bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5573: A high-affinity Optide (optimized peptide) inhibitor of the Hippo pathway’s YAP-TEAD interaction
Authors: Crook Zachary R. , Bradley Philip , Sevilla Gregory , Friend Della , King Chris , Mhyre Andrew , Strong Roland , Baker David , Olson James M.
Published: July 2017

The HIPPO pathway plays a critical role in contact inhibition, a pathway that is commonly dysregulated in many human cancers (including liver, colon, ovarian, and lung). The signaling pathway culminates in the intranuclear interaction of the transcriptional co-activator YAP and the transcription factor TEAD. This is representative of a number of cancer driving pathways that have proven nearly impossible to drug, as they are mediated by intracellular protein-protein interactions. High throughput screening campaigns with small molecule libraries have failed to provide specific, high affinity binders capable of disrupting larger protein-protein interfaces (such as YAP-TEAD), while at the same time, antibodies cannot penetrate the cell membrane to access cytosolic and nuclear targets. Optides are small disulfide-knotted peptides (knottins) that are large enough to interfere with protein-protein interactions, but small enough to access compartments beyond the reach of antibodies. Examples include the calcines, activators of sarcoplasmic reticulum ryanodine receptors, and BLZ-100, a knottin-fluorophore conjugate that is capable of accumulating in a wide range of tumor types. Using the computational design software Rosetta, we created a library of Optides designed to interact with TEAD in locations that overlap YAP binding. Mammalian surface display screening against soluble TEAD yielded a candidate (Hit1) that binds TEAD with nanomolar affinity and inhibits YAP binding. Affinity maturation, using site saturation mutagenesis, produced an improved sub-nanomolar variant (IV1) with potent YAP inhibition. This variant was also found to be highly resistant to reduction and proteolysis, crucial for a disulfide-knotted peptide with a cytosolic target in the proteinase-rich tumor milieu. With this highly potent YAP inhibitor, efforts are now focused on cell penetration and biodistribution with the long-term goal of advancing a clinical development candidate.

Citation Format: Zachary R. Crook, Philip Bradley, Gregory Sevilla, Della Friend, Chris King, Andrew Mhyre, Roland Strong, David Baker, James M. Olson. A high-affinity Optide (optimized peptide) inhibitor of the Hippo pathway’s YAP-TEAD interaction [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 5573. doi:10.1158/1538-7445.AM2017-5573
Abstract 5576: BPM 31510, a clinical stage candidate demonstrates potent anti-tumor effect in an immune-competent syngeneic pancreatic cancer model
Authors: Kazerounian Shiva , Sarma Aishwarya , Gaur Nidhi , Nastke Maria D. , Dadali Tulin , Diers Anne R. , Gesta Stephane , Vishnudas Vivek K. , Sarangarajan Rangaprasad , Narain Niven R.
Published: July 2017

BPM 31510, a clinical stage nanodispersion of ubidecarenone, demonstrates anti-tumor effects by eliciting an anti-Warburg metabolic switch in cancer. Previous studies in an immune compromised PaCa2 xenograft model has unequivocally demonstrated significant efficacy of BPM 31510 on tumor volume and survival. The fundamental property of BPM 31510 to influence mitochondrial bioenergetics and the recognized interplay between T cell metabolism and maturation prompted investigation into the effects of BPM 31510 on T lymphocyte functions in eliciting anti-cancer effects. In this study BPM 31510 selectively influenced activation and maturation of T cells in murine peripheral blood mononuclear cells (PBMCs). Moreover, in addition to determining changes in the CD3+ population, changes in surface expression of PD-1 and CTLA4 along with the IFN-γ secretion were examined. Murine cancer cell lines exposed to BPM 31510 were associated with variable sensitivity with highly metabolic tumor types being most sensitive. Next, the anti-cancer activity of BPM 31510 in an in vivo immunocompetent syngeneic Pan02 rodent model was investigated. Murine Pan02 pancreatic cancer cells were implanted subcutaneously into C57BL/6 mice. Tumors with mean volume of 80 mm3 were treated twice a day with vehicle control or BPM 31510 at 25, 50, 100 mg/kg, administered intraperitoneal. Tumor volumes were measured every 4 days. At day 21 post treatment, tumors were harvested and analyzed for the level of infiltrating immune cells by immunofluorescent staining with CD8+ for T cells and F4/80 for tumor macrophages. These results demonstrate a dose-dependent reduction in tumor volume following 21 days of BPM 31510 treatment. In summary, BPM 31510 exerts potent anti-tumor effects through its dual function of modulating tumor cell metabolism and potentially influencing immune check-point to improve overall survival outcomes.

Citation Format: Shiva Kazerounian, Aishwarya Sarma, Nidhi Gaur, Maria D. Nastke, Tulin Dadali, Anne R. Diers, Stephane Gesta, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. BPM 31510, a clinical stage candidate demonstrates potent anti-tumor effect in an immune-competent syngeneic pancreatic cancer model [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 5576. doi:10.1158/1538-7445.AM2017-5576
Abstract 5587: Novel monoclonal antibodies block N-glycosylation Sites of the MUC16 ectodomain in ovarian carcinoma
Authors: Thapi Dharmarao , Fernandez-Tejada Alberto , Axelrod Abraham J. , Rosales Nestor , Yan Xiujun , Stasenko Marina , Thapi Sahityasri , Wang Amy , Danishefsky Samuel J. , Spriggs David R.
Published: July 2017

The MUC16 gene encodes the CA125 antigen that imparts a negative survival impact on patients with high-grade serous ovarian cancer. Short elements from the C-terminal region of MUC16 transform 3T3 cells. Introduction of the 114 amino acid C-terminal portion of MUC16 promotes invasive growth in xenograft models. This effect is dependent on N-glycosylation sites of MUC16 and interactions present on the cell surface. Mechanisms by which the ectodomain promotes MUC16 oncogenic behaviors, the function of N-glycosylation and the implicated cell surface receptors are not fully delineated.

