bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-067: Identifying novel and druggable targets in a triple negative breast cancer cell line using the Invitrogen LentiArray Human Kinase CRISPR Library
Authors: Gokirmak Tufan , Sharp Jason , Revankar Chetana , Piper David R.
Published: August 2018

Breast cancer is the most commonly diagnosed cancer among women in worldwide and the second leading cause of death by cancer after lung cancer. Breast cancer can be broadly grouped into three main groups: HR+ (estrogen receptor positive and/or progesterone receptor positive), HER2+ (human epidermal growth factor receptor 2 positive) and triple-negative (HR- and HER2-). Hormone and anti-HER2 targeted therapies are available for receptor positive breast cancer types. However, because triple-negative breast cancer (TNBC) does not have estrogen, progesterone or HER2 receptors, there are no such targeted therapies available for this type of cancer. Currently, oncologists are resort to treating TNBC patients with traditional chemotherapy drugs, surgery and radiation. Thus, due to its poorer prognosis and fewer treatment options in comparison to other type of breast cancers, search for specific molecular targets and developing novel treatments for TNBC is one of the highest priorities of current breast cancer research. Protein kinases are involved in cell signaling cascades leading to cell proliferation, migration and survival in all cell types, and their dysregulation often leads to cancer. Due to their oncogenic roles, protein kinases have become a main focus of the targeted drug discovery efforts. Therefore development of novel high-throughput screening platforms that enable the identification of protein kinases or for that matter any gene, essential for tumor proliferation and viability is expected to advance oncogenic drug discovery efforts. In this study, we developed a loss-of-function screen for identifying essential kinases in a TNBC cell line, MDA-MB-231, using the Invitrogen™ LentiArray™ Human Kinase CRISPR Library. This arrayed lenti virus library targets 840 kinases with up to 4 different gRNAs per protein kinase for complete gene knockout. Our initial pilot screen identified polo-like kinase 1 (PLK1), checkpoint kinase 1 (CHK1) and cyclin-depended kinase 9 (CDK9) as essential kinases in MDA-MB-231 cell line and our current efforts focus on expanding this loss-of-function screen to the full kinase library and validation of the hits. Target identification and validation remains a significant challenge to translate to clinical trial outcomes and these generalized, functional genomic approaches may provide the next-generation platform to effectively bridge these gaps.

Citation Format: Tufan Gokirmak, Jason Sharp, Chetana Revankar, David R. Piper. Identifying novel and druggable targets in a triple negative breast cancer cell line using the Invitrogen LentiArray Human Kinase CRISPR Library [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-067.
Abstract LB-070: Design and modular development of I-PARTS: An integrated platform for anti-cancer radiation therapeutics screening
Authors: Papineni Rao V. , Costes Sylvain , Ray Shayoni , McLaughlin William , Lukaniec Brendan , Kruger Kurt , Kanugi Keith
Published: August 2018

Tumor irradiation during cancer treatment induces cell death by apoptosis, mitotic death, and clonogenic death. With expanded global access to cancer radiotherapy, there is a need to discover agents with anti-cancer and radiation sensitizing properties. in vitro high-throughput screening (HTS) systems routinely used to screen agents for cytotoxicity assays unfortunately do not directly measure clonogenic potential and fail to accurately predict the efficacy of an agent either in subsequent preclinical animal model testing or in clinical trials. Clonogenic assays unlike colorimetric and metabolic assays for determining cell viability and apoptosis, are less susceptible to artifacts. We are addressing these requirements through an integrated I-PARTS platform development and optimization. Here, the design and the prototype of a fully enclosed imaging/X-ray irradiation system will be presented. The proof of principle experimentation to obtain high content information on the cellular response to specific primary human cells (normal or tumors) to a variety of radiation doses and fraction modality will be described. The initial approaches taken towards the establishment of an integrated platform to screen anti-cancer drugs will be discussed.

Citation Format: Rao V. Papineni, Sylvain Costes, Shayoni Ray, William McLaughlin, Brendan Lukaniec, Kurt Kruger, Keith Kanugi. Design and modular development of I-PARTS: An integrated platform for anti-cancer radiation therapeutics screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-070.
Abstract LB-075: Structural stabilization of protein fragments by membrane anchoring for generation of cell-permeable protein mimetics
Authors: Tarasova Nadya , Stefanisko Karen , Khavrutskii Lyuba , Tarasov Sergey
Published: August 2018

Targeting protein-protein interactions can provide plentiful opportunities for the discovery of novel drug candidates and powerful chemical biology tools. However, the majority of these interactions are ‘undruggable”, and we still know very little about the structural mechanisms and functions for the vast majority of them. We have developed a rational approach that allows for the straightforward development of cell-permeable metabolically stable inhibitors of protein-protein interactions. The approach is based on structural stabilization of peptides by membrane anchoring. Biophysical data suggests that proximity to the membrane facilitates interactions of hydrophobic side chains of amphiphilic peptides with the bilayer and stabilizes natural fold of not only helical but also stretched and hairpin-type peptides. Membrane tethering is achieved through straightforward derivatization of peptides with fatty acids of appropriate length that depends on peptide's hydrophobicity. Lipidation facilitates cell entry and allows intracellular delivery of peptides up to 14-16 residues long. Negative charges can interfere with the entry, while positive charges generally have little effect. Wide applicability of this method was confirmed by generation of selective and highly potent dominant negative inhibitors of RAS oncogenes, β-catenin, STAT1, STAT3 and STAT5 N-domains, Il10R1, IFNGR1 and other non-druggable targets. Rational design of inhibitors can be accomplished even in the absence of the target tertiary structure by using conservation of certain sequence parts during the evolution. High throughput generation of selective chemical biology tools allows for effective interrogation of protein-protein interactions leading to discovery of mechanistic details of molecular signaling that could not be obtained with the help of genetic approaches.

