bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3729: Developing cetuximab-staurosporine conjugate as the therapeutic medicine in KRAS/BRAF mutated colon cancer cells
Authors: Chao Wei-Ting , Sun Wei-Ting , Chiu Hsiang-Ling , Wei Wan-Chen , Wu Yi-Che , Kao Ling-Yi , Chung Yuan-Chiang , Chuang Shih-Hsien
Published: August 2018

Epidermal growth factor receptor (EGFR) is the key receptor for cancer cell growing, cetuximab is the monoclonal antibody that targets EGFR and was approved for treatment of colorectal, head and neck cancer. However, patients with KRAS /BRAF mutation are screened for the resistance. Kinases activities in the signal axes have been demonstrated the role in drug resistance in colon cancer cells. In this study, cetuximab was conjugated with kinases inhibitor staurosporine and the efficacy was examined in KRAS/BRAF mutated colon cancer cells. The cetuximab and staurosprone conjugate was prepared based on the amide bond formation or the thioether bond formation. Both cleavable and non-cleavable linkers were applied to the conjugate. KRAS and BRAF mutated colon cancer cell lines SW480 and HT-29 were treated with cetuximab, staurosporine, cetuximab and Staurosporine combination, cetuximab-staurosporine conjugate, cell viability and the molecular expression were monitored. In the result, staurosporine was demonstrated to suppress cetuximab induced Src activation which may contribute to drug resistance. Wild type cell (SW48) is sensitive (63% viability) to cetuximab treatment compared to BRAF and KRAS mutated cells. Cetuximab-staurosporine conjugate showed twice efficacy than cetuximab on wild type cell, and also resulted in greater toxicity (50% viability) in BRAF/KRAS mutated cells which are resistant to cetuximab treatment. Taken together, cetuximab-staurosporine conjugate has therapeutic potential in BRAF/KRAS mutated colon cancer cells.

Citation Format: Wei-Ting Chao, Wei-Ting Sun, Hsiang-Ling Chiu, Wan-Chen Wei, Yi-Che Wu, Ling-Yi Kao, Yuan-Chiang Chung, Shih-Hsien Chuang. Developing cetuximab-staurosporine conjugate as the therapeutic medicine in KRAS/BRAF mutated colon 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 3729.
Abstract 3733: Combined targeting of estrogen receptor alpha and nuclear transport pathways remodel metabolic pathways to induce autophagy and overcome endocrine resistance
Authors: Erdogan Zeynep Madak , Cotul-Kulkoyluoglu Eylem , Wrobel Kinga , Sahoo Sunati , Haley Barbara , Landesman Yosef
Published: August 2018

Majority of breast cancer specific deaths in women with ERα (+) tumor occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to resensitize recurrent ERα (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERα and XPO1, a nuclear transport protein in overcoming endocrine resistance. Selinexor (SXR), an XPO1 antagonist, has been evaluated in multiple later stage clinical trials in patients with relapsed and /or refractory hematological and solid tumor malignancies. Using Cignalfinder to profile kinase signaling pathways, we found that 4-OH-Tam, SXR or their combination induced differential Akt phosphorylation profiles, changing the localization and activity of the kinase. Since we observed dramatic changes in Akt activity we hypothesized that metabolic profile of breast cancer cells would change in the presence of 4-OH-Tam and SXR. Using Seahorse metabolic profiler and cell viability experiments in limited media conditions we showed that tamoxifen resistant cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SXR and cells were more dependent on glutamine as the mitochondrial fuel source. In order to examine metabolic pathways that might result in the observed phenotype we performed transcriptomics and GC/MS whole metabolite profiling and identified aminoacid metabolism pathways to be upregulated when cells were treated with SXR+4-OH-Tam. We demonstrated that combined targeting of XPO1 and ERα rewires metabolic pathways and shuts down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Remodelling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERα(+) tumors, our findings show great promise for uncovering the role ERα-XPO1 crosstalk plays in reducing cancer recurrences.

Citation Format: Zeynep Madak Erdogan, Eylem Cotul-Kulkoyluoglu, Kinga Wrobel, Sunati Sahoo, Barbara Haley, Yosef Landesman. Combined targeting of estrogen receptor alpha and nuclear transport pathways remodel metabolic pathways to induce autophagy and overcome endocrine resistance [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 3733.
Abstract 3740: Functional cross-talk between nuclear receptor LRH-1 and androgen receptor signalling in prostate cancer
Authors: You Wenxing , Wang Yuliang , Chan Leung
Published: August 2018

Liver Receptor Homolog 1 (LRH-1, NR5A2) is an orphan nuclear receptor that is over-expressed in cancers in tissues such as the breast, colon and pancreas. In prostate cancer, LRH-1 is reported as a potent transcription factor to facilitate the formation of CRPC (castration-resistant prostate cancer cells) which is resistant to androgen deprivation therapy (ADT). However, it is still elusive how LRH-1 responses to ADT and facilitates the growth of CRPC. After deprivation of androgen in mouse prostate cancer xenograft model, LRH-1 mRNA was upregulated dramatically at a very low androgen level. After CRPC formation, LRH-1 mRNA was downregulated when androgen level recovered. KLK3/PSA is an important downstream target of activated AR (Androgen receptor), whose expression level at 4 days after castration is downregulated, while upregulated after relapse. Since PSA level/AR signaling activity showed an opposite expression pattern to LRH-1, we hypothesized that LRH-1 was suppressed by androgen in a negative-feedback manner. With androgen treatment of an androgen-sensitive prostate cancer cell line LNCaP, LRH-1 was downregulated in both dose- and time-dependent manner, while PSA shows an opposite expression pattern. And knocking down of AR blocked the suppression of androgen on LRH-1, indicating that androgen suppresses LRH-1 through AR. Furthermore, AR antagonist Enzalutamide can block the binding of androgen to AR thus upregulate LRH-1. According to ChIP-seq data, AR binds to the promoter and enhancer of LRH-1,and the binding intensity increased with androgen treatment. Through ChIP-qPCR, several binding sites of AR on LRH-1 promoter and enhancer were validated. The transcription activity of luciferase reporter with these AR binding sites was also suppressed by treatment of androgen. In the future work, to find out the epigenetics modifiers in the AR complexes when suppressing LRH-1 will be critical for the understanding of the detailed mechanism.

