bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1504: Opposing roles of FoxM1 and the hepatic specification genes FoxA1/A2 dictate differentiation state of liver cancer cells
Authors: Chand Vaibhav , Pandey Akshay , Kopanja Dragana , Guzman Grace , Raychaudhuri Pradip
Published: August 2018

The pro-proliferation transcription factor FoxM1 is over-expressed mainly in high-grade cancers. However, the significance of its over-expression in cancers, including poorly differentiated hepatocellular carcinoma (HCC), is not known. Here we provide evidence for a causal role of the over-expressed FoxM1 in driving high-grade progression of HCC. Immunohistochemical staining of human HCC specimens revealed opposite expression patterns of FoxM1 and the liver differentiation genes FoxA1/A2. Moreover, using a transgenic mouse model for oncogenic Ras-driven HCC as well as cell-based studies, we provide in vivo genetic evidence for a direct repression of FoxA1/A2 by FoxM1. Interestingly, FoxM1 represses these differentiation genes mainly in G1 phase, a phase in the cell cycle in which cells can undergo differentiation. Moreover, repression of FoxA1/A2 in G1 phase is important, as these genes are capable of inhibiting expression of the pluripotency genes that are mainly expressed in S/G2 phases. Also, we show that FoxA1/A2 inhibit expression of FoxM1 by inhibiting binding of FoxM1 to its own promoter and thus blocking its auto-activation. Our observations identify a significant new mechanism in which FoxM1 and the FoxA genes play opposing roles that determine the differentiation state of the HCC cells.

Citation Format: Vaibhav Chand, Akshay Pandey, Dragana Kopanja, Grace Guzman, Pradip Raychaudhuri. Opposing roles of FoxM1 and the hepatic specification genes FoxA1/A2 dictate differentiation state of liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1504.
Abstract 1508: Cloning, expression and purification of Cionasavignyi polypeptide CS5931 and its antitumor activity
Authors: Zhang Yangyang , Lai Honglin , An Weixiao , Cao Shousong , Lin Xiukun
Published: August 2018

Previous study in our laboratory has shown that a novel polypeptide CS5931 (molecular weight 5931 Da) derived from Ciona savignyi possesses potent cytotoxicity against a variety of human cancer cells including cervical carcinoma Hela cells, colorectal carcinoma HCT-116 and RKO cells, lung carcinoma A549 cells, and leukemia HL60 cells ,with IC50 values of 4.17, 2.50, 5.54, 4.69 and 4.98uM,respectively. This result suggested that HCT116 cells are the most sensitive to the treatment of CS5931. We also found that CS5931 markedly induced apoptosis in a dose-dependent manner in HCT116 cells by Annexin V-FITC/PI double staining analysis. However, its content in Ciona savignyi is very low thus developing a novel method for increase of the production of the polypeptide is promising. GRN polypeptides typically consist of 56-58 amino acids with six disulfide bonds and four beta-sheets to make the GRN polypeptide structurally compact and complex so large-scale preparation of natural polypeptide CS5931 is very difficult. Therefore, recombinant expression of CS5931 by genetic engineering is one of the effective ways to solve this problem. In the present study, we cloned a full length cDNA of CS5931 precursor by rapid amplification of cDNA ends (RACE) and obtained the amino acid sequence of CS5931. We further made the polypeptide expression with high efficiency in E. coli. The expressed protein was purified through affinity chromatography. The gene fragment coding CS5931 was successfully cloned and then linked into prokaryotic expression carrier pET28a(+) and pPICZαa, respectively. Two expression vectors pET28a(+)-CS5931 and pPICZαa-CS5931 were transformed into E. coli and Pichia pastoris, respectively. Then recombinant polypeptide CS5931 were successfully induced with high expression . To investigate the expression and anticancer effect of polypeptide CS5931 in different vectors and strains, we purified the expressed product developed suitable methods for CS5931 great expression while maintaining its potent anticancer activity and identified the role of CS5931 target proteins. Moreover, the purified polypeptide native CS5931 and recombinant CS5931 equally display great antitumor activity against HT29 cells. Our studies indicate that we have successfully cloned, expressed and purified Ciona savignyi polypeptide CS5931 and demonstrated its potent antitumor activity against colorectal cancer cells. Therefore, CS5931 may be developed as a novel anticancer agent in the treatment of colorectal cancer clinically.

Citation Format: Yangyang Zhang, Honglin Lai, Weixiao An, Shousong Cao, Xiukun Lin. Cloning, expression and purification of Cionasavignyi polypeptide CS5931 and its antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1508.
Abstract 1509: Cachexia: Leveraging transcriptomics to identify potential therapeutics
Authors: Ung Matthew , Fowler Kevin D. , Funt Jason M. , Escalante-Chong Renan , Koytiger Gregory , Kolitz Sarah , Kusko Rebecca L. , Zeskind Benjamin
Published: August 2018

Cachexia is a common cause of morbidity across multiple cancer types, yet remains largely under-studied especially in the realm of transcriptomics. An essential component of cancer cachexia is the induced transition of adipocytes from white to brown, which increases energetic inefficiency and contributes to systemic loss of adipose tissue. This is likely caused by the tumor secreting thermo-stimulatory factors into the extracellular space.