Citation Format: Nadya Tarasova, Karen Stefanisko, Lyuba Khavrutskii, Sergey Tarasov. Structural stabilization of protein fragments by membrane anchoring for generation of cell-permeable protein mimetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-075.
Abstract LB-082: ONC201 sensitizes resistant breast cancer cells to TRAIL through death receptor 5 upregulation
Authors: Ralff Marie D. , Wagner Jessica , El-Deiry Wafik S.
Published: August 2018

ONC201 is a potent inducer of cancer cell death through the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. ONC201 and its structural analogs comprise a novel class known as the imipridones. The compound is being tested clinically against a range of solid tumors and hematological malignancies. We found that both triple negative and non-triple negative breast cancer cells are sensitive to ONC201. This was initially surprising, as the majority of non-triple negative breast cancer cells are resistant to TRAIL. In TRAIL-sensitive cells, ONC201 induced cell death in a TRAIL-dependent manner. In TRAIL-resistant cells, the effects of ONC201 are not pro-apoptotic but are rather anti-proliferative. Here we further investigate the effects of ONC201 in TRAIL-resistant breast cancers from multiple molecular subtypes. ONC201 has been previously shown to induce transcriptional upregulation of TRAIL receptor death receptor 5 (DR5). A known mechanism of TRAIL resistance in breast cancer is low cell surface DR5 expression. We hypothesized that pre-treatment with ONC201 would upregulate DR5 and lead to TRAIL sensitization in resistant cells. Our results confirm our hypothesis and show that treatment with ONC201 transcriptionally upregulates DR5 and increases its expression at the cell surface. In addition, pre-treatment with ONC201 sensitizes TRAIL-resistant breast cancer cells to the pro-apoptotic effects of TRAIL. Caspase-8 and PARP are cleaved when cells are treated with the combination of ONC201 and TRAIL, but not when treated with either compound alone. Annexin-V PI staining and quantitation of subG1 DNA content via flow cytometry further confirmed these results. Knockdown of DR5 abrogated TRAIL sensitization by ONC201. Natural killer (NK) cells are known to use TRAIL to kill targets like tumor cells. We are exploring the hypothesis that pre-treatment of tumor cells with ONC201 would increase their killing by NK cells. We are currently investigating the importance of the TRAIL pathway and DR5 upregulation by imipridone compounds in this sensitization to NK cell induced apoptosis. In summary, these results describe a novel mechanism by which ONC201 sensitizes breast cancer cells to the effects of apoptosis inducing ligand TRAIL. They also provide preclinical rationale for the testing of the compound against breast cancers in the clinic.

Citation Format: Marie D. Ralff, Jessica Wagner, Wafik S. El-Deiry. ONC201 sensitizes resistant breast cancer cells to TRAIL through death receptor 5 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-082.
Abstract LB-090: Activation of KITENIN/ErbB4-mediated signaling contributes to acquisition of cetuximab resistance in colorectal cancer cells
Authors: Park So-Yeon , Yang Yi , Zhou Rui , Bae Woo Kyun , Chung Ik Joo , Kim Kyung Keun , Kim Hangun
Published: August 2018

ErbB4/KITENIN signaling plays a role in epidermal growth factor receptor (EGFR)-independent EGF pathways mediating the invasiveness and tumorigenesis of colorectal cancer cells. However, whether alterations in ErbB4/KITENIN signaling play a role in the resistance to anti-EGFR therapy remains unclear. Here, we established cetuximab-resistant DLD1 and HT29 cells, and analyzed changes in ErbB4/KITENIN signaling. c-Jun, a final effector in ErbB4/KITENIN-mediated signaling, was upregulated, whereas KITENIN levels remained constant in both cetuximab-resistant cell lines. The phosphorylation of EGFR and ErbB4 was increased in cetuximab-resistant cells, suggesting that ErbB4/KITENIN signaling contributed to the acquisition of cetuximab resistance in the cells. Silencing of KITENIN and/or ErbB4 increased cetuximab sensitivity in cetuximab-resistant cells. This study is the first to report the activation of ErbB4/KITENIN-mediated signaling in cetuximab-resistant colorectal cancer cells and the potential clinical application of ErbB4/KITENIN-targeting therapy for overcoming anti-EGFR resistance.

