bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1084: The role of tumor-associated neutrophils (TAN) and CD8 positive T lymphocyte in the progression of hepatocellular carcinoma (HCC)
Authors: Yusa Toshihiko , Okabe Hirohisa , Yamashita Yo-ichi , Yamao Takanobu , Umezaki Naoki , Miyata Tatsunori , Nakagawa Shigeki , Hayashi Hiromitsu , Imai Katsunori , Chikamoto Akira , Ishiko Takatoshi , Baba Hideo
Published: August 2019

[Background]Neutrophils are known as immune cells involved in biological defense against foreign substances. On the other hand, in the tumor microenvironment, it is said that neutrophils affect on tumor progression as a tumor-associated neutrophil (TAN). Although, TAN and other immune cells in the progression of HCC is unclear.

[Method]One hundred twenty eight patients who underwent curative hepatectomy for HCC in our institution were enrolled. Immunohistochemistry was performed with CD66b and CD8 antibody to evaluate TAN and CD8+ T lymphocytes, respectively. Expression high or low was determined by cut-off value of each number of immune cells. We counted the number of each immune cells in both tumor (intra-tumor) and the tumor marginal area (extra-tumor). The relationship between the infiltration of each immune cell and clinicopathological factors was examined.

[Result]Comparing the number of immune cells of intra-tumor and extra-tumor, there were significantly more cells in extra-tumor area (p<0.001). There was no difference in between background clinicopathological factors and the number of TAN or CD8+ T lymphocytes in either intra-tumor or extra-tumor areas. Overall survival was significantly better in the group with high CD8+ T lymphocytes in extra-tumor area compared to the low group (p = 0.0491), intriguingly though CD8+ T lymphocytes in intra-tumor area was not associated with prognosis. In the contrast, overall survival was significantly poor in the group with high TAN in extra-tumor area compared to the low group (p = 0.0296), though TAN in intra-tumor area was not associated with prognosis.

[Conclusion]Infiltration of TAN and CD8+ T lymphocytes not in intra-tumor but in the extra-tumor area may be involved in the progression of HCC.

Citation Format: Toshihiko Yusa, Hirohisa Okabe, Yo-ichi Yamashita, Takanobu Yamao, Naoki Umezaki, Tatsunori Miyata, Shigeki Nakagawa, Hiromitsu Hayashi, Katsunori Imai, Akira Chikamoto, Takatoshi Ishiko, Hideo Baba. The role of tumor-associated neutrophils (TAN) and CD8 positive T lymphocyte in the progression of hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1084.
Abstract 1092: V-ATPase family regulates lysosomal exocytosis and neutralizes with tumor-infiltrating lymphocytes for glioblastoma cells
Authors: Chang Yu-Chan , Hsiao Michael
Published: August 2019

Immunotherapy based on checkpoint inhibitors provides substantial clinical benefit, but only to a minority of cancer patients. Targeted immunological checkpoint pathways have demonstrated anti-tumor cytotoxicity in the treatment of refractory cancers. However, cancer cells release specific “drones”, such as small vesicles, which increase their ability in the blood and released the proteins such as PD-L1 to block the attack from T cells then they have a change to reach the tumor forming. To characterization of exosomes forming, trafficking and secretion can be a novel therapeutic approach to improve immunotherapy in cancer patients. The ATPase H+ Transporting V0 and V1 subunits (V-ATPase, also called ATP6 family) are critical for acidifying a variety of intracellular compartments and lysosomal exocytosis. Nevertheless, the dysfunctional roles of V-ATPases in glioblastoma has not yet been elucidated. For this purposed, we investigated V-ATPase members in lysosomal vesicles and found several candidates are key factors in regulating lysosomal exocytosis in glioblastoma cells. We established transcriptomics datasets by various glioblastoma cell lines, the results showed V-ATPases are highly expressed in malignant cell lines compared to benign or normal immortalized cells. Immunohistochemistry also performed a trend of consistency that increased of V-ATPase expression in tumor parts than normal adjacent tissues. The expression level of several V-ATPase subunits is also associated with clinical parameters and survival rate. Moreover, we observed the number of tumor infiltrating lymphocyte (TILs) was decreased in clinical cohorts and two-way cell models. Therefore, we established V-ATPases related proteomics datasets and found some interaction partners could stabilized protein structure of V-ATPase. Specific peptide can compete between V-ATPase and the interacting partners and further modulate TILs in glioblastoma. This approach can be a novel therapeutic strategy that combines immunotherapy to against glioblastoma in clinical patients.

Citation Format: Yu-Chan Chang, Michael Hsiao. V-ATPase family regulates lysosomal exocytosis and neutralizes with tumor-infiltrating lymphocytes for glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1092.
Abstract 1094: Lipopolysaccharide-induced chronic inflammation promotes lung tumorigenesis in the context of an immunosuppressive microenvironment
Authors: Liu Chia-Hsin , Chen Kong , Di Yuanpu
Published: August 2019