Here, we integrate publicly-available data to pinpoint actionable gene targets that enable cancer cells to induce fat “browning”. In particular, we identify genes differentially expressed between Lewis lung carcinoma subclones that can induce thermogenesis in primary adipocytes at high or low levels in vitro (GSE57797). We then evaluate whether the expression of these thermo-stimulatory genes predict patient survival in an independent gene expression dataset of primary tumors from advanced stage gastric cancer patients (GSE15459). Finally, we leverage drug gene expression data from CMAP and LINCS to identify molecules that reverse the thermo-stimulatory signature.

Our analysis reveals that the thermo-stimulatory signature predicts prognosis in patients with advanced gastric cancer (p=0.005), with the low thermo-stimulatory group exhibiting improved prognosis. The thermo-stimulatory signature is most strongly reversed by expression signatures from inhibitors of MEK (MAP2K1), including pd-0325901, AZD-8330 (ARRY-704), CI-1040, trametinib, and selumetinib. Interestingly, we observe a time-dependent effect of these MEK inhibitors, with shorter durations of exposure consistently reversing thermo-stimulatory signatures more effectively than longer durations of exposure.

Analysis of cachexia-related transcriptomic data reveals key marker genes and pathways which can be used to optimize MEK inhibitors for cachexia. Results from this study support the development of MEK inhibitors to treat cachexia, and suggest that careful optimization of pharmacokinetics will be important for success in the clinic.

Citation Format: Matthew Ung, Kevin D. Fowler, Jason M. Funt, Renan Escalante-Chong, Gregory Koytiger, Sarah Kolitz, Rebecca L. Kusko, Benjamin Zeskind. Cachexia: Leveraging transcriptomics to identify potential therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1509.
Abstract 1511: Differential expression analysis and biomarker identification in five salivary gland carcinoma types
Authors: Brayer Kathryn J. , Mitani Yoshitsugu , El-Naggar Adel , Ness Scott A.
Published: August 2018

Significant morphologic overlap can exist between distinct salivary gland neoplasms, making differential diagnosis challenging, even with the use of new immunohistochemistry stains and FISH probes (Griffith et al., Arch Pathol Lab Med 2017). RNA-sequencing (RNAseq) technology is increasingly being used as a tool for identify diagnostic molecular signatures in a wide variety of diseases (Byron et al., Nat Rev Genet 2016). Furthermore, transcriptomics data are being used to develop biomarkers that can be used in drug discovery assays. Using an unbiased RNAseq analysis pipeline (Brayer et al., Cancer Discov 2016; Brown et al., PLos One 2017), we developed molecular profiles for 126 salivary gland tumors with accompanying clinical data–68 adenoid cystic carcinoma (ACC), 23 acinic cell carcinoma (Acinic), 5 basal cell adenoma (BCA), 20 basal cell adenocarcinoma (BCAC), and 10 secretory carcinoma (SC)–and have identified distinct gene expression patterns for each neoplasm. Additionally, a detailed analysis within each cancer type has revealed heterogeneous gene expression patterns, with altered signaling pathways, suggesting molecular subtypes exist. In both ACC and Acinic samples we identified unique molecular signatures found in patients with poor overall survival. Transcripts within each molecular signature that are independent of gender, age, and cancer stage, but correlated with clinical outcome data, are being identified for use as diagnostic or prognostic biomarkers and for use in drug discovery assays. Preliminary results to identify biomarkers for these neoplasms will also be presented.

Citation Format: Kathryn J. Brayer, Yoshitsugu Mitani, Adel El-Naggar, Scott A. Ness. Differential expression analysis and biomarker identification in five salivary gland carcinoma types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1511.
Abstract 1518: Gene expression of trefoil proteins, gastrokines and mucins in Indian gastric cancer patients
Authors: Basak Dipjit , Mondal Pronoy Kanti , Mondal Brishti , Ghosh Ranajoy , Halder Aniket , Dhali Gopal Krishna , Chowdhury Abhijit , Mukhopadhyay Indranil , Datta Shalini
Published: August 2018

Background: Gastric cancer is the (GC) third most cause of cancer related mortality worldwide. Histopathologically, GC is sub classified into two major histopathological subtypes- diffuse and intestinal. The trefoil factor family - TFF1, TFF2 and TFF3 is known to play a pivotal role in gastrointestinal mucosal defence and repair. Gastrokines (GKN1, GKN2) are stomach specific secretary proteins which play fundamental roles in regulating gastric epithelial homeostasis. Similarly, secreted mucins are large O-glycosylated proteins that participate in the protection/defence of underlying mucosa in normal adults. The loss of expression of trefoil proteins, gastrokines and several mucins in retinoblastoma and colorectal cancer are reported in recent studies.

Aims: The molecular underpinnings and gene expression of Indian GC patients are largely unknown. In this study, we evaluated the gene expression of TFF1, TFF2, TFF3, GKN1, GKN2, MUC3A, MUC5AC, MUC13 in gastric cancer patients from India. Additionally, we wanted to see whether these gene expressions are different in two histopathological groups.

Materials and Methods: Endoscopic biopsy tissue from gastric tumour and adjacent normal regions were collected with ethical approval and informed consent from 42 patients . After histopathological confirmation by two pathologists, total RNA were isolated from tumour and corresponding normal tissue and gene expression was measured by qRT-PCR. Statistical analysis was performed by R package.