Citation Format: So-Yeon Park, Yi Yang, Rui Zhou, Woo Kyun Bae, Ik Joo Chung, Kyung Keun Kim, Hangun Kim. Activation of KITENIN/ErbB4-mediated signaling contributes to acquisition of cetuximab resistance in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-090.
Abstract LB-093: ERK mutations and amplification confer resistance to ERK-inhibitor therapy
Authors: Jaiswal Bijay S. , Durinck Steffen , Stawiski Eric W. , Yin Jianping , Wang Weiru , Lin Eva , Moffat John , Martin Scott E. , Modrusan Zora , Seshagiri Somasekar
Published: August 2018

MAPK pathway inhibitors targeting BRAF and MEK have shown clinical efficacy in patients with RAF and/or RAS mutated tumors. However, acquired resistance to these agents has been an impediment to improved long-term survival in the clinic. In such cases, targeting ERK downstream of BRAF/MEK has been proposed as a potential strategy for overcoming acquired resistance. Preclinical studies suggest that ERK inhibitors are effective at inhibiting BRAF/RAS mutated tumor growth and overcome BRAF or/and MEK inhibitor resistance. However, as with other MAPK pathway inhibitors, treatment with ERK inhibitors is likely to cause resistance in the clinic. In this study, we have used in vitro modeling, structural biology and genomic analysis to understand development of resistance to ERK inhibitors and the mechanisms leading to it. We have identified mutations in ERK1/2, amplification and overexpression of ERK2, and overexpression of EGFR/ERBB2 as mechanisms of acquired resistance. Structural analysis of ERK showed that specific compounds that induced on-target ERK mutations were impaired in their ability to bind mutant ERK. We show that in addition to MEK inhibitor, ERBB-receptor and PI3K/mTOR pathway inhibitors are effective in overcoming ERK-inhibitor resistance. These findings suggest that combination therapy with MEK or ERBB-receptor or PI3K/mTOR and ERK inhibitors may be an effective strategy for managing the emergence of resistance in the clinic.

Citation Format: Bijay S. Jaiswal, Steffen Durinck, Eric W. Stawiski, Jianping Yin, Weiru Wang, Eva Lin, John Moffat, Scott E. Martin, Zora Modrusan, Somasekar Seshagiri. ERK mutations and amplification confer resistance to ERK-inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-093.
Abstract LB-097: Linking prostate cancer cell AR heterogeneity to distinct castration and Enzalutamide responses
Authors: Li Qiuhui , Chen Xin , Deng Qu , Chao Hsueh-Ping , Tracz Amanda , Kirk Jason , Zhao Ruizhe , Rycaj Kiera , Tang Dean G
Published: August 2018

Expression of androgen receptor (AR) in prostate cancer (PCa) is heterogeneous but the functional significance of AR heterogeneity in regulating PCa biology and response to androgen/AR-targeted therapies remains unclear. Screening of ~200 castration-resistant PCa (CRPC) cores identified 3 AR expression patterns: nuclear (nuc-AR), mixed nuclear/cytoplasmic (nuc/cyto-AR), and low/no expression (AR-/lo). Xenograft CRPC modeling demonstrated that, although the AR+ CRPC responded to Enzalutamide, the AR-/lo CRPC was completely refractory to Enzalutamide. Genome editing-derived isogenic AR+ and AR-KO (knockout) PCa cells showed contrasting tumor-regenerating capacities and responses to castration and Enzalutamide. Whole-genome RNA-Seq and biochemical analyses coupled with experimental combinatorial therapy identified novel signaling pathways and BCL-2 as a critical therapeutic target in both AR+ and AR-/lo CRPC. Our study links AR heterogeneity to distinct castration/Enzalutamide responses and suggests the urgency in developing therapeutics to target AR-/lo PCa cells/clones.

Citation Format: Qiuhui Li, Xin Chen, Qu Deng, Hsueh-Ping Chao, Amanda Tracz, Jason Kirk, Ruizhe Zhao, Kiera Rycaj, Dean G Tang. Linking prostate cancer cell AR heterogeneity to distinct castration and Enzalutamide responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-097.
Abstract LB-118: A conserved checkpoint molecule signature offers novel targets for tumor specific regulatory T cell manipulation
Authors: Freeman Zachary T. , Hovleson Daniel H. , Pearson Alexander T. , Zhai Jingyi , Ghasemzadeh Ali , Nirschl Thomas R. , Jiang Hui , Tomlins Scott A. , Drake Charles G.
Published: August 2018

Regulatory T (Treg) cells play an important role in modulating the tumor microenvironment, leading to the suppression of anti-tumor immune responses. Tumor Tregs could be targeted by taking advantage of the differential expression of immune checkpoints on these cells as compared to other T cell subsets. While the elevated expression of checkpoints such as CTLA-4 on Tregs compared to conventional T cells is well-characterized, less is known about the collective landscape of Treg specific checkpoint expression specific in the tumor microenvironment. We identified a network of Treg-associated checkpoint markers including those previously associated with Treg function (CTLA4, ICOS) as well as several additional checkpoints (BTLA, LAG3, TIGIT, TNFRSF9) by characterizing the expression of 28 checkpoint molecules across 7,608 bulk tumor samples (31 tumor types) from TCGA. We then utilized whole transcriptome RNA sequencing of purified peripheral and tumor Treg cell populations across 4 different tumor types (glioblastoma multiforme, prostatic adenocarcinoma, urothelial transitional cell carcinoma, renal clear cell carcinoma) to characterize the Treg specific expression of checkpoint molecules. Peripheral and tumor Tregs demonstrate differential checkpoint expression landscapes, with tumor Tregs uniquely discriminated by collective expression of TNFRSF9 (4-1BB), HAVCR-2 (TIM-3), and LAG-3. This discriminatory signature was conserved across the 4 profiled tumor types, was associated with changes in Treg biology, and was confirmed in published data from three additional cancer cohorts (breast, colorectal, and non-small-cell-lung cancers). Furthermore, normalized TNFRSF9 expression in TCGA was also associated with a worse survival outcome, suggesting that elevated Treg TNFRSF9 expression may be a poor prognostic factor across a wide range of tumors. Taken together, these data highlight the potential of targeting TNFRSF9 expressing Tregs in the tumor as a novel immunotherapeutic approach for several types of cancer.

