBC. Histone modifiers, which are frequently dysregulated in cancer development, are excellent drug targets for cancer treatment. Here, we found that G9a, one of the histone H3 methyltransferases, was overexpressed significantly in human UBC patients, using the expression data from public databases. In the TCGA Provisional dataset, the average expression level of G9a in primary UBC samples (n=408) was 1.6-fold as much as that in normal bladder samples (n=19; P < 0.001). Knockdown G9a by small interfering RNA decreased cell viability and induced apoptosis in human UBC cell lines. Thus, UNC0642, a small molecule inhibitor targeting G9a with high selectivity, low cytotoxicity, and excellent in vivo pharmacokinetic properties, was chosen to test the inhibitory effect on UBC cells. UNC0642 decreased the level of histone H3K9me2, the downstream target of G9a, and increase apoptosis of UBC cells in vitro in a dose-dependent manner. UNC0642 exhibited similar inhibitory effects on the J82 xenografts, without the loss of body weight of mice. Targeting G9a in vivo reduced Ki67 expression and increase the level of cleaved Caspase 3 and BIM protein by immunohistochemistry and Western blot analysis, respectively. Taken together, our data indicate that G9a may be a promising therapeutic target for UBC, and an epigenetics-based therapy by UNC0642 is suggested.

Citation Format: Ruimin Huang, Jingya Sun, Meiqian Li, Yu Dong, Yuanheng Zhang, Jun Yan. UNC0642 inhibits G9a and induces apoptosis of human bladder 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 4315.
Abstract 4326: Epigenetic control of chromosome instability by STAT3 in gastric cancer
Authors: Chuang Yu Ming , Hung Sheng-Jou , Chou Jiang Liang , Huang Wan-Hong , Yan Pearlly S. , Liu Tsunglin , Chan Michael W.Y.
Published: August 2019

Gastric cancer is one of the most common causes of cancer death worldwide. Aberrant activation of JAK/STAT3 signaling is frequently observed in gastric cancer and associated with cancer progression. Our previous studies demonstrated that several STAT3 targets and tumor suppressors such as NR4A3 and GATA6 are found to be epigenetically silenced by DNA methylation in gastric cancer. However, the role of aberrant JAK/STAT3 signaling in the epigenetic changes of downstream targets is not fully understood. In this study, by using Methylation EPIC array, we compared the global methylation changes in gastric cancer patient samples with different STAT3 activation level. In silico analysis was also performed to identify STAT3 binding sites in those differentially methylated loci. Hypermethylated loci which are functionally relate carcinogenesis and mediated by STAT3 expression were examined. Interestingly, SMARCAL1, which was involved in chromosome stability and alternative lengthening of telomeres was identified. Unexpectedly, differentially methylated region (DMR) was not occurred in the promoter, but enhancer region located in the CpG island shore. Additionally, we also found a transcription factor binding site, YY1, which was a methylation-sensitive enhancer-promoter regulator, in this DMR region. By using a statistic shuffling model, we found that STAT3 related methylation changes were significantly associated with H3K4me1 and H3K27me3 at that region. TCGA dataset also indicated that this DMR with dramatic changes was occurred in chromosome instability (CIN) subtype of gastric cancer, whereas the expression of SMARCAL1 was decreased in precancerous lesion in previous published data. Taken together, STAT3-related methylation changes may regulate the activity of enhancer and affect the expression of SMARCAL1, during gastric cancer progression.