Result and Discussion: We identified that TFF1, TFF2, TFF3, GKN1, GKN2 are significantly differentially expressed in gastric tumour tissue with respect to adjacent normal tissue whereas mucins had no significant deregulation. TFF1, TFF2, GKN1 and GKN2 are found to be down regulated in most tumour tissues compared to adjacent normal. Only GKN2 expression is significantly different among diffuse and intestinal subtypes . Our statistical analysis also revealed that gene expression of TFF1, TFF2, GKN1 and GKN2 are highly correlated with the gene expression of MUC5AC. Further study with larger patient sample is required to fully understand the complex interplay of trefoil, gastrokines and mucins in GC.

Citation Format: Dipjit Basak, Pronoy Kanti Mondal, Brishti Mondal, Ranajoy Ghosh, Aniket Halder, Gopal Krishna Dhali, Abhijit Chowdhury, Indranil Mukhopadhyay, Shalini Datta. Gene expression of trefoil proteins, gastrokines and mucins in Indian gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1518.
Abstract 1530: A multifunctional role for adjuvant anti-4-1BB therapy in augmenting antitumor responses by CAR T cells
Authors: Mardiana Sherly , John Liza B. , Kershaw Michael H. , Beavis Paul A. , Darcy Phillip K.
Published: August 2018

Adoptive immunotherapy utilising chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematological cancers, with up to 90% complete remission being reported in patients with acute lymphoblastic leukemia (ALL). However, this success is not currently transferrable to solid malignancies due in part to the immunosuppressive tumor microenvironment limiting CAR T cell responses. Given that activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong co-stimulatory signals for augmenting and diversifying T cell responses, we hypothesized that the combination of α-4-1BB and CAR T cell therapy would result in improved anti-tumor responses. Using a human-Her2 self-antigen mouse model, we found that α-4-1BB significantly enhanced CAR T cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Investigation into the mechanism revealed increased expression of IFNγ and the proliferation marker Ki67 by tumor-infiltrating CAR T cells when combined with α-4-1BB. The importance of IFNγ was demonstrated in vivo where synergistic effects of the combination therapy were abrogated when IFNγ was depleted. Strikingly, we also found that α-4-1BB significantly reduced host immunosuppressive cells at the tumor site including regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs), correlating with an increased therapeutic response. Investigation in immunocompromised RAG -/- mice showed reduced synergistic effects following the combination therapy, suggesting some contribution by host T cells. Furthermore, we found increased frequency of endogenous T cells and dendritic cells in the tumor-draining lymph nodes, supporting potential engagement of host immune cells. We therefore conclude α-4-1BB has a multifunctional role for enhancing CAR T cell responses including engagement of endogenous immune cells, and this combination approach has high translational potential given that CAR T cell therapy has recently been FDA approved for pediatric ALL, and that α-4-1BB is currently being tested in clinical trials against various types of cancer.

Citation Format: Sherly Mardiana, Liza B. John, Michael H. Kershaw, Paul A. Beavis, Phillip K. Darcy. A multifunctional role for adjuvant anti-4-1BB therapy in augmenting antitumor responses by CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1530.
Abstract 1533: CD4+ T cells reprogram tumor metabolism and drive oxidative stress-induced tumor destruction
Authors: Zhou Gang , Habtetsion Tsadik , Ding Zhi-Chun
Published: August 2018

The inhibitory effects of cancer on T cell metabolism have been well established, but the metabolic impact of immunotherapy on tumor cells is poorly understood. Here, we developed a CD4+ T cell-based adoptive immunotherapy protocol that was curative for mice with implanted colorectal cancer. By conducting metabolic profiling on resected tumors, we show that adoptive immunotherapy profoundly altered tumor metabolism, resulting in glutathione depletion and accumulation of reactive oxygen species (ROS) along with increased oxidative DNA damages. We further demonstrate that T cell-derived TNFα can synergize with chemotherapy to drive heightened oxidative stress and tumor cell death. Reduction of oxidative stress, by neutralizing TNFα or scavenging ROS, antagonized the therapeutic effects of adoptive T-cell therapy. Conversely, provision of pro-oxidants after chemotherapy can partially recapitulate the antitumor effect of T-cell transfer. Furthermore, T cell-induced oxidative stress in tumor was observed in several different tumor models. These findings imply that tumor redox modulation represents an important mechanism underlying the efficacy of adoptive immunotherapy.