Citation Format: Wenxing You, Yuliang Wang, Leung Chan. Functional cross-talk between nuclear receptor LRH-1 and androgen receptor signalling in 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 3740.
Abstract 3743: Translational regulation by ERα in hormone-dependent cancers
Authors: Lorent Julie , Hoef Vincent van , Rebello Richard , Lawrence Mitchell , Topisirovic Ivan , Larsson Ola , Furic Luc
Published: August 2018

The estrogen receptor α (ERα) activities are complex: in the cytoplasm ERα can directly stimulate survival signalling at the cell membrane, while in the nucleus ERα activates and represses the transcription of target genes. We recently showed that in prostate cancer ERα expression is associated with increased proliferation and higher clinical grade.

Here we explore the role of ERα in coordinating transcription and mRNA translation. Unexpectedly, loss of ERα expression leads to decoupling of transcription and translation events. Namely, mRNAs whose levels are induced by ERα loss exhibit reduced translation efficiency and, vice versa, mRNAs whose levels are reduced by ERα loss exhibit enhanced translation efficiency. Such regulation is manifested at the protein level and targets a range of key cellular functions including translation and metabolism.

Our detailed mechanistic assessment reveal that while ERα-regulated microRNA levels contribute to global changes in mRNA levels, translational buffering is explained by changes in ribosome processivity and elongation rate.

Overall, we have recently identified a process by which ERα drastically impacts the translation of a subset of mRNAs in cancer cells. We propose that this new regulatory pathway plays a major role in mediating biological effects of ERα in neoplastic tissues. Moreover, our findings have important implications in understanding alterations in gene expression programs following treatment with ERα antagonists.

Citation Format: Julie Lorent, Vincent van Hoef, Richard Rebello, Mitchell Lawrence, Ivan Topisirovic, Ola Larsson, Luc Furic. Translational regulation by ERα in hormone-dependent cancers [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 3743.
Abstract 375: Origins of fractional control in regulated cell death
Authors: Santos Luis C. , Vogel Robert , Chipuk Jerry E. , Birtwistle Marc , Stolovitzky Gustavo , Meyer Pablo
Published: August 2018

Fractional killing of a population of cancer cells is often linked to cell-to-cell variability due to stochastic gene expression and fluctuations in protein levels. We found that mitochondria abundance is a major source of cell-to-cell variability that determines the fraction of cells that live or die in response to TNF-related apoptosis-inducing ligand (TRAIL). We show that the IC50 of TRAIL of clonogenic cancer cell populations correlates with mitochondria abundance, and cell-to-cell variability in mitochondria abundance controls up to 40% of the response to TRAIL. Our coarse-grained quantitative model of apoptosis reveals that increasing the mitochondrial surface area decreases the density of proapoptotic Bax pores, which in turn decreases the sensitivity of cells to TRAIL. We demonstrate that the clinically relevant small-molecule inhibitor of antiapoptotic Bcl-2 proteins ABT-263 reduces the IC50 of TRAIL by reducing the contribution of mitochondria to variability in apoptosis.

Citation Format: Luis C. Santos, Robert Vogel, Jerry E. Chipuk, Marc Birtwistle, Gustavo Stolovitzky, Pablo Meyer. Origins of fractional control in regulated cell death [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 375.
Abstract 3751: Inhibition of A2AR by AZD4635 induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical models
Authors: Borodovsky Alexandra , Wang Yanjun , Ye Minwei , Shaw Joseph C. , Sachsenmeier Kris , Deng Nanhua , Goodwin Kelly , Clarke James D. , Goodwin Richard , Strittmatter Nicole , Hay Carl , Sah Vasu , Deborah Lawson , Reimer Corinne , Congreve Miles , Mason Jonathan , Marshall Fiona , Lyne Paul , Woessner Richard
Published: August 2018

