bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3991: Profiling the immune tumor microenvironment in primary and recurrent epithelial ovarian cancer
Authors: Ojalvo Laureen S. , Thompson Elizabeth D. , Wang Tian-Li , Meeker Alan K. , Shih Ie-Ming , Fader Amanda N. , Cimino-Mathews Ashley , Emens Leisha A.
Published: July 2017

Clinical trials targeting the immune tumor microenvironment (TME) in epithelial ovarian cancer (EOC) typically have included patients with heavily pre-treated advanced disease and demonstrated only marginal efficacy. A better understanding of how the EOC TME evolves with progression from primary to recurrent disease may inform future immunotherapy trials. Here, we evaluate the immune TME in primary and recurrent EOC using tissue microarrays. Our cohort included matched primary and recurrent tumors from 17 patients, and additional non-matched primary tumors from 20 patients and recurrent tumors from 15 patients. We stained for CD8, FOXP3 (regulatory T cells (Tregs)), CD68 (tumor associated macrophages (TAMs)), programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) by immunohistochemistry to interrogate the immune composition of the TME. Tregs increased in recurrent tumors compared to primary tumors (8.0 vs 14.2/HPF, p=0.0210). Higher TAM density was associated with higher levels of Treg and CD8+ T cell infiltrates in recurrent tumors (p=0.001 and p<0.001, respectively), and with higher Treg but not CD8+ T cell infiltrates in primary tumors (p=0.027 and p=0.200). TAM-dense recurrent tumors had increased PD-L1 on tumor cells and immune cells, whereas TAM-dense primary tumors had increased PD-L1 only on immune cells. Increased Tregs in primary tumors correlated with decreased time to first recurrence (17.0 vs 28.5 months, p=0.022). Conversely, increased Tregs in recurrent tumors correlated with longer overall survival (OS) from recurrence (median not met vs 20.0m, p=0.022). Although TAM density did not affect patient survival, analysis of matched primary and recurrent tumors revealed that patients with increased TAMs at recurrence (n=5) had a longer median OS from recurrence than patients without increased TAMs at recurrence (n=12). Tregs increased at recurrence in the majority of matched tumor pairs (n=12), but there was no correlation with survival. In conclusion, the TME of EOC is immunologically active. TAM-dense recurrent disease had higher CD8+ T cell and Treg infiltrates and PD-L1 expression. In this study, patients with increased cellular recruitment to the TME at recurrence had improved survival. Larger, more detailed studies characterizing the evolution of the TME with progression from primary EOC to recurrence are warranted.

Citation Format: Laureen S. Ojalvo, Elizabeth D. Thompson, Tian-Li Wang, Alan K. Meeker, Ie-Ming Shih, Amanda N. Fader, Ashley Cimino-Mathews, Leisha A. Emens. Profiling the immune tumor microenvironment in primary and recurrent epithelial ovarian 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 3991. doi:10.1158/1538-7445.AM2017-3991
Abstract 3999: Cross talk between tumor cell derived HMGB1 and adaptive B cells in the tumor microenvironment of esophageal squamous cell carcinoma
Authors: Kam Ngar Woon , Guan Xin-Yuan , Kwong Dora Lai-Wan
Published: July 2017

Esophageal squamous cell carcinoma (ESCC) is the most aggressive type of cancer among gastrointestinal cancers. The lack of effective anti-tumor immunity strategy for ESCC patients imposes urgent demand for new treatment paradigm. Tumor microenvironment (TME) is characterized by diverse cell populations (cancerous, immune cells, and pleiotropic nature of cytokines and inhibitory receptors) whose alternations are closely related with tumor progression. High mobility group box chromosomal protein 1 (HMGB1) has been recognized to be expressed in various tumor cells, and importantly it has also been identified as one of the key factors regulating inflammatory reactions.

By comparing differentially expressed genes between ESCC tumors (T) and their corresponding non-tumorous (NT) tissues high-throughput transcriptome sequencing system (RNA-Seq, Illumina HiSeq 2500), we found that the ligands of TIM-3 (an inhibitory receptor), HMGB1 and galectin-9, but not, cell-surface phosphatidylserine, were overexpressed in all ESCC samples when compared to their NT counterparts. We further characterized HMGB1 and galectin-9 gene expression changes in 37 matched primary ESCC T and NT control samples by qRT-PCR. We found that HMGB-1 and galectin-9 mRNA is constitutively expressed in all samples, while ESCC T tissue significantly expressed ~2fold higher of HMGB-1, but not galectin-9, mRNA as compared to paired NT tissue. Histopathological characterization of ESCC tissue by sequential single staining for B-cells (CD20) and plasma cells (CD138), demonstrating that B cell infiltrate was dominated by CD20+ B cells lacking of terminal differentiation into plasma cells. Additionally, CD20+ B cells infiltrates were negative for CD27 (marker for memory B cells) and IgD (marker for naïve B cells). Additionally, we isolated CD20+ B cells from healthy peripheral blood mononuclear cells (PBMC), and stimulated with/without HMGB-1/IgM. Conditional medium (CM) were collected for foci formation and we found that ESCC cell lines KYSE30 and KYSE180 have better capacity for proliferation when culture with activated B cells CM as compared to unstimulated B cells. Collectively, we believe that the suboptimal clinical outcomes relies on CD20+HMGB1 receptor+ B cells that are exposed to high HMGB1 concentrations in the ESCC microenvironment may eventually become pro-tumor B cells subset.

