bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3980: Ly6G+ neutrophils and polymorphonuclear myeloid derived suppressor cells promote the survival of tumor cells
Authors: Herlihy Sarah , Lin Cindy , Ozeriva Maria , Garfall Alfred , Vogl Dan , Gabrilovich Dmitry , Nefedova Yulia
Published: July 2017

A heterogeneous group of immature myeloid cells which suppress the immune system are termed myeloid derived suppressor cells (MDSC). Collectively a MDSC population is comprised of monocyte-like MDSC and polymorphonuclear MDSC (PMN-MDSC). MDSC are increased in the presence of a number of different tumors. We previously demonstrated that PMN-MDSC represent the majority of MDSC in tumor-bearing mice. In a mouse model of multiple myeloma (MM) depletion of myeloid cells in vivo results in increased survival. Direct co-culture of neutrophils (CD11b+Gr1+ cells from tumor-free mice) or PMN-MDSC (CD11b+Gr1+ cells from tumor-bearing mice) and MM cells increases the survival of the MM cells from chemotherapeutic-induced death. Cell-cell contact is not necessary for the protective effect. Neutrophil and PMN-MDSC supernatants protect MM cells from cell death, suggesting the factor protecting tumor cells is soluble. Results in the mouse were confirmed using human MM cells and replicated on breast cancer cells with neutrophils or PMN-MDSC. Several cytokines and growth factors, such as IL-6 and fibroblast growth factor, are implicated in chemoresistance. The role of these neutrophil and PMN-MDSC soluble factors and the identification of novel contributors to chemoresistance are under investigation.

Citation Format: Sarah Herlihy, Cindy Lin, Maria Ozeriva, Alfred Garfall, Dan Vogl, Dmitry Gabrilovich, Yulia Nefedova. Ly6G+ neutrophils and polymorphonuclear myeloid derived suppressor cells promote the survival of tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3980. doi:10.1158/1538-7445.AM2017-3980
Abstract 3985: NCoR2 regulates glioblastoma progression and treatment resistance
Authors: Kaushik Shelly , Phillips Joanna J. , Weaver Valerie M.
Published: July 2017

Glioblastoma Multiforme (GBM, WHO Grade IV glioma) is one of the deadliest adult CNS tumors with a median survival of less than two years. It is largely resistant to aggressive therapeutic regimes, which combine radiation, chemotherapy and surgery. Thus, it is imperative to identify the molecular players and pathways that facilitate GBM resistance and recurrence. Nuclear co-repressor protein NCoR2 is upregulated in GBM and its increased expression correlates with poor prognosis in GBM patients (Alrfaei. Plos one. 2013). NCoR2, through its binding to the histone deacetylase protein HDAC3, mediates transcriptional repression (Zhu et al. Brain Res. 2013). NCoR2 is also involved in maintenance of neural stem cells (Jepson et al. Nature. 2007), which contribute to treatment resistance. Our study employs two distinct lineages of glioma - the less aggressive proneural and the highly aggressive mesenchymal subtypes. We show that NCoR2 is differentially expressed across these subtypes, suggesting a role for NCoR2-mediated transcriptional reprogramming in GBM progression. Moreover, knockdown of NCoR2 leads to phenotypic changes both in vitro and in vivo - including effects on neurosphere formation and proliferation, differential expression of stem-like markers as well as regulation of tumor growth and progression in mice. We also demonstrate that NCoR2 expression is increased upon treatment with temozolomide (used for GBM therapy), indicating a protective pro-tumor role for NCoR2. Finally, we explore the contribution of the GBM microenvironment to GBM progression by varying the three-dimensional properties as well as the rigidity of the extracellular matrix and investigating changes in NCoR2 expression and its regulation of tumor progression. We conclude that NCoR2 regulates GBM progression and resistance to therapy potentially via inducing a stem-like phenotype as well as responding to the tumor-promoting mechanical cues within the GBM microenvironment.

Citation Format: Shelly Kaushik, Joanna J. Phillips, Valerie M. Weaver. NCoR2 regulates glioblastoma progression and treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3985. doi:10.1158/1538-7445.AM2017-3985
Abstract 4001: Infiltration of neutrophils in the tumor was associated with poor prognosis of patients with gastric cancer
Authors: Tanaka Hiroaki , Hiramatsu Soichiro , Tamura Tatsuro , Nagahara Hisashi , Kimura Kenjiro , Shibutani Masatsune , Ohira Go , Toyokawa Takahiro , Yamazoe Sadaaki , Amano Ryosuke , Muguruma Kazuya , Maeda Kiyoshi , Hirakawa Kosei , Ohira Masaichi
Published: July 2017

Background:Recent evidence shows that neutrophils often migrate into the tumor and are associated with tumor progression. These tumor associated neutrophils (TANs) neutrophils have potential to induce angiogenesis, lymphangiogensesis and immunosuppression by inhibiting cytotoxic T lymphocytes. Immune status is one of the important prognostic factor of gastric cancer. We previously reported that lymphangiogenesis was augmented in the primary tumor of gastric carcinoma.

Aim:The aim of this study was to investigate the correlation between CD15+ TANs and clinicopatological features of patients with gastric cancer.