Adenosine signaling through the high affinity adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which can promote tumor growth and limit the efficacy of immune checkpoint inhibitors. AZD4635 (HTL-1071) is a potent and selective oral A2AR antagonist, currently in a Phase 1 clinical trial as a single agent and in combination with durvalumab (anti-PD-L1 Ab) in patients with solid malignancies. In functional in vitro assays, the IC50 of AZD4635 for inhibition of A2AR signaling is dependent on adenosine concentrations and ranges from 1, 10.0, 143 nM in the presence of 0.1, 1, 10 μM adenosine, respectively. However, full understanding of the impact of adenosine on AZD4635 mediated anti-tumor responses requires characterization of intratumoral adenosine concentration and spatial heterogeneity. Using a novel LC/MS based method, measurement of intratumoral adenosine concentrations in a panel of syngeneic tumor models demonstrated a wide range of adenosine levels (5-122 μM). Additionally, measurement of intratumoral adenosine by desorption electrospray ionisation - mass spectroscopy (DESI-MS) demonstrated that adenosine levels are spatially heterogeneous, with levels varying up to 50-fold among regions of a single tumor. High intratumoral adenosine concentrations were correlated with decreased intratumoral CD8 infiltration in vivo. The therapeutic benefit of A2AR blockade alone and in combination with anti-PD-L1 was evaluated in a panel of syngeneic mouse tumor models with varying adenosine concentrations. In sensitive models, inhibition of A2AR signaling with AZD4635 led to a dose dependent reduction in tumor growth alone and in combination with checkpoint inhibitors. Tumors harvested from the treated mice exhibited increases in the functional activity of T cell and myeloid subsets. These results demonstrate that AZD4635 is a potent and selective A2AR inhibitor, that blockade of A2AR signaling with an inhibitor such as AZD4635 can reduce tumor burden and enhance antitumor immunity.

Citation Format: Alexandra Borodovsky, Yanjun Wang, Minwei Ye, Joseph C. Shaw, Kris Sachsenmeier, Nanhua Deng, Kelly Goodwin, James D. Clarke, Richard Goodwin, Nicole Strittmatter, Carl Hay, Vasu Sah, Lawson Deborah, Corinne Reimer, Miles Congreve, Jonathan Mason, Fiona Marshall, Paul Lyne, Richard Woessner. Inhibition of A2AR by AZD4635 induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical models [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 3751.
Abstract 3763: IDO vaccine enhances antigen-specific anti-tumor effects by reducing IDO-expressing antigen presenting cells and MDSCs
Authors: Nandre Rahul , Verma Vivek , Gaur Pankaj , Vir Pooja , Wang Hua , Kang Baolin , Pedersen Ayako Wakatsuki , Zocca Mai-Britt , Andersen Mads Hald , Gupta Seema , Khleif Samir
Published: August 2018

Tumors evade immune surveillance using several mechanisms such as induction of regulatory T-cells (Tregs) and upregulation of indolemine-2,3-dioxygenase (IDO) enzyme. IDO is an intracellular enzyme that mediates its immunosuppressive effects by depleting tryptophan leading to accumulation of kynurenine and in turn reducing the proliferation of effector cells while activating Tregs. IDO is expressed by several suppressive immune cells including Tregs, suppressive antigen presenting cells (APCs), as well as by tumor cells. These immunosuppressive effects of IDO make it an attractive target for cancer immunotherapy, including a target for vaccine therapy. To test the anti-tumor immune effect of IDO vaccine, we evaluated the anti-tumor effect of an IDO-derived MHC I restricted peptide (IDO-pep) vaccine in the low IDO-expressing (TC-1) and high IDO-expressing B16 (melanoma) mouse tumor models. For this, mice were treated with three doses of IDO-peptides in combination with the respective tumor-specific vaccines (E7 for TC-1 and gp100 for B16) at an interval of seven days, followed by assessment of tumor growth and mice survival. We observed a significant delay in the tumor growth and prolonged survival in both tumor models when IDO vaccine was combined with tumor-specific antigen vaccine. We found that tumor-infiltration of antigen-specific CD8+ T-cells was significantly enhanced following the addition of IDO vaccine. Furthermore, while CD4+ T-cells increased in the tumor microenvironment (TME), there was a significant reduction in the numbers of Treg cells. We also found a significant reduction in the numbers of IDO-expressing myeloid-derived suppressor cells (MDSCs), dendritic cells as well as macrophages out of the total CD45+CD11b+ myeloid cells in the TME after the administration of IDO-pep in combination with tumor antigen-specific vaccine. In conclusion, our findings suggest that IDO-pep vaccine enhances the specific anti-tumor immune response by increasing the tumor antigen-specific effector T-cell infiltration in the TME through inhibition of immune suppressive cells. These results also suggest that MHC I restricted IDO peptide treatment is a promising strategy to improve cancer immunotherapy.

Citation Format: Rahul Nandre, Vivek Verma, Pankaj Gaur, Pooja Vir, Hua Wang, Baolin Kang, Ayako Wakatsuki Pedersen, Mai-Britt Zocca, Mads Hald Andersen, Seema Gupta, Samir Khleif. IDO vaccine enhances antigen-specific anti-tumor effects by reducing IDO-expressing antigen presenting cells and MDSCs [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 3763.
Abstract 3773: RORγ agonist enhances antitumor effects of agonist anti-OX40 antibody
Authors: Gaur Pankaj , Verma Vivek , Nandre Rahul , Vir Pooja , Wang Hua , Kang Baolin , Carter Laura , Hu Xiao , Liu Xikui , Gupta Seema , Khleif Samir
Published: August 2018