Clinical and epidemiological evidence suggest that chronic infection and inflammation increase risk of lung cancer. Pseudomonas aeruginosa infection is frequently found in patients with chronic obstructive pulmonary disease (COPD) and is associated with increased lung inflammation and acute exacerbations. However, the mechanism of chronic bacterial infection-induced lung inflammation in promoting lung tumorigenesis remains unclear. To elucidate this mechanism, we established a murine lung cancer model by treating mice with or without recurrent lipopolysaccharides (LPS) from Pseudomonas aeruginosa in combination with nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Interestingly, combined LPS and NNK exposure significantly increased tumor number, tumor incidence, and tumor area compared to NNK treatment alone. In addition, the inflammatory cell counts in the bronchoalveolar lavage (BAL) including macrophages, neutrophils, and lymphocytes were significantly increased in the LPS/NNK treatment group. The BAL fluid of chemokines/cytokines, as analyzed by luminex assays, revealed higher levels of IL-17, CXCL10, GM-CSF, G-CSF, MIP-1a, and KC in LPS/NNK than in NNK treatment group. Flow cytometry analysis of the mouse lung tissue revealed that combined LPS and NNK exposure significantly increased CD4+ T cells including Th1, Th17, and Tregs and myeloid-derived suppressor cells recruitment in the lung. Real-time polymerase chain reaction of mouse lung tissue showed T cell exhaustion related genes, including Pdcd1, Ctla-4, Tim-3, Lag-3, and Foxp3, were significantly upregulated in the LPS/NNK treatment than NNK treatment. Moreover, immunohistochemical staining of LPS/NNK-exposed lung tumors showed higher PD-L1 expression than NNK-exposed lung tumors. Our data suggest that chronic LPS exposure-promoted and NNK-induced lung tumorigenesis is associated with immunosuppressive tumor microenvironment. The changes include recruitment of Tregs and MDSCs, increased T cell exhaustion, and upregulated PD-1/PD-L1 pathway, which may be used as the therapeutic target for chronic inflammation-associated lung cancer treatment.

Citation Format: Chia-Hsin Liu, Kong Chen, Yuanpu Di. Lipopolysaccharide-induced chronic inflammation promotes lung tumorigenesis in the context of an immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1094.
Abstract 1104: Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment
Authors: Ravishankar Buvana , Adusumilli Lavanya , Pookot Deepa Pookot , Huang Emily , Sreenivasan Raashi , Marshall Lisa , Kaveri Deepika , Talay Oezcan , Suthram Silpa , Miakicheva Svetlana , Okal Abood , Zibinsky Mikhail , Jackson Jeffrey , Shibuya Grant , Leger Paul , Tivitmahaisoon Parcharee , Jacobson Scott , Wong Steve , Wadsworth Angela , Sanchez Jerick , Brovarney Martin , Chian David , Marubayashi Sachie , Jorapur Aparna , Bradford Delia , Colas Christophe , Cutler Gene , Schwartz Jacob , Wustrow David , Kassner Paul , Brockstedt Dirk
Published: August 2019

The tumor microenvironment (TME) is characterized by deficiencies in oxygen and key nutrients, such as glucose and amino acids, resulting in an overall immune-suppressive environment. Key suppressive cell types in the TME include tumor, stromal and myeloid-derived suppressor cells (MDSC) which create a nutrient-poor environment that supports tumor growth and limits immune surveillance. General control nonderepressible 2 (GCN2), a stress response kinase, plays a key role in sensing and modulating the cellular response to amino acid deprivation. GCN2 activation in T cells triggers the integrated stress response pathway and promotes T cell anergy and apoptosis. We have developed small molecule GCN2 inhibitors (GCN2i) that are highly potent and selective in vitro. Culturing primary mouse or human immune cells under low nutrient conditions activates the GCN2 pathway limiting T cell proliferation and function. Treatment of these nutrient-deprived T cells with GCN2i resulted in rescue of CD8+ T cell proliferation and effector functions. In addition, GCN2 inhibition in MDSC alone fully reversed CD33+MDSC-induced T cell suppression and effector functions. Our GCN2 inhibitors are orally bioavailable with drug like in vivo ADME properties. Our GCN2i is currently being evaluated in vivo, in murine syngeneic tumor models. Our results demonstrate that inhibition of GCN2 is an attractive approach for relieving T cell suppression and promoting effector function, demonstrating GCN2 as a promising therapeutic target for the treatment of cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Buvana Ravishankar, Lavanya Adusumilli, Deepa Pookot Pookot, Emily Huang, Raashi Sreenivasan, Lisa Marshall, Deepika Kaveri, Oezcan Talay, Silpa Suthram, Svetlana Miakicheva, Abood Okal, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Parcharee Tivitmahaisoon, Scott Jacobson, Steve Wong, Angela Wadsworth, Jerick Sanchez, Martin Brovarney, David Chian, Sachie Marubayashi, Aparna Jorapur, Delia Bradford, Christophe Colas, Gene Cutler, Jacob Schwartz, David Wustrow, Paul Kassner, Dirk Brockstedt. Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1104.
Abstract 1107: LAG3 in Solid Tumors as a Potential Novel Immunotherapy Target
Authors: Lee Su Jin , Byeon Sun-ju , Lee Jeeyun , Park Se Hoon , Park Joon Oh , Park Young Suk , Kang Won Ki , Lim Ho Yeong , Kim Kyoung-Mee , Kim Seung Tae
Published: August 2019