Citation Format: Zachary T. Freeman, Daniel H. Hovleson, Alexander T. Pearson, Jingyi Zhai, Ali Ghasemzadeh, Thomas R. Nirschl, Hui Jiang, Scott A. Tomlins, Charles G. Drake. A conserved checkpoint molecule signature offers novel targets for tumor specific regulatory T cell manipulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-118.
Abstract LB-124: Promoting an anti-tumor immune environment with a novel, exquisitely selective CSF1R inhibitor
Authors: Suzuki Erika , Kovacs Jeffrey J. , Spencer Nakia D. , Sonal Sonal , Feng Ningping , Harris Angela L. , Mullinax Robert A. , Zuniga Andy M. , Johnson Sarah B. , Mahendra Mikhila , Khor Tin Oo , Mseeh Faika , Liu Zhen , Burke Jason P. , Mikule Keith , Tremblay Martin , Heffernan Timothy P. , Jones Philip , Czako Barbara , Marszalek Joseph R.
Published: August 2018

Tumor-associated macrophages (TAMs) are critical drivers of tumor progression and immunosuppression within the tumor microenvironment. The dominant TAM phenotype is broadly characterized as harboring M2-like macrophage properties, which are anti-inflammatory and pro-tumor, as opposed to M1-like macrophages, which possess tumoricidal and pro-inflammatory characteristics. The dependence of M2 TAMs on CSF1 receptor (CSF1R) kinase signaling has made CSF1R a desirable therapeutic target, and the need for highly selective therapies for use in combinations. Through an extensive medicinal chemistry campaign, we identified a series of orally bioavailable, highly potent, exquisitely selective inhibitors of CSF1R (IC50 < 10 nM) with excellent pharmacologic properties that are appropriate for evaluation as a cancer therapy. The aim of our study was to assess the biological impact of our lead CSF1R inhibitor (CSF1Ri) on macrophage populations and the consequent effect on T effector cells. In vitro biochemical activity was evaluated in various kinase assays comparing our CSF1Ri to BLZ945. The compound was evaluated using syngeneic murine models of colorectal cancer (MC38) and pancreatic adenocarcinoma (PANC02). Tumors were immune profiled using NanoString, immunohistochemistry and flow cytometric analysis establishing that there was a depletion of macrophages and a reduction in the relative amount of M2+ (CD206+MHCII-) cells, with a concomitant increase in M1+ (MHCII+CD206-) cells. Myeloid-derived suppressor cells (MDSCs), CD4, and CD8 cell infiltration were also altered with an elevation of cytotoxic immune cells. In conclusion, we have identified and characterized a novel potent and selective inhibitor of CSF1R with highly favorable PK/PD properties, that compares favorably to BLZ945. We also have clear evidence that our novel CSF1R inhibitors modulates TAM infiltration and phenotype altering the immune cell milieu toward a more favorable anti-tumor environment.

Citation Format: Erika Suzuki, Jeffrey J. Kovacs, Nakia D. Spencer, Sonal Sonal, Ningping Feng, Angela L. Harris, Robert A. Mullinax, Andy M. Zuniga, Sarah B. Johnson, Mikhila Mahendra, Tin Oo Khor, Faika Mseeh, Zhen Liu, Jason P. Burke, Keith Mikule, Martin Tremblay, Timothy P. Heffernan, Philip Jones, Barbara Czako, Joseph R. Marszalek. Promoting an anti-tumor immune environment with a novel, exquisitely selective CSF1R inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-124.
Abstract LB-125: Atovaquone suppresses triple negative breast tumor growth by inhibiting myeloid derived suppressor cells
Authors: Gupta Nehal , Wright Stephen , Srivastava Sanjay
Published: August 2018

Triple negative breast cancer (TNBC) is considered to be the most aggressive and malignant neoplasm and is highly metastatic in nature. Metastatic TNBC is difficult to treat thus leading to extremely poor survival. The major contributing factor in TNBC progression and resistive nature is its ability to evade the immune surveillance. Modulating the immune system in breast tumors could be an important strategy for cancer therapeutics. The present study evaluates the immune response of atovaquone, an antiprotozoal drug in various TNBC cell lines and its efficacy in three independent in vivo models. In the first experiment, HCC1806 human breast cancer cells were orthotopically implanted in mammary fat of female mice. Oral administration of atovaquone reduced HCC1806 breast tumor growth by 45%. On the other hand, oral administration of atovaquone inhibited the growth of highly aggressive CI66 breast tumors by 70%. Paclitaxel is the first line chemotherapeutic agent for metastatic breast cancer. In third in vivo experiment, 4T1 paclitaxel resistant (4T1R) cells were injected in mice and atovaquone suppressed paclitaxel resistant tumor growth by 42%. Myeloid derived suppressor cells (MDSCs) are known to suppress immune function. Our current results demonstrated 70% and 30% suppression of tumor and blood MDSCs respectively, with atovaquone treatment in mouse bearing HCC1806 tumors. We also observed about 25% reduction in MDSCs with atovaquone treatment in mice with CI66 as well as 4T1R tumors. MDSCs induce regulatory T cells (Treg cells) which are immunosuppressive. Our results showed reduction of Treg cells by 12% and 17% with atovaquone treatment in HCC1806 tumors and in 4T1R tumors respectively. In addition, decrease in TGF-beta (13%) and IL-10 (40%) in HCC1806 tumor lysates was observed by atovaquone, an evidence of reduction in immunosuppressive cytokines produced by MDSCs. It appears that the mechanism of immune suppression of atovaquone by MDSCs in our model may be through TGF-β and IL-10, which are potent immunosuppressive cytokines and induce Treg cells that allow cancer cells to escape from immune surveillance. This is the first report demonstrating that immune modulation by atovaquone treatment is associated with breast tumor growth suppression, prompting further investigation to establish atovaquone as a treatment option for TNBC patients.