Citation Format: Ngar Woon Kam, Xin-Yuan Guan, Dora Lai-Wan Kwong. Cross talk between tumor cell derived HMGB1 and adaptive B cells in the tumor microenvironment of esophageal squamous cell carcinoma [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 3999. doi:10.1158/1538-7445.AM2017-3999
Abstract 40: Development of a novel antibody-drug conjugate targeting endosialin/TEM-1: potent antitumor activity in sarcoma
Authors: Sala Gianluca , Iacobelli Stefano , Capone Emily , PIccolo Enza , Sauniere Jean-Fred , Douet Vanessa Vannucci
Published: July 2017

The TEM-1/Endosialin/CD248 receptor is expressed in the cell surface of tumor-associated stroma cells, as well as in sarcoma and neuroblastoma cells. This receptor is emerging as an attractive molecule in diagnostics and therapeutics because of its expression across the stroma of many human tumors, the low to absent expression in normal tissues and accessibility from the vascular circulation. In this study, we present evidence of the preclinical efficacy of a novel Antibody-Drug Conjugate (ADC). It consists of a humanized TEM-1 monoclonal antibody (E.8-3) conjugated to a highly potent payload (TEM-1-ADC). In TEM-1 expressing cancer cell lines, this TEM-1-ADC demonstrated a powerful, specific and target-dependent killing activity. High expression levels of TEM-1 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Efficacy studies demonstrated that TEM-1-ADC treatment leads to a long lasting tumor growth inhibition of cell line-based models of human sarcoma. Taken together, our results demonstrated that TEM-1 is an attractive target in sarcoma and suggest that TEM-1-ADC has the potential to be developed into a biotherapeutic agent in these malignancies.

Citation Format: Gianluca Sala, Stefano Iacobelli, Emily Capone, Enza PIccolo, Jean-Fred Sauniere, Vanessa Vannucci Douet. Development of a novel antibody-drug conjugate targeting endosialin/TEM-1: potent antitumor activity in sarcoma [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 40. doi:10.1158/1538-7445.AM2017-40
Abstract 4000: Establishing the natural history of the immunosuppressive myeloid microenvironment in an inducible model of lung adenocarcinoma
Authors: Lagraoui Mouna , Dalgard Clifton L. , Sukumar Gauthaman , Huaman Celeste , Summers Thomas , Carter Corey A. , Schaefer Brian C.
Published: July 2017

Tumorigenesis is accompanied by broad changes to the surrounding tissue microenvironment in a tumor- and patient-specific manner. Phenotype shifts in tissue-resident immune cells promote cancer progression by establishing a proliferative inflammatory environment, activating angiogenesis, stimulating tumor cell invasion and suppressing anti-tumor immunity. While the former mechanisms are well appreciated and often targeted by current cancer therapeutics, the nature of the mechanisms controlling establishment of the local immunosuppressive state that contribute to a failure of anti-tumor immunity are less clearly defined. Blockade of tumor antigen-specific T cell killing is clearly a major contributor to the failure to control tumorigenesis. However, evidence suggests that phenotypic shifts in innate immune cell populations also contribute to the failure of anti-tumor immunity. We hypothesize that the emergence of early lung adenocarcinomas is accompanied by a shift in myeloid phenotypes, rapidly establishing a microenvironment favorable to tumor growth, survival and vascularization, and hostile to cell-mediated anti-tumor immune responses. Through the utilization of a Cre-inducible mouse model of p53-null, Kras-G12D+ lung adenocarcinoma, we have begun to define the natural history of the myeloid component of the tumor microenvironment, from tumor initiation to a point equivalent to human stage I adenocarcinoma. Histological, flow cytometry and real-time PCR approaches collectively demonstrate that tumor-associated myeloid phenotypes emerge at a very early stage of disease. These data are consistent with our hypothesis that the myeloid component of the tumor microenvironment plays a crucial role in establishing an immunosuppressive state during early tumorigenesis. Implications of these findings for current and emerging immunotherapies will be discussed.

Citation Format: Mouna Lagraoui, Clifton L. Dalgard, Gauthaman Sukumar, Celeste Huaman, Thomas Summers, Corey A. Carter, Brian C. Schaefer. Establishing the natural history of the immunosuppressive myeloid microenvironment in an inducible model of lung adenocarcinoma [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 4000. doi:10.1158/1538-7445.AM2017-4000
Abstract 4010: Tonggyu-tang, a traditional Korean medicine, suppresses inflammation, potential implications in tumor microenvironment
Authors: Kim Hyoin , Ko Seong-Gyu , Shin Yong Cheol , Kim Ji Hye , Seo Hye-Sook , Kim Tai Young , Hong Se Hyang , Lee Kangwook , Ku Jin Mo , Kim Myeong-Sun , Choi Yu-Jeong , Kang Soo-yeon , Cheon Chunhoo , Ko Youme , Wen Huang Ching , Sasaki Yui , Kang Sohyeon
Published: July 2017