We performed prospective immunohistochemical analysis of LAG3 for 430 consecutive patients with advanced gastrointestinal, genitourinary, or rare cancers between June 2012 and March 2016. Most patients (428/430, 99.5%) were evaluable for LAG3 expression by immunohistochemistry. In total, 18.5% (79/428) of the evaluated cancers expressed LAG3, including pancreatic (33.3%, 2 of 6), gastric (24.7%, 21 of 85), colorectal (23.6%, 48 of 203), melanoma (12.5%, 1 of 8), genitourinary (9.5%, 4 of 46), biliary tract (6.3%, 1 of 16), and sarcoma (5.4%, 2 of 37), but not miscellaneous (0.0%, 0 of 14) or hepatocellular (0.0%, 0 of 15) cancer. Among 149 metastatic CRC patients, there was no statistically significant difference in gender, age, primary tumor site, pathologic differentiation, KRAS and NRAS status, BRAF status, and microsatellite instability according to LAG3 status (expressed vs. non-expressed). Among 53 metastatic GC patients, LAG3 was only significantly associated with EBV status (P = .042). Our results add to the emerging literature on LAG3 expression in various cancer types and support the need for extended clinical exploration of this target for immunotherapy.

Citation Format: Su Jin Lee, Sun-ju Byeon, Jeeyun Lee, Se Hoon Park, Joon Oh Park, Young Suk Park, Won Ki Kang, Ho Yeong Lim, Kyoung-Mee Kim, Seung Tae Kim. LAG3 in Solid Tumors as a Potential Novel Immunotherapy Target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1107.
Abstract 111: Tumor microenvironment derived NRG1 promotes antiandrogen resistance in prostate cancer
Authors: Zhang Zeda , Karthaus Wouter , Mota Jose Mauricio , Mu Ping , Wu Chao , Abida Wassim , Linton Eliot , Lee Young Sun , Lee Eugine , Mao Ninghui , Adams Elizabeth , Choi Danielle , Rathkopf Dana E. , Carver Brett , Gopalan Anuradha , Jiang Xuejun , Watson Philip , Sawyers Charles
Published: August 2019

Despite the improved clinical outcomes for patients with advanced prostate cancer due to the use of second generation antiandrogens, acquired drug resistance inevitably occurs and remains the major challenge for prostate cancer therapy. While several cell-autonomous mechanisms of drug resistance have been elucidated previously, for a large number of patients the mechanism of resistance remains unclear. Recent studies point to the importance of the tumor microenvironment (TME), and cancer associated fibroblasts (CAF) in the TME, in mediating tumor progression and resistance to therapy, but whether CAFs specifically contribute to antiandrogen resistance in prostate cancer is not known. Using a preclinical model that faithfully mimics the typical progression of patients on antiandrogen therapy, we found that antiandrogen resistance develops significantly faster when cells are grown in the presence of their cognate CAFs. By carrying out biochemical purification and mass spectrometry analysis, we identified NRG1 as a CAF secreted factor, and found that it can promote antiandrogen resistance in tumor cells, via activation of HER3 in the tumor. Importantly, blocking either NRG1 or HER3 can re-sensitize tumors to antiandrogen treatment in this model. Moreover, NRG1 expression is up-regulated in CAFs after antiandrogen exposure or in androgen deprivation condition. Clinically, increased stroma-NRG1 expression was observed in patients post androgen deprivation therapy but not in hormonally intact men. Taken together, this work has revealed a novel, NRG1-mediated non-cell autonomous mechanism of antiandrogen resistance in prostate cancer, and suggests that therapeutically targeting NRG1 in the setting of metastatic, antiandrogen-resistant prostate cancer with elevated NRG1 could provide significant benefit to patients.

Note: This abstract was not presented at the meeting.

Citation Format: Zeda Zhang, Wouter Karthaus, Jose Mauricio Mota, Ping Mu, Chao Wu, Wassim Abida, Eliot Linton, Young Sun Lee, Eugine Lee, Ninghui Mao, Elizabeth Adams, Danielle Choi, Dana E. Rathkopf, Brett Carver, Anuradha Gopalan, Xuejun Jiang, Philip Watson, Charles Sawyers. Tumor microenvironment derived NRG1 promotes antiandrogen resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 111.
Abstract 1117: The molecular basis of LIF-mediated NPC metastasis
Authors: Liu Shu-Chen
Published: August 2019

Distant metastasis and local recurrence are frequently observed in nasopharyngeal carcinoma (NPC) but the mechanisms remain largely unclear. Here, we report that higher levels of leukemia inhibitory factor (LIF) and LIF receptor (LIFR) are correlated with poorer metastasis/recurrence-free survival. LIF reprograms the invasive mode from collective to mesenchymal migration via acquisition of EMT and invadopodia-associated characteristics. Higher cytoplasmic LIF enhances cancer vascular dissemination and local invasion mechanistically, both in vitro and in vivo, through modulation of LIFR-YAP1-focal adhesion signaling. Pharmaceutical intervention with AZD0530 markedly reverses LIF-mediated cancer dissemination and local invasion through promotion of cytoplasmic accumulation of YAP1 and suppression of focal adhesion kinases. Given the significant role of LIF/YAP1-focal adhesion signaling in cancer progression, targeting of this pathway presents a promising opportunity to block metastasis.