Materials and Methods: We investigated the association of the density of TANs in primary tumor with clinicopathological features by immunohistochemistry. We used specimens from a total of 143 primary tumor and 497 lymph nodes from 120 patients underwent gastrectomy between 2007 and 2008 in our department. We examined prognostic value of TANs identified as positive CD15 cells in both of the primary tumor and tumor draining lymph nodes.

Results:The median number of TANs in primary tumor and tumor draining lymph nodes were 18.4 and 23 cells/HPF, respectively. High TANs in both of the primary tumor and lymph nodes had correlation with depth of invasion (primary tumor P<0.001, lymph nodes P=0.036) and lymph nodes metastasis (primary tumor P<0.001, lymph nodes P=0.002). Kaplan-Meier analysis showed that patients with high TANs in both primary tumor and lymph nodes had worse prognosis than patients with a low TANs (primary tumor P=0.031, lymph nodes P=0.014). Multivariate analysis revealed that high TANs in lymph nodes were independent prognostic factors. (P=0.038)

Conclusion: Our results showed that patients with high CD15+TANs infiltration in gastric cancer tissues had poorer prognosis because of increasing lymph nodes metastasis. These findings suggested the TANs might have important role of tumor lymphangiogenesis in microenvironment of gastric carcinoma.

Citation Format: Hiroaki Tanaka, Soichiro Hiramatsu, Tatsuro Tamura, Hisashi Nagahara, Kenjiro Kimura, Masatsune Shibutani, Go Ohira, Takahiro Toyokawa, Sadaaki Yamazoe, Ryosuke Amano, Kazuya Muguruma, Kiyoshi Maeda, Kosei Hirakawa, Masaichi Ohira. Infiltration of neutrophils in the tumor was associated with poor prognosis of patients with gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4001. doi:10.1158/1538-7445.AM2017-4001
Abstract 4004: Rapamycin selectively targets obesity-polarized macrophages in the prostate tumor microenvironment
Authors: Galván Gloria C. , Pham Tommy , Harlow Brittany , Johnson Christian , Patodia Riddhi , Quach Duan , Liss Michael A. , deGraffenried Linda A.
Published: July 2017

Background: Obesity is associated with prostate cancer progression and mortality. Previous studies in our laboratory suggest that obesity drives prostate cancer progression in part by increasing macrophage recruitment and by polarizing macrophages in the tumor microenvironment into a tumor promoting M2/TAM phenotype. Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has been used for several decades in transplant patients, and in the last several years has been shown to be an effective disease suppressor in certain cancer types. Intriguingly, mTOR has been shown to be especially important for M2 polarization and stabilization.

Hypothesis: Based upon published data and our preliminary studies, we hypothesize that rapamycin will selectively target obesity-polarized macrophages and will provide a survival benefit to the obese prostate cancer patient. Methods: To address this hypothesis, we used our in vitro model of obesity-induced macrophage polarization that includes two different prostate cancer cell lines, macrophages, and sera from obese and non-obese men. qPCR was used to measure expression levels of markers associated with an M2/TAM phenotype. MTT assays were conducted to measure cell viability, and flow cytometry and Western blot analyses were used to determine cell cycle status and apoptosis.

Results: Obese conditions increased expression of M2/TAM markers in macrophages and rapamycin selectively decreased viability of obesity-activated M2/TAMs compared to M1 macrophages.

Conclusions: Our in vitro study suggests that obesity promotes a tumor-associated phenotype in macrophages and that the mTOR pathway is involved in the survival of M2/TAM macrophages. This study offers a novel mechanistic approach to treat obese patients with prostate cancer.

Citation Format: Gloria C. Galván, Tommy Pham, Brittany Harlow, Christian Johnson, Riddhi Patodia, Duan Quach, Michael A. Liss, Linda A. deGraffenried. Rapamycin selectively targets obesity-polarized macrophages in the prostate tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4004. doi:10.1158/1538-7445.AM2017-4004
Abstract 4007: Differential macrophage programming is associated with the aggressiveness of gliomas
Authors: Yao Nai-Wei , Liao Fang , Chang Chen , Chen Joanne Jeou-Yuan
Published: July 2017

Tumor-associated macrophages (TAMs), a major component of immune cells in the tumor microenvironment, have been shown to be associated with enhanced malignancy of gliomas. The polarization of macrophages toward a pro-inflammatory phenotype M1 or an anti-inflammatory phenotype M2 governed by niche factors in the tumor microenvironment may dictate the malignant outcome of gliomas. In this study, we examined the effects of differential programming of TAMs on the development of C6 gliomas in different host microenvironment using SD and Wistar rats. Using multi-parametric MRI measurements, we show that C6 cells implanted into SD rats developed larger tumors as compared to those in Wistar rats (p = 0.0199), which was accompanied with a shorter survival of SD rats (p = 1.06×10-9). The increased tumor growth in SD rats was associated with increased angiogenesis and higher levels of VEGF and VEGFR2 expression. We also observed a significantly increased ratio of M2/M1 in the tumors of SD rats while a decreased ratio of M2/M1 in those of Wistar rats, suggesting that the milieu of C6 gliomas in SD rats favors for the polarization of macrophages toward the pro-tumorigenic M2 phenotype. Most importantly, Induction of M1 polarization by IFNγ treatment greatly reduced tumor burden in SD rats, and induction of M2 polarization by IL-4/IL-10 treatment or blockade of M1 polarization by anti-IL12 treatment significantly promoted tumor growth in Wistar rats. These results demonstrate an important role of TAMs in glioma pathogenesis and the crucial role of microenvironment in dictating the polarization of TAMs into M1 or M2 phenotypes. We conclude that macrophage programming plays an important role in glioma development and that targeting glioma-associated macrophages may serve as a promising therapeutic approach.