Combination immunotherapy strategies are being developed to improve the clinical outcome in cancer patients. T-cell costimulation through OX40 has been shown to promote expansion and proliferation of effector T-cells leading to enhanced effector functions, memory generation and immune inflammatory antitumor responses. However, treatment with anti-OX40 as a single agent has not led to major positive clinical outcomes. Therefore, anti-OX40 is an ideal candidate for combination immunotherapy. In preclinical models, we have recently shown that combining anti-PD-1 concurrently with anti-OX40 negates the effects of agonist anti-OX40, making identification of combination partners crucial for antitumor therapy. RORγt, a master transcription factor, is known to drive Type 17 T-cell differentiation. Synthetic, small-molecule RORγ agonists have been shown to enhance Type 17 T-cell effector functions and survival, decrease immune suppressive mechanisms and modulate expression of a number of costimulatory and coinhibitory molecules. We hypothesized that combining RORγ agonist could enhance the antitumor effects of anti-OX40. Using a murine model where vaccine is used to prime the immune system, we assessed the effects of anti-OX40 antibody combined with a RORγ agonist (LYC-54143) on growth of established tumors and survival. We found that the combined treatment resulted in a significantly delayed tumor growth and prolonged mice survival. Analysis of the tumor microenvironment revealed that combination therapy significantly increased the numbers of total CD4+ T-cells including RORγt+ and highly activated INFγ+ cells, decreasing Treg numbers. Moreover, we found that RORγ agonist resulted in an increase in the numbers of total, antigen-specific, granzyme B+, and IFNγ+ CD8+ T-cells in the TME. These results show that inducing Th17 cells within the TME produces better anti-OX40 effect, suggesting that agonist anti-OX40 and RORγ agonist treatment is a novel potent combination for cancer immunotherapy.

Citation Format: Pankaj Gaur, Vivek Verma, Rahul Nandre, Pooja Vir, Hua Wang, Baolin Kang, Laura Carter, Xiao Hu, Xikui Liu, Seema Gupta, Samir Khleif. RORγ agonist enhances antitumor effects of agonist anti-OX40 antibody [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 3773.
Abstract 3774: BGB324, a selective small-molecule inhibitor of receptor tyrosine kinase AXL, targets tumor immune suppression and enhances immune checkpoint inhibitor efficacy
Authors: Davidsen Kjersti , Wnuk-Lipinska Katarzyna , Du Wenting , Blø Magnus , Engelsen Agnete , Terry Stephane , D´mello Stacey , Lie Maria , Kang Jing , Hodneland Linn , Bougnaud Sebastien , Aguilera Kristina , Straume Oddbjørn , Chouaib Salem , Brekken Rolf A. , Gausdal Gro , Lorens James B.
Published: August 2018

The AXL receptor tyrosine kinase is associated with poor overall survival in a wide spectrum of cancers. AXL signaling is important for tumor cell plasticity related to epithelial-to-mesenchymal transition (EMT), immune escape and intrinsic resistance to cytotoxic lymphocytes. AXL is expressed on several cells associated with the tumor immune microenvironment, including natural killer (NK) cells, dendritic cells and a subset of tumor-associated myeloid cells. AXL signaling enhances secretion of immune-suppressive cytokines from innate immune cells that limit antitumor immunity. Hence AXL resides uniquely at the nexus between tumor and microenvironmental antitumor immune suppression mechanisms. BGB324, a selective clinical-stage small-molecule Axl kinase inhibitor, is currently being evaluated in combination with pembrolizumab in three phase II clinical trials in patients with TNBC (NCT03184558), NSCLC (NCT03184571) and melanoma (NCT02872259). We show that BGB324 targets immune suppression mechanisms in the tumor microenvironment that improve immunotherapy in different murine tumor models. BGB324 treatment reduces myeloid-derived suppressor cells and tumor-associated macrophages, and lowers CCL11, IL-7, IL-1β, and IL-6 in murine pancreatic cancer models. This altered immune landscape is associated with increased tumor infiltration of NK and CD8+ T cells and enhanced therapy responses. Further, BGB324 targets tumor intrinsic immune resistance and enhances human CD8+ T cell and NK-cell mediated NSCLC tumor cell lysis. We are currently using high-dimensional mass cytometry analysis (CyTOF) to map adaptive Axl-dependent immune suppression during immune checkpoint blockade. Collectively these results highlight a prominent function for AXL in resistance to immune therapy and support continued clinical translation of combining BGB324 with immune checkpoint inhibitors to improve cancer treatment.

Citation Format: Kjersti Davidsen, Katarzyna Wnuk-Lipinska, Wenting Du, Magnus Blø, Agnete Engelsen, Stephane Terry, Stacey D´mello, Maria Lie, Jing Kang, Linn Hodneland, Sebastien Bougnaud, Kristina Aguilera, Oddbjørn Straume, Salem Chouaib, Rolf A. Brekken, Gro Gausdal, James B. Lorens. BGB324, a selective small-molecule inhibitor of receptor tyrosine kinase AXL, targets tumor immune suppression and enhances immune checkpoint inhibitor efficacy [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 3774.
Abstract 3784: Intraoperative changes of low density granulocyte (LDG) in gastrointestinal cancer surgery
Authors: Kumagai Yuko
Published: August 2018