Citation Format: Yu Ming Chuang, Sheng-Jou Hung, Jiang Liang Chou, Wan-Hong Huang, Pearlly S. Yan, Tsunglin Liu, Michael W.Y. Chan. Epigenetic control of chromosome instability by STAT3 in gastric 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 4326.
Abstract 4356: Menin and ATF4 cooperate to drive serine biosynthesis in Ewing sarcoma
Authors: Jimenez Jennifer , Svoboda Laurie K. , Sud Sudha , Kerk Samuel , Grembecka Jolanta , Lyssiotis Costas A. , Lawlor Elizabeth R.
Published: August 2019

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor. We previously reported that the scaffolding protein menin is overexpressed by ES, and that menin inhibition results in impaired ES cell proliferation, survival, and tumorgenicity. Additionally, our recent studies revealed a previously undescribed role for menin in the activation of the serine biosynthetic pathway (SSP), a critical metabolic pathway that is aberrantly activated in many human cancers. The biologic functions of menin are largely determined by its protein-binding partners, the best characterized of which is the histone methyltransferase MLL. In the current study, we are investigating the mechanistic link between menin and the SSP to determine whether menin activates the SSP via epigenetic trithorax complexes.

Our data show that the core SSP enzymes, PHGDH, PSAT1, and PSPH are highly expressed by ES. Moreover, expression of all three genes is highly correlated in primary tumors, suggesting that they may be coordinately regulated by an upstream factor. In other cancer types, activation of the SSP has been shown to result from amplification of the PHGDH locus or activation of the master transcriptional regulator, ATF4. We have found that PHGDH is not amplified in ES but ATF4 is overexpressed, and ATF4 loss of function leads to growth inhibition. Q-RT-PCR and western blot of ES cells following ATF4 knockdown reveals down-regulation of PHGDH, PSAT1, and PSPH. In addition, ChIP-qPCR shows enrichment of ATF4 binding at SSP gene promoters, which is diminished by treatment with MI-503, a menin:MLL interaction inhibitor. Menin inhibition with MI-503 also leads to loss of ATF4 expression, coincident with loss of SSP expression. Additionally, ChIP-qPCR shows enrichment of menin binding at the ATF4 gene promoter, which is associated with H3K4me3 enrichment. Preliminary studies show that ATF4 over-expression via lentiviral transduction may rescue the loss of SSP gene expression induced by MI-503. Together these findings support the hypothesis that ATF4 acts downstream of menin to drive SSP activation in ES. Ongoing studies are assessing whether this is mediated by trithorax-dependent or –independent functions of menin.

Citation Format: Jennifer Jimenez, Laurie K. Svoboda, Sudha Sud, Samuel Kerk, Jolanta Grembecka, Costas A. Lyssiotis, Elizabeth R. Lawlor. Menin and ATF4 cooperate to drive serine biosynthesis in Ewing sarcoma [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 4356.
Abstract 4366: Characterization of diacetylspermine as a metabolic urinary biomarker in breast cancer using patient-derived xenografts
Authors: Velenosi Thomas J. , Krausz Kristopher W. , Gonzalez Frank J.
Published: August 2019

Breast cancer is a heterogeneous disease and is currently diagnosed using clinical biomarkers to classify tumors and guide therapy. However, drug resistance is a major cause of treatment failure and current methods can only detect resistance after several months following drug therapy. Thus, there is a need for novel biomarkers to track breast cancer prognosis, diagnosis, and to monitor therapeutic efficacy. Polyamines are polycations that support the production of nucleic acids and are necessary for transcription, DNA replication and cell cycle progression. The rate-liming step in polyamine catabolism is the acetylation of spermine and spermidine by spermine/spermidine acetyl-transferase (SAT1). Indeed, diacetylspermine has been identified as a clinical urinary biomarker of early and late stage breast cancer. Despite multiple clinical studies citing the potential for diacetylspermine as a cancer biomarker, the oncogenic events contributing to increased diacetylspermine production are largely unknown. To identify metabolic biomarkers of breast cancer progression and therapeutic efficacy, an untargeted metabolomics study was performed using urine samples from mice implanted with breast cancer patient derived xenografts (PDXs). NSG mice were engrafted with a doxorubicin sensitive (Dox-Sens) or a doxorubicin resistant (Dox-Res) PDX. When tumors reached 100-200 mm3, mice were administered intravenous doxorubicin (2 mg/kg) or vehicle control once weekly for three weeks. Mice were placed in metabolic cages for 24 hours after each treatment to collect urine. At 28 days, a final 24-hour urine collection was performed in the absence of drug treatment followed by euthanization (n = 9). Untargeted metabolomics revealed diacetylspermine as the most significantly altered metabolite in urine samples by two-way repeated measures ANOVA (p = 1.26x10-11, q = 4.82 x 10-8). Urinary diacetylspermine was correlated with growth of the Dox-Sens tumors and undetectable in urine from mice with Dox-Res tumors. Remarkably, when Dox-Sens mice were treated with doxorubicin, there was a further increase in diacetylspermine despite the decrease in tumor size. These results demonstrate the potential utility of urinary diacetylspermine as a biomarker for monitoring tumor growth and doxorubicin treatment efficacy. Furthermore, these breast cancer PDX models have provided an opportunity to characterize the mechanism of oncogenic diacetylspermine production and response to drug treatment.