To unravel the complex MUC16 cancer biology, in this study we investigated the glycosylation-based mechanisms and potential partners involved in MUC16 oncogenic effects, and show that site-specific glycosylation of MUC16 plays a key role in mucin-related transformation by mediating complex cell surface interactions. These effects are mediated through MGAT5-dependent N-glycosylation of two proximal N- glycosylation sites within the 58 amino acid retained MUC16 ectodomain acting in combination with Galectin-3 and growth factor receptors. Neither N- nor O-glycosylation sites in the more distally located MUC16 regions can functionally substitute for those two sites. The tumor-enhancing properties of MUC16 depend on co-localization of MUC16, Galectin-3, and growth factors receptors on lipid rafts. Loss of either Galectin-3 expression or the glycosylation enzyme MGAT5 completely abrogates MUC16 tumor promotion. Using synthetic glycopeptides, we have developed novel monoclonal antibodies (mAbs) directed at the crucial ectodomain N-glycosylation sites to inhibit the glycosylation- dependent effects of MUC16 on metastasis and invasion. These novel antibodies were characterized in vitro and in vivo studies using various ovarian cancer cell lines. All of the antibodies against the N- glycosylation sites closest to the cell membrane of MUC16 block Galectin-3-mediated polymerization to cell surface signaling molecules and inhibit the tumor-promoting effects of MUC16. The implications are broad: both for the biological understanding of cancer mucin biology and for potential therapeutic strategies.

Citation Format: Dharmarao Thapi, Alberto Fernandez-Tejada, Abraham J. Axelrod, Nestor Rosales, Xiujun Yan, Marina Stasenko, Sahityasri Thapi, Amy Wang, Samuel J. Danishefsky, David R. Spriggs. Novel monoclonal antibodies block N-glycosylation Sites of the MUC16 ectodomain in ovarian 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 5587. doi:10.1158/1538-7445.AM2017-5587
Abstract 5594: High throughput combinatorial profiling of checkpoint inhibitor antibodies on the iQue® Screener PLUS
Authors: Hasaka Tom
Published: July 2017

Checkpoint inhibitors have become valuable immuno-modulatory targets in the advancement of cancer treatment. Looking for the synergy between new checkpoint inhibitor antibodies and known inhibitors is an important aspect of this research. The iQue Screener PLUS platform is a powerful tool to simultaneously assess these interactions in a single well of a microtiter plate. ForeCyt® software provides plate-level analytics and high content visualization to generate deep insight rapidly. Using a mixed lymphocyte reaction (MLR) model, we profiled potential synergies of several known checkpoint inhibitors antibodies. Responses of PD-1, PD-L1, and CD73 inhibitors both individually, and in combination with CTLA-4 inhibitors, were assessed for proliferation, viability and cytokine secretion simultaneously in the same well. MultiCyt® cell-based and bead-based reagents were used for this analysis. Synergies ranging from 2-10 fold increase over CTLA-4 alone were observed in the secretion of TNFα and IL-1β. Results were obtained and analysis completed in a 384-well plate in 30 minutes. In conclusion, this study highlights the power of the iQue Screener PLUS platform to rapidly characterize multiple endpoints and the ForeCyt software to provide high content visualization that reveals actionable insights.

Citation Format: Tom Hasaka. High throughput combinatorial profiling of checkpoint inhibitor antibodies on the iQue® Screener PLUS [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 5594. doi:10.1158/1538-7445.AM2017-5594
Abstract 5598: Adenosine signaling through A2AR limits the efficacy of anti-CTLA4 and chemotherapy in preclinical models
Authors: Ho Po , Hsieh Meng-Yin , Hotson Andrew , Miller Richard , McCaffery Ian , Willingham Stephen
Published: July 2017

Elevated levels of extracellular adenosine within the tumor microenvironment create an immunosuppressive niche that promotes tumor growth and metastasis. Adenosine signaling via the A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and has been shown to limit the efficacy of immunotherapies such as anti-PD-L1 or anti-PD-1 monoclonal antibodies (mAbs).

CPI-444 is a potent and selective oral A2AR antagonist that is currently being investigated in Phase 1/1b clinical trials alone and in combination with an anti-PD-L1 antibody (Atezolizumab) in selected solid tumors. New preclinical data suggests that combining CPI-444 with anti-CTLA-4 mAbs as well as chemotherapy treatment are also promising therapeutic strategies in solid tumors, suggesting a broad role for adenosine as an immune suppressive mechanism.

The efficacy of CPI-444 + anti-CTLA-4 mAb treatment was evaluated in MC38 and CT26 syngeneic mouse tumor models. In CT26, combination treatment eliminated established tumors in up to 90% of mice approximately 2 weeks after treatment was initiated. In MC38, combination CPI-444 and anti-CTLA-4 mAb treatment prolonged survival of 80% of mice compared to only 40% of mice that received CPI-444 or anti-CTLA-4 mAbs alone. The effect of CPI-444 + anti-CTLA-4 treatment on T-cell proliferation, T cell activation, and TREG function will be discussed.