Citation Format: Gang Zhou, Tsadik Habtetsion, Zhi-Chun Ding. CD4+ T cells reprogram tumor metabolism and drive oxidative stress-induced tumor destruction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1533.
Abstract 1538: Analysis of the paired TCRα- and β-V(D)J full-length chains of single-cell sequence from human naïve and antigen-experienced T cells
Authors: Wang Fei , Zhou Qing , Chao Cheng-chi
Published: August 2018

The unbiased and paired characterization of T-cell receptor (TCR) alpha and beta chains is critical to understand the TCR repertoire and adaptive immunity. However, the direct cloning of the single human full-length TCRα- and TCRβ-chains has very few reports. We present a new methodology enabling capture of natively paired, full-length rearranged T-cell receptor (TCR) sequences at single-cell level of human T cells. More than 50% paired TCRα- and β- chains were captured in 100 single naïve human CD8+ T cells using 5'SMART and 3' TCRαβ constant region nested gene-specific primers (GSP). Furthermore, we sequenced the TCR of the specific T cells that were stimulated by representative tumor-associated antigens (TAA), virus-related antigens (VRA) and neoantigens. The highest proportion of TCR subtypes were extracted in the TCR abundance ranking by IMGT blast. Finally, we obtained the paired, full-length V(D)J TCR from melanoma antigen Melan A epitope 27-35 that was TRAV(12-2)J(47), TRBV(7-6)J(2-1), and the TCR from antigen EBV LMP2-FLY that was TRAV(26-1)J(7), TRBV(27)J(2-1). These results suggest that the method provides accurate identification of the paired, V(D)J rearrangements for each individual human naïve and antigen-experienced T cells. The information could be used in the construction of an engineered T-cell receptor for cancer immunotherapy and infectious diseases.

Citation Format: Fei Wang, Qing Zhou, Cheng-chi Chao. Analysis of the paired TCRα- and β-V(D)J full-length chains of single-cell sequence from human naïve and antigen-experienced T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1538.
Abstract 154: Inhibition of WEE1 expression reduces lipid metabolism and stemness in hepatocellular carcinoma
Authors: Park Keon Uk , Ko Min Ji , Hwang Ilseon , Ryoo Hun-Mo , Lee Yun-Han
Published: August 2018

Background: Classical therapeutic regimens predominantly target proliferating cells, which are unlikely to be cancer stem cells (CSCs). Similarly, new generation therapies, e.g. sorafenib for hepatocellular carcinoma (HCC), do not seem to target CSCs as evidenced by frequent tumor relapse and resistance after therapy. Thus, identification and characterization of signaling pathways and biomarkers associated with CSC biology are therefore priorities for developing new paradigms of molecular cancer therapeutics. Increase of WEE1 kinase activity through an epigenetic regulation plays an important role in the development of HCC. However, the functional role of WEE1 in HCC progression remains to be clarified.

Methods: Human HCC cell lines were transfected with WEE1 siRNA and tested for growth inhibition, apoptotic induction, molecular changes in both RNA and protein levels, and changes in CSC phenotype using various methods such as MTS, FACS, microscopic analysis, Real-time PCR, Western blotting, sphere forming assay. To obtain insights into the molecular changes in response to WEE1 knockdown, global changes in gene expression were examined using RNA sequencing.

Results: Here we demonstrated that WEE1 siRNA silencing caused inhibition of HCC cell growth through blockade of cell cycle progression and induction of apoptosis. The anti-proliferative effects were driven by a subset of molecular alterations including the upregulation of cdk inhibitor p21 and the downregulation of AKT1, CDK2, cyclin B1 (CCNB1), PARP1 and GPAM which are functionally involved in control of cell cycle, apoptosis and lipid metabolism. Wee1 silencing in tumor cells also resulted in a strong inhibition of lipogenesis (SREBP1C, FAS) and caused a marked decrease in fat accumulation. Of importance, knockdown of WEE1 dramatically reduced the portion of liver CSC population through co-downregulation of cancer stemness genes and weakened the capacity of sphere formation and the ability of cancer cell migration. Systemic delivery of a modified WEE1 siRNA encapsulated in lipid nanoparticles significantly inhibited human HCC growth in murine xenograft models, and increased mice survival. Our findings suggest that the epigenetic modifier WEE1 functionally involve to HCC lipid metabolism and CSC-like phenotype maintenance and that molecular targeting of WEE1 may be an effective systemic therapy for prevention of tumor recurrence via elimination of CSCs in liver tumor microenvironment.

Citation Format: Keon Uk Park, Min Ji Ko, Ilseon Hwang, Hun-Mo Ryoo, Yun-Han Lee. Inhibition of WEE1 expression reduces lipid metabolism and stemness in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 154.
Abstract 1543: Upregulation of mannose 6-phosphate receptor (MPR) has a potential to increase the efficacy of combined therapy for melanoma patients
Authors: Atay Cigdem , Lavilla-Alonso Sergio , Gabrilovich Dmitry
Published: August 2018

BRAF -targeting therapy demonstrates impressive clinical response in patients with metastatic melanoma. However, patients quickly develop resistance associated with rapid tumor progression. Therefore, additional therapeutic approaches are necessary. Our group has previously shown that transient induction of cation-independent mannose 6-phosphate receptor (MPR) is important for the antitumor effect of chemo-immunotherapy. In this study, we aimed to decipher the role of MPR during possible combination therapy of BRAF inhibitor with adoptive T-cell therapy of BRAF inhibitor-resistant melanoma. For this aim, we have used PLX4720 as a BRAFV600E inhibitor and observed that in vitro PLX4720 treatment of human V600E-positive melanoma cells, including WM-35, SK-Mel-624 and WM983B, resulted in a dose-dependent upregulation of MPR on the cell surface. Importantly, increased levels of MPR on the cell surface induced the uptake of recombinant Granzyme B, which is a major mechanism of cell killing by cytotoxic T cells (CTL). We found that PLX4720 treatment rendered WM35 cells more prone to lysis by HLA-matching tumor-infiltrating lymphocytes (TILs) and increased cell killing is not due to PLX4720 treatment itself but increased levels of MPR on the cell surface. In order to prove this, we generated MPR-overexpressing WM35 cell line and showed that upregulation of MPR, independently of PLX4720 treatment, sensitizes melanoma cells for T cell killing in vitro. We further showed that PLX4720 treatment leads to upregulation of MPR in vivo by a mouse model bearing xenograft WM35-derived subcutaneous tumors as well. According to our data, 3 days of PLX4720 treatment is enough to induce MPR in tumors and this effect continues up to 6 days after 5 days treatment ends. Unexpectedly, we found that PLX4720 caused upregulation of MPR in resistant cell lines and that those cell lines became more sensitive to cytotoxic effect of TILs. Thus, our data suggest that adoptive T-cell therapy can be potentially effective in melanoma resistant to BRAF inhibitor. (This study is supported by NIH grant P50 CA168536.)