Citation Format: Shu-Chen Liu. The molecular basis of LIF-mediated NPC metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1117.
Abstract 1120: Proinflammatory milieu promotes leptomeningeal metastasis by activation of LCN2
Authors: Chi Yudan
Published: August 2019

The tumor microenvironment is emerging as a critical regulator of cancer progression, especially in metastatic brain malignancies. The unique properties of leptomeningeal metastasis (LM) require a specific framework for acquisition of metastatic traits within the fluid-filled leptomeningeal space. Here, we discuss a number of the distinct features of the proinflammatory environment, including cancer cells and relevant immune cells. We also highlight the exact role of its contribution to cancer growth in the development of LM. Proinflammatory cytokines induce the secretion of cancer-derived LCN2. Mechanistically, we have uncovered that iron, transported by LCN2, supports cancer cell growth. By pharmacologic interference with iron uptake, we will prove its therapeutic beneficence for targeting LM in pre-clinical models.

Citation Format: Yudan Chi. Proinflammatory milieu promotes leptomeningeal metastasis by activation of LCN2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1120.
Abstract 1128: Metastasis suppressor RKIP modulates gene expression variance in triple negative breast cancer
Authors: Yang Dongbo , Pott Sebastian , Rosner Marsha
Published: August 2019

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer with a highly metastatic phenotype. Conventional chemotherapy and radiotherapy are generally ineffective at treating metastasis. Natural metastasis suppressors such as the Raf kinase inhibitory protein (RKIP) are not functional in many TNBC cases and associated with poorer relapse-free survival. Although tumor cell heterogeneity is strongly implicated in contributing to metastasis, it is unknown whether metastasis suppression by RKIP reduces heterogeneity. Therefore, we performed single-cell RNA sequencing of mouse TNBC tumors as well as cultured cells to study the effect of RKIP overexpression on gene expression variance in tumor cells. Elucidating RKIP’s effect on gene expression variance at the single cell level reveals gene sets that are important in tumor cell transitions from non-metastatic to metastatic states. We predict that the gene sets whose variance is decreased by RKIP are effective drug targets for metastatic breast cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Dongbo Yang, Sebastian Pott, Marsha Rosner. Metastasis suppressor RKIP modulates gene expression variance in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1128.
Abstract 1137: Unraveling spatially-dependent interactions of tumor-associated macrophage in the tumor microenvironment
Authors: Wang Victor , Martinek Jan , Brookes Hannah M. , Kim Kyung In , Palucka Karolina , Chuang Jeff
Published: August 2019

Tumor-infiltrating lymphocytes (TIL) are a strong prognostic factor in cancer patient outcomes. This includes immune checkpoint blockade (ICB) of the PD-1/PD-L1 axis, where patients with a higher abundance of pre-treatment TILs respond more frequently. However, tumors often elaborate mechanisms to exclude TILs and/or suppress their function. Identifying negative regulators of TILs is thus of great importance to improve patient outcomes and increase the patient population that may benefit from ICB. Tumor-associated macrophages (TAM) are a known modulator of TIL activity but the interaction between the two cell types is typically characterized with in vitro experiments and spatially-agnostic sequencing, ignoring location-specific factors contributing to the interaction’s net effects. Interrogating the TAM-TIL interactions in an intact tumor microenvironment (TME) will uncover novel mechanisms responsible for TAM function and may be key to reversing TIL immunosuppression. Here we leverage bulk RNA-sequencing with histocytometry, a multiplex quantitative tissue imaging method, to spatially-resolve the TAM-TIL interactions in intact human metastatic melanoma microenvironments. Bulk sequencing distinguishes tumor samples by high and low enrichment of lymphocyte gene sets, with additional sub-stratification by macrophage enrichment. Modular repertoire analysis implicates an interferon response in the differential lymphocyte enrichment, but does not address macrophage-dependent effects. Drawing from physical chemistry concepts, our spatial analyses of cell-cell interactions from histocytometry reveals distinct TAM populations potentially regulating TIL activity. Phagocytic TAMs in the tumor and non-phagocytic TAMs in the stroma physically contact T-cells, with the former interaction strongly correlated with lymphocyte enrichment and T-cell infiltration. Local and regional TAM phagocytosis affinities estimated by the Langmuir adsorption model further detail the nature of this interaction within the tumor, as well as the role of macrophage phagocytosis in regulating TILs. We demonstrate linear models integrating bulk sequencing and spatial metrics to quantify the relationships between genetics, cell-cell interactions, and T-cell invasiveness. Our work unravels important TAM interactions that shape the TME which have not been previously appreciated, providing novel insight into the forces driving T-cell exclusion and revealing new TAM biology to explore further.

Citation Format: Victor Wang, Jan Martinek, Hannah M. Brookes, Kyung In Kim, Karolina Palucka, Jeff Chuang. Unraveling spatially-dependent interactions of tumor-associated macrophage in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1137.
Abstract 1141: Preclinical analysis of biodistribution and PET imaging of a zirconium-89 labeled PSMA-targeted antibody-chelator conjugate
Authors: Hammer Stefanie , Zitzmann-Kolbe Sabine , Oden Felix , Jannsen Joerg , Bickel Eva , Mueller Andre , Everz Lothar , Hagemann Urs B. , Karlsson Jenny , Ryan Olav B. , Hennekes Hartwig , Cole Patricia E. , Mumberg Dominik
Published: August 2019

The PSMA targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) is a targeted alpha therapy approach for metastatic castration resistant prostate cancer that has shown strong anti-tumor activity in PSMA-positive prostate cancer models (Hammer et al. AACR 2017/2018/2019). PSMA-TTC consists of a fully human IgG1 PSMA-targeted antibody covalently linked to a 3,2-HOPO chelator moiety, stably complexing the alpha emitter thorium-227. As this chelator can also complex zirconium-89, positron emission tomography (PET) imaging using the zirconium-89 labeled variant of the PSMA antibody-chelator conjugate could support clinical development of PSMA-TTC by enabling monitoring of organ distribution.