Chemotherapy releases adenosine and ATP into the tumor microenvironment (TME). Multiple chemotherapies have also been shown to up-regulate the ecto-enzymes CD39 and CD73 that produce adenosine and further suppress immune function. In the MC38 model, CPI-444 treatment synergized with doxorubicin and eliminated established tumors 80% of treated mice. CPI-444 treatment was also synergistic with cyclophosphamide, inhibiting the growth of RENCA tumors, a model that is considered resistant to chemotherapy. Ongoing studies are evaluating the effect of CPI-444 + chemotherapy on tumor infiltrating lymphocyte localization, activation, and expression of CD73 and CD39.

These results suggest that blockade of the adenosine signaling pathway may be vital for enhancing anti-tumor responses in solid tumors that show an incomplete response to anti-CTLA4 therapy or chemotherapy.

Citation Format: Po Ho, Meng-Yin Hsieh, Andrew Hotson, Richard Miller, Ian McCaffery, Stephen Willingham. Adenosine signaling through A2AR limits the efficacy of anti-CTLA4 and chemotherapy in preclinical models [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 5598. doi:10.1158/1538-7445.AM2017-5598
Abstract 5611: Use of ecto-domain vimentin to target brain cancers
Authors: Babic Ivan , Mukthavaram Rajesh , Jiang Pengfei , Glassy Eric , Nomura Natsuko , Pingle Sandeep , Glassy Mark C. , Kesari Santosh
Published: July 2017

Pritumumab is a natural human IgG1 kappa antibody derived from a regional draining lymph node of a patient with cervical carcinoma. The recognized antigen is an altered form of vimentin, called ecto-domain vimentin (EDV), that is expressed on the cell surface of epithelial tumor cells. For brain cancers, immunohistological analysis showed the binding of pritumumab to EDV to be restricted to tumor cells and not normal cells and tissues suggesting EDV may be a useful biomarker. A recombinant version of the mAb was made using the GPEx® system in CHO cells that showed comparable binding activity by flow cytometry, immunohistochemical analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC; EC50 of 39.6ng/ml) activity. Further studies include establishing xenograft models with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth of both brain cancer stem cells as well as pancreatic cancer in nude mice but not in SCID mice suggesting an active immune response is necessary for optimal treatment. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Overall, these data together suggest that EDV is a suitable target and biomarker for brain cancers.

Citation Format: Ivan Babic, Rajesh Mukthavaram, Pengfei Jiang, Eric Glassy, Natsuko Nomura, Sandeep Pingle, Mark C. Glassy, Santosh Kesari. Use of ecto-domain vimentin to target brain 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 5611. doi:10.1158/1538-7445.AM2017-5611
Abstract 5627: Uncovering novel signaling pathways that mediate tumor cell killing by T effector cells
Authors: Kuilman Thomas , Vredevoogd David , Peeper Daniel S.
Published: July 2017

Recent advances in the understanding of tumor immunology have led to the development of various immunotherapeutic strategies, some of which show unprecedented clinical efficacies. Two of the most effective treatments, ipilimumab and pembrolizumab / nivolumab target the immune checkpoints factors CTLA-4 and PD-1, respectively. While not all patients respond to immunotherapy, those that do frequently show long-lasting clinical responses. These accomplishments have led to a surge in research focusing on the question how the immune system can be stimulated for more common durable therapy responses.

A current focus of tumor immunological research is the identification of mechanisms allowing cancer cells to evade recognition and/or killing by the immune system while facing immunotherapy. For example, abrogation of interferon-γ signaling pathway has been shown to result in escape of immune surveillance after pembrolizumab treatment. However, it is likely that other tumor cell-intrinsic mechanisms play a role, too.

To uncover novel pathways that are involved in resistance to immune checkpoint inhibitors, we aim to identify transcriptional changes in tumor cells that result from effector T-cell exposure. To this end, we established a co-culture platform of a panel of Mart1-expressing melanoma cell lines with primary CD8+ T cells expressing a matching TCR. The cell lines display varying degrees of sensitivity to T cells, allowing us to bioinformatically uncover differential expression depending on this range of sensitivities. Rather than concentrating on single genes, we have focused on pathways, gene sets and ontologies. These analyses have been integrated with various publically available transcriptomic data sets to identify a set of mechanisms that may contribute to T-cell mediated killing. We will systematically abrogate key factors using CRISPR technology in our co-culture platform and assess whether this affects T-cell mediated killing. Our ultimate goal is to test whether these factors, or their signaling pathways, are involved in resistance to immunotherapy in patients, and to develop new strategies to prevent immunotherapy resistance.

Note: This abstract was not presented at the meeting.