The critical roles of inflammation in the development of cancer have long been appreciated. A growing body of evidence supports the notion that infiltrates of inflammatory cells into tumor microenvironment influence the tumor progression by providing bioactive molecules including pro-inflammatory cytokines. Importantly, the increased number of mast cells within tumor microenvironment has been associated with a poor survival in cancer patients. Moreover, keratinocyte inflammation is known to be crucial for skin tumor development. The use of natural products to reduce inflammation in tumor microenvironment is gaining an interest, because of their reduced toxicity toward normal cells. In this study, we tested the effects of Tonggyu-tang (TGT) which is composed of 14 different herbal extracts on the activity of mast cells. We found that TGT significantly reduced the expression and production of inflammatory cytokines such as IL-4, IL-6, IL-8, and TNF-α in PMA and ionomycin- stimulated HMC-1 (human mast cell line-1). In an attempt to determine molecular mechanism underlying the inhibitory effects of TGT on cytokine expression, we revealed that TGT suppressed MAPK signaling pathway including ERK, p38, and JNK as well as NF-κB pathway, which are known to regulate inflammatory cytokine expression. Similar results were obtained from the LPS-stimulated HaCaT cells, immortalized human keratinocytes. Taken together, our results demonstrate that TGT suppresses inflammation by inhibiting the expression of pro-inflammatory cytokine in both mast cells and keratinocytes, thereby potentially leading to inhibition of tumor progression.

Citation Format: Hyoin Kim, Seong-Gyu Ko, Yong Cheol Shin, Ji Hye Kim, Hye-Sook Seo, Tai Young Kim, Se Hyang Hong, Kangwook Lee, Jin Mo Ku, Myeong-Sun Kim, Yu-Jeong Choi, Soo-yeon Kang, Chunhoo Cheon, Youme Ko, Huang Ching Wen, Yui Sasaki, Sohyeon Kang. Tonggyu-tang, a traditional Korean medicine, suppresses inflammation, potential implications in 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 4010. doi:10.1158/1538-7445.AM2017-4010
Abstract 4011: Validation of novel continuous live-cell assays for immune cell activation and killing of blood cell cancers
Authors: Bevan Nicola , Campwala Hinnah , Szybut Clare , Patel Kalpana , Appledorn Dan , Dale Tim , Trezise Derek
Published: July 2017

The blood cancers leukaemia, lymphoma and myeloma are expected to cause the deaths of > 55,000 people in the US in 2016. New immunological approaches afford great promise for improved therapies. Here, we describe novel high throughput live-cell image-based assays for immune cell activation and killing of target cells that are geared toward screening for new treatments for these malignancies. Myeloid and lymphoid cells (Jurkats, Raji, Ramos, WIL2-NS, THP-1, PBMCs) were plated on poly-L-ornithine (PLO) or fibronectin coated 96-well flat plates and monitored over time (h to days) using non-invasive live-cell analysis (IncuCyte). The dynamics of proliferation were quantified via phase-contrast image analysis (% confluence), and validated as a robust measure of cell number by correlating to direct cell counts (Scepter, Millipore) and ATP assays (PerkinElmer). Anti-CD3/IL-2 (0.1-100/10 ng/mL) or anti-CD28 activation (1-100 ng/mL) of PBMCs evoked time-dependent proliferation (0-5d) that was sensitive to the initial cell density and concentration of stimulus. L-Kynurenine (4.69 - 300 µM), a metabolite of the amino acid L-tryptophan caused concentration and time-dependent inhibition of proliferation of PBMCs. To quantify immune-cell killing in co-cultures, WIL2Ns and Ramos B-cell myelomas were first transduced with nuclear-targeted RFP (NucLight Red) to enable direct cell counting. PBMCs, either pre-activated or activated in situ (IL-2/CD3), were then added and the time-course of killing quantified through live (RFP) and dead/apoptotic (annexin-V) cell counting. Together, these protocol developments and validation data illustrate non-invasive continuous measurement of proliferation, activation, clustering and immune-cell killing of non-adherent tumour cells at industrial scale. Unlike flow cytometry, this approach follows the full time-course of the biology without perturbing the cells and allows cell-cell interactions to be visualised. These assays are amenable to testing new therapeutic antibodies, small molecules and genetic T-cell modulation such as CAR-T.

Citation Format: Nicola Bevan, Hinnah Campwala, Clare Szybut, Kalpana Patel, Dan Appledorn, Tim Dale, Derek Trezise. Validation of novel continuous live-cell assays for immune cell activation and killing of blood cell cancers [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 4011. doi:10.1158/1538-7445.AM2017-4011
Abstract 4012: Visualization of EPR effect and active targeting by using microscopic mass spectrometry
Authors: Yasunaga Masahiro , Furuta Masaru , Ogata Koretsugu , Fujiwara Yuki , Koga Yoshikatsu , Matsumura Yasuhiro
Published: July 2017