Citation Format: Nehal Gupta, Stephen Wright, Sanjay Srivastava. Atovaquone suppresses triple negative breast tumor growth by inhibiting myeloid derived suppressor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-125.
Abstract LB-139: Panel-based NGS identifies prognostic and actionable genes in childhood acute lymphoblastic leukemia and suits for clinical sequence
Authors: Ishida Hisashi , Iguchi Akihiro , Aoe Michinori , Matsubara Takehiro , Tsukahara Hirokazu , Shimada Akira
Published: August 2018

Background: Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Whereas the cure rate has been much improved, considerable number of patients suffered from leukemia relapse. The cure rates of relapsed ALL patients remained poor as low as 40%, thus ALL is leading cause of cancer death during childhood. To improve the cure rates of these patients, precise re-stratification and newer targeted therapy are needed. For those reasons, detecting genes that had prognostic value and that are potentially actionable for in a short time is desired in clinical settings.

Methods: We perform next generation sequence of matched diagnosis, remission, and relapse samples of eighteen relapsed ALL patients. We use the desktop sequencer MiSeq (Illumina, Inc., San Diego, CA, USA) and HaloPlex custom panels (Agilent Technologies, Santa Clara, CA, USA). These HaloPlex custom panels include more than 150 cancer-related genes. In the same sequencing run, we checked single nucleotide variations (SNV), insertion/deletion, and copy number variations (CNV) simultaneously. We also compare the genetic alterations between diagnosis and relapse samples, and presume the type of clonal evolutions.

Results: We identified total 16 SNV and insertion/deletion and 19 CNV at diagnosis, and 28 SNV and insertion/deletion and 22 CNV at relapse. With these gene alterations, we could re-stratify many patients otherwise without any high-risk features (4/8 B-cell precursor ALL patients, 50%). We could also detect potentially actionable gene variants in most patients (13/17 of all patients, 76.5%). Among them, one patient harbored widely reported germline TP53 R248Q mutation, thus regarded as Li-Fraumeni syndrome. Most relapse samples (9/13 analyzed cases, 69.2%) acquire new lesions and lost some lesions present at diagnosis, suggesting their clonal evolution from ancestral clones. Remaining four cases (4/13 cases, 30.7 %) showed clonal evolution direct from diagnosis clones.

Conclusion: This inexpensive, rapid, sequential next generation sequence method will immediately apply as clinical sequence, and lead to better management of these patients and potential improvement in survival rate in childhood ALL.

Citation Format: Hisashi Ishida, Akihiro Iguchi, Michinori Aoe, Takehiro Matsubara, Hirokazu Tsukahara, Akira Shimada. Panel-based NGS identifies prognostic and actionable genes in childhood acute lymphoblastic leukemia and suits for clinical sequence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-139.
Abstract LB-144: Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy
Authors: Hong Xin , Sullivan Ryan J. , Kalinich Mark , Kwan Tanya , Pan Shiwei , LiCausi Joseph A. , Milner John D. , Nieman Linda T. , Wittner Ben S. , Ho Uyen , Chen Tianqi , Kapur Ravi , Lawrence Don , Flaherty Keith T. , Sequist Lecia V. , Ramaswamy Sridhar , Miyamoto David T. , Lawrence Michael , Giobbie-Hurder Anita , Toner Mehmet , Isselbacher Kurt J. , Maheswaran Shyamala , Haber Daniel A.
Published: August 2018

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-L1 expression and mutational burden, are insufficiently precise to guide treatment selection and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma Circulating Tumor Cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC-Score) applied to microfluidically-enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (N=42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC-Score within 7 weeks of therapy correlates with marked improvement in progression-free survival (Hazard Ratio (HR): 0.17, P=0.008) and overall survival (HR: 0.12, P=0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies.