Citation Format: Thomas Kuilman, David Vredevoogd, Daniel S. Peeper. Uncovering novel signaling pathways that mediate tumor cell killing by T effector 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 5627. doi:10.1158/1538-7445.AM2017-5627
Abstract 5629: Modeling cyclophosphamide induced ovarian toxicity in a novel translational canine model
Authors: Basu Puja , Egbert Rebecca , Pasternak Evan , Petroff Brian , Songsasen Nucharin
Published: July 2017

Reproductive failure following chemotherapy and/or ionizing radiation is a major adverse event, for which there are no established preventive measures. In previous studies, our laboratory has documented that the selective estrogen receptor modulator, tamoxifen (TAM) is effective in both prevention and reversal of ovarian toxicity from cyclophosphamide (CTX) in rodent models. To validate the effectiveness of this approach in a more relevant model system, we have developed a canine ovarian explant tissue model system. Dogs present with many cancers spontaneously and their pathology and treatment approaches mirror those of the human cancers. Thus, canine cancers and their treatments are excellent translational models for human diseases. Furthermore, dogs share the same environment as humans and have longer life spans than rodents. Our laboratory has recently established a dog ovarian explant tissue model system using fresh ovaries collected at the time of ovariohysterectomy. In the current study, we hypothesized that ovarian toxicity induced by CTX will be reduced by co-administration of TAM. Using this system, freshly prepared ovarian cortical tissue sections are cultured in the presence of TAM and CTX. Ovarian tissue viability is assessed by primordial and primary follicle morphometry, where the number of primordial, primary and apoptotic follicles were counted in 9 sections of 1 mm3 of ovarian cortical tissue from each dog, using CTX (at 0, 1 and 10µM) concentrations combined with TAM (0 and10µM). The number and percentage of each follicle was assessed. In addition, granulosa cells proliferation is assessed using bromodeoxyuridine (BrdU) incorporation. Further, we show that, as in women, anti-mullerian hormone (AMH) can be used as an indicator of follicle reserves in the dog, thereby establishing their use as an in-vivo monitoring system to test ovarian viability in future clinical studies. This study is intended to underpin a subsequent canine clinical trial testing TAM as a countermeasure against infertility from cancer treatment in dogs.

Note: This abstract was not presented at the meeting.

Citation Format: Puja Basu, Rebecca Egbert, Evan Pasternak, Brian Petroff, Nucharin Songsasen. Modeling cyclophosphamide induced ovarian toxicity in a novel translational canine model [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 5629. doi:10.1158/1538-7445.AM2017-5629
Abstract 5664: PLAG prevents the loss of circulating neutrophils in the chemotherapy induced neutropenia model
Authors: Kim Yong-Jae , Jung Jinseon , Lee Ha-Reum , Sohn Ki-Young , Kim Heung-Jae , Yoon Sun Young , Kim Jae Wha
Published: July 2017

Chemotherapy-induced neutropenia (CIN) is a common dose-limiting toxicity in cancer patients undergoing cytotoxic chemotherapy. It is generally understood that myelosuppression is the major mechanism resulting in CIN. However, an excessive extravasation (diapedesis) of neutrophils in response to tissue damage-caused by anti-cancer agents is also a possible mechanism. In this study, we have investigated the effects of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) for the maintenance of circulating neutrophil in the gemcitabine induced neutropenia. In CIN mouse model using anticancer drugs, PLAG effectively protected the loss of circulating neutrophils as similar to CXCR2 antagonist, reparixin. And succeeded in vitro neutrophil migration assay also revealed that chemo-attractive activity of neutrophil induced by treatment of anticancer drugs was completely interrupted by PLAG addition. Chemokine (C-X-C motif) ligand 2 (CXCL2) and CXCL8 were effectively induced by treatment of gemcitabine with dose dependent manner in monocyte cell lines and mainly mediated via the signal transducer and activator of transcription 3 (STAT3) signaling pathway. PLAG effectively attenuated gemcitabine induced CXCL2 and CXCL8 expression via interrupting STAT3 activity and also regulated gemcitabine induced macrophage 1 antigen (MAC-1) and L-selectin expression, which means that PLAG interrupted the acceleration of neutrophil extravasation under CIN. Also we have verified that PLAG analogues and metabolites were not affected on neutrophil migration unlike PLAG, suggesting that acetylated diacylglycerol has a key role to regulate on neutrophil migration. Collectively, modulating effect of PLAG on excessive neutrophil extravasation by chemotherapy via attenuating STAT3/CXCL2(8) pathway could be used as very powerful regimen for preventing of neutropenia during diverse chemotherapy.

Citation Format: Yong-Jae Kim, Jinseon Jung, Ha-Reum Lee, Ki-Young Sohn, Heung-Jae Kim, Sun Young Yoon, Jae Wha Kim. PLAG prevents the loss of circulating neutrophils in the chemotherapy induced neutropenia model [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 5664. doi:10.1158/1538-7445.AM2017-5664
Abstract 5672: Quantitative cell-based assays for characterization of antibodies targeting co-stimulatory immune checkpoint receptors
Authors: Gilden Julia K. , Wang Jun , Beck Michael , Grailer Jamison , Hartnett Jim , Fan Frank , Cong Mei , Cheng Zhe-jie Jey
Published: July 2017