Microscopic mass spectrometry (MMS), in which a microscope is coupled with an atmospheric pressure matrix-assisted laser desorption/ionization (MALDI) and quadruple ion trap time-of-flight (TOF) analyzer has been developed for the investigation of the distribution of various molecules including drugs. The matrix-coated drug sample is ionized and then separated on the basis of its mass-to-charge ratio (m/z). Images were acquired from imaging mass spectrometry (IMS) or tandem mass spectrometry (MS/MS) data. Recently, pharmacokinetic (PK) and pharmacodynamic (PD) studies have become important to evaluate the efficacy and toxicity of the drugs. In these analyses, tissue homogenates are generally used for the quantification by high-performance liquid chromatography (HPLC) or liquid chromatography mass spectrometry (LC-MS). However, they lack the information regarding the drug distribution in a specific anatomical area. The information of the drug distribution would allow us to optimize the drug design enabling more efficient drug delivery. (1) We studied the tissue distribution of paclitaxel (PTX) and its micellar formulation (NK105) using a MMS. NK105 showed much stronger antitumor effects on a human pancreatic cancer BxPC3 xenograft than PTX. In the drug imaging, we demonstrated that NK105 delivered more PTX to the whole tumor tissue. In the mouse model, PTX caused the peripheral neurotoxicity but NK105 did not. Multiple high drug-signal areas surrounding and inside the caudal nerve were observed in the case of PTX, whereas the signals after NK105 administration were quite low. (2) The tissue distribution and controlled drug release of ADC (antibody-drug conjugate) consisting of a tissue factor specific antibody (TF) linked to the anticancer agent monomethyl auristatin E (MMAE) was evaluated in comparison with control-ADC (control antibody-MMAE conjugation). TF-ADC showed stronger antitumor effect on BxPC3 xenograft than control-ADC. We then established the selective detection method of MMAE for distinguishing free MMAE and its conjugated form. The released MMAE signal detected following the accumulation of TF-ADC was greatest 24 h after the administration, compared with the control-ADC at the same time (P<0.01). In summary, we succeeded in visualizing (1) the EPR (Enhanced permeability and retention) effect and (2) active targeting using MMS. The data obtained will facilitate more efficient drug development in the preclinical setting.

Citation Format: Masahiro Yasunaga, Masaru Furuta, Koretsugu Ogata, Yuki Fujiwara, Yoshikatsu Koga, Yasuhiro Matsumura. Visualization of EPR effect and active targeting by using microscopic mass spectrometry [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 4012. doi:10.1158/1538-7445.AM2017-4012
Abstract 402: Biomarker-based pooled CRISPR-Cas9 screening: Platform development and validation
Authors: Scales Tim M. , Cross Benedict C.
Published: July 2017

Functional genomic screening with CRISPR-Cas9 has provided a powerful and precise new way to interrogate the phenotypic consequences of gene loss in high-throughput, unbiased analyses. Rapid development of pooled lentivirus and deep-sequencing-led approaches have allowed us and others to exploit this technology in target ID, target validation, drug MOA analysis and patient stratification. Adaptation of the Cas9 nuclease by domain fusion and catalytic inactivation has further provided a novel way to both up and down-regulate gene expression using targeted, high-throughput CRISPRi and CRISPRa tools. To date, the vast majority of screens have been conducted using sgRNA abundance in surviving cell populations as an NGS-based proxy for proliferation, for example for in vitro resistance analysis to a cytotoxic treatment regimen. However, where cellular pathophysiology is uncoupled from cell health, the enormous promise of pooled CRISPR-Cas9 screening has been largely untapped. We now report on the development of a FACS-based platform that can be used to monitor biomarker-based cellular response in combination with high-throughput genetic screening. The endogenous interaction between CD28 and CD80 was used as a proof-of-concept paradigm and the performance of our platform was measured in screen-simulation conditions. We find that FACS-linked NGS was able to robustly identify the CD28 deleted cells from a complex population using a FITC-labelled CD80-Fc. Pseudo-blinding of the data yielded unambiguous identification of the expected target guides, with robust screening metrics, validating a new platform for myriad discovery programmes.

Citation Format: Tim M. Scales, Benedict C. Cross. Biomarker-based pooled CRISPR-Cas9 screening: Platform development and validation [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 402. doi:10.1158/1538-7445.AM2017-402
Abstract 4021: Donor-specific primary monocyte-derived dendritic cell maturation model for high-throughput screening of immunostimulatory adjuvants
Authors: Chen Qin , Lawrenz Anthony , Wang Alan , Kelly Erin , Harberts Erin , Ernst Robert K. , Tong Jay
Published: July 2017

Dendritic cell (DC) based immunotherapy is a promising treatment alternative for various solid tumor and hematological malignancies. DCs are capable of initiating and propagating antigen-specific adaptive immune responses through their intrinsic maturation process. Identifying novel, effective adjuvant drug candidates that can stimulate DC maturation while understanding and accounting for the variability of patient-specific DC cell responses to the immunostimulatory adjuvants is essential for development of such immunotherapies. Reliable, high throughput assay platforms that characterize patient-specific modulatory effects during the DC maturation process in vivo and in vitro are also highly desirable. In this study, we present a high throughput in vitro primary cell assay to evaluate dose-dependent DC maturation in a panel of human leukocyte antigen (HLA) typed individual donors. CD14+ monocytes from four healthy donors were isolated from leukapheresis derived peripheral blood mononuclear cells (PBMC) and cultured for seven days with GM-CSF and IL-4 to achieve differentiation to DC. Cells were then stimulated for 24 hours with lipopolysaccharide (LPS), CpG oligonucleotides, or nine different novel toll-like receptor 4 (TLR4) ligands at three concentrations. To quantify the differentiation of monocytes into DC and subsequent maturation, flow cytometry was used to measure the expression of biomarkers, MHC-II, CD40, CD80, CD83, and CD86. Greater than 99% MHC-II expression was observed across all donors and confirmed successful differentiation to DC. The TLR4 ligands showed reduced biomarker expression, as compared to pathogenic E. coli LPS but similar to PHAD, a synthetic TLR4 agonist currently used in adjuvants. While monocytes from all donors responded to the ligands tested, the intensity of activation marker expression varied among donors with LPS/TLR4 ligand induced maturation process showing 43-91% CD40 and 27-82% CD83 expression, highlighting the necessity of multi-donor screening for drug development. In conclusion, a high throughput in vitro primary monocyte derived DC differentiation assay has been developed and is commercially available to assist researchers in characterizing donor-specific DC responses to immunostimulatory adjuvants.