Background: Low density granulocytes (LDG) have been shown to be increased in peripheral blood of autoimmune disease, severe sepsis and cancer. In this study, we examined the frequency and function of the LDG in patients who received abdominal surgery for gastrointestinal malignancy. Method: In 57 patients who received laparotomy due to the gastrointestinal cancer ( 5 esophageal, 25 gastric , 27 colorectal cancer), peripheral blood was collected before and after operative procedure in operation room. After Ficoll-Hypaque centrifugation, intermediate layer which consists mainly of mononuclear cells was collected and immunestained with mAbs to CD66b, CD45 as well as 7AAD and their expressions were examined with flow cytometer. CD66b(+) CD45(+) cells were defined as LDG and the ratios of LDG in total CD45(+) leukocytes were calculated in 7AAD(-) live cell fraction. Next, CD66b(+) LDG and CD66b(-) mononuclear cells were separated with MACS method, and LDG were cultured and examined for extracellular traps (NETs) formation in vitro. Finally, CD66b(-) mononuclear cells were cultured on anti-CD3 mAb coated plate with or without autologous LDG, and proliferation of T lymphocytes was examined with CFSE dilution method. Results: The ratio of the LDG is low (median= 1.14%, 0.62 ~1.66%, n=57) at a beginning of surgery. In contrast, the ratio was markedly increased (median= 8.10%, 5.91~10.3%, n=57, p<0.001) at the end of surgery. The ratio of postoperative LDG showed significant correlation with operation time and positive trend with intraoperative bleeding. The LDG showed relatively week expression of CD11b and CD66b, but highly expressed in IL8 receptor and CD62L, suggesting immature LDG. The short-term culture of the LDG produced massive SYTOX-positive web like stricture which was characteristic for NETs. The mixture of tumor cells on the LDG culture demonstrated that many tumor cells were trapped in the NETs within 5 min incubation and vigorously grew thereafter. Finally, addition of the LDG on mononuclear cell culture strongly suppressed the anti-CD3 stimulated T cell division. Conclusion: LDG with immature phenotype increase in circulating blood of the patients who underwent gastrointestinal cancer surgery, probably due to the surgical stress. The LDG produce NETs which may efficiently trap circulating tumor cells, and can inhibit T lymphocyte mediated-immune reaction. Thus, the LDG provide tumor permissive microenvironment in target organ and have supportive roles on the development of postoperative hematogenous metastasis in gastrointestinal cancer.

Citation Format: Yuko Kumagai. Intraoperative changes of low density granulocyte (LDG) in gastrointestinal cancer surgery [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 3784.
Abstract 3786: ILC-k: Human innate lymphoid cells displaying unique metabolic features and KIR-independent cytotoxicity, impaired in acute myeloid leukemia
Authors: Salomé Bérengère , Gomez-Cadena Alejandra , Loyon-Bonato Romain , Suffiotti Madeleine , Salvestrini Valentina , Curti Antonio , Tentorio Paolo , Mavilio Domenico , Riether Carsten , Ochsenbein Adrian , Marcenaro Emanuela , Gfeller David , Romero Pedro , Trabanelli Sara , Jandus Camilla
Published: August 2018

Innate Lymphoid Cells (ILCs) are a recently identified family of lymphocytes, divided into 3 different groups that mirror the functional specialization of helper CD4 T cells. However, it is now clear that the spectrum of ILC diversity is much broader than originally appreciated. ILCs have been shown to play important roles in inflammatory and autoimmune diseases, and their involvement in anti-tumor responses has also recently been evidenced by us and others. Here, we report on the identification of a previously undescribed human ILC subset, named hereafter “ILC-killer” (ILC-k), characterized by a Lineage negative, CD127+ cKIT- CRTh2- CD56+ phenotype, thus being distinct from conventional helper ILCs, e.g ILC1 (CD56-), ILC2 (CRTh2) and ILC3 (cKIT+). Transcriptomic profiling of ex-vivo highly pure ILC-k, conventional ILC subsets and conventional Natural Killer (NK) cell subsets showed closeness between ILC-k, ILC3 and CD56bright NK cells. However, ILC-k display a specific metabolism (low glucose uptake, high mitochondrial activity) and cytotoxic gene signature, also confirmed at protein level. By their functional evaluation, ILC-k showed specific ability to kill tumor cell lines in a KIR-independent, but NKp30- and Trail-dependent manner. Their relative frequency is decreased and both their metabolic fitness and cytotoxic potential are impaired in Acute Myeloid Leukemia (AML) patients at diagnosis (N=51). Upon remission, ILC frequencies and functions are restored to normal levels. Overall, we identified cytotoxic ILCs, distinct from conventional NK cells, that are functionally impaired in AML. Given their KIR-independence, ILC-k might represent promising therapeutic candidates to improve NK-cell based immunotherapy, that currently necessitates KIR-HLA mismatch between donor and recipient for full efficiency.

Citation Format: Bérengère Salomé, Alejandra Gomez-Cadena, Romain Loyon-Bonato, Madeleine Suffiotti, Valentina Salvestrini, Antonio Curti, Paolo Tentorio, Domenico Mavilio, Carsten Riether, Adrian Ochsenbein, Emanuela Marcenaro, David Gfeller, Pedro Romero, Sara Trabanelli, Camilla Jandus. ILC-k: Human innate lymphoid cells displaying unique metabolic features and KIR-independent cytotoxicity, impaired in acute myeloid leukemia [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 3786.
Abstract 3796: Substrate specificity of human kallikrein-related peptidase 2 (KLK2) towards beta chain of interleukin-10 receptor
Authors: Neto Luiz J. , Juliano Maria A. , Oliveira Juliana R.
Published: August 2018