Herein, we describe radiolabeling of the PSMA antibody-chelator conjugate with zirconium-89 and its quality control using bioanalytical assays. In addition, the zirconium-89 labeled PSMA antibody-chelator conjugate was injected into mice bearing LNCaP xenograft tumors and biodistribution was assessed by measurement of radioactivity in tumor and organs as well as PET imaging at different time points after injection.

The PSMA antibody-chelator conjugate was successfully labeled with zirconium-89 at high yield, purity and specific activity. Target binding was confirmed by determination of the immunoreactive fraction. Biodistribution experiments revealed high, long lasting uptake of zirconium-89 in PSMA-positive LNCaP tumors after single injection of the zirconium-89 labeled conjugate. An uptake of 40-50% of the injected dose per gram of tumor was measured at 24, 48, 72, 168 and 240h after injection. The PET images showed that the zirconium-89 labeled PSMA antibody-chelator conjugate provided an excellent image contrast for the delineation of LNCaP xenografts between 48 and 168 h after administration. Other organs showed only very little background and tumor-to-heart ratio was greater than 10 when PET imaging was performed between 48 and 168 h after injection.

These results indicate that the zirconium-89 labeled PSMA antibody-chelator conjugate shows promise for PET imaging studies. These properties may allow its use to support clinical development of PSMA-TTC.

Citation Format: Stefanie Hammer, Sabine Zitzmann-Kolbe, Felix Oden, Joerg Jannsen, Eva Bickel, Andre Mueller, Lothar Everz, Urs B. Hagemann, Jenny Karlsson, Olav B. Ryan, Hartwig Hennekes, Patricia E. Cole, Dominik Mumberg. Preclinical analysis of biodistribution and PET imaging of a zirconium-89 labeled PSMA-targeted antibody-chelator conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1141.
Abstract 1160: Endoplasmic reticulum stress disrupts stemness-related transcriptional regulatory network: Implication for therapy response in cancer
Authors: Olou Appolinaire , Mehla Kamiya , Singh Pankaj
Published: August 2019

Pancreatic cancer is the third leading cause of cancer-associated deaths in the United States. We have recently demonstrated that endoplasmic reticulum (ER) stress correlates with improved survival in human pancreatic cancer patients on gemcitabine therapy. Cancer cell stemness is a significant contributor of poor response to therapy and disease recurrence. We also observed that inducing ER stress can diminish stemness in cancer cells. Hence, to investigate the mechanism of ER stress-mediated alterations in diminishing cancer stemness, we investigated the impact of ER stress on transcriptomic alterations by performing RNAseq studies. We subjected cancer cells to treatment with thapsigargin and the harvested mRNA was utilized for RNAseq analysis. We observed significant alterations in mRNA expression levels of the stemness-associated gene set, which also showed significant enrichment in Ingenuity pathway analysis. Hence, our studies demonstrate that ER stress regulates stemness at the transcriptional level. Our ongoing and future studies will target individual genes/pathways to identify their relative contributions to ER stress-mediated regulation of stemness. Considering the role of stemness in disease recurrence, our studies will provide novel mechanistic insights that may lead to novel therapies for targeting disease recurrence in pancreatic cancer.

Citation Format: Appolinaire Olou, Kamiya Mehla, Pankaj Singh. Endoplasmic reticulum stress disrupts stemness-related transcriptional regulatory network: Implication for therapy response in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1160.
Abstract 1162: CD24 regulates cancer stem cell like traits in bladder cancer
Authors: Oki Akira , Hoque Mohammad Obaidul
Published: August 2019

Tumors are hierarchically organized by a rare population of cancer stem cells (CSCs) that contributes to cancer initiation, progression, and treatment failure. A better understanding of the molecular mechanisms underlying urothelial CSC regulation and the identification of key molecules associated with CSC generation and maintenance are pivotal for the molecular-targeting therapy. CD24 is overexpressed and acts as a lynchpin of tumorigenesis and metastatic progression in urothelial carcinoma of the bladder (UCB). However, its contribution to cancer stem-like traits of CD24 have not been determined. The functional relevance of CD24 was evaluated using in vitro and in vivo approaches. The knockdown of CD24 attenuated cancer stemness properties including sphere formation, self-renewal, invasion, chemo-resistance, and tumor initiation. The high-CD24-expressing cells, isolated from patient-derived UCB xenograft tumors, exhibited their enhanced stemness properties. Biologically, CD24 was associated with the expression of several key CSC-related molecules, including CD133, ABCG2, and YAP1. In human UCB samples, CD24 was overexpressed not only in primary tumors, but also in urine from UCB subjects. Conclusion: CD24 plays a crucial role in maintaining the urothelial cancer stem-like traits.

Note: This abstract was not presented at the meeting.