The human immune system is regulated by a broad network of co-inhibitory and co-stimulatory receptors that control the type, scale, and duration of immune responses. These receptors are now recognized as promising immunotherapy targets for the treatment of many cancers and autoimmune diseases. Immunotherapies that block co-inhibitory receptors such as PD-1 and CTLA-4 are showing unprecedented efficacy in the treatment of some tumors, and have generated interest in the characterization of additional immunotherapy targets that may broaden the number of patients who can be helped by these drugs. Immunotherapy research programs are now exploring a wide range of both co-inhibitory (e.g. LAG-3, TIGIT, Tim-3) and co-stimulatory (e.g. GITR, 4-1BB, OX40) receptors, individually and in combination. Quantitative and reproducible functional bioassays are essential tools in the development of biologics for cancer immunotherapy. Most existing assays rely on primary cells and suffer from lengthy protocols and high day-to-day and donor-to-donor variability. These approaches are cumbersome, error-prone, and do not produce data of the quality required for drug development in a quality control environment. To address this, we have developed a suite of cell line-based reporter bioassays for co-stimulatory immune checkpoint targets including GITR, 4-1BB, OX40, and CD40. In these assays, stable cell lines express luciferase reporters driven by response elements under the precise control of intracellular signals mediated by each co-stimulatory receptor. These bioassays reflect mechanisms of action for drug candidates designed for each co-stimulatory receptor and demonstrate high specificity, sensitivity and reproducibility. Reporter-based bioassays can serve as powerful tools in immunotherapy drug development for antibody screening, potency testing and stability studies.

Citation Format: Julia K. Gilden, Jun Wang, Michael Beck, Jamison Grailer, Jim Hartnett, Frank Fan, Mei Cong, Zhe-jie Jey Cheng. Quantitative cell-based assays for characterization of antibodies targeting co-stimulatory immune checkpoint receptors [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 5672. doi:10.1158/1538-7445.AM2017-5672
Abstract 5674: Cancer immunogenomics for the development of personalized ovarian cancer vaccine
Authors: Want Muzamil Y. , Tsuji Takemasa , Koya Richard , Battaglia Sebastiano
Published: July 2017

Ovarian cancer (OC) is the fifth leading cause of cancer death in the United States with approximately 20,000 women being diagnosed every year. Since OC development is mainly asymptomatic, patients are often diagnosed at late stage and present local and distal metastases. This offers a clinical challenge, as roughly 70% of the patients develop chemoresistant disease. With the advent of cancer immunogenomics it is now possible to identify tumor specific mutations, or neoantigens/neoepitopes, that can be exploited for the development of personalized T cell therapies via DC vaccines or adoptive cell therapy (ACT). We therefore hypothesize that tumor cells isolated from malignant ascites reflect the genomic landscape of the primary tumor, offering a unique opportunity to identify clinically actionable tumor neoantigens. We utilized primary tumor, tumor cells isolated from malignant ascites and metastatic samples to create patient-derived xenografts (PDX) mice and to interrogate the tumor mutational landscape. Somatic mutations were identified by whole exome sequencing (WES) and RNASeq analysis in tumor samples, using PBMCs as germline control. In parallel 20 PDX of ovarian cancer were established in NSG mice by injecting 2×106 cells/mouse subcutaneously (SQ, n=10) and intraperitoneally (IP, n=10). Tumor growth was monitored weekly via caliper measurement for mice injected SQ and via abdominal circumference for mice injected IP. Mutational analysis and prediction of high affinity peptides was done using bioinformatics approach. We have utilized different algorithms that are based on Artificial Neural Networks or published motifs for predicting binding of 8-11 mer peptides to number of Human MHC alleles (HLA) and out of 168 mutated peptides identified, 30 were found to have high affinity for different HLAs compared to wild type peptide. Mutated and wild type peptides were pulsed into dendritic cells and co-cultured with CD8+ T cells for 20 days. Validation of stimulated CD8+ T cells by these mutated peptides using ELISPOT is underway. TCR specific for neoantigen from CD8+T cells will be identified and cloned using retroviral vectors. Transfected CD8+ T cells with neoantigen specific TCR will be confirmed by co-culturing them with dendritic cells pulsed with wild type and mutated peptides. Further engineered CD8+ T cells specific for neoantigens will be tested for adoptive cell therapy in the established PDX models of ovarian cancer.

Citation Format: Muzamil Y. Want, Takemasa Tsuji, Richard Koya, Sebastiano Battaglia. Cancer immunogenomics for the development of personalized ovarian cancer vaccine [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 5674. doi:10.1158/1538-7445.AM2017-5674
Abstract 5675: A novel PI3K inhibitor suppresses tumor progression by immune modulation
Authors: Xu Zusheng , Lou Yangtong , Qu Yingying , Wang Wei , Wu Xiaodong , Ge Xiaomei , Gu Ying , Zhang Yingjia , Zhou He
Published: July 2017

Phosphoinositide 3-kinase delta (PI3Kδ) inhibitors have shown anti-tumor efficacy in leukemia in the clinic and one such compound, idelalisib, was approved by the FDA for the treatment of relapsed chronic lymphocytic leukemia (CLL) in 2014. A novel PI3Kδ inhibitor, YY-20394, was developed with specific selectivity against PI3Kδ and inhibited PI3Kδ expressing human tumor cells growth both in vitro and in vivo. In addition, our previous data have shown YY-20394 significantly inhibited primary tumor growth in immune-competent mice with 4T1 and CT26 tumors, as well as 4T1 lung metastasis with dose dependency. The anti-tumor efficacy of YY-20394 was largely mediated by T cells. More importantly, YY-20394 synergistically enhanced the anti-tumor efficacy of anti-PD-L1 antibody in CT26 model and achieved long-term immune memory which is specific for CT26 tumors, but not for unrelated A20 tumors. In vitro mechanistic studies showed YY-20394 significantly inhibited T cell differentiation into Treg both in mouse splenocyte and in human primary CD4+ T cells, and was especially potent inhibiting their IL-10 secretion in vitro. Analyses of tumors treated with YY-20394 reviewed that YY-20394 decreased the percentage of Treg in the tumor infiltrating leukocytes, as well as IL-10 secretion. It also showed some effects on the myeloid populations in the tumors. These data suggest the YY-20394, a novel PI3Kδ inhibitor, represents a promising and safe immune modulator and shows great potential as a cancer immuno-therapeutics.