Citation Format: Thomas J. Velenosi, Kristopher W. Krausz, Frank J. Gonzalez. Characterization of diacetylspermine as a metabolic urinary biomarker in breast cancer using patient-derived xenografts [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 4366.
Abstract 4374: The role of glycine decarboxylase in neuroblastoma
Authors: Alptekin Ahmet , Ding Jane , Ye Bingwei , Ding Han-Fei
Published: August 2019

Neuroblastoma is the most common cancer in infants. While the low and intermediate risk groups of neuroblastoma patients have ≥90% five-year-survival rates, the high-risk group has only 40-50% five-year-survival rates despite multimodal therapies. Genomic amplification of the oncogene MYCN (a member of the MYC family of transcription factors) is a major cause of high-risk neuroblastoma and is strongly correlated with poor prognosis. A key function of MYCN is to promote cell growth and proliferation, which requires increased cellular metabolism to meet the biosynthetic demand for growth. Serine-Glycine-One-Carbon (SGOC) metabolism is particularly important in sustaining cell proliferation by producing amino acids for protein synthesis and one-carbon units for nucleotide production. We investigated the role of glycine decarboxylase (GLDC) in neuroblastoma. GLDC catalyzes the first reaction in glycine cleavage, which generates one-carbon unit and reduced NADH. Analysis of gene expression data from two cohorts of neuroblastoma patients showed that GLDC expression is correlated with advanced stages, high-risk disease and poor prognosis in neuroblastoma. We found that GLDC is required for neuroblastoma cell proliferation as silencing GLDC expression by shRNA induced G1 cell cycle arrest. Consistent with this observation, microarray gene expression profiling revealed that GLDC knockdown resulted in a significant decrease in the expression of E2F target genes. In addition, we obtained evidence that GLDC is a direct target gene of MYCN. Knockdown of MYCN expression reduced GLDC mRNA and protein expression, and overexpression of MYCN increased GLDC mRNA and protein levels. Moreover, chromatin immunoprecipitation with quantitative PCR demonstrated that MYCN binds to the GLDC promoter region. These findings suggest an important role of GLDC in driving neuroblastoma cell proliferation, which could be exploited as a therapeutic strategy against high-risk neuroblastoma with MYCN amplification.

Citation Format: Ahmet Alptekin, Jane Ding, Bingwei Ye, Han-Fei Ding. The role of glycine decarboxylase in neuroblastoma [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 4374.
Abstract 4382: Epigenetic deregulation in glycolysis genes for colorectal cancer
Authors: Chen KuoHsing , Lai Liang-Chuan , Lin Liang-In , Chang Hsien-Fang , Chao Yu-Liang , Lin Been-Ren , Liang Jin-Tung , Cheng Ann-Lii , Chuang Eric Y. , Yeh Kun-Huei
Published: August 2019

Background

Energy restriction may have influence on methylation of glycolysis genes and also be associated with decreased incidence of colorectal cancer (CRC) in Dutch Hunger Winter cohort. This study aimed to explore the roles of epigenetic deregulation in energy/glycolysis pathway for CRC.