Citation Format: Nai-Wei Yao, Fang Liao, Chen Chang, Joanne Jeou-Yuan Chen. Differential macrophage programming is associated with the aggressiveness of gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4007. doi:10.1158/1538-7445.AM2017-4007
Abstract 4009: Characterization of M2 macrophage subtype content in cancer and normal tissues
Authors: Dauffenbach Lisa M. , Sia Gela C. , Zheng Jianping , Jun Natalia , Olsen Eric P. , Kerfoot Christopher A.
Published: July 2017

Tumor-associated macrophages play an important role in presenting tumor antigens to T cells and are being investigated for their role in sensitivity to immune checkpoint inhibitors such as PD-1 inhibitors. The M2 macrophage subtype, characterized by CD163 expression, is immunosuppressive and associated with poor prognosis. A multiplex CD68/CD163 immunohistochemical assay was developed to characterize the density of cells expressing each marker and determine the percentage of macrophages that are of the M2 subtype. The assay was used to analyze 22 normal tissue types and 50 tumor tissues within 5 different indications. The results were analyzed by image analysis and results were analyzed as percent positive and density per mm2. Cancer tissues expressed a greater percentage of CD163+ macrophages than normal tissues.

Citation Format: Lisa M. Dauffenbach, Gela C. Sia, Jianping Zheng, Natalia Jun, Eric P. Olsen, Christopher A. Kerfoot. Characterization of M2 macrophage subtype content in cancer and normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4009. doi:10.1158/1538-7445.AM2017-4009
Abstract 401: Designing for success: the right CRISPR design strategies for the right experiment
Authors: Brody Leigh , Humphryes-Kirilov Neil , Dillard Victor , Doyle Riley
Published: July 2017

CRISPR design is a complex multivariate problem. The weight of each variable differs depending on the experiment under consideration. Current methods of designing CRISPR experiments are based on a single-variable, resulting in poor experimental outcomes. From the numerous experiments enabled by the DESKGEN platform, we can demonstrate key design improvements to maximise experimental outcome.

Our current scoring methods were tested and validated using an sgRNA library of over 3,500 sgRNAs in essential genes with a variety of predicted sgRNA activities and specificities. The predicted 'good' sgRNAs achieved significantly higher read depletion for essential genes, with fewer failing sgRNAs, compared to sgRNAs that didn't meet the scoring criteria. Our scoring algorithm incorporates multiple published observations for choosing effective sgRNAs, which differs from other CRISPR tools that tend to focus on one factor such as sgRNA sequence or specificity. We were unable to find correlations for some currently accepted sgRNA selection criteria, such as position within a gene, which demonstrates the need to constantly challenge and improve sgRNA design rules.

Citation Format: Leigh Brody, Neil Humphryes-Kirilov, Victor Dillard, Riley Doyle. Designing for success: the right CRISPR design strategies for the right experiment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 401. doi:10.1158/1538-7445.AM2017-401
Abstract 4013: A novel multifunctional protein tag enables simple and sensitive bioluminescent quantification of tagged proteins
Authors: Eggers Christopher , Butler Braeden , Hurst Robin , Hall Mary , Binkowski Brock , Encell Lance , Schwinn Marie , Machleidt Thomas , Wood Keith , Fan Frank
Published: July 2017

Dysregulation of protein expression is a key mechanism of tumorigenesis. The most commonly used approach to monitor changes in expression is to perform SDS-PAGE, followed by immunoblotting, a labor-intensive process that requires high-quality antibodies to detect proteins at endogenous levels of expression. We have developed a novel protein tag utilizing NanoLuc Binary Technology (NanoBiT), a binary complementation system based on NanoLuc luciferase. The tag, designated High BiT (HiBiT), is only 11 amino acids in length, which minimizes potential interference with protein function. The amount of HiBiT-tagged protein is measured using a lytic detection reagent containing Large BiT (LgBiT), which binds with high affinity to HiBiT (KD~1 nM) to reconstitute a bright, luminescent enzyme. HiBiT-tagged proteins can be quantified in cell lysates over 7 orders of magnitude of linear dynamic range with a limit of detection of less than 10-19 moles (3 fg of 30 kDa protein). The simple add-mix-read assay protocol can be completed in minutes, providing an assay that is compatible with high-throughput applications. The sensitivity of the assay allows quantification of expression at endogenous levels, and the small tag size is ideal for CRISPR-mediated genome editing. HiBiT-tagged proteins separated by SDS-PAGE can be detected on blots at sub-picogram levels with a detection reagent containing LgBiT. By eliminating the multiple steps of blocking, binding, and washing of traditional blotting techniques, the protocol takes minutes instead of hours. Additionally, the cell surface expression, internalization, or secretion of HiBiT-tagged proteins can be measured in minutes using a non-lytic detection reagent containing the cell-impermeable LgBiT protein. HiBiT represents a next-generation protein tag that allows simple quantification of proteins of interest in their cellular context or following SDS-PAGE.