Citation Format: Zusheng Xu, Yangtong Lou, Yingying Qu, Wei Wang, Xiaodong Wu, Xiaomei Ge, Ying Gu, Yingjia Zhang, He Zhou. A novel PI3K inhibitor suppresses tumor progression by immune modulation [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 5675. doi:10.1158/1538-7445.AM2017-5675
Abstract 5689: Identify tissue-of-origin in cancer cfDNA by whole genome sequencing
Authors: Liu Yaping , Reed Sarah , Choudhury Atish D. , Parsons Heather A. , Stover Daniel G. , Ha Gavin , Gydush Gregory , Rhoades Justin , Rotem Denisse , Freeman Samuel , Adalsteinsson Viktor , Kellis Manolis
Published: July 2017

Cell free DNA (cfDNA) has been shown to be an emerging non-invasive biomarker to monitor tumor progression in cancer patients. Elevated cfDNA has been found not only from tumors, but also from normal tissues. Thus, the identification of cfDNA’s tissue-of-origin is critical to understand the mechanism of cfDNA release and tumor progression. Recent efforts to identify cfDNA’s tissue-of-origin begin to utilize cfDNA’s epigenomic status, such as DNA methylation and nucleosome spacing. However, both of these methods have limitations: (1) For nucleosome positioning, lack of reference nucleosome maps in different tumor and normal tissues has limited its application to tissue-of-origin deconvolution; (2) For DNA methylation, large DNA degradation during whole genome bisulfite sequencing (WGBS) library preparation, even with current low-input DNA technology, is still the major hurdle for its clinical application, although extensive DNA methylation studies by WGBS in tumor and normal tissues during the last decade have provided many reference maps. Very recently, a pioneer study showed significant differences between DNA fragment lengths of methylated and unmethylated cfDNA. Taking advantage of this experimental observation, we developed a machine learning approach to infer the base pair resolution DNA methylation level from fragment size information in whole genome sequencing (WGS). The predicted DNA methylation, from not only high coverage but also dozens of ultra-low-pass WGS (ULP-WGS), showed high concordance with the ground truth DNA methylation level from WGBS in the same cancer patients. Furthermore, by using hundreds of WGBS datasets from different tumor and normal tissues/cells as the reference map, we deconvoluted cfDNA’s tissue-of-origin status by inferred DNA methylation level at ULP-WGS from thousands of breast/prostate cancer samples and healthy individuals. The cfDNA’s tissue-of-origin status in cancer patients showed high concordance with confirmed metastasis tissues from physicians. Interestingly, some clinical information, such as cancer grades/stages, seemed to be correlated with cfDNA’s tissue-of-origin status. Overall, our methods here pave the road for cfDNA’s application in clinical diagnosis and monitoring.

Citation Format: Yaping Liu, Sarah Reed, Atish D. Choudhury, Heather A. Parsons, Daniel G. Stover, Gavin Ha, Gregory Gydush, Justin Rhoades, Denisse Rotem, Samuel Freeman, Viktor Adalsteinsson, Manolis Kellis. Identify tissue-of-origin in cancer cfDNA by whole genome sequencing [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 5689. doi:10.1158/1538-7445.AM2017-5689
Abstract 5692: Cross-platform detection and quantification of actionable mutations in cell-free DNA shows high concordance and correlation between next-generation sequencing and droplet digital PCR
Authors: Ulrich Bryan C. , Nagy Rebecca J. , Odegaard Justin I. , Lanman Richard B. , Oxnard Geoffrey R. , Paweletz Cloud P.
Published: July 2017

Background: Non-invasive genotyping of cell-free DNA (cfDNA) provides physicians with the ability to identify genomic alterations, evaluate response to treatment, and uncover potential mechanisms of resistance. Here, we analyze the concordance of next-generation sequencing (NGS) and droplet digital PCR (ddPCR) in detecting and quantifying actionable mutations from cfDNA of lung, colorectal, and breast cancer patients.

Methods: We studied a cohort of 221 advanced cancer cfDNA samples with completed Guardant360 digital sequencing results. Variant allele frequencies (% AFs) ranged from less than 0.2% to greater than 90% for common driver and resistance mutations. Blinded to plasma NGS results, we performed ddPCR using our validated assays for EGFR L858R, exon19 del, T790M (113 presumed lung cases); BRAF V600E, KRAS G12X (77 colorectal cases); ESR1 D538G, Y537C and PIK3CA H1047R (31 breast cases). Following unblinding, qualitative and quantitative accuracy were compared.

Results: Qualitative results (Table 1) show a high concordance between NGS and ddPCR, with 98.3% (576/586) of all assays run being concordant. 9/22 of samples that had an AF below 0.4% were discordant, while only one sample above 0.4% AF was discordant. AFs calculated by NGS and ddPCR were strongly correlated by linear regression (R between 0.951 and 0.999). Notably, the slope of the ddPCR AF versus NGS AF graph for EGFR ex19del was 0.725, which improved to 0.95 with the adoption of an alternate calling method.