Materials and methods

To perform a comprehensive analysis of methylation alterations in glycolysis pathway (67 genes in KEGG), we profiled 27 CRCs and 5 adjacent normal colon tissues (NTs) by using a powerful methylation array (Illumina Methylation EPIC Beadchips). We used Wilcoxon rank-sum test (p < 0.05) and β value difference (≧ 0.2 or ≦ -0.2) to define the differential methylated genes (DMGs). We also searched the open datasets to find the shared DMGs in CRC. The shared DMGs were validated by pyrosequencing in our samples.

Results

We found 17 hypomethylation genes and 2 hypermethylation genes involved in

glycolysis pathway in our cohort. After comparing to the methylation profiles of CRC in 3 datasets (GSE42752, GSE25062 and TCGA), only hypermethylation of ALDH1A3 and hypomethylation of HKDC1 remained as shared DMGs. These findings were validated well by pyrosequencing in our cohort. The mRNA expression of HKDC1 and ALDH1A3 was negatively associated with the methylation status of each of them in TCGA dataset. RT-qPCR also showed the corresponding expression of HKDC1 and ALDH1A3 between CRCs and NTs.

Conclusion

Our study reveals that hypomethylation of HKDC1 and hypermethylation of ALDH1A3 exist in CRCs worldwide. Further exploratory study of biologic roles of them in CRC is ongoing.

Citation Format: KuoHsing Chen, Liang-Chuan Lai, Liang-In Lin, Hsien-Fang Chang, Yu-Liang Chao, Been-Ren Lin, Jin-Tung Liang, Ann-Lii Cheng, Eric Y. Chuang, Kun-Huei Yeh. Epigenetic deregulation in glycolysis genes for colorectal 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 4382.
Abstract 4419: Cell cycle analysis during TTF to exploit novel targets for increasing treatment efficacy
Authors: Slangen Paul , Gooijer Mark de , Tellingen Olaf van , Giladi Moshe , Borst Gerben
Published: August 2019

Cell cycle distribution and cell deathCell cycle distribution (index,%, t=12hrs)TreatmentControlTTFS10,5 ± 0.410.8 ± 0.5NSG229.9 ± 1.248.4 ± 0.9p<0.05M6.3 ± 0.62.0 ± 0.2p<0.05G153.0 ± 1.238.0 ± 0.8p<0.05Cell death (%)TreatmentcontrolWee1TTFTTF+Wee1Cell count100+/-3.274.4 +/- 4.150.5 +/- 4.221.5 +/- 3.2SynergisticColony formation100 +/- 3.261.6 +/- 6.675.5 +/- 11.217.0 +/- 5.3Synergistic

While the effectiveness of TTFields is initially attributed to the effects of TTFields during cell division, recent studies have shown that TTFields additionally induce DNA damage and replication stress. We want to further exploit the working mechanism of TTFields, its effect on the cell cycle and identify targets to increase its efficacy.

A172 cells were synchronized in S-phase by double thymidine block. Six hours post-release (0h), cells were subjected to TTFields treatment (200kHz; 4,5V/cm pk-pk). Cell cycle analysis was performed by flow cytometry (PI, α-phospho-histone H3Ser10). Treatment of A172 cells synchronized in S phase with TTFields showed that TTFields cause an accumulation of cells in G2 phase, leading to delayed entry into mitosis and, subsequent lower entry into G1 (see table). Because this indication of G2 checkpoint activation we hypothesized the possible synergism between TTFields and Wee1 inhibition. A172 cells were simultaneously treated with TTFields (200kHz; 4,5V/cm pk-pk, 72hrs) and the Wee1 inhibitor (300nM, 72hrs). Cell survival was determined by automated cell counting (including Trypan Blue) and colony formation assay. We observed that abrogation of the G2 checkpoint by Wee1 inhibtion synergistically increases the efficacy of TTFields treatment (see table).

The underlying mechanism of the G2 checkpoint activation is subject of ongoing research. By any means, the combination of TTFields with the Wee1 inhibitor bypasses the G2 activation and causes a synergistic decrease in cell survival.