Citation Format: Christopher Eggers, Braeden Butler, Robin Hurst, Mary Hall, Brock Binkowski, Lance Encell, Marie Schwinn, Thomas Machleidt, Keith Wood, Frank Fan. A novel multifunctional protein tag enables simple and sensitive bioluminescent quantification of tagged proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4013. doi:10.1158/1538-7445.AM2017-4013
Abstract 4022: Validation of label-free impedance analysis as a versatile tool for cancer molecular therapeutics screening
Authors: Cerignoli Fabio , Xi Biao , Lamarche Brandon , Zhao Leyna , Abassi Yama
Published: July 2017

In vitro monitoring cancer cells response to treatment often involves laborious sample processing and collects single data points that are against the dynamic nature of cancer cells. Here we present the adaptation of an impedance-based methodology to dynamically monitor cancer cell behavior and therapeutic response. The technology detects cell death, proliferation or migration by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. It avoids sample labeling and processing and allows continuous monitoring of cell response. Our data shows validation with cancer cell lines and primary cells monitored for cell invasion/migration, anticancer drug response and receptor signaling activation, with similar results over end point assays. Easy experiment set up and minimal sample processing make the technology ideal for applications in large screening campaigns, while label-free technology allows further analysis on the same samples through orthogonal assays.

Citation Format: Fabio Cerignoli, Biao Xi, Brandon Lamarche, Leyna Zhao, Yama Abassi. Validation of label-free impedance analysis as a versatile tool for cancer molecular therapeutics screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4022. doi:10.1158/1538-7445.AM2017-4022
Abstract 4026: Bioluminescent metabolite assays enable easy measurement of changes in tumor biology
Authors: Valley Michael , Sobol Mary , Leippe Donna , Vidugiriene Jolanta
Published: July 2017

Tumor cells rely on metabolic reprogramming to meet their increased demands for energy and biomass. Such reprogramming is exemplified by increased rates of glucose uptake, greater utilization of glycolysis, and a reliance on glutaminolysis. Cancer cells also have a strong dependence on lipids and cholesterol and thus feature enhanced rates of biosynthesis and uptake of these molecules as well. We have developed a suite of bioluminescent assays that can monitor metabolites in all of these important pathways. These assays share a core technology that couples the production of NAD(P)H to the generation of light through a variety of metabolite specific dehydrogenases. Changes in glycolysis and glutaminolysis can be followed by measuring glucose, lactate, glutamate, and/or glutamine through their respective dehydrogenases (and glutaminase). Glucose uptake is typically monitored through the accumulation and phosphorylation of 2-deoxyglucose to produce 2-deoxyglucose-6-phosphate, and this can be measured by employing glucose-6-phosphate dehydrogenase. These bioluminescent assays are homogeneous and can be multiplexed with viability, cytotoxicity, and protein quantitation assays. Metabolites can be measured extracellularly (i.e. from media) or intracellularly and the assays can be performed in 96- or 384-well plates. These assays are simple and sensitive and can provide a greater understanding of how various treatments affect the metabolic state of a tumor cell.

Citation Format: Michael Valley, Mary Sobol, Donna Leippe, Jolanta Vidugiriene. Bioluminescent metabolite assays enable easy measurement of changes in tumor biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4026. doi:10.1158/1538-7445.AM2017-4026
Abstract 4028: Analysis and recovery of functionally defined single immune cell clones through opto-electro-positioning technology
Authors: Wang Xiaohua , Bronevetsky Yelena , Beaumont Kristin , Stadler Guido , Bao Xiaoyan R. , Smith Duane , Beemiller Peter , Chapman Kevin
Published: July 2017

Cancer patients mount protective innate and adaptive immune responses against tumor cells. Such responses are able to control or even eliminate tumor burden if harnessed properly. Identifying clonal adaptive immune cells targeting tumor cells with designated function is extremely valuable as these cells are able to either fight the tumor directly or provide tumor targeting immune receptors to engineer other effector cells like autologous CD8 T cells or NK cells. The process of isolating cancer specific immune cells and their receptors, however, remains technically challenging and labor intensive.

Opto-Electro-Positioning (OEP) technology employs a light actuated dielectrophoretic force to maneuver live cells in their culture environment. We have combined OEP with a nanofluidic cell culture system to produce an integrated microfluidic platform that enables multi-dimensional investigation of lymphocyte specificity and function. The system is able to 1) deterministically capture designated lymphocytes, 2) actively maintain and observe colony growth, and 3) conduct cytokine release assays, all in a single enclosed microfluidic chip. The new platform is suitable for high throughput screening to identify and harvest antigen specific and/or functionally defined T cells. We have successfully applied it to isolate primary human T cells targeting either EBV/Tetanus toxin antigens or tumor associated antigens such as MART-1 through tetramer staining or a proliferation assay. The system is also exceptionally efficient for investigating immune cells from clinical specimens typically of low cellularity. From tumor biopsies, we are able to capture tumor infiltrating T cells, stimulate proliferation, investigate their TNF-α secretion on chip, and obtain full transcriptomic analysis after export.