Citation Format: Cigdem Atay, Sergio Lavilla-Alonso, Dmitry Gabrilovich. Upregulation of mannose 6-phosphate receptor (MPR) has a potential to increase the efficacy of combined therapy for melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1543.
Abstract 1580: Separation of circulating biomarkers by nanoDLD lab-on-a-chip technology
Authors: Gifford Stacey M. , Wunsch Benjamin H. , Smith Joshua T. , Dogra Navneet , Kim Sungcheol , Stolovitzky Gustavo
Published: August 2018

Efficient isolation of circulating biomarkers is key for enabling liquid biopsies for cancer diagnosis and prognosis. Circulating biomarkers such as exosomes and cell-free DNA hold promise for non-invasive early cancer diagnosis, determining cancer stage and prognosis, as well as monitoring treatment progression and the development of drug resistance. However, isolation of these biomarkers has proven challenging in the clinic. Exosomes range in size from approximately 30 to 150 nm and the current gold standard isolation method includes large-scale ultracentrifugation, which is time-consuming and lacks reproducibility. Circulating cell-free DNA is found in a variety of sizes and states and while clinical applications for prenatal testing have emerged, applications in oncology remain elusive in part due to high background and challenges in isolating relevant molecules. We have developed a lab-on-a-chip technology based on deterministic lateral displacement at the nanoscale (nanoDLD) which separates and concentrates particles as small as 20 nm from smaller particles in continuous flow. Analysis of nanoDLD isolation of exosomes and DNA show improved resolution, recovery, and concentration compared to standard techniques. Current efforts to scale nanoDLD technology to clinically relevant volumes will provide a reproducible and automated tool for isolation, study, and clinical use of these important non-invasive biomarkers.

Citation Format: Stacey M. Gifford, Benjamin H. Wunsch, Joshua T. Smith, Navneet Dogra, Sungcheol Kim, Gustavo Stolovitzky. Separation of circulating biomarkers by nanoDLD lab-on-a-chip technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1580.
Abstract 1581: Circulating cell-free DNA for metastatic cervical cancer detection, genotyping and monitoring
Authors: Kang Zhigang , Stevanović Sanja , Hinrichs Christian , Cao Laing
Published: August 2018

Purpose: Circulating cell-free (ccf) human papillomavirus (HPV) DNA is a unique tumor marker for metastatic cervical cancers. We developed a method to genotype and quantify circulating HPV DNA in patients with HPV16- or HPV18-positive metastatic cervical cancer for disease monitoring and treatment-related decision making.

Patients and Methods: In this retrospective study, HPV ccfDNA was measured in serum samples from metastatic cervical cancer patients by duplex digital droplet (dd) PCR. Nine patients had received tumor-infiltrating lymphocyte (TIL) immunotherapy. ccfDNA data were aligned with the tumor HPV data, drug treatment, and clinical outcome.

Results: In blinded tests, HPV ccfDNA was detected in 19/19 (100%) patients with HPV-positive metastatic cervical cancer but not in any of the 45 healthy blood donors. The HPV genotype harbored in the patients' tumors was correctly identified in 87/87 (100%) sequential patient serum samples. In three patients who experienced objective responses after TIL immunotherapy, a transient HPV ccfDNA peak was detected 2-3 days after TIL infusion. Furthermore, a persistent clearance of HPV ccfDNA was observed in only two patients with a complete response (CR) after TIL immunotherapy.

Conclusions: HPV ccfDNA represents a promising tumor marker for non-invasive HPV genotyping and may be used in selecting patients for HPV type-specific T-cell-based immunotherapies. It may also have value in detecting the anti-tumor activities of therapeutic agents and in the long-term follow-up of cervical cancer patients in remission.