Citation Format: Paul Slangen, Mark de Gooijer, Olaf van Tellingen, Moshe Giladi, Gerben Borst. Cell cycle analysis during TTF to exploit novel targets for increasing treatment efficacy [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 4419.
Abstract 4434: Th1 cytokines promotes E3 ubiquitin ligase Cullin 5 expression via STAT1 signaling cascade and enhance cul5 mediated proteasomal degradation of HER2 in HER2+/ER- breast cancer
Authors: Jia Yongsheng , Ramamoorthi Ganesan , Kodumudi Krithika , Basu Amrita , Wiener Doris , Czerniecki Brian
Published: August 2019

The proto-oncogene HER2/ErbB2 overexpression occurs in 30% of invasive breast cancer patients and its critically associated with aggressive disease, metastasis and tumor recurrence. Despite advances in the treatment of HER2+ breast cancers, resistance and metastatic disease are potential obstacles. Cullin 5 (Cul 5), a scaffold protein mediates formation of the Cullin 5-RING E3 ubiquitin ligase functional complex. This complex acts on protective molecular chaperone heat shock protein (HSP90) complex and subsequently leads to polyubiquitination and proteasomal degradation of its client protein HER2/ErbB2. Reduced expression of Cul 5 in breast cancer patients can abrogates its inhibitory function and favor HSP90 mediated stabilization of HER2/ErbB2. More recently, we showed that Th1 cytokines (IFN-γ and TNF-α) decreased HER2 expression and induced senescence and apoptosis in HER2 overexpressing breast cancer cell lines. However, the role of Th1 cytokines on Cul 5 mediated regulation of HER2/ErbB2 remains unknown. In the present study, low level expression of Cul 5 was observed in various human (SKBR3, HCC1419 and JIMT1) and mouse (Tubo) HER2+/ER- breast cancer cell lines. Interestingly, treatment of HER2+/ER- breast cancer cells with Th1 cytokines increased the expression of Cul 5 and downregulated the expression of HER2. We knocked-down Cul 5 expression by specific siRNA found that treatment of Th1 cytokines failed to decrease HER2 expression in HER2+/ER- breast cancer cells. To investigate whether the mechanism of Th1 cytokines on Cul5 is via downstream signaling molecule STAT-1, we knock-down STAT-1. Silencing of STAT-1 expression prevented Th1 cytokines mediated activation of Cul 5. We also observed no effect on the inhibition of HER2 expression after Th1 cytokines treatment in HER2+/ER- breast cancer cells in which the expression of STAT-1 was knocked-down. Collectively, our findings demonstrate a novel mechanism that Th1 cytokines promote Cul 5 expression via STAT1 signaling and enhance Cul 5 mediated proteasomal degradation of HER2 in HER2+/ER- breast cancer. Enhancing Cul 5 levels may represent a new therapeutic avenue for the inhibition of HER2 overexpression and prevention of metastasis and tumor relapse.

Citation Format: Yongsheng Jia, Ganesan Ramamoorthi, Krithika Kodumudi, Amrita Basu, Doris Wiener, Brian Czerniecki. Th1 cytokines promotes E3 ubiquitin ligase Cullin 5 expression via STAT1 signaling cascade and enhance cul5 mediated proteasomal degradation of HER2 in HER2+/ER- 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 4434.
Abstract 444: Continuous, high throughput microfluidic device to monitor circulating tumor cells in cancer patients
Authors: Smith Kaylee , Kim Tae Hyun , Paoletti Costanza , Thamm Douglas H. , Hayes Daniel F. , Nagrath Sunitha
Published: August 2019