Overall, our data suggest the OEP nano-fluidic environment provides a powerful high throughput screening tool to identify rare lymphocytes against tumor associate antigens or neo-antigens and to investigate precious clinical samples for biomarker discovery.

Citation Format: Xiaohua Wang, Yelena Bronevetsky, Kristin Beaumont, Guido Stadler, Xiaoyan R. Bao, Duane Smith, Peter Beemiller, Kevin Chapman. Analysis and recovery of functionally defined single immune cell clones through opto-electro-positioning technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4028. doi:10.1158/1538-7445.AM2017-4028
Abstract 403: CRISPR/Cas9 genome-wide sgRNA libraries for loss-of-function and gain-of-function genetic screens
Authors: Tedesco Donato , Diehl Paul , Makhanov Mikhail , Baron Sylvain , Chenchik Alex
Published: July 2017

Genome-wide loss-of-function screening is a fundamental method to identify genes responsible for driving biological processes. Complex pooled lentiviral-based libraries expressing large numbers of genetic disruptors, such as shRNAs (RNAi) or sgRNAs (CRISPR), make large-scale cell screening practical. While RNAi-based approaches have proven to be an effective strategy for these screens, recent work has showed CRISPR-based technologies offer not only an effective alternative, but distinct advantage. We will present unpublished data from genetic screens in human cancer cell lines using CRISPR-Knockout (KO), CRISPRi and CRISPRa technologies, as well as RNAi. Although loss-of-function shRNA and sgRNA pooled library screens are similar in concept, the gene loss of function is achieved by different mechanisms (mRNA degradation with RNAi, full gene disruption with CRISPR-KO, transcriptional inhibition with CRISPRi), so some divergences are expected and indeed observed when comparing results obtained using one method versus the other. Furthermore, contrary to RNAi, CRISPR technology can be modified to activate gene expression (CRISPRa), thus enabling the use of genome-wide gain-of-function screening in gene function studies.

Citation Format: Donato Tedesco, Paul Diehl, Mikhail Makhanov, Sylvain Baron, Alex Chenchik. CRISPR/Cas9 genome-wide sgRNA libraries for loss-of-function and gain-of-function genetic screens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 403. doi:10.1158/1538-7445.AM2017-403
Abstract 4035: A novel combi-molecule engineered to target the putative synthetic lethal interactions between the epidermal growth factor receptor (EGFR) and poly(ADP-ribose)polymerase (PARP)
Authors: Mouhri Zhor Senhaji , Rupp Martin , Jean-Claude Bertrand J.
Published: July 2017

Over the past decade, PARP inhibition has been actively pursued as a novel approach for the selective therapy of tumors with BRCA1/2 mutations. The therapeutic benefits of PARP inhibitors have now been proven in the clinic against BRCA1/2 mutant ovarian cancers. This is hitherto limited to BRCA1/2 mutations, which only accounts for 5-10% of all cancers with hereditary mutations in the homologous recombination pathway. Therefore, new strategies are not only required to enhance the potency of PARP inhibitors but also to expand their use beyond BRCA mutation. While several combination modalities have been reported for PARP inhibitors, the concept of targeted PARP inhibitor has not yet been explored. Here using our novel combi-targeting approach, we report on the design of PARP inhibitors targeted to EGFR, a tyrosine kinase receptor overexpressed in several solid tumors. Recently, reports on the relationship between EGFR, PARP and BRCA have begun to emerge, one of which described a contextual synthetic lethality between EGFR and PARP (PloS one 7.10 (2012)). Here we report on the design and synthesis of novel PARP-EGFR combi-molecule based on structural modification of olaparib as a PARP inhibitor warhead and the quinazoline moiety for targeting EGFR. The results showed that: (a) it is capable of inducing a dose-dependent inhibition of PARP in isolated enzyme assay, (b) it induced a dose-dependent inhibition of EGFR in an isolated kinase assay, (c) it showed a dose-dependent inhibition of EGFR phosphorylation and downstream signaling in whole-cell assay, (d) it was selectively potent towards BRCA2 mutant and also EGFR-overexpressing cell lines, (d) it was extremely potent with activities superior to that of olaparib or gefitinib alone and their corresponding equimolar combination in three established triple negative breast cancer cell lines, (e) subcellular distribution analysis showed that it was abundantly localized in the perinuclear region. These results in toto suggest that this new combi-molecule could be developed as a single drug modality emulating the combination of PARP and EGFR inhibitors with the added benefit of being targeted to EGFR-expressing tumor cells.