Citation Format: Qin Chen, Anthony Lawrenz, Alan Wang, Erin Kelly, Erin Harberts, Robert K. Ernst, Jay Tong. Donor-specific primary monocyte-derived dendritic cell maturation model for high-throughput screening of immunostimulatory adjuvants [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 4021. doi:10.1158/1538-7445.AM2017-4021
Abstract 4024: Physiologically relevant target engagement using the cellular thermal shift assay (CETSA)
Authors: Karen Jakob , Sioberg Catrine , Shalaly Nancy Dekki , Molina Daniel Martinez
Published: July 2017

Targeted therapies are dependent on adequate occupancy and target engagement to reach their efficacy measure. Yet, more than half of novel candidate drugs entering clinical trial stage fail before proof of concept is achieved. Inadequate target engagement is often at the root cause of these failures. Therefore, we have developed a versatile label-free method to quantify the target engagement of drugs on their native physiological relevant targets. The method is validated in multiple sample matrices, using different technical platforms to allow studies of target engagement from bench to bedside: in living cells and tissue from rodents and man.

CETSA(®) is a patented biophysical method measuring the thermal stability of drug target proteins and the shift induced by their ligands. By quantifying the melting temperature and shift induced by the ligand we can quantify the potency of target engagement. This potency determination allows filtering of candidate drugs by their ability to engage the target in its physiologically relevant form, in relevant sample formats such as intact cells or tissue.

We present several applications of the method and the utility in preclinical in vitro studies and translation to in vivo systems. In addition, new powerful detection formats are being evaluated and will be presented.

The utility and value of the CETSA(®) method enabling preclinical development of novel drugs is substantiated by the examples included here. We show the value of the method both for compound/drug profiling, for target profiling and for unbiased drug profiling on whole proteomes in response to drug treatments.

Such datasets can be generated from in vitro systems to clinical samples and may reduce the high attrition rate in proof of concept studies by facilitating the discovery, development and evaluation of novel drugs.

Citation Format: Jakob Karen, Catrine Sioberg, Nancy Dekki Shalaly, Daniel Martinez Molina. Physiologically relevant target engagement using the cellular thermal shift assay (CETSA) [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 4024. doi:10.1158/1538-7445.AM2017-4024
Abstract 4025: Accelerating drug repurposing for cancer therapy using multiplexed viability assays
Authors: Corsello Steven M. , Mader Christopher C. , Bryan Jordan , Roth Jennifer , Peck David , Davis John , Bender Samantha , Wang Li , Wang Alice , Bittker Joshua , Vazquez Francisca , Subramanian Aravind , Tsherniak Aviad , Golub Todd R.
Published: July 2017

New therapies are desperately needed for patients with advanced cancer, but making safe and efficacious drugs remains expensive and time consuming. Meanwhile, multiple non-oncology drugs have been successfully repurposed for cancer, including thalidomide for multiple myeloma and aspirin for prevention of colorectal cancer. While the benefits of repurposing are clear, successes to date have been largely serendipitous. We have created two foundational tools to identify drug repurposing opportunities at a much greater scale: a new world-class screening library of more than 4,000 drugs/clinical candidates and a multiplexed method to rapidly conduct cellular viability assays across hundreds of cell lines.

PRISM, a recently developed high-throughput assay, employs DNA-barcoded cell lines to enable rapid testing of many drugs against pools of cancer cell lines. We have tested 4,100 compounds against 578 genomically characterized cancer cell lines using PRISM. Our experiment is among the largest cell line screens ever undertaken. Global analysis has revealed multiple strong clusters of active drugs, including vitamin D agonists, bromodomain inhibitors, and statins. In addition, many novel and established drug-biomarker relationships were identified, including alkylating agent response and low MGMT expression, BRAF inhibitor response and BRAF mutation, and MDM2 inhibitor response and TP53 wild-type status. Surprisingly, more than 100 non-oncology drugs unexpectedly killed multiple cancer cell lines, making them attractive repurposing candidates. For example, the FDA-approved phosphodiesterase inhibitor anagrelide selectively kills PDE3A-high lung cancer and melanoma cell lines. Confirmatory experiments are underway to validate screening results, evaluate putative biomarkers, and test drug activity in preclinical models. Importantly, this approach is expected to enable rapid initiation of clinical trials, dramatically accelerating patient access to potential new therapies.

Citation Format: Steven M. Corsello, Christopher C. Mader, Jordan Bryan, Jennifer Roth, David Peck, John Davis, Samantha Bender, Li Wang, Alice Wang, Joshua Bittker, Francisca Vazquez, Aravind Subramanian, Aviad Tsherniak, Todd R. Golub. Accelerating drug repurposing for cancer therapy using multiplexed viability assays [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 4025. doi:10.1158/1538-7445.AM2017-4025
Abstract 4029: Robust Ras inhibition assay utilizing fully synthetic Ras-binding domain
Authors: Khavrutskii Lyuba , Tarasov Sergey G. , Fields Colin , Stefanisko Karen , Tarasova Nadya
Published: July 2017