Analysis of circulating tumor biomarkers, designated “liquid biopsies” are less invasive and lower risk to the patient and might replace or complement standard tissue biopsies to determine prognosis, predict benefit from specific therapies, or monitor patients with cancer. Circulating tumor cells (CTCs) are extremely rare, with only 1-10 CTCs per mL of blood. Current technologies typically process 1-10 mL of blood ex vivo. We report an in vivo, indwelling CTC-capture device that is analogous to a Holter monitor used to interrogate cardiac rhythm abnormalities over long periods of time. The system incorporates a dual lumen intravenous catheter that directs blood from the vein into a small (~ the size of a wristwatch) capture carriage by virtue of a peristaltic pump. The carriage contains a CTC capture chip which separates CTCs from whole blood based on flow differences and solid phase anti-EpCAM capture, while the remaining whole blood is returned to the venous system via the dual lumen catheter. The chip is then removed from the carriage for further enumeration and characterization of the CTCs. In a proof of principle set of experiments, the system was placed into investigational canines into which fluorescently labeled human breast cancer cells were injected intravenously. Although the vast majority of these xeno-cancer cells were rapidly removed from circulation, the system captured “CTCs” over a 2 hour time period more successfully than was seen in simultaneous blood draws for ex vivo enumeration over the same time. No adverse effects were observed in the subjects at the time of the experiments or over the succeeding 48 hours after removal of the IV system.Our system permits interrogation of a much larger volume of blood than can be processed with standard blood draws. The larger processed volume will drastically increase the number of cells isolated which in turn increases the number of downstream assays that can be performed. This study is focused on developing a high throughput microfluidic device that can detect CTCs and implementing it in a canine model.

Citation Format: Kaylee Smith, Tae Hyun Kim, Costanza Paoletti, Douglas H. Thamm, Daniel F. Hayes, Sunitha Nagrath. Continuous, high throughput microfluidic device to monitor circulating tumor cells in cancer patients [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 444.
Abstract 4495: Role of base excision repair (BER) pathway in regulation of KRAS expression in pancreatic cancer
Authors: Pramanik Suravi , Roychoudhury Shrabasti , Harris Hannah , Song Heyu , Bhakat Kishor K.
Published: August 2019

Activating mutations in KRAS proto-oncogene, a signature event driving the development, progression and therapeutic resistance of pancreatic ductal adenocarcinoma (PDAC), remains undruggable. The occurrence of guanine oxidation (8-oxoguanine) in the KRAS promoter and up-regulation of KRAS gene transcription under oxidative stress has been shown to be associated with KRAS expression and cancer development and progression. However, the molecular mechanism by which 8-oxoguanine damage regulates KRAS expression is largely unknown. Here, we show that the base excision repair (BER) pathway, a fundamental DNA damage repair pathway that processes most of the endogenous damages including oxidative base damage is involved in regulation of KRAS expression and survival of PDAC. We show that Apurinic/apyrimidinic endonuclease (APE1), a key enzyme of the BER pathway, is highly elevated in pancreatic cancer tissue samples. To elucidate the role of active BER pathway in the regulation of KRAS expression, we used real-time PCR (RT-PCR) analysis. Inflicting cells with oxidative damage using glucose oxidase increased KRAS gene expression in control cells but not in APE1 downregulated cells. ChIP assay showed enhanced occupancy of APE1 in KRAS promoter upon oxidative stress. Consistent with this, using synthetic oligonucleotide containing the KRAS promoter region, we showed that APE1 could bind and cleave AP site in KRAS promoter. Further, ChIP-qPCR analysis showed decreased occupancy of MAZ, a transcription factor, to the KRAS promoter in APE1 downregulated cells. Down-regulation of APE1 also resulted in decreased KRAS expression and increased sensitivity of pancreatic cancer cells to routinely used chemotherapeutic agents such as Gemcitabine, 5-Fluorouracil, Oxaliplatin, etc., suggesting that targeting APE1 and in turn, BER can sensitize pancreatic cancer cells. Our study suggests that BER pathway or APE1 plays a significant role in the tumor-selective regulation of gene expression and sensitization of cancer cells to chemotherapy, and supports the further investigation of novel treatments that target this pathway for cancer therapy.

Citation Format: Suravi Pramanik, Shrabasti Roychoudhury, Hannah Harris, Heyu Song, Kishor K. Bhakat. Role of base excision repair (BER) pathway in regulation of KRAS expression in pancreatic 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 4495.