The kallikrein-related peptidases (KLKs) comprise a family of 15 homologous secreted trypsin- or chymotrypsin-like serine proteases. KLK2 and KLK3 have the most organ restricted expression profile of all KLKs; specifically, they are abundantly expressed in the luminal epithelium of the prostate. The expression of KLK2 gene is regulated by the androgens and androgen receptor (AR) signaling and like human PSA, serum KLK2 can also function as a biomarker for prostate cancer (PCa). The present work provides biochemical data related to the hydrolytic activities of KLK2 on libraries of fluorescence resonance energy transfer (FRET) peptides in the presence of sodium citrate, a kosmotropic salt that is present in normal prostate tissue and seminal fluid in high concentration. The peptide SYR↓IF (cleaved at↓) was the highly hydrolyzed and best substrate so far reported for KLK2. The sequence YRIF is present in the N-terminal segment (59YRIF62) in the β chain of interleukin-10 receptor. In order to verify the effect of KLK2 in the IL10RB surface expression in the bone marrow-derived macrophages (BMDM) cells, they were incubated with KLK2 at 37ºC for 2 hours. BMDM treated with KLK2 showed a significant reduction in the IL10RB surface expression compared with control group, indicating that KLK2 acts directly on this receptor, decreasing its expression. The effect of this reduction was evaluated trough BMDM activation assay. After incubation of BMDM cells with KLK2, they were washed and incubated with or without LPS, IFN-γ and recombinant IL10. When incubated with recombinant IL10, control macrophage showed a significant reduction of NO, TNF-α and IL-12p40 levels indicating the IL10RB functionality and according to IL10 immunosuppressive effect. However, KLK2 pretreated macrophage maintained NO, TNF-α and IL-12p40 levels, even in the presence of recombinant IL10, leading a proinflammatory status in BMDM culture, while KLK3 doesn't show the same effect. Taken together, the data indicates that KLK2 decreases the surface expression of IL10RB on macrophage cells and contributes to a proinflammatory environment.

Citation Format: Luiz J. Neto, Maria A. Juliano, Juliana R. Oliveira. Substrate specificity of human kallikrein-related peptidase 2 (KLK2) towards beta chain of interleukin-10 receptor [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 3796.
Abstract 3800: Inhibition of Polycomb Repressive Complex 2 EZH2 lysine methyltransferase improves tumoricidal activity of macrophages towards mesothelioma cells
Authors: Hamaidia Malik , Hoyos Clotilde , Willems Luc L.
Published: August 2018

Rationale: Clinical evidence indicates that tumor infiltration by tumor associated macrophages (TAMs) correlates with poor prognosis in malignant mesothelioma (MM). By attenuating the immune response, TAMs indeed promote survival of MM cells. TAMs share properties with alternative macrophages (M2) that are activated by anti-inflammatory (e.g. IL-10) or Th2-associated (i.e. IL-4, IL-13) cytokines.

Objectives: We hypothesized that macrophage activation is mediated by a transcriptional program tightly regulated by epigenetic modifications. We focused on the Polycomb Repressive Complex 2 (PRC2) EZH2 lysine methyltransferase responsible for trimethylation of histone H3 at lysine 27 (H3K27me3). We investigated the effect of a selective EZH2 inhibitor (EPZ005687) on tumoricidal activity of primary human monocyte-derived macrophages.

Results: Our data show that inhibition of EZH2 reduces CD206 expression and phagocytic activity of macrophages conditioned in MM pleural effusions (PE-macrophages). Moreover, EZH2 inhibition enhances the cytotoxic potential of PE-macrophages towards MM cells. Inhibition of EZH2 also increases the ability of macrophages to support proliferation of autologous T-cells.

Conclusion: Induction of cytotoxic activity of PE-macrophages via PRC2 EZH2 lysine methyltransferase could be of therapeutic value in MM.

Citation Format: Malik Hamaidia, Clotilde Hoyos, Luc L. Willems. Inhibition of Polycomb Repressive Complex 2 EZH2 lysine methyltransferase improves tumoricidal activity of macrophages towards mesothelioma 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 3800.
Abstract 3803: Immune escape from NK cells and activated CD8 T cells by miR-183 repression of MICA/B expression in human lung cancer
Authors: Tu Nhan , Trinh Thu Le , Donatelli Sarah S. , Tejera Melba M. , Gilvary Danielle L. , Zhou Jun-Min , Egsioglu Erika A. , Wei Sheng , Coppola Domenico , Djeu Julie Y.
Published: August 2018

Immune escape is a hallmark of cancer. In human lung cancer, we have identified a unique microRNA (miR)-based pathway employed by tumor cells to repress detection by immune cells via the NKG2D-MICA/B receptor-ligand system. MICA/B is readily induced by cell transformation and serves as a danger signal and ligand to alert NK and activated CD8+ T cells. However, immunohistochemical analysis indicated that human lung adenocarcinoma and squamous cell carcinoma specimens express little MICA/B while high levels of miR-183 were detected in both tumor types in a TCGA database. Human lung tumor cell lines confirmed the reverse relationship in expression of MICA/B and miR-183. Importantly, a miR-183 binding site was identified on the 3' untranslated region (UTR) of both MICA and MICB, suggesting its role in MICA/B regulation. Luciferase reporter constructs bearing the 3'UTR of MICA or MICB in 293 cells supported the function of miR-183 in repressing MICA/B expression. Additionally, anti-sense miR-183 transfection into H1355 or H1299 tumor cells caused the upregulation of MICA/B. Abundant miR-183 expression in tumor cells was traced to transforming growth factor-beta (TGFβ), as evidenced by anti-sense TGFβ transfection into H1355 or H1299 tumor cells, which subsequently lost miR-183 expression accompanied by MICA/B upregulation. Most significantly, anti-sense miR-183 transfected tumor cells became more sensitive to lysis by activated CD8+ T cells that express high levels of NKG2D. Thus, high miR-183 triggered by TGFβ expressed in lung tumor cells can target MICA/B expression to circumvent detection by NKG2D on immune cells.