Citation Format: Zhigang Kang, Sanja Stevanović, Christian Hinrichs, Laing Cao. Circulating cell-free DNA for metastatic cervical cancer detection, genotyping and monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1581.
Abstract 1584: Comparison of circulating tumor cell (CTC) capture/identification methods and NanoString evaluation of gene expression in CTCs and cell-free circulating tumor mRNA (cctmRNA) in patients with metastatic lung cancer
Authors: Boumber Yanis , Aggarwal Charu , Thrash-Bingham Catherine , Fittipaldi Patricia , Guseva Polina , Vlasenkova Ramillya , Rao Chandra , Pei Jianming , Egleston Brian L. , Borghaei Hossein , Cristofanilli Massimo , Mehra Ranee , Serebriiskii Ilya , Alpaugh R. Katherine
Published: August 2018

Circulating tumor cells (CTC) and cell free circulating tumor mRNA (cctmRNA) can serve as biomarkers for prognosis and treatment response in lung and other cancers. A barrier to CTC use in some cancers is the low numbers of CTC captured, and the low percent of patients with detectable CTCs. Past comparisons of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients have shown larger cohorts of patients with detectable CTCs, and higher CTC numbers, for SCLCs. We compared methods for CTC capture/identification, and profiled mRNA from both the CTCs and cell free plasma RNA (cctmRNA) for to identify tumor-associated biomarkers. Peripheral blood was collected from 10 SCLC and 10 NSCLC patients prior to therapy, and from 4 normal controls. Capture using the standard EpCAM CellSearch reagent (unicapture) was run in parallel with EpCAM plus Her2, EGFR and MUC-1 combined ferrofluid (quadcapture) plus the use of anti-cytokeratin 7 (CK7) to assist in CTC identification. Unicapture detected CTCs in 40% of NSCLC and 60% of SCLC; whereas, quadcapture/CK7 identified 20% CTCs in NSCLC and 80% in SCLC. Overall, the quadcapture did not improve CTC capture efficiency. Exploratory NanoString analysis was performed on cell-free plasma from these patients and the ferrofluid isolated CTCs. Cancer-specific biomarkers were detected in mRNA from plasma and CTCs by NanoString included CCL5, CLU and SRGN. In summary, quadcapture ferrofluid reagent did not improve the capture efficiency, although the addition of CK7 may have the potential to identify additional CTCs. Nanostring analysis of cctmRNA and CTCs mRNA suggested potential new lung cancer biomarkers that require validation in larger studies.

Citation Format: Yanis Boumber, Charu Aggarwal, Catherine Thrash-Bingham, Patricia Fittipaldi, Polina Guseva, Ramillya Vlasenkova, Chandra Rao, Jianming Pei, Brian L. Egleston, Hossein Borghaei, Massimo Cristofanilli, Ranee Mehra, Ilya Serebriiskii, R. Katherine Alpaugh. Comparison of circulating tumor cell (CTC) capture/identification methods and NanoString evaluation of gene expression in CTCs and cell-free circulating tumor mRNA (cctmRNA) in patients with metastatic lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1584.
Abstract 1646: Discovery and characterization of BAY-6035, a novel benzodiazepine-based SMYD3 inhibitor
Authors: Gradl Stefan , Steuber Holger , Weiske Jörg , Schmees Norbert , Siegel Stephan , Stoeckigt Detlef , Christ Clara D. , Li Fengling , Organ Shawna , Barsyte-Lovejoy Dalia , Szewczyk Magdalena M. , Kennedy Steven , Trush Viacheslav , Vedadi Masoud , Arrowsmith Cheryl H. , Brown Peter J. , Husemann Manfred , Fernandez-Montalvan Amaury E. , Badock Volker , Bauser Marcus , Haegebarth Andrea , Hartung Ingo V. , Stresemann Carlo
Published: August 2018

SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase (PKMT) which was initially described as H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has recently been reported to methylate and regulate several non-histone cancer relevant proteins such as mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition overexpression of SMYD3 has been linked to poor prognosis in certain cancers, thus supporting a possible oncogenic role for SMYD3 and making it an attractive target for anticancer drug development. Here we report the discovery of a novel potent and selective SMYD3 inhibitor series. We performed a thermal shift assay based (TSA) high throughput screening followed by extensive biophysical validation resulting in identification of a benzodiazepine-based SMYD3 inhibitor series. The co-crystallization structures revealed that this series binds to the substrate binding site and occupies the hydrophobic pocket for lysine binding using an unprecedented hydrogen bond pattern. The competitive behavior of the inhibitor in biochemical assays was consistent with the binding mode observed in the crystal structure. Further optimization generated BAY-6035, which showed improved nanomolar potency and was selective against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibited methylation of MAP3K2 by SMYD3 in a cellular assay with similar potency. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe and will foster the exploration of the biologic role of SMYD3 in diseased and non-diseased tissues.

Citation Format: Stefan Gradl, Holger Steuber, Jörg Weiske, Norbert Schmees, Stephan Siegel, Detlef Stoeckigt, Clara D. Christ, Fengling Li, Shawna Organ, Dalia Barsyte-Lovejoy, Magdalena M. Szewczyk, Steven Kennedy, Viacheslav Trush, Masoud Vedadi, Cheryl H. Arrowsmith, Peter J. Brown, Manfred Husemann, Amaury E. Fernandez-Montalvan, Volker Badock, Marcus Bauser, Andrea Haegebarth, Ingo V. Hartung, Carlo Stresemann. Discovery and characterization of BAY-6035, a novel benzodiazepine-based SMYD3 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1646.
Abstract 1660: Unraveling isoform selectivities of a broad class of HDAC inhibitors with QM/MM molecular dynamics simulations
Authors: Lin Jung-Hsin
Published: August 2018