Citation Format: Akira Oki, Mohammad Obaidul Hoque. CD24 regulates cancer stem cell like traits in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1162.
Abstract 1163: HR23A expression modulates drug resistance through regulation of autophagy and stem cell properties in cancer cells
Authors: Chuang Show-Mei , Yu Chung-Yun , Liu Bang-Hung , Lin Sheng-Chih , Tang Shao-Yi
Published: August 2019

DNA-damaging agents are commonly used as anticancer therapeutics. Unfortunately, drug resistance arises through a combination of mechanisms, with the sensitivity of cells to apoptosis depending on the balance between apoptotic and survival signals. Our previous studies show that knockdown of HR23A, an accessory protein involved in nucleotide-excision repair (NER) at an early lesion-recognition step, induces autophagy, increases the resistance to cisplatin, oxaliplatin, 5-FU and docetaxel. However, knockdown of HR23A contributed no effect to the sensitivity of tamoxifen or irinotecan. In present study, we found that knockdown of HR23A displayed more sensitivity to the treatment of doxorubicin in A549 cell line. Knockdown of HR23A enhanced cell death via increased ER stress-mediated apoptosis but not autophagy, suggesting a different role of HR23A in response to doxorubicin. Further investigation revealed that HR23A-knockdown cells appear to undergo epithelial-mesenchymal transition and take on certain attributes of cancer stemness. Our experiments show that depletion of HR23A enhances epithelial-mesenchymal transition, EMT regulator protein levels such as Twist1, Slug and Zeb1, cancer cell migration and various cancer stemness properties, including the expression of major pluripotency factors, the capacity for tumor-sphere formation in culture and the expression of cancer stem cell surface markers. The increases of these stemness properties are reversed by ectopic expression of HR23A or further knockdown of Twist1 in HR23A-depleted cells. Furthermore, HR23A directly targets the Twist1 protein and contributes to regulating Twist1 protein stability without changing its gene transcription. Together, our data suggest that knockdown of HR23A induces autophagy, undergoes epithelial-mesenchymal transition and promotes stemness properties, thus leading to the resistance to DNA-damaging agents.

Citation Format: Show-Mei Chuang, Chung-Yun Yu, Bang-Hung Liu, Sheng-Chih Lin, Shao-Yi Tang. HR23A expression modulates drug resistance through regulation of autophagy and stem cell properties in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1163.
Abstract 119: The influence of CD44/HA signaling on CML survival and resistance to tyrosine kinase inhibitor treatment
Authors: Humphries Kylie , Uchakina Olga , Hostetler Bryan , McKallip Robert
Published: August 2019

The development of tyrosine kinase inhibitors (TKIs) such as imatinib has greatly improved the prognosis and survival of patients diagnosed with chronic myeloid leukemia (CML). However, many CML patients relapse due to the presence of minimal residual disease and the development of TKI resistance. It is unknown exactly what causes the relapse, but based on research from our laboratory we hypothesize that hyaluronic acid (HA) produced by fibroblasts in the microenvironment of the bone marrow provide for a protective environment preventing the tumor cells from therapy-induced cell death or senescence. The purpose of this study is to determine the influence that HA and its binding to its receptor (CD44) has on CML survival and resistance development. Initially, we examined the effect that stromal cell derived and/or exogenously added HA has on CML resistance to imatinib. Cell survival, apoptosis, HA production, and signaling events elicited by the interactions of HA with CD44 was examined using methods such as ELISA, western blotting, and MTT analysis. Furthermore, we examined the potential therapeutic use of compounds known to alter HA production and/or signaling for the treatment of CML. Our study demonstrates increased cell survival when the tumor cells are co-cultured with fibroblasts or have exogenous HA added to them after imatinib treatment. Additionally, we show that knocking out hyaluronic acid synthases reduces this protective effect. This data supports our hypothesis and we believe it could help lead to improved therapeutic treatment for CML.

Note: This abstract was not presented at the meeting.

Citation Format: Kylie Humphries, Olga Uchakina, Bryan Hostetler, Robert McKallip. The influence of CD44/HA signaling on CML survival and resistance to tyrosine kinase inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 119.
Abstract 12: A novel Hsp90 machine inhibitor combining chemotherapy and immunotherapy
Authors: Chadli Ahmed , Eisa Nada , Crowley Vincent , Lu Sumin , Jilani Yasmeen , Korkaya Hasan , Blagg Brian
Published: August 2019

Molecular chaperones are guardians of the proteome and of cellular homeostasis. Dysregulation in the function of the Hsp90 chaperoning machine in particular has been implicated in metabolic, oncological, neurodegenerative, and cardiovascular diseases. Targeting Hsp90 has been shown to have a combinatorial impact on dysfunctional circuitries that underlie human cancers. Thus, several inhibitors targeting the N-terminal ATP-binding pocket of Hsp90 are undergoing clinical trials as anticancer agents. They inactivate the ATPase activity of the chaperone, causing proteasomal degradation of its “client” oncogenic proteins. Although these inhibitors provided a proof-of-principal that Hsp90 is a valuable therapeutic target, they exhibit modest activity in the clinic, in part because they concomitantly induce a heat shock response (HSR), which turns on pro-survival pathways. Consequently, new mechanisms to inhibit the Hsp90 chaperone machine that do not induce the heat shock response are needed.

To address this, we have developed a unique high throughput screen (HTS) based on the progesterone receptor (PR) and the five purified chaperones required for folding steroid receptors, i.e., Hsp90, Hsp70, Hsp40, Hsp70/Hsp90 organizing protein (Hop), and p23. During our preliminary screening of natural products, we discovered that the compound AD07 kills cancer cells through inhibition of the Hsp90 machine without a significant induction of HSR. In addition, AD07 induces a powerful T cell-mediated immune response, resulting in highly efficient tumor killing in a syngeneic mouse model and long-term memory against the primary tumor. At the molecular level, AD07 reduces the protein levels of two key mediators of tumor-induced immune tolerance: programmed cell death ligand-1 (PDL-1) and indolamine 2,3 dioxygenase (IDO). We therefore propose that AD07 is a promising anti-tumor agent targeting the Hsp90 machine through a novel mechanism of action involving cancer cell toxicity, which increases its immunogenicity, and modulation of the tumor microenvironment to reduce immunotolerance.