Citation Format: Zhor Senhaji Mouhri, Martin Rupp, Bertrand J. Jean-Claude. A novel combi-molecule engineered to target the putative synthetic lethal interactions between the epidermal growth factor receptor (EGFR) and poly(ADP-ribose)polymerase (PARP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4035. doi:10.1158/1538-7445.AM2017-4035
Abstract 4041: SUV39H2 inhibition enhances sensitivity of breast cancer cells to doxorubicin through downregulation of γ-H2AX production
Authors: Vougiouklakis Theodore , Saloura Vassiliki , Matsuo Yo , Nakamura Yusuke
Published: July 2017

Accumulating evidence has demonstrated the biological importance of protein methyltransferases in human tumorigenesis, and several small molecular inhibitors targeting these enzymes have been developed. Moreover, inhibiting pathways involved in DNA repair has been shown to enhance the cytotoxicity of DNA-damaging agents. Herein, we report the development of a potent SUV39H2 inhibitor (OTS193320) that decreases global histone H3 lysine 9 tri-methylation (H3K9me3) levels in cancer cells and attenuates cancer cell proliferation, and suppresses the tumor growth in mouse xenograft models. SUV39H2 (Suppressor of variegation 3-9 homolog 2), is a protein methyltransferase known to methylate histone H3 at lysine 9 (H3K9), and recently reported to methylate histone H2AX at lysine 134, which enhances the accumulation of phosphorylated H2AX (γ-H2AX) and regulates the DNA repair pathway in human cancer. SUV39H2 is highly expressed in many cancer types, including lung and breast cancers, while its expression levels are restricted to testis in normal adult tissues. Exposure of OTS193320 to two triple negative breast cancer cell lines attenuated H3K9me3 levels in a dose-dependent manner and triggered apoptotic cell death. Combination of OTS193320 and doxorubicin (DOX) resulted in reduction of γ-H2AX levels compared to single agent DOX, as visualized on western blot and immunocytochemical analysis. Furthermore, combination therapy attenuated the levels of p53-binding protein 1, which is reported to co-localize with γ-H2AX foci, compared to single agent DOX. Cell viability assays demonstrated a significant growth suppressive effect when OTS193320 was combined with DOX, compared to single agent treatment of either drug, suggesting chemosensitization to DOX. Importantly, in a mouse xenograft model of A549 lung cancer cells, we observed a tumor growth inhibition of 60.8% at day 14 with 25mg/kg intravenous administration of the inhibitor, without significant body weight loss or toxicity. Immunohistochemical staining of tumors treated with the inhibitor demonstrated a significantly lower number of Ki-67 positive cells and attenuated distribution patterns of H3K9me3 compared to the control tumors. Collectively, our results demonstrate that SUV39H2 inhibition may be a promising approach to develop a novel class of anti-cancer treatment.

Citation Format: Theodore Vougiouklakis, Vassiliki Saloura, Yo Matsuo, Yusuke Nakamura. SUV39H2 inhibition enhances sensitivity of breast cancer cells to doxorubicin through downregulation of γ-H2AX production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4041. doi:10.1158/1538-7445.AM2017-4041
Abstract 4043: Design of a novel MGMT inhibitor targeted to EGFR overexpressing tumor cells: a new approach to selectively potentiate temozolomide in refractory tumors
Authors: Rupp Martin , Jean-Claude Bertrand J.
Published: July 2017

Temozolomide (TMZ) is an oral alkylating agent commonly used as a first-line chemotherapeutic agent for the treatment of high-grade gliomas and melanoma. Alkylation of the O6-position of guanine in DNA is mainly responsible for the antitumor effect of TMZ. The primary mechanism of chemoresistance to TMZ has been shown to be the overexpression of the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) that specifically removes the methyl lesions from the O6-position of guanine. While O6-benzylguanine (O6-BG), a potent inhibitor of MGMT, was shown to sensitize tumor cells to TMZ in vitro and in vivo, the O6-BG+TMZ combination failed in clinical trial due to acute hematologic toxicity. To circumvent this problem, we wish to target O6-BG to the epidermal growth factor receptor (EGFR), a receptor that is not generally expressed in the hematological cells but overexpressed in many solid tumors. We have now successfully designed and synthesized one such molecule termed MR30 and showed that: 1) MR30 is capable of blocking EGFR and depleting MGMT levels in whole cells; 2) MR30 is a unique molecule capable of inducing stronger growth inhibition than the 2-drug combination involving clinical EGFR and MGMT inhibitors in a panel of melanoma, lung, prostate and ovarian cancer cell lines; 3) MR30 in vitro potentiated the effect of TMZ on MGMT positive cell lines; 4) its kinase inhibitory profiling over 25 different kinases demonstrated selectivity for EGFR, HER2 and EGFR mutant forms; 5) an isogenic model showed more than 70-fold selectivity towards EGFR expressing cells in growth inhibitory assay; 6) MR30 showed good cell penetration with distribution in the perinuclear region. The results in toto suggest that MR30 has the potential to be developed as a tumor selective potentiator of TMZ.