Therapeutics that have been used by large populations and showed limited or little adverse effects can be good candidates for cancer chemo-prevention. As an example of such therapeutics, statins, i.e., HMG-CA reductase inhibitors, have been widely prescribed for lowering the blood cholesterol level and for treating cardiovascular diseases. In 2008, we discovered that statins are moderate inhibitors of histone deacetylases (HDACs), which provides an epigenetic mechanism for their efficacy in cancer prevention. HDACs have been long recognized for as an important class of therapeutic targets, and these enzymes utilize a Zn2+ ion at its active site for catalysis. Subsequently, in our 2013 J. Med. Chem. article, we showed that we are able to strengthen this epigenetic mechanism and designed more potent and effective therapeutic agents for cancer prevention and treatment, including cancer metastasis, and published in Clin. Cancer Res. in 2016. To investigate the structure-activity relationships of a broad class of HDAC inhibitors with distinct chemical moieties, we have conducted QM/MM molecular dynamics simulations of several systems of HDAC isoforms bound with various ligands. In contrast to molecular dynamics simulations with classical force fields, which always rendered over-coordinated configurations of the Zn2+ at the active site, with the coordination numbers (CN) to be either 6 or 7, in our QM/MM molecular dynamics simulations, the CNs of Zn2+ remain 4 or 5. With the structural and energetics analysis from these QM/MM molecular dynamics simulations, we are able to delineate the origins of isoform-selectivity of different HDAC inhibitors, including the aforementioned HMGR-HDAC dual-action inhibitors that we recently developed.

Citation Format: Jung-Hsin Lin. Unraveling isoform selectivities of a broad class of HDAC inhibitors with QM/MM molecular dynamics simulations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1660.
Abstract 1663: Identification and characterization of a small-molecule inhibitor of the human uracil-DNA glycosylase for use in combination with DNA damage-based anticancer therapies
Authors: Nguyen Mya T. , Yan Yan , Fedorvo Yuriy , Pink John , Adams Drew , Gerson Stanton
Published: August 2018

Our research aims to develop therapeutically active inhibitors of the human uracil-DNA glycosylase (UNG). UNG, a highly conserved enzyme, plays a pivotal role in preventing abnormal bases, uracil and 5-fluorouracil, from incorporation into DNA as a result of treatment with chemotherapeutic drugs, floxuridine (5-FdU) and pemetrexed (PEM). Our previous publication reports that the depletion of UNG by shRNA causes uracil and 5-FU incorporation into DNA following 5-FdU or PEM exposure, and highly sensitizes certain human cancer cells to these chemotherapeutic drugs. These data suggest that UNG specific inhibitors could improve the anticancer effect of 5-FdU or PEM. However, a few known inhibitors have not been shown to function at therapeutic concentrations in human cells. Therefore, in order to identify a novel, small-molecule compound targeting the human UNG enzymatic activity, we have previously optimized a biochemically high-throughput screening (HTS) assay that measures the removal of uracil from a DNA hairpin based on the un-quenching of fluorescence. This assay has been successfully employed in a pharmacologically active compound collection screen (>3,000 small-molecules+ approved drugs). In 384-well plate runs using control wells in which enzyme is not added, the average assay provides signal to background ratios of 3.0 to 4.0. Typical Z factors range from 0.6-0.7, indicating a robust assay. We have identified five bioactive compounds with IC50 values < 1uM and have selected a nanoMolar inhibitor of the human UNG enzyme termed, UNGi-A. By using an in vitro DNA glycosylase activity assay, this compound displays a significant inhibitory activity towards the purified human UNG enzyme over time, in a dose-dependent manner. Further characterization shows a significant effect of this inhibitor on UNG activity in cellular extract that expresses endogenous UNG from DLD1 human colon cancer cell line. The result suggests that UNGi-A compound diminishes UNG activity in cell extracts. In addition, thermal shift assay (TSA) data suggest that UNGi-A binds directly to the human UNG enzyme. Together, this compound shows potential activity that blocks the human UNG activity and we are currently evaluating the effect of UNGi-A in combination with 5-FdU on UNG activity in human cancer cells.

Citation Format: Mya T. Nguyen, Yan Yan, Yuriy Fedorvo, John Pink, Drew Adams, Stanton Gerson. Identification and characterization of a small-molecule inhibitor of the human uracil-DNA glycosylase for use in combination with DNA damage-based anticancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1663.
Abstract 1665: AZ304, a novel dual BRAF inhibitor, exerts antitumor activity in colorectal cancers independent of BRAF status
Authors: Ma Rui , Xu Ling , Che Xiaofang , Qu Xiujuan , Drew Lisa , Shen Minhui , Cheung Tony , Liu Yunpeng
Published: August 2018

Background: BRAF mutation is associated with poor clinical outcome of malignant tumors and mediates resistance to chemotherapy and targeted therapy. We aimed to determine whether wild type and V600E mutant BRAF colorectal cancers exhibit distinct sensitivities to dual BRAF inhibitor, AZ304.

Methods: We measured kinase activity by AlphaScreen assay. MTT assays, colony-formation assays, and Western blot were performed to evaluate the in vitro anti-tumor effects of AZ304. In vivo efficacy was investigated by xenograft study and Immunohistochemistry.