Conclusions: This interlaboratory comparison of actionable mutations in cfDNA from lung, colorectal, and breast cancers by orthogonal genotyping platforms demonstrates a substantial concordance in detection and quantification between NGS and ddPCR. Our cross-platform study establishes the feasibility of standardization of qualitative and quantitative measurements of cfDNA-detected alterations.

Table 1LungEGFR L858REGFR Ex19 delEGFR T790M(n=113)G360+G360-G360+G360-G360+G360-ddPCR+580510530ddPCR-055458456BreastESR1 Y537CESR1 D538GPIK3CA H1047R(n=31)G360+G360-G360+G360-G360+G360-ddPCR+1022020ddPCR-03009029ColorectalBRAF V600EKRAS G12X(n=78)G360+G360-G360+G360-ddPCR+220571ddPCR-055118

Citation Format: Bryan C. Ulrich, Rebecca J. Nagy, Justin I. Odegaard, Richard B. Lanman, Geoffrey R. Oxnard, Cloud P. Paweletz. Cross-platform detection and quantification of actionable mutations in cell-free DNA shows high concordance and correlation between next-generation sequencing and droplet digital PCR [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 5692. doi:10.1158/1538-7445.AM2017-5692
Abstract 570: Transparent tumor tomography (T3): 3D spatial immunoanalysis for PD-L1 immune checkpoint blockade therapy
Authors: Lee Steve Seung-Young , Bindokas Vytautas P. , Kron Stephen J.
Published: July 2017

Untangling the complexity of programmed death-ligand 1 (PD-L1) expression within a heterogeneous tumor microenvironment is an urgent challenge in PD-1/PD-L1 immune checkpoint blockade therapy. Here, we address this challenge with a method, termed transparent tumor tomography (T3), facilitating three-dimensional (3D) visualization and spatial analysis of distributions of multiple biomarkers regarding to cancer cells, vasculature, and immune cells in context in the tumor microenvironment. With T3 analysis of transgenic mouse mammary tumors immunostained against Her2, CD45, Ki-67, CD31, and PD-L1, we reveal that PD-L1 expression within the tumor microenvironment is highly adaptable for efficiently preventing immune cell infiltration into the tumor. Stronger correlation of Her2 and PD-L1 expression in the tumor periphery where has a high CD45+ immune infiltrate density is determined by tumor-wide analysis. Also, tomographic analysis shows blood vessels expressing PD-L1 in the tumor core, where PD-L1 expression is lower. Furthermore, high-resolution T3 image localizes PD-L1 expression to a region between the endothelium and the surrounding smooth muscle cells in blood vessels. We investigate spatial pharmacokinetics of anti-PD-L1 antibody in the whole mouse mammary tumor in the context of hypoxia, CD31+ blood vessels, and target PD-L1+ cells. The 3D anti-PD-L1 antibody distribution is fit to a two-compartment pharmacokinetic model, yielding estimated distribution half-life of 4.7 min and terminal half-life of 2.5 days. We also evaluate anti-tumor immune responses after PD-L1 blockade therapy using T3. We observe broad distribution of tumor infiltrating CD3+CD8+ cytotoxic T cells in 3D tumor section following combination therapy of radiation and anti-PD-L1 antibody compared to PBS, anti-PD-L1 antibody alone, or radiation alone treatment group. Moreover, we apply T3 for immunoanalysis of whole core needle biopsy. We spatially map PD-L1 expression and granzymeB-producing CD3+CD8+ cytotoxic T cells in pre-treatment and in-treatment core needle biopsies at cellular resolution and in three dimensions. Meanwhile, T3 analysis is nondestructive, allowing secondary analysis by IHC and/or IF. We anticipate that T3 can be applied broadly to facilitate preclinical studies of immunotherapy and also find use in spatial, multiparameter analysis of patient biopsies, particularly to improve predictive testing and analysis of immune responses to tumor immunotherapy.

Citation Format: Steve Seung-Young Lee, Vytautas P. Bindokas, Stephen J. Kron. Transparent tumor tomography (T3): 3D spatial immunoanalysis for PD-L1 immune checkpoint blockade therapy [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 570. doi:10.1158/1538-7445.AM2017-570
Abstract 5701: Innovative approach for isolation of circulating cell-free DNA from liquid biopsies
Authors: Malykh Andrei , Evtushenko Vladimir
Published: July 2017

We developed a new approach for isolation of circulating cell-free DNA (cfDNA) from liquid biopsies (plasma or serum). Our technology is based on using the proprietary bi-functional substance X (SubX®) that binds DNA under physiological conditions (e.g. directly in biological liquids), followed by adsorption of [DNA- SubX®] complex on a solid phase matrix. Since SubX®) captures DNA via phosphate groups it allows for elimination of bias related to both AT/CG content and DNA fragments length, thus improving extraction efficacy and accuracy of downstream applications. All currently available commercial kits are [silica-chaotropic salt]-based and exhibit bias for either short or long DNA fragments, as well as for the GC content. In addition, such systems require at least 4- fold dilution of starting volume of bio liquid with concentrated (~5.0 M) guanidine thiocyanate (GTC) to keep resulted GTC concentration at about least 3.0 M. Thus, already low concentrations of cfDNA are significantly reduced and may negatively influence extraction efficacy of low abundance DNA fragments. Since our method does not require chaotropic salt for DNA capture, no dilution of starting material (and thus cfDNA) takes place. DNA-binding matrix SubX® allows either scaling the procedure to either large volumes of starting material or extra low volumes and adaptation of the protocol for 96/384 well format. Separation of cfDNA from the bulk of proteins occurs in a single vortex-spin step without employing chaotropic agents thus speeding-up isolation procedure and reducing the costs. The whole procedure can be completed within an hour and cfDNA is eluted in small volume of TE buffer. Average DNA yield from 0.2 ml of double centrifuged blood plasma from normal donors, cancer and multiple sclerosis patients was 7.5±4.2 ng (n=96).