Citation Format: Nhan Tu, Thu Le Trinh, Sarah S. Donatelli, Melba M. Tejera, Danielle L. Gilvary, Jun-Min Zhou, Erika A. Egsioglu, Sheng Wei, Domenico Coppola, Julie Y. Djeu. Immune escape from NK cells and activated CD8 T cells by miR-183 repression of MICA/B expression in human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3803.
Abstract 3806: The pattern of NKG2DL expressions determines immunogenicity and antitumor effect of protein-bound polysaccharide-K in tumor-bearing mouse models
Authors: Takehara Tomohiro , Soejima Kenzo , Betsuyaku Tomoko , Yoshimura Kiyoshi
Published: August 2018

Background and Aim: Natural killer group 2, member D ligands (NKG2DLs) have paradoxical immunological characteristics, exerting both immune-stimulatory and immune-suppressive effects on T cell activation. First, we attempted to determine the relationship between the antitumor effect of protein-bound polysaccharide-K (PSK) in a tumor-bearing mouse model and the expression profile of NKG2DLs on a tumor. Next, we investigated whether NKG2DL expression on tumor cells affects T cell activation by examining the expression of NKG2D on and IFN-γ production in CD8+ T cells in mice bearing NKG2DL–expressing and NKG2DL–nonexpressing tumors.Materials and Methods: PSK was administered to evaluate its effectiveness against tumor growth. The expression of Rae-1 and H60 were analyzed in the cell lines. Results: PSK showed the highest antitumor effects in mice implanted with cells expressing neither Rae-1 nor H60. PSK had little antitumor effect in mice implanted with cells expressing both Rae-1 and H60. A correlation between the expression of NKG2DLs and the antitumor effect of PSK was observed. After PSK administration, INF-γ production in CD8+ T cells increased in mice with cells expressing neither Rae-1 nor H60, but did not change in mice implanted with cells expressing both Rae-1 and H60.Conclusion: we demonstrated that the expression pattern of NKG2DLs affects tumor immunity and the efficacy of immunotherapy in a tumor-bearing mouse model. In particular, the expression of H60 and Rae-1 family members may be an immunosuppressive factor. Further analysis is required to gain a better understanding of the mechanism of interaction between NKG2D expressed on immune effector cells and H60/Rae-1 expression on tumors. Although NKG2DL expression may represent a marker predicting response to immunotherapy, further studies in humans are required to validate this finding in the clinical setting.

Citation Format: Tomohiro Takehara, Kenzo Soejima, Tomoko Betsuyaku, Kiyoshi Yoshimura. The pattern of NKG2DL expressions determines immunogenicity and antitumor effect of protein-bound polysaccharide-K in tumor-bearing mouse models [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 3806.
Abstract 3808: Interrogation and modulation of the immunosuppressive activity of human TAM-like macrophages using in vitro cultures
Authors: Gallagher Laura E. , Hall Andrew , Patience Lauren A. , Janicova Lucia , Anderton Stephen
Published: August 2018

Tumor Associated Macrophages (TAMs), characteristic of an M2-like immune-suppressive phenotype, can induce proliferation and survival of tumor cells, facilitate angiogenesis, and suppress anti-tumor immune responses via expression of co-inhibitory molecules (e.g. PD-L1) and cytokines (e.g. IL-10, TGF-β). TAMs are therefore a highly attractive target of innovative cancer immunotherapies. Understanding the ability of pre-clinical candidate compounds to reverse TAM (M2-like)-mediated immune suppression and the potential for reprogramming of M2-like macrophages to a M1-like phenotype is key in the development of effective TAM-targeted cancer immunotherapies. Here we outline development of an assay to assess the capabilities of pre-clinical compounds to reverse M2 macrophage-mediated immune suppression.

Monocytes were isolated from whole blood obtained from healthy volunteers and cultured under M2-polarising conditions. The resulting macrophages were phenotypically characterized and then used in co-culture with autologous PBMC, stimulated through T cell receptor ligation. Resulting cytokine production was assessed, alongside CD4+ and CD8+ T cell viability and cell cycle status (flow cytometry).

M2-like macrophages polarized with M-CSF displayed an immune suppressive phenotype as shown by their production of IL-10 and their inhibition of IFN-γ production by, and cell cycle status of, T cells in co-culture assays. This suppressive activity was only partially reversed by PD-1-blockade. Modifications to the macrophage polarization protocol were seen to alter the resulting macrophage cell surface phenotype (e.g. expression levels of PD-L1, TIM-3 and CD200R) and their suppressive activity in the co-culture assay. These cell surface phenotypes broadly reflected those seen amongst tumor-derived macrophages from renal and ovarian carcinoma patients (CD14+ CD163+ with expression of TIM-3 and LAG-3). Moreover, compound-mediated changes in functionality seen with macrophages polarized from healthy PBMC monocytes could also be seen using patient-derived material.

The assay outlined here therefore provides a M-o-A human in vitro system to test novel compound activity (either singly or in combination) upon TAM-like macrophage generation, phenotype and suppressive function. This allows selection of the most efficacious compounds for further investigation using patient-derived immune cells.