Citation Format: Xin Hong, Ryan J. Sullivan, Mark Kalinich, Tanya Kwan, Shiwei Pan, Joseph A. LiCausi, John D. Milner, Linda T. Nieman, Ben S. Wittner, Uyen Ho, Tianqi Chen, Ravi Kapur, Don Lawrence, Keith T. Flaherty, Lecia V. Sequist, Sridhar Ramaswamy, David T. Miyamoto, Michael Lawrence, Anita Giobbie-Hurder, Mehmet Toner, Kurt J. Isselbacher, Shyamala Maheswaran, Daniel A. Haber. Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-144.
Abstract LB-151: Prophylactic TNFα blockade unplugs toxicity and efficacy in immunotherapy anti-PD-1 + anti-CTLA-4 combinations
Authors: PEREZ-RUIZ ELISABETH , MINUTE LUNA , OCHOA MARIA CARMEN , AZPILICUETA ARANTXA , GARASA SARAY , RODRIGUEZ-RUIZ M E , BELSUE VIRGINIA , MOLINA CARMEN , BERRAONDO PEDRO , MELERO IGNACIO
Published: August 2018

The combination of anti-CTLA-4 and anti-PD-1 checkpoint inhibitors ipilimumab and nivolumab (DIT) has improved survival of patients with metastatic melanoma and renal cell carcinoma in exchange for frequent immune-mediated serious side effects (mainly gastrointestinal), leading to treatment cessation in about one third of the patients and the need to reduce doses of these immunotherapy agents when used in combination. Tumor necrosis factor-α (TNF-α) is an important actionable mediator in inflammatory bowel disease (both ulcerative colitis and Crohn's disease) and its blockade with monoclonal antibodies has been recommended to treat steroid-resistant colitis following checkpoint inhibitors. We have studied whether prophylactic TNFα blockade is able to reduce immune toxicity associated with DIT without altering antitumor activity. Experiments were performed in C57BL/6 mice harboring subcutaneous tumors derived from the MC38 and B16OVA cell lines. Acute immune-mediated colitis was induced by exposure to 3% dextran sulfate sodium (DSS) in drinking water for 3 days. For TNFα blockade etanercept (hTNFR2-IgG chimera) or a mouse anti-TNF-α monoclonal antibody were used. Anti-tumor efficacy was tested following individual tumor sizes upon combined treatment with anti-mPD-1 + anti-mCTLA4 mAbs. Severity of DSS colitis was assessed and scored by daily weight, ultrasound evaluation of intestinal wall thickness and histological assessment on day 7. We observed that the antitumoral efficacy of PD-1+CTLA-4 blockade against MC38 of B16OVA established tumors was not reduced with the prophylactic association of TNF-α blockade. Intriguingly, an improvement of antitumor activity was observed in those mice in which DSS colitis was induced. Importantly, in mice suffering DSS-induced colitis that were co-treated with DIT and TNF-α blockade, digestive toxicity ameliorates as shown by body weight, ultrasound examination and histology. Therefore, TNF-α blockade can be prophylactically associated with anti-PD1 + anti-CTLA-4 mAb combination regimens to prevent toxicity without hindrance of anti-tumor efficacy. These results in mouse models warrant a clinical trial testing profilactic etanercept to improve the safety profile of the nivolumab + ipilimumab combination widening its therapeutic window.

Citation Format: ELISABETH PEREZ-RUIZ, LUNA MINUTE, MARIA CARMEN OCHOA, ARANTXA AZPILICUETA, SARAY GARASA, M E RODRIGUEZ-RUIZ, VIRGINIA BELSUE, CARMEN MOLINA, PEDRO BERRAONDO, IGNACIO MELERO. Prophylactic TNFα blockade unplugs toxicity and efficacy in immunotherapy anti-PD-1 + anti-CTLA-4 combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-151.
Abstract LB-153: KY1055, a novel ICOS-PD-L1 bispecific antibody, efficiently enhances T cell activation and delivers a potent anti-tumour response in vivo
Authors: Sainson Richard C. , Parveen Nahida , Borhis Gwenoline , Kosmac Miha , OKell Tracey , Taggart Emma , Carvalho Joana , McCourt Matthew , Ali Hanif , Craig Hannah , Labokha Aksana
Published: August 2018

Antibodies blocking the PD-1/PD-L1 axis present significant breakthroughs in the treatment of hitherto untreatable cancers. However, despite the highly encouraging progress that has been made, multiple tumour types demonstrate limited response to these therapies. The emerging picture is that combination with other therapies is required to improve response rates and/or the durability of response. Additional benefit could be achieved by combining an anti-PD-L1 approach with another modulator of T cell activation. ICOS is a co-stimulatory molecule expressed on activated T cells such as TEff and TReg cells present in the tumour microenvironment (TME). ICOS agonism has been demonstrated to improve TEff cell survival and IFNγ release, resulting in the activation of T cell-dependent eradication of the tumour. In addition, the high expression of ICOS observed on TReg cells in the TME allows these highly immunosuppressive cells to be selectively depleted when using the appropriate antibody format. With the aim of targeting both ICOS and PD-L1, we have developed a bispecific mAb² (KY1055) as a human IgG1 format that consists of fully human Fab arms targeting ICOS and a modified Fc (Fcab) containing binding sites to PD-L1. We demonstrate potent (single digit nM) binding to ICOS via the Fab and to PD-L1 via the Fcab. Furthermore, in vitro studies reveal that KY1055 can bridge together ICOS- and PD-L1-expressing cells and can activate ICOS signalling through this cross-presentation. Functionality of the Fcab was validated by demonstrating that it blocks the PD-1-PD-L1 inhibitory pathway, resulting in the up-regulation of IFNγ in monocyte:T cell co-culture assays. We also demonstrate the ability of KY1055 to deplete ICOShigh cells in vitro by ADCC. In vivo, we confirm the anti-tumour efficacy of KY1055 in several syngeneic models that are poorly responsive to the respective monotherapies and further demonstrate improved anti-tumour efficacy when combined with anti-CTLA4 and anti-PD-1 in vivo. Finally, we demonstrate that KY1055 depletes TRegs and improves the TEff : TReg ratio in vivo in a CT26 model of tumour growth. Altogether, these data warrant further development of KY1055 for the treatment of solid tumours.