Intensifying efforts to develop clinically useful inhibitors of Ras proteins resulted in increasing need for quantitative, reliable and robust in vitro assays of Ras activity. The vast majority of detection methods used in the assays require fusion, chemical modification or immobilization of the protein. We have found that K-Ras modifications have significant impact on its activity and function because of the highly allosteric nature of the protein. We have attached three different fluorescent moieties to two sites distant from the active site of the protein in a site-selective manner. All six modifications resulted in significant drops in affinity towards Ras-binding domain of one of Ras's major effectors c-Raf as determined by Microscale Thermophoresis (MST). Fluorescent labeling of recombinant Raf1 RBD also resulted in inhibition of K-Ras binding. To produce a pair that interacted as unlabeled proteins, we have generated RBD with a single-site attached fluorescent moiety utilizing microwave-assisted high efficiency solid phase peptide synthesis. Optimization of coupling and deprotection protocols and the type of the solid support allowed conducting the synthesis of 76 residues long protein in fully-automated manner in one run. Refolding of the product yielded RBD that was indistinguishable from the recombinant one in CD spectra, melting temperature and K-Ras binding affinity as determined by isothermal calorimetry and microscale thermophoresis. Fluorescent RBD produced significant changes in both thermophoretic signal and fluorescence anisotropy upon binding to K-Ras. Both MST and fluorescence polarization assays have proven sensitive, reproducible and straightforward for determination of Ras inhibition constant for Ras inhibitors identified by virtual screens and rational design. They can be easily adapted to high throughput screening formats. Developed synthetic protocols allow for straightforward generation of proteins with various natural and non-natural modifications.

Citation Format: Lyuba Khavrutskii, Sergey G. Tarasov, Colin Fields, Karen Stefanisko, Nadya Tarasova. Robust Ras inhibition assay utilizing fully synthetic Ras-binding domain [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 4029. doi:10.1158/1538-7445.AM2017-4029
Abstract 4032: Synergistic effects of Bortezomib and JQ1 for human and canine osteosarcoma treatments
Authors: Yang Ya-Ting , Yuzbasiyan-Gurkan Vilma
Published: July 2017

Osteosarcoma (OS) is the most common primary malignant bone tumor of both humans and dogs, with the incidence in dogs being nearly 15 times that observed in humans. Despite advances in treatment of other cancers, the clinical outcome of OS remains poor for the past several decades. Therefore, identifying novel and effective treatments for osteosarcoma is an urgent need. Among the small molecular compounds we tested in a drug screening study, we identified a bromodomain inhibitor (JQ1) that targets bromodomains which modify chromatin and block transcription of key factors, and a proteasome inhibitor that induces cell death through various mechanisms (bortezomib) as having good growth inhibition effects on three canine OS cell lines (D17, Abrams, and Gracie) and two human OS cell lines (SAOS2 and U2OS). We then determined the IC50 values of these two drugs, the concentration capable of inhibiting proliferation by 50%. The IC50 values of bortezomib and JQ1 are both within achievable plasma concentrations and tolerable levels. JQ1 inhibited OS cell proliferation and survival by inducing G2 cell cycle arrest, while bortezomib induced G1 cell cycle arrest. In addition, these two drugs decreased cell migration ability in wound healing assay as well as invasion ability in a Matrigel assay. Furthermore, in combination studies using both JQ1 and bortezomib, we identified synergistic effects. With these promising results from in vitro studies, we further examined JQ1 as therapeutic agent in an orthotopic tumor xenograft mouse model. Using OS cells stably transfected with a lucerifase vector, we were able to track the tumor progression during the treatment period. Mice treated with JQ1 did not show any signs of adverse effect to the drug, indicating that JQ1 was safe and well tolerated at a daily dosage of 50 mg/kg (IP). While some of the mice in treatment group showed decreasing tumor growth compared to the control group, tumor growth trends in the two groups were not consistent, suggesting that we have to optimize the model and the treatment protocol as well as to evaluate combination treatments with bortezomib and JQ1 in in vivo models.

Citation Format: Ya-Ting Yang, Vilma Yuzbasiyan-Gurkan. Synergistic effects of Bortezomib and JQ1 for human and canine osteosarcoma treatments [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 4032. doi:10.1158/1538-7445.AM2017-4032
Abstract 4054: Development of dual function small molecules as therapeutic agents for cancer treatment
Authors: Grindrod Scott , Velena Alfredo , Jung Mira
Published: July 2017

Inhibitors of histone deactylases (HDACs) are emerging as potent anti-cancer agents for treatment of patients with hematopoietic and solid tumors. However, most of these compounds exhibit limitations, including off-targets and toxicity. To improve the efficacy and target specificity of drugs, we developed the compound, SP-1-161 conferring HDAC inhibition activity and ataxia-telangiectasia mutated (ATM) activation. The purpose of this study is to develop a therapy that optimizes the protective effects of ATM activation in normal tissue with the sensitizing effects of HDAC inhibition in cancerous tissue into a single molecule. The compound SP-1-161 was rationally designed by optimizing ATM activation by the cap domain of a hydroximic acid, screened against a panel of Class I and Class II HDAC enzymes, and identified as a pan-HDAC inhibitor with nanomolar potency (IC50 = 8 nM). Western analysis confirms that SP-1-161 increases acetylated histone H3/H4 and α-tubulin and ATM activation in MCF7 cells. Phosphorylated ATM was gradually increased within 1 - 4 hrs in a time-dependent manner. SP-1-161 was then tested in normal breast epithelial (184A1) and breast cancer (MCF7) cell lines to determine its cytotoxicity and effect on radiation clonogenic survival in combination with graded radiation exposure. The data showed that the cytotoxicity values (IC50) were low micro-molar ranges. Furthermore, SP-1-161 protected 184A1 cells (from D0 = 1 Gy to D0 = 1.4 Gy) while increasing sensitivity of MCF7 cells to IR (from D0=1.6 Gy to D0=1.12 Gy). Together, our results demonstrate that SP-1-161 is an unprecedented radio-chemo therapeutic agent for treatment of cancers while protecting normal cells.