Citation Format: Martin Rupp, Bertrand J. Jean-Claude. Design of a novel MGMT inhibitor targeted to EGFR overexpressing tumor cells: a new approach to selectively potentiate temozolomide in refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4043. doi:10.1158/1538-7445.AM2017-4043
Abstract 4050: Multifaceted targeting of drug-resistant EGFR+ and HER2+ breast tumors
Authors: Law Brian K. , Law Mary E. , Ferreira Renan B. , Wang Mengxiong , Davis Bradley J. , Castellano Ronald K. , Heldermon Coy D.
Published: July 2017

Unmet needs in the breast cancer field include the development of effective treatments for 1) drug-resistant HER2+ breast cancers, 2) Triple-Negative (HER2-, ER-, PR-) Breast Cancers (TNBCs), and 3) Inflammatory Breast Cancers (IBCs). Treatment-refractory HER2+ tumors retain HER2 expression after acquiring drug resistance, and large fractions of TNBCs and IBCs overexpress either HER2 or EGFR. Thus, drugs that selectively kill breast tumors that overexpress EGFR or HER2 could benefit patients suffering from these three classes of treatment-refractory tumors. Our team has identified a class of compounds termed Disulfide bond Disrupting Agents (DDAs) that selectively kill cancer cells that overexpress HER2 or EGFR. DDA-mediated cancer-selective cytotoxicity results from A) downregulation of the EGFR/HER2/HER3 proteins, B) inactivation of the Akt oncogene, and C) activation of the Unfolded Protein Response (UPR). Importantly DDAs only elicit these responses in cancer cells that overexpress EGFR or HER2. DDAs exhibit striking anticancer activity in human HER2+ xenograft tumors grown orthotopically in mice, with no evidence of toxicity. DDAs function through mechanisms distinct from those of HER2 specific monoclonal antibodies and tyrosine kinase inhibitors, and biochemical studies indicate that DDAs are complementary with Lapatinib with respect to blockade of HER2 driven proliferative and survival signaling. Current efforts are directed toward identifying mechanism-based drugs that synergize with DDAs to potentiate HER1-3 downregulation, Akt inhibition, and UPR, and to kill HER2+/EGFR+ patient-derived xenograft tumors that are refractory to current therapies.

Citation Format: Brian K. Law, Mary E. Law, Renan B. Ferreira, Mengxiong Wang, Bradley J. Davis, Ronald K. Castellano, Coy D. Heldermon. Multifaceted targeting of drug-resistant EGFR+ and HER2+ breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4050. doi:10.1158/1538-7445.AM2017-4050
Abstract 4060: Pharmacological targeting of the Aurora A and Histone 3 lysine 9 methyltransferase pathways in pancreatic cancer induces mitotic catastrophe
Authors: Mathison Angela , Salmonson Ann , Missfeldt Mckenna , Assuncao Thiago de , Bintz Jennifer , Christensen Trace , Kossak Sarah , Nair Asha , Iovanna Juan , Huebert Robert , Lomberk Gwen
Published: July 2017

By regulating gene expression networks that mediate neoplastic behavior, epigenetic protein complexes are the ultimate effectors of oncogenic pathways. This hierarchical position of epigenetic pathways within the mechanisms that regulate cancer makes them ideal candidates for therapeutic targets. The current work was designed to characterize the role of the K9H3 Histone Methyltransferase (HMT) pathway in mediating oncogenic signals downstream of Aurora Kinase A (AurkA), with the goal of designing efficient combinatorial therapies against PDAC. Affinity protein purification, combined with mass spectrometry, demonstrates that HP1γ isolated from mitotic cells interacts with AurkA and the HMTs, G9a and GLP. We also find that this complex is critical for mitotic progression and cell proliferation. Congruently, treatment of PDAC cells with individual drugs against AurkA or HP1-HMT inhibits cell growth by inducing senescence, a cytostatic response. However, the combination of these agents has a synergistic effect of reducing cell growth in both, monolayer and spheroid cultures to result in a cytotoxic effect. Confocal and electron microscopy, along with cell cycle analysis, demonstrate that the cytotoxic effect of this combination is due to induction of mitotic catastrophe, a distinct form of cell death that occurs during mitosis. Molecularly, the combination of AurkA and HP1-HMT inhibition bypasses G2/M arrest with downregulation of the Chk1-Cdc25c pathway. In vivo treatment of PDAC orthotopic xenografts with the HP1-HMT inhibitor alone demonstrated reduced PDAC growth, and increased efficacy in PDAC growth reduction was observed in combination with the AurkA inhibitor over either individual treatment. Thus, our data demonstrate a novel AurkA-HP1-HMT mitotic pathway that holds promise for potential pharmacological targeting in combinatorial therapy for this malignancy.

Citation Format: Angela Mathison, Ann Salmonson, Mckenna Missfeldt, Thiago de Assuncao, Jennifer Bintz, Trace Christensen, Sarah Kossak, Asha Nair, Juan Iovanna, Robert Huebert, Gwen Lomberk. Pharmacological targeting of the Aurora A and Histone 3 lysine 9 methyltransferase pathways in pancreatic cancer induces mitotic catastrophe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4060. doi:10.1158/1538-7445.AM2017-4060
Abstract 4061: Computational exploration of mechanistic determinants of antibody drug-conjugate pharmacokinetics using quantitative systems pharmacology modeling strategies
Authors: Wilkins A. Katharina , Matteson Andrew , Gruenbaum Lore , Park Jennifer , Burke John M. , Apgar Joshua
Published: July 2017

The pharmacokinetics of antibody drug conjugate (ADC) therapeutics typically show a discrepancy between the PK of total antibody (conjugated and unconjugated antibody) and that of conjugated antibody, carrying one or more payload molecules. This discrepancy is often attributed to deconjugation (Kamath, 2014), however recent evidence suggests that the underlying mechanisms may be more complex. This work employs a computational quantitative systems pharmacology (QSP), or mechabistic PK/PD approach to understand the impact of drug antibody ratio (DAR) and the resulting changes in molecular properties on overall PK and relative payload disposition as observed in preclinical and clinical studies. Our work establishes the benefit of using computational models to design novel ADCs and to optimize the discovery and development of existing ADCs.