Citation Format: Richard C. Sainson, Nahida Parveen, Gwenoline Borhis, Miha Kosmac, Tracey OKell, Emma Taggart, Joana Carvalho, Matthew McCourt, Hanif Ali, Hannah Craig, Aksana Labokha. KY1055, a novel ICOS-PD-L1 bispecific antibody, efficiently enhances T cell activation and delivers a potent anti-tumour response in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-153.
Abstract LB-164: Global study of chromatin interactions reveals biologically relevant candidate target genes at endometrial cancer risk loci
Authors: Glubb Dylan M. , Kho Pik Fang , Consortium Endometrial Cancer Association , Thompson Deborah , Spurdle Amanda , O'Mara Tracy
Published: August 2018

We have previously used genome-wide association studies and meta-analyses to uncover 17 genetic risk loci for endometrial cancer. However, the regulatory gene targets of these loci have not been systematically evaluated. To identify candidate gene targets at endometrial cancer risk loci, we have assessed chromatin looping interactions between endometrial cancer risk variants and neighboring genes using the global HiChIP chromatin conformation capture technique. To perform HiChIP, estrogen-stimulated Ishikawa endometrial cancer cells were cross-linked and proximity ligation performed. Chromatin associated with RNA polymerase II was isolated and HiChIP libraries prepared before sequencing using the Illumina HiSeq 4000 system. HiChIP paired-end reads were aligned using the HiC-Pro pipeline and filtered for valid reads. The hichipper package was used to identify and characterise global chromatin interactions (FDR < 0.01). At 11 of the 17 endometrial cancer risk loci, we identified chromatin interactions between risk variants and genes (37 coding and 10 non-coding). Interaction distances ranged from 8 kb to 1.7 Mb. Expression of candidate target genes CUX2, CYP19A1, SKAP1, SNX11 and EIF2AK4 was found to associate with endometrial cancer risk in whole blood transcriptome-wide association studies (TWAS). Ingenuity Pathway Analysis revealed that candidate genes were enriched for relevant functional annotations such as tumorigenesis, cell migration, necrosis and viability. Furthermore, enrichment of candidate genes (p=0.001) was observed in the CXCR4 signaling pathway, which is involved with endometrial cancer development, invasion and progression. In summary, we have performed the first global study of chromatin interactions in endometrial cancer cells. This analysis has revealed biologically relevant candidate target genes at endometrial cancer risk loci, five of which are supported by TWAS. Transcriptional regulation of candidate target genes by endometrial cancer risk variants will be verified in future studies.

Citation Format: Dylan M. Glubb, Pik Fang Kho, Endometrial Cancer Association Consortium, Deborah Thompson, Amanda Spurdle, Tracy O'Mara. Global study of chromatin interactions reveals biologically relevant candidate target genes at endometrial cancer risk loci [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-164.
Abstract LB-174: Novel AC electrokinetic platform for rapid isolation and characterization of extracellular vesicles from NSCLC patients
Authors: Krishnan Rajaram , Hinestrosa Juan P. , Searson David , Turner Robert , Kovelman Robert , Balcer Heath , Madsen James , Bodkin David
Published: August 2018

We have developed a novel Alternating Current Electrokinetics (ACE) platform to isolate macromolecules such as circulating cell-free DNA (ccfDNA) and nanoparticles, including cell-derived extracellular vesicles (EVs). ccfDNA and EV isolation, and antibody staining using the platform required less than two hours to complete. EVs isolated by ultracentrifugation from the pancreatic cancer cell line ASPC-1 were used to validate the ACE platform's performance. EVs from pancreatic cancer ASPC-1 cells were detected using an anti-CD63 antibody and an antibody to the pancreatic cancer specific membrane protein Glypican-1, confirming that the ACE platform could isolate EVs and identify membrane-bound proteins. Recovered EVs were isolated and EV-associated RNA was amplified by RT-PCR. EVs from isolated from NSCLC patient plasma on the ACE platform contained higher levels of PD-L1 compared to healthy individuals (p<0.001). In parallel on the ACE platform, ccfDNA was isolated and quantified from the same samples, with increased levels measured in cancer patients. Improved differentiation between the NSCLC patients and the healthy normals was achieved when both markers were included in the analysis. Therefore, we believe the ACE platform has the potential to become a multimodal analytics tool for cancer diagnostics and treatment monitoring.