Citation Format: Scott Grindrod, Alfredo Velena, Mira Jung. Development of dual function small molecules as therapeutic agents for cancer treatment [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 4054. doi:10.1158/1538-7445.AM2017-4054
Abstract 4055: Enhancing anti-PD-1 immune blockade in melanoma through selective inhibition of histone deacetylase 6
Authors: Knox Tessa , Sahakian Eva , Nair Jayakumar , Kim Jennifer , Banik Debarati , Hadley Melissa , Powers John , Cheng Fengdong , Shen Sida , Pinilla Javier , Weber Jeffrey , Kozikowski Alan , Sotomayor Eduardo , Villagra Alejandro
Published: July 2017

Histone deacetylases (HDACs), were originally described in a limited context as histone modifiers. New evidence has demonstrated that HDACs are also involved in a diverse range of cellular processes that are not restricted to the chromatin environment, such as the regulation of the cell cycle/apoptosis and, more recently, a modulator of immune response. However, much remains unknown about the mechanism of action of HDACs and their roles in the immune-biology of cancer. The non-specific nature of pan-HDAC inhibitors results in a narrow therapeutic window of use, limiting the dose and duration due to toxicity. Our group has focused in one specific HDAC, HDAC6, and shown that both the genetic abrogation and pharmacological inhibition of this HDAC modulates the expression of a variety of immune-regulatory proteins in the tumor microenvironment, including PD-L1, PD-L2, MHC class I, B7-H4 and TRAIL-R1. In particular, we have previously demonstrated that both pharmacological inhibition and/or genetic abrogation of HDAC6 plays a critical role in the immune check point blockade by down-regulating the expression of PD-L1 and other check-point modulators such as PD-L2, B7-H4, etc. Moreover, we have also observed that in vivo inhibition of HDAC6 reduces tumor growth in B16 and SM1 murine melanoma models within syngeneic immunocompetent hosts. Additionally, we have found that the combination of low doses of the HDAC6i Nexturastat A and checkpoint immune blockade, including anti-PD-1 and anti-CTLA4, results in an important improvement in anti-tumor immune responses as evidenced by the reduction of tumor growth when compared to treatment with individual stand-alone agents. In these studies we also evidenced an increased production of IFNγ and IL-2 in the stand-alone check-point inhibitor treatments, which leads to an upregulation of PD-L1 and PD-L2. Similar levels of IFNγ and IL-2 were found in the combination groups. However, the expression level of PD-L1 and PD-L2 were comparable to the non-treated group. Taking all together, we have found that HDAC6i could be used as a potential adjuvant in ongoing therapeutic options involving immune check-point blockade.

Citation Format: Tessa Knox, Eva Sahakian, Jayakumar Nair, Jennifer Kim, Debarati Banik, Melissa Hadley, John Powers, Fengdong Cheng, Sida Shen, Javier Pinilla, Jeffrey Weber, Alan Kozikowski, Eduardo Sotomayor, Alejandro Villagra. Enhancing anti-PD-1 immune blockade in melanoma through selective inhibition of histone deacetylase 6 [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 4055. doi:10.1158/1538-7445.AM2017-4055
Abstract 4056: A modulator of multidrug resistance protein 1 selectively depletes glutathione and synergizes with L-buthionine sulfoximine to sensitize MRP1-expressing cancer cells to chemotherapy
Authors: Gana Christine C. , Hanssen Kimberley , Yu Denise M. , Flemming Claudia , Norris Murray , Haber Michelle , Fletcher Jamie
Published: July 2017

Multidrug resistance protein 1 (MRP1) is frequently overexpressed in tumors where it effluxes chemotherapeutic agents, protecting tumor cells from chemotherapy. This is clearly illustrated in the pediatric solid tumor neuroblastoma, where MRP1 expression is highly prognostic of clinical outcome (1,2) and contributes to chemoresistance in mouse models (3). MRP1 also effluxes low levels of glutathione (GSH), a crucial intracellular antioxidant necessary to avoid damage from the reactive oxygen species induced by radiotherapy and chemotherapy and an important component of both Phase II and Phase III drug metabolism. Depletion of GSH in cancer cells would be of therapeutic value, however the GSH synthesis inhibitor L-buthionine sulfoximine (BSO) has only limited selectivity for tumor over normal cells. We have identified a novel MRP1 inhibitor, 7-methyl-5-phenyl-2-[(4-phenyl-1-piperazinyl)carbonyl]pyrazolo[1,5-a]pyrimidine, that blocks MRP1-mediated drug efflux while greatly enhancing MRP1- mediated GSH efflux and are investigating the utility of this dual-function inhibitor for the treatment of MRP1-overexpressing cancers.

Methods: Cells expressing high levels of MRP1 were treated with MRP1 drug substrates in combination with MRP1 inhibitors. Viability was assessed by short-term cytotoxicity or clonogenic assays and GSH levels determined by glutathione recycling assay.

Results: Our MRP1 modulator depleted intracellular GSH in an MRP1-dependent manner and sensitized MRP1-expressing human neuroblastoma, lung and ovarian cancer cell to chemotherapeutics. This effect was highly synergistic with BSO, both in depleting GSH (CI<0.36, p<0.05) and abolishing colony formation (CI<0.01, p<0.05).