Results: AZ304 exhibited potent inhibitory activity against both wild type and V600E mutant forms of the serine/threonine-protein kinase BRAF with enzyme IC50values of 79nM and 38nM, respectively. By suppressing the ERK phosphorylation, AZ304 led to effective cell proliferation inhibition against a panel of human cancer cell lines with different BRAF and RAS generic status. In selected colorectal cancer cell lines, AZ304 significantly inhibited cell growth in vitro and in vivo, regardless of BRAF genetic status. Additionally, combination with the EGFR inhibitor Cetuximab enhanced the potency of AZ304 independent of BRAF mutational status.

Conclusions: The BRAF inhibitor, AZ304, had a broad spectrum of antitumor activities and the combination with Cetuximab significantly enhanced AZ304 potency against colorectal cancers in vitro and in vivo.

Citation Format: Rui Ma, Ling Xu, Xiaofang Che, Xiujuan Qu, Lisa Drew, Minhui Shen, Tony Cheung, Yunpeng Liu. AZ304, a novel dual BRAF inhibitor, exerts antitumor activity in colorectal cancers independent of BRAF status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1665.
Abstract 1669: Identification of mutant KRas-Raf-1 binding disruptors
Authors: Vangipurapu Rajanikanth , Chen Liwei , Kennedy Perry , Morrison Karen C. , Hergenrother Paul J. , Sebti Said M.
Published: August 2018

Mutant KRas is a major driver of human cancer, yet currently no clinically approved therapies that directly target mutant KRas are available. In order to identify novel drugs that bind either mutant KRas or its effector Raf-1, we used alpha-screen and GST-pull down assays to identify disruptors of KRas G12D binding to the Ras-binding domain of Raf-1. From a novel, natural product-inspired chemical library called Complexity To Diversity (CTD), we have identified a series of 5 hits that belong to a family of gibberellic acid-derived compounds and 2 hits from a family of pleuromutilin-derived compounds. At present we are using Differential Scanning Fluorimeter (DSF) and Isothermal calorimetry (ITC) assays to determine whether these hits bind to KRas G12D or Raf-1 RBD. These studies may lead to identifying chemical probes that can be developed to target mutant KRas-driven cancers.

Citation Format: Rajanikanth Vangipurapu, Liwei Chen, Perry Kennedy, Karen C. Morrison, Paul J. Hergenrother, Said M. Sebti. Identification of mutant KRas-Raf-1 binding disruptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1669.
Abstract 1672: Preclinical evaluation of WXFL1024H251, a potent second-generation HSP90 inhibitor
Authors: yan Xiaobing , Ding Charles Z. , Hu Lihong , Huang Wei , Chen Shuhui , Wu Dongdong , Wu Jiahu , Ma Guoqiang , Zhang Qiyao , Gao Rui , Zhu Yusong , Dong Tiantian , Zhu Yizhong , Yan Caixia , Yan Yan
Published: August 2018

Objective: Heat Shock Protein 90 (Hsp90) is a molecular chaperone that mediates the post-translational stability of its protein substrates, many of which are onco-proteins. Hsp90 is emerging as an important target for cancer therapy because its inactivation may abrogate multiple signaling pathways simultaneously, irrespective of the mutational status of proteins involved. We disclose here our clinical candidate WXFL1024H251, a novel second-generation small-molecule Hsp90 inhibitor with a chemical structure unrelated to the first generation, the ansamycin family of Hsp90 inhibitors.

Method: The anti-proliferative activity of WXFL1024H251 was evaluated in a variety of cancer cell lines in a 96-hour viability assay. QW, Q2W or TIW intravenous administration of at doses of 5~50 mg/kg was used to evaluate the in vivo anti-tumor activity of WXFL1024H251 in BT474 cell line-derived xenograft (CDX) breast cancer model. Effects of WXFL1024H251 on HSP90 function were assessed by western blot analysis of PD markers HSP70, Her2 and p-Her2 in the BT474 tumors.

Result: A panel of 20 cancer cell lines were screened for sensitivity to WXFL1024H251. Proliferation of 2 cancer cell lines A549 and BT474 were potently inhibited by WXFL1024H251, with IC50 of 0.007 μM and 0.009 μM, respectively. A549 cell line has KRAS mutation and medium to high EGFR expression. BT474 is an ER+ and ERBB2+ breast cancer cell line. In the BT474 tumor model, 3 week treatment with WXFL1024H251 showed good antitumor efficacy with T/C values of 36% at 7.5 mg/kg Q2W and 45% at 10 mg/kg QW; toxicity were acceptable with both dosing regiments.

Conclusion: These preclinical studies showed good antitumor efficacy of WXFL1024H251, which was associated with client protein maturation and HSP70 up-regulation in the tumor tissue. WXFL1024H251 may represent a novel promising clinical antitumor agent for treating breast cancer and other cancer types with high expression of HSP90.

Citation Format: Xiaobing yan, Charles Z. Ding, Lihong Hu, Wei Huang, Shuhui Chen, Dongdong Wu, Jiahu Wu, Guoqiang Ma, Qiyao Zhang, Rui Gao, Yusong Zhu, Tiantian Dong, Yizhong Zhu, Caixia Yan, Yan Yan. Preclinical evaluation of WXFL1024H251, a potent second-generation HSP90 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1672.