Citation Format: Rajaram Krishnan, Juan P. Hinestrosa, David Searson, Robert Turner, Robert Kovelman, Heath Balcer, James Madsen, David Bodkin. Novel AC electrokinetic platform for rapid isolation and characterization of extracellular vesicles from NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-174.
Abstract LB-178: Oxidation markers in relation to prostate cancer and BPH and progression of prostate cancer
Authors: Wu Tianying , Bracken Bruce , Kasper Susan
Published: August 2018

Prostate cancer (PCa) is the leading cancer affecting men of all races in the U.S. Benign prostatic hyperplasia (BPH) is prostate enlargement and is not malignant. BPH and PCa have similar symptoms and both have elevated PSA levels. A biomarker which can distinguish PCa from BPH will help the diagnosis of PCa and avoid unnecessary biopsies. Further, many PCa patients after prostatectomy can have recurrence. Currently, PSA is the only monitoring marker for recurrence; however, the underlying mechanism for recurrence is unknown. A mechanistic marker that can predict the progression and recurrence this will allow us to design prevention strategies. Many animal studies have linked oxidative stress with PCa progression and recurrence. The aim of this study is to compare the levels of several oxidation markers between PCa and BPH, and compare the levels of oxidation marker in different stages of PCa and determine their associations with PSA velocities after prostatectomy. We have measured multiple oxidation markers including lipid, protein, DNA, carbohydrate and global oxidation markers. We have collected blood samples among 15 BPH and 22 prostate cancer patients who have prostatectomy. We have found that levels of advanced glycation end products (AGEs), a marker of glucose oxidation, formed under nonenzymatic glycation of lipids and proteins, are higher in PCa patients than BPH. However, among PCa patients, AGEs levels are lower among those with Gleason score higher than 7 than those with Gleason score less than 7. Finally, we have found F2-isoprostanes, a well-established lipid oxidation marker, are associated with PSA velocities among PCa patients who had prostatectomy. Our results suggest that AGEs can help distinguish BPH and PCa but are associated with lower grade PCa. Measurement of F2-isoprostanes, a marker of lipid oxidation can predict PCa progression among men with prostatectomy.

Citation Format: Tianying Wu, Bruce Bracken, Susan Kasper. Oxidation markers in relation to prostate cancer and BPH and progression of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-178.
Abstract LB-183: Investigating the role of polarity protein, SCRIB, in hematopoiesis
Authors: Novita Novita , Dew Alysha , Johnson Chad , Walkley Carl R. , Purton Louise E. , Humbert Patrick O. , Darcy Phillip K. , Ellis Sarah L.
Published: August 2018

Acting as both a polarity regulator and a scaffolding protein, Scribble (SCRIB), coordinates intracellular signalling pathways that regulate cell proliferation, migration and apoptosis. Deregulation of SCRIB has been linked to multiple epithelial cancers but the role of SCRIB in hematopoiesis and hematopoietic malignancies remains relatively unexplored. Using different approaches, we show that SCRIB is expressed in hematopoietic stem and progenitor cells and in their differentiated progenies, implying a role for SCRIB in blood production. We investigated the effect of SCRIB loss in hematopoiesis using two conditional knockout mouse models in conjunction with an extensive phenotypic labelling regime and multi-color flow cytometry. We reveal a role for SCRIB in T cell development although subtle differences between the two mouse models were noted. Compensatory mechanisms often mask the effects of single protein deletion during steady-state so stress hematopoiesis was induced through a transplant assay to minimise these effects. Lethally irradiated recipient mice receiving a transplant of SCRIB knockout bone marrow revealed a more striking phenotype with significantly altered populations of granulocyte, macrophage, erythroid, T and B cell progenitors, and mature macrophage fractions compared to controls. Collectively, our data shows SCRIB is required for the development of multiple hematopoietic fractions and studies are currently underway to identify the underlying mechanism/s and elucidate how deregulation of SCRIB impacts on hematopoietic malignancies.

Citation Format: Novita Novita, Alysha Dew, Chad Johnson, Carl R. Walkley, Louise E. Purton, Patrick O. Humbert, Phillip K. Darcy, Sarah L. Ellis. Investigating the role of polarity protein, SCRIB, in hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-183.
Abstract LB-191: BET inhibitors kill colorectal cancer cells by activating death receptor 5 through the ER stress response pathway
Authors: Tan Xiao , Tong Jingshan , Yu Jian , Shen Liangfang , Zhang Lin
Published: August 2018

Bromodomain and extra-terminal (BET) family proteins are epigenetic "readers" that are frequently activated in cancer and play an important role in regulating gene expression and oncogenic signaling. Targeting BET proteins using small-molecule inhibitors represents an attractive therapeutic strategy. Several small-molecule BET inhibitors have shown promises in recent preclinical and clinical studies. However, the mechanisms by which BET inhibitors selectively kill cancer cells are not well understood. In this study, we found that the BET inhibitors JQ1 and I-BET151 induce mRNA and protein expression of Death Receptor 5 (DR5) in a variety of colorectal cancer (CRC) cells with different genetic backgrounds. This is an on-target effect as knockdown of the BET family member BRD4 also induces DR5 expression. The induction of DR5 by the BET inhibitors is mediated by C/EBP homologous protein (CHOP) through the endoplasmic reticulum (ER) stress response pathway. Furthermore, knockout of DR5 in colon cancer cells suppresses apoptosis and caspase activation induced by the BET inhibitors, and abrogates their chemosensitization effects in CRC cells. Collectively, our results uncover a key role of DR5 in apoptosis induced by the BET inhibitors in CRC cells, and suggest activation of the death receptor pathway as a novel anticancer mechanism of BET-targeting agents.

Citation Format: Xiao Tan, Jingshan Tong, Jian Yu, Liangfang Shen, Lin Zhang. BET inhibitors kill colorectal cancer cells by activating death receptor 5 through the ER stress response pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-191.