Citation Format: Andrei Malykh, Vladimir Evtushenko. Innovative approach for isolation of circulating cell-free DNA from liquid biopsies [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 5701. doi:10.1158/1538-7445.AM2017-5701
Abstract 5710: PGRMC1 is a biomarker for breast cancer cell proliferation and early relapse
Authors: McDonald Elizabeth S. , Pant Dhruv K. , Pan Tien-chi , Mankoff David A. , Mach Robert H. , Chodosh Lewis A.
Published: July 2017

Background: Breast cancers may recur following a long latency period, implying a dormant or quiescent phase, however little is known about the biology of cellular quiescence and few markers exist for this clinically important state. The purpose of this study was to investigate whether progesterone receptor membrane component family 1 (PGRMC1), which is expressed in both proliferating and quiescent tumor cells, is a prognostic biomarker in human breast cancer and to define its expression in breast cancer subtypes.

Methods: Seventeen publicly available data sets were combined to analyze PGRMC1 expression in 4,463 invasive breast cancers as a function of established molecular and phenotypic markers, estimates of cellular proliferation, and recurrence-free survival data. A gene expression signature-based assay was utilized to estimate cellular proliferation. Association between gene expression and relapse-free survival was assessed using Cox proportional hazards regression while controlling for the effect of proliferation.

Results: PGRMC1 expression was analyzed stratified by immunohistochemical and molecular subtype, tumor grade and size and compared with a known triple negative breast cancer biomarker, BCL11a. PGRMC1 and BCL11a each exhibited a robust positive correlation of comparable magnitude with proliferation across all breast cancer subtypes (r=0.27, p=6.5x10-17 and r=0.29, 1.7x10-41, respectively), and PGRMC1 was strongly associated with proliferation within the basal subtype (r=0.42, p=2.4x10-37 PGRMC1 versus 0.31, p=1.1x10-8 BCL11a). PGRMC1 expression was associated with a higher risk of early breast cancer recurrence (HR=1.25, 95% CI [1.12,1.39], p=6.4x10-5) and this association was dependent upon its association with proliferation.

Conclusions: PGRMC1 is a breast cancer proliferation biomarker. Further study of the function of this protein is warranted.

Citation Format: Elizabeth S. McDonald, Dhruv K. Pant, Tien-chi Pan, David A. Mankoff, Robert H. Mach, Lewis A. Chodosh. PGRMC1 is a biomarker for breast cancer cell proliferation and early relapse [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 5710. doi:10.1158/1538-7445.AM2017-5710
Abstract 5713: PIGN gene expression aberration weakens chromosomal stability via altering its interaction with the spindle assembly checkpoint protein complex during leukemogenesis
Authors: Pu Jeffrey J. , Teye Emmanuel K. , Sido Abigail , Kawasawa Yuka I. , Xin Ping , Finnberg Niklas K. , El-Deiry Wafik S. , Shimko Sara
Published: July 2017

Previous studies have linked increased frequency of glycosylphosphatidylinositol-anchor protein (GPI-AP) deficiency with genomic instability and the risk of carcinogenesis. Recently, Phosphatidylinositol Glycan Anchor Biosynthesis; Class N (PIGN), a gene participating in GPI-AP synthesis, was suggested as a cancer chromosomal instability suppressor in a colon cancer model. We investigated the association of PIGN with genomic instability and leukemogenesis. A Random Forest analysis of the gene expression array data from 55 MDS patients (GSE4619) demonstrated a significant (p = 0.0007) correlation (Pearson r =-0.4068) between GPI-anchor biosynthesis gene expression and genomic instability, in which PIGN was ranked as the third most important in predicting risk of MDS progression. We observed that PIGN gene expression aberrations (increased transcriptional activity but diminished to no protein production) were associated with increased frequency of GPI-AP deficiency in leukemic cells during leukemic transformation/progression. The PIGN gene expression aberrations were attributed to partial intron retentions between exons 14 and 15 resulting in frameshifts and premature termination which were confirmed by examining the RNA-seq data from a group of AML patients (phs001027.v1.p1). PIGN gene expression aberration correlated with the elevation of genomic instability marker expression that was independent of the TP53 regulatory pathway. PIGN protein expression suppression/elimination caused a similar pattern of genomic instability that was rescued by PIGN restoration. Furthermore, PIGN bound to the spindle assembly checkpoint proteins and regulated their expression during the cell cycle. In conclusion, PIGN gene is crucial in the regulation of mitotic integrity to maintain chromosomal stability and prevents leukemic transformation/progression.

Citation Format: Jeffrey J. Pu, Emmanuel K. Teye, Abigail Sido, Yuka I. Kawasawa, Ping Xin, Niklas K. Finnberg, Wafik S. El-Deiry, Sara Shimko. PIGN gene expression aberration weakens chromosomal stability via altering its interaction with the spindle assembly checkpoint protein complex during leukemogenesis [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 5713. doi:10.1158/1538-7445.AM2017-5713