Citation Format: Laura E. Gallagher, Andrew Hall, Lauren A. Patience, Lucia Janicova, Stephen Anderton. Interrogation and modulation of the immunosuppressive activity of human TAM-like macrophages using in vitro cultures [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 3808.
Abstract 3810: Ovarian tumor-induced changes in macrophage phenotype are associated with tumor metastasis
Authors: Yellapa Aparna , Bitterman Pincas , Sharma Sameer , Basu Sanjib , Barua Animesh
Published: August 2018

Background: Ovarian cancer (OVCA) differs from other gynecologic malignancies in its dissemination pattern where the tumor migrates in a diffuse intrapelvic and -abdominal manner. OVCA also triggers a variety of cellular and/or humoral immune responses to a variety of tumor-associated proteins. OVCA is mostly diagnosed at late stages where tumor has already metastasized, indicating an escape from immune responses. Despite several immune response factors, ovarian tumor metastasizing to omentum indicates presence of one or more immune evasion mechanisms. Immune system recognizes and eliminates pathogenic products and tumor cells and macrophages play a critical role in this process. Macrophages in tumor condition express anti-inflammatory cytokines that induce immune suppression. Cellular immune response maximizes killing efficacy of macrophages and proliferation of CD8+ T cells. M2 macrophages are tumor-associated macrophages involved in active growth and progression of tumors. However, the mechanism of ovarian tumor-induced changes in M1 macrophages to M2 phenotype is not well understood. Therefore, the goal of our study was to determine the tumor-induced changes in macrophage phenotype and cytotoxic T cells in association with OVCA metastasis.

Materials and Methods: This study was performed in exploratory design using ovarian specimen from normal subjects (60-80 years, n=10) and ovarian malignant tumors at early (n=16, 4 from each histologic type of OVCA) and late stages (n =20) as well as omental tissues from normal (n=3) and OVCA patients (n= 3). Tissue specimen were processed for immunohistochemistry (IHC), Western blotting (WB) and gene expression studies. Expression of M1 and M2 macrophages, CD8 and PI3K was examined by immunohistochemistry (IHC), Western blotting (WB) and qPCR.

Results: Compared with normal ovaries, more M1 and M2 macrophages were localized in tumor tissues. However, compared with early-stage OVCA, the frequency of M2 macrophages was significantly higher in ovarian tumors and omental tissues at late OVCA. Intensity of PI3K was also higher in ovarian tumors and omental tissues at late OVCA compared with normal ovarian tissues. Frequency of CD8+T cells was higher in tumor ovarian samples in comparison with normal ovarian tissues.

Conclusion: Results of this study suggest that the growth and metastasis of ovarian tumors are associated with increased conversion of M1 macrophages to M2 phenotype. This change in macrophage phenotype may be mediated by the increase in PI3K expression.

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Citation Format: Aparna Yellapa, Pincas Bitterman, Sameer Sharma, Sanjib Basu, Animesh Barua. Ovarian tumor-induced changes in macrophage phenotype are associated with tumor metastasis [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 3810.
Abstract 3814: TriTACs are novel T cell-engaging therapeutic proteins optimized for the treatment of solid tumors and for long serum half-life
Authors: Wesche Holger , Aaron Wade , Austin Richard J. , Baeuerle Patrick A. , Jones Adrie , Lemon Bryan , Sexton Kenneth , Yu Timothy
Published: August 2018

T cell engagers are antibody-based therapeutics that transiently tether T cells via the T cell receptor complex (TCR) to surface antigens on tumor cells. This leads to activation of T cells and redirected lysis of the attached target cell. The therapeutic potential of this modality was demonstrated by blinatumomab, a CD19/CD3-bispecific T cell engager approved for the treatment of adult patients with relapsed/refractory acute lymphoblastic leukemia. Despite success of this T cell-engaging therapy in a hematologic malignancy, clinical studies in solid tumors with other T cell engagers have been less encouraging so far. The TriTAC (Tri-specific T cell Activating Construct) platform was developed to address shortcomings of existing T cell engagers, including short serum half-life, limited tissue penetration, and suboptimal activity. TriTAC constructs are made of a single polypeptide designed to bind to a cancer surface antigen, the CD3 epsilon subunit of the TCR, and to human serum albumin. CD3 is bound by a single-chain variable fragment (scFv) while both tumor targeting and albumin binding are achieved by single domain antibodies. The latter allow TriTACs to be very small, stable, and easily produced and purified. Noncovalent binding to serum albumin has been validated as an effective way to extend the serum half-life of other proteins up to several weeks. Even though TriTACs have three binding domains, their overall size is only ~50 kDa, one third of the size of a monoclonal antibody. This is expected to allow for faster diffusion into human tumor tissues than is possible with antibodies given the high interstitial pressure and dense extracellular matrix in solid tumors. TriTACs can induce T cell to kill tumor cells in vitro at single-digit picomolar to femtomolar concentrations with concomitant induction of inflammatory cytokine release and T cell proliferation. TriTACs can diffuse much faster across an extracellular matrix than antibodies, and eradicate tumors in mouse xenograft models supplemented with human T cells. In nonhuman primates, TriTAC molecules have serum half-lives of approximately 4 days, and appear well tolerated.

Citation Format: Holger Wesche, Wade Aaron, Richard J. Austin, Patrick A. Baeuerle, Adrie Jones, Bryan Lemon, Kenneth Sexton, Timothy Yu. TriTACs are novel T cell-engaging therapeutic proteins optimized for the treatment of solid tumors and for long serum half-life [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 3814.