Citation Format: Ahmed Chadli, Nada Eisa, Vincent Crowley, Sumin Lu, Yasmeen Jilani, Hasan Korkaya, Brian Blagg. A novel Hsp90 machine inhibitor combining chemotherapy and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 12.
Abstract 1206: Inhibition of GPR4 attenuates intestinal inflammation in a mouse colitis model
Authors: Sanderlin Edward J. , Marie Mona , Velcicky Juraj , Loetscher Pius , Yang Li V.
Published: August 2019

Inflammatory bowel disease (IBD), a chronic inflammatory disorder of the digestive tract, is associated with a higher risk of colorectal cancer development. Current therapeutic approaches for IBD are limited and primarily include biologics and steroids such as anti-TNFα monoclonal antibodies and glucocorticoids. Chronic usage, however, can cause significant adverse drug effects such as increased risk of infection and lymphoma in some patients. GPR4, a pH-sensing G protein-coupled receptor, has recently emerged as a potential therapeutic target for intestinal inflammation. Previous studies demonstrate that GPR4 is a pro-inflammatory receptor expressed in vascular endothelial cells (EC) and stimulates leukocyte-EC adhesion and extravasation. Knockout of GPR4 alleviates intestinal inflammation in IBD mouse models. In this study, we have assessed the effects of a recently developed GPR4 antagonist, 2-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)methyl)phenyl)-5-(piperidin-4-yl)-1,3,4-oxadiazole (GPR4 antagonist 13, also known as NE 52-QQ57), in the dextran sulfate sodium (DSS)-induced acute colitis mouse model. The GPR4 antagonist 13 inhibited intestinal inflammation. All colitis parameters, such as body weight loss, fecal score, colon shortening, splenic expansion, and mesenteric lymph node enlargement, were reduced in the GPR4 antagonist 13 treatment group compared to the vehicle group. Histopathological features of active colitis and inflammatory gene expression of colon tissues were reduced in the GPR4 antagonist 13 treatment group compared to vehicle control. Our results indicate that the GPR4 antagonist 13 provides a protective effect in the DSS-induced colitis mouse model and inhibition of GPR4 can be explored as a novel anti-inflammatory approach.

Citation Format: Edward J. Sanderlin, Mona Marie, Juraj Velcicky, Pius Loetscher, Li V. Yang. Inhibition of GPR4 attenuates intestinal inflammation in a mouse colitis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1206.
Abstract 1207: Reversing lactate-driven immunosuppression using the novel, potent and selective MCT4 inhibitor AZD0095
Authors: Critchlow Susan E. , Hughes Gareth , Staniszewska Anna , King Matt , Michopoulos Filippos , Hopcroft Lorna , Brown Martin R. , Bradley Jenna , Hammond Beverley , MorentinGutierrez Pablo , Carnevalli Larissa , Hardaker Elizabeth , Goldberg Frederick W.
Published: August 2019

Metabolic adaptation to a Warburg phenotype is a hallmark of cancer, where tumors rely on glucose to produce ATP and building blocks required to support rapid tumor growth. Glycolytic metabolism requires continuous export of lactate from cells by monocarboxylate transporters (MCTs) resulting in accumulation of lactic acid in the tumor microenvironment. Tumor-derived lactate is immune-suppressive and has been shown to modify immune cell function. Inhibition of the MCT4 lactate transporter has the potential to reduce lactate in the tumor microenvironment and reverse immune suppression. AZD0095 is a highly selective, potent inhibitor of MCT4 with a cellular activity of 1-3 nM with a >1000-fold selectivity over MCT1. We confirm that lactic acid inhibits T cell proliferation and viability. AZD0095 does not inhibit T cell proliferation or survival (IC50 > 10 μM). Oral administration of AZD0095 (10-100 mg/kg BID) modulates lactate transport in mouse syngeneic models which do not express the MCT1 lactate transporter. AZD0095 treatment causes an increase in tumor-infiltrating lymphocytes and an increase in cytotoxic T cells in combination with α-PD1. AZD0095 treatment suppresses myeloid infiltration as a monotherapy and in combination with checkpoint inhibitor. Consistent with the observed changes in immune cell infiltration, the combination of AZD0095 with checkpoint inhibitors α-PD1 or α-CTLA4 (10 mg/kg 3x week) enhances the anti-tumor activity of checkpoint inhibitor monotherapy in MC-38 and EMT6 (MCT1 knock-out) mouse syngeneic models. These data demonstrate that AZD0095 can reverse lactate-driven immunosuppression and enhance response to checkpoint inhibition in pre-clinical models.