Conclusion: Our MRP1 modulator blocks MRP1- mediated drug efflux while greatly enhancing MRP1- mediated GSH efflux in MRP1 expressing cells, potentially providing an increased therapeutic window for chemotherapeutics in MRP1- overexpressing cancers. Synergy with BSO could widen the range of drugs and cancers that this could be beneficial for.

References 1. Haber, M., et al., (2006) J.Clin Oncol 24, 1546-1553 2. Norris, M. D., et al., (1996) New Engl J Med 334, 231-238 3. Burkhart, C. A., et al., (2009) Cancer Res 69, 6573-6580

Citation Format: Christine C. Gana, Kimberley Hanssen, Denise M. Yu, Claudia Flemming, Murray Norris, Michelle Haber, Jamie Fletcher. A modulator of multidrug resistance protein 1 selectively depletes glutathione and synergizes with L-buthionine sulfoximine to sensitize MRP1-expressing cancer cells to chemotherapy [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 4056. doi:10.1158/1538-7445.AM2017-4056
Abstract 4062: A computational driven protocol of metronomic oral vinorelbine in metastatic non small cell lung cancer
Authors: Imbs Diane-Charlotte , Cheikh Raouf El , Ciccolini Joseph , Serre Raphaël , Barlesi Fabrice , Barbolosi Dominique
Published: July 2017

Over the last decade, metronomic chemotherapy (MC) has emerged as an alternative way to administrate chemotherapy. MC can be defined as regular administration of low/less-toxic doses without extended drug-free breaks. Because countless schedules are possible, testing them empirically looks like an unachievable goal. Therefore, mathematical modeling can be an effective tool to determine appropriate metronomic strategies.

A phase Ia/Ib study based on a mathematical model of oral vinorelbine in metastatic NSCLC and malignant pleural mesothelioma was designed to confirm safety and evaluate efficacy of a new metronomic regimen (Elharrar et al. 2016). The optimized schedule providing weekly Day1 (D1), D2 and D4 with respective doses of 60, 30 and 60 mg was suggested by in silico simulations using a Pharmacokinetic-Pharmacodynamic (PKPD) model taking into account vinorelbine PK, PD toxicities and PD efficacy (Faivre et al. 2013). Trial recruitment will be two-staged, as 12 patients have been enrolled to confirm safety and consolidate the calibration of model parameters. An extension phase on 20-extra patients will be conducted to evaluate the efficacy of the optimized protocol. Monolix 4.3.3 was used for population PKPD analysis.

A three-compartment model best described oral vinorelbine PK data. PK analysis revealed a high inter-individual variability on drug exposure (CV=73%). PD toxicity (neutropenia) was adequately described by a modified Friberg model (Friberg et al. 2002), considering continuous administrations of metronomic dosing regimen. On the 12 recruited patients, 3 (25%) presented either a stable disease (SD) or a partial response (PR). No direct link has been established between toxicity (grade 4 neutropenia) and drug exposure. However, responders to treatment were those presenting a higher vinorelbine area under the curve (p=0.02). Tumor assessments were used to adjust efficacy parameters. The model was used to simulate alternative metronomic scenarios targeting higher efficacy with similar toxicity. Simulations showed that the initial proposed schedule was the most appropriate in terms of efficacy/toxicity balance. Its efficacy will be evaluated in the second part of the study.

This trial can be considered as a proof-of-concept study demonstrating the feasibility to run a computational-driven protocol to ensure an optimal efficacy/toxicity balance in cancer patients.

Citation Format: Diane-Charlotte Imbs, Raouf El Cheikh, Joseph Ciccolini, Raphaël Serre, Fabrice Barlesi, Dominique Barbolosi. A computational driven protocol of metronomic oral vinorelbine in metastatic non small cell lung 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 4062. doi:10.1158/1538-7445.AM2017-4062
Abstract 4063: Design and synthesis of indole derivatives as ABCG2 mediated resistance reversal agents
Authors: Cai Chao-Yun , Zhai Hong , Lei Zi-Ning , Chen Bao-Li , Du Zhao-Yi , Zhang Yun-Kai , Wang Yi-Jun , Wang Bo , Chen Zhe-Sheng
Published: July 2017

Multidrug resistance is a phenomenon that cancer cells become resistant to anticancer drugs. Multidrug resistance has been shown to be related with the overexpression of ATP-binding (ABC) transporters, including ABCG2. Therefore, ABCG2 is a therapeutic target for the development of multidrug resistance reversal agents. Fumitremorgin C, with an indole group which is an important pharmacophore in many drugs, is a known ABCG2 inhibitor. Inspired by this, we synthesized two series of indole derivatives as ABCG2 mediated resistance reversal agents. With MTT assay, we found that two compounds from the two series of indole derivatives respectively exhibited good reversal activity against ABCG2. The two compounds did not significantly alter the expression of ABCG2 in ABCG2-overexpressing cell lines and they stimulated the basal ATP hydrolysis of ABCG2.

Citation Format: Chao-Yun Cai, Hong Zhai, Zi-Ning Lei, Bao-Li Chen, Zhao-Yi Du, Yun-Kai Zhang, Yi-Jun Wang, Bo Wang, Zhe-Sheng Chen. Design and synthesis of indole derivatives as ABCG2 mediated resistance reversal agents [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 4063. doi:10.1158/1538-7445.AM2017-4063