Citation Format: A. Katharina Wilkins, Andrew Matteson, Lore Gruenbaum, Jennifer Park, John M. Burke, Joshua Apgar. Computational exploration of mechanistic determinants of antibody drug-conjugate pharmacokinetics using quantitative systems pharmacology modeling strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4061. doi:10.1158/1538-7445.AM2017-4061
Abstract 4065: Association study between iron and cholesterol for the treatment of drug resistant neuroblastoma
Authors: Lee Sang Y. , Slagle-Webb Becky , Schengrund Cara-Lynne , Connor James
Published: July 2017

Neuroblastoma is the most common childhood extracranial solid malignancy. Although cancer cells need more iron and lipids for their active cell metabolism, possible links between iron, mutations in genes involved in iron metabolism (e.g., HFE) and lipid metabolism have not been studied well. We determined the HFE genotype in human neuroblastoma cells as well as patients. Neuroblastoma cells and patients have C282Y HFE mutation rates (~12%) that are similar to those of the general Caucasian population. While cultured cells derived from drug resistant neuroblastomas had less cholesterol, they expressed more ferritin, and contained more iron, than cells derived from drug susceptible tumors. In drug resistant neuroblastoma CHLA-171 cells, siRNA induced alteration of HFE expression had no effect on their iron levels but decreased their cholesterol. Results of in vitro experiments indicated that simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR), was cytotoxic to drug resistant cells as was the iron chelator di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). In vivo, the anti-tumor effect of simvastatin administered orally was minimal. In summary, we found i) an inverse association between ferritin and HMG-CoAR expression in neuroblastoma cells that was reflected in their iron and cholesterol levels, and ii) that the most cytotoxic cholesterol inhibitor of neuroblastoma cells was simvastatin, while the most cytotoxic iron chelation agent was DpC. Our results indicate that it may be possible to use HFE genotype to develop a precision medicine approach to use certain iron chelators and/or cholesterol decreasing agents in the treatment of drug resistant neuroblastomas. Our results also indicate that alterations in iron and cholesterol metabolism are part of the cell’s drug resistance mechanism in neuroblastomas.

Citation Format: Sang Y. Lee, Becky Slagle-Webb, Cara-Lynne Schengrund, James Connor. Association study between iron and cholesterol for the treatment of drug resistant neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4065. doi:10.1158/1538-7445.AM2017-4065
Abstract 4069: Trimethylation and acetylation of Histone H3K27 modulates 5-fluorouracil response by regulating DPYD expression
Authors: Wu Rentian , Nie Qian , Tapper Erin E. , Jerde Calvin R. , Offer Steven M. , Diasio Robert B.
Published: July 2017

The antimetabolite 5-fluorouracil (5-FU) is one of the most widely used chemotherapy drugs. Dihydropyrimidine dehydrogenase (DPD) initiates the catabolic degradation of 5-FU to inactive metabolites and is widely accepted as a major determinant of 5-FU response and toxicity. While genetic variants in DPYD have been shown to variants may affect 5-FU metabolism, they are rare events and do not completely explain the reported variability in DPD function or the resultant differences in treatment response. Here, we report that DPYD expression is epigenetically regulated by histone modification at relevant promoter and enhancer regions. Using both chemical inhibitors and genetic approaches, we show that that H3K27 tri-methylation (H3K27me3) at the DPYD promoter is regulated by the epigenetic modifiers Ezh2 and UTX. Promoter H3K27me3 suppresses DPYD expression by inhibiting the binding of the transcription factor PU.1 bindingto the promoter and ,increases sensitivity leading to increased resistance to 5-FU. In a cohort of healthy volunteers, H3K27me3 levels were inversely correlated with An enhancer that regulates DPYD expression was also identified. Modifying the H3K27 status of the DPYD promoter or enhancer modulates DPYD gene expression. Deletions and mutations on the DPYD promoter or enhancer abolish their transcription regulation function. Enrichment of H3K27me3 at the DPYD promoter was negatively correlated with both DPYD expression and DPD enzyme activity in peripheral blood mononuclear cellsspecimens from healthy volunteers. Lastly, tumor expression data suggests that DPYD repression by Ezh2 is a strong predictor ofs poor survival in 5-FU treated cancers. By using a combination of in silico prediction and in vitro reporter assays we also identified a functional enhancer region for DPYD that is also likely to be clinically relevant to 5-FU treatment outcome. Collectively, the presentthese findings suggest that a previously uncharacterized mechanisms regulates DPD expression and may likely contribute to tumor resistance to 5-FU.

Citation Format: Rentian Wu, Qian Nie, Erin E. Tapper, Calvin R. Jerde, Steven M. Offer, Robert B. Diasio. Trimethylation and acetylation of Histone H3K27 modulates 5-fluorouracil response by regulating DPYD expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4069. doi:10.1158/1538-7445.AM2017-4069