Citation Format: Susan E. Critchlow, Gareth Hughes, Anna Staniszewska, Matt King, Filippos Michopoulos, Lorna Hopcroft, Martin R. Brown, Jenna Bradley, Beverley Hammond, Pablo MorentinGutierrez, Larissa Carnevalli, Elizabeth Hardaker, Frederick W. Goldberg. Reversing lactate-driven immunosuppression using the novel, potent and selective MCT4 inhibitor AZD0095 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1207.
Abstract 1210: Targeting the IDO1-Kynurenine-AhR pathway for cancer immunotherapy
Authors: Pinto Sheena , Steeneck Christoph , Albers Michael , Anderhub Simon , Birkel Manfred , Buselic-Wölfel Larisa , Eisenhardt Gisela , Kremoser Claus , Hoffmann Thomas , Deuschle Ulrich
Published: August 2019

The aryl hydrocarbon receptor (AhR) is a ligand-controlled transcription factor that is primarily known to be a sensor of xenobiotics and tumor-promoting activities of halogenated hydrocarbons and polycyclic aromatic hydrocarbons. Several endogenous ligands such as metabolites produced by commensal microorganisms on skin and in gut and metabolites of L-Tryptophan - produced under control of the Tryptophan dioxygenases IDO1 and TDO2 such as L-Kynurenine and Kynurenic acid are known to modulate the transcriptional activity of AhR. The activity of AhR is predominant in many different immune and epithelial cells, thereby balancing immune responses towards various signals.

Activation of the IDO1-Kyn-AhR pathway and accumulation of nuclear AhR protein is frequently seen in different tumor types and possibly linked to the observed diminished anti-tumor immune response. AhR agonizing ligands produced by cancer cells and/or lymphocytes recruited to the TME reduce the anti-tumor immune response through increasing the numbers and function of regulatory T cells while reducing that of cytotoxic CD8+ T cells.

In order to reduce AhR-mediated immune-suppression in cancer patients, Phenex Pharmaceuticals initiated a program to identify small molecule AhR antagonists to block activated downstream signaling of AhR. To this end, we have identified novel AhR antagonists, which showed strong antagonistic activity against agonist-activated human AhR in a cell based CYP1A1 promoter-driven luciferase reporter assay in HepG2 cells. The compound series display good oral bioavailability and low clearance in mice. Our AhR antagonist molecules show single agent anti-tumor activity and increase the efficacy of Gemcitabine and anti-PD-L1 checkpoint inhibition in different syngeneic mouse tumor models. Overall, AhR antagonist treatment increased CD8+ T cell and M1 macrophage numbers, and decreased IL22 levels, which might be of functional relevance in the tumor context. Future research is directed to determine PK-PD markers suitable for clinical development and identification of human malignancies likely of responding to AhR antagonist treatment.

Citation Format: Sheena Pinto, Christoph Steeneck, Michael Albers, Simon Anderhub, Manfred Birkel, Larisa Buselic-Wölfel, Gisela Eisenhardt, Claus Kremoser, Thomas Hoffmann, Ulrich Deuschle. Targeting the IDO1-Kynurenine-AhR pathway for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1210.
Abstract 1213: RNA-based screens in primary human immune cells
Authors: Joubert Bronwyn , Ghirelli Cristina , Nett Isabelle , Prime John , Martin Glynn , Moore Jonathan , Cross Benedict , McCarthy Nicola J.
Published: August 2019

Genetic screens have long been used as an approach to identify and validate new targets for drug discovery. The vast majority of these have been carried out in cell lines: mostly cancer cell lines. However, with improvements in tissue culture techniques, the increasing interest in using the immune system to tackle disease and the discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 mediated genome editing, screening primary cells that have not been subverted by transformation into immortal lines, is both appealing and feasible.

We have successfully carried out a CRISPR-Cas9 screen in primary T cells using a combined lentivirus and electroporation protocol. Freshly isolated primary T cells are stimulated with anti-CD3 and anti-CD28 antibodies and then transduced with a pooled sgRNA library. After antibiotic selection, T cells successfully transduced with sgRNAs are electroporated to introduce Cas9 mRNA.

We chose to validate this approach by carrying out a screen similar to that published by Birsoy et al., 2015, in which they ran a CRISPR-Cas9 screen in Jurkat T cells in the presence and absence of the electron transport chain inhibitor phenformin. In our screen, we exposed the CRISPR-Cas9 edited pool of primary T cells to a dose of phenformin that resulted in growth inhibition to a similar degree to that used by Birsoy and colleagues. Our data are in agreement with the published screen, showing that loss of the cytosolic aspartate aminotransferase GOT1 sensitises primary T cells to phenformin. We took multiple time points in our primary T cell screen and used T cells isolated from three different donors, allowing for the analysis of guide drop-out kinetics and reproducibility between donors.

We anticipate that these data will be useful in building more complex screens that assess T cell biology in the presence of additional cells, such as myeloid derived suppressor cells (MDSCs). With a view to this, we have also carried out an arrayed siRNA screen in MDSCs to look for genes that when knocked down reduce the capacity of MDSCs to inhibit T cell proliferation. The endpoint for this screen is based on co-culture of siRNA transfected MDSCs with proliferating primary T cells.

Using this complex data set, we have identified several potential targets, which when validated could provide new therapeutic targets through which the immunosuppressive nature of MDSCs in the tumour microenvironment can be mitigated.

Birsoy, K., et al. (2015) http://dx.doi.org/10.1016/j.cell.2015.07.016

Citation Format: Bronwyn Joubert, Cristina Ghirelli, Isabelle Nett, John Prime, Glynn Martin, Jonathan Moore, Benedict Cross, Nicola J. McCarthy. RNA-based screens in primary human immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1213.